Nanobio Pharmaceutical Technology Elsevier PDF

Nanobio Pharmaceutical Technology
Applications and Perspectives

Editors
Latha Subbiah
Selvamani Palanisamy
Subramanian Natesan
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Reed Elsevier India Pvt. Ltd.
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Organising Committee
CHIEF PATRON
Dr. M. Rajaram, Hon’ble Vice-Chancellor, Anna University, Chennai
PATRON
Dr. S. Ganesan, Registrar, Anna University, Chennai
CHAIR
Dr. T. Senthil Kumar, Dean, Anna University, BIT Campus, Tiruchirappalli
ADVISOR
Dr. K. Ruckmani, Professor & Head, Dept. of Pharmaceutical Technology,
Director, Centre for Excellence in Nanobio Translational REsearch (CENTRE),
Anna University, BIT Campus, Tiruchirappalli
ORGANIZING SECRETARIES
Dr. S. Latha & Dr. P. Selvamani, Assistant Professor, Dept. of Pharmaceutical Technology, Anna University, BIT
campus, Tiruchirappalli
MEMBERS
Dr. A. Puratchikody
Dr. N. Subramanian
Dr. E. Sanmugarpriya
Dr. R. Vijaya
Mr. A. Shanmugarathinam
Mrs. A. Umamaheswari
Mr. R. Suriyakanth
Dr. P. SenthamilSelvan
Mrs. K. Akilandeswari
Dr. K. Kavitha
Dr. S. Lakshmanaprabu
Mrs. M. Vijayalakshmi
PROCEEDINGS EDITORIAL BOARD CHAIRPERSON

Mr. A. Shanmugarathinam
PROCEEDINGS EDITORIAL BOARD MEMBERS

Mrs. M. Arputha Bibiana
Mr. C. Prabu
Ms. P.S. Dhivya
Mr. S. Rajkumar
Mrs. M. Poornima
Ms. S. Monisha
Ms. C. SherlinaDaphny
Ms. T. Supassri
ADVISORY BOARD MEMBERS
INTERNATIONAL SILVIO DUTZ TANAJI T. TALELE

Technische UniversitätIlmenau, Germany St. John’s University, USA
JAGAT KANWAR RAVI PALANIAPPAN
Deakin University, Australia Mercer University, Atlanta, USA
MANIKAM SIVAKUMAR JITKANG LIM
The Nottingham University, Malaysia Universiti Sains Malaysia, Malaysia
R. THIRUMURUGAN KARINA POMBO GARCIA
International Medical University, Helmholtz-Zentrum Dresden-Rossendorf,
Malaysia. Germany
R. RAJAN SHEEBA DAVID
International Medical University, International Medical University,
Malaysia. Malaysia.
L. MANIKANDAN DEEPAK BALAJI
Forma Therapeutics, Singapore Thimiri Consulting Group, France
RENGANATHAN ARUN, NIRMESH JAIN
University of Zurich, Switerland University of Sydney, Australia
ASHOK KUMAR VASUDEVAN MANI
ASIA Metropolitan University, Malaysia. Universiti Teknologi MARA, Malaysia
NATIONAL R. MANAVALAN RAJIV DAHIYA
Annamalai University, Tamil Nadu, India Global College of Pharmacy, UP, India
T.K. PAL T.N.K. SURIYA PRAKASH
Bioequivalence Study Centre, Al-Shifa College of Pharmacy, Kerala,
West Bengal, India India
DHIRENDER BAGADHUR P. GOPINATH
IIT, Mumbai IIT, Roorkee
P.JAISANKAR RANU DUTTA
Indian Institute of Chemical Biology, West University of Allahabad, Uttar Pradesh,
Bengal, India India
J.K. GUPTA L.K. GHOSH
Jadavpur University, West Bengal, India Jadavpur University, West Bengal, India
P. GAUTAM B.S. LAKSHMI
Anna University, Tamil Nadu, India Anna University, Tamil Nadu, India
M. KRISHNAN G. MATHAN
Bharathidasan University, Tamil Nadu, Bharathidasan University, Tamil Nadu,
India India
P. RAJAGURU R. SIVAKUMAR
Anna University, Tamil Nadu, India Grace College of Pharmacy, Kerala, India
G.P. MOHANTA G. MARIAPPAN
Annamalai University, Tamil Nadu, India Kamla Nehru Institute of Management
and Technology,Uttar Pradesh, India
S. KAVIMANI R. NETHAJI
Mother Teresa Post Graduate and Research Devakiammal College of Pharmacy,
Institute, Pondicherry, India Kerala, India
C. DHANAPAL R.S. JEYA PRAKASH
Annamalai University, Tamil Nadu, India Manipal College of Pharmaceutical
Sciences, Karnataka, India
M.V. RAO M. SIVAKUMAR
Bharathidasan University, Tamil Nadu, India Anna University, Tamil Nadu, India
M.B. VISWANATHAN K. NEHRU
Bharathidasan University, Tamil Nadu, India Anna University, Tamil Nadu, India
T.K. RAVI V. RAJESHKANNAN
Sri Ramakrishna Institute of Paramedical Bharathidasan University, Tamil Nadu,
Sciences, Tamil Nadu, India India
J. NIRMAL K. ASOKKUMAR
Dr.HarisinghGour University, Madhya Sri Ramakrishna Institute of Paramedi-
Pradesh, India cal Sciences, Tamil Nadu, India
D.C. SUNDARAVELAN C. VIJAYARAGHAVAN
Sri Ramakrishna Institute of Paramedical PSG College of Pharmacy, Tamil Nadu,
Sciences, Tamil Nadu, India India
Contents
SECTION-I NANOMATERIALS
1 In vitro Antimicrobial Efficacy of Zinc Doped Nano Hydroxyapatite

Against Human Pathogens 3
R. Baskar, G. Devanand Venkatasubbu and K. Umamaheswari
2 Synergistic Antibacterial Efficacy of Two Drugs in Addition With Biosynthesized
AgNPs From an Allergenic Fungus 8
Chitra. N, B. K. Nayak, and Anima Nanda
3 Synthesis and Characterization of PAMAM Dendrimer and its Application
for Removal of Heavy Metals 14
E. Gomathi, P. Rajesh Prasanna & P. Selvamani
4 Bio-Toxicity of Silver Nanoparticles Against Multidrug Resistant Pathogens
and Human Epidermoid Larynx Carcinoma (HEp-2) Cells 21
Muthukrishnan Lakshmipathy and Anima Nandaa
5 Design and Evaluation of Polyvinyl-Gum Ghatti Self-Assembled Nanoscale Particles |
for Oral Delivery of Simvastatin 25
Bibek Laha, Leena Kumari, Arpana Kashyap and Sabyasachi Maiti
6 Green Synthesis and Characterization of Herbal Mediated Silver Nano Particles
Using Gymnema Sylvestre (Linn.) 31
Kalakotla Shanker and Gottumukkala Krishna Mohan
7 Synthesis and Characterization of Green Silver Nanoparticles Mediated by
Aegle marmelos (L.) Leaf Extract 38
Sukumar Dandapat, Manoj Kumar and M. P. Sinha
8 Invitro Cytotoxicity Studies of Nickel Oxide Nanoparticles on Cancer Cells Using MTT Assay 45
K. Perumal Raj, V. Thangaraj, Dr. A. P. Uthirakumar, P. Sivakarthik
9 Characterization Studies of Sunlight Induced Biosynthesis of Silver
Nanoparticles Using Solanum melongena (Egg Plant) 50
V. Aravindhan, C. Senthil Kumar, H. Linda Jeeva Kumari and K. Ruckmani
10 Thermal, Anti-Fungal and Primary Electrochemical Studies of Palladium Nanoparticles 59
N. John Sushma, K. Mallikarjuna, D. Prathyusha, G. Narasimha, G. Swathi, B.V. Subba Reddy and
B. Deva Prasad Raju
11 Optimization Studies on Bioinspired Green Synthesis of Silver Nanoparticles
Using Clitoria Ternatea66
N. John Sushma, G. Swathi, D. Prathyusha and B. Deva Prasad Raju
12 Biosynthesis and Characterization of Silver and Gold Nanoparticles
in Piper Nigrum L. (Piperaceae) 73
M. Manogaran and M. B. Viswanathan
13 Synthesis of Gsh & Fa Conjugated Chitosan Functionalized Gold Nanospheres:
Physicochemical Properties and Applications in Cancer Therapy 84
Kalpana Haria, Hemamalini Vedagirib, and Premkumar Kumpati
14 Antimicrobial, Antioxidant and Angiogenic Potential of Silver Nanoparticles
From Leaves of Murraya Paniculata (L.) Jack 91
Rama. P, Vignesh. A and K. Murugesan
15 Biosynthesis of Silver Nanoparticles Using Tephrosia Tinctoria for
Antibacterial Activity Against Multidrug Resistant Pathogens 105
D.C. Aiswaryaa, K. Rajaram and P. Sureshkumar
xContents
16 Green Synthesis of Silver Nanoparticles From Leaf Extracts of Breynia Vitis-Idaea111
B. Anandaraj, T. P. Rajesh, P. Suresh Babu and C. Narendhar
17 Characterization of Solid State Forms of Pioglitazone 117
B. Poornima, K. V. S. R. G Prasad and K. Bharathi
18 Synthesis of Metaloxide Nano Composites for Solar Cells 125
S. Shanthi and Dr. M. Dharmendirakumar
19 Phytogenic Synthesis of Silver Nanoparticles and its Potential Foron Blue Dye Decolorisation 133
P. Balashanmugam, P. Arthi and P. T Kalaichelvan
20 Aristolochia Indica L. Mediated Synthesis of Nano-Silver Particles for its Antimicrobial Activity
Against Human Pathogens 139
G. Sathishkumar, C. Rajkuberan, K. Ravindran and S. Sivaramakrishnan
21 Pharmacokinetics of Enrofloxacin Loaded Solid Lipid Nanoparticles Following
Oral Administration in Emu (Dromaius Novaehollandiae) Birds 149
P. Senthil Kumar, A. Arivuchelvan, A. Jagadeeswaran, N. Subramanian, C. Senthil Kumar
and P. Mekala
22 Antidiabetic Activity of Chromium (iii) Nanoparticle Against Streptozotocin-Induced
Diabetis in Rats 157
Kanakalakshmi Annamalai, Anjali Mohan Nair and Shanthi kuppusamy
23 Formulation of Soap and Cream From Biosynthesised Silver Nanoparticles
of Adathoda Vasica Nees. Leaf Extract 162
H. Linda Jeeva Kumari, S. Sonia, R. Krishnamoorthy, M. Sivakumar and K. Ruckmani
24 Invitro Cytotoxicity Assessment of Silver Nanoparticles Synthesized Using
Aspergillus Terreus Against Breast Cancer Cell Line 173
M. D. Bala Kumaran and P. T. Kalaichelvan
25 Larvicidal and Antimicrobial Potency of Silver Nanoparticles Synthesized
Using an Actinomycete, Saccharopolyspora Erythraea178
Prabha S. B. and K. Murugesan
26 Biomimetic Production and Cytotoxic Effect of Silver Nanoparticles From Fungi 187
S. Akila and Anima Nanda
27 Synthesis and Characterization of Andrographis Paniculata Loaded
Nanoparticles for the Development of Antimicrobial Poly Cotton Fabrics 193
R. Rajendran, K. Hemalatha, A. Manikandan and R. Radhai
SECTION-II NANO DRUG DELIVERY SYSTEMS
1 M odified Release Multiparticulate Delivery System for Tramadol HCL

Using Gelucire 43/01 as Rate Retarding Material 201
Divya Priya. S, Sruthi. S, Hema. R, Rajasekar. K, Ganesan. V, Lakshamana Prabu. S,
Puratchikody. A, Shanmugarathinam. A.
2 Extraction and Characterization (Both Physico-Chemical and Analytical) of Pectin
Obtained From Dillenia Indica and Abelmoschus Esculentus and Compatibility
Study With Glipizide for Application in Novel Drug Delivery Systems 207
Sivasankar Mohanty, G. Krishna Mohan and M. Sunitha Reddy.
3 Preparation and Characterization of Leflunomide Loaded Nanosuspensions
for Rheumatoid Arthritis 214
S. Lakshmana Prabu, S.P. Sharavanan, A. Shanmugarathinam, K. Ruckmani and A. Bhuvaneswari
4 Diclofenac Sodium Nanosuspension: An Approach To Improve Anti-Inflammatory Therapy 220
S. Lakshmana Prabu, S.P. Sharavanan, A. Shanmugarathinam, K. Ruckmani, S. Aravindan,
A. Bhuvaneswari and V. Manikandan
Contentsxi
5 Formulation and In Vitro - In Vivo Evaluation of Wedelolactone Loaded Nanosuspension

for Hepatoprotective Activity in CCl4 Induced Significant Hepatic Damage
and Oxidative Stress Model 226
S. Brito Raj, P. Sucharitha, T. Murali1, S. Wasim Raja and K. Bhaskar Reddy
6 Enhanced Oral Bioavailability of Naringenin Using Polymeric Nanoparticles:
Formulation, Characterization and in Vivo Studies 237
P. Suseelaa, K. Premkumarb, S.D. Saraswathya
7 Design and Characterisation of Baclofen Sustain Release Tablet Using Hydrophilic Matrix 245
R. Mohan Kumar, M. Balakumaran, M. Annapoorni, P. Selvamani,
N. Subramanian and K. Ruckmani.
8 Preparation and Evaluation of Miconazole Nitrate Nanoemulsion Using
Tween 20 As Surfactant for Effective Topical and Transdermal Delivery 252
P. Jaya Sree and Dr. C. Thirumal Azhagan
9 Development and Characterization of Enrofloxacin SLNs and its Pharmacokinetics
Following Oral Administration in Emu (Dromaius Novaehollandiae) Birds 258
P. Senthil Kumar, A. Arivuchelvan, A. Jagadeeswaran, N. Subramanian,
C. Senthil Kumar and P. Mekala
10 Redox Environment Cleavable Polymeric Nanoparticles for Drug Delivery 271
M. Gover Antoniraj, C. Senthil Kumar, Angeline Tisha and K. Ruckmani
11 Preparation of Prednisolone Loaded Caco3 Microparticles for Sustained Release 278
C. Prabu, S. Latha and P. Selvamani
12 Development and Evaluation of Artemisinin Magnetic Nanosponges for
Targeting Breast Cancer 289
S. P. Saravanan , P. Chandrasekar, K. Sanjai, Padma @ Rajam, R. Suriyakanth
and N. Subramanian
13 Quercetin Loaded Chitosan/ Peg Nanoparticles: Formulation and
In-Vitro Characterisation 296
Saravanakumar P, Vinoth J, Chandrasekar P and Subramanian N
14 Cancer Targeting With Artesunate Magnetic Nanoparticles Encapsulated
With Thermo-Responsive Polymers 302
Vinoth. J, Sharavanan. S.P, Senthil Kumar. C, Dhinakaran. P and Subramanian. N
15 Formulation and In Vitro Evaluation of Controlled Release Tablets of
Norfloxacin Using Natural Polymers 310
V.Ganesan, S.R.Senthilkumar
16 Exploration of Quail’s Egg Lecithin in Development and Evaluation of
Novel Celecoxib Loaded Lecithin Organogel 316
S. Balaguru, Ramya Devi.D, B.N. Vedha Hari
17 Development of Response Surface Methodology for Tenofovir Disoproxil
Fumarate Microparticles Using Solvent Evaporation Method 326
Fathima A, Vedha Hari BN, Ramya Devi D
18 Formulation and Evaluation of Mouth Dissolving Film Leflunomide
S. LakshmanaPrabu, A. Shanmugarathinam, S.P. Saravanan, M. Elakkiya, V. Manikandan,
A. Bhuvaneswari, S. Aravindan
19 Development and Sustainable Release Evaluation of Enrofloxacin
Solid Lipid Nanopartilces 343
P. Senthil Kumara, A. Arivuchelvanb, A. Jagadeeswaranb, N. Subramanianc,
C. Senthil Kumard and P. Mekalab
xiiContents
20 Formulation and Evaluation of Stavudine Loaded Niosomes and Quantitative

Estimation By HPLC351
S. Latha, P. Selvamani, R. Bharathi , R. Benaseer Begam, T. Keerthana, M. Ponmalar,
K. Hari Gopala Reddy
21 Optimization and Formulation of Drug Loaded Magnetic Microcapsules 358
C. Prabu, S. Latha, P. Selvamani
22 Formulation and Characterization of Carisoprodol Magnetic Nano-Emulsion
A. Stephen Prawin Kumar, G. Phonnavan, G. VinothKumar, M. Neelumathi, P. Selvamani, S. Latha
23 Lipid Nanocarriers of Methotrexate in the Topical Treatment of Psoriasis 376
Vanaja.K, Shailesh Biradar, Narendra C, Shobha Rani RH and Paradkar AV.
SECTION-III BIOTECHNOLOGY
1 Comparison of Cell Surface Glycosylation on Apoptotic Cells 387

Keerthivasan Ambigapathy and Deepak Balaji Thimiri Govinda Raj
2 Packed Bed Column Adsorption of Cr(VI) onto Acid Treated Codium
Tomentosum Biomass396
P. Suresh Babu, S. Eswaramoorthi, T.P. Rajesh and B. Anandaraj
3 Biotransformation of Clozapine to Norclozapine an Active Metabolite
by Cunninghamella Elegans 404
S. Varalaxmi and M. Vidyavathi.
4 Production of Electricity From Municipal Waste Water Using Microbial Fuel-Cells 411
J. Jayabarath, K. Gobika, R. Yuvashri and K. Jeyaprakesh
5 Production of Antimicrobial Secondary Metabolites From Marine Associated
Fluorescent Pseudomonas418
J. Jayabarath, J. Jeny Joseline and S. Sivakavi,
6 Hla G 14Bp Indel Polymorphism Implicated in Genetic Predisposition
to Asthma in Kodaikanal 426
Renuka S, Thenmozhi M and V.J. Kavitha
7 Production and Characterization of Biosurfactant From Hydrocarbon Contaminated Soil 431
S. Monisha, T. Supassri, M. Dhivya, D. Venkatesan, P. Selvamani and S. Latha
8 Optimization of Media for the Production of Keratinase Enzyme 440
B. Anandaraj, T.P. Rajesh, N. Ilavarasan and P. Saranya Devi
9 Enhancementofpost-Harvest and Shelf Life of Carica papaya Using Aloe
Vera Gel Based Coatings 450
Nandakumar. A, Ebenezer Samuel King. J and Arulvel. R
10 Biological Control of Damping Off and Stem Rot of Tomato (Lycopersicon Esculentum Mill.)
Using an Actinomycete, Saccharopolyspora Erythraea457
Prabha S.B and K. Murugesan
11 Exploration for Antimicrobial Proteins From Marine Bivalve 467
G. Vinoth Kumar, M. Arputha Bibiana, C. Sherlina Daphny, P. Selvamani and S. Latha
SECTION-IV PHARMACEUTICAL TECHNOLOGY
1 Phytochemical Investigation and Invitro Anti Diabetic Activity of Myxopyrum

serratulum A. W. Hill 475
K. Kavitha, K. Sujatha, S. Manoharan and Sundara Ramaprabhu
Contentsxiii
2 In Vitro Antioxidant Activities, Total Flavonoids and Total Phenolic Content
of Ethanolic Extract From Whole Plant of Lactuca Runcinata (Dc). 482
Jeyaraman Amutha Iswarya Devi and Arumugam Kottai Muthu
3 GC-MS Characterization of Volatile Odorous Compounds In Allium Cepa488
N.C.J. Packia Lekshmi, S. Viveka, M.B. Viswanathan, G. Manivannan and T. Mini Shobi
4 Antimicrobial Activity Study of Flavonoids and Salicylic Acid Extracted
From Tagetes Erecta Linn.495
R. Devika and Y. Justin Koilpillail
5 Therapeutic Potential of Pleurotus ostreatus: An Edible Mushroom in Human
Cancer Cell Line (Mcf-7) and In Rat Mammary Carcinoma 500
K. Deepalakshmi and S. Mirunalini
6 A Single Chemical Entity to Balance Anti-Amyloid and Anti-Cholinesterase
Capacity: In Silico Drug Design 508
Pavadai Parasuraman, Ramalingam Suresh and Manathusamy Gopalakrishnan
7 A Calcium Channel Blocker - Amlodipine Attenuates Acetic Acid Induced
Ulcerative Colitis in Mice 514
Rajinikanth. B and Venkatachalam. V
8 Antioxidant Activity and Mosquitocidal Activity of Allium Sativum
of Kodaikanal Hilly Areas 521
M. Razia, K. Lavanya and B. Sri Shaila Devi
9 Phytochemical and Antimicrobial Investigations In Pseudarthria Viscida (Fabaceae) 527
S. Tamil Selvi, M. B. Viswanathan, and M. Venkatesan
10 Extraction and Antimicrobial Evaluation of Buffer Extracts of Marine
Invertebrate Donax Cuneatus532
M. Arputha bibiana, C. Sherlina Daphny, M. Umamageshwari, P. Selvamani, S. Latha
11 Evaluation of Anticholinesterase Activity of Cadaba Indica by TLC Bioautography
Method and Isolation of Active Phytoconstituents By Bioassay Guided Fractionation 540
P.S. Dhivya, P. Selvamani, S. Latha, M. Omrohini and M. Sobiya
12 Energy Conservation Opportunities In Paper Plant Using Combined Heat
and Power for Pharmaceutical Application 546
N. Stalin
13 Energy Management and Integration of Pem Fuel Cell With UAV for
Pharmaceutical Application 555
N. Stalin
14 Pharmacognostical Evaluation of Duranta Repens Leaves 567
Emdad Hossain, Ranadheer Rai and Rishikant Tripathi
15 Screening of Aegle Marmelos for Antidiabetic Activity In Streptozotocin
Induced Diabetic Rats 575
S. Jebaseelan, Dr. P. Ramasubramanian and Dr. Venkatesh
16 Anthelminthic Activity of Acalypha Indica Belongs to The Family Euphoriaceae582
S. Kalimuthu, V. Balasubramanian, R. Xavier Arulappa
17 Spectroscopic Studies of Plant Extract of Ixora Coccinea (L.f) Belongs
to the Family Rubiaceae 590
V. Balasubramanian, S. Kalimuthu, N. Balakrishnan
18 Diuretic Activity of Ethanolic Extract of Aristolochia Indica Linn. In Rats. 606
R. Mohan Kumar, M. Balakumaran, P. Selvamani, N. Subramanian and K. Ruckmani
xivContents
19 Invitro Antioxidant and Hepatoprotective Activity of Ethanolic Extract of

Sesbania Grandifolia Linn Leaves 609
K. S. Sridevi Sangeetha and S. Umamaheswari
20 Synthesis and Characterization of Novel Amino Acid Prodrug of Rosiglitazone 615
S. Vijayaraj, G. Kalyana Chakravarthi and R. Shanmugam
21 Assessment of Carcinogenicity Potential By In Vitro Methods: Towards Better Strategy 623
Darshan T. Valani, S. Rajesh Sundar, Mukul R. Jain and Sonal S. Bakshi
22 Antimicrobial Activity of Asystasia Gangetica (L.) T. Anderson Flower Extracts 632
R. Jayalakshmi, P. Selvamani, K. Ruckmani and N. Subramanian
23 Computational Graphical User Interface Tool Development for Personalized
Drug Selectivity and Designed Pharmacology 637
M. Poornima, P. Selvamani and S. Latha
Preface
Multidisciplinary knowledge and skills are required to bring an approved drug product for clinical use
which includes identification of molecular structures, creation of active molecules and development of drug
delivery vehicles for comprehensive therapies.
Recent understanding of chemistry and complex biology has served as a tool for enormous development of
biotechnology which plays an important role to eradicate disease, to maintain good health and to provide
essential requirements for all the life forms.
Nanotechnology is a revolutionary technology of the 21st century and found applications in all facets of
life. Products of nanotechnology are expected to modernize current trends in drug delivery and molecular
medicine which will have significant contributions on the future healthcare system.
Under the realm of unimaginable breakthroughs occurring in bio and nanotechnologies with promising
applications in the area of drug development and delivery generated great research thrust on “Applications
and Perspectives of Nanobio Pharmaceutical Technology.”
We made an attempt to bring down the expertise of renowned academic and industrial researchers here
to provide a comprehensive treatise on this subject through the TEQIP II and DBT, New Delhi sponsored
International Conference on Innovations and Future Research Dimensions on Nanobio Pharmaceutical
Technology. It is organized by the Department of Pharmaceutical Technology of the Centre for Excellence
in Nanobio Translational REsearch (CENTRE) of the Anna University in its Bharathidasan Institute of
Technology Campus, Tiruchirappalli, Tamil Nadu, India.
Among the research reports attracted by the conference, selected research reports in the form of full papers
has been incorporated in this book under various sections namely, Nanomaterials, Nano Drug Delivery
Systems, Biotechnology & Pharmaceutical Technology.
We hope this compilation of recent research reports will offer newer opportunities for the realization of the
promises of Nanobio Pharmaceutical Technology.
Latha Subbiah
Subramanian Natesan
Acknowledgments
We thank the authorities of Anna University, for providing us with the permission and assistance through
TEQIP II grants to produce this proceedings based on the International Conference on Innovations
and Future Research Dimensions on Nanobio Pharmaceutical Technology. The editors acknowledge
the assistance of their doctoral scholars for their editorial assistance of the received research reports.
Specifically, we thank all the individuals who have contributed papers and those who took time from
their busy schedules to review these papers and for their valuable comments and helped to shape this
book. We are grateful to M/s. Bridge People Consultancy Services Pvt. Ltd., Chennai and Grammarly
INC; that helped us to remove many errors of grammar, spelling and related errors in all the published
research reports. Also, we are truly indebted to our family members for their encouragement, patience,
and support during all of our professional endeavors. Finally, we appreciate the support extended by
Mr. K.V.C. Satish Kumar, Knowledge Curve, Chennai and Elsevier for the fine production of this
proceedings.
Latha Subbiah
Subramanian Natesan
SECTION-I
NANOMATERIALS
In Vitro Antimicrobial Efficacy of Zinc Doped Nano
Hydroxyapatite against Human Pathogens
R. Baskar1, G. Devanand Venkatasubbu2 and K. Umamaheswari3
1
Department of Biotechnology, University of Madras, Guindy Campus, Chennai-600025, TN
2, 3
Centre for Biotechnology, Anna University, Chennai-600025, TN
e-mail: rk_uma@yahoo.com, Mob: 09940326510
Abstract:
Hydroxyapatite (HA) nano crystals were synthesized by wet chemical precipitation reaction and doped
with Zinc (Zn) ions at 2 % and 5 % concentration. The morphology, crystallinity and Zn incorporation
into HA nanocrystals were studied using High Resolution Transmission Electron Microscopy (HRTEM),
X-ray Diffraction (XRD) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES)
respectively. The Minimum Inhibitory Concentration (MIC) of Zn-HA nanocrystals was determined against
various human pathogens by micro broth dilution assay. The synthesized Zn-HA nanocrystals exhibited a
uniform morphology with size of 10-20 nm and crystalline in nature. The weight percentage of Zn in 2%
and 5 % Zn-HA nanocrystals were found to be 0.4990 and 1.3740 respectively. The Zn-HA nanocrystals
showed inhibition against E. coli, P. aeruginosa, K. pneumonia, S. aureus and C. albicans at a concentration
between 15 – 500 µg/mL. The results of the present study shows that 5 % Zn-HA nanocrystals shows good
antimicrobial activity than 2 % Zn-HA nanocrystals.
Keywords: Hydroxyapatite, Zinc, Antimicrobial activity, Nanocrystals
INTRODUCTION
Nanoscale structures and materials have been explored in many biological applications because their novel
properties and functions drastically differ from their bulk counterparts. In particular, their high volume/
surface ratio, surface tailorability, improved solubility and multifunctionality open many new possibilities
for biomedical application [1]. Calcium phosphate nanoparticles have gained increasing interest in medicine
because of their high biocompatibility and good biodegradability which is because calcium phosphate is the
inorganic mineral of human bone and teeth [2]. Hydroxyapatite (HA), Ca10(PO4)6(OH)2, is one of the most
stable forms of the calcium phosphates. The recent trend in biomaterials research is focused on overcoming
the limitations of HA ceramics (low bioresorbability, surface area and bioreactivity) and on improving
their biological properties by exploring the unique advantages of nanotechnology. The use of nano-HA in
orthopedics is considered to be very promising, due to its dimensional similarity with the bone crystals [3].
HA coated implant is more susceptible to bacterial infection as the micro-structure surface which is beneficial
for osseointegration, could also become a reservoir for bacterial colonization [4]. However proteins, amino
acids, and other organic substances are adsorbed easily on HA, which in turn favors the adsorption and
replication of the bacteria in HA [5]. In order to reduce the incidence of implant-associated infections,
several biomaterial surface treatments have been proposed [6].
It has been reported that incorporation of trace ions such as Ag, Zn, Ti, and Cu into HA structures not
only provides crystallinity, but also improves their antimicrobial property [7]. Among the various elements
that can be substituted, zinc (Zn) seems to be a potential candidate [8]. Zn is an essential element for cells.
However, its increase above certain threshold level has been shown to inhibit some of the bacterial enzymes,
including NADH dehydrogenase and protective enzymes such as thiol peroxidase and glutathione reductase
[9]. Since, Zn ions has both direct proliferative effect on bone tissues and excellent antimicrobial effects
it would be the suitable candidate to dope with HA nanoparticles to improve its biological potentials. In
this study, Zn-HA nanocrystals were synthesized using a simple wet chemical precipitation reaction using
two different weight percentage concentrations of Zn ions. The antimicrobial efficacy of the synthesized
nanocrystals against various human pathogens was tested quantitatively by determining Minimum Inhibitory
Concentration (MIC).
MATERIALS AND METHODS

Preparation of Zinc doped Hydroxyapatite Nanocrystals
The nanocrystals of HA were prepared involving the following reaction by wet chemical precipitation
method:
10Ca (OH)2 + 6 H3PO4 → Ca10(PO4)6(OH)2 + 18H2O
Calcium hydroxide and Orthophosphoric acid were used as precursors for the preparation of HA
nanocrystals. One liter of an aqueous suspension of 85% orthophosphoric acid (0.6 M) was slowly injected
to one litre of an aqueous suspension of calcium hydroxide (1.0 M) with continuous stirring for 2 h at room
temperature. The final pH of the solution was adjusted to 11. The solution was centrifuged and washed using
deionized water and dried [10]. Zn ions were doped with HA nanocrystals by adding zinc nitrate to a solution
with Zn concentration of 2 and five Wt. %.
Characterization of Zinc doped Hydroxyapatite Nanocrystals

The synthesized Zn-HA nanocrystals were characterized for their crystalline phase using powder X-ray
diffractometer (Seifert, JSO-DE BYEFLEX 2002, Germany).The average size and shape of the sample were
determined by High Resolution Transmission Electron Microscope (JEOL 2000Fx-II, Tokyo, Japan). The
samples were analyzed by ICP-OES spectroscopy (Perkin Elmer Optima 5300 DV) to determine the amount
of Zn present in the 2 % and 5 % Zn-HA nanocrystals.
Antimicrobial Testing by Microbroth dilution Technique

The antimicrobial activity of 2% and 5% Zn-HA nanocrystals were tested against five bacterial and one
fungal strains including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus
aureus and Candida albicans. Minimum Inhibitory Concentration (MIC) was determined as per Clinical
and Laboratory Standards Institute (CLSI) guidelines. Muller-Hinton broth and RPMI 1640 was used as a
medium. The Zn-HA nanocrystals were serially diluted and were inoculated with a suspension of standard
inoculum whose density was adjusted to that of 0.5 McFarland standard and were incubated at 37°C for 24 h.
MIC80 was recorded as the lowest concentration of the nanocrystals at which growth was inhibited by 80%
in an ELISA reader (Bio-Rad, [11]).
RESULTS AND DISCUSSION

The formation of single phase nanocrystals of Zn-HA was confirmed from the XRD pattern shown in
Figure 1a and 1b. The spectrum matches with the JCPDS values (09-0432) and the major peaks indicate the
crystalline form. The crystalline size of the pure HA crystals was calculated to be 52.00 nm using Scherrer
formula. The peak in the XRD pattern of Zn-HA is identical to the XRD pattern of pure HA, and no other
crystalline phase is detected. As Zn concentration increases, the XRD peak of the samples become broader
indicating lower crystallinity due to the addition of Zn.
4
Figure 1: XRD pattern of (a) 2% Zn-HA nanocrystals (b) 5% Zn-HA nanocrystals
The morphology of the 2 % and 5 % Zn-HA nanocrystals were shown in Fig. 2a and 2b. The nanocrystals
were needle-like in shape with sharp edges, and its average sizes range from 10 to 20 nm. The nanoparticles are
crystalline as seen from XRD. As the concentration of Zn increases, agglomeration of particles also increases.
Figure 2: HRTEM image of (a) 2% Zn-HA nanocrystals (b) 5% Zn-HA nanocrystals.
The total amount of Zn present in the 2 % and 5 % Zn-HA nanocrystals were calculated using ICP-OES
spectroscopy and the weight percentage of Zn ions were tabulated in Table 1. The amount of Zn increases
with the increase in the amount of Zn ions added in the mother liquid. Whereas the Zn content of the final
samples were lower than those of a corresponding amount of starting material that implies that some of the
Zn ions remain in the mother solution after precipitation.
Table 1: ICP-OES values of (Weight %) of Zn in Zn-HA nanocrystals.
S. No Sample Zn Concentration (Weight %)

1. 2% Zn-HA nanocrystals 0.4990
2. 5% Zn-HA nanocrystals 1.3740
Antimicrobial property is one of the most considerable characters of any nanomaterials that are
predominately applied in biomedical applications. The in-vitro antimicrobial efficacy of synthesized Zn-HA
was assayed quantitatively using microbroth dilution method and the minimum inhibitory concentration
80 % (MIC80) was determined. The MIC values ranged between 15 µg/mL and 500 µg/mL for both the
5
bacterial and fungal strains tested. The concentration-dependent inhibition was observed. The MIC80 values
of 2 % and 5 % Zn-HA nanocrystals were shown in Table 2. C. albicans and K. pneumoniae followed by
S.aureus were more susceptible to both 2 % and 5 % Zn-HA nanocrystals whereas P. aeroginosa, and E.
coli was less susceptible. The reports on antimicrobial activity of Zn-HA nanoparticles were limited, and the
previous studies were based on the principle of diffusion. Thian et al. [8] reported the 6-log reduction in the
numbers of S. aureus treated with ZnHA at the day six from the log reduction assay whereas Stanic et al. [7]
reported that S. aureus were less susceptible than E. coli to the Zn-HAP. Our study shows that S. aureus was
more susceptible than E. coli. To Zn-HA nanocrystals. The results of the present study on the antimicrobial
activity of the Zn-HA nanocrystals correlate well with the earlier documented reports. The antimicrobial
mechanism of Zn-HA nanocrystals may be due to the formation of bonds between the Zn ions and cell
membrane proteins, thereby causing structural changes causing cell death.
Table 2: Minimum Inhibitory Concentration (MIC) (µg/mL).
S. No Organism Tested MIC80 (µg/mL)

2% Zn-HA nanocrystals 5% Zn-HA nanocrystals
1. Escherichia coli 453.0 427.8
2. Pseudomonas aeroginosa 500.0 468.0
3. Klebsiella pneumoniae 250.0 218.7
4. Staphylococcus aureus 343.7 234.3
5. Candida albicans 66.4 16.5
CONCLUSION
The present study involves wet chemical precipitation method for the synthesis of Zn-HA nanocrystals due
to its high reproducibility and simplicity. The nature of nanoparticles was evident from the results of XRD,
HRTEM and ICP-OES. The synthesized particles were crystalline and needle-shaped, and the size ranged
from 10-20 nm. The antimicrobial efficacy of Zn-HA nanocrystals was studied against human bacterial and
fungal pathogens using Microbroth dilution assay. The result showed that 5 % Zn doped samples inhibit the
growth at lower concentration than 2 % Zn doped samples against all the tested pathogens. Furthermore,
in vivo studies on microbial toxicity and cytotoxicity of Zn-HA nanocrystals will give better understanding
about the long-term performance and use of HA as effective ceramic for biomedical applications.
REFERENCE
1. Gao J and Xu B, Applications of nanomaterials inside cells, Nano Today 2008 Oct 29; 4:37-51.
2. Epple M, Ganesan K, Heumann R, Klesing J, Kovtun A, Neumann S and Sokolova V, Application of
calcium phosphate nanoparticles in Biomedicine, Journal of Material Chemistry 2010; 20:18-23.
3. Roveri N and Iafisco M, Evolving application of biomimetic nanostructured Hydroxyapatite,
Nanotechnology, Science and Applications 2010 Nov 8; 3(1):107–125.
4. Saidin S, Chevallier P, Abdul Kadir MR, Hermawan H and Mantovani D, Polydopamine as an intermediate
layer for silver and hydroxyapatite immobilisation on metallic biomaterials surface, Materials Science
and Engineering: C 2013 Dec 1; 33(8):4715-4724.
5. Rameshbabu N, Sampath Kumar TS, Prabhakar TG, Sastry VS, Murty KVGK and Prasad Rao K,
Antibacterial nanosized silver substituted hydroxyapatite: Synthesis and characterization, Journal of
Biomedical Material Research A 2007 Mar 1; 80:581–591.
6
6. Chen W, Liu Y, Courtney HS, Bettenga M, Agrawal CM Bumgardner JD and Ong JL, In vitro anti-
bacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating,
Biomaterials Jul 26 2006; 27:5512–5517.
7. Stanic V, Dimitrijevic S, Stankovic JA, Mitric M, Jokic B, Plecas IB and Raicevic S, Synthesis,
characterization and antimicrobial activity of copper and zinc-doped hydroxyapatite nanopowders,
Applied Surface Science 2010 Apr 1; 256:6083-6089.
8. Thian ES, Konishi T, Kawanobe Y, Lim PN, Choong C, Ho B and Aizawa M, Zinc-substituted
hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties, Journal of Materials
Science: Materials in Medicine 2013 Nov 16; 24:437–445.
9. Kumar A, Pandey AK, Singh SS, Shanker R and Dhawan A, Engineered ZnO and TiO2 nanoparticles
induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli, Free Radical
Biology & Medicine 2011Aug 28; 51 :1872–1881.
10. Pataquiva Mateus AY, Ferraz MP and Monteiro FJ, Microspheres based on hydroxyapatite nanoparticles
aggregates for bone regeneration, Key Engineering Materials 2007; 330-332:243-246.
11. Annadhasan M, SankarBabu VR, Naresh R, Umamaheswari K and Rajendran N, A sunlight-induced
rapid synthesis of silver nanoparticles using sodium salt of N-cholyl amino acids and its antimicrobial
applications, Colloids and Surfaces B: Biointerfaces 2012 Apr 8; 96: 14– 21.
7
Synergistic Antibacterial Efficacy of Two Drugs
in Addition with Biosynthesized AgNPs
from an Allergenic Fungus
Chitra N,1 B.K. Nayak* and Anima Nanda
1
Department of Botany, K. M. Centre for P.G. Studies (Autonomous), (Govt. of Puducherry),
Lawspet, Pondicherry-605008, India
Department of Biomedical Engineering, Sathyabama University, Rajiv Gandhi Salai,
Chennai - 600119, India
*Corres. author
e-mail: bijuknayak@gmail.com, Tel.: +91 9443653844
Abstract:
Efficacy of silver nanoparticles in order to control the growth and epidemics of pathogenic bacteria has been
known from time immemorial. In recent days, bacteria are making themselves resistant to varied antibiotics
based on their genetic affinities. In the present study, a green, simple and effective approach was performed
to synthesize potent silver nanoparticles (AgNPs) from an airborne allergenic fungus, Alternaria sp. using
both reducing and stabilizing agent. The appearance of yellowish brown color in the conical flask suggested
the formation of AgNPs. The supernatant of the fungus culture changed the solution into brownish color
upon the completion of 10 minute reaction. The characterization of silver nanoparticles was confirmed by
Uv-VIS spectrophotometer, Field emission scanning electron microscopy (FESEM) and XRD analysis. Size
of the nanoparticles measured between 20 nm to 30 nm by FESEM. Silver nanoparticles showed good
antimicrobial activity against the selected pathogens, but when the nanoparticles were tested against the
test pathogens in combined with the two drugs viz., Amoxyclav/Clavulanic Acid (30 mcg) and Oxacillin (1
mcg); the efficacy of the drugs was increased at some extent by one fold in the case of Oxacillin.
Keywords: AgNPs, Alternaria sp., FESEM, XRD, Uv-vis Spectrophotometer
INTRODUCTION
Nanosized particles have attracted worldwide attention in recent times due to their promising interdisciplinary
fields of science that offers valuable nanomaterials having wide application in a range of areas, including
catalysis, optics, mechanics, magnetics, energetics, and biomedical sciences [1]. Compared to physical and
chemical process, biological methods have an increasing interest because of the necessity to develop new
clean, cost-effective and efficient synthesis techniques. Synthesis of nanoparticles by biological systems
such as bacteria, fungi, yeast and several plant extracts have been investigated due to their ability to reduce
metal ions [2]. Silver nanoparticles (AgNPs) have drawn special attention owing to its immense potential
as an antimicrobial agent in biomedical and other health care applications. They are an attractive option
because they are nontoxic to the human body at low concentrations and have broad spectrum antibacterial
actions. Silver ions are very reactive and are known to bind to the vital cell components, inducing cell death
[3]. Duran et al. [4] described the extracellular synthesis of silver nanoparticles from Fusarium oxysporum
strain of fungus by the presence of the hydrogenase enzyme that had exceptional redox properties, acting as
an electron shuttle for reduction of metal ions. Fungal strains have the ability to resist environmental stresses
and have the capability of growing in the presence of high metal concentrations.
The aims and objectives of the recent study is to biosynthesize silver nanoparticles by extracellular
method from an aero-allergenic fungus, Alternaria sp. isolated from vegetable market in order to confirm the
formation of silver nanoparticles by UV-vis spectroscopy followed by various microscopic characterization
and to evaluate its efficacy as a bactericide in order to combat the growth of selected bacterial pathogens viz.,
Staphylococcus aureus, Bacillus cereus, Proteus vulgaris, E. coli and Vibrio cholerae.
Isolation of Alternaria sp.
The airborne fungi were collected from indoors of a vegetable market by exposing Sabouraud Dextrose agar for 5
minutes on media plates based on gravitation method and the plates were incubated in BOD incubator at 25 ± 3°C
for 3 7 days in the Microbiology research Laboratory, Sathyabama University, Chennai for their enumeration.
Alternaria sp was isolated and identified from the mixed culture of airborne fungi [5,6] put on pure culture and
stored in a refrigerator at 4°C for further studies.
Synthesis of Silver Nanoparticles
Isolated Alternaria sp. fungus was subjected to biosynthesis of silver nanoparticles. Fungal biomass was
grown aerobically in a specific liquid medium containing (g/L): KH2PO4 7.0; 2.0 K2HPO4 MgSO4. 7H2O
0.1; (NH4)2SO4 1.0; yeast extract 0.6; glucose 10.0 at 25 ± 3°C and incubated at 25°C in a shaker at 140 rpm
for 72 hours. After incubation, the biomass was filtered using Whatman filter paper No.1 and extensively
washed with distilled water to remove all residual media components. The resulting fresh and clean biomass
was taken into the Erlenmeyer flasks, containing 100 ml of deionized Milli-Q water. The flask was again
incubated at 25° C in a shaker at 140 rpm for 72 hours. The biomass was filtered again with Whatman filter
paper No.1, and the cell free extract was used in the following experiment. 1mM AgNO3 was prepared, and
50 ml was added to the cell free extract and kept in a dark condition for 48 hrs.
Characterization of silver nanoparticles
The solution in the flask was observed for color change and maximum absorbance was analyzed using
UV-visible spectrophotometer. 1 ml of sample supernatant was taken after 24 hours and absorbance were
measured by using UV-visible spectrophotometer (T-60, PG Instruments Ltd. Lutterworh, United Kingdom)
between 300-600 nm. FESEM analysis was used to determine the surface morphology and particle size
of the silver nanoparticles. The AgNPs samples were sonicated and later centrifuged at 15000 rpm for 20
minutes. Before the process for FESEM analysis, the samples were further sonicated to get the uniformity
and better observation. Later the supernatant were discarded, and pellet was washed with the Milli-Q water
for three to four times. Later on the sample were transferred into the Petri plate and dried for about two hours
at 50° C, after that the sample were subjected to FESEM analysis.
XRD analysis was used to determine the crystallinity, and metallic nature and face centered cubic
structure of silver nanoparticles. For XRD analysis, the sample was prepared by centrifugation of the silver
nanoparticle solution at 15000 rpm for 20 minutes. The supernatant was discarded, and the pellet was washed
with Milli-Q water three to four times and then dried in petri plates. The powder form of the sample was
subjected for XRD analysis at International Research Centre, Sathyabama University, Chennai, Tamilnadu,
India.
Antibacterial study of AgNPs
The silver nanoparticles were checked for its antibacterial activity by disc diffusion method [7]. The
antimicrobial activity of the prepared silver nanoparticles from Alternaria sp was tested against the pathogenic
bacteria such as Staphylococcus aureus, Bacillus cereus, Proteus vulgaris, Vibrio cholerae and Escherichia
9
coli. The Amoxyclav (AMC-30mcg) and Oxacillin (OX-1mcg) were taken separately as control parallel
to the AgNPs to find a comparative assessment of the antibiotic efficacy over the pathogenic bacteria. The
combined effects of AgNPs with antibiotics were used to find out synergistic effect against above bacterial
pathogens. The zone of inhibition was measured after overnight incubation at 37° C.

The fungal strain, Alternaria sp used in this study was isolated from indoor air of the vegetable market and used
for the biosynthesis of silver nanoparticles. AgNPs were synthesized by the reaction of Ag+ ions from AgNO3
with the supernatant of Alternaria sp under dark conditions. After 48 h incubation, appearance of yellowish
brown color in the conical flask indicated the formation of AgNPs [8]. The supernatant of the Alternaria sp
culture changed the solution to a brownish color upon completion of the 24 h reaction with Ag+ (Fig.1)
Figure 1: Synthesis of silver nanoparticles from Alternaria sp.
The AgNPs were characterized by Uv-vis spectroscopy, which has proved to be very useful for the
analysis of nanoparticles. As illustrated in Fig.2, Uv-Vis spectra, a strong surface plasmon resonance were
centered at approximately 430 nm indicating the presence of silver nanoparticles. The exact mechanism for
the synthesis of silver nanoparticles has not been clear yet, but it has been attributed that the presence of
NADH dependent nitrate reductase enzyme in the fungal biomass is responsible for the reduction reaction.
When the silver ions come in contact with the cell wall of the fungal biomass, the nitrate reductase secreted
by the fungus causes the reduction of silver ions into silver nanoparticles [9].
Figure 2: UV–vis spectrum of silver nanoparticles synthesized from Alternaria sp.
Field emission scanning electron microscopy (FESEM) was used to understand the surface topology and
the size of silver nanoparticles. Analysis of AgNPs by FESEM showed spherical shaped silver nanoparticles
which were well dispersed within the diameter ranges of 34 nm and 47 nm (Fig.3).
10
Figure 3: FESEM analysis of silver nanoparticles synthesized from Alternaria sp.
These biologically synthesized silver nanoparticles were further characterized by X-ray diffraction
(XRD) technique to determine the metallic nature of nanoparticles. The XRD pattern clearly showed
that silver nanoparticles have been formed resulting in the diffraction peaks at 38, 45, 64 and 77
respectively confirming the metallic nature of nanoparticles and peak was specific for the silver
nanoparticles (Fig.4). The results obtained were similar to the earlier studies made by the following
workers [10, 11].
Figure 4: XRD analysis of silver nanoparticles synthesized from Alternaria sp.
The antimicrobial activity of synthesized silver nanoparticles was studied by disc diffusion method
against different clinically isolated pathogens viz., Staphylococcus aureus, Bacillus cereus, Proteus vulgaris,
Vibrio cholerae and Escherichia coli. Synthesized silver nanoparticles showed good antimicrobial activity
against the selected pathogens except Proteus vulgaris. While the antibiotics, Amoxyclav and Oxacillin, on
their own didn’t show any impressive result over the test pathogens, the combined formulations of AgNPs
with antibiotics showed remarkable results. Each disc is impregnated with 25µg silver nanoparticle solution.
Vibrio cholerae was found to be more susceptible followed by Bacillus cereus in the combined formulation
of Oxacillin and AgNPs (Table 1).
11
Table 1: Zone of inhibition (mm), Antibacterial Activity of AgNPs against bacterial pathogens
Pathogens AgNPs AMC AMC+AgNPs OX OX+AgNPs
Staphylococcus aureus 14 10 15 07 15
Bacillus cereus 15 08 18 10 17
Proteus vulgaris 13 09 15 07 15
Escherichia coli 18 11 18 08 11
Vibrio cholerae 18 10 17 20 25
The antimicrobial studies showed that combination formulation of Oxacillin and AgNPs were
significantly effective compared to Amoxyclav and AgNPs combination. The studies confirmed that the
biologically synthesized AgNPs from Alternaria sp amplified the antibacterial property of commercial
antibiotics when used in combination. Further investigation is required to study its effect in vivo
cytotoxicity for accessing its biocompatibility before administrating as antimicrobial drug for animals and
human beings.
CONCLUSION
During our study, in vitro biosynthesis of silver nanoparticles was made by extracellular method from
Alternaria sp. Synthesized silver nanoparticles showed good antimicrobial activity against the selected
pathogens and its activity were further enhanced in combination with antibiotics. Hence it may be concluded
that combined formulation of available drugs with silver nanoparticles would be an alternative approach in
order to treat the multi drug resistant pathogenic bacteria and also to minimize the antibiotic doses to cure
the dreaded diseases.
REFERENCE
1. Chauhan A, Zubair S, Tufail S, Sherwani A, Sajid M, Raman SC and Azam A, Fungus-mediated

biological synthesis of gold nanoparticles: potential in the detection of liver cancer, Int J Nanomed,
2011; 6: 2305–19.
2. Ghosh S, Patil S, Ahire M, Kitture R, Gurav DD, Jabgunde AM, Kale S, Pardesi K, Shinde V, Bellare J,
Dhavale DD and Chopade BA, Gnidia glauca flower extract mediated synthesis of gold nanoparticles
and evaluation of its chemocatalytic potential, J Nanobiotech, 2012; 10: 1–9.
3. Kulkarni N and Muddapur U, Biosynthesis of Metal Nanoparticles: A Review, J Nanotech 2014, doi.
org/10.1155/2014/510246.
4. Duran N, Marcato PD, Alves OL, De Souza GIH and Esposito E, Mechanistic aspects of biosynthesis of
silver nanoparticles by several Fusarium oxysporum strains, J Nanobiotech, 2005; 3: 1–7.
5. Barnett HL and Hunter BB, Illustrated genera of imperfect fungi, 3rd Ed. Burgess Publishing Co.
Minnepolis. Minnesota, 1972.
6. Onions AHS, Allsopp D and Eggins HOW, Smith’s introduction to industrial Mycology, London,
Edward Arnold, 1986.
7. Bauer AW, Kirby WM, Sherris JC and Turck M, Antibiotic susceptibility testing by a standardized single
disk method, Am J Clin Pathol. 1966; 45: 493–96.
12
8. Majeed S, Nanda A and Thirunavukarasu K, Evaluation of antimicrobial activity of biologically

synthesized silver nanoparticles from filamentous fungi, International Journal of PharmTech Research.
2014; 6: 1049–53.
9. Duran N, Marcapto PD, Duran M, Yadav A, Gade A and Rai M, Mechanistic aspects in the biogenic
synthesis of extracellular metal Nanoparticles by peptides, bacteria, fungi and plants, Appl Microbiol
Biotechnol, 2011; 90: 1609–24.
10. Kathiresan K, Manivannan S, Nabeal MA and Dhivya B, Characterization and antibacterial analysis
of silver nanoparticles synthesized by a marine fungus, Penicillium fellutanum, isolated from coastal
mangrove sediment, Colloids Surf B Biointerfaces 2009; 71: 133-7.
11. Ingle A, Rai M, Gade A and Bawaskar M, Fusarium Solani: A navel biological agent for extracellular
synthesis of silver nanoparticles.
13
Synthesis and Characterization of Pamam Dendrimer and
its Application for Removal of Heavy Metals
E. Gomathi1, P. Rajesh Prasanna2 and P. Selvamani3
1
Department of Petrochemical Technology, 2Department of Civil Engineering, 3Department of Pharmaceutical
Technology, BIT Campus - Anna University, Tiruchirappalli – 620 024, Tamil Nadu, India
e-mail: gomathipetro@gmail.com
Abstract:
The objective of the present study is to synthesize silica based polyamidoamine (PAMAM) dendrimer with
ester and amino groups at the outer surface. The synthesis of dendrimer is usually carried out in two ways
namely convergent technique and divergent technique. PAMAM is normally synthesized by divergent
methods starting from ammonia or ethylenediamine initiator core reagents. Amine terminated PAMAM
dendrimers exhibit a high affinity for adsorption of metal ions to their surface via co ordination to the amine
or the acid functionality. The structures were characterized by FTIR, SEM and AAS. FTIR methods were
employed to monitor amidation reaction in order to judge the optimum reaction time. The experiments
showed that both ester- and amino-terminated dendrimer-like PAMAM grafted silica-gel exhibited better
adsorption capabilities for base metal ions: Cr, Hg,. It was shown that the adsorption data of the composite
could be fitted using the Langmuir equation with a maximum adsorption capacity.
Keywords: Silica-gel, Polyamidoamine-typed hyper branched polymer Preparation, Adsorption, Metal Ions.
INTRODUCTION
The pollution of water resources due to the disposal of heavy metals has been increasing worldwide concern.
High levels of heavy metals can damage soil fertility and may affect the flora and fauna [1]. Industrial effluents
discharged from various industries like textile, tannery, sugar processing, dye and distilleries contain higher
amount of metals like chromium, copper, nickel, lead, zinc, mercury and cadmium and results in a series of well
documented problems in living beings because the heavy metals cannot be completely degraded [2]. Hence it is
necessary to treat the effluent to reduce the concentration of heavy metal contamination and different systems like
mechanical systems, aquatic systems and terrestrial systems were primarily used [3]. There are four main methods
of wastewater treatment: physical, chemical, physical-chemical and biological methods. Despite the large number
of methods, most are either too expensive to be applied in small plants or inefficient [4]. Low initial investment,
simple design, large quantity of wastewater treatment and ease of use are only some advantages that make sorption
one of the most advantageous methods for treatment of waste water containing heavy metals [5]
Dendrimers represent a novel class of three –dimensional, highly branched, globular macromolecules, which
fall a category of dendritic polymers. [6]. There are generally two synthesizing methods to fabricate dendrimers
[7] the divergent method and the convergent method. Both methods can effectively contribute to generation
growth. PAMAM dendrimers are dendrimer polymers synthesized with ethylenediamine as a monomer. PAMAM
dendrimers have a 3-D stereoscopic structure, with a particle diameter of approximately 2 20 nm. Due to its
unique spherical structure, PAMAM dendrimers have some excellent features, such as large internal cavities, high
solubility, low viscosity, and the diversification of structure design. The notable features of PAMAM are that both
the surface -NH2 groups and internal cavities play important roles in the adsorption of contaminants [8].

Reagents
Reagents included Ethanol, Tetra Hydro furan, methanol, ethylene diamine, methyl acrylate, toluene was
purchased from Loba Chemicals, and Amino Propyl Triethoxysilane (APES) was purchased from Synergy
Scientific Services. All the other solvents and reagents were of analytical or high performance liquid
chromatography grade.
Preparation of Ester and Amino Terminated PAMAM Dendrimers

The general scheme of synthesizing ester and amino terminated PAMAM dendrimers is by the introduction
of amino groups onto the silica gel surface followed by the Michael’s addition of methyl acrylate to amino
groups on the silica gel surface and ends with the terminal ester group amidation by ethylene diamine, given
as [8, 9, 10, 11].
Preparation of SiO2-G0
Under an N2 atmosphere the reaction was carried out with 50.0 g of silica-gel and 50 ml of APES were stirred
at 700 C in the 150 ml of toluene solution for 6 hours in a round bottomed flask mounted on a magnetic stirrer
provided with a heater. The rate of mixing was controlled by the RPM controller provided. Condensing
system was used to prevent toluene loss due to vaporization, since it is a highly volatile compound. The
product was then filtered off, packed in a thimble bag and then transferred to a Soxhlet extraction apparatus
for reflux-extraction in toluene and ethanol for 10 hours, respectively. The product thus extracted was dried
under vacuum at 500 C over 48 hours.
Preparation of SiO2-G0.5
A mixture of 40 g of SiO2-G0 and 31 ml of Methyl Acrylate (MA) were added to a 500 ml flask with 240
ml of methanol as solvent. The mixture was stirred at 500 C for 3 days to react sufficiently under nitrogen
atmosphere condition, brought about by purging the round bottomed flask with nitrogen gas after the
reactants are added followed by the continuous passage of nitrogen gas into the round bottomed flask, at
the time of reaction. The solid product was then filtered off, packed in a thimble bag, and transferred to the
Soxhlet extraction apparatus for reflux extraction in ethanol and tetrahydrofuran for 24 hours, respectively.
After extracting, the product was dried under vacuum at 500C over 48 h and SiO2-G0.5 was obtained.
The reaction was carried out under a nitrogen atmosphere, a suspension of 30 g of SiO2-G0.5 and 300 ml of
EDA was stirred at room temperature about 250 C in a flask using 200 ml of methanol as solvent for 5 days.
The product was filtered off, packed in a thimble bag and transferred to the Soxhlet extraction apparatus for
reflux-extraction in ethanol and tetrahydrofuran for 24 hours, respectively and then was dried under vacuum
at 500 C over 48 hours. The product SiO2-G1.0 was obtained.
Under a nitrogen atmosphere, the reaction involved mixing of a suspension of 19.4 g of SiO2-G1.0 and 62 ml
of Methyl acrylate in 120 ml of methanol solvent. The reactant mixture was stirred for 4 days at 500 C and
then the product SiO2- G1.5 was filtered off, packed in a thimble bag and transferred to the Soxhlet extraction
apparatus for reflux-extraction in ethanol and tetrahydrofuran for 24 hours, respectively and then was dried
under vacuum at 500 C over 48 hours. The product SiO2-G1.5 was obtained.
The reaction was carried out under a nitrogen atmosphere and 11.4 g of SiO2-G1.5, 150 ml of Ethylene
Diamine were mixed with 70 ml of methanol as solvent. The mixture was stirred at 250 C for 7 days .The
15
solid product was then filtered, packed in a thimble bag and transferred to the Soxhlet extraction apparatus
for reflux-extraction in ethanol and tetrahydrofuran for 24 hours, respectively and then was dried under
vacuum at 500C over 48 hours. The product SiO2-G2.0 was obtained.
Characterization of Ester and Amino Terminated PAMAM Dendrimer

Fourier Transforms Infra Red Spectrophotometer (FTIR)
FTIR was used to measure changes in the chemical structure of the silica gel based ester and amino group
terminated PAMAM dendrimer on which all the functional groups are added in a step wise manner. Infrared
spectra were recorded on a Nicolet MAGNA IR 550 (series II) spectrophotometer, using ATR attachment
at room temperature. The spectrum was acquired at 400 4000 cm-1 wave numbers with a 4 cm-1 resolution
using EZOMNIC 6.0 (Thermo Nicolat) software.
Scanning Electron Microscope (SEM)

The shapes and the surface morphology of the samples were examined on a SEM, to make sure that the
synthesized particles are of nano size.
RESULT AND DISCUSSION

Characterization of the PAMAM Dendrimer
Nanoadsorbants have been characterized by using a wide range of techniques. The characterization of
nanoadsorbants is a difficult task owing to their complexity, variety of structures and components involved
in these systems, as well as the limitations associated with each technique, but such knowledge is essential
for their successful commercial exploitation.
Scanning Electron Microscope (SEM)

Scanning Electron Microscopy analysis was carried out at 10 Kv. The results of the scanning electron microscopy
can be observed in the Figures 1 and 2.The figures reveal that the particle appearances of the G1 and G2 den-
drimers were very similar, thus demonstrating that the particles of the dendrimer samples have good mechanical
stability, and they had not been destroyed during the whole reaction.
Figure 1: SEM image of G1 PAMAM Dendrimer Figure 2: SEM image report of G2 PAMAM dendrimer
Fourier transforms infrared spectroscopy

The surface attachment of the amino groups on the surface of the silica gel was characterized by FTIR spectrum
for the G0 and G 0.5 of the PAMAM dendrimer (Fig. 3 and 4) Thirteen characterization peaks at 3378.84, 3164.05,
3612.23, 3718.89, 3942.27, 3812.66, 2677.37, 1408.30, 1078.38, 1602.21, 727.43, 626.35 and 487.43 were ob-
served to be NH bending (Primary and Secondary amides), NH stretching vibrations (Primary amides and Secondary
16
amides), C-N stretching band, conforming primary amine, C-N Stretching vibrations indicating primary and secondary
amides, OH stretching vibrations showing the vibrations of the SiOH group and the torisonal oscillation of NH3 group.
Figure 3: FTIR Spectrum of G0 PAMAM Dendrimer Figure 4: FTIR Spectrum of G0.5 PAMAM Dendrimer
Figure 5: FTIR Spectrum of G1 PAMAM Dendrimer Figure 6: FTIR Spectrum of G1.5 PAMAM Dendrimer
Figure 7: FTIR Spectrum of G2 PAMAM Dendrimer
Characterization of Synthetic Solutions

Atomic Absorption Spectroscopy (AAS):
Atomic Absorption Spectrophotometer was used to measure the concentration of metal ions were detected
by means of AAS. The adsorption amount was calculated according to the equation:
17
(Co − C )V
Q=
W
Q- Adsorption amount (mmol/g);
C0- Initial concentrations of metal ions (mmol/ml);
C- Final concentrations of metal ions (mmol/ml);
V- Volume (ml);
W- Weight of SiO2-G0–SiO2-G2.0 (g).
Adsorption properties of SiO2-G0–SiO2-G2

Static adsorption experiment was employed to determine the adsorption capabilities of SiO-G0–SiO-G2.0 for
different kinds of metal ions. A typical way was that: a dose of desired amount of the metal ions solution were
added to a 50 ml Pyrex glass tubes and then the glass tubes were placed in a thermo statcum-shaking assembly.
A known amount of SiO-G0–SiO-G2.0 (0.05 g) was charged, and the mixture solutions were mechanically
shaken at room temperature for 24 h, then the solutions in the tubes were separated from the adsorbent and the
concentration of metal ions were detected by means of atomic absorption spectrometry (AAS).
Adsorption of Mercury
The feed taken for mercury adsorption’s studies was mercury synthetic solution. And the initial concentration
was found to be 7.036 mg after treated with SiO2-G1.5 and SiO2-G2 was found to be 4.301mg/l and1.153mg/l
respectively.
Table 1: Shows decrease in concentration of Mercury
Generation Concentration of Mercury (mg/l)

Feed 7.036
SiO2-G1.5 4.031
SiO2-G2 1.153
Figure 8: Generation of Dendrimers Vs Concentration of Mercury
18
Adsorption of Chromium
Table:2
Generation Concentration of Chromium (mg/l)

Feed 0.325
SiO2-G0.5 0.275
SiO2-G1 0.210
SiO2-G1.5 0.027
SiO2-G2 0.043
The Table. 2, shows the decrease in concentration of chromium, when dendrimer generation increases.
Comparing all adsorption studies of SiO2-GO.5 to SiO2-G2, it is clear that, dendrimer’s capacity with respect
to different metal ions are, mercury >> chromium.
Figure 9: Generation of Dendrimers Vs Concentration of Chromium
CONCLUSION
Ester and amino terminated PAMAM dendrimer was synthesized by divergent method of synthesis and
they were grafted successfully on the surface of the silica gel. The percentage of grafting of the ester and
amino group terminated PAMAM dendrimer on the surface of the silica gel increased with the increase in
the number of generations as given by the FTIR reports. SEM analysis report makes it is evident that the
size range of the PAMAM dendrimer is around 1nm and the pore surface diameter decreased after the series
of grafting reactions. All of the ester and amino terminated PAMAM dendrimer presented regularities in
adsorption of metals like chromium and mercury. It was found that the absortion efficiency increased with
the generation of dendrimers, further the time to absorb the metals decreases with respect to the generation.
The adsorption of ester and the amino terminated products increased with the increase in the increase in the
grafting percentage and the addition of the surface functional groups. From this it can be concluded that the
amine and the ester groups are alone responsible for the easy and efficient adsorption of the metal ions, the
amine terminated groups exhibiting higher adsorption due to its coordination and acid functionality.
19
REFERENCES
1. Mohammad Reza Hadj Mohammadi, Mina Salary Removal of Cr from aqueous solution using Pine
needles powder as an adsorbant, 6, 1-2, (2011).
2. R. Muhammad and S.S. Ashraf, Chem.Engg.J. 209, 520 (2012).
3. Jameel M. Dhabab, Removal of Fe(II), Cu(II), Zn(II), and Pb(II) ions from aqueous solutions by
duckweed, Journal of Oceanography and Marine Science. 2(1), 17-22 ,(2011).
4. K. Xie, W. Zhao and X. He, Carbohydr.polym, 83, 1516 (2011).
5. V.V. Panic, Z.P. Madzarevic, T. Volkov-Husovic and S.J. Velickovic, Chem.Eng.J., 217, 192 (2013).
6. D.A. Tomalia, H. Baker, J.R. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder and P. Smith,
polym.J., 17, 117(1985).
7. B. Klajnert and M. Bryszewska, Acta, 37, 39 (2004).
8. Chih-Ming Chou and Hsing-Lung Lien., Dendrimer-conjugated magnetic nanoparticles for removal of
zinc (II) from aqueous solutions., J Nanopart Res. 13, 2099-2107, (2011).
9. Yuzhong Niu, Haifeng Lu, Dengxu Wang, Yuanzhi Yue and Shengyu Feng, Synthesis of siloxane-based
PAMAM dendrimers and luminescent properties of their lanthanide complexes, Elseiver Journal of
Organometallic Chemistry, 696, 544-550,( 2011).
10. Rongjun Qu, Yuzhong Niu, Changmei Sun and Chunnuan Ji, Syntheses, characterization, and adsorption
properties for metal ions of silica-gel functionalized by ester-and amino-terminated dendrimer-like
polyamidoamine polymer, Microporous and Mesoporous Materials, 97, 58–65, (2006).
11. Dey R.K., Tanushree Patnaik, Singh V.K., Sanjay K. Swain and Claudio Airoldi, Attachment of linear
poly (amido amine) to silica surface and evaluation of metal-binding behavior, Elsevier Applied
Surface Science, 255, 8176–8182, (2009).
20
Bio-Toxicity of Silver Nanoparticles Against Multidrug
Resistant Pathogens and Human Epidermoid Larynx
Carcinoma (HEp-2) Cells
Muthukrishnan Lakshmipathy and Anima Nandaa*
1
Faculty of Bio & Chemical Engineering, Sathyabama University, Rajiv Gandhi Road,
Chennai, Tamil Nadu 600119, India.
e-mail: animananda72@gmail.com, Tel.: +914424503804, Fax: +914424502344
*
Abstract:
The nanotechnological advancement in reducing the size of noble metals (Ag, Au, Ti) with enhanced
electrochemical properties has been a breakthrough in materials science research. Biological means of
synthesis especially microbe-mediated synthesis of silver nanoparticles (AgNPs) using the soil bacterium,
Bacillus subtilis A1 and its bio-toxicity against multidrug resistant strains and DNA damage have been
reported. The AgNPs showed maximum absorbance in the range 420 – 450 nm, crystal structure and
spherical shaped particles of size ~30.45 nm. The AgNPs showed potential antibacterial activity against
multidrug-resistant strains with MIC ≤ 32 μg/mL. Further study on the dose-dependent DNA damage
using comet assay revealed cleaved DNA fragments in the form of a comet with olive tail moment 6.4%
and 1.1 % respectively at 100 μg/mL and 10μg/mL respectively. This toxicological potential plays a key
role in inducing apoptosis, a limiting factor in cell death that could be harnessed for improved biomedical
application.
Keywords: Bio-toxicity, Silver nanoparticles, HEp-2 cells, MIC, Comet assay.
INTRODUCTION
Nanobiotechnology, an interdisciplinary field has opened new vistas converging technological insights
of nano applied at bio-molecular level. These wide applications of nanomaterials remain inevitable as far
as healthcare industry, and biomedical field is concerned. Besides an application, focus on reliable, eco-
friendly, pharmaceutically effective synthesis protocol using biological machinery draws much attention
[1]. Micro-organisms, the so-called nature’s treasure have been involved in nullifying the adverse effects of
metal ions released in the environment since time immemorial. The hunt for such novel route has attracted
scientists across the globe implying microbes for preparing nanoparticles such as silver, gold, titanium, etc.
Silver, known for their broad spectrum antibacterial and antifungal activity has been employed in sensors,
biomedical imaging, etc. [2]. However, its level of toxicity needs to be assessed. This study investigates on
bacteria-mediated synthesis of silver nanoparticles using environmental isolate, and Bacillus subtilis A1 and
its antibacterial effect (inhibitory concentration) against multidrug-resistant clinical isolates. Next, the DNA
damage induced by silver nanoparticles was demonstrated using single cell gel electrophoresis on HEp-2
cells (Human epidermoid larynx carcinoma).

Bacterial Strains
The clinical strains of bacteria were procured from M/s. Sharp Laboratory, Chennai and the antibiotic
sensitivity of the clinical isolates was determined using Kirby-Bauer-disc diffusion method on Mueller-
Hinton agar (MHA). The zone of inhibition was measured prior to incubation and maintained at 4° C.
Synthesis & Characterization

The silver nanoparticles were synthesized using an environmental isolate Bacillus subtilis A1 extract and
characterized by UV-visible spectroscopy, X-ray diffraction, field emission scanning electron microscopy as
reported in our previous study [3].
Antimicrobial Activity
The antimicrobial activity of silver nanoparticles was performed using the redox Resazurin microtiter assay
plate (REMA) method [4] and the minimum inhibitory concentration determined as per the CLSI norms
[5]. Susceptibility to silver nanoparticles was tested (1mg/mL), and the isolates were considered susceptible
if the MIC was < 8 µg/mL and resistant if the MIC was > 8 µg/mL. The cultures were maintained at an
inoculum of 1.5 x 107 CFU/mL.
Single Cell Gel Electrophoresis (Comet Assay)

The single cell gel electrophoresis has been performed as per Singh et al. [6] In brief, 1% of normal
melting point agarose was prepared on frosted slides along with Hep-2 cells mixed with 0.5 % low
melting point agarose. The suspension was pipetted onto pre-coated slides and kept immersed in
chilled lysis buffer (pH 10) for 60 min. The slides were then placed in alkaline buffer (pH > 13) in an
electrophoresis tank and left for 30 min. Electrophoresis was performed at 25 V for 25 min at 4° C.
The slides were neutralized in 0.4 M Tris (pH 7.5) and stained with Ethidium bromide (25 µL in 50µg/
mL) and visualized under fluorescent microscope. DNA damage was quantified by tail moment, tail
length and Olive tail moment (OTM).

In the present study, antimicrobial efficacy of biogenic silver nanoparticles toward multidrug-resistant
clinical isolates and its DNA fragmentation (single strand breaks) potential were investigated. The
metabolic products of the B. subtilis A1 behaved as reducing an agent in the synthesis and growth of
silver nanoparticles as evidenced from a color change to brown showing characteristic absorbance
for silver. Further, the crystalline nature and fcc symmetry of the particles proved its nano-size of ~30.45
nm. The particles showed spherical to roughly spherical in shape with 97 % (w) purity of silver as observed
from FESEM and EDX spectrum [3].
The antimicrobial activity of silver nanoparticles was investigated because of the increase in the drug
resistant clinical strains to fourth-generation antibiotics. The clinical strains used in this study showed partial
to complete drug resistance pattern as in Table 1. The emergence of resistance to imipenem and ciprofloxacin
is of particular concern as these antibiotics remain as the drug of last resort. For the isolates CI 1 to 5, the MIC
of silver nanoparticles was in the range 4 – 32 µg/mL (Table 2). The isolate CI 5, Acinetobacter baumannii was
found highly resistant to antibiotics whereas susceptible on exposure to AgNPs. It is noteworthy to mention that
all the strains exposed to silver nanoparticles were found susceptible. The modus operandi by which the NPs
paralyze the cell remains unexplored. It was hypothesized that upon treatment, DNA replication and ribosomal
subunit protein expression gets interrupted leading to loss of ATP production [7]. Moreover, Ag+ has a greater
affinity toward membrane-bound enzymes involved in the respiratory chain [8]. It may be anticipated that
22
the generation of ROS (reactive oxygen species) and the tendency of soft acid (silver) to react with soft bases
present in sulphur containing proteins in the membrane serve as a binding site accounting for cell damage [9].
Table 1: Showing the antibiogram pattern of clinical isolates
Clinical Isolates AMP AK AMX CAZ CI CP IMP

CI 1 (E. coli) R R S S R S S
CI 2 (Ps. aeruginosa) S S R R R S S
CI 3 (S. aureus) R R R S S S S
CI 4 (Enterococcus faecalis) R R R I R I R
CI 5 (Acinetobacter baumanniii) R R R R R R I
CI - Clinical isolates; AMP - Ampicillin, AK - Amikacin, AMX - Amoxicillin, CAZ - Ceftazidime,

CI - Ceftriaxone, CP - Ciprofloxacin, IMP - Imipenem, R - Resistant, S - Sensitive, I - Intermediate
Table 2: Showing the minimum inhibitory concentration of the clinical isolates
Test Bacterial strains MIC range (µg/mL)

E. coli 4–8
Ps. aeruginosa 8 – 16
S. aureus 16 – 32
E. faecalis 8 – 16
Acinetobacter baumannii 16 – 32
Silver nanoparticles induce significant DNA damage in HEp-2 cells through apoptosis as evidenced from
comet assay. Fig. 1 shows single strand breaks in DNA induced by the nanoparticles account for the
oxidative damage in treated cells. The level of DNA damage upon pre-treatment could be appreciated
from the % of DNA in tail, tail moment and olive tail moment whereas no change was observed in
untreated cell. The increase in concentration of AgNPs (10 µg to 100 µg/mL) significantly increases the
level of DNA damage during single cell gel electrophoresis, indicative of apoptotic cell death. The anti-
proliferative effect of AgNPs toward HEp-2 cells at 31.25 µg/mL was determined as its IC50 concentration
[10], whereas enhanced internucleosomal DNA fragmentation was observed in a dose dependent manner.
This is another evidence for the generation of ROS as a result of oxidative stress induced by NPs forecasting
apoptotic cell death [11]. It may be anticipated that the loss of mitochondrial membrane potential (ΔΨm)
was regarded as a limiting factor in the apoptotic pathway [12].
Figure 1: Comet assay showing the DNA damage induced by AgNPs on Hep-2 cells (A) Control and Cells treated with
AgNPs at 10 µg/mL (B) and 100 µg/mL (C)
Table 3: Showing data on the percentage of DNA damage induced by AgNPs at various concentrations
HEp-2 cells
Control % Nanoparticles (10 µg/mL) % Nanoparticles (100 µg/mL) %
Head DNA 97.5 92.5 77.5
Tail DNA 2.5 7.5 22.5
Tail Moment 0.1 0.67 8.1
Olive Tail Moment 0.6 1.1 6.4
23
CONCLUSION
The present study validates the potential antibacterial and apoptotic influence of silver nanoparticles.
The broad spectrum antibacterial efficacy toward drug-resistant clinical isolates proves its size dependent
physicochemical properties in bringing about cell death. Moreover, DNA damage induced by AgNPs remains
one of the limiting factors in surpassing the specificity exhibited by antibiotics. This study would open new
vistas of using AgNPs with improved biomedical applications.
ACKNOWLEDGEMENT
The authors acknowledge Department of Biotechnology (DBT), Government of India for the financial support.
The authors are grateful to Hon’ Chancellor, Managing Directors and the Department of Biomedical Engineering
for providing infrastructural facilities.
REFERENCES
1. Nanda A and Saravanan M, Biosynthesis of silver nanoparticles from Staphylococcus aureus and its
antimicrobial activity against MRSA and MRSE, Nanomedicine: Nanotechnology, Biol Med. 2009;
5(4):452 456.
2. Huang T, Nallathamby PD, Gillet D and Xu XHN, Design and Synthesis of Single Nanoparticle Optical
Biosensors for Imaging and Characterization of Single Receptor Molecules on Single Living Cells, Anal
Chem 2007 Sep; 79(20):7708–7718.
3. Muthukrishnan L and Nanda A, Geno-toxic study of silver bio-nanoparticles toward Gram-positive and
Gram-negative clinical isolates, J Pharm Res 2013 Jul; 6(7):725 729.
4. Juan-Carlos P, Anandi M, Mirtha C, Humberto G, Jean S and Francoise P, Resazurin Microtiter Assay
Plate: Simple and Inexpensive Method for Detection of Drug Resistance in Mycobacterium tuberculosis,
Antimicrob Agents Chemother 2002 Aug; 46(8):2720–2722.
5. NCCLS, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically,
Approved standard, 6th ed. NCCLS document M7 A6. NCCLS, Wayne, Pa; 2003.
6. Singh NP, McCoy MT, Tice RR and Schneider EL, A simple technique for quantification of low levels
of DNA damage in individual cells, Exp Cell Res 1988 Mar; 175:184 191.
7. Yamanaka M, Hara K and Kudo J, Bactericidal actions of a silver ion solution on Escherichia coli,
studied by energy-filtering transmission electron microscopy and proteomic analysis, Appl Environ
Microbiol 2005 Nov; 71:7589–7593.
8. Bragg PD and Rainnie DJ, The effect of silver ions on the respiratory chains of Escherichia coli, Can J
Microbiol 1974 June; 20(6):883–889.
9. McDonnell G and Russell AD, Antiseptics and disinfectants: activity, action and resistance, Clin
Microbiol Rev. 1999 Jan; 12(1):147–179.
10. Lakshmipathy M and Nanda A, In vitro oncogenic influence of silver nanoparticles on carcinoma model,
Proc Inter Conf Mathemat Sci 2014 Jul: 664–667.
11. Haruna S, Kuroi R, Kajiwara K, Hashimoto R, Matsugo S, Tokumaru S and Kojo S, Induction of
apoptosis in HL-60 cells by photochemically generated hydroxyl radicals, Bioorg Med Chem Lett 2002
Feb; 12(4):675–676.
12. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT & Thompson CB, Bcl-xL regulates
the membrane potential and volume homeostasis of mitochondria. Cell. 1997 Nov; 91(5): 627–637.
24
Design and Evaluation of Polyvinyl-Gum Ghatti Self-
Assembled Nanoscale Particles for Oral Delivery of
Simvastatin
Bibek Laha, Leena Kumari, Arpana Kashyap and Sabyasachi Maiti
Department of Pharmaceutics, Gupta College of Technological Sciences, Ashram More, G.T Road, Asan-
sol-713301, West Bengal, India
e-mail: sabya245@rediffmail.com, Mob: +919474119931
Abstract:
This work describes the synthesis of a novel gum ghatti-based amphiphilic copolymer and its ability to
form nanostructured particles in water. Hydrophobic polyvinyl chain was conjugated in the backbone of
hydrophilic gum ghatti by etherification reaction mechanism to impart amphiphilic character. Simvastatin,
a poor water soluble LDL-cholesterol lowering drug was incorporated by solvent evaporation technique by
varying drug: copolymer weight ratio (1:2, 1:4 and 1:6). Microscopic analysis of the aqueous copolymer
dispersion revealed spherical morphological structures. Thus it was suggested that the copolymer self-
assembled in water and formed nanomicellar structures. The size of nanoparticles were characterized
by Malvern Zetasizer Nano ZS 90 apparatus and was found to be in the range of 710.2 802.5 nm. The
copolymer dispersion exhibited negative zeta potential values (-20.6 25.3 mV) suggesting physical stability
of the dispersion. Even after an observation period of 3 months, no signs of aggregation were evident either
macro- or microscopically. Compared to saturation water solubility of the drug, about 50 fold increase in
drug solubility was observed after copolymer micellization. The drug loading was found to decrease with
increasing amount of the copolymer. Only a small amount of drug (<10 %) released into simulated gastric
fluid (pH 1.2) and the drug release extended in simulated intestinal fluid (pH 6.8) up to 6 h releasing about 95
% of the incorporated drug. The drug release mechanism assumed Fickian diffusion in simulated intestinal
fluids. Preliminary results obtained with the copolymer nanoparticles were encouraging and thus could be
useful as controlled drug delivery carriers.
INTRODUCTION
In past few years, a large number of biopolymers and their derivatives have been investigated for their
potential application as nanoparticle drug delivery systems. Natural biomaterials are safe, non-toxic,
hydrophilic and biodegradable [1]. Further, most of the natural polymers are hydrophilic due to the presence
of hydroxyl, and carboxyl groups in their structures. They can be chemically modified to design novel
materials for drug delivery [2]. All these merit endow biopolymers a promising future in controlled drug
delivery applications. As drug delivery system, nanoparticles can entrap drugs into their interior structures
and could show controlled release properties due to pH, ion and/or temperature sensitivity of materials and
can improve the utility of drugs and reduce dose-related toxic side effects [3].
In case of poorly water-soluble drugs, quite often the dissolution of drugs is the rate-limiting step which,
ultimately, controls oral bioavailability of the drug [4]. This poses a major challenge for effective delivery
of poorly water-soluble therapeutics via the oral route. Under such circumstances, polymeric self-assembled
nanocarriers could be beneficial.
Polymeric amphiphiles spontaneously form micelles or micelle-like aggregates via undergoing intra- or
intermolecular associations between hydrophobic moieties, primarily to minimize interfacial free energy.
Polymeric micelles have been recognized as a promising drug carrier since their hydrophobic domain,
surrounded by a hydrophilic outer shell, can serve as a preservative for various hydrophobic drugs [5]. The
grafting of hydrophobic segments onto biopolymer backbone by chemical reaction can be used to synthesize
new amphiphilic copolymers. In recent years, numerous studies have been carried out to investigate the
synthesis and the application of polysaccharide-based self-aggregate nanoparticles as drug delivery systems.
These include but not limited to HPC-g-PEO10-hexadecyl, DEX-g-PEO10-hexadecyl [6-7]; stearic acid
grafted chitosan [8]; and poly (ε-caprolactone) grafted dextran [9].
Gum ghatti is non-starch polysaccharides, exudates of the plant Anogeissuslatifolia (Combretaceae,
Myrtales). On the basis of toxicity, mutagenicity, and teratogenicity, it is generally recognized as safe
since 1976 by the regulatory authorities of USA. It is composed of L-arabinose, D-galactose, D-mannose,
D-xylose and D-glucuronic acid in a molar ratio of 10:6:2:1:2 plus traces less than 1% of 6-deoxyhexose.
Pharmaceutically, gum ghatti so far, has been investigated to limited applications in the dosage form design
(as an emulsifier, thickener and binder) [10]. In light of the above, an emphasis on drug delivery applications
of gum ghatti could be a novel attempt.
To enrich the resources on self-assembled biopolymer-based nanoparticle carriers, hydrophobic
polyvinyl segments were conjugated onto hydrophilic gum ghatti polymer chain to obtain novel amphiphilic
copolymer. The objective of this study was to synthesize polyvinyl-gum ghatti self-assembled copolymer
nanoparticles and evaluate controlled drug delivery potential. Simvastatin, a practical water insoluble BCS
Class II lipid-lowering drug was used as a model drug in this investigation.

Materials
Simvastatin was obtained as gift sample from Mylan Laboratories, Hyderabad, India. Gum ghatti was
purchased from HiMedia Pvt. Ltd., Mumbai, India. Polyvinyl alcohol (MW 14,000) was supplied by SD
Fine Chem Ltd., Mumbai, India. Sodium hydride and thionyl chloride was procured from Spectrochem
Pvt. Ltd., Mumbai, India. N, N-dimethyl formamide (DMF) was a product of Merck Specialities Pvt. Ltd.,
Mumbai, India. All others were of analytical grade and used as received.
Synthesis of Copolymer
Polyvinyl alcohol (2.5 %, w/v) was chlorinated in presence of 33 % v/v thionyl chloride mixture in chloroform
by refluxing for 2h at room temperature. Then the solvents were removed under vacuum. To this, 5 ml water
was added, stirred with a glass rod, and then the product was placed in a water bath for the removal of
gaseous by-products (HCl and H2). Excess polyvinyl alcohol was removed by washing with hot water (2×25
ml) (80° C). The percentage yield of the solid transparent polyvinyl chloride was 10.6 %. A moist mass of
gum ghatti powder (1 gm) was prepared with the addition of a small amount of water and then 20 ml DMF
was added. The gum dispersion was cooled in an ice bath (10° C) and to this sodium hydride (1 gm) was
added. After 20 min, a dispersion of polyvinyl chloride in DMF (1 % w/v) was mixed with the polymer
mixture and stirred continuously for 30 min. The reaction product was poured into 25 ml of absolute alcohol,
and the precipitated product was collected. The precipitate was re-dissolved in water and adjusted to pH 7.0
with the controlled addition of 0.01N glacial acetic acid. The copolymer was precipitated by the addition of
50 ml absolute alcohol, separated by filtration and air-dried. The product yield was above 60 %.
26
Determination of Water Solubility of Simvastatin

Saturation water solubility of simvastatin was determined by shake-flask method. In 25 ml volumetric flask,
an excess amount of drug was dispersed in water and agitated in a water bath shaker (Remi Instruments,
Mumbai, India) for 24 h at 37 ± 0.5° C.The drug suspension was filtered through 0.45 μ membrane filter,
diluted in distilled water, and assayed by UV-Vis spectrophotometer (UV1, Thermo Scientific, UK) at a
wavelength of 238 nm.
Preparation of Drug-Loaded Nanoscale Particles

Accurately weigh 10mg of the copolymer was dissolved in 10ml of distilled water under magnetic stirring
for 10 min. The required amount of simvastatin was dissolved in 2ml of chloroform and slowly added to the
copolymer solution. The organic solvent was completely evaporated by continuous stirring of the systems
for 4h. Thereafter, the dispersion was filtered by Whatman filter paper (No. 1) to remove the un-dissolved
drug, if any. The filtrate containing the drug-loaded particles were preserved and evaluated in subsequent
studies. Different drug: copolymer weight ratio (1:2, 1:4 and 1:6) were tested for evaluating their effect on
actual drug loading of the nanoparticles and were designated as NP1, NP2 and NP3, respectively.
Estimation of Actual Drug Loading

An aqueous copolymer filtrate (0.5 ml), loaded with simvastatin was evaporated to dryness and dissolved in
5 ml methanol for drug extraction. Then the solution was filtered through 0.45 µ membrane. The absorbance
of the filtrate was noted after suitable dilution in UV-Vis spectrophotometer (UV1, Thermo Scientific, UK)
at 238 nm. The drug content was measured by using the slope of the standard curve of simvastatin.
Microscopic Features of Nanoparticles

The aqueous copolymer dispersion was examined under Magnus Digital microscope (Magnus MLX,
Olympus, India) under 100×magnifications. The photographs were captured by Moticam 1000 camera (1.3
mega pixel) using Motic Images Plus 2.0 software.
Size and Zeta Potential

Malvern Zetasizer Nano ZS 90 apparatus (Malvern Instruments, Worcestershire, UK) equipped with a DTS
1060 cell was used for the determination of hydrodynamic diameter and zeta potential of nanoparticulate
systems. A 1:11 (v/v) dilution of the sample in 1 mM NaCl was done. The following conditions were
maintained during analysis: temperature of 25°C, dielectric constant (78.5); refractive index (1.33); viscosity
(0.8872 cP); and cell voltage (150 V).
In vitro Drug Release Study

The release of simvastatin from the nanoparticle formulations were tested by dialysis method. Five milliliters
of particulate dispersion was placed in dialysis bag (MWCO 12-14 kDa), tied at both ends and dipped into
simulated intestinal fluid (phosphate buffer, pH 6.8). The volume of the dissolution medium was adjusted to
200 ml, and the temperature of the medium was set at 37 ± 0.5° C. The magnetic rotation was set at150rpm. At
definite time intervals, 5 ml of aliquot was withdrawn from the dissolution fluid and replenished immediately
with the same volume of fresh medium. The samples were analyzed (UV1, Thermo Scientific, UK) at 238
nm. The drug release study was conducted in simulated gastric fluid (HCl solution, pH 1.2) under the similar
experimental conditions for 2 h. All studies were carried out in triplicate.
Statistical Analysis
The drug release data of nanoparticle systems was analysed by GraphPad Prism Version 3.00 (Trial) software.
One way analysis of variance (ANOVA): single factor was applied for statistical interpretation of data.
Significant difference was indicated at p<0.05.
27

In this research work, gum ghatti-based novel copolymer was synthesized by three main chemical reactions:
chlorination of polyvinyl alcohol, formation of gum ghatti alkoxide and final reaction between alkoxide and
polyvinyl chloride leading to the synthesis of polyvinyl-gum ghatti copolymer. The copolymer was soluble
in water, and the copolymer solution showed sphere-like micellar nanostructures under microscope (Fig 1a).
This clearly suggested amphiphilic nature of the copolymer. Simvastatin was loaded into micellar structures
by solvent evaporation technique using different drug: copolymer ratio. No difference in the appearance of
the particle structure was evident, and hence a representative photograph was displayed in Fig 1b.
Figure 1: Morphological structures of (a) drug-free; and (b) drug-loaded copolymer nanoparticles
Increasing drug: copolymer ratio caused a drastic reduction in actual drug loading into the nanoparticles
structures. The mean hydrodynamic diameter of the particles lied in the range of 710.2 802.5nm. Zeta potential
values were negative probable due to the presence of anionic carboxyl groups in gum ghatti (Table 1). Higher
loading was associated with smaller particle diameter and higher negative zeta potentials. Presumably, the
encapsulation of simvastatin enhanced the hydrophobic interactions in the core and produces more compact
particles. It was noteworthy that the particle diameter was relatively higher than other copolymer micellar
particles. The high molecular weight of polysaccharide was considered to be responsible for the increase of
particle size [11]. Indeed, long polysaccharide chains would be more deployed in water, thus contributing
to an increase of the size of the particles. The magnitude of zeta potential usually gives an indication of the
potential stability of any dispersion system.
Table 1: Properties of simvastatin-loaded copolymer nanoparticles
Formulation code Drug: copolymer Drug loading (mg Z-average Zeta Potential
weight ratio of drug/10 mg of diameter (nm) (mV)
copolymer)
NP1 1:2 1.30 710.2 -25.3
NP2 1:4 0.59 745.9 -24.5
NP3 1:6 0.35 802.5 -20.6
If all the particles in a dispersion have a large negative zeta potential then they will tend to repel each
other, and there will be no tendency for the particles to come together. No sign of particle aggregation was
also evident even after an observation period of three months. The saturation water solubility of simvastatin
was 2.54µg/ml and compared to this, about 50 times higher solubility was envisaged.
In order to evaluate the controlled drug release potential, the copolymer particles were subjected to
simulated gastric (pH 1.2) and intestinal fluid (pH 6.8). Only a very limited amount of drug (<10%) liberated
in acidic fluid at the end of 2 h. It was observed that the drug release rate became faster at the lowest drug:
copolymer ratio (1:2). The drug release profiles were illustrated in Fig 2. The slowest drug release profile was
28
evident at the highest drug: copolymer ratio (1:6). A burst release of drug (21 34%) was noted at each drug:
copolymer ratio within 20min and this could be attributed to quick dissolution of a drug adsorbed onto the
surfaces of nanoparticles. One way ANOVA did reveal any statistically significant differences in their mean
percentage drug release data (p>0.05).
Figure 2: In vitro drug release profiles of simvastatin-loaded copolymer nanoparticles in phosphate buffer solution of
pH6.8. Key: (●) NP1; (■) NP2 and (▲) NP3
The drug release data up to 60% was fitted into an empirical equation of Korsmeyer and Peppas [12]:
where Mt/M∞ was the fractional drug release at time t, k was a constant characteristic of the spherical device
and the values of ‘n’ indicated diffusion exponent (n).
Table 2: Korsmeyer-Peppas modelling of in vitro drug release data obtained in phosphate buffer solution (pH 6.8)
Formulation code Korsmeyer-Peppas model parameters

Diffusion exponent (n) Release rate constant (k) Correlation coefficient (r2)
NP1 0.270 0.4764 0.963
NP2 0.282 0.3872 0.968
NP3 0.357 0.3280 0.975
Fickian diffusion was indicated when n is 0.43 or less. As was evident from Table 2, the value of diffusion
exponent was less than 0.43 and thus the drug release was said to follow simple diffusion mechanism.
CONCLUSION
This study revealed that hydrophobic polyvinyl chain could be successfully grated onto the gum ghatti
polymer backbone via etherification reaction. The copolymer exhibited amphiphilic property because
it self-assembled and formed spherical, nanometric micellar structures in water. Irrespective of the
drug: copolymer ratio, a maximum 50 folds increase in drug solubility was achieved by copolymer
micellization. The nanoparticles were able to control the drug release rate for a longer duration which
could be advantageous in terms of reduction of frequency of administration and dose-related side effects.
Thus, the design of copolymer particles could be a promising approach for improving therapeutic
efficacy of the hydrophobic drug and thereby achieving better treatment for the patients suffering from
hypercholesterolemia.
29
ACKNOWLEDGMENTS
We would like to express our sincere gratitude to all management members of Trinity Trust, Asansol, West
Bengal, India for their kind support for successful completion of this work.
REFERENCES
1. Liu Z, Jiao Y, Wang Y, Zhou C and Zhang Z, Polysaccharides-based nanoparticles as drug delivery
systems, Adv Drug Deliv Rev 2008; 60: 1650–1662.
2. Bhardwaj TR, Kanwar M, Lal R and Gupta A, Natural gums and modified natural gums as sustained-
release carriers, Drug Dev Ind Pharm 2000; 26:1025–1038.
3. Jung T, Kamm W, Breitenbach A, Kaiserling E, Xiao JX and Kissel T, Biodegradable nanoparticles for
oral delivery of peptides: is there a role for polymers to affect mucosal uptake? Euro J Pharm Biopharm
2000; 50: 147–160.
4. Charman WN and Stella VJ, Transport of lipophilic molecules by the intestinal lymphatic system, Adv
Drug Deliv Rev 1991; 7: 1–14.
5. Letchford K and Burt H, A review of the formation and classification of amphiphilic block copolymer
nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes, Euro J Pharm
Biopharm 2007; 65:259–269.
6. Francis MF, Cristea M and Winnik FM, Polymeric micelles for oral drug delivery: Why and how? Pure
Appl Chem 2004; 76:1321–133.
7. Francis MF, Cristea M, Yang Y and Winnik FM, Engineering polysaccharide-based polymeric micelles
to enhance permeability of cyclosporin a across caco-2 cells, Pharm Res 2005; 22:209-219.
8. Li X, You J, Cui F, Du Y, Yuan H and Hu F, Preparation and characteristics of stearic acid grafted
chitosan oligosaccharide polymeric micelle containing 10-hydroxycamptothecin, Asian J Pharm Sci
2008; 3: 80-87.
9. Bajgai MP, Aryal S, Lee DR, Park S-J and Kim HY, Physicochemical characterization of self-assembled
poly (ε-caprolactone) grafted dextran nanoparticles. Colloid Polym Sci 2008; 286:517–524.
10. Deshmukh AS, Setty CM, Badiger AM and Muralikrishna KS, Gum ghatti: A promising polysaccharide
for pharmaceutical applications, Carbohydr Polym 2012; 87: 980-986.
11. Chauvierre C, Labarre D, Couvreur P and Vauthier C, Novel polysaccharide-decorated poly (isobutyl
cyanoacrylate) nanoparticles, Pharm Res 2003; 20: 1786–1793.
12. Korsmeyer RW, Gurny R, Doelker EM, Buri P and Peppas NA, Mechanism of release from porous
hydrophilic polymers, Int J Pharm 1983; 15: 25-35.
30
Green Synthesis and Characterization of Herbal Mediated
Silver Nano Particles Using Gymnema sylvestre (Linn.)
*Kalakotla Shanker and Gottumukkala Krishna Mohan
Centre for Pharmaceutical Sciences, IST, JNT University, Kukatpally, Hyderabad-500085

*Corresponding author
e-mail: shankerkalakotla@gmail.com, Mobile: 08185990001
Abstract:
Current study focuses on a novel approach towards the synthesis and characterization of herbal mediated
silver nanoparticles (SNPs) using aqueous and alcoholic extracts of Gymnema sylvestre Linn. Belonging
to the family: Asclepiadaceae. Phytochemical screening of plant extracts reveals presence of phenolic
compounds such as flavonoids, tannins and saponins which may play an important role in the formation
of herbal mediated SNPs which shows anti-bacterial and anti-diabetic activities according to previous
literature. Standard protocols have followed to synthesize SNPs, further the synthesized herbal mediated
SNPs characterized by the various instruments like SEM, EDAX, FT-IR, UV, X-ray diffraction, PSA and
TGDTA. Morphology and metal composition of synthesized nanoparticles were determined by SEM and
EDAX respectively. Size, weight variation & thermal stability of SNPs were analyzed by using PSA and
TGDTA. Further the SNPs were also characterized by UV spectroscopy, a sharp peak was observed in
between 422 nm to 470 nm indicates formation of silver nanoparticles. Simultaneously FT-IR analysis
showed that the biosynthesized silver nanoparticles were capped with biomolecular compounds which
were responsible for the reduction of silver, the bands seen at 3396.8 cm-1 and 2924.9 cm-1 were assigned
to the stretching and bending vibrations of secondary amines respectively. The bands seen at 1384.8 cm-1
and 1067.8cm-1 corresponds to –C-N- stretching vibrations. X-ray diffraction was carried out to confirm
the crystalline structure of SNPs. The synthesized SNPs were predominately spherical in shape and
polydispersed. Novelty of the current research: It can be concluded that the leaves of G. Sylvestre can be a
good source for the synthesis of silver nanoparticles. Plant extracts have been used eco-friendly and thus
can be cost effective. This study may be used in the development of value-added research in the biomedical
and nanotechnology fields.
Keywords: Herbal mediated silver nanoparticles (SNPs), Gymnema sylvestre Linn, SEM, EDAX, FT-IR
INTRODUCTION
Nanoparticles have been synthesized and stabilized through chemical and mechanical methods [1, 2],
electrochemical techniques [3], photochemical reactions in reverse micelles (4) and nowadays green
synthesis methods [5]. Synthesis of nanoparticles through biological method is a good, environment
friendly and economically alternative method. Synthesis of green nanoparticles and their characterization
is emerging fields of nanotechnology from the past few decades, due to nanoparticles have applications
various fields like physics, chemistry, biology and medicine. Application of natural methods to the synthesis
of nanoparticles has vital importance in medicinal and technological aspects [6, 7]. Biologically synthesized
silver nanoparticles (SNPs) have a wide range of applications because of their remarkable physical, chemical
and biological properties. There is a very little literature on the biosynthesis and pharmacological activities
of SNPs using plants and phytoconstituents from plants [8-10]. In this article, we describe a simple method
for the synthesis of SNPs.
Gymnema sylvestre is a valuable herb belonging to the family Asclepiadaceae, and widely distributed
in India, Malaysia, Srilanka, Australia, Indonesia, Japan, Vietnam, tropical Africa and the southwestern
region of the China. The plant is commonly known as Periploca of the woods (English); Gurmar (Hindi);
Meshashringi, madhunashini (Sanskrit); Kavali, kalikardori (Marathi); Dhuleti, mardashingi (Gujarati);
Adigam, cherukurinja (Tamil); Podapatri (Telgu) and Sannagerasehambu (Kannada) [11-15]. The word
“Gymnema” is derived from the Hindu word “Gurmar” meaning “destroyer of sugar” and it is believed that
it might neutralize the excess of sugar present in the body in Diabetes mellitus [16]. Present study is the first
report on the synthesis and characterization of herbal mediated silver nanoparticles using medicinally active
Gymnema Sylvestre.
Figure 1: Gymnema Sylvestre leaves
Extraction
Soxhlet extraction
The leaves of G. sylvestre were initially rinsed thrice in distilled water and dried on fresh paper. 250 g of
powdered leaves of G. sylvestre were packed in the soxhlet apparatus and allowed to successive solvent
extraction using methanol and water for 18 hours. The methanol and water extracts were concentrated using
rota evaporator further crude extract has kept in desiccator. Further preliminary phytochemical analysis has
done using standard test procedures to confirm the availability of active phytochemicals in plant extract.
Synthesis of Herbal mediated silver nanoparticles

The 10 3 M Silver nitrate solution was prepared and stored in brown bottles. 10 ml of herbal extracts was
taken in 250 ml conical flask/beakers separately and to this 90 ml of AgNO3 solution was added. The conical
flasks were incubated at room temperature. A color change of the leaf extracts from pale yellow to dark
brown was checked periodically. The brown color formation indicates that the silver nanoparticles were
synthesized from the herbs, and they were centrifuged at 5000 rpm for 15 minutes in order to obtain the
pellet that is used for further study.
32
Figure 2: Silver nitrate solution colour changes after addition of plant extract from white (left) to dark brown (dark)
CHARACTERIZATION OF SYNTHESIZED SILVER NANO PARTICLES
UV-Vis spectral analysis

The colour change in reaction mixture (metal ion solution + seaweed extract) was recorded through visual
observation. The bio reduction of silver ions in aqueous solution was monitored by periodic sampling of
aliquots (0.5 ml) and subsequently measuring UV-vis spectra of the solution. UV-vis spectra of these aliquots
were monitored as a function of time of reaction on UV-vis spectrophotometer UV-2450 (Shimadzu).
XRD analysis and PSA

The synthesized herbal mediated SNPs obtained were purified by repeated centrifugation at 5000 rpm for
20 min followed by redispersion of the pellet of SNPs in 10 ml of deionized water. After drying of purified
SNPs, the crystalline structure was analyzed by XRD. The dried mixture of SNPs was collected for the
determination of the formation of SNPs. The instrument operated at a voltage of 40 kV and a current of 30
mA with Cu Kα radiation in a θ- 2 θ configuration. The crystallite domain size was calculated from the width
of the XRD peaks, assuming that they are free from non-uniform strains, using the Scherrer’s formula: D=
0.94 λ / β Cos θ 1) where D is the average crystallite domain size perpendicular to the reflecting planes, λ is
the X-ray wavelength, β is the full width at half maximum (FWHM), and θ is the diffraction angle. Particle
size measurement was determined by using Particle size analyzer. SNPs samples were taken in 10 ml of
ethanol and water test tubes, both test tubes were ultra sonicated (15 minutes) for proper distribution of
nanoparticles in solution. The average particle sizes of SNPs were between 20-60 nm.
FESEM and EDAX Analysis

FESEM analysis was done using thin films of the sample were prepared on a carbon coated copper grid by
just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting
paper and then the film on the FESEM grid were allowed to dry by placing it under a mercury lamp for 5
min. Further the sample of SNPs was taken and subjected to EDAX instrument for Ag and other compound
analyses.
FTIR and TGDTA

Buker FTIR instrument was used to determine the sample functional groups. FTIR measurement was done
to identify silver ions and capped plant compounds which could account for the reduction of silver nitrate
into silver nanoparticles. The sample was mixed with KBr. Thin sample pellet was prepared by pressing
with the Hydraulic Pellet Press and subjected to FT-IR analysis. The reaction type and weight loss have
been confirmed using TGA/DTA. The reaction type and weight loss have been confirmed using TGA/DTA
thermal system (DTG-60, Shimadzu, Kyoto, Japan).
33
RESULTS AND DISCUSSIONS
UV-visible Spectroscopy
The formation of silver nanoparticles was followed by measuring absorbance at a wavelength range from
400–800 nm. The characteristic bands were detected around 400–450 nm (Fig. 3). These absorption bands
were assumed to correspond to the silver nanoparticles.
Figure 3: UV spectrum of synthesized SNPs
XRD and PSA

Figure 4 shows the XRD pattern of powder silver nanoparticles. The presence of peaks at 2θ values 38.1°,
44.09°, 64.36°, 77.29°, 81.31°, 97.92°, 110.81° and 114.61° corresponds to (111), (200), (220), (311), (222),
(400), (331), and (420) planes of silver, respectively. Thus, the XRD spectrum confirmed the crystalline
structure of silver nanoparticles. No peaks of other impurity crystalline phases have been detected. The
particle size was analyzed using particle size analyzer. A small drop of the sample was dispersed in dispersant,
water and analyzed under laser light beams in a disposable sizing cuvette. The particle size was analyzed
under the category of intensity of laser light on the sample particle. Laser diffraction revealed that the
particles obtained are aggregated mixture with size ranging between nanometers and micrometers as shown
in the Figure 5. The average particle diameter was found between 10-70 nm.
Figure 4: XRD graph of synthesized SNPS
34
Figure 5: Particle size analysis of SNPs
FESEM and EDAX

Figure 6 shows the FESEM image and XRD micrograph of silver nanoparticles. It exhibits that almost all
the nanoparticles were of spherical shape with no agglomeration. The elemental analysis of the sample has
been performed using EDX spectroscopy. Inset of Figure 6 shows EDX spectrum of silver nanoparticles.
The peaks observed at 3.0, 3.2, and 3.4 keV correspond to the binding energies of Ag La, Ag Lb, and Ag Lb2
respectively; while the peaks situated at the binding energies of 0.85, 1.0, 8.05. Therefore, the EDX profile
of sample (inset of Figure 6) indicates that the silver nanoparticles sample contain pure silver, with no oxide.
Figure 6: FESEM and EDAX micrographs of SNPs
FTIR & TGDTA

FTIR measurement was carried out to identify the biomolecules for capping and stabilization of the metal
nanoparticles synthesized by G. sylvestre FT-IR analysis showed that the biosynthesized silver nanoparticles
were capped with bimolecular compounds which were responsible for reduction of silver, the bands seen
at 3396.8 cm-1 and 2924.9 cm-1 were assigned to the stretching and bending vibrations of secondary amines
respectively. The bands seen at 1384.8 cm-1 and 1067.8cm-1 corresponds to –C-N- stretching vibrations
35
(fig.7). To investigate the thermolytic and weight loss behavior of silver nanoparticles TG/DTA analysis
was performed. Figure shows TG/DTA curves of Ag nanoparticles prepared with a solvent ratio of 75
vol% ethanol and 100 g/L concentration of silver. An exothermic peak was observed around 400 °C with
decreasing weight. This peak was due to thermal decomposition of material that retained on the surface of
the SNPs (fig.8).
Figure 7: FTIR spectra of synthesized SNPs Figure 8: DGDTA graph of SNPs
CONCLUSION
The rapid biological synthesis of silver nanoparticles using Gymnema sylvestre leaves extract provides
environmental friendly, simple and efficient route for the synthesis of benign nanoparticles. The synthesized
nanoparticles were of spherical; the estimated sizes were 10-70 nm. According to the results obtained the
nanoparticles were surrounded by a thin layer of metabolites such as terpenoids having functional groups
of amines, alcohols, ketones, aldehydes, etc., which were found from the characterization using UV-vis
spectrophotometer, FESEM, Particle Analyzer, XRD and FTIR techniques. TGDTA was performed to
know the weight loss on temperature enhancement. Hazardous organic solvents and surfactants which are
often employed in chemical synthesis of nanoparticles can be avoided through green synthesis techniques.
The present protocol is an eco-friendly and cost-effective way of synthesis of silver nanoparticles under
laboratory as well as room conditions. All these techniques were proved that the synthesized herbal mediated
silver nanoparticles having appropriate size, shape and compounds of plant extracts that may be useful drug
delivery systems. These results give us an opportunity to conduct in-vitro and in-vivo pharmacological
activities which are useful for mankind.
ACKNOWLEDGMENTS
I am thankful for the Science and Research Board-DST for providing research funding I am grateful to the
Head CPS JNTU Hyderabad for providing facilities to carry out this research work.
REFERENCES
1. Balantrapu K and Goia D, Silver nanoparticles for printable electronics and biological applications,
Journal of Material Research 2009; 24(9):2828-2836.
36
2. Tripathi RM, Saxena A, Gupta N, Kapoor H and Singh RP, High antibacterial activity of silver nanoballs
against E.coli MTCC 1302, S.typhimurium MTCC 1254, B.subtilis MTCC 1133 and P.aeruginosa
MTCC 2295, Dig J Nanomater Bios 2010; 5(2):323-330.
3. Patakfalvi R and Dekany I, Preparation of silver nanoparticles in liquid crystalline systems, Colloid
Polym Sci 2010; 280(5):461-470.
4. Rodriguez-Sanchez L, Blanco MC and Lopez-Quintela MA, Electrochemical synthesis of silver
nanoparticles, Journal of Physics and Chemical Biology 2000; 104:9683-9688.
5. Taleb A, Petit C and Pileni MP, Optical properties of self-assembled 2D and 3D superlattices of silver
nanoparticles, J Phys Chem B 1998; 102(12):2214-2220.
6. Mondal S, Roy N, Laskar RA, Sk I, Basu S, Mandal D, et al. Biogenic synthesis of Ag, Au and bimetallic
Au/Ag alloy nanoparticles using aqueous extract of Mahogany (Swietenia mahogani JACQ.) leaves,
Colloids Surf B: Biointerfaces 2011; 82(2): 497-504.
7. Begum NA, Mondal S, Basu S, Laskar RA and Mandal D, Biogenic synthesis of Au and Ag Nanoparticles
using aqueous solutions of black tea leaf extracts, Colloids Surf B Biointerfaces 2009; 71(1):113-118.
8. Kattumuri V, Katti K, Bhaskaran S, Boote EJ, Casteel SW, Fent GM, et al. Gum arabic as a phytochemical
construct for the stabilization of gold nanoparticles: in vivo pharmacokinetics and X-raycontrast-
imaging studies, Small 2007; 3(2):333-341.
9. Song JY and Kim BS, Biological synthesis of bimetallic Au/Ag nanoparticles using Persimmon
(Diopyros kaki) leaf extract, Korean J Chem Eng 2008; 25 (4):808-811.
10. Gilaki M, Biosynthesis of Silver nanoparticles using plant extracts, Journal of Biological Sciences
2010; 10(5):465-467.
11. Anonymous, The Wealth of India: A Dictionary of Indian Raw materials and Industrial products,
Council of Scientific and Industrial Research, New Delhi, 1956, 276-77.
12. P. Kanetkar, R. Singhal and M. Kamat, J Clin Biochem Nutr, 2007, 41, 77-81.
13. R. Paliwal, S. Kathori and B. Upadhyay, Ethno-Med, 2009, 3(2), 133-135.
14. P.R. Rachh, M.R. Rachh, N.R. Ghadiya, D.C. Modi, K.P. Modi, N.M. Patel and M.T. Rupareliya, Int J
Pharmacol, 2010, 1-4.
15. S.E. Potawale, V.M. Shinde, L. Anandi, S. Borade, H. Dhalawat and R.S. Deshmukh, Pharmacologyonline,
2008, 2, 144-157.
16. K.R. Keshavamurthy and S.N. Yoganarasimhan, Flora of Coorg – Karnataka, Vimsat publishers,
Banglore, 1990, 282
37
Synthesis and Characterization of Green Silver
Nanoparticles Mediated by Aegle marmelos (L.)
Leaf Extract
Sukumar Dandapat1, Manoj Kumar and M.P. Sinha
Department of Zoology, Ranchi University, Ranchi, Jharkhand-834008

1
Corresponding author
e-mail: scholar.sukumar27@gmail.com
1
Abstract:
Synthesis of green silver nanoparticle madiated by medicinal plant extract is easier, cheaper and eco-friendly
and the bioactive phytochemicals like phenols, saponins, flavonoids, tannin terpenois, alkaloids etc. of A.
marmelos are acts as reducing and capping agents during synthesis of green silver nanoparticles AgNPs
for target specific action and delivery of drugs. Colour change from pale yellow to dark brown and highest
absorption of spectrum at 200 nm and a broad spectrum at 474 nm of UV-visible spectroscopy provides
the first confirmation about the synthesis of green nanoparticle. FT-IR spectroscopy showed transmission
peak at 3275 cm-1 corresponding to O-H and H- stretch, 1604 cm-1 corresponding to C = C stretch, 1384
cm-1 corresponding to N = O bend, 1072 cm-1 corresponding to C = N stretch, 825 cm-1 corresponding
to symmetric P-O-C stretching and 750 cm-1 corresponding C-Cl and = C-H bending were provided the
conformation about the presence of alcohols and phenols, represents alkenes, as aliphatic nitro compound,
represents aliphatic amines, aliphatic phosphate, chloro alkane respectively. Final confirmation obtained
from Scanning electron microscopy showed the spherical and cubical in shapes green AgNPs with diameter
of 60 nm – 120 nm in and the average diameter of the particles were of 70 nm.
Keywords: Drug, Nano, Plants, Disease.
INTRODUCTION
Antibiotics, other synthetic drugs and antibiotic chemotherapy has been one of the most important medical
achievements use against pathogenic microbial diseases and other diseases since their introduction. However,
over the past few decades commonly used antibiotics such as streptomycin, amoxicillin, tetracycline, etc.
have become less effective due to the emergence of multi-drug resistant bacteria and also they associated
with side effects 1, 2.
However, many infectious diseases and disorders, especially intracellular infections, neurological
disorder, cancer etc. remain difficult to treat with the antibiotics and other chemotherapeutic agents
because they are difficult to transport through cell membranes and have low activity inside the cells,
thereby imposing negligible inhibitory or bactericidal effects on the intracellular matrix of bacteria 3. It is
very challenging to target the drug in the central nervous system and another nervous tissue due to blood-
brain barrier, which strictly restrict the delivery of most drugs to the brain because they do not cross the
BBB in sufficient amount 4.
Over the last few decades, the applications of nanotechnology in medicine have been extensively
explored a broad area in the field of pharmacology. Nanotechnology in the field of medicine, concerns the
size of matters in the range between 1- 100 nm are drug or natural or synthetic polymer loaded material
acts as carrier and within this scale materials have unique physicochemical properties including ultra-small
size, large surface to volume ratio, high reactivity and unique interactions with structural components such
as core, emulsion to works as carrier and functional groups includes the therapeutic molecules and ligands
for targeting location of biological systems, which are significantly improved the pharmacokinetics and
therapeutic index of the drugs in contrast to the free drug counterparts 5-7.
Within few decades many advantages of nanoparticle-based drug delivery have been recognized,
including improving serum solubility of the drugs, prolonging the systemic circulation lifetime, releasing
drugs at a sustained and controlled manner, preferentially delivering drugs to the tissues and cells of interest,
and concurrently delivering multiple therapeutic agents to the same cells for combination therapy 8-9.
Medicinal plants have been used as the chief source of treatment and disease management almost in
all countries of the world because the herbal medicines are inexpensive, easily available and do not possess
side effect 10-11. Medicinal plants play an important role to improve the immunological response against
many pathologies due to the presence of a wide variety of secondary metabolites, which are associated with
therapeutic efficacy against various diseases and disorders 12-13.
However the delivery and efficacy of many herbal drugs is often limited to reach the site of therapeutic
action and they require few modifications such as changing the molecular structure of the drug or their
proper distribution by incorporation in carrier system 14. Mathur and Govind15 reported that, when the
materials are incorporated into nano carriers, they require in lesser quantity to exert the action in target area,
and this is useful, when dealing with effective phytomolecules.
Aegle marmelos commonly known as bael, belonging to the family rutaceae have been tested against the
pathogenic bacteria. These plants are frequently used as folk medicine for treatment of various diseases like,
antidiabetic, antihyperlipidemic, cardioprotective, radioprotective, antiulceranticancerous, antimicrobial
and male contraceptives 16, 17.
In the last two decades, a number of nanoparticle-based therapeutic and diagnostic agents have been
developed for the treatment of cancer, diabetes, pain, asthma, allergy, infections, and so on 18, 19.
Therefore, the present study was carried out the process of synthesis of green silver nanoparticles using
aqueous extract of Aegle marmelos leaf extract.
Collection of Plant Material

The fresh tender leaves were collected from Ranchi district, washed and disinfected by treating with HgCl2
and washed again. The leaves were dried in the shade under room temperature for six to seven days, powered
and sieved 20.
Extract Preparation
50 g of a fine powder was subjected to extraction by soxhlet using methanol and distilled water for the aqueous
extract. The extract obtained was filtered, concentrated and dried in rotary flash evaporator maintained at 45 ºC
for proper dehydration methanol free because methanol induce toxicity to living organisms. Percentage yield
of each extract was calculated, and the dried extract was stored in air tight containers at room temperature
for further studies 21.
Phytochemical Screening
Estimation of phyto phenols, tannins, flavonoids, alkaloids, steroids, terpenoids, saponins, carbohydrates,
protein, cumarins content of fowling Sfowara 22. The details have been described elsewhere Kumar et al. 23.
39
Synthesis of Green AgNPs

The reaction mixture was prepared by adding 1 mL of the plant extract to 99 mL of 1 mM AgNO3 (169.87
mg) solution in a 250 mL round-bottom flask, which was mounted with a cooling condenser and magnetic
stir bar. The mixture was allowed to stir for 2 hours at 90 °C (immediate color change was observed from
light yellow to dark brown, and thereafter no further color change was observed even after 2 hours). After 2
hours, the mixture was allowed to cool down before being centrifuged. The centrifugation was performed at
room temperature and a speed of 9000 rpm. After washing three times with distilled water, a black powder
was obtained that was dried overnight in an oven at 80 °C 24-26.
Characterisation of Silver Nanoparticles

UV-visible spectra analysis: The reduction of pure Ag+ ions was monitored by measuring the UV-visible
spectrum of the reaction medium at 5 h after diluting a small aliquot of the sample into Milli-Q water. UV-
visible spectral analysis was done by using Parkin Elmer lambda 25 UV-Vis spectrophotometer.
Fourier Transforms Infrared Spectroscopy (FT-IR analysis) Measurements

FT-IR analysis was carried out on IPR resting-21 (Shimadzu) in the diffuse reflectance mode operated at a
resolution of 4 cm-1 in the range of 400 to 4 000 cm-1 to evaluate the functional groups that might be involved
in nanoparticle formation.
SEM Analysis of Silver Nanoparticles

SEM (Scanning electron microscope) analysis was done using JEOL JSM-6390 LV (Japan) SEM machine.
Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount
of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid
was allowed to dry by putting it under a mercury lamp for 5 min and was coated with gold using ion sputter.

Synthesis of green nanoparticles mediated by aqueous A. marmelos leaf extract and AgNO3 solution were
presented in Fig-1, showed the change in light yellow colour of plant extract into dark brown indicate the
formation of green nanoparticle 26, 27. Mohan et al. 28 also reported the changes in light colour of solution
contain AgNO3 and Plant extract into dark brown increases with an increase in temperature and incubation
period.
Figure 1: (A) Photograph of Plant extract and (B) AgNO3 and Plant extract mediated silver nano particle solution after
2 hours of heating at 80 ºC.
40
Phytochemical Screening
Results of phytochemical screening revealed the presence of alkaloids, saponins, flavonoids, phenols,
etc. presented in Table 1. Major bioactive components presents in phytochemicals are acts as reductant to
react with silver ions, and, therefore, leaf extract has been used as a reducing and stabilizing agent for the
biosynthesis of silver nanoparticles 29.
Table 1: Qualitative phytochemical composition of Aegle marmelos leaf extract
Phytochemicals Prresent (+) / Absent (-)

Alkaloids +
Steroids +
Terpenoids +
Flavonoids +
Saponins +
Phenolic compounds +
Tannin +
Carbohydrates +
Protein +
Cumarins +
UV-visible spectra analysis

UV- vis absorption spectroscopy is an important bio-physical technique to monitor the formation and
stability of green nanoparticles with the help of the absorption spectrum. The absorption spectrum
of nanoparticles obtained from UV-visible absorption spectroscopy was presented in Figure-2, which
showed a broad peak at 474 nm and highest absorption of spectra represent highest peak at 200nm
corresponds to the plasmon resonance. Kumar et al. 27 reported the highest absorption of spectrum at
200nm of alion mediated AgNPs solution by UV- vis absorption spectroscopy. Khan et al. 26 reported
broad peak at higher wavelength indicates and increases the nanoparticle size and narrow line at shorter
wavelength represents smaller particle size.
Figure 2: UV-Visible spectrum of A. marmelos and AgNO3 mediated green nanoparticles.
FTIR- Analysis
FT-IR analysis was carried out to analyse the dual role of plant extract as capping agent and highly
bioreductant 26 and to analyse common types of molecular bonds and functional groups 30, 31. FT-IR
41
absorption spectra of green nanoparticle mediated A. marmelos leaf extract and AgNO3 is presented
in Figure 3. The spectra showed broad transmission peak at 3275 cm-1 corresponding to hydrogen
bonded hydroxyl group (O-H and H- stretch) of alcohols and phenols, 1604 cm-1 corresponding to C =
C stretch represents alkenes, 1384 cm-1 corresponding to N = O bend as aliphatic nitro compound, 1072
cm-1 corresponding to C = N stretch represents aliphatic amines, 825 cm-1 corresponding to aliphatic
phosphate symmetric P-O-C stretching and 750 cm-1 corresponding chloro alkane ( C-Cl) and = C-H
bending 32, 33.
Figure 3: FTIR Spectrum of A. marmelos and AgNO3 mediated green nano particles.
SEM analysis of green nano particles

Scanning electron microscopy was provided the final conformation about the morphology of synthesized
green nano particles. SEM image of A. marmelos and AgNO3mediated green nano particles was shown
in figure -4. The green nano particles were of spherical and cubical in shapes and were formed with
a diameter of 60 nm – 120 nm in diameter and the average diameter of the particles were of 70 nm.
Kumar et al. 27 reported the size of green nano particles synthesized from alion of Aloe vera and
AgNO3 in the range of 287–293 nm and average size of nanoparticls were70 nm synthesized from.
Firdhouse et al. 25 reported the size and shape of green nano particle were 20 nm -150 nm in diameter
and spherical respectively.
Figure 4: SEM image of A. marmelos and AgNO3 mediated green nano particles.
42
CONCLUSION
This is the first ever reported; green nano particles synthesized mediated by aqueous leaf extract of A.
marmelos and AgNO3. Which is ecofriendly and cheap, and easy to synthesize and can be used in the
preparation of new pharmaceuticals due to its tiny size, capping ability of bioactive compounds, and possess
therapeutic efficacy against various disorders and diseases.
ACKNOWLEDGEMENT
The authors acknowledged the facilities provided for the whole experiment by the Department of Zoology,
Ranchi University, Ranchi. Authors also thankful to CIF, BIT Mesra, Ranchi, for their technical co-operations.
REFERENCES
1. Service RF, Antibiotics that resist resistance, Science 1995; 270: 724-727.
2. Olowe OA, Olayemi AB, Eniola KIT and Adeyeba AO, Aetiological agents of diarrhoea in children
under five years of age in Osogbo, African Journal of Clinical and Experimental Microbiology 2003;
4(3): 62 – 66.
3. Zhang L, Pornpattananangkul D, Hu C.-MJ and Huang C.-M, Development of Nanoparticles for
Antimicrobial Drug Delivery Current Medicinal Chemistry 2010; 17: 585-594.
4. Pardridge WM, Blood - the brain barrier delivery, Drug Discovery Today 2007; 1(2): 54–61.
5. Moghimi SM, Hunter AC and Murray JC, Nanomedicine; Current status and future prospect, The
FASEB Journal 2005; 19: 311-330.
6. Shoaib A, Nano technology in drug delivery, Introduction and recent developments, The Int. J. Nano
Technol, 2007; 2(1): 54-56.
7. Zhang L, Gu FX, Chan JM, Wang AZ, Langer RS and Farokhzad OC, Nanoparticles in medicine:
therapeutic applications and developments, Clin.Pharmacol.Ther., 2008; 83: 761-9.
8. Davis ME, Chen ZG and Shin DM, Nanoparticle therapeutics: an emerging treatment modality for
cancer, Nat. Rev. Drug Discov., 2008; 7: 771-82.
9. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R and Langer R, Nanocarriers as an emerging
platform for cancer therapy, Nat. Nanotechnol., 2007; 2: 751-60.
10. Brahmachari UN, The role of science in recent progress of medicine, Curr.Sci. 2001; 81: 15- 16.
11. Dandapat S, Kumar M, Kumar A and Sinha MP, Antipathogenic efficacy of methanolic leaf extract of
Cinnamomumtamala and Aegle marmelos (L.) with their nutritional potentiality, The Bioscan, 2013;
8(2): Supplement on Medicinal Plants, 635-641.
12. Bandow JE, Botz H, Leitchert LIO, Labischinski H and Hecker M, Proteomic approach to understanding
antibiotic action, Antimicrob, Agents are Chemother, 2003; 47: 948-955.
13. Dandapat S, Kumar M, Kumar A and Sinha MP, Therapeutic efficacy and nutritional potentiality of
Cinamo mumtamala (Buch.-Ham) leaf. Int. J. Pharm., 2013; 3(4): 779-785.
14. Atmakuri LR and Dathi S, Current trend in herbal medicines, J. Pharma Science, 2010; 3(1): 109-113.
15. Mathur M and Govind V, Role of nanoparticle for production of smart herbal drug- an overview, Indian
Journal of Natural Product Resources, 2013; 4(4): 329-338.
16. Kirtikar KR and Basu BD, Indian Medicinal Plants, International book distributors, Dehardun, India,
1995; 5: 830-832.
43
17. Gupta D, John PP, Pankaj K, Kaushik R and Yadav R, Pharmacological review of Aegle marmelos
Corr. fruits. Int. J. Pharma. Sci. Res., 2011; 2(8): 2031-2036.
18. Brannon-Peppas L and Blanchette JO, Nanoparticle and targeted systems for cancer therapy, Adv. Drug
Deliv.Rev., 2004; 56: 1649–1659.
19. Kawasaki ES and Player A, Nanotechnology, nanomedicine, and the development of new, effective
therapies for cancer, Nanomedicine, 2005; 1: 101–109.
20. Kumar M, Dandapat S, Kumar M and Sinha MP, Determination of nutritive value and mineral Elements
of Five- Leaf Chaste Tree (Vitex negundo) and Malabar Nut (Adhatoda vasica Nees), Acad. J. Plant
Sci., 2013; 6(3): 103-108.
21. Dandapat S, Kumar M, Sinha MP and Sinha, Therapeutic efficacy of Cinnamomumtamala (Buch.-
Ham.) and Aegle marmelos (L.) leaf.Balneo Research Journal, 2014; (5)3: 113-122.
22. Sofowara A, Screening Plants for Bioactive Agents, Medicinal Plants and Traditional Medicinal in
Africa, 3rd Ed. Spectrum Books Ltd, Sunshine House, Ibadan, Nigeria, 2008; pp: 134-156.
23. Kumar M, Dandapat S, Kumar A and Sinha MP, Anti-typhoid activity of Adhatoda vasica and
Vitexnegundo, Persian Gulf crops protection, 2013; 2(3): 64-75.
24. Song JY and Kim B, Rapid biological synthesis of silver nanoparticles using plant leaf extracts,
Bioprocess Biosyst. Eng., 2008; 32: 79-84.
25. Firdhouse MJ, Lalitha P and Sripathi SK, Novel synthesis of silver nanoparticles using leaf ethanol
extract of Pisonia grandis (R. Br). Der Pharma Chemica, 2012; 4(6): 2320-2326.
26. Khan M, Khan M, Adil SF, Tahir MN, Tremel W, Alkhathlan HZ, et al. Green synthesis of silver
nanoparticles mediated by Pulicaria glutinosa extract, Int J Nanomedicine, 2013; 8: 1507–1516.
27. Kumar TVC, Prasad TNVKV, Adilaxmamma K, Alpharaj M, Muralidhar Y and Prasad PE, Novel
synthesis of nanosilver particles using plant active principle aloin and evaluation of their cytotoxic
effect against Staphylococcus aureus. Asi. Pecific J. Tropic.Diseas., 2014; 4 (Supp-1): S92-S6.
28. Mohan KK, Sinha M, Mandal BK, Ghosh AR, Siva KK and Sreedhara PR, Green synthesis of silver
nanoparticles using Terminalia chebula extract at room temperature and their antimicrobial studies,
Spectrochim.ActaA Mol. Biomol. Spectrosc., 2012; 91: 228-33.
29. Vilchis-Nestor AR, Sanchez-Mendieta V, Camacho-Lopez MA, Gomez-Espinosa RM, Camacho-Lopez
MA and Arenas-Alatorre J, Solventless synthesis and optical properties of Au and Ag nanoparticles
using Camellia sinensis extract, Materials Letters, 2008; 62, pp.3103–3105.
30. Larkin P, Infrared and Raman Spectroscopy; Principles and Spectral Interpretation, Elsevier, ISBN
978-0-12-386984-5, Retrieved 5 December 2012.
31. George Socrates, Infrared and Raman Characteristic Group Frequencies: Tables and Charts, John
Wiley & Sons, ISBN 978-0-470-09307-8, 2004; Nework.
32. Silverstein RM, Bassler GC and Morrill TC, Spectrometric Identification of Organic Compounds, 4th
ed. 1981, John Wiley and Sons, QD272.S6 S55, New York.
33. Stuart B, Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd ISBNs:
0-470-85427-8 (HB); 0-470-85428-6 (PB), 2004, New York.
44
Invitro Cytotoxicity Studies of Nickel Oxide Nanoparticles
on Cancer Cells Using MTT Assay
K. Perumal Raj*1, Dr. V. Thangaraj2, Dr. A.P. Uthirakumar3 and P. Sivakarthik4
1
Assistant Professor in Chemistry, VSB Engineering College, Karur
2
Assistant Professor in Chemistry, Anna University (BIT Campus), Tiruchirappalli
3
Assistant Professor in Chemistry, SONA College of Technology, Salem
4
Assistant Professor in Chemistry, University College of Engineering, Panruti
e-mail: 1perumalrajk@gmail.com
Abstract:
The aim of this study to observe the cytotoxicity of synthesized Nickel oxide nanoparticles on human
cancer cell lines using MTT assay. The precursor for the Nickel oxide nanoparticles was synthesized
using nickel sulphate by organic solvent assisted solution method. The synthesized NiO nanoparticles
(<100 nm) were characterized by X-ray diffraction analysis, Scanning Electron microscopy analysis,
UV and IR spectral techniques. The characterization studies revealed that growth of the nickel oxide
nanoparticles were regularized and perfect. The synthesized nickel oxide nanoparticles were subjected to
in-vitro cytotoxicity studies against various human cancer and normal cell lines using MTT assay model.
All the synthesized Nickel oxide nanoparticles showed moderately to significant cytotoxic activity against
the tested cell lines.
INTRODUCTION
The field of metal oxide nanoparticles has attracted the attention of Chemistry, Physics and Biological
researchers interested by the novel behavior of them with the sizes 1-100 nm. The nanocrystalline NiO
have many potential applications in various fields like magnetic carriers for drug targeting and catalysis
[1, 2], environmental remediation [3], nanoscale optoelectronic devices such as electrochromic display
[4], optical fibers, photovoltaic applications [5], sensors [6, 7] in biotechnology [8] and in medical
diagnosis [9]. Moreover, the nanocrystalline NiO shows novel and significant mechanical, electronic,
magnetic and optical properties in comparison with their bulk counterparts [10]. Hence, NiO has been
received considerable attention during the past decades. Some of the methods that have been used for
the preparation of NiO nanoparticles are Sol-Gel, thermal decomposition, Spray-pyrolysis and surfactant
mediated methods.
The need of size tunable, well dispersed, stable NiO nanoparticles tempted to generate a new method for
the synthesis. The NiO nanomaterials are synthesized by using a simple solution method [11-13]. This article
focused on the synthesis of NiO nanoparticles assisted by the organic solvent, their characterization and to
observe the cytotoxicity of synthesized NiO nanoparticles on human cancer cell lines using MTT assay [14].
Synthesis of Organic Solvent Assisted NiO Nanoparticles

All the chemicals used in this work were of analytical grade and were used without further purification. The
precursor materials were prepared by using 1 g of nickel sulphate (NiSO4) dissolved in 200 ml of double
distilled (DD) water. Ammonia (18 %) solution was added, dropwise, a pale green precipitate was formed.
Further addition of ammonia solution, the precipitate was completely dissolved and a clear blue colored
solution was obtained. At the time of color change, the pH of the content was measured using a pH meter
and the observed value was 10.2.
The clear blue colored solution (the Ni-ammonium complex ion solution) was used as a precursor for
the Ni source in this synthesis. 100 of this solution was diluted with 50ml of acetone, the organic solvent.
The mixture was stirred by a magnetic stirrer for about 5 minutes. Then the content was kept in a water bath
where the temperature is maintained at 50 °C and a constant shaking was given for 26 hours. Pale green
colored material were collected and washed with DD water again and again and finally with ethanol. The
resulting material was heated in a muffle furnace for 300 °C and hence the NiO nanoparticles are formed.
The resulting material was named as NiO-Ace. Here, the Ace represents acetone, the organic solvent which
is used to grow the NiO nanoparticles. The direct growth of NiO (NiO-std) without the addition of an organic
solvent was also carried out.
Characterization of Samples
The synthesized NiO nanoparticles were characterized by an array of techniques. The powder X-ray
diffraction (XRD) patterns of the samples were obtained with a Rigaku powder X-ray diffractometer. Cu-Kα
(λ = 1.5406 Å) radiation was used as the X-ray source (40 kV, 30 mA), the scan speed was 2 °/min and the 2
ranged from 20° to 80°. The particle size was estimated by the Scherrer equation. The surface morphologies
of the as synthesized NiO nanoparticles were analyzed by Scanning Electron Microscopy (SEM, Hitachi
S-4700) and Transition Electron Microscopy (TEM, JEOL -2010). The UV-Visible spectra were measured
by Bio-Spectrometer (UV-ELCO Bl-198) for the photocatalytic degradation study.
In vitro Cytotoxicity Studies

The cytotoxicity activities of the organic solvent assisted synthesis of Nickel oxide nanoparticles were also
studied using MTT assay method. The cytotoxicity studies involve the analysis of morphological damage or
inhibition of zone of outgrowth induced by the chemicals tested.
Antiproliferative Activity by MTT [(3-(4, 5-dimethylthiazol-2yl)-2, 5- diphenyl tetrazolium

bromide] Assay
MTT measures the metabolic activity of viable cells. The assay is nonradioactive and can be performed
entirely in a microtiter plate (MTP). It is suitable for measuring cell proliferation, cell viability and
cytotoxicity. This method is based on the principle that viable cells convert MTT into a formazan salt, which
is insoluble. It is solubilized and quantified. Increase in its concentration indicates increased number of
viable cells. The absorbance directly correlates with the cell number. This method is applicable for adherent
cells cultured in MTP.
Normal Human Dermal Fibroblasts (NHDF)), Human Cervical Cancer Cell Line (HeLa), Human Breast
Cancer cells (MCF-7), Human Liver cancer Cells (HepG2) and Human Lung cancer cells (A-549) were
obtained from National Centre of Cell Sciences (Pune, India).
Assay method
0.1ml of the cell suspension (containing 1x105cells) and 0.1ml of the test compounds (3.125-100 mg/ml)
in DMSO such that the final concentration of DMSO in media is less than 1%) were added to the 96 well
plates and kept in carbon dioxide incubator with 5 % CO2, at 370 C for 72 hours. Blank contains only cell
suspension and control wells contain 1 % DMSO and cell suspension. After 72 hours, 20ml of MTT was
added and kept in carbon dioxide incubator for 2 hours followed by 80ml of lysis buffer (15 % SLS in 1:1
46
DMF and water). The plate was covered with aluminum foil to protect it from light. Then the 96 well plates
are kept in rotary shaker for 8 hours. After 8 hours, the 96 well plates were processed on ELISA reader for
absorption at 562 nm. The readings were averaged and viability of the test samples was compared with
DMSO control.
The percentage growth inhibition was calculated using the following formula
Mean OD of Individual Test Group
% growth inhibition = 100- X 100
Mean OD of Control group

The pale green colored solution formed in the synthetic sequence was the nickel hydroxide [Ni(OH)2],
which was formed after the addition of ammonia solution to NiSO4. Further addition of ammonia solution
changed the color to blue. The color change was due to the formation of highly stable intermediate Ni-
ammonium complex ([Ni(NH3)4]2+).This complex was responsible for the growth of NiO nanoparticles.
Then the NiO seed particles were formed from the isolated [Ni(NH3)4]2+ complex ions and started to
grow further in a regular manner. Finally, when the sample was heated to 300 °C, NiO nanoparticles were
formed.
The powder X-Ray diffraction technique is the mostly used analytic method for the determination of
mean size and the quality of the NiO nanoparticles .The diffraction pattern appeared at 2Ɵ angles for the
synthesized Nickel oxide nanoparticles was shown in Fig. 1. The peaks revealed that the solvent assisted
growth of NiO nanoparticles are nanocrystalline in nature and are perfect when compared to that of the NiO
nanoparticles without the assistance of the organic solvent i.e. direct growth. The particle size was calculated
using Scherrer equation and the calculated value is 29 nm.
Figure 1: XRD pattern for NiO-Ace Figure 2 : SEM image of NiO-Ace Figure 3 :TEM image of NiO-Ace
The SEM images recorded for the NiO-Ace nanoparticles by organic solvent assisted solution method
was shown in Fig. 2. A regularised pattern was found in its morphology of the NiO nanoparticles. The
assistance of acetone for the growth of regularised and well defined NiO nanoparticles was further confirmed
by TEM analysis (Fig. 3).
Fig. 4 depicts the UV-VIS spectra for the acetone assisted synthesized Nickel Oxide nanoparticles. The
λmax values at 307 nm and 557 nm in Fig. 4a clearly revealed the formation of Nickel oxide nanoparticles,
which did not appear Fig. 4b and 4c, the UV spectra are for Nickel ammonium complex, the intermediate
and Nickel sulphate respectively.
47
Figure 4 :UV-Vis Spectrum of NiO-Ace Figure 5:IR Spectrum of NiO-Ace
The creation of chemical bond between Ni and O was investigated by FT-IR spectroscopy (Fig. 5). The
band at 620 cm-1 is due to the formation of Ni-O-Ni band in the Nickel oxide nanoparticles. The characteristic
bands at the wavelength range 1500-800 cm-1 is due to the C-N stretching vibration and 3434 cm-1 is due
to O-H stretching. The bands in the FT-IR clearly proved the formation of Ni-O bond in the Nickel oxide
nanoparticles.
In vitro Cytotoxicity Studies

The cytotoxicity activities of the organic solvent assisted synthesis of Nickel oxide nanoparticles studied
using MTT assay method were reported in Table 1 and 2. The cytotoxicity value for NiO on A549 was
91.05 %, but it was moderately active, but the IC50 value was 11.46 for MCF-7 (82.5 %) that has significant
activity. However, the cytotoxicity activities of the NiO on various cell lines have moderate to significant
when compared to the standard, namely, Cisplatin.
Table 1: In vitro cytotoxicity of Nickel oxide nanoparticles and a standard
Name of the Concentration % Cytotoxicity

Compound (µg/ml) NHDF HeLa MCF-7 HepG2 A-549
100 78.12 82.61 82.5 79.81 91.05
50 63.48 70.15 74.18 65.08 70.36
25 42.34 58.41 64.51 44.31 53.25
NiO-Ace 12.5 23.83 33.21 35.62 27.5 23.58
6.25 10.42 15.3 15.24 10.14 13.47
3.125 0 4.25 3.48 2.24 0
IC50 27.95 15.28 11.46 27.72 28.39
10 78.91 96.41 98.11 97.32 100
5 65.42 71.16 76.32 73.23 72.83
2.5 38.44 65.22 63.84 58.61 61.42
Cisplatin 1.25 26.42 54.23 52.73 41.72 43.76
0.625 15.13 29.25 30.15 25.86 24.12
0.312 8.21 13.91 15.61 12.97 16.27
IC50 4.45 1.15 1.6 2.87 3.12
48
Table 2: In Vitro Cytotoxic effect of NiO nanoparticles on Human Cancer cells and normal cell lines.
IC50 (µg/ml)*
Cell lines studied
NiO-Ace Cisplatin
NHDF (Normal Human Dermal Fibroblasts) 27.95 4.45
HeLa (Human Cervical Cancer Cells) 15.28 1.15
MCF7 (Human Breast Cancer Cells) 11.46 1.6
HepG2 (Human Liver Cancer cells) 27.72 2.87
A-549 (Human Lung Cancer Cells) 28.39 3.12
*Average of three determinations, three replicates. IC50, Drug concentration inhibiting 50 % cellular
growth following 72 h of drug exposure.
CONCLUSION
A simple, economical solution method was employed for the acetone assisted synthesis of Nickel oxide
nanoparticles. The various characteristic tools like Powder XRD, SEM, TEM analysis, UV-VIS and FT-
IR spectral methods were confirmed the size, morphology and the bond formation. The present study
demonstrates the moderate to significant Cytotoxic activity of acetone assisted synthesized Nickel oxide
nanoparticles on various human cancer cell lines and normal cell lines using MTT assay method.
REFERENCES
1. T. Hyeon, Chem.Commun, 8 (2003) 927.

2. Q.A. Pankhurst, J. Connolly, S.K. Jones and J. Dobson, J. Phys D: Appl. Phys. 36 (2003) 167.
3. K.T Ranjit, G. Medine, P. Jeevanantham, I.N. Martyanaov and K.J. Klabunde, Environmental catalysis,
(2005) CRC Press, Boca Raton, FL p-391.
4. S.H. Lin, F.R. Chen and J. Kai, J Appl. Surf. Sci. 254 (2008) 2017.
5. M. Brogstron, E. Blart, G. Boschloo and E. Mukhtar, J Phys. Chem B, 109 (2005) 22928.
6. A. Salimi, A. Noorbakhsh and A. Semnani, J Solid State Electrochem., 15 (2011) 2041.
7. G. Mattei, P. Mazzolidi, M. L. Post, D. Buso and A. Martucci, J. Am. Chem. Soc., 94 (2011) 2499.
8. C.A. Mirkin, R.L. Letsinger, R.C. Mucic and J.J. Storhoff, Nature, 382 (1996) 607.
9. J.J. Storhoff, R. Elghanian, R.C. Mucic, J. Am. Chem. Soc, 120 (1998) 1959.
10. D.L. Leslie-Pelecky and R.D. Rieke, Chem. Mater., 8 (1996) 1770.
11. Periyayya Uthirakumar, S. Muthulingam, Ji H. Kang, Current Nano Science, 9 (2013) 3.
12. P. Uthirakumar, C.H. Hong, Phys. Lett. A, 359 (2006) 223.
13. P. Uthirakumar, B. Karunagaran and C. H. Hong, J. Cryst. Growth, 304 (2007) 150.
14. Raju Senthil Kumar, Balasubramanian Rajkapoor and Perumal, Asian Pacific Journal of Tropical
Medicine 4 (2011) 379-385.
49
Characterization Studies of Sunlight Induced Biosynthesis
of Silver Nanoparticles using Solanum melongena
(Eggplant)
V. Aravindhan1, C. Senthil Kumar2, H. Linda Jeeva Kumari3 and K. Ruckmani4
National Facility for Drug Development for Academia, Pharmaceutical and Allied Industries,
1, 3, 4
2, 4
Department of Pharmaceutical Technology, Bharathidasan Institute of Technology, Anna University,
Tiruchirappalli 620 024, Tamil Nadu, India
e-mail: hodpharma@gmail.com, Mob: +91 98424 84568
Abstract:
The present work focuses on the synthesis of silver nanoparticles (AgNPs) using Eggplant (Brinjal), a
common vegetable that is preferred for cooking in the Indian household. The phytochemical analysis of
the aqueous fruit extract of Solanum melongena Linn. (Solanaceae) reveals that it consists of water soluble
phyto-constituents that can act both as reducing agent and capping agent for the conversion of silver
nitrate aqueous into silver nanoparticles. This process involves sunlight induced rapid green synthesis of
AgNPs that follows bottom-up self-assembly. The detailed characterization studies of the biosynthesised
nanoparticles were carried out with UV-Vis spectroscopy, Fourier Transform Infra-Red spectroscopy (FT-
IR), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) with particle size analysis, Field
Emission Scanning Electron Microscopy (FE-SEM) and Energy Dispersive X-ray spectroscopy (EDX). UV-
Vis spectroscopy shows Surface Plasmon Resonance (SPR) from 392–450 nm, confirming the presence of
AgNPs. FT-IR absorption spectrum reveals the compatibility of the synthesized AgNPs to that of the fruit
extract. AFM image shows linear irregular distribution of particles. Zeta sizer gives the average particle size
of AgNPs varied from 100 – 150 nm with zeta potential from -8 to -23.3 mV. -23.3 mV corresponds to stable
nanoparticles. FE-SEM reveals various shapes and sizes of AgNPs. EDX confirms the presence of Silver
metal ion. Thus, AgNPs were synthesised from silver nitrate by the reducing power of active constituents
present in Eggplant fruit extract.
INTRODUCTION
Nanotechnology is gaining tremendous impetus in the present century due to its capability of modulating
metals into their nanosize, which drastically changes their chemical, physical and optical properties.
The physical, chemical, electrical and surface properties of the material are found to vary with its size
and shape from its bulk material. It has earned its importance owing to its high surface area to volume
ratio, by which it has high contact area with the material, thus possessing good reactivity and catalytic
property. It is employed in the areas of imaging, drug delivery, sensing, medical implants and gene
delivery systems in the field of biology [1]. Nanoparticles are similar in size to biomolecules, which
make them suitable for applications like molecular imaging, bio-conjugates, biomimetic scaffold, intra-
cellular tagging and bio-responsive probes [2].
Silver nanoparticles are the most widely studied metallic nanomaterial with applications in medicine,
catalysis, biotechnology and so on. It has been reported that silver nanoparticles are non-toxic to humans
and most effective against bacteria, virus and other eukaryotic microorganism at low concentrations and
without any side effects [3]. Green synthesis methods of AgNPs have reduced hazards that are due to the
global efforts. Implantation of these sustainable processes should adopt the fundamental principles of
green chemistry. Bio-inspired synthesis of nanoparticles provides advancement over chemical and physical
methods as it is a cost effective and environment friendly and in this method there is no need to use high
pressure, energy, temperature and toxic chemicals [4]. Among various green synthetic methods proposed
for the preparation of metal nanoparticles, sunlight irradiated route is an important and economical one.
Processes for making nanoparticles using plant extracts are readily scalable and may be less expensive [5].
Plant extracts may act both as reducing agents and stabilizing agents in the synthesis of nanoparticles [6].
AgNPs synthesised from herbal extracts are reported to have excellent anti-microbial, anti-inflammatory
and anti-oxidant values [7]. There are many plants reported to have been used for the synthesis of silver
nanoparticles such as Phyllanthus amarus [8], Alternanthera sessilis [9], and Azadirachta indica [10], and
Catharan thusroseus [11]. Solanum melongena (Brinjals) is an herbaceous plant which is grown mainly
for medicinal and nutritional values. Eggplants do contain ascorbic acid and phenolic acids, both of which
are powerful antioxidants [12]. In this study, we exploit the phytochemical constituents of Brinjal for their
reducing power in the synthesis of AgNPs.
Reagents
Silver Nitrate was purchased from Sigma-Aldrich, Merck. All other reagents were of AR Grade.
Preparation of the Extract

Fresh Brinjals (Solanum melongena) were obtained from the fields and were authenticated in the Department
of Botany, St. Joseph’s College of Arts and Science, Tiruchirappalli. Solanum melongena (39.5 g) was
weighed and thoroughly washed twice in distilled water, dried, cut into fine pieces and smashed in mortar and
pestle with100 ml double distilled water. The extract was filtered through Whatmann No.1 filter paper (pore
size 0.45 μm) and was further filtered using 0.22 μm sized filters using a vacuum evaporator (Fig.1). The
extract was analysed for the major phytochemical constituents and used for the synthesis of nanoparticles,
and the rest was stored at 4 °C until further use.
Brinjal Maceration Extract
Figure 1: Preparation of the Extract
Synthesis of Nanoparticles
An aqueous solution of 1 mM Silver nitrate (AgNO3) was prepared and used for the synthesis of silver
nanoparticles. 100–500 µl of Solanum melongena fruit extract was added into 1 ml of aqueous solution of 1
mM silver nitrate for its reduction into Ag+ ions and kept for incubation period for maximum 10 minutes in
sunlight that passed through transparent glass window (Fig. 2). Here, the filtered extract act as a reducing and
stabilizing agent for AgNO3 solution. The colour changed from light yellow to bright reddish brown colour,
indicating the formation of silver nanoparticles. Time taken for the addition of extract into the aqueous
51
AgNO3 solution was considered as the start of the reaction. The reaction mixture was centrifuged at 7,500
rpm for 10 minutes at 32 °C in order to obtain the pellet that is used for further studies.
Figure 2: Schematic diagram of the synthesis of AgNPs
UV-Visible Spectra
The reduction of Silver nitrate solution to Silver nanoparticles as pure silver ions was confirmed by measuring
UV absorbance against double distilled water as blank. The spectral analysis was done using double beam
Shimadzu Spectrophotometer (UV-2600) at a resolution of 1 nm from 200 to 800 nm.
FT-IR
FTIR is the fact that most molecules absorb light in the infra-red region of the electromagnetic spectrum.
This absorption corresponds to the bonds present in the molecule. The frequency range is measured as wave
numbers typically over the range 4000 – 400 cm-1. FTIR spectrophotometer (JASCO FT/IR-6300) was used for
the analysis. The sample was mixed with KBr procured from Hi-Media Chemicals, Mumbai, India. Hydraulic
Pellet Press method was followed to produce thin sample pellet and was subjected to FT-IR analysis
AFM
Nanoparticles were diluted tenfold with ultrapure water, and a drop was deposited on mica sheet fixed on a
metallic stub. The drop was dried overnight. The AFM images were collected with Park Nano instruments
operating in non-contact mode.
Particle size measurement

The synthesized AgNPs were diluted with suitable solvents to an appropriate scattering intensity and are hit
with laser light, inducing Brownian movement of Silver nanoparticles. The experiment was carried out in
a computer controlled particle size analyser using Zeta sizer nano-series (Malvern) to find out the particles
size distribution and zeta potential.
SEM analysis
Morphological characterization of the samples was done using FE-SEM (FEI Quanta MKII). A pinch of
dried sample was coated on a carbon tape. It was again coated with platinum in an auto fine coater and then
the material was subjected to analysis. The freeze dried sample of AgNP solution was sonicated with distilled
water. Small drop of this sample was placed on a glass slide and allowed to dry. A thin layer of platinum was
coated to make the samples conductive. FEI Quanta MKII FE SEM machine was operated at a vacuum of the
order of 10-5 Torr. The accelerating voltage of the microscope was kept in the range 10 – 20 kV.
52
EDX analysis
Compositional analysis on the sample was carried out by the Energy Dispersive X-ray spectroscopy (EDX)
attached with the FE-SEM. The EDX analysis of Ag sample was done by the FE-SEM (FEI Quanta MKII
FE SEM). EDX normally reveals the presence of phases.
Synthesis of AgNPs
When the fruit extract of Solanum melongena at varying concentrations was mixed with 1mM silver nitrate
solution, the pale yellow colour of aqueous extract changed to brownish red colour immediately within 10
minutes of incubation in sunlight, indicating the formation of silver nanoparticles (Fig. 3)
Figure 3: Synthesis of AgNPs (before and after)
Phytochemical Analysis
The phytochemical analysis of the aqueous fruit extract shows the presence of alkaloids, saponins, tannins,
phytosterols, flavonoids, proteins and carbohydrates whereas synthesized AgNPs shows the presence of
saponins, tannins, flavonoids, proteins and carbohydrates (Table.1). These secondary metabolites act as
reducing and capping agents for the efficient reduction of Silver nitrate solution to silver nanoparticles.
Table 1: Phytochemical Screening of plant extract and synthesised nanoparticles
Sl.No. Chemical constituents with Tests Phytochemical Screening

Plant Extract AgNPs
1 Alkaloids (Mayer’s test) + -
2 Saponins (Foam test) + +
3 Tannins (Lead Acetate test) + +
4 Phytosterols (Salkowski test) + -
5 Flavonoids (Shinoda test) + +
6 Proteins (Ninhydrin test) + +
7 Carbohydrates (Molisch’s test) + +
+ Present - Absent
UV-Vis Spectrophotometric Analysis

The reduction of pure silver ions was confirmed by UV-Vis absorption spectra taken for varying concentrations
of fruit extract in which the maximum absorbance is seen at 430 nm. 500 µL of the extract was found to be
stable after which the particles tend to agglomerate.
53
Figure 4: UV-Vis spectra of synthesized AgNPs
FT-IR ANALYSIS
The FT-IR spectra of fruit extract taken before and after the synthesis of AgNPs were analysed for the
determination of possible functional groups. Fig. 5 shows the FT-IR analysis of fruit extract and AgNPs using
Solanum melongena. Table 2 shows the band observed at 2931 cm-1 and 2842 cm-1 in the fruit extract, AgNPs
respectively arises from Alkane C-H stretching, and 1653 cm-1 is shifted to 1617 cm-1 in the nanoparticles
suggest that (C = C) groups present in the fruit extract interact with the nanoparticles. The peak at 1617 cm-1
corresponds to (NH) 10 Amine ring and peak at 1384 cm-1 corresponds to Alkane germinal methyl. The FT-IR
spectrum confirmed the amine and proteins be strongly attached to the metal nanoparticles, and the role of
protein is to prevent the agglomeration and thereby stabilize the nanoparticles. These indicate the function of
biological compounds has performed a dual function for formation and stabilization of metal nanoparticles
in an aqueous medium.
Figure 5: FT-IR absorption spectra of fruit extract and bio-moieties with synthesized AgNPs
54
Table 2: Corresponding functional groups from the FT-IR absorption spectra
Frequency (cm-1) Bond Functional group

2931(Extract, AgNPs), 2842(AgNPs) C-H stretch Alkanes
1652(Extract) C = C stretch Alkene
1617(AgNPs) NH bend 10 Amine
1384(AgNPs), 1321(Extract) C-H rock Alkane
1151(Extract) C-N stretch Aliphatic amine
1017(Extract) C-N stretch Aromatic amine
AFM
Fig. 6 shows the AFM images (2D and 3D) of silver nanoparticles deposited on hydrophilic silicon substrate.
The nature of the substrate plays a very important role in influencing the surface coverage. Our data suggest
that the mica and hydrophilic silicon are more appropriate substrates for the AFM imaging of Silver
nanoparticles, since they spread more homogeneously on these solid substrates.
Figure 6: AFM 2D and 3D images for synthesized AgNPs.
Particle size distribution

Fig. 7 shows the average particle size distribution of the synthesized AgNPs of 500 µl fruit concentration to
be 108 nm.
Figure 7: Particle size distribution of synthesized AgNPs
55
Zeta potential
Particles will repel each other if the systems have high positive or negative value of zeta potential, and a system
having value ± 30 mV is considered a stable formulation if dispersed in a liquid as a colloidal dispersion.
Fig.8 shows the zeta potential value of the synthesized silver nanoparticles 500 µl fruit concentration to be
-23.3 mV and is found to be stable.
Figure 8: Zeta potential of synthesized AgNPs
FE-SEM Analysis
The FE-SEM image of Silver nanoparticles synthesized by green synthesis process is shown in Fig.9. It gave
a clear image of highly dense silver nanoparticles. The SEM image showing silver nanoparticles synthesized
using Solanum melongena extract confirmed the development of mostly spherical and some irregular silver
nanostructures.
Figure 9: SEM Micrograph analysis of synthesized AgNPs
56
EDX analysis
Fig. 10 shows the EDX plot of SEM image of Ag nanoparticles. The EDX reading proved that the required phase of Silver
(Ag) is present in the sample. The graph also shows the presence of Carbon (C), Oxygen (O) in the EDX picture of silver
nanoparticles. This is probably due to the presence of substrate over which the AgNP sample was held during SEM microscope
Figure 10: EDX report of synthesized AgNPs
CONCLUSION
“There is a plenty of room at the bottom” – Richard Feynman, 1959
Anything with nanometric size 10−9 in one dimension is said to be in nanoscale. The history of
nanomaterials traces back to the Roman period. The Lycurgus cup is a great example that appears red in
transmitted light and green in reflected light that is due to gold and silver nanocrystals in the wall of the
cup. Recent researches prove that nanomaterials have prospective applications in the medical field which
gives newer pathways in the development of green synthesis. This report demonstrates sunlight induced
synthesis of nanoparticles for the first time from Brinjal fruit extract which provides a natural, efficient
and cost-effective mode of applications. The synthesized nanoparticles will be further studied for various
pharmaceutical applications to analyse their medicinal properties.
ACKNOWLEDGEMENT
The authors kindly acknowledge the Department of Pharmaceutical Technology, Bharathidasan Institute
of Technology, Anna University, Tiruchirappalli and the Department of Science and Technology, New
Delhi, Govt. of India for its efforts in establishing the National Facility for Drug Development for
Academia, Pharmaceutical and Allied industries at Bharathidasan Institute of Technology, Anna University,
Tiruchirappalli which was a great source for utilizing the facilities and to carry out the research work.
REFERENCES
1. Suri S.S., Fenniri H. and Singh B., (2007): “Nanotechnology-based drug delivery systems.” Journal of
Occupational Medicine and Toxicology, 2: 16 -21.
2. Koo O.M., Rubinstein I. and Onyuksel H., (2005): “Role of nanotechnology in targeted drug
delivery and imaging: a concise review”, Nanomedicine: Nanotechnology, Biology and Medicine,
1(3): 193-212.
3. Jeong S.H, Yeo S.Y. and Yi S.C., (2005): “The effect of filler particle size on the antibacterial properties
of compounded polymer/ silver fibres.” J. Mat. Sci. 40: 5407-5411.
57
4. Dubey M., Bhadauria S. and Kushwah B.S., (2009): “Green Synthesis of Nano silver Particles From
Extract of Eucalyptus Hybrida (Safeda) Leaf.” Digest Journal of Nanomaterials and Bio structures, 4(3):
537 – 543.
5. Iravani S., (2011): “Green Synthesis of Metal Nanoparticles Using Plants.” Green Chemistry 13: 2638
– 2642.
6. Song J.Y. and Kim B.S., (2009): “Rapid biological synthesis of silver nanoparticles using plant leaf
extracts”. Biosyst Eng. 32:79–84.
7. Elumalai E.K., Prasad T.N.V.K.V., Kambala V., Nagajyothi P.C. and David E., (2010): “Green synthesis
of silver nanoparticle using Euphorbia hirta L and their antifungal activities.” Scholars Res. Library. 2
(6): 76-81.
8. Subbaiya R., Lavanya R.S., Selvapriya K. and Selvam M.M., (2014): “Green Synthesis of Silver
Nanoparticles from Phyllanthusamarus and their Antibacterial and Antioxidant Properties.” Int.J.Curr.
Microbiol.App.Sci. 3(1): 600-606.
9. Niraimathi K.L., Sudha V., Lavanya R. and Brindha P., (2012): “Biosynthesis of silver nanoparticles
using Alternantherasessilis (Linn.) extract and their antimicrobial, antioxidant activities.” Colloids and
Surfaces B: Biointerfaces.102: 288– 291.
10. Tripathi A., Chandrasekaran N., Raichur A.M. and Mukherjee A., (2009): “Antibacterial applications
of silver nanoparticles synthesized by aqueous extract of Azadirachtaindica (Neem) leaves”. J Biomed.
Nanotechnol. 5(1):93-98.
11. Ponarulselvam S., Panneerselvam C., Murugan K., Arthi N., Kalimuthu K. and Thangamani S.,
(2012): “Synthesis of silver nanoparticles using leaves of Catharanthusroseus Linn. G. Don and their
antiplasmodial activities.”, Asian Pac. J Trop. Biomed. 2(7): 574 – 580.
12. Das M. and Barua N., (2013): “Pharmacological activities of Solanum melongena Linn. (Brinjal
plant)”. International Journalof Green Pharmacy. 7(4): 274-277.
58
Thermal, Anti-Fungal and Primary Electrochemical
Studies of Palladium Nanoparticles
N. John Sushma1, K. Mallikarjuna2, D. Prathyusha3, G. Narasimha4, G. Swathi5,

B.V. Subba Reddy6 and B. Deva Prasad Raju7*
Department of Biotechnology, Sri Padmavati Women’s University, Tirupati-517502, India

1, 3, 5
2
Department of Physics, 4Department of Virology, Sri Venkateswara University, Tirupati – 517 502, India
6
Indian Institute of Chemical Technology, Hyderabad – 500 007, India
7
Department of Future Studies, Sri Venkateswara University, Tirupati - 517 502, India
e-mail: drdevaprasadraju@gmail.com, Tel: +91-94402 81769
Abstract:
Green nanotechnology is one of the potential breakthroughs of biology, medicine, chemistry, physics,
sensors and material science. In this report, an attempt has been made to demonstrate a single step method
for palladium nanoparticles of the size less than 5 nm using anti-cancer potent Piper longum leaf extract.
The synthesized PdNPs were confirmed and characterized by several analytical techniques such as UV-Vis
spectra, XRD, TEM, SAED, FT-IR, TG-DTA and primary electrochemical analysis. The morphology and
size obtained PdNPs are spherical in size and the order of 3–5 nm and the crystalline facets (111), (200),
(211), (311) and (222) face centered cubic respectively. The presences of the bio-organic moieties, which are
responsible for the formation of PdNPs, were analyzed with the Fourier transform infrared spectroscopy. The
thermal behavior of synthesized PdNPs was studied with TG-DTA analysis. The bioactivity demonstrated by
synthesized PdNPs was lead to an excellent clinical use as anti-fungal material. We have demonstrated the
modified carbon paste electrode using palladium nanoparticles by means of cyclic voltammetry in a solution
of 1 M KCl and 1 mM [Fe(CN)6]3-/4-.
Keywords: Biogenic preparation, Palladium nanoparticles, TG-DTA analysis, Anti-Fungal activities,
Electro-chemical studies.
INTRODUCTION
In recent years, proliferation of green nanotechnology is gaining importance due to the elimination or
reduced hazardous reagents and effective synthesis of expected products in low price manner [1]. Nobel
metal nanoparticles have been shown to inter-disciplinary potential application in the area of catalysis,
bio-labeling, medical and sensors [2, 3]. In particular, palladium belongs to platinum group materials
which are widely used in automotive catalytic converters in order to reduce gaseous emission vehicles
exhaust for the sake of environment protection [4]. A number of methods, such as chemical and physical
methods is based on the availability and feasibility of protocols to achieve the required applications of
palladium nanoparticles [5, 6]. These methods are quite expensive equipment or hazardous chemicals
which have harmful effects on the environment as well as on the human health. Utilization of inexpensive
and nontoxic chemicals, environment friendly solvents and renewable or biodegradable materials of
biomolecules is a central tenant in material synthesis and processing when considering green chemical
procedures [7, 8] Greener aspects in molecular design now invariably include the use of bio-renewable raw
materials in benign reaction media and recyclable nanocatalysts in atom-economical syntheses as thrust
areas. Several green methods have been applied for the preparation of green nanoparticles, including the
application of natural materials such as microorganisms, marine organisms, biomolecule extracts and
plant materials [9, 10]. The Piper longum extract is a rich source of flavonoids, amides, lignans, long and
short chain esters, terpenes, steroids, prophenylphenols, and alkaloids [11]. In this article, we put efforts
for synthesizing palladium nanoparticles by biogenic procedure, a facile, rapid and one-pot aqueous
synthesis and characterization and antifungal studies of the palladium nanoparticles by using anti-cancer
potent Piper longum leaf extract.

Palladium Chloride was obtained from Sigma-Aldrich (USA) and used without further purification. The
fresh Piper longum leaves were collected from the Green House in Sri Venkateswara University Campus,
Tirupati, Andhra Pradesh, India. The fine graphite powder, KCl, Potassium ferrocyanide was obtained from
Merck chemicals. Silicon oil, acetone (GR grades) was procured from Himedia chemicals.
Preparation of Piper longum Leaf Extract

About 20 g of fresh leaves were thoroughly washed and then finely chopped into leaf pieces and placed in
a 250 ml Erlenmeyer flask containing 100 ml of double distilled water. The resulting leaf suspension was
boiled for 5 min, and the extract was cooled and then filtered through the whatman no. 41 filter paper (pore
size 20 µm) and stored at 4 °C for further experiments.
Synthesis of Palladium Nanoparticles

The PdNPs were prepared by treating of 30ml of 0.001 M PdCl2 with 2 ml of aqueous extract and the mixture
was stirred at room temperature for 4 h. The visual appearance of a dark grey color in the reaction vessel
indicates the formation of palladium nanoparticles.
Characterization
The UV-vis absorption spectroscopic analysis was carried out on Genesys 10S UV-Vis spectrophotometer
with resolution of 1nm between 200-800 nm. The particle size and morphology of the PdNPs were studied
with Philips Tecnai F 12 model TEM. The TEM grids were prepared by placing a drop of the bio-reduced
diluted solution onto a carbon coated copper grid and later dried it under the lamp. The X-ray diffraction
(XRD) measurement of a thin film of the bio-reduced colloidal palladium solution was drop coated
onto a glass slide and dried it. XRD patterns of the prepared thin film was analyzed by using Inel C120
X-ray diffractometer. The IR spectrum analyzed on Thermo-Nicolet IR 200 spectrophotometer, operated
at a resolution of 4 cm-1 in the region of 4000-500 cm-1. The Thermogravimetric and differential thermal
analysis (TG-DTA) of powdered PdNPs were analyzed between 30-800 °C in air at heating rate 20 °C/min
with the Exstar 6000 series analyzer. Electrochemical measurements were performed on CHI model 660c
electrochemical workstation with a connection to a personal computer that was used for electrochemical
measurements.
UV-Vis absorption studies

The bio-reduction of palladium ions was studied by monitoring changes in color with UV-Vis absorption
spectroscopy. The growing of PdNPs was studied as a function of time reaction shown in Fig 1. The
change in color is attributed to the collective oscillation of conduction band electrons induced by the
60
interaction of electromagnetic wave with metallic nanoparticles. It is evident that, a small absorption
band 600-700 nm was observed which indicates either formation of stable aggregates of the palladium
nanoparticles in the solution or shape anisotropy in the particles. The broad SPR peak at 320 nm is well
known for the metal nanoparticles in the range of 2-100 nm and this absorption peak corresponds to the
real and imaginary parts of dielectric function of metals almost vanish [12]. Thus, the phytochemicals
within Piper longum broth not only result in an effective reduction of PdII to stable Pd nanoparticles but
also their effective wrapping around the palladium nanoparticles to provide excellent stabilizing material
against agglomeration.
Figure 1: Optical properties of PdNPs at different time intervals.
TEM and SAED studies

Fig. 2 shows the TEM image of the biosynthesized palladium nanoparticles. From the image, it is interesting
to note that almost all the particles are not in physical contact, equally distribution and found to be surrounded
by a thin layer of biomolecules, which seems to be responsible for stabilizing the nanoparticles. The SAED
pattern suggests that the particles are highly crystalline nature as shown in the inset of the Fig. 2. From the
figure, the diffraction rings from inner to outer, clearly indicating the FCC phases of palladium with (111),
(200), (220), (311) and (222) reflections, respectively.
Figure 2: TEM image and SAED pattern of the synthesized PdNPs.
XRD analysis
The structural determination of PdNPs was characterized with XRD patterns as shown in Fig. 3. The number
of strong Bragg’s diffracted peaks was observed at 46.74°, 54.81°, 81.39°, 99.09°, and 104.75° corresponding
to the (111), (200), (220) (311) and (222) facets of the face centered cubic lattice of palladium nanoparticles,
it was good agreement with JCPDS-89-4897 file.
61
Figure 3: XRD patterns of synthesized PdNPs.
FTIR analysis
The FTIR spectrum of synthesized colloidal palladium nanoparticles using Piper longum leaf broth is shown
in Fig.4. It is confirmed by the fact that, the identified functional groups in bio-organic moieties are acted as
the reducing and efficient stabilizing agents for the formation of PdNPs. The band at 3425 cm-1 corresponds
to amine the group stretching vibrations superimposed on the side of the hydroxyl group and the peak at 2923
cm-1 indicates hydrogen bonded O-H stretch carboxylic acid (41). The absorption peak at 1630 cm-1 is due
to the C=C stretching and aromatic stretching vibrations, respectively. It indicates the presence of flavonoids
in the leaf extract. The rutin like flavonoids are to play an important role in the synthesis of PdNPs through
the reduction of PdII ions to stable Pd nanoparticles with interaction of carbonyl groups, and glycosides act as
capping agents. The band at 1464 cm-1 corressponds to geminal methyl group stretching vibrations, and 1180
cm-1 indicates -O- vibrations. In addition to that, the peaks at 3425, 2923, 2374 and 1464 cm-1 are indicating
the presence of the protein molecules in the process of formation of nanoparticles [13].
Figure 4: FTIR spectrum of synthesized PdNPs
TG-DTA studies
The thermal stability of the synthesized PdNPs was studied at higher temperature through TG-DTA of the
powder sample. The results of TG-DTA studies of the PdNPs were shown in Fig. 5. From the TG profile, it
is evident that successive weight losses in the temperature region 30-800 °C. The initial weight loss between
30-200 °C was attributed to loss of the bound water molecules in the powder sample. The second step
62
200-500 °C was most likely the consequence of the thermal degradation of the bio-organic moieties which
are wrapping around the nanoparticle. While in third step 500-800 °C weight was raised could be due to
remaining bioorganic materials and palladium residue (44, 45). From the DTA curve, the exothermic peak
was observed at 495 °C.
Figure 5: Thermal studies of PdNPs
Anti-fungal activities
For Antifungal studies, Mueller-Hinton agar plates were swabbed with sterile cotton tipped swab that was
first dipped in the freshly prepared diluted culture. A 5 mm hole was bored aseptically with a sterile cork
borer. The agar plugs were taken at carefully without disturbing the surrounding medium. The holes were
filled with 50, 100, 150 µl of 0.01M PdNPs and allowed to stand for 1 h for the perfusion of the nanoparticles.
The plates were kept for further incubation at 30 °C for five days. The colloidal PdNPs inhibited the growth
of the fungus Aspergillus niger which was grown in the czapek dox agar medium and formed a clear zone
around the cavity is an indication of anti-fungal activity shown in Fig. 6. The maximum zone of inhibition
of diameters was determined 2.5, 3.0 and 3.4 cm respectively, and we used a control 50 µl (1 mg/1 ml)
of antifungal clotrimazole. It is evident that the concentration of the PdNPs increases, the diameter of the
inhibition zone is also increases.
Figure 6: Anti-fungal activity of PdNPs against Aspergillus niger at different concentrations
Electrochemical studies
The electrochemical measurements of palladium nanoparticles were carried out at different scan rates in
aqueous solution. The Cyclic voltammetry responses of 1 mM [Fe(CN)6]3-/4- in a 1M KCl solution at the
Bare-CPE and modified electrode doped with 10 µl and 20 µl of 0.001 M PdNPs and their electron transfer
kinetics of the redox couple in the solution on PdNPs doped electrode and Bare-CPE as shown in Fig. 7(a).
63
The cyclic voltammograms show highly separated redox peaks for PdNPs as compared with Bare-CPE
due to the electrocatalytic nature of the PdNPs. It is evident that the oxidation peak potential shifted by
increasing the different volume of PdNPS towards more potential confirms the electrochemical reaction.
The redox peak currents were increased gradually with increasing of PdNPs volume in the bare carbon paste
and showed maximum redox current value at 20 µl. The redox peak currents were increased gradually with
increasing scan rate shown in Fig. 7(b) [14, 15]
Figure 7(a): Cyclic voltammograms of bare and different volumes (1 mM 10 and 20 µl of PdNPs) of doped CPE
electrodes (b) Cyclic voltammograms of bare and different scan rates of 1 mM 20 µl of PdNPs doped CPE electrode.
CONCLUSIONS
A simple, efficient, low-price, environmentally friendly, single step green chemical method is used for
synthesizing the stable and colloidal palladium nanoparticles by using Piper longum leaf extract. The
morphology and size of the obtained PdNPs be spherical, size in the range of 3-5 nm and the crystalline
facets (111), (200), (211), (311) and (222) face-centered cubic respectively. The palladium nanoparticles
are highly stable at high temperatures, and weight loss is very less. Green technology represents a new
era of the innovative method to develop and test for modern drug formulations based on biosynthesized
nanoparticles with different bioactivities such as antibacterial, antifungal and anticancer properties. The
cyclic voltammograms show highly separated redox peaks for PdNPs as compared with Bare-CPE due to the
electrocatalytic nature of the PdNPs.
REFERENCES
1.
R.A. Sperling, P.R. Gil, F. Zhang, M. Zanella and W.J. Parak, Biological applications of gold
nanoparticles, Chem. Soc. Rev. 37, 1896 (2008).
2. R. Chinchilla and C. Najera, Chemicals from Alkynes with Palladium Catalysts, Chem. Rev. 114, 1783
(2014).
3. W.R. Algar, D.E. Prasuhn, M.H. Stewart, T.L. Jennings, J.B. Blanco-Canosa, P.E. Dawson and I.L.
Medintz, The controlled display of biomolecules on nanoparticles: a challenge suited to the bioorthogonal
chemistry, Bioconjugate Chem. 22, 825 (2011).
4. M. Sathishkumar, K. Sneha, I.S. Kwaka, J. Mao, S.J. Tripathy and Y.S. Yun, Phyto-crystallization of
palladium through reduction process using cinnamom zeylanicum bark extract; J. Hazard. Materials
171, 400 (2009).
5. E. Abdellatif, K. Said El and B. Mosto, Review on palladium-containing perovskites: synthesis, physico-
chemical properties and applications in Catalysis, J. Nanoscience, Nanotechnol, 14, 2012 (2014).
64
6. Ch. Sreelakshmi, K.K.R. Datta, J.S. Yadav and B.V. Subba Reddy, Honey derivatized Au and Ag
nanoparticles and evaluation of its antimicrobial activity, J. Nanoscience, Nanotechnol, 11, 6995 (2011).
7. S. Iravani, Green synthesis of metal nanoparticles using plants, Green Chem. 13, 2638 (2011).
8.
M.A. Faramarzi and A. Sadighi, Insights into biogenic and chemical production of inorganic
nanomaterials and nanostructures, Adv. Colloid. Interface. Sci. 189, 1 (2013).
9. A. Chakraborty, Kr. D. Das, M. Sinha, S. Dey and S. Bhattacharjee, Moringa oleifera leaf extract
mediated green synthesis of stabilized gold nanoparticles, J. Bionanoscience 7, 415 (2013).
10. Y. Kai, L. Todd, L. Jiayou and X. Xianmei, Facile green synthesis of palladium nanoparticles for
efficient liquid-phase hydrogenation of biomass-derived furfural, Sci. Adv. Materials 6, 135 (2014).
11. R.N.S. Yadav and M. Agarwala, Phytochemical analysis of some medicinal plants, J. Phytology 3, 10
(2011).
12. S. Madhusudana Reddy, K.K.R. Datta, Ch. Sreelakshmi, M. Eswaramoorthy and B.V. Subba Reddy,
Honey mediated green synthesis of Pd nanoparticles for suzuki coupling and hydrogenation of
conjugated olefins, Nanosci. Nanotechnol. Lett. 4, 420 (2012).
13. R. Vijayakumar, V. Devi, K. Adavallan and D. Saranya, Green synthesis and characterization of gold
nanoparticles using extract of anti-tumor potent crocus sativus, Physica E 44, 665 (2011).
14. S. Chen and K. Huang, Electrochemical studies of water-soluble palladium nanoparticles, J. Cluster
Sci. 11, 405 (2000).
15. R.W. Raut, A.S.M. Haroon, Y.S. Malghe, B.T. Nikam and S.B. Kashid, Rapid biosynthesis of platinum
and palladium metal nanoparticles using root extract of asparagus racemosus Linn., Adv. Mat. Lett. 4,
650 (2013).
65
Optimization Studies on Bioinspired Green Synthesis of
Silver Nanoparticles using Clitoria Ternatea
N. John Sushma1*, G. Swathi2, D. Prathyusha3 and B. Deva Prasad Raju4
Department of Biotechnology, Sri Padmavati Mahila Visvavidyalayam, Tirupati – 517 502

1, 2, 3
4
Department of Future Studies, Sri Venkateswara University, Tirupati – 517 5024
Abstract
Plant mediated synthesis process was more advantageous over the chemical mediated synthesis method
because it was more environmental favorable, less time-consuming, large scaled up and low cost. Keeping
this in mind, the present investigation has been taken up with the biological synthesis of Silver nanoparticles
(AgNps) using whole plant methanolic extract of Clitoria ternatea for the bioreduction of silver ions to
nanoparticles. The bioreduction process was carried out to study the various factors affecting the nanoparticles
synthesis via changing the silver ion concentration, pH and temperature. 5 mM silver ion concentration, pH
9 and temperature 50 °C is more favorable for maximum production of silver nanoparticles. Synthesis of
AgNps was confirmed by UV-Visible spectroscopy, surface plasmon resonance (SPR) peak was observed
at 470nm which indicates the polydispersion of particles. The pH and temperature of the medium plays an
important role in the synthesis of control shaped and sized nanoparticles. The colour intensity of the aqueous
solution varied with pH. In this study, at pH 9, the colour of the aqueous solution was dark brown, whereas
in pH 11 the colour was light brown; the colour difference in the aqueous solution occurred due to the
higher production of silver nanoparticles and as the temperature increased absorbance was decreased which
is inversely proportional to time. The antioxidant activity of the silver nanoparticles was carried out using
DPPH assay and Reducing power assay.
Keywords: Silver nanoparticles; Clitorea ternatea; Temperature; pH
INTRODUCTION
Nanotechnology can be defined as the designing and production of materials and devices of nanoscale that
are less than 1000nm, or 1 micrometer size has gained tremendous improvement in human life [1]. Green
technology is the most effective field in nanoparticle synthesis with expected products and economical
manner among various fields of nanotechnology [2]. Currently various types of metal inorganic nanoparticles
zinc, titanium, magnesium, copper, gold, alginate [3], silver has been widely utilizing as a health additive in
Indian and Chinese ayurvedic medicines due to its small size and large surface area compared to bulk metals
[4]. Green synthesis method of silver nanoparticles using plants is environment friendly, cost effective, no
involvement of cell culture maintenance and no requirement of an aseptic environment [5]. Plants containing
the phytochemical compounds may be used to reduce silver ions to silver nanoparticles and also used as
capping and stabilizing agents [6] controlled nanopaaricle synthesis depends on varying temperature and pH
conditions of the reaction mixture [7].
In this study, Clitoria ternatea whole plant methanolic extract was used for the silver Nanoparticles
synthesis. The optimum silver ion concentration, pH and temperature for silver nanoparticles synthesis was
studied by UV-vis spectrophotometer.
Chemicals and Plant Material

Silver nitrate, deionised water, hydrochloric acid, sodium hydroxide, methanol, Clitoria ternatea were
collected from Ramapuram village of Chittoor district, AP.
Figure 1: Clitoria ternatea
Preparation of Leaf Extract

Whole plant of Clitoria ternatea was washed with tap water and again washed thoroughly by double distilled
water for three times and shade dried about 7 days. Thoroughly dried plant was cut into fine pieces and made
a fine powder. 5 grams of fine powder were taken in 100 ml of 70 % methanol and stirred for 4 days. The
extract was filtered through Whatman No 1 filter paper, and supernatant collected was stored at 4 °C for
further nanoparticles synthesis process.
Synthesis of Silver Nanoparticles

200 µl of leaf extract was added into 2 ml of 5 mM silver nitrate aqueous solution and incubated at room
temperature. Formation of brown colour solution was observed. Later, the experiments were carried out
at different concentrations of silver nitrate (1, 2, 3, 4 and 5 mM), pH (3, 5, 7, 9 and 11) and temperature
(40 °C, 50 °C, 60 °C, 70 °C and 80 °C). The pH of the reaction mixture was adjusted by using 0.001 N
sodium hydroxide and 0.001 N Hydrochloric acid. The effect of these parameters on the synthesis of silver
nanoparticles was monitored by UV-Vis spectrophotometer (GENESYS 10S UV-Vis v4.002 2L9P112005).
(a) (b) (c)
Figure 2: a) silver nitrate solution b) silver nitrate + plant extract c) Nanoparticles
Characterization of green synthesized silver nanoparticles

The reduction of silver ions (Ag+) to silver nanoparticles (Ag0) was spectrometerically identified by double
beam UV-Vis spectrophotometer (GENESYS 10S UV-Vis v4.002 2L9P112005) at different wavelength
(400- 700 nm). The graph of wavelength on X-axis and absorbance on Y-axis was plotted.
67
Visual observation
Formation of silver nanoparticles was preliminarily confirmed by the change of light green to brown
colour by adding leaf extract with silver nitrate solution due to the excitation of free electrons in the
nanoparticles [8].
UV-Vis spectrophotometer analysis
Effect of silver ion concentration:

The UV-Vis spectrum (Fig. 1) shows the effect of silver nitrate concentration in the silver nanoparticles
synthesis by using the whole plant extract of Clitoria ternatea. Characteristic surface plasmon absorption
band was observed at 470 nm for the brown coloured silver nanoparticles synthesized from 1 mM silver
nitrate. From 1 mM concentration to 3 mM concentration showed increased absorbance whereas 4 mM
concentrations showed decrease in absorbance and again raised in absorbance at 5 mM concentration. The
absorption was increased while increasing the concentration of silver nitrate from 1 mM to 3 mM. In 5 mM
concentration, the nanoparticles synthesis and size reduction was started quickly than other concentrations
due to the more availability of functional groups in the plant extract. Colour intensity was less from 1 to
4 mM when compared to 5 mM concentration. Though 3 and 5 mM showed very near absorbance our
investigation concludes that 5 mM concentration is suitable for nanoparticle synthesis because of quick
colour change.
Figure 3: UV-Visible analysis of silver nanoparticle synthesis at different concentrations of AgNO3
Effect of Temperature
Temperature is a physical parameter which plays an important role in controlled synthesis of nanoparticles
by nucleation process. On an addition of methanolic extract of Clitoria ternatea to the aqueous 5 mM silver
nitrate solution. The reaction medium quickly changed from light green to brown colour. Experimental
temperature ranges from 40 to 80 °C and maximum synthesis and increase in absorbance observed increase
in temperature at 50 °C and thereafter decrease in absorbance observed at high temperatures (i.e) at 60,
70, 80 °C indicates crystal formation around the nucleus [9]. It was observed that maximum absorbance
was observed at 50 °C at 470 nm [10]. At moderate temperature, the peak was narrow which indicates that
formed nanoparticles are small in size and at high temperatures broadening of the peak was observed which
indicates large size nanoaprticles are formed. Our observation concludes that 50 °C temperature was suitable
for nanoparticle synthesis.
68
Figure 4: UV-Visible analysis of nanoparticle synthesis at different temperatures
Effect of pH
Effect of pH on synthesis of silver nanoparticles by Clitoria ternatea plant extract was tested over varying pH
range controlling shape and size of the particle [9]. The UV-Visible spectra showed the maximum absorption
at pH 9, as increase in pH observes decrease in absorbance. Larger size nanoparticles were synthesized at
low pH (acidic conditions), whereas at alkaline conditions smaller size nanoparticles were observed. At low
pH (3, 5) at high pH of 11, the surface plasmon resonance of the peak was broadened resulted in large size
particles whereas at pH 9 there observed a narrow peak indicating formation of smaller size particles. At
alkaline pH, the greater number of nuclei formation occurs instead of aggregation leads to the synthesis of
more nanoparticles with smaller diameter due to the high concentration of hydroxyl ions and domination of
repulsive forces. The optimum pH was found to be 9 as soon as the addition of AgNO3 to plant extract and
synthesis occurred in 30 minute’s duration [11]. The present investigation indicates alkaline pH of 9 is more
suitable for synthesis of silver nanoparticles.
Figure 5: UV-Visible analysis of nanoparticle synthesis at different pH
Effect of Time
Absorbance of silver nanoparticles increased as the incubation times increases for about 12 hrs of time. The
absorbance of silver colloidal solution increased with span of time and maximum absorption was observed
at 12 hrs of reaction [9]. The SPR band increases as the time increases (30 mins to 12 hrs) obviously
confirmed the silver nanoparticle synthesis was accomplished in 30 min and stabilized at 12 hrs of time. In
our observation, the optimum time was 30 min and at 150 and 180 mins there is a decrease in absorbance as
time increased and again raised and stabilized at 12 hrs. More silver nanoparticles are formed as the reaction
time increases due to OH groups are being transferred to carbonyl groups through air oxidation as a result
silver ion reduction takes place [12].
69
Figure 6: UV-Visible analysis of nanoparticle synthesis at different time intervals
Concentration Ratio of Silver Nitrate and Plant Extract

Optimization of concentration ratio of silver nitrate and plant extract is (5 Mm) 2 ml:200, 400, 600, 800, 1000
µl. as the concentration of the plant extract increasing the absorbance of the SPR band increased at 1000 µl
concentration at 470 nm. Synthesis was started at 200 µl concentration, so we have taken the optimum plant
concentration as 200 at this concentration optimum brown colour of the solution was observed.
Figure 7: UV-V analysis of nanoparticle synthesis at different plant concentrations
Antioxidant Activity of Silver Nanoparticles by DPPH and Reducing Power Assay
DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging assay

Scavenging activity on DPPH was assessed according to the method [13] with a slight modification. Briefly,
50 to 150 µl of extracts (1 mg/ml) were mixed with 3ml of the methanolic solution of 0.1 mM DPPH. The
mixture was shaken well and incubated at room temperature for 30 min and absorbance was measured at 517
nm in a spectrophotometer. Experiment was performed in triplicate and average was taken for determination
of percentage inhibition.
Reducing Power Assay

The reducing power of extracts was determined by following method 0.5 ml of extracts (0.2 to 1 mg/ml)
was mixed with 0.5 ml of 0.2 M phosphate buffer (pH 6.6) and 0.5 ml potassium ferrocyanide (1 %). After
incubating the mixture at 50 ºC for 20 min., 0.5 ml of 10% trichloroacetic acid was added and then mixture
was centrifuged at 3000 rpm for 10 min. 1ml of supernatant was mixed with 1ml of distilled water and 0.2
ml FeCl3 (0.1 %) and the absorbance was measured at 700 nm.
70
Figure 8: (a)Antioxidant activity of dpph (b)Reducing power assay Activity
CONCLUSION
Nanotechnology is ecofriendly approach for the synthesis of nanoparticles using biological material with
medicinal values and cost effective approach. In this study, we used Clitoria ternatea for the synthesis and
optimized the various parameters for effective synthesis were:Temperature: 50 °C, Time:12hrs, concentration
of silver nitrate:5mM, pH:9, concentration of silver to plant extract ratio:(10:90).
REFERENCES
1. Cristina B., I.P.B. Ivan and K. Robbie, 2007, Nanomaterials and nanoparticles: Sources and toxicity,
Biointerphases, 2(4): MR17 - MR172.
2. EK Elumalai, TNVKV Prasad, V Kambala, PC Nagajyothi and E David, Archives of Applied Science
Research, 2010, 2 (6) 76-81.
3. K.S.H. Naveen, G. Kumar, L. Karthik and K.V.B. Rao, “Extracellular biosynthesis of silver nanoparticles
using the filamentous fungus penicillium sp,” Archives of Applied Science Research, vol. 2, no. 6, pp.
161–167, 2010.
4. M Rai, A Yadav and A Gade, Biotechnol. Advances, 2009, 27, 76–83.
5. Ravichandran V., Z.X. Tiah, G. Subashini, F.W.X. Terence, F.C.Y. Eddy, J. Nelson and A.D. Sokkalingam,
2011, Biosynthesis of silver nanoparticles using mangosteen leaf extract and evaluation of their
antimicrobial activities, Journal of Saudi Chemical Society, 15: 113-120.
6. JL Gardea-Torresdey, E Gomez, J Peralta-Videa, JG Parsons and HE Troiani, Nano. Lett., 2002, 2,
397–401.
7. M Gericke, A Pinches, Gold Bull., 2006, 39, 22-28.
8. M Safaepour, AR Shahverdi, HR Shahverdi, MR Khorramizadeh and AR Gohari, Avicenna J Med
Biotech, 2009, 2, 111-115.
9. Amit K.M., K. Abhishek and C.B. Uttam, 2012, Free Radical Scavenging and Antioxidant Activity of
Silver Nanoparticles Synthesized from Flower Extract of Rhododendron dauricum, Nano Biomedicine
Engineering, 4(3): 118-124.
10. Sougata G., P. Sumersing, A. Mehul, K. Rohini, K. Sangeeta, P. Karishma, S.C. Swaranjit, B. Jayesh,
D.D. Dilip, J. Amit and A.C. Balu, 2012, Synthesis of silver nanoparticles using Dioscorea bulbifera
71
tuber extract and evaluation of its synergistic potential in combination with antimicrobial agents,
International Journal of Nanomedicine, 7: 483-496.
11. Sathishkumar M., K. Sneha and Y.S. Yun, 2010, Immobilization of silver nanoparticles synthesized
using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity, Bioresource
Technology, 101: 7958-65.
12. Ravichandran V., Z.X. Tiah, G. Subashini, F.W.X. Terence, F.C.Y. Eddy, J. Nelson and A.D.
Sokkalingam, 2011, Biosynthesis of silver nanoparticles using mangosteen leaf extract and evaluation
of their antimicrobial activities, Journal of Saudi Chemical Society, 15: 113-120.
13. Blois M.S (1958) Nature, 181, 1199-1200.
72
Biosynthesis and Characterization of Silver and Gold
Nanoparticles in Piper nigrum L. (Piperaceae)
M. Manogaran1 and M. B. Viswanathan2, *
1, 2
Centre for Research and Development of Siddha-Ayurveda Medicines (CRDSAM), Department of Plant
Science, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India
*Corresponding author.
e-mail: vinaabdu@gmail.com, Tel: +91 431 2407061, fax: +91 431 2407045
Abstract
The synthesis of nanoparticles or nanocrystals is in the modern nanotechnology. Biosynthesis of
nanoparticles by plant extracts is currently under exploitation. Not only could silver and gold nanoparticles
ranging from 90 to 40 nm in size be fabricated, but also spherical shape silver nanopartilces or rod,
triangular, oval- and circle-shaped gold nanoparticles could be easily modulated by reacting the biomass
of Piper nigrum leaf with aqueous silver or gold precursors at ambient temperature. The marked
difference of shape between gold and silver nanoparticles was attributed to the comparative advantage
of protective biomolecules and reductive biomolecules. The polychlorinated compounds and the water-
soluble heterocyclic components were mainly responsible for the reduction of silver ions or chloroaurate
ions and the stabilization of the nanoparticles, respectively. The Piper nigrum leaf in this work was very
suitable for simple synthesis of nanoparticles.
INTRODUCTION
Nanotechnology (and nanoscience) originates from the Greek word meaning “dwarf”. It is the science
of the extremely tiny and involves the study and use of materials on an unimaginable small scale.
Proliferation of nanotechnology has opened up novel fundamental and applied frontiers in materials
science and engineering, such as quantum dots [1], surface-enhanced Raman scattering (SERS) [2]
and nanobiotechnology [3]. Among them, nanobiotechnology is the multidisciplinary integration of
biotechnology, nanotechnology, chemical processing, physical methodology and systems engineering
into biochips, molecular motors, nanocrystals and nanobiomaterials [3]. In the last decade, biosynthesis
of nanoparticles as an emerging highlight of the intersection of nanotechnology and biotechnology has
received increasing attention due to a growing need to develop environmentally benign technologies
in material syntheses. The significance of such a synthetic protocol has been well demonstrated [4, 5].
This is particularly important for noble metals such as Au and Ag which have strong surface plasmon
resonance (SPR) oscillations. The shape selective metal NPs such as rods, tubes, wires, triangles, prisms,
hexagons and cubes can be regularly synthesized by chemical, biological and physical methods [6-10].
The syntheses of metal nanoparticles (NPs) and nanostructured materials are attracting attention in recent
research because of their valuable properties which make them useful for catalysis [11]. In addition,
they synthesized gold nanotriangles using Tamarind leaf extract and studied their potential application in
vapour sensing [12]. In the last decade, biosynthesis of NPs have received considerable attention due to
the growing need to develop clean, non-toxic chemicals, environmentally benign solvents and renewable
materials [13]. Very recently, they have demonstrated synthesis of gold nanotriangles and silver NPs using
Aloe vera plant extracts [14]. This scientific study leads to manipulate, measure, manufacture and make
predictions at the scale of 1-100 nm. Nano refers to a nanometer (nm), i.e. one nanometer is a millionth of
a millimeter or about one eighty thousandth the width of a human hair. At this dimension, materials exhibit
novel properties that differ from both the isolated atom and bulk material, and largely depend on size of
the particles. It is interdisciplinary involving chemistry, physics, biology, engineering, toxicology, etc.
Common nanoparticles that are distributed in nature are such as proteins, enzymes and nanosized particles
present in the atmosphere that are useful for the survival of organisms.
One thing that nanotechnologies share is the dimensions that they operate on. They exploit the fact that,
at this scale, materials can behave very differently from when they are larger. Nanomaterials can even change
their color; particles of gold can appear red, blue or yellow depending upon their size [15].
Nanoparticles have greater commercial availability in the world market. Therefore, research in
this direction is under exploration at present. Synthesis of nanoparticles is from physical and chemical
means and thus it may cause considerable environmental pollutions and consume relatively high
production cost.
Nanoparticles are being viewed as fundamental building blocks of nanotechnology. The most
important and distinctive property of the nanoparticles is that they exhibit larger activity. Synthesis
of nanoparticles can be carried out using various deals of efforts that have been put up for the search
of methods utilizing the biological systems such as microorganisms and plants for the synthesis of
metal nanoparticles. Various microorganisms such as bacteria, fungi, and yeasts have come-up as
nanofactories that synthesize metal nanoparticles. Metal nanoparticles have tremendous applications in
the areas of catalysis, optoelectronics, diagnostic biological probes and display devices [16]. Effectively
studied nanoparticles today are those made from noble metals, in particular Ag and Au that play a
significant role in the field of biology and medicine. The use of environmentally benign materials like
plant leaf extract, bacteria and fungi for the synthesis of silver nanoparticles offers numerous benefits
of eco-friendliness and bio-medical application as they do not use toxic chemicals in the synthesis.
Development of nanoparticles forms green chemistry as they are free from pollution and efficient in
activity. Chemical synthetic methods lead to the presence of some toxic chemical species adsorbed on
the surface that may have effects in medical applications.
Gold nanoparticles have been in use for more than 400 years for the treatment of certain illness, and the
staining of glass enamels [17]. But nowadays, the preparation of nanoscaled gold materials has become very
important due to their unique properties of medicinal usages. Silver (Ag) is known as a powerful disinfecting
agent for killing unicellular microorganisms and has the strongest antimicrobial effects. Also, it is known
to exhibit superb inhibitory effects of algal growth. Presently available silver-based inorganic antimicrobial
agents are produced in the form of silver-supported inorganic powders, silver colloids, and metal silver
powders. The silver-supported inorganic powders are the most used and thus are representative of a typical
inorganic antimicrobial agent.
In addition, heavy metals such as copper [18] or zinc may exert the same action. Gold nanoparticles
have been considered as an important area of research due to their unique and tunable surface plasmon
resonance and their application in biomedical sciences including drug delivery, tissue or tumor imaging,
photothermal therapy and immunochromatographic identification of pathogens in clinical specimens.
A traditional medicine, well-known for curative properties, as well as one of the food ingredients used
as digestive in India is Piper nigrum L. (Piperaceae) which was investigated for the synthesis of silver and
gold nanoparticles.
74
EXPERIMENTAL DETAILS
Collection of Plant Material

The plant material, Piper nigrum (MM and MBV 1328) was collected at Yercaud in the Shervarayan hills,
Salem District, Tamil Nadu, India, under cultivation.
Preparation of Leaf Extracts

The leaves were collected individually from the plant, washed thoroughly thrice with distilled water,
shade-dried up to 5 days and prepared fine powder by grinding. The fine powder was sterilized at 121°C
for 15 min and weighed from which 20 g each was taken, mixed with 200 ml of Milli Q water and kept
in boiling water bath at 60°C for 10 min. The extracts were filtered using Whatman No.1 filter paper
and the extracts thus collected were stored in a Refrigerator at 4°C for further studies to avoid microbial
contamination.
Biosynthesis of Nanoparticles
For the synthesis of silver and gold nanoparticles, silver nitrate and chloroauric acid prepared at the
concentration of 10-3 M with pre-sterilized Milli Q water were used respectively. A quantity of 1.5 ml
of each extract was mixed with 30 ml of 10-3 M of silver nitrate and chloroauric acid respectively for
the synthesis of silver and gold nanoparticles. Silver nitrate and chloroauric acid were taken in similar
quantities of 1.5 ml each without adding plant extracts to main respective controls. The saline bottles
were tightly covered with aluminium foil in order to avoid photo reduction of silver and gold ions,
incubated at room temperature under dark condition and observations were recorded at 0.5, 1, 2, 5, 12,
and 24 h (Fig. 1).
Characterization of Nanoparticles
After the prescribed incubation, the solutions were characterized on the basis of spectroscopic and microscopic
analyses.
UV-Vis Spectra analysis

A small quantity of biosynthesized nanoparticles was characterized by UV-vis spectroscopy. After color
development, a small aliquot of the solution was absorbed between 200 and 900 nm under UV-vis
spectroscopy.
Fourier Transform-Infra Red (FT-IR) Spectroscopy

The analysis of bio-reducing agent present in each of the extracts was measured by FT-IR. After the reaction,
a small aliquot of the concentrated reaction mixture was measured in the transmittance mode at 4000 to
400 cm-1 (Fig. 2). The spectra of the extracts taken before and after the biosynthesis of nanoparticles were
analyzed.
Scanning Electron Microscopy (SEM) with Energy Dispersive Spectroscopy (EDS)

The aqueous solution containing silver and gold nanoparticles was subjected to cooling centrifugation at
6000 rpm for 10 min. Supernatant solution was decanted and the remains present as thin-layer solid material
which was collected, dried in hot air oven at 60°C until complete drying and examined under scanning
electron microscopy (MODEL JEOL, JSM-5610) at different magnifications (10,000 X and 40,000 X). The
nature of elements was identified with the help of EDS in EDAX mode (Figs.4 & 5).
75

Silver nitrate and chloroauric acid were used because of their high antibacterial property and commercially
more viable to synthesize gold and silver nanoparticles.
Biosynthesis of silver and gold nanoparticles by Piper nigrum leaf

After incubation, biosynthesis of nanoparticles was indicated by the change of colors from water color
to brown for silver nanoparticles and light yellow to red for gold nanoparticles Spectroscopic data were
analyzed to characterize silver and gold nanoparticles.
UV-VIS Spectroscopy
The UV-vis spectroscopic studies revealed the presence of beard peaks at 540 nm. In case of silver nitrate
solution, the peaks were stronger than chloroauric acid solution. The plasmon resonance of the silver
nanoparticles was recorded. When the precursor silver nitrate and chloroauric acid solutions were mixed
with the plant extracts they were reduced into silver (Ag) and gold (Au) nanoparticles respectively. When the
leaf extract of Piper nigrum was mixed at 0.1% concentration of the respective silver nitrate (AgNO3) and
chloroauric acid (HAuCl4) aqueous solutions the solutions changed their color from white to brown for silver
nanoparticles and light yellow to red for gold nanoparticles. The change in color is due to the excitation of
surface plasmon vibration, which is indicated by the formation of silver and gold nanoparticles at different
time intervals such as 30 min, 1, 2, 5, 12, and 24 h (Fig. 1).
Figure 1: Absorption spectra of silver (a), and gold (b) nanoparticles after bioreduction in Piper nigrum leaf at 121°C
when 0.1 g each leaf fine powder exposed to 100 ml each of 1 mM aqueous solution of AgNO3 and HAuCl4 respectively
The UV-vis absorption spectroscopy is one of the main techniques followed to examine size and shape of
the nanoparticles in the aqueous suspensions [19]. Optical response was recorded under UV-vis spectroscopy
in relation to increase in time duration. The observation of brown and red colors is a characteristic feature
for the surface plasmon resonance (SPR) band due to the formation of different sizes of silver and gold
nanoparticles in the respective solutions. The transverse plasmon resonance absorption peak appeared at 540
nm is slightly shifted to shorter wavelength along with increase in intensity.
The observation of reduction of silver ions present in the aqueous solution of silver complex during
reaction with the ingredients of the plant extract may be correlated by the formation of silver nanoparticles
in the solution under UV-vis spectroscopy. This observation could be attributed to the excitation of surface
plasmon vibrations and it has resulted in the formation of silver nanoparticles. The UV-vis spectrograph of
colloidal solution of silver nanoparticles by changing water to yellowish brown colour was recorded as a
function of time using a quartz cuvette with water as reference in 1:1 ratio of silver ion complex (1 mM) and
leaf extract in Parthenium hysterophorus [20].
76
Silver nanoparticles were formed in Cinnamomum camphora when constant aqueous AgNO3 at 50 ml, 1
mM with 0.1 g bio-mass produced silver nanoparticles as indicated by sharp absorbance at around 440 nm [21].
Gold nanoparticles were produced in Coriander leaf extract at 10-3 M aqueous HAuCl4 after 12 h due to
the excitation of surface plasmon vibrations as indicated by color change to pink-ruby red [22]. The surface
plasmon resonance (SPR) bond of gold occurs initially at about 541 nm after 5 min, increases in intensity as
a function of time of reaction and centers at about 536 nm. The plasmon bond of gold nanoparticles is broad
with an absorption tail in the longer wavelength that extends well in to the near infrared region attributing
to the excitation of in-plane surface plasmon resonance and indicates significant anisotropy in the shape of
gold nanoparticles.
Fourier Transform-Infra-Red Spectroscopy (Table 1; Fig. 2)

The FT-IR spectrum of the crude leaf extract is given wherein some pronounced absorbances were recorded
in the region between 4000 and 400 cm-¹. They include 3432 (secondary amine, free, N-H asymmetric
stretching), 2830 (alkyl ethers for C-H stretching), 2085 (isothiocyanates, aromatic N=C=S stretching), 1632
(β-dikeone, enolic form, C = O), 1381 (alkanes, R-CH3 symmetric bending), 1353 (deformation bending and
652 (C-S, R-C-CH3 stretching for sulphur compounds), cm-¹.
The FT-IR spectrum of the leaf extract with silver nitrate solution after 5 h is 3435 (secondary amine
(free) N-H asymmetric stretching), 2829 (alkyl ethers, C-H stretching), 2728 (aldehyde, C-H stretching), 2091
(isothio-cyanates, aromatic N=C=S stretching), 1631 (β-diketone (enolic form) C=O), 1353 (deformation
bending, R-C-CH3) and 644 (sulphur compounds, C-S stretching) cm-¹. Further, the spectrum revealed the
disappearance of alkanes at 1381 (alkanes, R-CH3 symmetric bending) and appearance of C-H stretching
of aldehyde at 2728 (aldehyde, C-H stretching) and the functional groups were as that of the crude extract.
In the case of silver nitrate solution at 12 h, aldehyde disappeared and absorptions were noticed at 3432
(secondary amine (free), N-H asymmetric stretching), 2831 (alkyl ethers, C-H stretching), 2081 (isothio-
cyanates, aromatic N=C=S stretching), 1632 (β-diketone (enolic form) C=O), 1358 (deformation bending,
R-C-CH3) and 653 (sulphur compounds, C-S stretching) cm-¹ as that of the functional groups recorded for
the crude extract.
The FT-IR spectrum of the leaf extract with chloroauric acid solution after 5 h revealed absorptions at
3465 (dimeric associated (intermolecular), O-H / NH stretching bonded), 2828 (alkyl ethers, C-H stretching),
2730 (aldehyde, C-H stretching), 2094 (isothio-cyanates, aromatic N=C=S stretching), 1630 (β-diketone
(enolic form), C=O) 1382 (alkanes, CH3 symmetric bending, R-CH3), 1353 (deformation, R-C-CH3), 771
(polychlorinated compounds, C-Cl Stretching) and 650 (sulphur compounds, C-S stretching) cm-¹. The
absorptions at 2730 (C-H stretching of aldehyde) is different from the crude extract but similar as that of
silver nitrate solution after 5 h, and at 1382 (alkanes, CH3 symmetric bending, R-CH3) it is similar as that
of the crude extract. The absorptions at 3465 (dimeric associated, intermolecular O-H stretching) and 771
(polychlorinated compounds, C-Cl stretching) are different from that of the crude extracts and silver nitrate
solutions at 5 and 12 h. The remaining functional groups are as that of the crude extract. After 12 h, the FT-
IR spectrum showed absorptions at 3400 (polymeric hydroxyl compounds, O-H/NH stretching bonded 2832
(alkyl ethers, C-H stretching 2083 (isothio-cyanates, aromatic N=C=S Stretching), 1633 (β-diketone (enolic
form), C=O), 1356 (deformation (bending), R-C-CH3) and 649 (sulphur compounds, C-S stretching) cm-¹.
Absorptions at 3465 (dimeric associated (intermolecular), O-H/NH stretching bonded), 2730 (aldehyde,
C-H stretching), 1382 (alkanes, CH3 symmetric bending, R-CH3) and 771 (polychlorinated compounds,
C-Cl stretching) at 5 h were disappeared at 12 h but a lone new absorption appeared at 3400 (polymeric
hydroxyl compounds, O-H/NH stretching bonded) indicating the presence of polymeric hydroxyl compounds
expressing O-H stretching. The remaining functional groups are similar as that of the crude extract and other
solutions at different intervals.
77
Figure 2: Typical FT-IR absorption spectra of the leaf Piper nigrum (1), before bioreduction (2, 3) and after bioreduction
of silver ions and chloroaurate ions (4, 5).
Functional groups such as alkyl ethers showing C-H stretching between 2828 and 2932, isothiocyanates
showing aromatic N=C=S stretching between 2081 and 2094, β-dikeone (enolic form) showing C=O
stretching between 1630 and 1633, deformation bending R-C-CH3 between 1353 and 1358, sulphur
compounds showing C-S stretching between 644 and 653 are present in the crude extract and silver nitrate
(AgNO3 and chloroauric acid (HAuCl4) aqueous solutions at 5 and 12 h. Secondary amines are present
between 3432 and 3435 in the crude and silver nitrate (AgNO3) aqueous solutions at 5 and 12 h. In the
case of chloroauric acid (HAuCl4) aqueous solution, dimeric associated (intermolecular) O-H stretching
recorded at 3465 at 5 h. The same solution has polymeric hydroxyl compounds showing O-H stretching
at 3400. Aldehyde bond between 2728 and 2730 is present in both the solutions at 5 h. The crude extract
and chloroauric acid (HAuCl4) aqueous solution at 5 h have alkanes CH3 symmetric bending for R-CH3 at
1381 and 1382 respectively. Only chloroauric acid (HAuCl4) aqueous solution at 5 h has polychlorinated
compounds showing C-Cl stretching (Fig. 2).
Leaves of Piper nigrum contain compounds such as piperine (an alkaloid), capsaicin, limonene and
pinene (cyclic terpenes), linalool (a terpene alcohol), caryophyllene (a bicyclic sequiterpene), and sabinene.
Capsaicin is an amphipilic molecule composed of a hydrophilic aromatic ring (A region), a dipolar amide (B
region) and a lipophilic (hydrophobic) octanyl side chain [23]. Nitrogen is present only in two compounds
such as piperine and capsaicin. In piperine, nitrogen is present in an aromatic ring whereas in capsaicin it is
present in the dipolar amide of aliphatic chain. The values recorded such as 3432 cm-1 in the crude extract,
3435 cm-1 and 3432 cm-1 in the silver nitrate solution after 5 h and 12 h respectively, and 3465 cm-1 and 3400
cm-1 in the gold chloride solution after 5 h and 12 h respectively. These values mostly correspond to NH/
OH stretching in capsaicin [23] and pinene [24]. The absorbance at 2829 and 2832 cm-1 is for alkyl ethers
CH stretching corresponded to 2840 cm-1 in piperine (https://www.erowid.org/archive/.../ piperine/IR.analysis.
piperine.pepper.pdf). Absorbance at 2728 and 2730 cm-1 in silver nitrate and gold chlorides solutions after 5
h is for aldehyde CH stretching corresponded to 2730 cm-1 in linalool (http://riodb01.ibase.aist.go.jp/sdbs/cgi-
bin/direct_frame_top.cgi). The absorbance at 1630 – 1633 cm-1 is for β-diketone, enolic form, C=O, present
in all the extracts corresponded to 1638 cm-1 of piperine [25] and 1633 and 1634 cm-1 of caryophyllene [26]
in all the extracts. Absorbance at 1381 and 1382 cm-1 in the crude extract and gold chloride solution after
5 h respectively is for alkanes R-CH3 symmetric bonding corresponded to 1376 cm-1 in linalool (http://
riodb01.ibase.aist.go.jp/sdbs/cgi-bin/direct_frame_top.cgi) and 1377 cm-1 in limonene (http://riodb01.ibase.aist.
go.jp/sdbs/ cgi-bin/direct_frame_top.cgi). Absorbance at 1353 - 1358 cm-1 in all the extracts is for deformation
bending corresponded to 1362 cm-1 in limonene R-C-CH3 (http://riodb01. ibase.aist.go.jp/sdbs/cgi-bin/
direct_frame_ top. cgi). Absorbance at 771 cm-1 in gold chloride solution after 5 h is for polychlorinated
compounds with C-Cl stretching corresponded to 783 cm-1 in limonene (http://riodb01. ibase.aist.go.jp/sdbs/
cgi-bin/ direct_frame_top.cgi). Absorbance at 644 – 653 cm-1 in all the extracts is for sulphur compounds
78
with C-S stretching corresponded to 667 cm-1 in linalool (http://riodb01. ibase.aist.go.jp/sdbs/cgi-bin/direct_

frame_ top.cgi).
The mechanisms involved in the uptake of metal ions may be intracellular accumulation and surface
adsorption. The former one is an active process because the plant must be active to carry out. In the case
of surface adsorption, it is a passive process because the chemical groups attached to the cell walls of the
plant can bind with metal ions even though when the plant is dead. It is considered as an advantage in
phytoremediation technologies by which metal contaminants are removed. If the chemical groups attached
to the cell walls are the binding sites then these groups can be adsorbed as metal ions. Therefore, there may
be a possibility to use the plant tissues to filter such ions out of the aqueous solutions. This technology is
called phytofiltration.
Scanning Electron Microscope (SEM) (Fig. 3)

The leaf extract of Piper nigrum is a promising one for the development of silver and gold nanoparticles. The
SEM absorption of the products was recorded as synthesized nanoparticles in the form of spherical structures
measuring about 90 nm in diameter to silver nanoparticles and rod, triangle and circular structures of about
40 nm to gold nanoparticles.
Figure 3: Scaning electron microgrographs of silver and gold nanoparticles after bioreduction. The biomass
(a) and (b) 0.1 g was exposed to 100 ml in 1mM aqueous solution of AgNO3 and HAuCl4 at 60°C, Scale bars:
(a)-(b) 90-40 nm.
The size of nanoparticles has been reported variously such as 2-3 nm in Medicago sativa [27], 10-20 nm
in Emblica officinalis [28], 16-40 nm in Pelargonium graveolens [7], 30-80 nm in Parthenium hysterophorus
[20], 60-80 nm in Carica papapya [29] for Ag nanoparticles, 5-20 nm in Avena sativa at pH 3&4 [30], <20
nm in Oat [30], 15-25 nm in Emblica officinalis [28], 20-40 nm in Tamarindus indica [12] and Alfalfa,
Medicago sativa [31] and 6.75-57.91 nm in Coriander sativum [22] for Au nanoparticles, 55-80 nm in
Cinnamomum camphora [21] and 15.2 ± 4.2 nm in Aloe vera for Ag and Au nanoparticles [14], 18-38 nm
in Phyllanthus amarus [32] for Ag/Au nanoparticles, 1-4 nm in Medicago sativa [31] for Ti/Ni bimetallic
nanoparticles, and 50-100 nm in Azadirachta indica [9] for Ag, Au and Ag/Au bimetallic nanoparticles.
Energy Dispersive Spectroscopy (EDS)

The EDS revealed the presence of higher percentages of pure silver and gold nanoparticles. Silver peak is
higher than that of gold peak. The SEM micrographs of Alfalfa biomass showed a corresponding elemental
composition of Na, Mg, K, S, Ca, P, Fe, Co, Mn, Cu, Mo, B, Cl as well as traces of Zn, Si, Ni and Pb analysis
by EDS [33] wherein the highest being Ca, Mg and S with 63.5%, 11.9% and 10.72% respectively and the
absence of Fe although it is found in very low propositions. As EDS equipment works at low vacuum (1-270
pa) it allows to observe non-conducting samples without the need to cover them with a thin conductive film,
and consequently no evidence of noise by the coating material.
79
Biosynthesis of silver and gold nanostructures in the leaf extract of Piper nigrum is further demonstrated
and confirmed by the characteristic peaks observed in the EDS image. Analysis showed the presence of Ag,
Cl, O and Mg, or Au, Cl, O, Zn and Cu as the elemental composition of the silver nitrate and gold chloride
solutions respectively, the highest being Ag and Au (Figs. 4 & 5).
The nanoparticles formed in the plant extracts have high toxicity to the pathogens of Staphylococcus
epidermidis and Bacillus cereus of gram-positive bacteria and Salmonella paratyphi of gram-negative
bacteria ranging from 7-11 mm. The results revealed that the plant extracts may be used as therapeutic agents
in biomedical applications.
Figure 4: EDS-Spectroscopy view of the leaves in Piper nigrum showing synthesis of silver nanoparticle and elemental
silver signal at higher percentage
Figure 5: EDS-Spectroscopy view from Piper nigrum showing synthesis of gold nanoparticles and elemental silver
signal at higher percentage
4.. CONCLUSION
A bioreductive approach to the synthesis of silver and gold nanoparticles in the leaves of Piper nigrum
demonstrates the formation of spherical silver nanoparticles and triangular, rod, oval-and circle-shaped gold
nanoparticles to the aqueous solutions of AgNO3 and HAuCl4 in the range of 90 and 40 nm respectively. The
marked difference in the shape of the gold and silver nanoparticles may be attributed to the comparative
80
advantage of protective biomolecules and reductive biomolecules. The compounds such as piperine, capsaicin,
limonene and pinene, linalool, caryophyllene, and sabinene present in the leaves of Piper nigrum are mainly
responsible for the reduction of silver ions or chloroaurate ions and their stabilization. Instead of the boiled
leaf broth method followed in the previous studies, leaves of Piper nigrum appear to be environment-friendly
and low-cost candidate as a reductant for synthesizing either silver or gold nanoparticles. This procedure can
be extended to the synthesis of other nanoparticles from different chemical compositions.
ACKNOWLEDGEMENTS
The authors express their special thanks to the Head, National Facility for Marine Cyanobacteria,
Bharathidasan University, Tiruchirappalli-24, Archbishop Casmier Instrumentation Centre (ACIC), St.
Joseph’s College (Autonomous), Tiruchirappalli-2 and the Director, Centralized Instrumentation and Service
Laboratory, Annamalai University, Chidambaram, for their help in spectral studies.
REFERENCES
1. Chan WCW and Nie S, Quantum dot bioconjugates for ultrasensitive nonisotopic detection, Science
1998; 281:2016-2018.
2. Tian Z and Ren B, Adsorption and reaction at electrochemical interfaces as probed by surface-enhanced
Raman spectroscopy, Annu Rev Phys Chem 2004; 55:197-229.
3. Klefenz H, Nanobiotechnology: from molecules to systems, Eng Life Sci 2004; 4:211-218.
4. Bhattacharya D and Gupta RK, Nanotechnology and potential of microorganisms, Crit Rev Biotechnol
2005; 25:199-204.
5. Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G and Mukherjee P, The use of microorganisms for
the formation of metal nanoparticles and their application, Appl Microbiol Biotechnol 2006; 69:485-
492.
6. Lim JK, Kim Y, Lee SY and Joo SW, Spectroscopic analysis of l-histidine adsorbed on gold and silver
nanoparticle surfaces investigated by surface-enhanced Raman scattering. Spectrochim Acta A, 2008;
69:286–289.
7. Shankar SS, Ahmad A, Pasricha R and Sastry M, Bioreduction of chloroaurate ions by Geranium leaves
and phytic fungus yields Gold nanoparticles of different shapes, J Mater Chem, 2003a; 13:1822.
8. Shankar SS, Ahmad A and Sastry M, Geranium leaf assisted biosynthesis of silver nanoparticles,
Biotechnol Prog 2003b; 19:1627-1631.
9. Shankar SS, Rai A, Ahmad A and Sastr M, Rapid synthesis of Au, Ag and bimetallic Au core-Ag shell
nanoparticles using Neem (Azadirachta indica) leaf broth, J Colloid Interface Sci 2004a; 275:496-502.
10. Shankar SS, Rai A, Ankamwar B, Singh A, Ahmad A and Sastry M, Biological synthesis of triangular
Gold nanoprisms, Nat Mater 2004b; 3:482.
11. Narayanan R and EI-Sayed MA, Shape-dependent catalytic activity of platinum nanoparticles in
colloidal solution, Nano Lett 2004; 4:1343-1348.
12. Ankamwar B, Chaudhary M and Sastry M, Gold nanotriangles biologically synthesized using tamarind
leaf extract and potential application in vapor sensing, Synth React Inorg Met-Org Nano-Metal Chem
2005; 35:19-26.
13. Harris AT and Bali R, On the formation and extent of uptake of silver nanoparticles by live plants, J
Nanopart Res 2008; 10:691-695.
81
14.
Chandran SP, Dhadudhary M, Pasricha R, Ahamad A and Sastry M, Nanotriangles and silver
nanoparticles using Aloe vera plant extract, Biotech Prog 2006; 22:577-583.
15. Wagner J and Kohler JM, Continuous synthesis of gold nanoparticles in a microreactor, Nano Lett
2005; 5:685.
16. Wagner J, Kirner T, Mayer G, Albert J and Kohler JM, Generation of metal nanoparticles in a
microchannel reactor, Chem Eng J 2004; 101:251.
17. Tanaka K., Nanotechnology towards the 21st century, J Thin Solid Films 1999; 341:120-125.
18. Arru L, Rognoni S, Baroncini M, Bonatti PM and Perat P. Copper localization in Cannabis sativa L.
grown in a copper-rich solution. Euphytica 2004;140:33-38.
19. Wiley BJ, Im SH, Mc Lellan J, Seikkinin A snd Xia Y, Maneuvering the surface plasmon resonance
of silver nanostructures through shape-controlled synthesis, J Phys Chem B, 2006; 110:15666-15675.
20. Parashar V, Parashar R, Sharma B, Avinash C and Pandey, Parthenium leaf extract mediated synthesis
of silver nanoparticles: A novel approach towards weed utilization, Dig. J Nanomater and Biostruct
2009; 4:45-50.
21. Huang J, Li Q, Sun D, Lu Y, Su Y, Yang X, Wang, Wang Y, Shao W, He N, Hong J and Chen C,
Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum campora leaf, Nanotech
2007; 18:104-105.
22. Badri Narayan K and Sakthivel N, Coriander leaf mediated biosynthesis of gold nanoparaticles,
Materials Lett 2008; 62:4588-4590.
23. Alberti A, Galasso V, Kovac B, Modelli A and Pichierri F, Probing the molecular and electronic structure
of capsaicin: a spectroscopic and quantum mechanical study, J Phys Chem A 2008; 112:5700-5711.
24.
Farooq A, Tahara S, Choudhary MI, Atta-ur-Rah-man, Ahmed Z, Baser KHC and Demirci F,
Biotransformation of (D)-α-pinene by Botrytis cinerea. Z Naturforsch 2002; 57:303-306.
25. Ee GCL, Lim CM, Lim CK, Rahmani M, Shaari K and Bong CFJ, Alkaloids from Piper sarmentosum
and Piper nigrum. Nat Prod Rep 2009; 23:1416-1423.
26. Demirci F, Iscan G, Guven K, Kırımer N, Demirci B and Baser KHC, Antimicrobial activities of
Ferulago essential oils. Z. Naturforsch. 2000; 55:886-889.
27. Gardea-Torresdey JL, Tiemann KJ, Armendariz V, Bess-oberto L, Chianelli RR, Rios J, Parsons JG and
Gamez G, Charachterization of Cr (VI) Binding and Reductinon to Cr (III) by agricultural by products
of Avena mondia (Oat) biomass, J Hazard Materials 2000; 80:175-188.
28. Ankamwar B, Damle C, Ahmad A and Sastry M, Biosynthesis of gold and silver nanoparticles using
Emblica officinalis fruit extract, their phase transfer and transmetallation in an organic solution, J
Nanosci Nanotech 2005; 5:1665-1671.
29. Mude N, Ingle A, Gede A and Rai M, Synthesis of silver nanoparticles using callus extract of Carica
papaya — A First Report. J Plant Biochem & Biotech 2009; 18:83-86.
30.
Armendariz V, Herrera I, Peralta-Videa JR, Yacaman MJ, Troiani H, Santiago P and Gardea-
Torresday JL, Size controlled gold nanoparticle formation by Avena sativa biomass: use of plants in
nanobiotechnology, J Nano Res 2004; 6:377-382.
31. Gardea-Torresdey JL, Tiemann KJ, Gamez G, Dokken K, Cano-Aguilera I, Furenlid LR and Renner
MW, Reduction and accumulation of gold (III) by Medicago sativa alfalfa biomass: X-ray absorption
spectroscopy, pH, and temperature dependence. Environ Sci Technol 2000; 34:4392-4396.
82
32. Kasthuri J, Katjoravem K and Rajendran N, Phyllanthin-assisted biosynthesis of silver and gold

nanoparticles: a novel biological approach, J Nanopart Res 2009; 11:1075-1085.
33. Herrera-Becerra R, Zorrilla C, Rius JL and Ascencio JA, Electron microscopy characterization of
biosynthesized iron oxide nanoparticles, Appl Phys A 2008; 91:241-246.
83
Synthesis of GSH & FA Conjugated Chitosan
Functionalized Gold Nanospheres : Physicochemical
Properties and Applications in Cancer Therapy
Kalpana Hari1, Hemamalini Vedagiri2 and Premkumar Kumpati3
Cancer Genetics and Nanomedicine Laboratory, Department of Biomedical Science,

1, 3
Bharathidasan University, Tiruchirappalli-620024, Tamil Nadu, India

2
Department of Biotechnology, Jamal Mohamed College, Tiruchirappalli-620020, Tamil Nadu, India
e-mail: pkumpati@hotmail.com, Phone: +91 8056589893, Fax: +91-431-2407 045
Abstract:
Gold nanoparticles (AuNPs) have been widely used in various biomedical applications due to their unique
size tunable optical, electronic properties and enhanced permeation and retention effect. In addition, the
external characteristics of AuNPs possess a strong surface chemistry that aids them suitable for the attachment
of biomolecules and drugs. The conjugation of biomolecules with AuNPs provides the high affinity and
bioavailability to target the various diseases. In this research, we have synthesized and characterized stable
monodispersive gold nanospheres (AuNS) by environmental benign green method using the chitosan
biopolymer via simple wet chemical reduction. The synthesized AuNS were characterized by assessing their
size, zeta potential, and surface characteristics. Folic acid was then conjugated by using activated folic
acid via terminal amino groups of the chitosan, and were purified from unreacted products. Conjugation of
glutathione (GSH) was successfully carried out through thiol chemistry. The specific interaction between the
folic acid / GSH with AuNS was evaluated by surface plasmon resonance, which confirmed specific binding
of the folate (FA) and GSH on the surface of AuNS. From the results, it has been concluded that chitosan
reduced AuNS possess suitable surface and functional groups to conjugate biomolecules for the targeted
therapy. The target specificity of AuNS coated with GSH and Folic acid was tested using breast cancer
cell lines and both cell types exhibit significant uptake of GSH &FA conjugated AuNS. Thus, FA and GSH
conjugated AuNS represent a potential new drug carrier for tumor cell-selective targeting.
INTRODUCTION
The nano carriers such as polymeric nanoparticles, metallic nanoparticles, liposomes play a crucial role in
drug delivery applications for intravenous administration to control the drug release, reduce toxic effects
and maintain the stability of the drug [1]. Among these drug delivery systems, colloidal nano carrier system
has a greater advantage due to their long half-life and specific targeting. Due to these applications metallic
colloidal nano carriers were widely used in the biomedical field for cancer and various diseases [2]. Currently
used nano carrier system possess numerous limitations such as short blood half-life (rapid elimination from
the bloodstream due to the immunological reactions) and nonspecific targeting (inducing severe side effects)
and also with the consequence of the adsorption of blood proteins (opsonins) onto the surface of colloidal
carriers [3]. To overcome these challenges functionalization of nanoparticles are usually necessary for their
biomedical applications.
However some surfactant such as cetyl trimethyl ammonium bromide (CTAB), sodium citrate
used for the conjugation, binds more strongly on the surface of the nanoparticles that leads to severe
toxicity, otherwise induces partial or complete agglomeration that affects physico chemical properties of
the nanoparticles thus limiting its potential in various applications [4]. In this context recent studies are
directed to modify the surface of the nanoparticles with biocompatible polymers such as polyethylene
glycol (PEG), PLGA and biomolecules including starch, glutathione, folic acid, etc. [5]. Previous studies
showed that these biologically functionalized AuNP using PEG possess good colloidal stability, non-
toxicity, increased bioavailability and also prevent non-specific adsorption with proteins based on both in
vitro and in vivo investigations [6].
Among these promising biomolecules, low molecular weight targeting agents, folic acid might be
exploited actively to target cancer cells due to frequently overexpressed on the surface of human cancer
cells. In addition, the folate receptor is efficiently cell internalized after binding with its ligand (folic acid)
[7]. Likewise, GSH is a small-sized natural tripeptide (c-Glu-Cys-Gly) that comprises an -SH group, an
amino group and two carboxyl groups. Moreover, it is the most abundant thiol species in the cytoplasm and
the major reducing agent in biochemical processes [8]. Hence, it has been conjugated on the surface of AuNS
used as a stabilizer as well as the coupling agent. Moreover, it has been well studied that FA and GSH have
the ability to enter the cancerous cells. Hence we have synthesized stable monodispersive gold nanospheres,
characterized and conjugated with biomolecules such as folic acid and glutathione to enhance the cellular
uptake of AuNS.

Gold (III) chloride trihydrate (HAuCl4. 3H2O, 99.99%), Medium molecular weight chitosan (composed
of b-(1 – 4)-linked d-glucosamine and N-acetyl-d-glucosamine, a degree of deacetylation of 75 – 85%
with Viscosity (200-800 cps)), glutathione (GSH), Folic acid, dicyclohexyl carbodiimide (DCC), were
purchased from Sigma-Aldrich. All chemicals were of analytical grade and used as received without
any further purification. All aqueous solutions were made with ultra-high-pure water system by Milli-Q
water (18.2 MZ).
Synthesis of Gold Nanoparticles (AuNs) Using Chitosan biopolymer

A monodispersive AuNS were synthesized well-known wet-chemical reduction method using a chitosan
biopolymer with some modification as of previously reported [9]. Briefly, the stock solution of chitosan
polymer was diluted to the required concentration using 1% acetic acid and allowed to kept in water bath at
80°C under magnetic reflux then an aqueous solution of HAuCl4 (1 mM) was quickly added and the mixture
was allowed to stir vigorously until the pale yellow color turned to dark ruby red color which confirms the
formation of AuNS. At the end of the reduction reaction, the synthesized AuNS were monitored periodically
by UV-visible spectrophotometer (Shimadzu, UV-2450, Japan) Fig (1). This process affords thus highly
stable, monodispersive AuNPs beneficial for the formation of water-soluble with an excellent reproducibility.
Characterization of AuNs
Characterization of nanoparticles was done using standard analytical techniques [10]. To analyze the SPR
spectrum of the synthesized, as well as surface modified AuNPs, the UV–VIS spectrophotometer (Shimadzu,
UV-2450, Japan) were performed using a Spectrum record (200 – 900) nm at room temperature. The size
and surface morphology of as synthesized AuNS were analyzed using high-resolution transmission electron
microscopy (HR-TEM, JOEL 2010) operated at 200kV. The concentration of gold atoms was directly
measured using (ICP – OES) Spectrometer (PerkinElmer, Optima 8x00). The number of AuNPs per Liter
was calculated according to the number of atoms present in each AuNPs. The size and surface charge of the
synthesized and modified AuNPs at different levels were examined using (Malvern Zetasizer, Nano (ZS90).
instrument (5 mW HeNe laser λ = 632 nm). The elemental analysis was done by EDS (Energy Dispersive
X-ray Spectroscopy) by placing a drop of the AuNPs solution and allowed to dry on a copper holder coated
85
with chromium film. The FT-IR investigations were carried out using (FTIR) Fourier Transforms Infrared
spectroscopy (Spectrum-RXI PerkinElmer), to confirm the functional groups of the chemical molecules with
the spectrum range of 400 – 4000 under transmittance mode operating at a wavenumber resolution of 4 cm-1.
Synthesis of Glutathione capped-Gold nanoparticles (GSH-AuNPs)

Glutathione capped AuNPs were prepared by as shown in previous methods [11]. Typically AuNPs were
allowed to stir about 5 minutes. And then equal volume of 10−3 mol L−1 concentrated aqueous solution of
glutathione was added in dropwise to the freshly prepared AuNPs. Then the solution was allowed to form
a monolayer on the nanoparticles by vigorous stirring about 2 h in a dark at room temperature. To remove
excess and uncapped GSH, the AuNPs solution was allowed to centrifugation at 13,000 rpm at 28°C for 15
minutes, and then the supernatant was removed, and the precipitant was resuspended in Milli Q water. The
ruby red color of the AuNPs solution turns to purple, indicating the self-assembly of the glutathione onto
the surface of AuNPs, and larger size of particles confirms the capping of glutathione. The adsorption of
GSH on the nanoparticle depends on the particle size and the ionic strength of the solution. The assembly of
glutathione was characterized by analyzing the progression of SPR band, and hydrodynamic sizes of GSH
capped AuNPs using spectrophotometric and dynamic light scattering technique.
Synthesis of Folic acid conjugated AuNS

Synthesis of FA-protected AuNPs was referenced to a literature [12]. The experimental process was as
follows: FA has a poor solubility in water. And it was dissolved by adding NaOH. In brief, 0.012 g FA
Powder was mixed with 10 mL H2O, and then approximately 100 mL 1.0 M NaOH solution was added to the
above turbid solution until it came to clear. During a typical synthesis, the prepared FA solution was added
drop by drop to the 1.0 mM HAuCl4 5 mL solution under magnetic stirring, and the light yellow HAuCl4
solution turned slightly darker and some precipitate emerged. The precipitate was regarded as FA, which was
insoluble at lower pH when encountering HAuCl4. For this reason, a further amount of 180 mL 0.1M NaOH
was subsequently added to obtain a clear solution with a bright orange color. Half an hour later, the system
was heated to 50°C, and the reaction continued for 6 h until a transparent red solution was obtained. The
as-prepared FA-AuNPs solution was centrifuged at 10,000 rpm for 10min to remove the free FA and then
washed three times for subsequent measurements.

Fig. 1 shows the absorption band at 520nm thus confirms the formation the formation of individual Au
nanospheres in solution which is the characteristic surface plasmon resonance band of AuNs and there was no
shifting in peaks as well as broadening of SPB, the narrow peak discloses the formation of gold nanoparticles
with uniform size and distribution. Our experiments revealed that the time taken to start reduction process
of Au (III) to Au (0) was very less [13]. The formed AuNS has size around 12 nm in diameter and well
dispersed in Milli Q water.
Table 1: Hydrodynamic diameter, poly dispersive index and zeta potential of AuNS, GSH-AuNS and FA-AuNS at
room temperature (Mean ± S.D., n=3).
Nanoparticles AuNS GSH-AuNS FA-AuNS

Hydrodynamic Diameter (r.nm) 24.09±0.349 71.78±0.99 74.06 ± 1.79
PDI 0.254±0.01 0.27±0.01 0.271±0.040
Zeta Potential (mV) 50.77±2.15 46.4±0.78 40.0±0.00
86
Figure 1: a UV-visible spectrum of Synthesized AuNS by Chitosan (λ max 520 nm). b. AFM images of synthesized
AuNS c. HR-TEM images of AuNS d. SAED pattern of AuNS.
Fig. 2 shows the SPR peak of assembled GSH and FA on AuNS and the SPR effect is sensitive to
conjugated analyte owing to their changes in original mass and their proportional increase in refractive
index that has been observed as a shift in the resonance angle of SPR and using FTIR spectrum. The
unmodified nanoparticles display characteristic surface plasmon band around 532 nm, while GSH and
FA coated AuNS shows a surface Plasmon band around 532 nm. These values are in accordance with
the colors of AuNS solution observed by naked eyes (purple color) [14]. This red-shift (10-12 nm)
in the position of the maximum absorption indicates that GSH and FA have interacted with AuNS.
The hydrodynamic diameters and zeta potentials of AuNS, GSH-AuNS and FA-AuNS, were measured
at pH 7 further to verify the presence of GSH and FA on the nanoparticles. Table 1 shows that the
hydrodynamic diameter of AuNS is smaller than GSH-AuNS and FA-AuNS that might be due to the
modification of GSH and FA onto the surface of AuNS which increases the particle size and further, the
amine and carboxyl groups of GSH and FA attract the counter ions from the chitosan to form electrical
double layer [15].
Figure 2: UV-visible spectrum of AuNS and functionalized AuNS a. GSH-AuNS b. FA-AuNS.
87
Figure 3: FTIR spectrum of free GSH and FA and conjugated GSH-AuNS and FA-AuNS
Therefore, the increased double layer thickness results in a larger particle diameter. The zeta potentials
of AuNS dispersed in water are positively charged due to the polycationic nature of amino groups
(D-Glucosamine) of chitosan polymer (Chitosan pKa = ~6.5) enables the attachment of the polymer to the
gold nanoparticle surfaces through electrostatic interactions [16]. Hence, AuNS carries net positive charge.
After conjugation with GSH through Au-SH bond, the chitosan molecules are slightly replaced by GSH,
and FA which has a free amine group and two carboxyl groups. According to the dissociation constant of
GSH and FA, the amine group is protonated, while the two carboxyl groups are deprotonated at neutral
pH. Since one carboxyl group is neutralized by the positive charge of an amino group, GSH- AuNS and
FA –AuNS carries a net negative charge [17]. The change in the surface charge alters the dielectric state
of the nanoparticles, consequently resulting in the decrease of the zeta potential values. And GSH-AuNS
confer less negative zeta values compared with AuNS because they carry one net charge. Fig.3 Shows the
FT-IR spectra of pure GSH & GSH-AuNS and FA &FA-AuNS [18]. From these spectra the characteristic
absorption peaks for –SH at 2524 cm−1 as found in pure GSH has disappeared in GSH-AuNS. The intense
difference between the FT-IR spectra, especially for –SH, suggests that GSH is modified onto the surface of
gold nanoparticles via the thiol group from the cysteine moiety of GSH. And the formed Glutathione coated
AuNS and FA-AuNS were stable over long time period. Both conjugated AuNS not only serve as a stabilizer
and drug reservoir, but also affords a long time circulation and low cytotoxicity [18].
Figure 4: Cellular uptake of free and conjugated AuNS treated MDA-MB-231 and MCF-7 cells
From the ICP-OES results, it has been concluded that the biodistribution of conjugated AuNS was high in
MDA-MB-231 and MCF-7 treated cells when compare to naked AuNS. Thus confirms the biocompatibility
of conjugated nanoparticles thus helps for the targeted drug delivery [20].
88
CONCLUSION
In this study, we examined the appropriateness of chitosan monolayer protected AuNS for the biomolecule
conjugation. In this work we have successfully conjugated the folic acid and glutathione on the surface of the
AuNS, thus confirmed by surface plasmon band spectrum and Fourier resonance energy transfer spectrum.
The cellular uptake studies confirmed that the conjugation of both GSH and FA showed higher cellular
distribution compared to naked AuNS. Thus, our results explain the suitability of utilizing chitosan protected
AuNS as a potential surface for conjugating various biomolecules and could support long time circulation
and biodistribution, which makes it potential for the targeted delivery in cancer therapy and also for various
biomedical applications.
REFERENCES
1. Wim H De Jong and Paul JA Borm, Drug delivery and nanoparticles: Applications and hazards, Int.
Jornal of nanomedicine, 3(2), 133-149 (2008).
2. Joao Conde, Goncalo Doria and Pedro Baptista, Noble Metal Nanoparticles Applications in Cancer,
Journal of Drug Delivery, 751075, 12 pages (2012).
3. Dan Peer, Jeffrey M. Karp, Seungpyo Hong, Omid C. Farokhzad, Rimona Margalit and Robert Langer,
Nature Nanotechnology, 2, 751-760 (2007).
4. Alaaldin M, Alkilany Catherine J and Murphy, Toxicity and cellular uptake of gold nanoparticles: what
we have learned so far? J Nanopart Res. 12, 2313–2333 (2010).
5. Rana S1, Bajaj A, Mout R and Rotello VM, Monolayer coated gold nanoparticles for delivery applications,
Adv Drug Deliv Rev. 64(2), 200-16 (2012).
6. Cheng J, Gu YJ, Cheng SH and Wong WT, Surface functionalized gold nanoparticles for drug delivery,
J Biomed Nanotechnol, 9(8), 1362-9 (2013).
7. Zhaowu Zhang, Jing Jia, Yanyan Ma, Jian Weng, Yanan Sun and Liping Sun, Microwave-assisted one-
step rapid synthesis of folic acid modified gold nanoparticles for cancer cell targeting and detection,
Med Chem Comm., 2, 1079 (2011).
8. Haibing Li, Zhimin Cui and Cuiping Han, Glutathione-stabilized silver nanoparticles as colorimetric
sensor for Ni2+ ion, Sensors and Actuators B: Chemical, 143, 87–92 (2002).
9. Huang H and Yang X, Synthesis of chitosan-stabilized gold nanoparticles in the absence/presence of
tripolyphosphate, Biomacromolecules; 5(6), 2340-6 (2004).
10. Suresh K. balasubramanian et al., Characterization, purification, and stability of gold nanoparticles,
Biomaterials, 31, 9023–9030 (2010).
11. Bao QY1, Geng DD, Xue JW, Zhou G, Gu SY, Ding Y and Zhang C, Glutathione-mediated drug release
from Tiopronin-conjugated gold nanoparticles for acute liver injury therapy, Int J Pharm, 446, 12-8
(2013).
12. Jun Aia, b, Yuanhong Xua, Dan Lia, Zuojia Liua and Erkang Wang. Talanta, Folic acid as delivery
vehicles: targeting folate conjugated fluorescent nanoparticles to tumors imaging, 101, 32–37 (2012)
13. Sun C, Qu R, Chen H, Ji C, Wang C, Sun Y and Wang B, Degradation behavior of chitosan chains in the
‘green’ synthesis of gold nanoparticles, Carbohydrate Research 343(15), 2595-2599 (2008).
14. Yunxue Zhao, Xiaohu Gu, Haizhang Ma, Xigan He, Min Liu and Yi Ding, Association of Glutathione
Level and Cytotoxicity of Gold Nanoparticles in Lung Cancer Cells, J. Phys. Chem. C 115, 12797–
12802 (2011).
89
15. Zhaowu Zhang, Jing Jia, Youqun Lai, Yanyan Ma, Jian Weng and Liping Sun, Conjugating folic acid to
gold nanoparticles through glutathione for targeting and detecting cancer cells, Bioorganic & Medicinal
Chemistry, 18, 5528–5534 (2010).
16. Devika R. Bhumkar, Hrushikesh M. Joshi, Murali Sastry and Varsha B. Pokharkar, Chitosan Reduced
Gold Nanoparticles as Novel Carriers for Transmucosal Delivery of Insulin, Pharmaceutical Research,
24 (8), (2007).
17. Teow Y and Valiyaveettil S., Active targeting of cancer cells using folic acid-conjugated platinum
nanoparticles, Nanoscale, 2(12), 2607-13 (2010).
18. Roopa Dharmatti, Chinmay Phadke, Ashmi Mewada, Sunil Pandey, Goldie Oza, Chetna Sharon and
Madhuri Sharon, Surface Orchestration of Gold Nanoparticles Using Cysteamine as Linker and Folate
as Navigating Molecule for Synaphic Delivery of Doxorubicin, Journal of Nanomedicine Research.1,
(2014).
90
Antimicrobial, Antioxidant and Angiogenic
Potential of Silver Nanoparticles from Leaves of
Murraya paniculata (L.) Jack
Rama P, Vignesh A and K Murugesan
Centre for Advanced Studies in Botany, Guindy Campus, University of Madras, Chennai
e-mail: deeparamya48@gmail.com, Mobile: 7708908195
Abstract:
Green synthesis of silver nanoparticles was carried out using leaves of Murraya paniculata and was used as
a reducing agent. Biosynthesized AgNPs were characterized by UV–Vis, FESEM, TEM-EDAX, XRD and
FT-IR spectroscopy techniques. It was tested for their antimicrobial activity against human pathogens. In
vitro radical scavenging assay and reducing power assay was tested. Cytotoxicity of biosynthesized AgNPs
against in vitro human endothelial vein cell (HUVEC) line was analysed. Enhancement of cell migration
was determined by scratch wound assay and expression of VEGF by HUVEC was examined using western
blotting analysis. UV–Vis spectroscopy of AgNPs showed absorption maxima at 438 nm. Characterization
of AgNPs revealed that it is face-centered cubic structure, spherical shape with an average particle size
of 23 nm. Among the human pathogens viz., Bacillus cereus, Staphylococcus aureus, Candida albicans
and Escherichia coli, AgNPs demonstrated highest sensitivity toward Bacillus cereus. AgNPs containing
leaf extract showed good antioxidant activity than extract and standard ascorbic acid. Cytotoxicity of
biosynthesized AgNPs against in vitro human endothelial vein cell line showed dose-dependent activity.
It was observed that AgNPs at 15.625 μg/ml concentration could stimulate proliferation and migration of
endothelial cells (EC). The expression of VEGF by EC in response to synthesized AgNPs might provide
an important mechanism by which EC migration and permeability is regulated. This result indicates that
AgNPs possess antimicrobial, antioxidant and angiogenic activities, which may lead to the development of
nanomedicine for the treatment of diseases.
Keywords: Murraya paniculata, X-ray diffraction analysis, Vascular endothelial growth factor.
INTRODUCTION
Nanotechnology is one of the most promising areas of research in modern medicinal science [1]. Nanoparticles
are of great scientific interest as they bridge gap between bulk level and atomic-molecular level. To utilize
and optimize the chemical and physical properties of nano-sized metal particles, a large spectrum of
research has been focused to control the size and shape which is crucial in tuning their properties [2].
Biological methods of nanoparticles synthesis using micro-organism, enzymes, fungus, and plants have been
suggested as alternatives to chemical and physical methods. Among the various known synthesis methods,
plant mediated nanoparticles synthesis is preferred as it is cost-effective, eco-friendly and safe for human
therapeutic use [3]. The use of plant extracts for the synthesis of nanoparticles is potentially advantageous
over micro-organisms due to the ease of scale up, the less biohazard and elaborate process of maintaining
cell cultures [4].
Murraya paniculata (Rutaceae) is a shrub and is commonly known as Orange Jasmine. It is distributed in
India, Australia, China and Bangladesh and known to possess a broad spectrum of medicinal, pharmacological
and therapeutic properties. A decoction of the leaves is a remedy for stomach ache, chronic dysentery,
bruises and a wash against certain fungoid skin troubles. The flavor and fragrance of leaves retain even after
drying [5]. This plant is known to possess anti-fertility [6] anti-noniceptive and anti-inflammatory [7]; anti-
diarroheal [8]; antidiabetic and antioxidant properties [9].
Among metal nanoparticles, silver nanoparticles have attracted intensive research interest because
of their advantageous application [10], not only in biomedical [11]; and also in drug delivery [12]; food
industries [13], agriculture [14] and textile industries [15].
Hence the present study was initiated to synthesize and characterize silver nanoparticles from M.
paniculata and to evaluate their antimicrobial, antioxidant activities and their toxicity towards HUVEC cell
line and angiogenic activity.

Preparation of plant material
Fresh leaves of M. paniculata, were collected inside Guindy campus, University of Madras, Tamil Nadu,
Chennai. The collected fresh leaves were surface cleaned with running tap water, followed by distilled water.
Biosynthesis of silver nanoparticles was carried out by microwave irradiation method.
Microwave irradiation method

Fresh leaves (8 g) were finely chopped and mixed with 100 ml of deionized water, boiled in microwave oven
for 10 minutes, then the samples were allowed to cool at room temperature and the extracts were filtered with
Whatman No. 1 filter paper.
Synthesis of Silver nanoparticles

About 10 ml of aqueous extract of M. paniculata, from microwave method was added to 90 ml of aqueous
silver nitrate (1 mM) solution in Erlenmeyer flask. This is mixed thoroughly and then the reaction mixture
was manually shaken well and kept under dark conditions, until the colour change was noticed. The primary
detection of synthesized silver nanoparticles was carried out in the reaction mixture by observing the colour
change of the medium from greenish to dark brown. After 24 hrs incubation, the silver nanoparticles were
isolated and concentrated by repeated (4-5 times) centrifugation of a reaction mixtures at 10, 000 rpm for
10 minutes. The supernatant was replaced by distilled water each time and the suspension was stored as
lyophilized powder for optical measurements.

The reduction of pure silver Ag+ ions was monitored by measuring the UV-Vis spectrum of the reaction
mixture was taken on the wavelength from 200 to 800 nm. 1 mM AgNO3 solution was used for the baseline
correction. The morphology of the AgNPs was examined using Field emission scanning electron microscope
(HITACHI SU6600 FESEM) with EDAX. Thin films of the samples were prepared on aluminium foil by
dropping a small amount of sample and placed on a copper grid, extra solution was removed using a blotting
paper and then allowed to dry prior measurements. For HRTEM analysis, sample was sputter coated on copper
stub and the images of nanoparticles were studied. TEM measurements were performed on instruments
operated at an accelerating voltage of 200 kV. The formation and quality of the compounds were checked by
XRD analysis using a XPERT-PRO with a diffractometer with operating voltage 40 kV at a current strength
of 30 mA. The samples were subjected Cu Kα radiation and the scanning was done in the region of 30°-
80°C. The images obtained were compared with Joint committee on power diffraction standards (JCPDS)
library to account for the crystalline structure. To identify the biomolecules present within the AgNPs after
the synthesis of silver nanoparticles, FTIR analysis was performed with KBr pellet and recorded in the range
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of 400-4000 cm-1. The various modes of vibrations were identified and assigned to determine the different
functional groups present in AgNPs.
Screening of antimicrobial activity for silver nanoparticles

The antimicrobial effect of synthesized AgNPs and aqueous extract was evaluated against human pathogens
such as gram positive Bacillus cereus, Staphylococcus aureus, gram negative Escherichia coli and Candida
albicans by agar well diffusion method. Active cultures for experiments were prepared by transferring a
loop full of cultures from stock cultures to nutrient broth and incubated for 18 hrs at 37°C. Each culture was
swabbed uniformly into the individual nutrient agar plates using sterile cotton swabs. A 6 mm hole was bored
aseptically with a sterile cork borer. Using sterile micropipette, 100 μl of plant extract, AgNPs and standard
antibiotic amphicillin were loaded into well and it was allowed to dry. Silver nitrate (1 mM) was used as a
control. After 24 hrs incubation, the diameter of zone of inhibition was measured [16].
Antioxidant potential of silver nanoparticles

DPPH radical scavenging activity
To assess the scavenging ability on DPPH, 1 ml of DPPH (0.1 mM) was added to different concentrations of
(50, 100, 150, 200 and 250 μg/ml) aqueous leaf extract and AgNPs [17]. The reaction mixture was shaken
and incubated in dark for 30 minutes. The absorbance at 517 nm was measured against blank in a UV-
Visible spectrophotometer. Ascorbic acid was used as a standard. The lower absorbance of reaction mixture
indicated a higher percentage of scavenging activity. The percentage of inhibition or scavenging of free
radicals was determined by the following formula;
RSA – (Absorbance of control - Absorbance of sample/Absorbance of control) × 100
Reducing potential activity

The reducing power assay was conducted based on the method of Oyaizu (1986) [18]. 1 ml of different
concentration of aqueous extract and AgNPs were separately mixed with 2.5 ml of phosphate buffer (0.2 M
pH 6.6) and 2.5 ml of potassium ferricyanide (1 %). The mixture was incubated at 50°C for 20 minutes. To
this mixture, 2.5 ml of trichloroacetic acid (10 %) was added, which was then centrifuged at 3000 rpm for
10 minutes. Finally 2.5 ml of the supernatant solution was mixed with 2.5 ml of distilled water and 0.5 ml
of ferric chloride (0.01 %). The absorbance was measured at 700 nm in a UV-Visible spectrophotometer.
Ascorbic acid was used as a standard and the reaction mixture instead of sample used as control.
Cytotoxicity of silver nanoparticles on human umbilical vein endothelial cell line
The cell viability of the AgNPs from M. paniculata was determined by MTT assay [19].
Cell line and drug preparation

Human umbilical vein endothelial cell line (HUVEC) was obtained from NCCS (National centre for
cell sciences), Pune, India. The cells were cultured in Dulbeco’s Modified Eagle’s Medium (DMEM)
supplemented with 10 % fetal bovine serum (Hi-media) and 2 mM glutamine (Sigma chemical co), 100 UI/
ml pencillin, 100 μg/ml streptomycin (GIBCO BRL). The cells were maintained at 95 % air humidity in
a biological incubator at 37°C with 5 % CO2, then the viability was assessed using MTT assay. The stock
solution was prepared in DMSO and was stored at -20°C until use. The concentrations used for the study
were freshly prepared for each experiment with a final dimethyl sulfoxide (DMSO) concentration of 0.1 %.
Assessment of Cell viability using MTT assay

HUVEC cell line was cultured and seeded in 96-well plates approximately (1×105 cells/well) was incubated
for 24 hrs at 95 % air humidity in a biological incubator at 37°C with 5 % CO2. Cells were replaced with
fresh media containing different concentration of (1000-3.925 μg/ml) of synthesized silver nanoparticles
93
and incubated for 48 hrs. Thereafter, the supernatant was removed and cells were washed with DMEM
medium. Then these plates were subjected for MTT (3-(4, 5-dimethylthiazol-2-yl), 2, 5-diphenyltetrazolium
bromide, a yellow tetrazole) assay. 10 μl of MTT was added in each well and incubated overnight at 37°C.
Purple color formazone crystals formed were, then dissolved in 100 μl of dimethyl sulfoxide (DMSO). The
suspensions were read at 570 nm in spectrophotometer. The effect of drug on growth is assessed as percent
of cell viability.
Cell migration analysis using silver nanoparticles

The cell migration of HUVEC was evaluated using scratch wound assay [20]. In brief, (1×105 cells/ml)
were added into 24-well culture plates and cultured overnight. The cells were starved with 1 % v/v FBS
medium for 24 hrs. The cells were then scraped with a cross in the middle of the well with 200 μl pipette
tips and the medium was changed with fresh medium containing IC50 concentration of silver nanoparticles.
The cells were then incubated for another 48 hrs. The cells around the wounds were visualized and imaged
under inverted microscope (4X magnification). The percentages of open wound area was measured and
calculated.
Expression of vascular endothelial growth factor using western blot analysis

Western blot analysis was carried out to detect VEGF expression on endothelial cells. HUVEC (1 ×105
cells/well) were seeded in 24-well culture plate and incubated for 24 hrs to allow attachment. Various
concentrations of silver nanoparticles were added to the well plates and incubated for 48 hrs. After treatment,
cells were harvested and washed twice with PBS. The cell pellets were then lysed with RIPA buffer (50
mM Tris-HCl pH 7.4; 150 mM sodium chloride; 0.1 % SDS; Triton-X-100; 5 mM EDTA; 0.25 % sodium
deoxycholate) for 15 minutes in ice. The samples were then centrifuged at 14,000 rpm for 5 minutes at
4°C. The protein concentrations were determined by BCA (Bicinchoninic acid) method. The supernatant
protein was resolved by 12 % SDS and transferred to 0.45 μM PVDF membranes (polyvinylidene fluoride).
The membrane was then blocked with 5 % non-fat milk in Tris buffered saline containing Tween 20 (20
mM Tris-HCl pH 7.6; 150 mM NaCl; 0.1 % Tween 20). The blots were incubated overnight with primary
antibodies against human beta actin (1:2000). After incubation with the secondary horseradish peroxidase-
conjugated antibodies for 2 hrs detection was performed using chemiluminescene assay kit. The densities
of the bands were evaluated using total lab software and signal was normalized for β-actin.

Metal nanoparticles were generally obtained from noble metals like silver, gold, copper, tin, titanium
and platinum. Among the most metals, silver nanoparticles are being extensively used in medicine for its
therapeutic values.
In this study, silver nanoparticle colloidal solution was synthesized using M. paniculata as a reducing
agent.
UV-Visible spectrum of silver nanoparticles

The successful synthesis of silver nanoparticles and its stability was explored by UV-Vis spectrometry. The
silver nanoparticles were formed by adding different concentration of extracts (5 ml, 10 ml, 15 ml and 20
ml) with aqueous silver nitrate (1 mM). After 24 hrs incubation, the colour of the solution changed from
pale yellow to deep brown, depending on the concentration of the extracts (Figure 1). It might be due to
the surface plasmon resonance effect and reduction of AgNO3 [21]. At 10 ml concentration of extract, the
characteristic absorption peak was observed at 438 nm in UV-Vis spectrum which confirmed the formation
of silver nanoparticles and their stability on the 30th day (Figure 2). According to the generalized theory, only
a single SPR band peak is expected in the absorption spectra of spherical nanoparticles, whereas anisotropic
94
nanostructures or aggregates of spherical nanoparticles could give rise to two or more SPR bands depending
upon the shape of the particles [22].
A – Silver nitrate (1 mM); B - 5 ml extract + 95 ml Silver nitrate (1mM)

C - 10 ml extract + 90 ml Silver nitrate (1mM); D - 15 ml extract + 85 ml Silver nitrate (1mM)
E – 20 ml extract + 80 ml Silver nitrate (1mM); F – 100 ml extract alone
Figure 1: Synthesis of silver nanoparticles using different concentrations of aqueous extracts from leaves of
M. paniculata.
Figure 2: Graph depicted synthesized silver nanoparticles and their stability.
High resolution transmission electron microscopic analysis

of silver nanoparticle (HRTEM)
The application of HR-TEM in nanoscience is significant to view the particles in nanoscale. The HR-TEM
images of different magnification of synthesized silver nanoparticles and Selected area electron diffraction
(SAED) patterns are depicted (Figure 3a-f), which gives clear indication regarding size, shape and
distribution of nanoparticles. The SAED pattern revealed its crystalline nature (white dots in Figure 3f). It
clearly indicates that the individual nanoparticles ranged from 5 nm to 23 nm. The shape of the nanoparticles
95
was almost spherical to cubical. The result of EDS gives a clear idea about the elements present in the
nanoparticles, which suggest the presence of silver as the ingredient element. The Copper (Cu) peak comes
from the TEM grid. Metallic silver nanoparticles typically showed an absorption peak at 3 keV due to the
surface plasmon resonance (Kaviya et al., 2011).
Figure 3 (a-f): HR-TEM image of silver nanoparticles fabricated by leaf extract of M. paniculata at different
magnification with SAED pattern.
Figure 4: EDAX analysis of silver nanoparticles from leaves of M. paniculata
Field emission scanning electron microscopic analysis of silver nanoparticles (FESEM)

The surface morphology of AgNPs was investigated using FESEM with EDAX. Formation of AgNP and its
agglomeration was clearly observed (Figure 5). The FESEM micrograph shows spherical shape with particle
size that ranged from 5-23 nm. The silver peak in the EDAX spectrum confirms the presence of elemental
silver (Figure 6). The samples for FESEM-EDAX were prepared in aluminium foil. The peak for aluminium
(Al) that was obtained in the profile was due to the aluminium foil used.
96
Figure 5: FESEM microscopic images of synthesized AgNPs at various resolutions
Figure 6: FESEM (EDAX) microscopic images of synthesized AgNPs showing chemical compositions.
X-ray diffraction analysis of silver nanoparticles (XRD)

The XRD pattern of synthesized nanoparticles from aqueous leaf extract of M. paniculata was shown in
Figure 7. The crystalline nature of synthesized nanoparticles was confirmed. A number of Braggs reflection
with 2θ values of 38.0°, 44.1° and 64.6° sets of lattice plan was observed which may be indexed to (111),
(200) and (220) facets of silver respectively confirms the FCC crystalline structure of metallic silver. The
values are in agreement with the JCPDS (Joint Committee on Powder Diffraction Standard) file No. 04-078.
Figure 7: X – Ray diffraction analysis of silver nanoparticles from M. paniculata
97
Fourier transform infrared spectrum of silver nanoparticles (FTIR)

An FTIR measurement was carried out to determine the biomolecules responsible for the reduction and
capping of synthesized silver nanoparticles. The differed peaks observed are as follows 3429 cm-1 (O-H
stretch, H- bonded alcohols, phenols groups), 2923 cm-1 (C-H stretch alkanes), 1626 cm-1 (N-H bend 1°
amines ), 1583 cm-1 (C-C stretch in ring aromatic), 1039 cm-1 (C-N stretch aliphatic amines), 728 cm-1 (C-Cl
stretch alkyl halides) and 589 cm-1 (C-Br stretch alkyl halides) respectively (Figure 8). These data indicated
that the involvement of phenols, alkanes, amines, aromatic, aliphatic amines, alkyl halides residues present
in M. paniculata in the nanoparticles synthesis. As mentioned earlier, leaves of M. paniculata is a rich source
of alkaloids, phenolics, flavonoids, polysaccharides and proteins. It is well known that proteins can binds
to AgNPs through either amino acids or cystein residues in the protein [24] and therefore stabilization of
AgNPs by the surface bound proteins is possible to in the present green synthesis. A similar observation is
noticed in biological synthesis of AgNPs using Jatropha curcas seed extract [25]
Figure 8: FTIR analysis of silver nanoparticles from leaves of M. paniculata
Biological applications of silver nanoparticles

Antimicrobial activity of silver nanoparticles
The synthesized silver nanoparticles tested for their efficacy against human pathogens (Staphylococcus aureus,
Escherichia coli and Candida albicans. Our present study shows that the synthesized nanoparticles have potent
activity against Bacillus cereus and Candida albicans followed by Escherichia coli, Staphylococcus aureus.
It shows that all the tested micro-organsims were completely inhibited at the concentration of 100 μg/ml of
synthesized silver nanoparticles. Antimicrobial activity of aqueous extract, silver nanoparticles and standard drug
were shown in Figure 9.The SNPs shows better activity against gram positive as compared to that of the gram
negative. The antimicrobial activities shown by SNPs synthesized using different plant systems may differ from
species to species due to variations in shape and size of SNPs, bacterial load, exposure time and nutrient media.
Figure 9: Antimicrobial activity of synthesized silver nanoparticles from leaves of M. paniculata.
98
The effects of silver nanoparticles on the bacterial cell are unclear and complicated [26]. However,
there are various mechanisms on the action of silver nanoparticles on the bacterial cell [27]. Some of
these mechanisms were summarized and presented as follows: (i) the ability of silver nanoparticles to
anchor to the bacterial cell wall and then penetrate it [28] (ii) the formation of free radicals by the silver
nanoparticles which can damage the cell membrane and make it porous [29, 30] (iii) releasing the silver
ions by the nanoparticles which can interact with the thiol groups of many vital enzymes and inactivate
them [31, 32] and (iv) the nanoparticles can modulate the signal transduction in bacteria which stops
the growth of bacteria [33].
All the human pathogens are resistant to standard drug amphicilin but they are susceptible to the
synthesized nanoparticle from leaves of M. paniculata. The results indicated that leaf extract alone did not
exhibit antimicrobial activity against human pathogens. Silver nitrate (1 mM) showed an appreciable positive
effect. However, plant AgNPs exhibited greatest antimicrobial activity against tested human pathogens.Thus,
we can conclude from the results, of this study that the AgNPs inhibited the growth and multiplication of all
the tested human pathogens.
Antioxidant activity of silver nanoparticles

In light of difference among the wide range of assays available, the results of a single antioxidant assay
can give only a reductive suggestion of the antioxidant potential. Moreover, the chemical complexity of
samples with a mixture of different functional groups and chemical behavior could lead to a scattered
result. Therefore, an approach with multiple assays in screening work is highly desirable. Thus to
ensure the better comparison of the results and covering a wide range of possible applications, in
vitro antioxidant activities was assessed by different methods like reducing power and DPPH radical
scavenging assay.
DPPH radical scavenging activity of silver nanoparticles

Free radical scavenging activity by DPPH assay is the most extensively used method to understand the
potentiality of AgNPs towards their bioactivity. DPPH (purple) is a protonated radical which has a
characteristic absorbance at 517 nm using spectrophotometer, which decolorizes (yellow) upon activation
with antioxidants. DPPH scavenging activity has been used by various researchers as a fast and reliable
parameter to assess the in vitro antioxidant activity of AgNPs solution [34].
Figure 10: DPPH radical scavenging activity of synthesized silver nanoparticles from aqueous extract of
M. paniculata.
99
The scavenging activity of DPPH was increased in dose dependent manner (Figure 10). The DPPH
scavenging ability of the AgNPs was significantly higher than aqueous extract of M. paniculata and
almost similar to that of standard ascorbic acid. The AgNPs solution exhibited a proton-donating ability
and could serve as a free radical inhibitor per scavenger, possibly acting as a primary antioxidant.
Reducing power assay using silver nanoparticles

Reducing power is associated with antioxidant activity [35] and the compounds with reducing power
indicate that they are electron donors and can reduce the oxidized intermediates of the lipid peroxidation
processes; thus they can act as primary and secondary antioxidants. The presence of antioxidant would
result in the reduction of Fe2+ to Fe3+ and the amount of Fe2+ complex was monitored by measuring
the formation of blue colour at 700 nm. The increased absorbance at 700 nm indicated an increase in
reductive potential.
Reducing potential of synthesized silver nanoparticles was found increases with increase in concentrations,
indicating that the reducing potential of AgNPs found to be lesser than that of the ascorbic acid (Figure 11).
It is evident from figure that the reducing powers of the aqueous extract of M. paniculata, AgNPs and the
standard drug were increasing with increasing concentrations.
Figure 11: Reducing potential of synthesized silver nanoparticles from aqueous extract of M. paniculata.
Cell viability assay using silver nanoparticles

Viability of HUVEC cells were checked using different doses (1000-3.906 µg/mL) of silver nanoparticles
from leaves of M. paniculata. At the concentration, 15.625 µg/mL of AgNPs from M. paniculata,
almost 89.03 % cells were viable (Figure 12). So synthesized silver nanopartcles from the Murraya
paniculata does not show toxicity to HUVEC cells. It is toxic, only when it exceeds the dosage level
ie., By increasing the concentration of AgNPs begans to show toxicity to HUVEC cell line. Based on
this result, we conclude that AgNPs is applicable to check whether it has angiogenic property by cell
migration assay.
100
Figure 12: Cell viability of HUVEC by MTT assay and cytotoxicity was expressed as the concentration of 50 % (IC50)
cell growth inhibition. The experiments were performed in triplicates. Images were taken by Inverted Phase contrast
microscope magnification 20X scale bar 50 µm.
Cell migration assay using silver nanoparticles

Angiogenesis is associated with several pathologies including cardiovascular diseases, chronic inflammation,
cancer, wound healing and depending on the circumstances; it can be beneficial and deleterious. To
explore the angiogenic potential of AgNPs from M. paniculata, HUVEC cells were treated with different
concentration of synthesized nanoparticles. At the concentration of 15.63 μg/ml, synthesized nanoparticles
from M. paniculata enhances the angiogenesis by cell migration after incubation for 48 hrs (Figure 13).
Figure 13: Cell migration potential by Scratch wound assay. Photos were taken 48 hrs after scratch wounding at 4X
magnification.
Wound healing involves regeneration of specialized cells by the proliferation of surviving cells and
connective tissue response characterized by the formation of granulation tissue [36]. It is also characterized
by hemostasis, reepithelialization and remodeling of the extracellular matrix. Thus, the effect of ethanolic
extract and the acetone fraction on wound contraction and epithelialization suggest it may enhance epithelial
cell migration and proliferation, as well as the formation, migration, and action of myofibroblasts. It is, likely
that in addition to enhancing wound contraction and epithelialization, AgNPs from M. paniculata may also
stimulate processes associated with tissue regeneration.
Expression of VEGF by western blotting analysis

VEGF is an important proangiogenic cytokine and improves angiogenesis during wound healing by
stimulating the migration and proliferation of endothelial cells through extracellular matrix [37].
101
The VEGF levels in HUVEC cells were significantly upregulated after 24 hrs of incubation (Figure 14a).
The highest upregulation of VEGF protein was detected at the concentration of 15.625 μg/ml (Figure 14b).
Figure 14a: Effect of silver nanoparticles from M. paniculata on the expression of VEGF in HUVEC cells (14b):
Quantitative expression of VEGF after normalization to β-actin
Angiogenesis is a critical component in wound healing. Delayed or abburent revascularization at

the wound site contribute to the etiology of chronic wounds. Induction of angiogenesis by VEGF can be
considered as a factor to improve wound healing. Western blotting analysis of HUVEC up-regulates the
VEGF expression similar to that of β-actin.
CONCLUSION
We have developed a simple green chemistry approach for the synthesis of silver nanoparticle by M.
paniculata leaf extract that demonstrate the multifunctional activities of biosynthesized AgNPs. We have
observed the biocompatible nature of AgNPs towards the normal cells and the results indicates the future
application as drug delivery vehicle to enhance angiogenesis. Apart from that, bio-synthesized silver
nanoparticles shows enhanced antimicrobial activity compared to that of the standard drug. Also synthesized
AgNPs possess good reducing potential and radical scavenging activity which confirms that AgNPs has
excellent antioxidant properties. We strongly believe that bio-synthesized AgNPs will open a new direction
toward various biomedical applications in future.
ACKNOWLEDGEMENT
The authors greatly acknowledging University Grant Commision (Grant Number GCCO/A-2/UGCBSR/
2012/384) for financial support of this work. The authors are also thankful to the Director, CAS in Botany,
University of Madras, Chennai, Tamil Nadu, India for providing lab facilities.
REFERENCES
1. Jae Y.S and Beom S.K., 2009, Rapid biological synthesis of silver nanoparticles using plant leaf extracts,
Bioprocess, Biosyst. Eng, 32: 79-84.
2. Thakkar K.N., Mahatre S.S. and Parikh R.Y., 2010, Biological synthesis of metallic nanoparticles,
Nanomedicine, 6(2): 257-262. doi: 10.1016/j.nano.2009.07.002. Epub 2009 Jul 16.
3. Kumar V and Yadav S.K., 2009, Plant mediated synthesis of silver and gold nanoparticles and their
applications, J. Chem. Technol. Biotechnol., 84: 151-157.
102
4. Kalishwaralal K., Deepak V., Ram Kumar Pandian S., Kottaisamy M., Barath Mani K.S., Kartikeyan
B. and Gurunathan S., 2010, Biosynthesis of silver and gold nanoparticles using Brevibacterium casei.,
Colloids Surf. B: Biointerfaces 77: 257-262.
5. Omankutty Amma M., Rajaraman K., Sankarikutty B., Sumathikutty M.A., Padmakumari K.P. and
Narayanan C.S., 1984, Processing of curry leaves, Indian Food Packer, 32-36.
6. Xiao P.G. and Wang N.G., 1991, Can ethnopharmacology contribute to the development of anti-fertility
drugs? J Ethnopharmacol, 32: 167-77. http://dx.doi.org/10.1016/0378-8741(91)90114-S.
7. Wu D., Lei Y., Tong Y., Tang F., Qian Y. and Zhou Y., 2010, Angiogenesis of the frozen-thawed human
fetal ovarian tissue at the early stage after xeno transplantation and the positive effect of Salviae
miltiorrhizae, Anat Rec (Hoboken) 293: 2154-2162.
8. Rahman M.A., Hasanuzzaman M., Uddin N. and Shahid I.Z., 2010, Antidiarrhoeal and anti-inflammatory
activities of Murraya paniculata (L.) Jack. Pharmacologyonline. 3: 768-776.
9. Gautam M.K., Anamika Gupta M., Vijaykumar C.V. and Rao R.K., 2012, Studies on the hypoglycemic
effects of Murraya paniculata Linn. extract on alloxan-induced oxidative stress in diabetic and non-
diabetic models. Asian Pacific Journal of Tropical Disease, S186-S191.
10. Nazeruddin G.M., Prasad N.R., Prasad S.R., Garadkar K.M. and Nayak A.K., 2014, In vitro bio-
fabrication of silver nanoparticle using Adhathoda vasica leaf extract and its anti-microbial activity,
Physica, E6: 156–61.
11. Chaloupka K., Malam Y. and Seifalian A.M., 2010, Nanosilver as a new generation of nanoproduct in
biomedical applications, Trends Biotechnol. 28: 580-588.
12. Prow T.W., Grice J.E., Lin L.L., Faye R., Butler M., Becker W., Wurm E.M.T., Yoong C., Robertson
T.A., Soyer H.P. and Roberts M.S., 2011, Nanoparticles and microparticles for skin drug delivery, Adv.
Drug Deliv. Rev. 63: 470-491.
13. Chaudhry Q and Castle L., 2011, Food applications of nanotechnologies: an overview of opportunities
and challenges for developing countries, Trends Food Sci. Tech. 22: 595.
14. Nair R., Varghese S.H., Nair B.G., Maekawa T., Yoshida Y. and Sakthi Kumar D., 2010, Nanoparticulate
material delivery to plants, Plant Sci. 179: 154-163.
15. Kelly F.M. and Johnston J.H., 2011, Colored and functional silver nanoparticle wool fiber composites,
ACS Appl. Mater. Interfaces, 3: 1083–1092.
16. Okeke M.I., Iroegbu C.U., Eze E.N., Okoli A.S. and Esimone C.O., 2001, Evaluation of extracts of the
root of Landolphia owerrience for antibacterial activity, Journal of Ethnopharmacology 78: 119-127.
17. Blois S., 1958, Antioxidants determinations by the use of a stable radical, Nature, 181: 1199 – 1200.
18. Oyaizu M., 1986, Studies on products of browning reaction: Antioxidative activity of products browning
reaction prepared from glucosamine, Japanese Journal of Nutrition, 44: 307-315
19. Mossman T., 1983, Rapid colorimetric assay for cellular growth and survival: application to proliferation
and cytotoxic assays, Journal of Immunological method, 65: 53-63.
20. Yue G.G.L., Fan J.T., Lee J.K., Zeng G.Z., Ho T.W., Fung K.P., Leung P.C., Tan N.H. and Lau C.B.S.,
2011, Cyclopeptide RA-V inhibits angiogenesis by down regulating ERK-1/2 phosphorylation in
HUVEC and HMEC-1 endothelial cells, Br. J. Pharmacol., 164(7): 1883-1898.
21. Das R., Nath S.S., Chakdar D., Gope G. and Bhattacharjee R., 2010, Synthesis of silver nanoparticles
and their optical properties, Journal of Experimental Nanoscience 5: 357-362.
103
22. Dwivedia P., Shahid S. Narvia and Ravi P. Tewari, 2014, Phytofabrication characterization and
comparative analysis of silver nanoparticles by diverse biochemicals from Elaeocarpus ganitrus Roxb.,
Terminalia arjuna Roxb., Pseudotsuga menzietii, Prosopis spicigera, Ficus religiosa, Ocimum sanctum,
Curcuma longa. Industrial crops and products, 54: 22-31.
23. Kaviya S., Santhanalakshmi J., Viswanathan B., Muthumary J. and Srinivasan K, 2011, Biosynthesis of
silver nanoparticles using citrus sinens is peel extract and its antibacterial activity, Spectrochim. Acta A
79: 594-598.
24. Gole A., Dash C., Ramakrishnan V., Sainkar S.R., Mandale A.B., Rao M. and Sastry M., 2001, Pepsin-
gold colloid conjugates: preparation, characterization, and enzymatic, Langmuir 17: 1674-1679.
25. Bar H., Bhui D.K., Sahoo G.P., Sarkar P., De P.S. and Misra A., 2009, Green synthesis of
silver nanoparticles using latex of Jatropha curcas, Colloid Surf. A: Physiocochem. Eng. Aspects 339:
134-139.
26. Kim S.H., Lee H.S., Ryu D.S., Choi S.J. and Lee D.S., 2011, Antibacterial activity of silver-nanoparticles
against Staphylococcus aureus and Escherichia coli, Korean J. Microbiol. Biotechnol 39: 77-85.
27. Prabhu S and Poulose E.K., 2012, Silver nanoparticles: mechanism of antimicrobial action, synthesis,
medical applications and toxicity effects, Int. Nano Lett. 2: 1–10.
28. Sondi I and Salopek-Sondi B., 2004, Silver nanoparticles as antimicrobial agent: a case study on E. coli
as a model for Gram-negative bacteria, J. Colloid Interface Sci. 275: 177-182.
29. Danilcauk M., Lund A., Saldo J., Yamada H. and Michalik J., 2006, Conduction electron spin resonance
of small silver particles, Spectrochim. Acta, Part A 63: 189–191.
30. Kim J.S., Kuk E., Yu K., Kim J.H., Park S.J., Lee H.J., Kim S.H., Park Y.K., Park Y.H., Hwang C.Y.,
Kim Y.K., Lee Y.S., Jeong D.H. and Cho M.H., 2007, Antimicrobial effects of silver nanoparticles,
Nanomedicine, 3: 95-101.
31. Feng Q.L., Wu J., Chen G.Q., Cui F.Z., Kim T.N. and Kim J.O., 2008, A mechanistic study of the
antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus, J. Biomed. Mater. Res.
52: 662–668.
32. Matsumura Y., Yoshikata K., Kunisaki S. and Tsuchido T., 2003, Mode of bacterial action of silver
zeolite and its comparison with that of silver nitrate, Appl. Environ. Microbiol. 69: 4278-4281.
33. Shrivastava S., Bera T., Roy A., Singh G., Ramachandrarao P. and Dash D., 2007, Characterisation of
enhanced antibacterial effects of novel silver nanoparticles, Nanotechnology, 18: 1-9.
34. Yagi A., Kabash A., Okamura N., Haraguci H., Moustafa S.M. and Khalifa T.I., 2002, Antioxidant, free
radical scavenging and anti-inflammatory effects of aloes in derivatives in Aloe vera, Planta Med, 68:
957-60.
35. Oktay M., Gulcin I. and Kufrevioglu O.I., 2003, Determination of in vitro antioxidant activity of fennel
(Foeniculum vulgare) seed extracts, Leb Wissen Technol, 36: 263-71.
36. Whaley K and Burt A.D., Inflammation, healing and repair In: MacSween RM, Whaley K, editors.
Muir’s Textbook of Pathology. 13th ed. London: Arnold; 1996. p. 112-65.
37. Ferrara N., 1999, Molecular and biological properties of vascular endothelial growth factor. Journal of
Molecular Medicine, 77(7): 527-543.
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Biosynthesis of Silver Nanoparticles using Tephrosia
tinctoria Pers. for Antibacterial Activity against Multidrug
Resistant Pathogens
D.C. Aiswarya1, K. Rajaram2, P. Sureshkumar3
1Department of Nanoscience and Nanotechnology, SRM University, Kattankulathur - 603 203,

Chennai, Tamil Nadu, India
2Department of Biotechnology, Bharathidasan Institute of Technology (BIT) Campus, Anna University,
Tiruchirappalli-620 024, Tamil Nadu, India
Email : archies.bioinfo26@gmail.com, biorajaram@gmail.com
#Corresponding author e-mail: drsureshbiotech2003@gmail.com
Abstract:
The aqueous extract of T. Tinctoria was dissolved with different concentration of silver nitrate and allowed
for 24hrs. Light yellow colour solution changed to brown colour. Because, the secondary metabolites present
in the plant extract reduces the silver nitrate. All reaction mixture was analysed under UV spectrophotometer.
The peak between 400-500nm was increased when increase the concentration of silver nitrate. Then the
reaction mixture was centrifuged and washed with distilled water. Further, it was characterized using TEM
and FTIR. The green synthesized AgNPs was showed higher inhibitory activity in the multidrug-resistant
pathogens (E.coli, P.aeruginosa, K.pneumoniae, E.faecalis, P.vulgaris and S.aureus).
Keywords: Tephrosia tinctoria, AgNPs, TEM, Antibacterial agent and FTIR.
INTRODUCTION
Nanoparticles are the choice for past decades, because of its unique properties in the application of biological
and electronics. There are many synthetic methods available to produce nanoparticles. But they are producing
many hazards by-products that produce health and environmental issues. Hence, there is a need to use
the biosynthetic method (green synthesis) which no longer produce environmental pollution and it also
cheapest source [1-2]. Primary and secondary metabolites of the plant extracts will reduce and also it bind
to the nanoparticles to establish the biological activity. Hence, plants are used to select for the large scale
production than microorganisms [3-4].
Silver contained materials were used even in ancient time. The nano form of silver is very effective
against microorganisms and also it is very effective against antibiotics resistance bacteria. The mechanism
of silver nanoparticle is partially known, that DNA damage through direct binding, still the mechanism
is unclear [5]. In the medical field, silver nanoparticles were used in the form of ointments to burn and
open wounds to avoid infection. Because, the green synthesized nanoparticles are very much active towards
various multidrug-resistant pathogens [6-7].
The present study designed to synthesise the silver nanoparticles using stem aqueous extract of
Tephrosia tinctoria Pers for the antimicrobial activity against multidrug resistance pathogens. T.tinctoria
has been reported for its higher amount of isoflavonoids and phenol [8] and also reported for antioxidant [6],
antimicrobial [9], antidiabetic [6] and anticancer [10] activities.

Chemicals
All chemicals and Media were obtained from Sigma chemical Co., MO, USA.
Medicinal plant
Tephrosia tinctoria Pers. was collected from Kolli hills in Tamilnadu, India and authenticated (BSI/
SRC/5/23/09-10/TECH.-1569) by Botanical Survey of India (BSI) Coimbatore, Tamil Nadu, India.
Synthesis of Silver nanoparticles (AgNPs)

The stems of T. Tinctoria were finely ground into powder. Then the powder was measured to 100 grams and
added into 500 mL distilled water and mixed well. The mix was left undisturbed for 2 hrs and then it was
filtered using Whatman filter paper. The filtrate was centrifuged in order to get a clear solution. Then it was
filtered again using a Whatman filter paper. Later the clear solution was poured into petri dish and left in the
oven for 48 hrs at 50°C. The aqueous extract 0.3g dissolved into 50mL of distilled water. Different volume
(0.2, 0.4, 0.6, and 0.8) of 1mM AgNO3 was added and left in dark condition at room temperature for 24 hrs.
The change of colour from yellow to dark brown confirmed the synthesis of AgNPs. This change of colour
showed that the aqueous silver ions could have been reduced by aqueous extract of the plant to generate
stable AgNPs. The UV-Vis Spectrum of the reaction mixture was observed using UV-Vis spectrophotometer.
The wavelength was set between 200 to 500 nm.
Purification and Characterization of synthesized AgNPs

The synthesized AgNps was separated by centrifuging the reaction mixture at 8000 rpm for ten min. Then the
pellet dissolved with distilled water and centrifuged at 8000rpm for ten min. The pellet again dissolved with
distilled water, and a washing step repeated for ten times to remove the plant extract and unutilized silver
nitrate. Finally, the purified pellet was used for the following characterization and antimicrobial assays. The
structure and shape of AgNPs were observed under Transmission Electron Microscopy (TEM), and the FT-
IR spectrum of AgNPs was recorded between 400 – 4000 Wavelength (cm-1).
Antimicrobial activity
Disc-Diffusion method
The antibacterial activity of AgNP and plant extract was carried out on human pathogenic E.coli, P.aeruginosa,
K.pneumoniae, E.faecalis, P.vulgaris and S.aureus, by standard disc diffusion method. Muller Hinton
broth/agar medium was used to cultivate bacteria. Fresh overnight inoculum (100 μl) of each culture was
spread on to Muller Hinton agar plates. Sterile paper discs of 5 mm diameter filled with plant extract (50
& 100 μg/ml), AgNP (50 & 100 μg/ml) and standard antibiotic (Cefotaxime 100 μg/ml) were placed on
separate plates. The plates were incubated at 37°C. After incubation, the plates were examined for zones of
inhibition. The clear area appeared around the well, and the diameters of such zones were measured using
meter ruler and expressed in centimetres.
Minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration

(MBC)
The bacterial culture growth was carried out by inoculating 10μl of the bacterial strains of E. Faecalis, S.
aureus and E. Coli in each test with plant extract (25, 50 and 100 μg/ml), AgNP (25, 50 and 100 μg/ml)
and standard antibiotic (Cefotaxime 100 μg/ml) and left for 12 hrs at 37°C. After incubation, the turbidity
was observed through naked eye (MIC) and 100μl of the respective mixture were spread into the plates
containing Muller Hinton agar to know any microbial growth after the treatment (MBC).
106

The synthesized silver nanoparticle with different concentrations of silver nitrate for constant volume of plant
extracts of Tephrosia tinctoria under suitable conditions responded to the following analyses. Reduction of
silver ions into silver nanoparticles using aqueous extract of leaves of Tephrosia tinctoria was evident by the
visual change of colour from light brown to reddish brown due to excitation of surface plasmon vibrations
in silver nanoparticles [11, 12]. The UV-visible spectra show an absorption band at 420 nm that corresponds
to the absorbance of silver nanoparticles [12]. Increased concentrations of silver nitrate resulted in a brown
solution of nano silver indicating the completion of the reaction. Hence, the peak between 400 – 500 nm is
increasing when increase the concentration of silver nitrate (Fig 1).
Figure 1: UV-Vis Spectrum of green synthesized AgNPs at difference volume of 1 mM Silver nitrate. A – 0.8 ml of
1mM silver nitrate; B – 0.6 ml of 1mM silver nitrate; C – 0.4ml of 1mM silver nitrate; D – 0.2 ml of 1mM silver nitrate.
After purification, the AgNPs were viewed under TEM. The TEM image showed that the silver nanoparticles
are spherical in shape. The size of AgNPs was observed below 50 nm and also they are well dispersed (Fig 2).
Figure 2: TEM image of the green synthesized AgNPs using T.tinctoria
107
Figure 3: FTIR Spectrum of green synthesised AgNPs using T.tincotria
AgNps are showed 13 peaks starts from 3438.46 (O-H), 2925.48 (C-H), 2856.06 (C-H), 1731.76 (C=C),
1637.76 (C=C), 1457.92 (CH3), 1373.07 (C-H), 1247.72 (C-C), 1186.01(C-O), 1124.3 (C-H), 1078.01(C-
O), 968.09 (C-C) and 655.679 (C-H) respectively (Fig 3).
The AgNPs and extract were tested against the selected clinical pathogens. The result showed that the disc
containing AgNPs effectively inhibited than the plant extract (Fig 4).
Figure 4: Antibacterial activity of AgNPs against clinical pathogens (A) P.aeruginosa, (B) K.pneumoniae, (C) S.aureus
(D) E.faecalis, (E) E.coli and (F) P.vulgaris. (Disc Diffusion Method)
108
Table 1: Zone of Inhibition of AgNPs against clinical pathogens (Disc Diffusion method)
S.No. Pathogens Cefotaxime 100μg/ml (cm) Extract 100μg/ml (cm) AgNPs 100μg/ml (cm)
1 P. aerginosa 2.6 0.7 0.8
2 K. pneumoniae 3.7 -- 0.7
3 E. faecalis 3.5 -- 1.2
4 P. vulgaris 3.3 0.7 1.15
5 S. aureus 3.6 -- 1.25
6 E. coli 4 -- 1.25
Maximum zone of inhibitions (1.25cm) were observed against S.aureus and E.coli.
The bactericidal activity due to the silver nanoparticle attachment towards the cell and create the free
radical which cause the denaturation final cell death [13, 14]. Both MIC and MBC analyses showed that the
AgNPs showed no turbidity and no bacterial growth at the 100μg/ml concentration of AgNPs against all
tested clinical pathogens.
CONCLUSION
The present study describes the green synthesis of silver nanoparticles using T.tinctoria. The particle was
found to be spherical in shape that was ranging from 20-50 nm with an average size of 23.1nm. FT-IR
spectroscopic studies confirmed that the carbonyl group of amino acid residues which has a strong binding
ability with silver, suggesting the formation of a layer covering silver nanoparticles. The green synthesized
AgNPs has significant antimicrobial activity against the selected clinical pathogens.
REFERENCES
1. Murphy CJ, Sustainability as an emerging design criterion in nanoparticle synthesis and applications, J
Mater Chem. 2008;18:2173–76.
2. Dahl JA, Maddux BL and Hutchison JE, Toward greener nanosynthesis, Chem Rev.
2007;107(6):2228-69
3. Parsons JG, Peralta-Videa JR and Gardea-Torresdey JL, In: Sarkar D, Datta R and Hannigan R, (Eds.).
Developments in Environmental Science, Elsevier Ltd., Oxford, UK. 2007; 5:Chapter 21.
4. Inbakandan D, Venkatesan R and Ajmal KS, Biosynthesis of gold nanoparticles utilizing marine sponge
Acanthella elongate, Colloids Surf., 2010; B81:634.
5. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN and Kim JO, A mechanistic study of the antibacterial effect
of silver ions on Escherichia coli and Staphylococcus aureus, J Biomed Mater Res., 2000; 52:662–668.
6. Ip M, Lui SL, Poon VKM, Lung I and Burd A, Antimicrobial activities of silver dressings: an in vitro
comparison, J Med Microbiol., 2006; 55: 59–63.
7. Ponarulselvam S, Panneerselvam C, Murugan K, Arthi N, Kalimuthu K and Thangamani S, Synthesis of
silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their antiplasmodial activities.
Asian Pacific Journal of Tropical Biomedicine 2011; 574-580.
8. Rajaram K and Sureshkumar P, In vitro antioxidant and antidiabetic activity of Tephrosia tinctoria Pers.
An endemic medicinal plant of South India, Journal of Pharmacy Research, 2011; 3(4):891-893.
9. Lakshmi P, Ganapaty S and Lakshminarayana K, Antimicrobial activity of the root extracts of T. pumila
and T. tinctoria on clinical and phytopathogens, Journal of Pharmacy Research. 2009; 2(11):1694–96.
109
10. Rajaram K, Moushmi M, Velayutham DPM, Kumpati P, Ganasaraswathi M and Sureshkumar P,

Comparative bioactive studies between wild plant and callus culture of Tephrosia tinctoria Pers. Applied
Biochemistry and Biotechnology, 2013; 178(8): 2105–20.
11. Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R and Sastry M, Extracellular
biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum, Colloids Surface B 2003;
28:313–318.
12. Sastry M, Mayyaa KS and Bandyopadhyay K, pH Dependent changes in the optical properties of
carboxylic acid derivatized silver colloidal particles, Colloid Surface A 1997;127:221–228.
13. Dibrov, Pavel, Judith D, Khoosheh KG and Claudia CH, Chemiosmotic mechanism of antimicrobial
activity of Ag+ in Vibrio cholerae., Antimicrobial Agents and Chemotherapy, 2002;46(8):2668–70.
14. Sondi, Ivan and Branka S, Silver nanoparticles as an antimicrobial agent: a case study on E. Coli as a
model for Gram-negative bact.
110
Green Synthesis of Silver Nanoparticles from Leaf
Extracts of Breynia Vitis-Idaea
B. Anandaraj1, T.P. Rajesh2, P. Suresh Babu3, C. Narendhar4
Department of Biotechnology, Anna University Trichy

1, 2
3
Department of Biotechnology, Sree Sastha Institute of Engineering and Technology,
Chembarambakkam, Chennai–600123, TN
4
Department of Biotechnology, Srivenkateshwara College of Engineering, Coimbatore
Abstract:
The development of the biological synthesis of nanoparticles using plant extracts plays an important role in
the field of nanotechnology as it is environmentally friendly and does not involve any harmful chemicals. In
this study, the synthesis of silver nanoparticles using the leaves extract of Breynia vitis-idaea is reported. The
synthesized nanoparticles were characterized using UV-visible spectroscopy, X-ray diffraction (XRD) and
Particle size analyzer. The XRD analysis shows that the synthesized silver nanoparticles are of face-centered
cubic structure. Well-dispersed silver nanoparticles with an approximate size of 50-70 nm were observed in
the particle size analysis studies. The application of the synthesized silver nanoparticles can be in the field
of cosmetics, food packaging industry, medicine for antimicrobial fabrics or surgical knives, sterile gloves,
optics as electrochromic devices, and electronics as conductive connections etc.
INTRODUCTION
In recent years, green synthesis of metal nanoparticles is an interesting issue of the nanoscience and
technology. Among the various metal nanoparticles, silver nanoparticles have rapidly increased due to
their unusual optical, chemical, electronic, photo-electrochemical, catalytic, magnetic, antibacterial,
and biological labelling properties [1]. A number of approaches are available for the synthesis of silver
nanoparticles, although bio inspired synthesis using plant sources offers several advantages such as cost-
effectiveness, ecofriendliness and the elimination of high pressure, energy, temperature, and toxic chemicals
necessary in the traditional synthesis methods [2]. Several plants have been utilized for the production of
silver nanoparticles [3-5].
Breynia vitis-idaea (Burm.f.) Fischer. (Euphorbiaceae) is an evergreen, glabrous tree or large shrub.
Found in the Gangetic plain, western peninsula, china, Malay Peninsula and Sri Lanka. These plants are
planted as ornamental hedge in garden. Bark is yellowish grey, leaves are alternate dark brown or black
when dry, flowers are small, greenish yellow or pink, and dull red, purple or white berries. Root, leaves
and bark contains the saponins, breynin and terpenic and phenolic glycosides. Roots decoction was used
as mouthwash [6]. In this present study, we have reported the green synthesis and characterization of silver
nanoparticles using B. vitis-idaea leaf extract. To the best of our knowledge, this is the first report for the
synthesis of silver nanoparticles using B. vitis-idaea leaf extract.

Preparation of plant extracts
The leaves and stem of B. vitis-idaea were collected from Pachaimalai hills region of Salem district,
Tamilnadu, India. The area falls within the latitudes 11° 10′ 48″ N and longitudes 78° 21′ 0″ E. These areas
consist of villages which are generally classified as rural area. The plant materials were thoroughly washed
with tap water to remove soil, dirt, etc., and finally with double-distilled water. The plant leaves were dried
in shade and powdered in a mechanical grinder. 2 g of powdered leaves were mixed with 100 mL doubled-
distilled water, and this mixture was heated in a clean glass beaker at a temperature of 60°C for a period of
10 mins. After cooling, it was filtered through Whatman Filter paper mesh no.1 to obtain a clear solution.
Silver nitrate and other required chemicals were procured from Sigma Alderich and used without further
purification.
Synthesis of silver nanoparticles

The filtered Leaf extract (5 mL) was added to 25 mL of 3 mM aqueous silver nitrate solution (1:5 ratio)
at room temperature. The incubation was done for 2 hours. The observations were made using a UV-
Spectrometer every 30 minutes, to encompass a change in color and absorption pattern. After completion of
the reaction, the mixture was centrifuged at 16,000 rpm for 5 mins at 25°C. The supernatant was discarded.
The remaining particles were then rinsed to remove any organic residue and re-suspended in 95% ethanol
for further characterization.

The crystallinity and phases of the silver nanoparticles were characterized by X-ray diffractometer (X’pert
Pro, PANalytical, Netherlands) with CuKα radiation (λ = 1.5418 Å) in the range of 20°–80° with 2°/min
scanning rate.
In addition, the optical property of prepared silver nanoparticles was analyzed via UV-visible (Specord
210 Plus, Analytik Jena, Germany) absorption double beam spectrophotometer with a deuterium and halogen
lamp in the range from 300–700 nm at room temperature.
The particle diameter of the reaction mixture was determined by Nanoparticle analyzer (SZ-100, Horiba
Scientific, Japan). Particle size analysis was performed in dynamic light scattering (DLS) mode.
RESULTS & DISCUSSION

The color changes were noted by visual observation in the beaker which contains silver nitrate solution with
B. vitis-idaea leaves extract. The color of the AgNO3 extract solution changed from colourless to reddish
brown after 5 min and eventually to dark gray (Figure 1).
Figure 1: Color changes of silver nitrate (colourless, Left) to silver nanoparticles by the addition of B. vitis-idaea leaf
extract (dark gray, right).
112
This color changes indicates the formation of silver nanoparticles in the solution. B. vitis-idaea leaves
extract without silver nitrate did not show any color changes.
UV–vis absorbance of the reaction mixture was taken from 0 till 120 mins (Figure 2). It was observed
that the absorbance peak was centered near 445 nm, indicating the reduction of silver nitrate into silver
nanoparticles. It was also observed that the reduction of silver ions into silver nanoparticles started at the
start of reaction and reduction was completed at 120 mins at room temperature, indicating rapid biosynthesis
of silver nanoparticles.
Figure 2: Nanoparticle size distribution in the reaction mixture and dry samples is measured using nano particle
analyzer.
Figure 3: Histogram of particle size analyser. a) Reaction mixture b) Dried sample
The XRD pattern of the biosynthesized silver nanoparticles using the leaves extract of B. vitis-
idaea was shown in the figure 4. The XRD pattern showed numbers of Bragg reflections that may be
indexed on the basis of the face-centered cubic (FCC) structure of silver. A comparison of our XRD
with the standard confirmed that the silver nanoparticles formed in our experiments were in the form of
nanocrystals as evidenced by the peaks at 2θ values of 38.19°, 44.25°, 64.33° and 77.21° corresponding
to (111), (200), (220) and (311) Bragg reflections, respectively, which may be indexed based on the FCC
structure of silver. X-ray diffraction results clearly showed that the silver nanoparticles formed by the
reduction of silver ions by the B.vitis-idaea leaf extract are crystalline in nature. Apart from these peaks
responsible for silver nanoparticles, the recorded XRD pattern shows additional unassigned peaks,
noted with stars. This may be due to the formation of the crystalline bio-organic phase occurs on the
surface of the silver nanoparticles.
113
Figure 4: XRD pattern of silver nanoparticles.
Previous studies reported that silver nanoparticles can be synthesized by plants such as Azadirachta
indica [7], Capsicum annuum [8], Carica papaya [9], Gliricidia sepium [10], Eucalyptus hybrid [11],
Pelargonium graveolens [12], Medicago sativa [13], Azadirachta indica [14], Lemongrass [15], Aloe vera
[16], Cinnamomum Camphora [4], Emblica officinalis [17], Diospyros kaki [18], Coriandrum sp. [19],
Boswellia ovalifoliolata [20], and Citrus aurantium [21]. In the current study, aqueous silver ions were
reduced to silver nanoparticles after mixing with B.vitis-idaea leaf extract followed by incubation for 120
mins in the dark. The color turned reddish brown to dark gray. These color change appeared due to the
surface plasmon resonance (SPR) of deposited silver nanoparticles. In the current study, the mechanism
by which the plant extract could be synthesized silver nanoparticles may be explained by the higher total
phenolics content in the plant. These plant phenolics are strong antioxidants with high reducing capacity [22]
which can be used for silver nanoparticles synthesis. The higher content of total phenolic content in B.vitis-
idaea leaf extract facilitates the reduction of silver ions to nano sized silver particles due to the electron
donating ability of these phenolic compounds.
Figure 2 shows UV-vis absorption spectrum of silver nanoparticles.Though the plasmon band
is broad due to the presence of components in B.vitis-idaea leaf extract which are also being read in
the spectrophotometric range, it is observed that the silver surface plasmon resonance (SPR) occurs at
445 nm.
Generally, biosynthetic methods are considered as time consuming when compared with chemical
methods. To the best of our knowledge, reaction time of at least 12 hrs is required in plant-mediated
nanomaterial synthesis. However, the time consumed in the present study for the reaction to complete is
several fold lesser than reported. Such alacrity in reaction time can be the outcome of potent antioxidant
activity of the B.vitis-idaea extract, which makes the reaction much more efficient than others.
Figure 3 shows the histogram of diameter versus frequency observed on Particle size analyser. It is clear
from the histogram that the mean particle size of silver nanoparticles is 60 nm.
Figure 4 shows the XRD patterns obtained for silver nanoparticles synthesized by B.vitis-idaea extract.
All major peaks can be indexed for the face centered cubic structure of silver. Minor but broad peaks at lower
2-𝜃, theta values can be assigned to the organic content of the B.vitis-idaea extract. The XRD pattern thus
clearly shows that silver nanoparticles formed by B.vitis-idaea extract are crystalline in nature. The average
diameter of the silver particles was calculated from the (111) diffraction peak using Scherrer’s formula:
0.9λ
t=
(β cos θ )
114
where 𝑡 is mean crystallite size, 𝛽 is the width of the peak at half maximum intensity of a specific
phase in radians, and 𝜆 is the wavelength of incident rays, 𝜃 is the center angle of the peak in radian. The
mean crystallite size for silver nanoparticles synthesized using B.vitis-idaea extract was determined to
be 16.87 nm.
CONCLUSION
The silver nanoparticles have been produced by B.vitis-idaea extracts, which is an economical, efficient and
eco-friendly process. Further characterisation (i.e., TEM, SEM, FTIR, and AFM) and determination of in
vivo toxicity of silver nanoparticles were under process. Nano particles by B.vitis-idaea may have persuasive
application in medicine therapeutics and diagnostics.
Conflict of Interest
The authors declare that there are no conflicts of interest.
REFERENCES
1. V.K. Sharma, R.A. Yngard and Y. Lin, “Silver nanoparticles: green synthesis and their antimicrobial
activities,” Advances in Colloid and Interface Science, vol. 145, pp. 83–96, 2009.
2. D. Goodsell, Bionanotechnology: Lessons from Nature, Wiley- Liss, Hoboken, NJ, USA, 2004.
3. S.S. Shankar, A. Ahmad and M. Sastry, “Geranium leaf assisted biosynthesis of silver nanoparticles,”
Biotechnology Progress, vol. 19, no. 6, pp. 1627–1631, 2003.
4. J. Huang, Q. Li, D. Sun, Y. Lu1, Y. Su1, X. Yang, H. Wang, Y. Wang, W. Shao1, N. He1, J. Hong and
C. Chen, “Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora
leaf,” Nanotechnology, vol. 18, no. 10, Article ID 105104, 2007.
5. M.D.A. Farooqui, P.S. Chauhan, P. Krishnamoorthy and J. Shaik, “Extraction of silver nanoparticles
from the leaf extracts of Clerodendrum inerme,” Digest Journal of Nanomaterials and Biostructures,
vol. 5, no. 1, pp. 43–49, 2010.
6. D.H. Meng, J. Wu, L.Y. Wang and W.M. Zhao, “Two new glycosides from Breynia vitis-idaea,” Journal
of Asian Natural Products Research, vol. 12, no. 6, pp. 535–541, 2010.
7. S.S. Shankar, A. Rai, B. Ankamwar, A. Singh, A. Ahmed and M. Sastry, “Biological synthesis of
triangular gold nonoprism,” Nature Materials, vol. 3, pp. 482–488, 2004.
8. H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S.P. De and A. Misra, “Green synthesis of silver nanoparticles
using latex of Jatropha curcas,” Colloids and Surfaces A, Vol. 339, pp. 134–139, 2009.
9. A.K. Jha and K. Prasad, “Green synthesis of silver nanoparticles using Cycas leaf,” International
Journal of Green Nanotechnology: Physics and Chemistry, Vol. 1, pp. 110–117, 2010.
10. R.W. Raut, N.S. Kolekar, J.R. Lakkakula, V.D. Mendhulkar and S.B. Kashid, “Extracellular synthesis
of silver nanoparticles using dried leaves of Pongamia pinnata (L) Pierre,” Nano-Micro Letter, Vol. 2,
pp. 106–113, 2010.
11. M. Dubey, S. Bhadauria and B.S. Kushwah, “Green synthesis of nanosilver particles from the extract
of Eucalyptus hybrid (Safeda leaves),” Digest Journal of Nanomaterials and Biostructures, Vol. 5,
pp. 537–543, 2009.
12. S.S. Shankar, A. Ahmad and M. Sastry, “Geranium leaf assisted biosynthesis of silver nanoparticles,”
Biotechnology Progress, vol. 19, no. 6, pp. 1627–1631, 2003.
115
13. J.L. Gardea-Torresdey, E. Gomez, J.R. Peralta-Videa, J.G. Parsons, H. Troiani and M. Jose-Yacaman,
“Alfalfa sprouts: a natural source for the synthesis of silver nanoparticles,” Langmuir, vol. 19, no. 4, pp.
1357–1361, 2003.
14. S.S. Shankar, A. Rai, A. Ahmad and M. Sastry, “Rapid synthesis of Au, Ag, and bimetallic Au core-
Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth,” Journal of Colloid and Interface
Science, vol. 275, no. 2, pp. 496–502, 2004.
15. S.S. Shankar, A. Rai, A. Ahmad and M. Sastry, “Controlling the optical properties of lemongrass
extract synthesized gold nanotriangles and potential application in infrared-absorbing optical coatings,”
Chemistry of Materials, vol. 17, no. 3, pp. 566–572, 2005.
16. S.P. Chandran, M. Chaudhary, R. Pasricha, A. Ahmad and M. Sastry, “Synthesis of gold nanotriangles
and silver nanoparticles using Aloe vera plant extract,” Biotechnology Progress, vol. 22, no. 2, pp.
577–583, 2006.
17. B. Ankamwar, C. Damle, A. Ahmad and M. Sastry, “Biosynthesis of gold and silver nanoparticles using
Emblica officinalis fruit extract, their phase transfer and transmetallation in an organic solution,” Journal
of Nanoscience and Nanotechnology, vol. 5, no. 10, pp. 1665–1671, 2005.
18. J.Y. Song and B.S. Kim, “Biological synthesis of bimetallic Au/Ag nanoparticles using Persimmon
(Diopyros kaki) leaf extract,” Korean Journal of Chemical Engineering, vol. 25, no. 4, pp. 808–811,
2008.
19. K.B. Narayanan and N. Sakthivel, “Coriander leaf mediated biosynthesis of gold nanoparticles,”
Materials Letters, vol. 62, no. 30, pp. 4588–4590, 2008.
20. S. Ankanna, T.N.V.K.V. Prasad, E.K. Elumalai and N. Savithramma, “Production of biogenic silver
nanoparticles using Boswellia ovalifoliolata stem bark,” Digest Journal of Nanomaterials and
Biostructures, vol. 5, no. 2, pp. 369–372, 2010.
21. P. Rajasekharreddy, P.U. Rani and B. Sreedhar, “Qualitative assessment of silver and gold nanoparticle
synthesis in various plants: a photobiological approach,” Journal of Nanoparticle Research, vol. 12, no.
5, pp. 1711–1721, 2010.
22. CG. Joshi, Gopal M and Vaigundan D, “In Vitro antioxidant activities of Breynia Vitis-Idaea extracts,”
Journal of Chemical and Pharmaceutical Research, vol. 3, no. 5, pp. 340-347, 2011.
116
Characterization of Solid State Forms of Pioglitazone
B. Poornima, K.V.S.R.G Prasad and K. Bharathi
Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Vishwa Vidyalayam,

Tirupathi, Andhra Pradesh, India
e-mail: poornimachandran7@gmail.com, Mobile No: 08790355248
Abstract:
Two new polymorphic forms of Pioglitazone with unique physical and spectroscopic properties.The two
polymorphs obtained by evaporation method and diffusion method had shown different IR spectra, X-ray
diffraction patterns, SEM analysis and also different DSC thermal graphs as represented above when
compared to its commercial form.According to results obtained it is confirmed that, the polymorphic form
prepared by evaporation method had shown better dissolution properties and solubility, different thermal
behaviour and spectroscopic properties and dissolution profile.
Keywords: Pioglitazone, Anti diabetic drug, Polymorphism.
INTRODUCTION
Polymorphism is the ability of a substance, or an element or a compound to crystallize in more than one
distinct crystal species. It is a major problem that has been of considerable importance in the pharmaceutical
industry in the development of new drug candidates [1]. Because the polymorphs have different crystal
structures, different physico - chemical properties such as colour, density, melting point, solubility, dissolution
rate etc., different chemical reactivity and different bioavailability, they can be interconverted by phase
transformations or a solvent- mediated process [2]. A full evaluation of possible variations in polymorphs that
might be encountered is now essential for the development of new drug compound; therefore, the physical
characterization of solids has become an extremely important area in pharmaceutics and has been the subject
of many studies involving different analytical methods [3]. The most common polymorph screening methods
are crystallization from a melt, vapour, and solutions through cooling or evaporating the solvent, addition
of an anti solvent to the solution [4], or slurrying the solid active pharmaceutical ingredient for an extended
period of time at different temperatures [5].
Pioglitazone hydrochloride is chemically [(±)- 5- [[ 4- [2- (5- ethyl- 2- pyridinyl)ethoxy ] phenyl]
methyl]-2,4-] thiazolidinedione hydrochloride (PGH) (Fig.1).which is an oral antihyperglycemic agent
that acts primarily by increasing insulin sensitivity in target tissues. It is used both as monotherapy and in
combination with a sulfonylurea or insulin in the management of type 2 diabetes mellitus (non- insulin-
dependent diabetes mellitus, NIDDM) [6]. Pioglitazone contains one asymmetric carbon, and the compound
is synthesized and used as racemic mixture. The two enantiomers of pioglitazone interconvert in vivo. No
differences were found in the pharmacologic activity of the two enantiomers. This drug has been investigated
by several authors with respect to polymorphism of various solid phases; however, numerous results reported
in the literature confirmed that the drug existed in two crystalline forms such as Form I and Form II [7- 11].
The purpose of this study is to develop various solid state forms of Pioglitazone by different methods.
They were characterized by thermal, crystallographic, microscopic, spectroscopic and elemental analytical
techniques.
EXPERIMENTAL
Materials
Commercial pioglitazone was gifted by Amoli organics Pvt. Ltd. (Baroda), India. All solvents used in the
study were of analytical grade.
Preparation of the polymorphs

Pioglitazone hydrochloride was crystallized from various solvents like acetone, ethanol, isopropanol, ether
(class III)N,N dimethyl formamide &methanol(class II).
Method I
Slow evaporation method: The saturated solution of the drug was prepared by dissolving the drug in excess
amount of solvent in a clean container. The container should have a large surface and covered with an
aluminium foil with some punched holes to facilitate the evaporation of the solvent at room temperature.
Formed crystals were collected and dried.
Method- II
Solvent diffusion method: Concentrated solution of the drug was prepared with suitable solvent (DMF).
Another solvent (ether) was slowly added drop wise along the walls of the container. Then the generated
polymorphs were collected at the junction of mixing of solvents.
Method III
Antisolvent method: The polymorphs were prepared by using the antisolvents Acetone and water
respectively. The drug was dissolved in DMF at 60°C and cooled to 4°C for 72 hrs to obtain the
polymorphs.
METHODS OF INVESTIGATION
Physical Characterization
Melting point determination

The melting point of generated polymorphs was measured by melting point apparatus (Stauart melting point
apparatus model SMP3).
Measurement of flow Properties

The bulk density and tap density of various solid state forms of pioglitazone were determined using Electrolab
tap density tester (ETD-1020, India). Accurately weighed samples of known volume were noted. The bulk
density, tap density, Carr’s index and Hausner ratio of polymorphs of pioglitazone were calculated.
Solubility Studies
The solubility of the polymorphic forms of PGH was determined in pH2 Kcl buffer. Excess amount of sample
was added to 20 ml of KCl buffer, continuously shaken mechanically using Orbital shaker (Electro lab
Orbital shaker) with 300rpm at room 25±0.5°C for 48 hrs. And the samples were sonicated using a sonicator
(Ultrasonicating bath, Spectrolab, Model UCB-30) for 2 hrs. Then the samples were filtered through a 0.45
µm filter. The filtrate was diluted suitably and then analyzed using UV spectrophotometer (UV-Double beam
spectrophotometer, UV- 1800 model, Shimadzu) at a λmax of 269nm.
118
MORPHOLOGICAL STUDY
Phase contrast microscopy

The particle characteristics of Pioglitazone were assessed by Phase contrast microscope.
Scanning Electron Microscopy

The morphological studies were performed using Scanning Electron Microscope (SEM) with Energy –
Dispersive X-ray Analysis (EDAX) System (model CARL- ZEISS EVO MA 15). The samples were observed
at 10,000x magnification with an accelerating voltage of 20kV.
THERMAL ANALYSIS
Differential scanning Calorimetry (DSC)

DSC analysis was performed using Metteler Toledo 821e DSC (Mettler Toledo, Switzerland) operating
with STARe software using (4-7 mg) sample in aluminium pans with pierced lids at heating rate of
10°C min -1and nitrogen purge (20 ml min-1). Temperature axis and cell constant were calibrated
using indium.
SPECTROSCOPIC AND ELEMENTAL ANALYSIS

Fouirer transform infrared spectroscopy (FTIR)
FTIR spectroscopy was used to investigate the chemical stability of the drug. Samples were measured
on BRUKER model 65 with OPUS software, in the interval 400-4000 cm-1, at 4 cm-1 optical resolution.
Standard KBr pellets were prepared from 100 mg of KBr pressed with 10 ton and samples containing 0.5mg
of pioglitazone polymorphs were used.
X-ray diffraction:
XRD is one of the most sensitive and foolproof method for solid-state characterization as the results is
obtained directly from the molecular arrangements of the crystalline material (Chao and Vial, 1987).
Fig shows the XRD patterns of solid state forms of Pioglitazone. Crystalline forms of Pioglitazone showed
sharp diffraction peaks.
Dissolution examinations:
Dissolution was studied by Paddle dissolution apparatus (Electro Lab, TDT-08L, Dissolution tester, USP)
at a speed of 75 rpm. 900ml of dissolution medium was placed in a 37°C (± 0.5°C) dissolution bath and the
examination was performed for 2 hrs. The dissolution medium used here is Kcl buffer with pH 2 (pH meter
– Digital pH meter 802 SYSTRONICS). The concentration of each polymorph along with its commercial
form was determined Spectrophotometrically at 269 nm (UV-Double beam spectrophotometer, UV- 1800
model, Shimadzu). The dissolution experiments were conducted triplicate and standard deviation was also
calculated.
RESULTS AND DISCUSSION:

Both polymorphs A, B, which are obtained from Antisolvent method, were exist similar as that of commercial
form. Form B obtained from anti solvent method is similar as that of reported Form II of Pioglitazone
indicating that polymorphic transition from commercial form.
119
Physical characterization
Melting point
The melting range of different polymorphic forms of Pioglitazone was noted. Table (1) Different polymorphic
forms show different melting range.
Morphology
Crystal morphology plays a major role in pharmaceutical processing and product development of solid
dosage forms. Differences in crystal habit may strongly influence the particle orientation; modify flow
ability, packing, compaction, compressibility and dissolution characteristics of the drug substance. Solid
liquid interaction may have impact on crystal growth. It may enhance or inhibit the crystal growth at certain
crystal faces resulting in different habits such as acicular, plates, tabular, bladed, prismatic, etc.
The different morphological features of various solid- state forms of pioglitazone were examined visually
by phase contrast microscopy (Fig. 1). The stable crystalline form of pioglitazone occurs as fine needles. The
other forms were found to exhibit flake like structures.
Figure 1: Phase contrast microscope photographs
(a) Evaporation method (b) diffusion method (c) commercial form
SEM photographs reveal that (Fig. 2) there are distinct differences in morphology of different solid
state forms of pioglitazone. The polymorphs of pioglitazone are obtained as needle shaped clusters with
antisolvent acetone and needle shaped crystals with antisolvent water. Rod shaped needles with smooth
end crystals were obtained from solvent diffusion method. Netted like lumps were obtained with solvent
evaporation method, where as irregularly shaped lumps were obtained from grinding method.
Diffusion method Evaporation method

Figure 2: SEM pictures of Pioglitazone polymorphs
Thermal analysis
DSC
The DSC curves and the relevant data of the polymorphs were shown in Fig.3. These polymorphs of
pioglitazone gave different DSC patterns, two endotherms were obtained in case of evaporation method,
120
the peaks are at 198 and 258. The other forms show single endothermic peaks with different melting range
as shown in figure. Similar to FT-IR spectroscopic findings, where identical spectral pattern were observed
in all cases, the DSC curves exhibit significant differences (Fig 3) both in the position and the shape of the
observed single endotherm below.
Figure 3: DSC spectra of Pioglitazone polymorphs
SPECTROSCOPIC AND ELEMENTAL ANALYSIS
Fouirer transform infrared spectroscopy (FTIR)

FTIR spectroscopy has been used for exploring the differences in molecular confirmations, crystal packing
and hydrogen bonding arrangements for different solid state forms of an organic compound. Spectral
variations originate due to alteration in bonds that exhibit characteristic vibrational frequencies, leading
to frequency shifts and splitting in absorption peaks. The FTIR spectra of pioglitazone (Fig. 4) showed
characteristic symmetric N-H stretch and aromatic C-H stretch at 3364 and 3084 respectively. The other
characteristic C-N ring symmetric stretch, C-S stretch and para di substituted aromatic ring are obtained at
1460, 1242 and 850 respectively.
Figure 4: FTIR Spectra of Pioglitazone polymorphs
121
The C-S stretch and C-N ring stretch was observed at lower frequencies in case of evaporation method
and solvent diffusion method at 1238, 1462 respectively.
Table 2 FTIR interpretated peaks of Pioglitazone polymorphs
X-ray diffraction (XRD)

XRD is a significant method of analysis for the determination of polymorphs. The XRD patterns of pioglitazone
along with its polymorphs are shown in Fig.5 and the data of diffraction peaks with 2θ angles are represented
in Table.3. The two polymorphs which are prepared from solvent evaporation method and solvent diffusion
method are different from each other with different diffraction peaks. Moreover, the differences either in
the position or in the intensity of peaks may not be attributed to preferred orientation crystal growth, but
suggested different arrangements of Pioglitazone molecules of crystal lattice of each form. The characteristic
angles of diffraction together with five main peak intensities for two polymorphic forms.
Figure 5: XRD spectra of Pioglitazone polymorphs
Table 3: XRD peaks of Pioglitazone polymorphs
Form 2θ angle (°) I/I° (%)

D 18.55 100
18.78 49
16.39 30
21.38 21
25.04 15
E 20.02 100
26.30 56
22.7 28
28.21 27
20.7 14
DISSOLUTION PROFILE
The prepared polymorphic forms were studied for dissolution. The fig. 6 shown that the polymorphic form
prepared by evaporation method had better dissolution than commercial form.
122
Figure 6: in vitro dissolution of Pioglitazone polymorphs

SOLUBILITY STUDY
The solubility of pioglitazone polymorph prepared by slow evaporation method had shown better solubility
than the commercial form (table 4)
Table 4: Solubility profile
Form Solubility in KCl buffer (mg/ml)

Diffusion method 26
Evaporation method 35
Commercial form 29
PHYSICAL PARAMETERS STUDIED

Accurately weighed samples of known volume were noted. The bulk density, tap density, Carr’s index and
Haussners ratio of polymorphs of pioglitazone were calculated were represented in table 1.
Table 1:
Polymorphic form Bulk Density Tab Density Haussner’s ratio Carr’s Index Melting point °C
Commercial form 1.4733 1.7 1.147 13.3433 181.8
Form D 2.7516 3.2133 1.166 14.326 171.5
Form E 1.17 1.5166 1.2866 0.2233 196.7
CONCLUSION
The two new polymorphic forms of Pioglitazone with unique physical and spectroscopic properties.
The two polymorphs obtained by evaporation method and diffusion method had shown different IR
spectra, X-ray diffraction patterns, SEM analysis and also different DSC thermal graphs as represented
above when compared to its commercial form.
According to results obtained it is confirmed that, the polymorphic form prepared by evaporation
method had shown better dissolution properties and solubility, different thermal behaviour and spectroscopic
properties and dissolution profile.
123
REFERENCES:
1. P. Lang, V. Kiss, R. Ambrus, G. Farkas, P. Szabo-Revesz, Z. Aigner and E. Varkonyi, Polymorph

Screening of an active material, J. Pharma. Biome. Anal. 84 (2013) 177–183.
2. C. Rustichelli, G. Gamberini, V. Ferioli, M.C. Gamberini, R. Ficarra and S. Tommasini, Solid- state
study of polymorphic drugs: Carbamazepine, J. Pharma. Biome. Anal. 23 (2000) 41–54.
3. H.G. Porter, in: D. M. Woodbury, J.K. Perny, C. E. Pippenger (Eds.), Antiepileptic Drugs, Raven Press,
New York (1982) 167–175.
4. C. Sun, Solid – state properties and crystallization behaviour of PHA-739521 polymorphs, Int.
J. Pharm.319 (2006) 114–120.
5. A.M. Campeta, B.P. Chekal, Y.A. Abramov, P.A. Meenan, M.J. Henson, B. Shi, R.A. Singer and K.R.
Horspool, Development of a targeted polymorph screening approach for a complex polymorphic and
highly solvating API, J. Pharm. Sci. 99 (2010) 3874–3886.
6. Yadollah Yamini, Elham Tahmasebi and Abolfazl Saleh, Extraction of trace amounts of pioglitazone
an antidiabetic drug with hallow fiber liquid phase microextraction and determination by high-
performance liquid chromatography- ultraviolet detection in biological fluids, J. Chromatogr. B 877
(2009) 1923–1929.
7. Wizel, Finogueev, Hildesheim and Pioglitazone Hydrochloride, World Intellectual Property Organization,
WO 03/026586 A2.
8. Mailatur Sivaraman Mohan, Indu Bhushan, Siva reddy and Kamlakar Golli Reddy, Pioglitazone
Composition, Patent Application Publication, Pub.No. US 2008/0182880 A1, (Jul.31, 2008).
9. Khanduri, Kumar and Panda, World Intellectual Property Organization, WO 2005/021542 A2,
(10 March 2005).
10. Chandra Has Khanduri, Yatendra Kumar, Atulya Kumar Panda, Suchitra Chakraborty and Mukesh
Kumar Sharma, Process for the preparation of Pioglitazone, Patent Application Publication, Pub. No:
US 2007/ 0078170 A1, (Apr. 5, 2007)
11. Shlomit W Wizel, Serguei Finoqueev and Jeva Hildesheim, Pioglitazone Hydrochloride, United States
Patent, Patent No: US 7,135, 485 B2, (Nov. 14, 2006).
124
Synthesis of Metaloxide Nano Composites for Solar Cells
S. Shanthi1 and Dr. M. Dharmendirakumar2
1
Research scholar, 2Assistant Professor, Anna University, Chennai
e-mail: shanthiphd2011@hotmail.com, Mob 9677268816
Abstract:
Synthesis of Titanium stannous chloride oxide nanocomposites was done by solvothermal method. This
method is to provide a simple economic and effective technique to produced nanocomposites. The absorbance
analysed with the help of UV-Visible spectroscopy. The particle size was measured by electron microscopy.
The structure, morphology were investigated by XRD. The the titanium stannous chloride nanocomposites
were used to measure the efficiency of solar cell.
Keyword: XRD, SEM, Nanocomposite.
INTRODUCTION
Titanium dioxide is a preferred system for experimentalists because it is well suited for many experimental
techniques. Polished crystals with a high surface quality can be purchased from various vendors. They
can be reduced easily, which prevents charging of this wide band gap semiconductor. One should also not
underestimate the “self-promoting” effect of popularity New phenomena are studied most easily on well-
characterised, often tested systems, and especially TiO2 the most stable rutile phase (1 1 0) surface, falls
certainly into this category. All these factors have contributed in making TiO2 the model system in the
surface science of metal oxides.
In recent years, much effort has been devoted to the research of metal oxide nanocomposites. This kind
of nanomaterials exhibits unusual physical and chemical properties in comparison with their bulk materials,
such as size-dependent variation of the band gap energy (1, 2). Titanium dioxide is one among the important
inorganic pigments used in the plastic and paint industry (3, 4). However, the wide energy band gap of TiO2
(3.2 eV) is frequently studied in optic fields, especially as an excellent photo catalytic material applied
in photo degradation and solar energy conversion (5, 6). TiO2 exists mainly in three naturally occurring
crystallographic forms, namely anatase, brooklite, and rutile. Combination of two or more materials will
provide the formed composites with several outstanding physical and chemical performances, especially for
oxides and semiconductors (7, 8).
Tin oxide (SnO2) is an important n-type semiconductor metal oxide with wide band gap energy (3.6eV).
Because of its Unique properties of electronic, optical, electrochemical, and catalytic properties, SnO2 were
extensively used in solar cell, transparent conducting electrodes, solid state sensors, rechargeable Li batteries
and optoelectronic devices (9). In this present study the main objective is to measure the efficiency of solar
cells using nanocomposite.
1. EXPERIMENTAL METHODS

Solvothermal synthesis method is very similar to the hydrothermal method, the only difference is in the
precursor solution which is usually non -aqueous or mixture of aqueous and non-aqueous. Making use
of the solvothermal method one gains the benefits of both the sol gel and hydrothermal method. So
solvothermal synthesis allows the precise control over size, shape distribution and high crystallinity of metal
oxide nanoparticles, because of these benefits solvothermal analysis method is carried out for synthesis of
nanocomposites.
2. MATERIALS USED

The precursor materials used for the preparation of semiconductor nanocrystalline TixSn(1-x)O2 are listed
below: All the precursor materials were purchased from Sigma Aldrich and used without further purification.
• Titanium tetrachloride (Analytical Reagent grade) TiCl4
• Tin (II) chloride (Analytical Reagent grade) SnCl2.2H2O
• Urea
• Ethylene glycol
3. SYNTHESIS
Titanium tetrachloride, Tin (II) chloride and urea along with ethylene glycol were used for the preparation of
TixSn(1-x)O2 (x = 0.0, 0.25, 0.50, 0.75 and 1.0) nanocomposites. Titanium tetrachloride and Tin (II) chloride
taken together in the required composition and urea in 1:2 molecular ratio were mixed and dissolved in
100 ml ethylene glycol and kept in a domestic microwave oven (ONIDA model number MO28CJS16B
operated with frequency 2.45 GHz and power 800W). Finally the solution was stirred for 30 minutes, the
precipitate was formed and washed by using double distilled water and acetone to remove the impurities.
The sample was then filtered and dried in an oven at 2.45 GHz. The absorbance were measured using UV-
Visible spectrophotometer and photoluminescence studies were carried out to measure the emission rate of
the prepares sample (nanocomposite).
4. CHARACTERIZATION
Characterizations of the nanocomposite materials is necessary to understand/analyse various facts of metal-
oxide nanocomposites.
5. RESULTS AND DISCUSSION
A. XRD Analysis-Estimation of structural parameters

XRD was used to investigate the phase structures & average particle size of the SnO2-TiO2 & various
concentrations of nanocomposites annealed at 400°C.That characteristic peaks of 2Ɵ degrees were
recognised corresponding to the h, k, l Miller indices respectively.(fig1).Presence of rutile, anatase &
Tetragonal structure is confirmed for pure TiO2,Ti0.75Sn0.25O2, Ti0.50Sn0.50O2, Ti0.25Sn0.75O2 & SnO2 nano
composites.The strongest peaks for anatase & rutile phase were observed at 2Ɵ=25.4°[10] & 2Ɵ=27.5°
[11] respectively. As can be noticed, the nanocomposites showed a low degree of crystallization and a
126
thermal treatment was necessary in order to increase it and to stabilize the nanoparticles. The result of
XRD powder patterns indicated that the experimental data are in good agreement with the simulated
XRD powder patterns based on the reference data. The Broadening based on the peaks indicated that the
particles were of nanometer scale.
The lattice constants ‘a’ and ‘c’ for the tetragonal phase structure have been determined by the relation
[12].
1 h2 + k 2 l2
= + 2 ---------------------(1)
d 2
a 2
c
where ‘d’ is the interplanar spacing and (h k l) are miller indices, respectively.
The lattice parameters are calculated from the indexed powder XRD pattern by using the software powder X [13].
Table 1: Calculated lattice parameters for alloyed Ti(1-x)SnxO2 (x = 0, 0.25, 0.5, 0.75 and 1) nanocrystals
(*JCPDS file no. 89-4921; #JCPDS file no. 41-1445)
Sample name Lattice parameter (Å) Unit cell volume
a=b c Å3
*TiO2 (anatase) 3.777 9.501 135.54
TiO2 3.7730 9.5067 135.33
Ti0.75Sn0.25O2 3.8232 8.8973 130.05
Ti0.5Sn0.5O2 3.9703 7.4865 118.01
Ti0.25Sn0.75O2 4.4506 3.9801 78.84
SnO2 4.7301 3.1784 71.11

#
SnO2 4.7382 3.1871 71.55
The crystallite size in each case was determined by using the Debye-Scherrer formula [14],
0.9 λ ………………(2)
D= ,
β cos θ
where
D is the mean size (diameter) of the crystallite,
b is the full width at half maximum of intensity (in radians),
l is the wavelength of the X- ray radiation used (1.540598 AU), and
q is half the angle at which maximum intensity was observed.
Four to five different diffraction peaks were chosen to calculate crystallite size from Debye-Scherrer
formula.
The average crystallite size (D) and average lattice strain (ε) of nanocrystalline material is determined
by Williamson and Hall (W-H) equation [15]
β cos θ 1 4ε sin θ
= + ...................(3)
kλ D kλ
where β is the full width at half maximum (FWHM), ε is the average strain in the material, k is the shape factor
(0.9) and λ is the wavelength of X-ray. The average crystallite size (D) and average microstrain (ε) of alloyed
alloyed Ti(1-x)SnxO2 (x = 0, 0.25, 0.5, 0.75 and 1) nanocrystals are determined by plotting a graph between β
cosθ/0.9λ vs 4 sinθ/0.9λ. The slope of the plot represents average strain (ε) in the material whereas inverse of the
intercept on β cosθ/0.9λ axis gives the average crystallite size (D).
127
Table 2: Calculated crystallite size and lattice strain for alloyed Ti(1-x)SnxO2 (x = 0, 0.25, 0.5, 0.75 and 1) nanocrystals
from Scherrer formula and W-H plots.
Sample name Crystallite size (nm) Lattice strain

From Scherrer formula From W-H plot
±0.37 ±0.45 x 10-3
TiO2 4.35 4.05 1.894
Ti0.75Sn0.25O2 7.15 6.58 1.303
Ti0.5Sn0.5O2 5.26 4.88 1.592
Ti0.25Sn0.75O2 6.28 6.03 0.633
SnO2 8.69 8.11 0.852
Figure 1: Powder XRD patterns of (a) bulk TiO2 (JCPDS file no. 89-4921) (b) TiO2 nanocrystals (c) Ti0.75Sn0.25O2
nanocrystals (d) Ti0.5Sn0.5O2 nanocrystals (e) Ti0.25Sn0.75O2 nanocrystals (f) SnO2 nanocrystals (g) bulk SnO2 (JCPDS file
no. 41-1445)
Figure 2: Williamson–Hall plot of TiO2 nanocrystals
128
Figure 3: Williamson–Hall plot of Ti0.75Sn0.25O2 nanocrystals
129
Figure 6: Williamson–Hall plot of SnO2 nanocrystals
B. Scanning Electron Microscope Analysis

Scanning Electron Microscope Analysis (SEM) Fig 7,8,9,10 and 11 shows the SEM image of pure
TiO2,Ti0.75Sn0.25O2, Ti0.50Sn0.50O2, Ti0.25Sn0.75O2 & SnO2.The surface morphology of the nanocomposite is
analysed by scanning electron microscope.Compared with the pure TiO2 and SnO2, the size of the various
concentrations of nanocomposites was enlarged and uniform.so its surface area was larger.
The growth of the fine particles of TiO2 in regular pattern is observed on the surface of the sample.
The size and morphological evaluations of various concentrations of nanocomposites is also dependent
on reaction time. To verify the morphology scheme obtained from XRD data from the scanning electron
microscopy was studied [16].
130

Figure 7: SEM image of pure TiO2 Figure 8: SEM image of Ti0.75Sn0.25O2

Figure 9: SEM image of Ti0.50Sn0.50O2 Figure 10: SEM image of Ti0.25Sn0.75O2
Figure 11: SEM image of SnO2
C. UV-Visible spectrum and band gap energy

Fig 12. Shows the optical absorption spectra for pure TiO2,Ti0.75Sn0.25O2, Ti0.50Sn0.50O2, Ti0.25Sn0.75O2 & SnO2.
The corresponding energy versus (αhν)2 x103 fig (13). The observed optical band gap energy values are
3.358ev, 3.423ev, 3.492ev, 3.665ev, 3.777ev.The band gap observed for pure TiO2 is lesser than increasing
concentration of SnO2.This result indicates that the new material may be good for solar cells [17].
Figure 12: UV-Vis-NIR spectrum of nanocomposites

131
Figure 13: Band gap determination of nanocomposites
7. CONCLUSION
Titanium stannous chloride oxide nano composites was successfully synthesized by solvothermal mehod.
Presence of rutile, anatase & Tetragonal structure is confirmed by XRD analysis. Low band gap energy of
the synthesized material indicates that it is very useful to prepare solor cells.
8. REFERENCES
1. D.J. Norris, N. Yao, F.I Charnock and T.A. Kennedy, Nano Lett 1 (2001) 3.
2. W. Chen, J-O. Malm, V. Zwiller, Y. Huang, S. Liu, R. Wallenberg, J-O. Bovin and L. Samuelson, Phy.
Rev B 61 (2000) 11021.
3. Terwillinger CD and Chiang YM, Nanostruct Mater 2:37 (1993).
4. Fukuji S. Kazuaki, H. Kazuaki, Y. Yakro and M. Yoshitomo, Preparation and optical properties of TiO2.
5. F. Bosc, A. Ayral, P.A. Albouy and C. Guizard, A. Simple ruote for low temperature synthesis of
mesoporous and nanocrystalline anatase thin films, chem, Mater 15:2463–2468 (2003).
6. H. Hansel, H. Zettl G. Krausch R. Kisseley M. Thelakkat and H.W. Schmidt, optical and electronis
contributions in double- heterojunction organic ihin films solar cells.15:2056–2060 (2003).
7. M.H. Liao, C.H. Hsu and D.H Chem, Preparation and properties of amorphous titania coated zinc oxide
nanoparticles, J. Solid State Chem 179: 2020–2026 (2006).
8. X.G. Peng, M.C. Schlamp, A.V. Kadavanich and A.P Alivasatos, Epitaxial growth of highly luminescent
cdse/cds core/shell nanocrystals with photostability and electronic accessibility, J.M. Chem Soc.
119:7019 (1997).
9. K. Anandan, S. Gnanam, J. Gajendran and V. Rajendiran, “Effect of temperature on SiO2 nanoparticles-
facile solvothermal synthesis and characterization 49Poster 26, NCMNN (2010).
10. Cullity BD, Elements of X-ray Diffraction, Addison-Wesley, New York, 1978.
11. Parikh KD, Dave DJ, Parekh BB and Joshi MJ, Bull. Mater. Sci., Vol. 30, pp. 105–112, 2007.
12. Cullity BD (Ed.), Elements of X-ray diffraction, Addison-Wesly, New York, pp.102, 1997.
13. Willamson GK and Hall WH, Acta Metall. Vol. 1, pp. 22–31, 1953.
14. H.J. Snaith and L.S. Mende, Adv. Mater., 19, 3187 (2007).
15. Willamson GK and Hall WH, Acta Metall. Vol. 1, pp. 22–31, 1953.
16. JCPDS PDF-2 Pattern 88-1172 & TiO2 - SnO2 78-1063, 81-1387.
17. F.A. Deorsola and D. Valluri J Mater Sci 43:3274 3278 (2008).
132
Phytogenic Synthesis of Silver Nanoparticles and its
Potential Foron Blue Dye Decolorisation
P. Balashanmugam1*, P. Arthi 2 and P.T Kalaichelvan3
1
Centre for Advanced Studies in Botany, University of Madras, Chennai 600 025, India
2
Department of Inorganic Chemistry, University of Madras, Chennai 600 025, India
3
ALKA Research Foundation, Coimbatore, 641 046, India
* Corresponding Author e-mail: biobala17@gmail.com, 91+9994633730
Abstract:
The ability of the Silver nanoparticles (AgNPs) to decolorize dyes, with special emphasis on the textile dyes
has been investigated. It is a novel bio approach for the synthesis and stabilization of AgNPs using aqueous
extract of Cassia Roxburghii leaves under ambient conditions. The instant formation of AgNPs was analyzed
by visual observation and UV–visible spectrophotometer. The synthesized AgNPs were characterized by HR-
TEM with EDX. Appearance of dark brownish color confirmed the formation of AgNPs. Phytoconstituents
present in the plant extract might have helped in the reduction and capping agent of AgNPs. The EDX profile
showed the presence of silver metal in the synthesized AgNPs. HR-TEM studies exposed that the diameter of
stable AgNPs was approximately 40 nm. These particles were then checked for their efficiency to decolorize
the textile dye foron blue. The AgNPs are observed to be have a good catalytic activity on the reduction of
foron blue. The nanoparticles treated effluent was tested on seed germination and growth of green gram.
The treated dyes also improved seed germination and growth of green gram when compared to the untreated
dyes. This indicated the non- toxicity of the AgNPs decolorized dye effluent.
Keywords: Bioreduction, silver nanoparticles, foron blue, decolorisation, seed germination.
INTRODUCTION
Textile industries are the major users of dyes in the world. A huge portion of dyes are discharged out from
the textile industries, causing serious spoil to the environment [1]. Nanotechnology based technologies has
been proved to be the most attractive and cost- effective method to counter textile dye pollution. The ability
of the Silver nanoparticles (AgNPs) to decolorize the textile dyes. Nanoparticles (NPs), which are generally
considered as particles with a size of 100 nm, exhibit completely new improved properties as compared to
the larger particles of the bulk material that they are composed of based on specific characteristics such as
size, distribution and morphology [2]. Biological methods for NPs synthesis using fungi, microorganisms,
enzymes, and plants have been suggested as possible ecofriendly alternatives to chemical and physical
methods [3]. Now a days bioreduction methods based on fungi, micro organisms, plants are being attempted
due to the ease of synthesis, and greater stability of NPs. AgNPs have received attention due to the surface
plasmon resonance, which can be easily monitored by UV-visible spectrophotometer [4]. The applications
of AgNPs in the field of medicine, optoelectronics, optics, catalysis, sensors are well known. Plant extracts
have been shown to produce nanoparticles with good stability due to the presence of reducing agents like
alkaloids, polyphenols and flavonoids which are the major phytoconstituents [5]. In the present study, the
direct synthesis of AgNPs using aqueous extract of Cassia Roxburghii leaves by the reduction of Ag+ ion has
been demostrated. The formation of AgNPs was characterized using UV-visible spectrophotometer, XRD,
HR-TEM with EDX. Moreover, its catalytic activity on reduction of foron blue (FB) in the presence of
silver nanoparticles has also been experimented. The foron blue dye decolorized water was utilized for the
germination of green gram seeds.

Collection of plant and Preparation of the plant extract
All the chemicals were purchased from Sigma Aldrich, Germany, and were of analytical grade. Silver nitrate
was purchased from Hi-media Lab Pvt. Ltd Mumbai, India. The healthy leaves of C. Roxburghii were
collected from the herbal garden located at CAS in Botany, University of Madras, Guindy campus, Chennai.
The collected leaves were washed twice with tap water and then rinsed with distilled water to remove the
dust on their surface. Then the leaves were shade dried for 3 days at room temperature. The air dried leaves
were ground to coarse powder using a blender. 4 g of the dried and powdered leaves were mixed with 100 ml
of distilled. This mixture was boiled for 15 min at 55°C the mixture, cooled and filtered through Whatman
No. 1 filter paper. The boiled extracts was refrigerated and used for further experiments.
Synthesis of silver nanoparticles

In the typical synthesis of silver nanoparticles, the leaf extract of C. roxburghii had been screened for the
formation of AgNPs. One ml of the aqueous leaf extract of C. roxburghii was added to 9 ml of 1 mM (10-3
M) solution of silver nitrate in 250 ml test tube. The reaction was performed in dark at room temperature to
minimize photo activation of silver nitrate. The aqueous leaf extracts of C. roxburghii and AgNO3 solution
were used as control.
CHARACTERIZATION OF SILVER NANOPARTICLES

UV-Visible spectroscopy
The formation of dark brown colour during the synthesis was found to be a confirmation for the formation of
silver nanoparticles. The reduction of the pure Ag+ ions was monitored by measuring the UV-Vis spectrum
of the reaction medium after overnight incubation by continuous scanning from 300 nm to 700 nm using
HITACHI U-2900 UV-Visible spectrophotometer.
HR-TEM and EDX

High-resolution transmission electron microscopy (HR-TEM) samples of the aqueous suspension of AgNPs
were prepared by placing a drop of the suspension on carbon-coated copper grids and the films on the grids
were allowed to stand for 2 min, and the extra solution was removed using a blotting paper and the grid
was allowed to dry prior to measurement. HRTEM observations were performed on a FEI-TECNAI G2
T-30, S TWIN instrument. EDX analysis was conducted with the same instrument to confirm the elemental
composition of the sample.
Decolorisation of foron blue dye

For decolorisation study, 250 mL Erlenmeyer flasks containing 125 mL solutions of Foron blue dye
was prepared in the media containing 30 g sucrose, 3 g NaNO3, 0.5 g KCl, 0.5 g MgSO4.7H2O, 0.01g
FeSO4.7H2O, 1 g K2HPO4 and 15 g agar per liter was used final pH of the medium is 7.3 ± 0.2 at 35°C.
[6]. The AgNPs was added to the above medium which is indicated as test and the prepared medium without
adding AgNPs serves as control. Foron blue dye of 50 μM concentration was used in this study. The flasks
were incubated at 35°C under shaking conditions. After 24 hrs interval, samples were withdrawn, filtered
and centrifuged at 5000 rpm for 5 mins and the supernatants was analyzed spectrophotometrically using
UV-Visible spectrophotometer at 498 nm. The decolorisation efficiency was expressed as per the following
equation;
134
Decolorisation (%) = [(Initial Absorbance – Final Absorbance) / Initial Absorbance] × 100.
Seed germination studies (Plate trail: Green gram)

The seeds were surface sterilized in a 10% sodium hypochlorite solution for 10 min, followed by rinsing
thrice with distilled water. The experiment was done in disposable plastic petri plates. The top and the bottom
of the Petri plates were padded with a layer of tissue paper. Green gram seeds were soaked in 25 ml of
colored/untreated dye and 25 ml of decolorized/treated dye for 8 hrs. After soaking seeds were placed in each
petri plates and the tissue paper was soaked with their respective dye mixtures. The plates were incubated
for 3 days. The tissue paper was kept moist by spraying water. After 3 days the germination percentage was
calculated using the following formula:
No. of seeds germinated
Germination index (GI ) = × 100
Total no of seeds sown

Synthesis of AgNPs and UV-Visible Spectrum
The study on extracellular green synthesis of silver nanoparticles through plant extracts were carried out. It is
well known that silver nanoparticles exhibit yellowish to dark brown color (fig 1) in aqueous solution due to
excitation of surface Plasmon vibrations in silver nanoparticles. [7] This important observation indicates the
reduction of the Ag+ ions which takes place extracellularly. The appearance of a dark brown color in solution
containing the C. roxburghii aqueous extract is a clear indication of the formation of silver nanoparticles in
the reaction mixture.

Figure 1: C-without silver nitrate (Control) Figure 2: UV-Vis Spectrum of AgNPs
T.-AgNO3 (1 mM) Reduction after 24 hrs (Test)
The silver nanoparticles were characterized by UV-Vis spectroscopy, one of the most extensively
used techniques for the structural characterization of silver nanoparticles [8]. Absorption spectra of silver
nanoparticles formed in the reaction solution has absorbance peak at 430 nm (Fig.2) after 24 hrs. The
broadening of peak indicated that the particles are mono dispersed and spherical in morphology.
HR-TEM and EDX Studies

Figure 3a represents the High-resolution transmission electron microscopy (HR-TEM) image recorded
from the drop coated films of silver nanoparticles synthesized with C. roxburghii leaves extract. The HR-
TEM image showed spherical and relatively uniform shape of nanoparticle formation with diameter range
135
30–40 nm and analysis through Energy dispersive X-ray (EDX) confirmed the presence of elemental silver
signals of the silver nanoparticles [9] (Fig.3b). The vertical axis displays the number of X-ray counts whilst
the horizontal axis displays energy in KeV. [10]
Figure 3a: HR-TEM Figure 3b: EDX
Decolorisation of foron blue

A significant decolorisation rate was observed for the foron blue dye. The AgNPs effectively decolorized
76% of the dye in 18 hrs incubation and the dye was fully decolorized after 24 hour incubation (Fig 4a).
Whereas the plant extract was able to degrade only 48% of the dye under the same incubation conditions and
complete decolorisation was observed after 48 hrs incubation. (Fig 4b)
Figure 4a: Decolonisation of Foron blue dye Figure 4b: % of decolorisation of the Foron blue dye after 24
hrs incubation
It can be seen from a previously report that malachite green was decolorised by Acremonium kiliense [11].
According to them 95.4% MG was decolorized within 72 hrs likewise among the azo dyes, the percentage
of decolorisation for foron blue dye was not as high as the other two azo dyes, but a 76% decolorisation
after 12 hrs was achieved for this dye [12]. In this study when compared with nanoparticle the plant extract
could degrade only 48% of the dye this might me due to the complex nature of the dye. A slower rate of
decolorisation was attributed to higher molecular weight, structural complexicity of the dyes [13].
Seed germination study (Plate trail)

The effect of untreated dyes and treated dyes on the germination of Green gram is shown in (Fig 5a and
5b) respectively. The Green gram showed good germination in the presence of treated dyes as compared to
untreated dyes. For instance, the treated Foron blue dye in Green gram seeds showed 60% germination while
the untreated Foron blue dye showed only 20% germination [14] (Fig 5c). From this experiment a significant
difference was observed during germination of seeds under treatment conditions [15].
136
Figure 5: Seed germination for green gram Figure 5c: Seed germination for green gram
TFb- Treated Foron blue dye 60% growth germination UFb- untreated Foron blue dye 20 % growth germination
CONCLUSION
A rapid green eco-friendly method to synthesize AgNPs using C. roxburghii aqueous extract has been
developed. The plant extract showed potential for extracellular synthesis of fairly monodispersed, spherical
AgNPs in the range of 30–40 nm. Phytoconstituents serve both as reducing and capping agents. This
study exposed the ability of the AgNPs to decolorize foron blue. From the results we conclude that the
NPs exhibited the ability to decolorize foron blue. better than the plant extract. These preliminary results
suggest that AgNPs can be used for treatment of textile effluents. The development of such particles may
be considered a breakthrough in the field for the efficient clean up of the dyes. This method is a promising
alternative to the traditional reduction routes to avoid usage of toxic chemicals for decolorisation
treatments.
ACKNOWLEDGEMENT
The authors wish to thank The Director, CAS in Botany, University of Madras, Chennai, for providing
the laboratory facilities .We also thanks to NCNSNT, University of Madras, for providing characterization
studies and UGC herbal science scheme for financial support.
REFERENCE
1. Van der Zee fp. 2002. Anaerobic azo dye reduction. [Ph D Dissertation] retrieved March 2007 from
http://www.docstoc.com/docs/8494166/THESIS-Anaerobicazo-dye–reduction.
2. Nithya R and Ragunathan R, Synthesis of silver nanoparticles using Pleurotus sajor caju and its
antimicrobial study, Digest journal of nanomaterials and Biostructures, 2009; 4 (4): 623–629.
3. Tratnyek PG and Johnson RL, Nanotechnologies for environmental cleanup, Nanotoday, 2006;
1(2): 44–48.
4. Willems, van den Wildenberg. Roadmap report on nanoparticles, Barcelona, Spain: W&W Espana
sl. 2005.
5. Jahn and W.J. Struct. Biol, 1999, 127, 106.
6. Hazrat Ali, Wesal Ahmad and Taqweemul Haq, Decolorization and degradation of malachite green by
Aspergillus flavus and Alternaria solani, African j.Biotechnol, 2009; 8 : 1574–1576.
137
7. Thirumurugan A, Jiflin GA, Rajagomathi G, Tomy NA, Ramachandran S and Jaiganesh, Biotechnological
synthesis of gold Nanoparticle of Azadirachta indica leaf extract, International Journal of Biological
Technology, 2010; 1: 75–77.
8. Seyedeh Masumeh Ghaseminezhad, Sepideh Hamedi and Seyed Abbas Shojaosadati, Green synthesis of
silver nanoparticles by a novel method: Comparative study of their properties, Carbohydrate Polymers,
2012; 89: 467–472.
9. Harekrishna Bar, Dipak Kr, Bhui, Gobinda P, Sahoo, Priyanka Sarkar, Sankar P De and Ajay Misra, Green
synthesis of silver nanoparticles using latex of Jatropha curcas, Colloids and Surfaces A: Physicochem.
Eng. Aspects, 2009; 339: 134–139.
10. Marimuthu Vivek, Palanisamy Senthil Kumar, Sesurajan Stusffi and Sellappa Sudha, Biogenic Silver
Nanoparticles by Gelidiella acerosa extract and their Antifungal Effects, Avicenna Journal of Medical
Biotechnology, 2011; 3(3): 143–148.
11. Youssef AS, Sherif MF and Assar SA, Studies on the decolorisation of malachite green by the local
isolate Acremonium kiliense, Biotechnology, 2008; 7: 213–223.
12. Avneesh D Singh, Vikineswary Sabaratnam, Noorlidah Abdullah, Annuar MSM and Ramachandran
KB, Decolourisation of chemically different dyes by enzymes from spent compost of Pleurotus sajor-
caju and their kinetics, Afr.J.Biotechnol, 2010; 9: 041–054.
13. Hu Tai-lee and Wu SC, Assessment of the effect of azo dye Rp2B on the growth of nitrogen fixing
cyanobacterium- Anabena sp. Biores Technol, 2001; 77: 3–95.
14. Vijaykumar Naranbhai Vora, Ramesh Kadvabhai Kothar and Charmy Ramesh Kothari, Decolorization
of Textile Dyes and its Effects on Seeds Germination, The FASEB Journal, 2007; 21: lb68.
15. Initha C, Lebno Ecy, Rajn S, Murgesan AG, Rajesh and Elayrjh B, Biodegration of textile azo
dyes and its bioremdiaton potential using seed germination efficiency, Int.JCurMicobl.ApSci, 2013;
(2) 10: 496-50.
138
Aristolochia Indica L. Mediated Synthesis of Nano-Silver
Particles for its Antimicrobial Activity against Human
Pathogens
G. Sathishkumar, C. Rajkuberan, K. Ravindran and S. Sivaramakrishnan
Department of Biotechnology and Genetic Engineering, School of life Sciences, Bharathidasan University,
Tiruchirappalli, Tamilnadu, India-620024
e-mail: Sivaramakrishnan123@yahoo.com, Mobile: +91-9894269100
Abstract:
Nano fabrications from biological entities have a widespread application and emerging as dynamic area
in material science. In this study silver nanoparticles (AIAgNPs) was successfully synthesised using the
leaf extract of Aristolochia indica L. (Aristlochiceae) for their potential applications put in antimicrobial
therapy. Synthesized AIAgNPs were confirmed through formation of yellowish brown colour due to the
excitation of surface plasmonic vibrations (SPR). In UV-Visible spectroscopy it produces an absorbance
spectrum at 420 nm. Morphometric features of synthesized AIAgNPs were studied with Field emission
Scanning electron microscopy (FESEM) and High resolution transmission electron microscope HRTEM.
It shows spherical AgNPs with the size range between 8-41nm with the mean size of 24 ± 9 nm. XRD
diffractogram planes confirms that the synthesized AgNPs were face cubic crystalline (Fcc) in nature.
EDAX analysis authorizes the presence of elemental metal signal for silver and FTIR results give an
idea about the possible biomolecule responsible for reduction of AgNO3. In addition, synthesized AgNPs
and aqueous leaf extract were screened for its antimicrobial activity against clinically isolated human
pathogens towards biomedical applications.
Keywords: Aristlochia indica L. Silver nanoparticles, Human Pathogens, HRTEM, Biosynthesis
INTRODUCTION
Nanotechnology encompasses fabrication, characterization and manipulation of structures into functional
values to improve the quality of environment and their associated life forms. In general fabricated nano
scale values with dimension smaller than 100nm have wide applications such as catalysis, electronics
and biomedical devices compared to macro scale counterparts. Fabrication of noble metal nanoparticles
with improved physio-chemical properties like stability, surface chemistry and morphology will provide
hybrid materials for various biotechnological applications [1-2]. Among diverse nanoparticles, silver
nanoparticles were known to possess stupendous antimicrobial and catalytic properties [3].Silver
nanoproducts (AgNPs) with the size range of <100 nm have been used in many household and industrial
applications, such as in tires, textiles, cosmetics, food packaging and processing, medical applications in
wound care products, therapeutic devices, diagnostics and drug delivery [4]. Biosynthesis and assembly
of nanoparticles is significant because of its clean, nontoxic and environmentally acceptable green
chemistry principles. Organisms ranging from bacteria to fungi and even plants have their potential to
generate nano scale particles. Hence, both unicellular and multicellular organisms are known to produce
inorganic materials either intra-or extracellular [5]. In particular, plant based synthesis of nanoparticles
received more attention owing to their single-step, facile and easy scaling up process [6-7]. India has
rich biodiversity in vegetation several plant species still to explore for their bioactive components, the
biomolecules like proteins, phenols and flavonoids play a vital role in the reduction of Ag+ ions [8]. There
are several medicinally valuable plant spices has been already explored for nanoparticle synthesis such
as Azardirachta indica [7], Ocimum sanctum [8], Acalypha indica [9], Zingiber officinale [10], Syzygium
Cumini [11], Macrotyloma Uniflorum [12], Desmodium triflorum [13], Morinda citrifolia [14], Curcuma
longa [15] and Couroupita guianensis [16].
Aristlochia indica L. owning phytochemicals such as aristolochic acid, terphenoids is known to medicinal
value traditionally used for the treatment of cholera, fever, bowel troubles, ulcers, leprosy, skin diseases,
menstrual problems and snake bites [17]. Ethanolic extract of A.indicia L. shows potent inhibitory action
against human pathogenic fungi and bacteria such as Aspergillus niger, A. fumigatus and P.aerginosa [18].
Therefore, the present study was aimed to fabricate silver nanoparticles through green chemistry route using
the leaf extract of A. indica. On the other hand synthesized silver nanoparticles were tested against clinical
isolates of bacterial human pathogens in view to develop a nano-drug formulation for various pathogenic
diseases.
Materials and Methods

Materials
Fresh and healthy leaves of A. indica L. were collected from in and around Bharathidasan university campus,
Tiruchirappali, Tamilnadu, India and transferred to laboratory for further studies. Silver nitrate (AgNO3) and
other chemicals were purchased from Himedia, Mumbai, India.
Preparation of leaf extract

Collected leaf samples were washed using running tap water followed by distilled water thrice to remove
contaminants. Further, they were shade dried for 5-10 days at the environmental temperatures (27-37°C
day time). The dried leaves were finely powdered mechanically using commercial electrical stainless steel
blender. For synthesis of AIAgNPs aqueous leaf extract was prepared by simple decoction method. Briefly,
20 g of dried leaf powder was mixed with 200 ml of distilled water and kept in boiling water bath at 60oC for
10 min. After cooling, the extract was filtered through Whatman filter paper No. 1 and stored in refrigerator
at 4oC for further studies.
Synthesis of AIAgNPs
For the synthesis of AIAgNPs the reaction mixture and physiological conditions such as temperature, pH
and time were fixed as per our earlier study [7 and 16]. Briefly, the reaction mixture was prepared by adding
5ml of the plant crude extracts to 95 ml of 1mM AgNo3 solution in a 250ml Erlenmeyer flask and pH of the
reaction medium was adjusted to 7 using 10% sodium hydroxide. The reaction mixture was kept in boiling
water bath at 90°C, a control set up was also maintained without addition of plant extract. Reduction of
was observed by change in their colour of the reaction mixture, it was monitored periodically at 10 minutes
interval by UV-Visible spectroscopy.
Characterization of AIAgNPs
UV-Visible spectrophotometer
Excitation of Surface Plasmonic vibrations due to the reduction of Ag+ ions were measured
spectrophotometrically at different time intervals. For that small aliquot of the samples were diluted with
distilled water and absorption maxima was scanned by UV– visible spectrophotometer, at the wavelength of
300 – 700 nm using JASCO V-650 UV-Visible Spectrophotometer.
140
FTIR and XRD analysis

FTIR spectroscopic studies were carried out to find possible bio-reducing agent present in the plant extract.
The wavelength spectrum of the leaf extract before and after the addition of AgNO3,the sample was mixed
with KBr powder and pelletized after drying the spectra were recorded using Perkin Elmer make model
spectrum RX1 (Wavelength range between 4000 cm-1 to 400 cm-1). X-ray diffraction was performed for
determination of the dimension of biologically synthesized AgNPs with h, k, l values. The diffraction
pattern was obtained with conditions at 40 kV and 30 mA in Cu, Ka radiation and particles size (L) using
(PAN analytical X pert PRO Model) of the Ag and Au was calculated using following Debye-Scherrrer’s
equation.
L = 0.9λ/β cos h θ
Where, λ is the wavelength of the X-ray, β is full width and half maximum and θ is the Bragg’s angle.
FESEM and HRTEM measurements

The reaction mixture was subjected to repeated ultracentrifugation at 28, 000 rpm for 20 min, resulting
pellet was mixed with deionised water and used for FESEM, HRTEM, and EDAX analysis. The purified
sample was freeze-dried and a thin film was prepared for FESEM (ZESIS) analysis, for HRTEM analysis
sample was coated on a copper grid and exposed to JEOL JEM 2100 high resolution transmission
electron microscope.
Screening of antimicrobial activity

The antimicrobial effect of synthesized AgNPs and crude plant extract were evaluated against human
pathogens such as Enterococcus sp. P.aeruginosa, B.cereus, E. coli, E.aerogens and S.aureus by disc
diffusion method. Cultures were maintained at −80oC on glycerol stock. Clinically isolated strains were
sub-cultured in nutrient broth for 24 h at 30oC. Each strain was swabbed uniformly into the individual
nutrient agar plates using sterile cotton swabs. Using sterile micropipette leaf extract (25 µl and 50
µl), crude AgNPs (25 µl) and purified AgNPs (25 µl) were loaded onto a sterile paper disc and it was
allowed to dry. The sample loaded discs along with standard antibiotic disc (kanamycin 100 mg/ml) were
impregnated in the nutrient agar medium. The doses were selected based on the preliminary data obtained
from the earlier studies in our laboratory. After 24 h incubation at 37oC, the different levels of zone of
inhibition were measured.
Results and discussion

In nature living organisms have been known to accumulate metals in their system that makes an intention
towards the employment of biological origins for eco-friendly synthesis of metallic nanoparticles. Plant as
autotroph is the renewable energy resource present in the earth, exploitation of plant resource for the nano
particle synthesis will be a sustainable green chemistry approach. Apart from its eco-friendly strategy, plants
own metabolic status which provides them strength to withstand such environmentally diverse habitats.
These plant products have enormous significance for the surviving humanity in various manners such as
pharmaceuticals and agriculture [19]. Plant phenolic compounds are well known because of its free radical
scavenging, antimicrobial and cytotoxic potential [20].
Synthesis of AIAgNPs
After addition of A.indica leaf extract into the reaction medium, it shows variation in the colour due to
the excitation of Surface Plasmon Resonance (SPR). Synthesized AIAgNPs exhibits yellowish brown
colour whereas control silver nitrate solution remain transparent. Hence this result agrees with the result
of Krishnaraj et al. 2010 [11] where the clear AgNO3 solution turned into yellowish brown colour within
30 min after the addition of Acalypha indica leaf extract. Intensity of colour increased in direct proportion
141
to the incubation period, it may be due to the excitation of SPR effect and reduction of metal ions. Efficient
synthesis of nanoparticles was observed at ambient conditions, Fig. 1a shows the intense SPR peak at
420 nm for AgNPs. Synthesis of AgNPs was improved with increasing the reaction time duration
from intensity of the SPR increases as the incubation time gets increased [16]. Intensity of the SPR
correlates with the yield of nanoparticles the maximum absorbance corresponds to the high yield of
nanoparticles.
Figure 1:Visible colour change and UV-Visible spectroscopy analysis of synthesized AIAgNPs displays intense
absorbance spectra at 420 nm
Characterization of AIAgNPs
Size and morphology of nanoparticles were characterized with microscopic studies (FESEM and
HRTEM), topographical image shows uneven and agglomerated spherical AgNPs with Size below 100 nm
(Fig. 2a). The topographical image of irregular nanoparticles is mainly due to agglomeration [21].
EDAX analysis indicates a strong metal signal for silver, the presence of copper signal because of the
grid used for the HRTEM analysis. Interestingly HRTEM micrograph displays the fine configuration of
spherical AgNPs with the size range of 8-41nm (Fig. 3a) with the mean of 24±9 nm. It was also found that
synthesized AIAgNPs having a thin layer of biomolecule on its surface, particles were polydispersed and
stable for long period of time. Individual particles were noticed in HRTEM micrograph, it was also found
that particles were not involved in direct contact because of the presence of biomolecule as stabilizing
agent [22]. XRD analysis shows the diffraction planes at (111), (200), (222) and (220) which corresponds
to face centered (Fcc) cubic crystalline silver (Fig.3b). This result authorize that synthesized AIAgNPs
were amorphous crystalline nature [18]. The average size of the particle was found to be 25nm. FTIR
analysis was performed to ascertain the involvement of possible biomolecule responsible for the reduction
and stabilization of nanoparticles. The IR spectrum of AgNPs manifests prominent transmittance located
at 3434, 2361,2075,1637,1324 and 677 cm−1. The strong bands at 1637 cm−1 corresponds to the –C=C–
stretches and broad peak at 3434cm−1 indicates the –O-H– stretches. The prominent band at 1324 in
AIAgNPs and leaf extract respectively may be attributed to –C–O stretching mode (Fig. 4). This results
highly corroborate with the results of [12 and 16] where the vibrational stretches clearly indicates that
water soluble phenolic compounds such as tannins and other polyphenols present in the extract influence
the reduction and stabilization of nanoparticles
142
Figure 2: a. FESEM micrograph of AIAgNPs b. EDAX analysis shows strong metal peak for silver
Figure 3: a. HRTEM micrograph at different magnification shows spherical AIAgNPs with the size range between b.
XRD diffractogram shows the planes for Face centred cubic crystalline AgNPs
143
Figure 4: Antimicrobial activity of synthesized AIAgNPs by disc diffusion method
Screening of antimicrobial activity

Bactericidal effect of synthesized nanoparticles (Zone of inhibition) were measured and compared with
standard antibiotic drug (Fig. 5). Aqueous leaf extract does not show any inhibitory action even at 50µl
quantity whereas synthesized particles as crude organosol and in purified exhibits efficient inhibitory
action against all the pathogens (Table. 1). The results of [23] explains that the size and morphology
of the nanoparticles significantly influence the bactericidal effect, smaller particles having the large
surface area available for interaction would give more bactericidal effect than the larger particles.
The mechanism of the bactericidal effect of silver colloid particles against bacteria is not very well
known. [24] demonstrated the possible bactericidal mechanism of AgNPs, it summarize that AgNPs
act similarly to the antimicrobial agents used for the treatment of bacterial infections. (1) Interference
with cell wall synthesis, (2) Inhibition of protein synthesis, (3) Interference with nucleic acid synthesis,
and (4) Inhibition of a metabolic pathway. Apart from this there are few more studies have been
reported on the bactericidal mechanism of AgNPs in particular, suggesting three possible mechanism of
bactericidal action. [25] emphasized AgNPs release silver ions, which make an additional contribution
to the bactericidal effect. In addition with that [26] disclosed that AgNPs will attach to the surface of
the cell membrane and disturb its power functions, such as permeability and respiration. Plasmolysis
(cytoplasm separated from bacterial cell wall) in P. aeruginosa and the inhibition of bacterial cell
wall synthesis in S. aureus bacteria were reported. Followed by [27] addressed that AgNPs can able to
penetrate the bacteria and cause further damage, possibly by interacting with sulfur- and phosphorus-
containing compounds such as DNA.
144
Table 1: Inhibitory action of silver nanoparticles synthesized using the leaf extract of A.indica against human
pathogenic bacteria
Clinical isolates of Zone of inhibition(mm)

Human Pathogen Standard antibiotic Purified AgNPs Crude AgNPs Leaf extract 25 µL Leaf extract 50 µl
(100mg/ml)
Enterococcus sp. 18 11 11 - -
P.aeruginosa 20 8 8 - -
B.cereus 19 7 9 - -
E. coli 18 11 11 - -
E. aerogenes 18 7 7 - -
S.aureus 14 8 8 - -
Figure 5: A-Aqueous leaf extracts 25µl and 50µl respectively, C-purified AgNPs, D-crude AgNPs E-Standard drug
(kanamycin)
145
CONCLUSION
Bio-fabricated silver nanoparticles using the leaf extract of A.indica showed potent antimicrobial activity
against human pathogens, it was well demonstrated with the inhibition of bacterial growth. Hence, the bio-
fabrication of AgNPs appears to be cost efficient and alternative to conventional physical and chemical
methods of silver nanoparticles synthesis. These noble metal nanoparticles provide an efficient means to
deliver drugs distribute in a targeted and controlled fashion with high site specificity to combat various
deadliest diseases.
REFERENCES
1. Moaveni P, Karimi K and Valojerdi ZM, The Nanoparticles in plants: Review Paper, Journal of
Nanostructure in Chemistry 2011; 2 (1):59–78.
2. Remya Nair, Varghese SH, Baiju Nair G, Maekawa T Yoshida Y and Sakthi Kumar D, Nanoparticulate
material delivery to plants, Plant Science 2010; 179:154–163.
3. Vilas V, Philip D and Mathew J, Catalytically and biologically active silver nanoparticles
synthesized using essential oil, Spectrochim Acta A Mol Biomol Spectrosc, 2014; 132
(11):743–50.
4. Khan SS, Srivatsan P, Vaishnavi N, Mukherjee A and Chandrasekaran N, Interaction of silver nanoparticles
(SNPs) with bacterial extracellular proteins (ECPs) and its adsorption isotherms and kinetics, J. Hazard,
Mater, 2011; 192: 299– 306.
5. Mohanpuria P, Rana NK and Yadav SK, Biosynthesis of nanoparticles: technological concepts and
future applications, J. Nanopart, Res. 2008; 10: 507–517.
6. Bar H, Bhui DK, Gobinda PS, Sarkar P, Pyne S and Misra A, Green synthesis of silver nanoparticles
using seed extract of Jatropha curcas, Colloids and Surfaces A: Physicochem. Eng. Aspects, 2009;
348:212–216.
7. Sathishkumar G, Gobinath C, Wilson A and Sivaramakrishnan S, Dendrophthoe falcata (L.f) Ettingsh
(Neem mistletoe): A potent bioresource to fabricate silver nanoparticles for anticancer effect against
human breast cancer cells (MCF-7), Spectrochim Acta A Mol Biomol Spectrosc, 2014; 128 (15):
285–290.
8. Narayanan KB and Sakthivel N, Green synthesis of biogenic metal nanoparticles by terrestrial and
aquatic phototrophic and heterotrophic eukaryotes and biocompatible agents, Advances in Colloid and
Interface Science, 2011; 169: 59–79.
9. Shankar SS, Rai A, Ahmad A and Sastry M, Rapid synthesis of Au, Ag, and bimetallic Au core-Ag
shell nanoparticles using Neem (Azadirachta indica) leaf broth, J. Colloid, Interface, Sci. 2004; 275:
496–502.
10. Singhal G, Bhavesh R, Kasariya K, Sharma RA and Singh. R.P., 2011. Biosynthesis of silver nanoparticles
using Ocimum sanctum (Tulsi) leaf extract and screening its antimicrobial activity, J. Nanopart, Res. 13,
2981–2988.
146
11. Krishnaraj C, Jagan EG, Rajasekar S, Selvakumar P, Kalaichelvan PT and Mohan N, Synthesis of
silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against water borne
pathogens, Colloids Surf, B: Biointerfaces, 2010; 76:50–56.
12. Kumar KP, Paul W and Sharma CP, Green synthesis of gold nanoparticles with Zingiber
officinale extract: Characterization and blood compatibility, Process Biochem, 2011; 46:
2007–2013.
13. Kumar V, Yadav SC and Yadav SK, Syzygium cumini leaf and seed extract mediated biosynthesis
of silver nanoparticles and their characterization, J. Chem, Technol. Biotechnol. 2010; 85:
1301–1309.
14. Vidhu VK, Aromal SA and Philip D, Green synthesis of silver nanoparticles using Macrotyloma
uniflorum. Spectrochim, Acta A, 2011; 83:392– 397.
15. Ahmad N, Sharma S, Singh VN, Shamsi SF, Fatma A and Mehta BR, Biosynthesis of silver
nanoparticles from Desmodium trifolium: A novel approach towards weed utilization, Biotechnol. Res.
Int. 2011; 1–8.
16. Sathishkumar G, Gobinath C, Karpagam K, Hemamalini V, Premkumar K and Sivaramakrishnan S,
Phyto-synthesis of silver nanoscale particles using Morinda citrifolia L. and its inhibitory activity
against human pathogens, Colloids Surf. B:Biointerfaces 2012; 95:235–240.
17. Sathishkumar M, Sneha K and Yun YS, Immobilization of silver nanoparticles synthesized using
Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity, Bioresource Technol.
2010; 101:7958–7965.
18. Vimala RT, Sathishkumar G and Sivaramakrishnan S, Optimization of reaction conditions to fabricate
nano-silver using Couroupita guianensis Aubl. (leaf & fruit) and its enhanced larvicidal effect,
Spectrochim Acta A Mol Biomol Spectrosc, 2015; 135:110-115.
19. Abhijit dey and Jitendranath de, Aristlochia indica L: a review. Asian journal of plant sciences. 2011;
10:108-116.
20. Surendrakumar M, Rajeswari and Astalakshmi N, Evaluation of antimicrobial activities of Aristolochia
indica (linn), International Journal of Pharmacy and Pharmaceutical Sciences. 2011; 3: 201.
21. Jeyaraj M, Sathishkumar G, Sivanandhan G, Mubarak Ali D, Rajesh M, Arun R, Kapildev G,
Manickavasagam M, Thajuddin N, Premkumar K and Ganapathi A, Biogenic silver nanoparticles
for cancer treatment: an experimental report, Colloids Surf B Biointerfaces, 2013; 106:86-92.
22. Rajakumar G and Rahuman AA, Larvicidal activity of synthesized silver nanoparticles using
Eclipta prostrata leaf extract against filariasis and malaria vectors, Acta Trop. 2011; 118:196–203.
23. Sharma, VK, Yngard RA, Lin Y. Silver nanoparticles: green synthesis and their antimicrobial activities,
Adv. Colloid. Interface. Sci. 2009; 145:83–96.
24. Guzman M, Dille J and Godet S, Synthesis and antibacterial activity of silver nanoparticles
against 3 gram-positive and gram-negative bacteria, Nanomedicine: Nanotechnol. Biol. Med. 2012;
8:37–45.
147
25. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN and Kim JO, A mechanistic study of the antibacterial
effect of silver ions on Escherichia coli and Staphylococcus aureus, J. Biomed, Mater. Res. 2000;
52:662–668.
26. Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri BJ, Ramrez, JT and Yacaman, MJ, The
bactericidal effect of silver nanoparticles, Nanotechnology 2005; 16:2346–2353.
27. Song HY, Ko KK, Oh IH and Lee BT, Fabrication of silver nanoparticles and their antimicrobial
mechanisms, Eur. Cells Mater. 2006; 11:58.
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Pharmacokinetics of Enrofloxacin Loaded Solid
Lipid Nanoparticles following Oral Administration
in Emu (Dromaius Novaehollandiae) Birds
P. Senthil Kumar1*, A. Arivuchelvan2, A. Jagadeeswaran2, N. Subramanian3,

C. Senthil Kumar4 and P. Mekala2
Department of Veterinary Pharmacology and Toxicology,

Veterinary College and Research Institute, Namakkal, Tamil Nadu
1
Veterinary College and Research Institute, Orathanadu-614625, Tamil Nadu, India.
2
Veterinary College and Research Institute, Namakkal, Tamil Nadu, India.
3
Department of Pharmaceutical Technology, Anna University, BIT campus, Trichy
4
Ph.D. Scholar, Dept. of Pharmaceutical Technology, Anna University, BIT campus, Trichy
*Corresponding author e-mail: p.senthilkumar@tanuvas.org.in , Phone: +91 94431 42359
Abstract:
The study was conducted to formulate the enrofloxacin SLNs and evaluate its pharmacokinetic (PK) behaviour
in emus. Enrofloxacin SLNs were prepared by a hot homogenization coupled with ultrasonication method
and characterized for further investigation in emus. PK of native enrofloxacin was studied after i.v. and
oral bolus administration at 10mg/kg in emus and it was compared with disposition kinetics of enrofloxacin
SLNs. Enrofloxacin and its metabolite ciprofloxacin in plasma were estimated using HPLC and PKs were
calculated by a noncompartmental analysis. The results demonstrated that the particle size, polydispersity
index, zeta potential, encapsulation efficiency and loading capacity of the SLNs were 154.72± 6.11nm,
0.42±0.11,-28.83±0.60mV, 59.66±3.22% and 6.13±0.32%, respectively. AFM image showed spherical to
circular particles with well defined periphery. Pharmacokinetic results showed that the t1/2β, AUC0-∞, Vdarea/F,
MRT and F were 3.107, 1.894, 1.594, 2.993 and 1.895 times enhanced, while CLB and β were significantly
decreased by 1.958 and 3.056 times compared to the values of native enrofloxacin. The t1/2β and MRT of the
metabolite were longer than those of parent substance. Based on the PK/PD indices, the SLNs extended the
enrofloxacin concentration upto 48 h.
Keywords: Enrofloxacin, Pharmacokinetics, SLNs, Tripalmitin, PK/PD integration
INTRODUCTION
Enrofloxacin is a fluroquionolone antimicrobial agent developed solely for use in animals. It has potent
bactericidal activity against a range of clinically relevant Gram negative and Gram positive pathogens as
well as Mycoplasma and Chlamydiae. The relative safety of enrofloxacin, its low minimum inhibitory
concentrations, broad spectrum of activity, long post-antibiotic effect and good tolerance has encouraged
their use in veterinary medicine (Scheer 1987). Despite the therapeutic potential of enrofloxacin, the very
poor aqueous solubility of enrofloxacin leads to difficulty in designs of pharmaceutical formulation and
variations in bioavailability (Martinez et al., 2006)
Emu (Dromaius novaehollandiae) belongs to ratite group. Bacterial infections are important causes of
morbidity and mortality in domestic emu birds (Sales, 2007). Restraining is not easy and it causes stress in
emu birds. Hence, enrofloxacin with sustained release profile is highly convenient to utilize in emu birds. But,
all the oral enrofloxacin formulations are available as conventional, immediate-release form that necessitates
administration twice daily or daily for several days or weeks.
The solid lipid nanoparticles (SLNs) introduced in 1991, are submicron-sized (50 to 1000 nm) carriers
composed of a lipid matrix stabilized by a surfactant. SLNs possess good tolerability, stability, scaling up
feasibility and the ability to incorporate hydrophilic/hydrophobic drugs (Muller et. al., 2000. Further, SLNs
formulation has the ability to prolong or sustain the release profile of the loaded molecules and hence reduce
need for the repeated administration and increase the therapeutic value of the treatment (Xie et al., 2011a).
Hence, the present research was premeditated with the objectives of preparation and characterization
enrofloxacin SLNs; determination of pharmacokinetics of enrofloxacin SLNs following oral bolus
administration in domestic emu birds. Based on these findings, recommendations on the dosage of
enrofloxacin SLNs are made.

Preparation of enrofloxacin SLNs
Enrofloxacin SLNs were prepared by hot homogenization followed by ultrasonication method. Enrofloxacin,
tripalmitin, span 80 were added at the ratio of 1:5:20 to get organic phase of preparation. The lipid content in the
organic phase was melted by heating at 70°C using magnetic stirrer with hot plate. The contents in the organic
phase were mixed properly by placing in the shaker (Spinix). An aqueous phase was prepared by dissolving
tween 80 and polyvinyl alcohol at the ratio of 20:20 by heating to the same temperature as the organic phase.
The hot aqueous phase was added to the organic phase under magnetic stirring (Remi, Mumbai, India) at 1000
rpm to form pre-emulsion. The hot pre-emulsion was then homogenised at 10,000 psi for 3min using the high
pressure homogenizer (Heidolph Electro, Germany) kept in a water bath maintained at 70°C.
The hot emulsion so obtained was ultrasonicated (Sonics Vibra Cell, USA) using high-intensity (5/64’’ 2
mm tip diameter) microprobe with amplitude 20% for 15 min to form nanoemulsion. Then, the nanoemulsion
was run under magnetic stirring at 1000 rpm for 4 h to obtain enrofloxacin loaded tripalmitin SLNs. All the
batches were prepared in triplicate and the average size was measured.
Characterization of enrofloxacin SLNs

Particle size, polydispersity index (PDI) and zeta potential of enrofloxacin SLNs were measured by Photon
Correlation Spectroscopy (PCS) using zetasizer nanoZS with the Malvern PCS software version 6.20.
Surface morphology and shape of the enrofloxacin SLNs were examined using Atomic force microscopy
(PARK XE-100). The loading capacity and encapsulation efficiency of enrofloxacin SLNs were calculated
as Xie et al. (2011a)
Pharmacokinetic study
Experimental design
The study was conducted in apparently healthy 8 emu birds (4 male + 4 female) aged 18 to 24 months with
a mean (±SE) body weight of 38.20 ± 1.03 kg. The use of the birds and experimental design was approved
by Institutional Animal Ethics Committee (IAEC), TANUVAS, Chennai.
Administration of drugs and collection of blood samples

Trial I: Native enrofloxacin was administered i.v. (bolus dose) to the emu through the jugular vein. Two
millilitre blood samples were drawn by jugular venipuncture into heparinized tubes immediately before and
at 0.083, 0.167, 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, 8, 12, 18, 24 and 36 hrs after dosing.
Trial II: After 2 weeks wash out period, the same birds were administered orally with the same dose of
native enrofloxacin directly using a thin plastic tube attached to a syringe. Then, 2ml of blood samples were
drawn as same method at 0.25, 0.50, 0.75, 1.5, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48 and 60 hrs after dosing.
150
Trail III: With 2 weeks wash out period, the same birds were administered orally with enrofloxacin SLNs
directly using a thin plastic tube attached to a syringe. Blood samples (2 mL) were drawn by jugular
venipuncture into heparinized tubes at 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48, 60, 72, 84
and 96 hrs after dosing. The birds were checked for observable signs of toxicity for up to 7 days after
administration of enrofloxacin SLNs.
The collected blood samples were centrifuged at 950 xg for 20 min to separate the plasma. The plasma
samples were stored at -40°C until assay.
Drug assay
Determination of enrofloxacin and ciprofloxacin was performed by high performance liquid chromatography
(HPLC). The method developed by Kung et al., (1993) was followed.
The HPLC system comprised of LC-20 AD double plunger pump, Rheodyne manual loop injector with
a 20 μL loop, column oven CTO-10 AS vp, SPD-M20A diode array detector and a software LC Solution
for data analysis. The compound separation was achieved using a reverse phase C18 column (Hibar 250-4,
6 RP-18 endcapped, Particle size 5μm, 4.6x250 mm, Merck, Darmstadt, Germany) as a stationary phase.
The column was protected with 2 to 8 mm Phenomenax guard column (KJO-4282). The mobile phase was
consisted a mixture of acetonitrile, methanol and water (containing 0.4% phosphoric acid (85%, v/v) and
adjusted to pH 3.0 using triethylamine in the ratio of 17:3:80 (v/v/v). The flow rate of mobile phase was 1
mL/min and samples were analysed for 10 min at 40°C. The scan range of PDA was 220 to 400 nm, and the
detection wavelength was 278nm. The mean (±SE) retention times for ciprofloxacin and enrofloxacin were
5.65±0.003 min and 7.16±0.006 min, respectively.
The standard curves of enrofloxacin and ciprofloxacin were linear in the range of 0.01 to 10.0µg/mL.
Absence of change in the retention time was considered the method found specific and selective. The mean
absolute recovery was within the range of 97.778 to 107.45% for plasma and the coefficient of variation
(CV) was 2.129 to 7.676%. The intra-day and inter-day CV were within the limits (<10%) specified and
hence the method was suitable for assay of both enrofloxacin and ciprofloxacin in emu plasma. The limit
of detection and quantification were 0.01 and 0.025 µg/mL for enrofloxacin and 0.025 and 0.05 µg/mL for
ciprofloxacin, respectively.
The concentrations of enrofloxacin and ciprofloxacin in the plasma samples were determined by
substituting the respective peak areas/peak heights in the linear regression formula.
Pharmacokinetic analysis
Non-compartmental pharmacokinetic analysis was used to describe the pharmacokinetics of enrofloxacin
and ciprofloxacin based on statistical moments theory using pharmacokinetic software PK function (Usansky
et al., 2011).
The elimination rate constant (β) was calculated from the log-linear portion of the elimination curve
using linear regression analysis. The elimination half-life (t1/2β) was calculated according t1/2β = ln 2/β, where,
ln2-0.693. The area under the plasma concentration-time curve (AUC) and the area under the first moment
curve (AUMC) were calculated using the trapezoidal rule and extrapolated to infinity by means of the
elimination rate constant. The mean residence time (MRT = AUMC/AUC), total body clearance (CLB=Dose/
AUC), volume of distribution to steady state (Vdss= CLB x MRT) and apparent volume of distribution (Vdarea
=Dose/ β x AUC0-∞) were calculated after i.v. administration.
Pharmacokinetic/Pharmacodymanic (PK/PD) integration

The ratios Cmax/MIC and AUC/MIC were calculated for hypothetical MIC90 (0.05, 0.125, 0.25 and 0.5 µg/
mL) values using the means of Cmax and AUC obtained in this study.
151
Statistical analysis
Statistical analysis of the data was performed by using SPSS 17.0 software. The results were expressed as
mean±SE. Harmonic mean was used with data not distributed normally. Test of significance such as t-test
and analysis of variance (one way ANOVA) were applied to find out difference between and among various
groups respectively (Snedecor and Cochran, 1989). Comparison of the means of different subgroups was
performed by Duncan‘s multiple range tests as described by Kramer (1957).
RESULTS
The mean (±SD) particle size, PDI, zeta potential, encapsulation efficiency and loading capacity of the
enrofloxacin SLNs are given in Table 1. AFM analysis showed that the enrofloxacin SLNs were spherical
and circular in shape (Fig.1). The particles were well dispersed with good particle size distribution. The
surfaces of the nanoparticle were smooth.
Table 1 Mean(±SD) particle size, PDI, zeta potential, EE and LC of selected enrofloxacin SLNs formulations
Particle size (nm) PDI Zeta potential (mV) EE (%) LC (%)

154.717± 6.149 0.422±0.109 -28.83±0.603 58.33±3.51 6.03±0.97
Figure 1: Atomic force microscopic image of enrofloxacin SLNs
The mean (±SE) plasma concentrations of enrofloxacin and its metabolite ciprofloxacin after native
enrofloxacin (i.v. and oral) and enrofloxacin SLNs (oral) administration are depicted graphically in
Fig.2. After i.v. administration of native enrofloxacin, enrofloxacin could be detected upto 18h in one bird
while in seven birds the drug was detected upto 24h. Detectable concentrations of enrofloxacin after oral
administration of native enrofloxacin were found upto 24h in seven birds while in one bird the drug was
detected upto 36h. The plasma concentration of the active metabolite ciprofloxacin was observed from 15
min to 24h for the both routes of i.v. and oral administration of native enrofloxacin.
Figure 2: Semilogarithmic plot of mean plasma enrofloxacin and its metabolite ciprofloxacin concentration (µg/mL)
vs. time in emus (n=8) following administration of native enrofloxacin and enrofloxacin SLNs (10mg/kg).
152
The mean (±SE) plasma concentration of enrofloxacin after oral administration of enrofloxacin SLNs
was 0.964±0.074 μg/mL at 15min, reached a significantly higher peak concentration of 3.721±0.128 μg/mL
at 1h, then decreased sharply to the same levels of native drug 2 h post-administration. Although the plasma
drug concentration decreased to 0.580±0.032 μg/mL at 6h, the concentration was maintained over 0.012 μg/
mL for up to 60h.
The pharmacokinetics of native enrofloxacin and enrofloxacin SLNs following oral administration in
emu birds are given in Table 2.
Table 2: Pharmacokinetic parameters of enrofloxacin after administration (10mg/kg) of native enrofloxacin (i.v. and
p.o.) and enrofloxacin SLNs (p.o.) in emus
Variable Unit Routes of administration

Native Enrofloxacin Enrofloxacin SLNs
Intravenous Oral Oral
Enrofloxacin Ciprofloxacin Enrofloxacin Ciprofloxacin Enrofloxacin Ciprofloxacin
β. h-1 0.159±0.007 0.152±0.006 0.162±0.015 0.129±0.004 0.054±0.003 0.044±0.005
AUC0-t µg.h/mL 19.553 ±3.518 1.518±0.258 15.756 ±1.416 1.423±0.130 29.511±0.880 2.675±0.081
AUC0-∞ µg.h/mL 20.085 ±3.493 1.561±0.262 16.056 ±1.436 1.496±0.128 30.420±0.760 2.970±0.153
AUMC0-t µg.h2/mL 90.670±19.068 10.591±2.058 102.756±16.766 10.575±1.106 538.882±36.290 46.035±1.673
AUMC0-∞ µg.h2/mL 104.619±19.920 11.889±2.058 109.083±17.395 12.892±1.063 603.401±27.268 75.657±10.032
MRT h. 5.105 ±0.216 7.454±0.223 6.616 ±0.475 8.625±0.173 19.807±0.590 25.463±3.288
MAT h. - - 1.511±0.475 - 14.702±0.590 -
Vd area/F L/kg - - 3.881±0.234 - 6.186±0.357 -
Vdarea L/kg 3.921 ±1.005 - 3.171 ±0.269 - 15.291±1.147 -
Vdss/F L/kg - - 4.168±0.191 - 6.520±0.196 -
CLB L/h.kg 0.629±0.164 8.256±2.385 0.507±0.003 6.897±0.509 0.3330±0.007 3.421±0.177
CLB/F L/h.kg - - 0.646±0.052 - - -
t1/2β h. 4.364±0.179 4.595±0.163 4.125±0.361 5.393±0.186 13.012±0.717 16..913±2.061
Cmax µg/mL - 0.197±0.029 2.397±0.052 0.169±008 3.815±0.059 0.358±0.011
tmax h. - 1.417±0.834 2.167±0.279 3.167±0.167 1.167±0.105 1.33±0.105
AF % - - 79.941±7.147 - 151.462±3.782 -
AUC0-t Cipro/ 7.764 9.031 9.063
AUC0-t Enro
The PK/PD integration parameters of Cmax/MIC and AUC0-24/MIC were calculated from the obtained PK
parameters is presented in Table 3.
Table 3: PK/PD parameters of native enrofloxacin and enrofloxacin SLNs considering MICs of 0.05, 0.125, 0.25 and
0.5 µg/mL
Ratio MIC (µg/mL) Intravenous (Native Oral (Native enrofloxacin) Oral (Enrofloxacin SLNs)
enrofloxacin)
Cmax/MIC 0.05 295.11±44.52 47.94±1.04 76.29±1.18
0.125 118.04±17.81 19.17±0.42 30.52±0.47
0.25 59.02±8.90 9.59±0.21 15.26±0.24
0.5 29.51±4.45 4.79±0.10 7.63±0.12
AUC0-24/MIC 0.05 391.06±70.35 315.11±28.31 374.43±10.97
0.125 156.42±28.14 126.05±11.32 149.77±4.39
0.25 78.21±14.07 63.02±5.66 74.89±2.19
0.5 39.11±7.03 31.51±2.83 37.44±1.10
*For Cmax, a value of 14.755 µg/mL (mean peak plasma concentration at 5 min) was used for the calculation
153
DISCUSSION
In this study, the temperature for the preparation of SLNs did not exceed the melting point of enrofloxacin
(2190C–2330C), hence the stability and antibacterial activity of the enrofloxacin are not affected. According
to Luo et al., (2006), the size of nanoparticles ranges from 100 to 200 nm was favourable for better per oral
performance of incorporated drugs. The particle size of the enrofloxacin SLNs obtained in this study are
within the accepted range for oral administration.
In the present study, the particle size distribution was monodisperse and homogenous as formulation
has less mean (±SE) PDI of 0.42±0.11. In this study, the mean (±SD) zeta potential of -24.90±1.00 mV was
recorded and it could provide proper stability to the enrofloxacin SLNs. The enrofloxacin SLNs obtained in
the present study had relatively medium drug entrapment efficiency (59.67%).
No overt signs of toxicity or abnormal behaviour were observed when enrofloxacin SLNs were
administered to emus through oral route. Published data regarding pharmacokinetics of drug loaded
SLNs in ratites and other domestic animals was limited. Hence, the results obtained in the present study
are interpreted by comparing pharmacokinetic results reported for laboratory animals administered with
SLNs. The plasma concentration of enrofloxacin after enrofloxacin SLNs administration in emus showed
biphasic release pattern. The initial fast (burst release) release of the drug could be due to desorption and
diffusion of enrofloxacin accumulated at the oil–water interface and in the outer shell of nanoparticles
(Xie et al., ,2008; Muller et al. 2000; Han et al.2009; Wang et al., 2011). The initial release should be
sufficiently rapid to ensure that the therapeutic drug levels are achieved in a timely manner in vivo.
The subsequent slow release was mainly due to the slow diffusion of drug molecules through the lipid
matrix of the nanoparticles (Mehnert and Mäder 2001; Muller et al., ,2000) which maintains the effective
therapeutic drug concentrations for a longer period. In the present study, the sustained release performance
of enrofloxacin loaded SLNs provided the plasma concentrations of enrofloxacin exceeding 0.012 μg/
mL for 60h which was therapeutically effective for many common pathogens (Prescott and Yielding
1990). The bi-exponential release of enrofloxacin from SLNs observed in this study are in accordance
with the reports of Xie et al., ,2011b (ofloxacin loaded palmitic acid SLNs in mice), Xie et al., ,2011a
(enrofloxacon loaded palmitic acid SLNs in mice), Kurtz et al., ,1994 (doxorubicin loaded SLNs in rats)
and Pandita et al., , 2011 (paclitaxel loaded in SLNs in mice).
Significantly higher (p<0.01) AUC0–∞ and Cmax value with shorter (p<0.01) tmax was observed for
enrofloxacin SLNs compared to native enrofloxacin after administration at the same dose in emus. The
relative bioavailability obtained in this study (189.47%) is comparable to that reported by Suresh et al.,
(2007) for lovastatin SLNs (173.0%). In concurrence with Xie et al., (2011a), the triglycerides utilized in
the SLNs formulation enhanced the lymph formation and simultaneously promoted the lymph flow rate.
The possible mechanisms for increased absorption of enrofloxacin SLNs discussed under AUC0-∞ could
be the reasons for higher bioavailability obtained in the present study. According to Suresh et al., (2007),
the increased relative bioavailability was due to transport of SLNs by intestinal lymph which avoided
first pass hepatic metabolism of drugs. Investigation of lymph at regular intervals for enrofloxacin could
have provided valuable information in this study and support suggestion of Suresh et al., (2007) regarding
lymphatic transport of SLNs.
The MRT for enrofloxacin SLNs was significantly increased compared to native enrofloxacin in this
study. According to Duchene and Ponchel (1997); Vasir et al., (2003), the adhesive properties of nanoparticles
with gastrointestinal tract wall increase their residence time in the gastrointestinal tract. Moreover, Xie et al.,
(2010b) explained that nanoparticles could protect the drug from chemical and enzymatic degradation and
gradually release drug from the lipid matrix into blood, resulting in a several-fold increase in MRT. Mehnert
and Mader (2001) reported that the drug transported as lipid vesicles remained intact for extended periods
and, thereby, resulted in prolonged release of the encapsulated drug.
154
The use of SLNs sustained the therapeutic concentrations up to 48h, and enhanced the bioavailability and
volume of distribution besides appreciably increasing the AUC/MIC ratio. However, these derived values do
not take into account the contribution made by the active metabolite ciprofloxacin, and therefore underestimate
enrofloxacin efficacy. From these results, it is obvious that use of enrofloxacin SLNs administration at 10mg/
kg every 48h is able to produce an ideal clinical outcome against pathogens susceptible to 0.125 µg/mL.
CONCLUSION
From the results of the executed experiments, it can be concluded that hot homogenization coupled with
ultrasonication method is suitable for producing SLNs with optimal particle size, shape, PDI, zeta potential,
drug loading and encapsulation efficiency. Enrofloxacin SLNs were rapidly absorbed after oral bolus
administration and therapeutic concentrations in plasma were achieved for extended period of time. SLNs
significantly improved the bioavailability, t1/2β, AUC0-∞, Vdarea/F and MRT while significantly decreased the
CLB and elimination rate constant compared to native enrofloxacin. In the present study, the SLNs played an
important role in the drug delivery system and significantly changed the in vivo pharmacokinetic behaviour
of drug molecules. The results of pharmacokinetic parameters of enrofloxacin SLNs strongly support the
potential application of SLNs in emus as sustained delivery system for enrofloxacin.
ACKNOWLEDGEMENT
Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai is gratefully acknowledged.
The authors wish to thank Dr. K. Rukumani, Professor and Head, Department of Pharmaceutical Technology,
Anna University BIT campus, Trichy for their support of this work.
Conflict of interest
The authors declare no conflicts of interest
REFERENCES
1. Anton N, Benoit JP and Saulnier P, (2008) Design and production of nanoparticles formulated from
nano-emulsion templates-a review J Control Release 128: 185–199.
2. Bunjes H, Koch MHJ and Westesen K, (2002) Effect of surfactants on the crystallization and
polymorphism of lipid nanoparticles Prog Colloid Polym Sci 121: 7–10.
3. Duchene D and Ponchel G, (1997) Bioadhesion of solid oral dosage forms, why and how?. Eur J Pharm
Biopharm 44: 15–23.
4. Han, C, Qi, C, Zhao and M B K, (2009) Hydrogenated castor oil nanoparticles as carriers for
the subcutaneous administration of tilmicosin: in vitro and in vivo studies, Journal of Veterinary
Pharmacology and Therapeutics 32: 116–123.
5. Ji J, Hao S, Wu D, Huang R and Xu Y, (2011) Preparation, characterization and in vitro release of
chitosan nanoparticles loaded with gentamicin and salicylic acid Carbohydrate, Polymers 85: 803–808.
6. Kramer CY, (1957) Extension of multiple range tests to group correlated adjusted means Biometrics, 13: 13-
18 doi:102307/3001898 Access date: 16032014.
7. Kurtz MB, Heath IB, Marrinan J, Dreikorn S, Onishi J and Douglas C, (1994) Morphological effects
of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan
synthase. Antimicrobial Agents and Chemotherapy 38: 1480-1489.
8. Luo YF, Chen DW, Ren LX, Zhao XL and Qin J, (2006) Solid lipid nanoparticles for enhancing
vinpocetine‘s oral bioavailability .J Control Release 114:53–59.
155
9. Martinez M, McDermott P and Walker R, (2006) Pharmacology of the fluoroquinolones: A perspective

for the use in domestic animals. The Veterinary Journal 172, 10–28.
10. Mehnert W and Mader K, (2001) Solid lipid nanoparticles: Production, characterization and applications.
Advanced drug delivery reviews 47:165–196.
11. Muller RH, Mader K and Gohla S, (2000) Solid lipid nanoparticles (SLN) for controlled drug delivery-a
review of the state of the art, Eur J Pharm Biopharm 50: 161–177.
12. Muller RH, Radtke M and Wissing SA, (2002) Solid lipid nanoparticles (SLN) and nanostructured lipid
carriers (NLC) in cosmetic and dermatological preparations, Adv Drug Deliv Rev 54: 131–55.
13. Olbrich C, Kayser O and Muller RH, (2002) Lipase degradation of Dynasan 114 and 116 solid lipid
nanoparticles (SLN) effect of surfactants, storage time andcrystallinity, Int J Pharm 237: 119–128.
14. Pandita D, Ahuja A and Lather V, (2011) Development of lipidbased nanoparticles for enhancing the oral
bioavailability of paclitaxel, AAPS PharmSciTech 12, 712–722.
15. Prescott JF and Yielding KM, 1990 In vitro susceptibility of selection veterinary bacterial pathogens to
ciprofloxacin, Canadian J Vet Res 54: 195–197.
16. Scheer M, (1987) Studies on the antibacterial activity of Baytril, Veterinary Medical Reviews 2: 90–98.
17. Schwarz C and Mehnert W, (1999) Solid lipid nanoparticles (SLN) for controlled drug delivery II Drug
incorporation and physicochemical characterization, J Microencapsul 16: 205–213.
18. Schwarz C, Mehnert W and Muller R, (1994) Influence of production parameters of solid lipid
nanoparticles (SLN) on the suitability for intravenous injection, Eur J Pharm Biopharm 40: 24–30.
19. Snedecor GW and Cochran WG, (1989) Statistical methods 8th Edn Iowa State University Press, Ames
Iowa, USA
20. 20. Suresh G, Manjunath K, Venkateswarlu V and Satyanarayana V, (2007) Preparation, characterization
and in vitro evaluation of lovastatin solid lipid nanoparticles, AAPS Pharm Sci Tech 8: E1–E9.
21. Usansky JI, Desai A and Tang-Liu D (2011), PK Functions for Microsoft Excel Department of
Pharmacokinteics and Drug Metabolism Allergan, Irvine, CA 92606, USA.
22. Vasir JK, Tambwekar K and Garg S, (2003) Bioadhesive microspheres as a controlled drug delivery
system, Int J Pharm 255: 13–32.
23. Wang XF, Zhang SL and Zhu LY, (2011) Enhancement of antibacterial activity of tilmicosin against
Staphylococcus aureus by solid lipid nanoparticles in vitro and in vivo, Vet J 55: 672–680.
24. Xie S, Zhu L, Dong Z, Wang X, Wang Y, Li X and Zhou W, (2011a) Preparation, characterization and
pharmacokinetics of enrofloxacin-loaded solid lipid nanoparticles: Influences of fatty acids. Colloids
and Surfaces B: Biointerfaces 83: 382–387.
25. 25. Xie SY, Wang SL, Zhao BK, Han C, Wang M and Zhou WZ, (2008) Effect of PLGA as a polymeric
emulsifer on preparation of hydrophilic proteinloaded solid lipid nanoparticles, Colloids Surf B:
Biointerfaces 67: 199–204.
26. 26. Xie Y, Zhu L, Dong Z, Wang X and Zhou WZ, (2011b) Preparation and evaluation of ofloxacin-
loaded palmitic acid solid lipid nanoparticles, International Journal Nanomedicine 5: 693–701.
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Antidiabetic Activity of Chromium (III) Nanoparticle
Against Streptozotocin-Induced Diabetis in Rats
Kanakalakshmi Annamalai, Anjali Mohan Nair and Shanthi kuppusamy
Department of Environmental Science, PSG College of Arts and Science, Coimbatore-641 014
e-mail: akanakalakshmi1988@gmail.com
Abstract:
The usage of micronutrients for medical complications are gaining more popularity in nowa days. Among
the micronutrients, chromium is an essential mineral which plays vital role in glucose metabolisms and
gaining new insights in to its versatile applications in treating metabolic disorders like diabetes mellitus. To
support this concept very few experiments had been conducted in animal models. In view of the above fact
the present study was aimed to explore the antidiabetic activity of chromium (III) nanoparticles against STZ
(65 mg/kg i.p) induced diabetes in rats. In diabetic rats, the chromium (III) nanoparticles were administrated
on daily basis at the dose range of 30 mg/kg p.o for about 28 days. The levels of blood glucose and body
weight of the animal were measured periodically. STZ treated diabetic rats showed significant alteration
in the biochemical parameters and this elevation were restored to near normal (p<0.5) by chromium (III)
nanoparticle treatment. In conclusion, the results from the present study indicates that chromium (III) possess
antidiabetic activity against STZ model in rats.
INTRODUCTION
The world Health Organization estimates globally 170 million individuals suffers from diabetes every year
[1]. The elevated serum concentrations are said to the initial for transformin the normal glucose metabolism
into diabetes.Therefore strategies to improve insulin resistance by either nutritional supplementation or by
pharmacologic means will represent an attractive approach. The role of metals like vanadium, magnesium,
chromium, zinc are likely to have an effect on biochemical, immunologic and physiological actions of the
body as micronutrient [2, 3]. Earlier findings suggested that chromium in the trivalent form (Cr+3) will
regulate carbohydrate, lipid, protein metabolism via the enhancement of insulin action [4] and the beneficial
role of chromium in diabetes has been implicated in many studies [5, 6]. Moreover, a landmark for the
discovery of chromium as a human nutrition which will potentiate the action of insulin in glucose uptake
was reported clearly in earlier studies [7]. But biologic action of chromium as a cofactor for insulin action
was not fully understood and some studies suggested the chromium will function as a part of the oligopeptide
low molecular weight chromium (LMCr) binding substance and it can influence the insulin metabolism also.
The new phenomena and properties of the nanoparticle have many potential application in biomedical
field. Therefore the present study is aimed to expand the effect of chromium (III) nanoparticle against
streptozotocin induced diabetes rats.

Size determination Chromium (III) nanoparticle
The powder of chromium (III) nanoparticle was procured from the Department of Environmental Science,
PSG College of Arts and Science, Coimbatore. The Transmission Electron Microscope (TEM, Philips CM20)
analysis was performed in order to know the morphology and average particle size of the nanopaticle. The
average particle size was found to be 80 nm shown in Fig 1.
Figure 1: TEM image of Chromium III nanoparticle
Chemicals
The estimation of biochemical parameters was carried using commercially available kits (Primal Healthcare
Limited, Mumbai, India) by using auto analyzer. STZ and other chemicals were analytical grade procured
from Himedia Laboratories, Mumbai India.
Experimental animal Management

Male Wister rats (150-200 g) were used to evaluate antidiabetic activity. The animals were housed in
polypropylene cages at standard laboratory conditions [temperature (22±2 0C) and humidity (45±5) % with 12
h day/12 h night cycle was maintained. Standard laboratory pellet and purified drinking water was provided
ad libitum.All the experimental procedure were approved by Institutional Animal Ethics Committee (IAEC)
and conducted as per the guide lines (KMCRET/M. PHARM/5a/2013-2014)
Induction of Experimental Diabetes

Experimental diabetes was induced by injecting an interaperitonial dose of STZ in 0.1 M citrate buffer pH
(4.5). The rats were received 5% glucose solution for the next 24 h to prevent STZ induced fatal hypoglycemia.
After injection STZ, fasting glucose was determined and the animal with a blood glucose level over 250mg/
dL were indicated as diabetic.
Experimental design for Antidiabetic activity

The rats were divided into fourgroups containing six rats in each group. Group 1: Control rats; group 2: STZ-
induced diabetic control rats; group 3: STZ-induced diabetic rat were treated with Pioglitazone (10 mg/kg);
group: 4 STZ-induced diabetic rat were treated with chromium (III) nanoparticle (30 mg/kg). The chromium
(III) nanoparticle and Pioglitazone were administered orally for28 days.
Sampling Protocol
The blood samples were collected through retro-orbital plexus puncture for all the experimental rats. The
serum was separated by centrifugation and used for the estimation of biochemical parameters
158
Estimation of biochemical parameters and antioxidant levels

The glycosylated haemoglobin(HbA1C) were estimated using whole blood using the commercially available
kits. The serum lipid profiles like high density lipoprotein (HDL) were estimated using commercially available
kits. The serum low density lipoprotein (LDL) and very low density lipoprotein (VLDL) were calculated.
The antioxidants, superoxide dismutase (SOD) were also determined by using standard procedures.
All the data expressed as mean ± SEM were evaluated by one –way analysis of variance (ANOVA) by using
SPSS software version 20.0
Effect of chromium (III) nanoparticle on blood glucose

The blood glucose levels were significantly increased after the administration of STZ compared to the control
rat. The treatment of chromium (III) nanoparticle (30 mg/kg) and Pioglitazone (10 mg/kg) significantly
reduced the (p<0.5) of blood glucose level compared to the diabetic control rats as shown in Table 1.
Table 1: Hypoglycemic activity of Chromium (III) nanoparticle in STZ-induced diabetic rats
Groups Blood Glucose level

Initial 1 week 2 week 3 week 4 week
Control (0.9% NS) 161.00±1.63 177.67±3.19 182.67±3.45 188.00±1.82 191.17±1.07
STZ 118.6±0.92a 107.4±0.85a 103.0±0.63a 98.0±0.96a 95.9±0.56a
Pioglitazone (10 mg/kg, 162.67±2.48*** 151.50±1.60*** 169.50±5.22*** 168.33±3.65*** 172.67±3.72***
p.o)
Chromium (III) 127.8±0.40*** 124.8±0.45** 125.3±0.32** 125.8±0.32** 126.4±0.56**
Nanoparticle (30 mg.kg,
p.o)
: p<0.001.Group III( pioglitazone standard), group IV( chromium III) was compared with group II.***P<0.001, **P<0.01.
a
Effect of chromium (III) nanoparticle on HbA1c and lipid profiles

HbA1c is a marker evaluation of long term glycemic control in patients with diabetes and it predicts the risk
for the development of complication associated with the diabetes [8]. The chromium complexes was found
to give superior results when compared to the in organic salts [9]. Diabetic rats treated with chromium (III)
nanoparticle and Pioglitazonesignificantly reduced the elevated HbA1c levels (Table 2). The treatment of
chromium for diabetes may lead to the improvement in the blood glucose and HbA1c levels.
Table 2: Effect of Chromium (III) nanoparticle on HbA1c and hypolipidemic activity in STZ-induced diabetic rats
Groups HDL LDL VLDL HBA1C

Control (0.9% NS) 56.70±1.90 48.37±7.86 13.37±1.35 4.84±0.01
STZ 35.36±0.42a 147.8±0.34a 36.81±0.14a 8.69 ± 0.24a
Pioglitazone (10 mg/kg, p.o) 54.90±0.67** 43.67±8.32*** 12.18±1.15*** 6.73±0.09***

Chromium (III) Nanoparticle (30 mg.kg, p.o) 40.28±0.38 **
92.28±1.04 **
28.36±0.12 ***
7.34±0.24**
: p<0.001.Group III( pioglitazone standard), group IV( chromium III) was compared with group II.***P<0.001, **P<0.01.
a
The disturbance of lipoprotein metabolism leads to the occurrence of vascular diseases in diabetic
patients [10]. In the present study, STZ induced diabetic rats showed increased LDL and VLDL whereas the
159
HDL level were decreased when compared to the normal rats. Administration of chromium (III) nanoparticle
showed a significant reduction in LDL and VLDL compared to the diabetic rats and it showed significant
increase in the HDL was observed shown in (Table 3).In our study the administration chromium (III)
nanoparticle and Pioglitazone reduced the HbA1c levels in diabetes induced rats revealed that chromium
(III) nanoparticle has an ability to prevent the diabetes associated complications and it can normalize the
lipid metabolism in diabetic rats.
Table 3: Antioxidant activity of Chromium (III) nanoparticle in diabetic rats STZ-induced diabetic rats
Groups SOD (Unit /min/mg/protein)

Control (0.9% NS) 13.81±0.59
STZ 7.03±0.46a
Pioglitazone (10 mg/kg, p.o) 15.21±0.69***
Chromium (III) Nanoparticle (30 mg.kg, p.o) 9.20±0.12
p<0.001.Group III( pioglitazone standard), group IV( chromium I) and group V(chromium II) was compared
a
with group II.***P<0.001.
Antioxidant activity of Chromium (III) nanoparticle

Earlier findings from various studies shows that enhanced blood glucose is susceptible to oxidation,
hyperglycemia causes excessive Reactive oxygen species (ROS) production which may implicate various
metabolic and neurodegenerative disorders. The antioxidant activity of chromium (III) nanoparticle
was studied in diabetic rats and the data was given in the Table 4. After the induction of diabetes with
STZ, the significantly reduced SOD was observed when compared to the normal control rats. The
condition was reversed when the diabetic rats were administered with Chromium (III) nanoparticle and
Pioglitazone.
CONCLUSION
The results from the present study is clearly indicating that chromium (III) nanoparticle showed significant
antidiabetic and antioxidant activity against STZ induced diabetic rats.
ACKNOWLEDGEMENTS
I thank Dr. Jayaraman, Dept of Pharamacology, KMCH college of Pharmacy, Coimbatore for guiding me
to carry out this work. Also I thank Johnson C John, student Dept of Pharamacology, KMCH college of
Pharmacy, Coimbatore for helping me during the laboratory work. Finally I thank Dr. K. T. Mani Senthil
Kumar, Head, Dept of Pharamacology, KMCH college of Pharmacy, Coimbatore for providing the laboratory
facility.
REFERENCES
1. Khanam R and Pillai KK, Effect of chromium picolinate on modified forced swimming test in diabetic
rats: involvement of serotonergic pathways and potassium channels, Basic Clin Pharmacol Toxicol.
2006 Feb;98(2):155-9.
2. Alkaladi A, Abdelazim AM and Afifi M, Antidiabetic Activity of Zinc Oxide and Silver Nanoparticles
on Streptozotocin-Induced Diabetic Rats, Int. J. Mol. Sci. 2014 Jan; 15: 2015-2023. doi:10.3390/
ijms15022015.
160
3. Shrivastava R, Upreti RK, Seth PK and Chaturvedi UC, Effects of chromium on the immune system,
FEMS Immunol Med Microbiol, 2002 Sep 6;34(1):1-7.
4. Laschinsky N, Kottwitz K, Freund B, Dresow B, Fischer R and Nielsen P, Bioavailability of chromium(III)-
supplements in ratsand humans. Biometals, 2012 Jul; 25: 1051-1060. DOI 10.1007/s10534-012-9571-5.
5. Rhodes NR1, McAdory D, Love S, Di Bona KR and Chen Y, et al. Urinary chromium loss associated with
diabetes is offset by increases in absorption, J Inorg Biochem. 2010 Jul;104(7):790-7. doi: 10.1016/j.
jinorgbio.2010.03.015.
6. Cefalu WT, Wang ZQ, Zhang XH, Baldor LC and Russell JC, Oral chromium picolinate improves
carbohydrate and lipid metabolism and enhances skeletal muscle Glut-4 translocation iin obese,
hyperinsulinemic (JCR-LA corpulent) rats. J. Nutr. 2002 Jun; 132(6): 1107-14.
7. Kim DS, Kim TW and Kang JS, Chromium picolinate supplementation improves insulin sensitivity in
Goto-Kakizaki diabetic rats, J Trace Elem Med Biol. 2004 Dec;17(4):243-7.
8. Tembhurne SV and Sakarkar DM, Protective effect of Murrayakoenigii (L) leaves extract in
streptozotocin induced diabetics rats involving possible antioxidant mechanism, J Med Plant Res. 2010
Nov; 4(22):2418-2423. DOI: 10.5897/JMPR10.349.
9. Lamson DW and Plaza SM, The safety and efficacy of high dose chromium Altern Med Rev, 2002
Jun;7(3):218-35.
10. Maser RE, Wolfson SK Jr, Ellis D, Stein EA and Drash AL, et al, Cardiovascular disease and arterial
calcification in insulin-dependent diabetes mellitus: interrelations and risk factor profiles, Pittsburgh
Epidemiology of Diabetes Complications Study-V. Arterioscler Thromb, 1991 Jul-Aug;11(4):958-65.
161
Formulation Using Soap and Cream from Biosynthesised
Silver Nanoparticles of Adathoda Vasica Nees. Leaf Extract
H. Linda Jeeva Kumari1, S. Sonia2, R. Krishnamoorthy3, M. Sivakumar4

and K. Ruckmani5
National Facility for Drug Development for Academia, Pharmaceutical and Allied Industries,
1, 5
1, 3, 5
Department of Pharmaceutical Technology, 2, 4Division of Nanoscience and Technology,
Bharathidasan Institute of Technology, Anna University, Tiruchirappalli 620 024, Tamil Nadu, India
Email: hodpharma@gmail.com, Mob: +91 98424 84568
Abstract:
Green synthesis of silver nanoparticles have gained much attention in the present days because of their efficacy,
low toxicity, easy and less expensive production compared to that of the conventional chemical synthesis.
In the present study, we focus to adopt an eco-friendly method for the biosynthesis of silver nanoparticles
(AgNPs) using aqueous Adhatoda vasica leaf extract with its phytoconstituents acting as reducing and
capping agents for the reduction of silver nitrate into pure silver ions. Various molar concentrations (1mM, 2
mM, 3 mM and 4 mM) of silver nitrate solution were prepared and 1 ml of each were added with 100 µl, 200
µl, 300 µl, 400 µl and 500 µl of plant extract at room temperature and observed for 24 h. The visible colour
change from yellow to reddish brown indicates the formation of AgNPs. Optimisation was done by physical
observation such as stability of the synthesised nanoparticles without agglomerating and obtaining the
Surface Plasmon Resonance peaks within the expected range of AgNPs by UV-Visible Spectroscopy. Its pH
was also determined (pH 5.2). This was further characterized by Fourier Transform Infra-Red spectroscopy
(FT-IR), Atomic Force Microscopy (AFM), High Resolution Transmission Electron Microscopy (HR-TEM)
and X-ray Diffraction (XRD). Furthermore, these nanoparticles can be used in various formulations to
exploit their goodness for human use. Hence, the biosynthesised AgNPs using Adathoda vasica leaf extract
was used for anti-bacterial soap and cream formulations. The antimicrobial activities of the AgNPs, Soap
and Cream were done against various clinical pathogens to prove their efficacy alongside standard antibiotic,
commercial antimicrobial soap and a commercial antimicrobial cream respectively. Hence, the obtained
formulations are a boon to the pharmaceutical industries.
INTRODUCTION
Nanotechnology, a growing field has diversified applications in Physics, Chemistry, Engineering and Biology
[1]. It shows great promise for providing us in the near future with many breakthroughs that will change the
direction of technological advances in a wide range of applications. It has gained its importance as the material
properties such as optical, electrical, magnetic, catalytic and reducing tend to change depending on its size
and shape [2]. Nanomaterials exhibit a high surface area to volume ratio which favours high contact area
with the reacting substance that increases the rate of reaction. Even though there are many ways of chemical
and physical methods available for synthesis, toxic chemicals are being used [3]. A suitable alternative
method is to use plants or microbes [4] as reducing agents for green synthesis as they are cost efficient, clean,
eco-friendly and they do not require maintenance [5, 6]. Also, the nanoparticles synthesized this way are
more stable and the rate of synthesis is faster. Biosynthesis of nanoparticles has been successively carried out
using extracts from several plants like Aloevera [7], Azadirachta indica (Neem)[8], Diopyros kaki, Magnolia
kobus [9], Acalypha indica [10], Capsicum annuum [11], Jatropha curcas [12], Cinnamon zeylanicum [13]
and so on. Metal nanoparticles are employed in catalysis, photonics, optoelectronics, biological tagging and
pharmaceutical applications [14-17]. Silver nanoparticles in particular have potential anti-microbial [18],
anti-oxidant [19] and anti-inflammatory properties [20]. Adathoda vasica, a commonly found shrub, along
the tropical regions of south-east Asia, has an excellent antibiotic property and is commonly used in the
treatment of respiratory infection especially in tuberculosis and upper respiratory infections. It has excellent
anti-rheumatic, anti-inflammatory [21], anti-diabetic, anti-hemorrhagic, anti-spasmodic, anti-fungal [22],
anti-tussive [23] and anti-oxidant [24] properties. In the present study, the aqueous leaf extract of Adathoda
vasica is used for the synthesis of AgNPs and the same has been used in the formulation of anti-bacterial
soap and cream for effective pharmaceutical applications.
Materials
Silver Nitrate was purchased from Sigma-Aldrich, Merck. Culture media for the cultivation of bacteria were
from Hi-media, Mumbai, India. Clinical pathogens were obtained from local hospitals. All other reagents
used were of AR Grade.
Preparation of the Extract

The fresh leaves of Adathoda vasica were obtained from the hostel premises of Anna University campus,
Tiruchirappalli, and were authenticated in the Department of Botany, St. Joseph’s College of Arts and
Science, Tiruchirappalli. Adathoda vasica leaves (20 g) were weighed and thoroughly washed twice in
distilled water and boiled in round bottom flask with100 ml double distilled water for an hour until the leaf
extract completely goes down into solution. The obtained extract was then filtered through Whatmann No.1
filter paper so as to separate from its residues and collect the pure extract. The extract was analysed for the
major phytochemical constituents and used for the synthesis of nanoparticles. The rest was stored at 4°C
until further use.
Synthesis of Nanoparticles
Various molar concentrations (1mM, 2 mM, 3 mM and 4 mM) of silver nitrate solution were prepared and
1 ml of each were used with 100 µl, 200 µl, 300 µl, 400 µl and 500 µl of plant extract at room temperature
and observed for 24 h. The colour changed from yellow to reddish brown indicating the presence of AgNPs.
This is due to the phytochemicals present in aqueous Adhatoda vasica leaf extract that act as reducing
and capping agents for the reduction of silver nitrate into pure silver ions. Then the nanoparticles were
centrifuged at 7,500 rpm for 10 min in order to obtain the pellet which was air-dried and used for further
studies. The chemical tests for these AgNPs were also done as that of the phytochemical analysis of the
extract to understand the bio-functionality of the synthesised nanoparticles. The pH of the sample was
also determined. The optimised concentration of nanoparticles was used for bulk synthesis to get the same
concentration and the procedure was repeated to get nanoparticles in larger amounts which were used for
further characterizations and formulations.
UV-Visible Spectra
The spectroscopy wavelength range is used between 200 to 800 nm and the speed of the spectrometer is 40/
min (JASCO V 650 UV-Visible spectroscopy and photomultiplier). The wavelength of the light source is 340
nm with bandwidth as 2 nm.The samples were diluted in 1:9 ratio of double distilled water and subsequently
analysed at room temperature.
163
FTIR spectroscopy
FT-IR spectral studies were performed to identify the possible bio molecules responsible for the reduction
and stability of the metal nanoparticles. FT-IR measurements were taken for the sample using Perkin-Elmer
spectrophotometer in the range of 4000 – 400 cm-1 for transmittance values. Hydraulic Pellet Press method
was done to produce thin sample pellet with potassium bromide for the analysis.
AFM Imaging analysis

Atomic Force Microscopy (AFM) was done to understand the pattern of distribution of particles for the
synthesised nanoparticles along with their size. Nanoparticles were diluted tenfold with milliQ water and
a drop was deposited on mica sheet fixed on a metallic stub. The drop was allowed to dry overnight. Non-
contact mode was followed in Park Nano Instruments to obtain 2D and 3D images of silver nanoparticles.
High Resolution Transmission Electron Microscopy (HR-TEM)

High Resolution Transmission Electron Microscopy (HR-TEM) is an imaging mode of the transmission
electron microscope (TEM) that allows for direct imaging of the atomic structure of the sample. The
structural morphology and size of the nanoparticles can be obtained along with the Selected Area Electron
Diffraction (SAED) pattern.
X-Ray Diffraction Pattern

The metal nanoparticles were coated on the glass substrate in liquid state. After drying, the sample was
analysed by X-Ray diffraction. This technique is used to determine the chemical composition, crystalline
and grain size.
Nano-based Soap Formulation

The basic saponification reaction for the production of soap is the reaction between a neutral fatty acid and
alkali to form soap and glycerol. This idea has been used to prepare a stable soap with a basic pH suitable for
skin that does not tend to change colour easily. Stearic acid is a saturated fatty acid which has been widely
used as skin cleansers in the soap-making process. Sodium hydroxide (lye) is used as an alkali to dissolve
grease and fats, dirt which is polar tends to attach to the lye for efficient cleansing, also, it is necessary to
curdle up the soap into a solid and stable soap bar. Hence, stearic acid as a neutral fat and lye as an alkali
has been used here for the basic saponification reaction along with the synthesized AgNPs for a nano-based
soap production.
Nano-based Cold Cream Formulation

The cold cream formulation was done as follows: 2 g bees wax and 6 ml liquid paraffin were taken in a beaker
and heated in a water bath at 70 °C. The aqueous phase was prepared for heating 0.1 gm of borax in 2 ml of
Milli-Q water in another beaker at 50 °C. The aqueous phase was transferred to a mixture of bees wax and
liquid paraffin. This preparation was done in two so that to administer lower and higher concentration of the
nanoparticles. At room temperature, 1 ml and 2 ml of the biosynthesized AgNPs were added to the respective
beakers of low and high concentration and continuous stirring was given until the solution is completely
homogeneous. These formulations were kept in airtight containers and preserved for further studies.
Antimicrobial susceptibility testing of AgNPs, Soap and Cold Cream

The anti-microbial action of the AgNPs on clinical pathogens such as Staphylococcus aureus, Escherichia
coli and Pseudomonas aeruginosa were studied. The micro-organisms were clinical isolates obtained from
local hospitals. The culture medium used was Mueller Hinton Agar (MHA) for antimicrobial susceptibility
testing. Well Diffusion technique was followed. Freshly grown overnight cultures were used. MHA plates
164
were prepared and 100 µl of the overnight cultures of the respective bacteria were spread plated evenly. The
plates were allowed to dry for 5 minutes in an inverted position inside the laminar hood. Then 4 wells were
punctured at equidistance using sterile pipette tips. The AgNPs (0.5 mg/ml) were prepared in duplicates
using sterile double distilled water and completely suspended them by ultrasonicator to make a homogeneous
solution. Standard antibiotic streptomycin (0.5 mg/ml) was used as a positive control and the sterile double
distilled water as a negative control. Well A was filled with 50 µl of positive control, well B with 50 µl of
negative control, well C with 50 µl of AgNPs duplicate 1 and well D with 50 µl of AgNPs duplicate 2.
The plates were incubated overnight at 37 °C in upright position. The diameter of the zones of inhibition
was recorded and tabulated. The same procedure was followed for the formulated soap and cream. For the
antimicrobial susceptibility testing of soap formulation, three wells were punctured. Commercial antibacterial
soap was used as a control. The soap was dissolved in sterile distilled water in the required concentration.
Well A was filled with 50 µl of control (100 mg/ml), well B with 50 µl of formulated nano-based soap (50
mg/ml) and well C with 50 µl of 100 mg/ml nano-based soap. For cream, commercial antibacterial cream
was used as a control. As the cream was semi-solid in nature, here we adopted to just check the efficacy of the
cream by applying a streak onto the cultured plates with a sterile loop. A is the control which is a commercial
antibacterial cream. B is the formulated nano-based cream in lower concentration whereas C is that of the
higher concentration.
Synthesis of AgNPs
The colour change was observed from yellow to reddish brown for all the twenty samples (silver nitrate
solution 1 mM, 2 mM, 3 mM and 4 mM concentrations with 100 µl, 200 µl, 300 µl, 400 µl and 500 µl of
plant extract) was observed which is due to the excitation of Surface Plasmon Resonance.
Phytochemical analysis
The phytochemical analysis of the aqueous leaf extract shows the presence of alkaloids, saponins, tannins,
proteins and carbohydrates whereas synthesised AgNPs shows the presence of alkaloids, proteins and
tcarbohydrates (Table.1). These secondary metabolites act as reducing and capping agents for the efficient
reduction of silver nitrate solution to silver nanoparticles.
Table.1 Phytochemical Screening of plant extract and synthesised nanoparticles
Sl.No. Chemical constituents with Tests Phytochemical Screening

Plant Extract AgNPs
1 Alkaloids (Mayer’s test) + +
2 Saponins (Foam test) + -
3 Tannins (Lead Acetate test) + -
4 Phytosterols (Salkowski test) - -
5 Flavonoids (Shinoda test) - -
6 Proteins (Ninhydrin test) + +
7 Carbohydrates (Molisch’s test) + +
+ Present - Absent
UV-Vis Spectrophotometric Analysis

Fig.1(a) represents the UV-Vis spectrophotometric analysis of the plain leaf extract which does not show
any peaks. The absorption spectra for all the twenty samples were studied. Fig.1(b) shows the nanoparticles
165
synthesised using 1 mM concentration of Silver nitrate solution and Fig.1(c) shows for 2 mM concentration
varying the plant extract from 100 µl to 500 µl. On the whole, the peaks were observed from 380 nm to 450
nm, confirming the presence of silver nanoparticles. Synthesis using 1 mM concentration of silver nitrate
showed better peaks when compared to 2 mM, 3 mM and 4 mM concentrations. Nanoparticles from 500 µl
of extract with 1 ml of 1mM concentration silver nitrate were found to be stable with maximum absorbance
at 430 nm. The rest of the samples tend to agglomerate within 48 hrs after reaction. Thus we optimise the
sample by physical observation and absorption spectral analysis. The pH of the sample was found (pH 5.2).
The optimised concentration of nanoparticles thus found (500 µl of plant extract with 1 ml of 1mM silver
nitrate (Ratio 1:2)) was used for bulk synthesis. 25 ml of plant extract was added to 50 ml of 1 mM silver
nitrate to get the same concentration which were used for further characterizations and formulations.
Figure 1(a): UV-Vis absorption spectra for leaf extract
Figure 1 (b): UV-Vis spectra of synthesised AgNPs with 1 mM concentration
Figure 1 (c): UV-Vis spectra of synthesised AgNPs with 2 mM concentration
166
FT-IR analysis
The FT-IR spectra of the leaf extract and the optimised silver nanoparticles were obtained (Fig. 2). Table
2 displays the peaks observed in the leaf extract and the metal nanoparticles and the possible functional
groups involved in the formation of the metal nanoparticles. The band observed at 3323 cm-1 in the
leaf extract arises from the O-H functional groups and it is shifted to the 3455 cm-1 in the nanoparticles
suggest that (O-H) alcohol groups present in the leaf extract interact with the nanoparticles. There is
also an non interactive C-O stretching alcohol group at 1069 cm-1.The strong absorption peaks arises at
1637 cm-1 & 1625 cm-1 were associated with the C=C stretching vibrations of alkene group apart from
these 934 cm-1,897 cm-1, 840 cm-1 also corresponds to =C-H bending of alkene group. The peak arising
at 2925 cm-1, 1396 cm-1, 1337 cm-1 corresponds to C-H stretching of alkane groups. Apart from these,
the plant extract contains alkyl halides and nitrile group 1241 cm-1, 718 cm-1 & 2352 cm-1 respectively.
Thus the biological compounds are responsible for the formation and stabilization of nanoparticles.
Table 2: Corresponding functional groups from the FT-IR absorption spectra
Frequency (cm-1) Bond Functional group

3323(Extract) 3455(AgNPs) O-H stretch Alcohol
2925(Extract) C-H stretch Alkane
2352(Extract, AgNPs) CN stretch Nitrile
1637(AgNPs) 1625(Extract) C=C stretch Alkene
1396,1337(Extract) C-H stretch Alkane
1241(Extract) C-F stretch Alkyl halide
1069(Extract) C-O stretch Alcohol
934,897,840(Extract) =C-H bend Alkene
718(Extract) CCl stretch Alkyl halide
Figure 2: FT-IR absorption spectra of leaf extract and biofunctionalised AgNPs
AFM Imaging analysis

2D and 3D AFM images of the synthesised AgNPs are shown in Fig.3. The nanoparticles were measured by
non-contact mode and the scan rate was 0.5Hz. The image shows the surface topography and average size of
the particles. The average size of the nanoparticles was obtained at around 30 nm. The pattern of distribution
of particles was found to be even and regular showing better characterization of the synthesised AgNPs.
167
Figure 3 A: FM 2D and 3D images of the synthesised AgNPs.
High Resolution Transmission Electron Microscopy (HR-TEM)

HR-TEM image of AgNPs is shown in Fig.4 (a). The colloidal solution was coated on the copper grid and
dried at room temperature for HR-TEM measurements. AgNPs were observed as irregular distribution of
spherical shaped structures in the surface area. The average particle size ranges from 20 nm to 30 nm. The
SAED (Selected Area Electron Diffraction) pattern was observed by the back scattering of electrons which
is shown in Fig. 4 (b).
Figure 4 (a): HR-TEM image of AgNPs Figure 4(b): SAED pattern
XRD Analysis
The XRD pattern of AgNPs is shown in Fig.5. The four diffraction peaks were recorded in the 2Ѳ range as
30° to 120° Cu-Kα radiation source as [111], [200], [220] and [311] planes for hexagonal structure.
Figure 5: X-Ray Diffraction pattern analysis of AgNPs
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Antimicrobial susceptibility testing

Fig. 6 shows the anti-microbial action of the AgNPs on clinical pathogens such as Staphylococcus aureus,
Escherichia coli and Pseudomonas aeruginosa respectively whereas Fig.7(a) for the formulated nano-based
soap and Fig.7(b) for the nano-based cold cream. Well diffusion technique was followed and the diameters
of zones of inhibition were measured in mm to analyse the antimicrobial activity of the nanoparticles and the
nano-based soap which are tabulated in Table 3 and Table 4 respectively. The cold cream was checked for
its antimicrobial activity in a streak method only because of its semi-solid nature.
Figure 6: Anti-microbial activity of the synthesised AgNPs Zones of inhibition observed around the wells accordingly
A– Streptomycin (0.5 mg/ml); B – Distilled water; C – AgNPs duplicate 1 (0.5 mg/ml) and D – AgNPs duplicate 2
(0.5 mg/ml).
Figure 7 (a): Anti-microbial activity of the soap Zones of inhibition observed around the wells accordingly A–
Commercial anti-bacterial soap (100 mg/ml); B – AgNPs based soap formulation (50 mg/ml) and C – AgNPs based
soap formulation (100 mg/ml)
Figure 7 (b): Anti-microbial activity of the cold cream Zones of inhibition observed around the lines of streak
accordingly A– Commercial anti-bacterial cream ; B – AgNPs based cold cream formulation (low concentration) and
C – AgNPs based cold cream formulation (high concentration)
169
Table 3: Antimicrobial activity of AgNPs

Micro-organisms Zone of inhibition
(diameter in mm)
A B C D
Staphylococcus aureus 10 - 12 12
Escherichia coli 9 - 16 16
Pseudomonas aeruginosa 13 - 16 16
Table 4 Antimicrobial activity of Nano-based soap
Micro-organisms Zone of inhibition (diameter in mm)

A B C
Staphylococcus aureus 9 10 12
Escherichia coli 9 10 11
Pseudomonas aeruginosa 9 11 13
From the figures 6, 7(a), 7(b) and tables 3, 4 it is found that the bio-functionalised nanoparticles
synthesised from Adathoda vasica leaf extract seems to be highly anti-bacterial when compared to the
standard antibiotic at the same concentration. Hence, these can be administered in various applications. The
formulated nano-based soap and nano-based cold cream does have their bactericidal property and seemingly
higher than that of the available commercial soap and cream.
CONCLUSION
Nanoparticles are having their wide applications in various fields. The average size of silver nanoparticles
synthesised using Adathoda vasica leaf extract was 30 nm. The natural and cost effective route of synthesis
with achieving smaller size nanoparticles has been used to formulate nano-based products such as soap and
moisturizing cold cream. The samples were tested against three pathogens namely Staphylococcus aureus, a
gram positive bacterium whereas Escherichia coli and Pseudomonas aeruginosa are gram negative bacteria.
Surprisingly, both have great anti-bacterial properties when compared to commercial anti-bacterial soap and
cream. Hence, the formulated products are prospective applications of nanotechnology which can be widely
used in the pharmaceutical industry.
ACKNOWLEDGEMENT
The authors kindly acknowledge the Department of Science and Technology, New Delhi, Govt. of India
sponsored National Facility for Drug Development for Academia, Pharmaceutical and Allied industries
(VI-D&P/349/10-11/TDT), Bharathidasan Institute of Technology, Anna University, Tiruchirappalli for its
valuable and sophisticated facilities to carry out the present study.
REFERENCES
1. Mnyusiwalla A, Daar AS and Singer PA. ‘Mind the gap’: science and ethics in nanotechnology.
Nanotechnology. 2003; 14(3):R9.
2. Suri SS, Fenniri H and Singh B. Nanotechnology-based drug delivery systems. J Occup Med
Toxicol. 2007; 2(1):16.
3. Yasin S, Liu L and Yao J. Biosynthesis of silver nanoparticles by bamboo leaves extract and their
antimicrobial activity. JFBI. 2013; 6(1):77-84.
170
4. He S, Zhang Y, Guo Z and Gu B. Biological Synthesis of Gold Nanowires Using Extract of

Rhodopseudomonas Capsulate. Biotechnol Prog. 2008; 24:476-480
5. Sondi I and Salopek-Sondi B. Silver nanoparticles as antimicrobial agent: a case study on E. coli as
a model for Gram-negative bacteria. J Colloid Interface. 2004; 275(1):177-182.
6. Lalitha A, Subbaiya R and Ponmurugan P. Green synthesis of silver nanoparticles from leaf extract
Azhadirachta indica and to study its anti-bacterial and antioxidant property. Int J Curr Microbiol
App Sci. 2013; 2(6):228-235.
7. Chandran SP, Chaudhary M, Pasricha R, Ahmad A and Sastry M. Synthesis of gold nanotriangles
and silver nanoparticles using Aloevera plant extract. Biotechnol. Progr. 2006; 22(2):577-583.
8. Shankar SS, Rai A, Ahmad A and Sastry M. Rapid synthesis of Au, Ag, and bimetallic Au core–Ag
shell nanoparticles using Neem Azadirachta indica leaf broth. J Colloid Interface. 2004; 275(2):496-
502.
9. Song JY, Jang HK and Kim BS. Biological synthesis of gold nanoparticles using Magnolia kobus
and Diopyros kaki leaf extracts. Process Biochem. 2009; 44(10): 1133-1138.
10. Krishnaraj C, Jagan EG, Rajasekar S, Selvakumar P, Kalaichelvan PT and Mohan N. Synthesis of
silver nanoparticles using Acalypha indica leaf extracts and its antibacterial activity against water
borne pathogens. Colloid Surface B. 2010; 76(1):50-56.
11. Li S, Shen Y, Xie A, Yu X, Qiu L, Zhang L and Zhang Q. Green synthesis of silver nanoparticles
using Capsicum annuum L. extract. Green Chem. 2007; 9(8):852-858.
12. Bar H, Bhui DK, Sahoo GP, Sarkar P, De SP and Misra A. Green synthesis of silver nanoparticles
using latex of Jatropha curcas. Colloid Surface A. 2009; 339(1):134-139.
13. Sathishkumar M, Sneha K, Won SW, Cho CW, Kim S and Yun YS. Cinnamon zeylanicum bark
extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal
activity. Colloid Surface B. 2009; 73(2):332-338.
14. Stoimenov PK, Klinger RL, Marchin GL and Klabunde KJ. Metal oxide nanoparticles as bactericidal
agents. Langmuir. 2002; 18(17):6679-6686.
15. Wickline SA and Lanza GM. Nanotechnology for molecular imaging and targeted therapy.
Circulation. 2003; 107(8):1092-1095.
16. Farokhzad OC and Langer R. Impact of nanotechnology on drug delivery. ACS nano. 2009; 3(1):16-
20.
17. Stephens-Altus JS and West JL. Nanotechnology for tissue engineering. Adv Tissue Eng. 2008;
333.
18. Maneerung T, Tokura S and Rujiravanit R. Impregnation of silver nanoparticles into bacterial
cellulose for antimicrobial wound dressing. Carbohyd polym. 2008; 72(1):43-51.
19. Kaviya S, Santhanalakshmi J, Viswanathan B, Muthumary J and Srinivasan K. Biosynthesis of
silver nanoparticles using citrus sinensis peel extract and its antibacterial activity. Spectrochim
Acta Mol Biomol. 2011; 79(3):594-598.
20. Elumalai EK, Prasad TNVKV, Hemachandran J, Therasa SV, Thirumalai T and David E. Extra-
cellular synthesis of silver nanoparticles using leaves of Euphorbia hirta and their antibacterial
activities. J Pharm Sci Res. 2010; 2(9):549-554.
21. Chakraborty A and Brantner AH. Study of alkaloids from Adhatoda vasica Nees on their
antiinflammatory activity. Phytother Res. 2001; 15(6):532-534.
22. Ramachandran J and Sankaranarayanan S. Antifungal Activity and the mode of Action of Alkaloid
Extract from the Leaves of Adhatoda Vasica. 2003;
171
23. Dhuley JN. Antitussive effect of Adhatoda vasica extract on mechanical or chemical stimulation-
induced coughing in animals. J Ethnopharmacol. 1999; 67(3):361-365.
24. Karthick V, Ganesh Kumar V, Maiyalagan T, Deepa R, Govindaraju K, Rajeswari A and Stalin
Dhas T. Green Synthesis of Well Dispersed Nanoparticles using Leaf Extract of Medicinally useful
Adhatoda Vasica Nees. Micro Nano. 2012; 4(3):192-198.
172
In vitro Cytotoxicity Assessment of Silver Nanoparticles
Synthesized Using Aspergillus Terreus Against Breast
Cancer Cell Line
M.D. Bala Kumaran and P.T. Kalaichelvan
CAS in Botany, University of Madras, Chennai – 600 025, India

e-mail: dakshinbala@gmail.com, Mob: +919500077165
Abstract:
Biosynthesis of nanoparticles is under exploration due to their wide range of biomedical applications and
research interest in nanotechnology. Nano-sized particles with antimicrobial and anticancer properties are
of great interest in the field of medical research. The present study focuses on the extracellular biosynthesis
of silver nanoparticles (AgNPs) from silver nitrate solution using Aspergillus terreus, isolated from soil.
The biologically synthesized silver nanoparticle was tested for its anticancer property against breast cancer
(MCF) cell line. The fungal extract was reacted with AgNO3 and the synthesis of nanoparticles was screened
using UV–Vis spectroscopy. The synthesized nanoparticles were then characterized using XRD and TEM
equipped with EDS and the size of the silver nanoparticles was found to be 5 to 20 nm with spherical shape.
The invitro cytotoxicity of silver nanoparticles toward MCF cell lines was assessed by MTT assay using
dose dependant approach. The apoptosis property was studied by acridine orange and eithidium bromide
staining method and the biologically synthesized silver nanoparticles showed good cytotoxicity activity
against breast cancer cell line with an IC50 value of 35.8 µg/ml.
Keywords: Silver nanoparticles, Aspergillus terreus, MCF Cell line
INTRODUCTION
Nanotechnology is an emerging field of science with immense scope in the disciplines of electronics,
nanomedicine, aeronautics, biomaterials and energy, cosmetics, and food [1]. Developments in the
organization of nanoscale structures into predefined superstructures ensure that nanotechnology will play
critical role in many key technologies [2-3].
Nanoparticles possess unique electrical, optical as well as biological properties and are thus applied in
catalysis, biosensing, imaging, drug delivery, nanodevice fabrication and in medicine [4]. In the past few
years, there has been an increasing interest in silver nanoparticles on account of the antimicrobial properties
that they display [5]. They are even being projected as future generation antimicrobial agents [6].
In recent years, the use of nanoparticles, particularly metal nanoparticles has expanded in biomedical
research. They are used in diagnosis and therapeutics due to their unique properties of small size, large surface
area to volume ratio, high reactivity to the living cells, stability over high temperatures and translocation into
the cells, etc. They are available in different sizes and shapes due to their ability to react and agglomerate
with other nanoparticles in their surroundings. They also exhibit exceptional optical properties making them
capable of producing quantum effects suitable for imaging applications. Most commonly studied metal
nanoparticles include gold, silver, titanium oxide and iron nanoparticles [7].
Nanoparticles (NPs) exhibit quite a number of interesting and unique properties based on their specific
characteristics, such as size, distribution and morphology that leads itself to various applications, for instance
in catalysis, microelectronics, sensors , biomaterials, therapeutics as well as antimicrobial applications [8].
The present study was aimed at investigating fungus mediated synthesis of silver nanoparticles and its
in vitro cyto toxicity activity against human breast cancer cell line.

Source of microorganisms
In the present study, Aspergillus terreus isolated from soil samples was subjected for screening of silver
nanoparticles synthesis. The fungus was maintained in potato dextrose agar (HiMedia, Mumbai, India) slant
at 27°C and used for further study.
Biomass Production and synthesis of AGNPs

To prepare the fungal biomass for synthesis process, the fungus was grown aerobically in potato dextrose
broth and incubated for 7 days at 27°C. After incubation, the fungal mat was extensively washed using
sterile double distilled water to remove the medium components. Further, 10g of biomass was taken in
100ml of sterile double distilled water in an Erlenmeyer flask and incubated in an orbital shaker at 27°C for
72 hours at 120 rpm. After incubation, the filtrate was collected using whatman No.1 filter paper and used
for further study. To the 100 ml fungal filtrate, calculated amount of silver nitrate was added to reach a final
concentration of 1mM and reaction was carried out under dark conditions [9].
Characterization of AgNPs
The time-dependent synthesis of silver nanoparticles was observed using ultraviolet-visible (UV-vis)
spectrophotometry (Hitachi 2900 UV-Vis spectrophotometer). The biologically synthesized silver
nanoparticles were further characterized using transmission electron microscopy (TEM) by drop-coating the
AgNPs solution onto the carbon grid for their morphology and size. The presence of ions were confirmed
using energy-dispersed spectroscopy (EDS) equipped along with TEM. Further the silver nanoparticles was
characterized using XRD to study crystalline nature of AgNPs [10].
Cell culture
Vero (African green monkey kidney cell line/normal cell line) and MCF (Breast cancer cell line) cell line
was obtained from NCCS, Pune, India and cells were grown at 37 °C in 5% carbon dioxide atmosphere
using Eagle’s minimal essential medium (EMEM) by supplementing with 10% fetal bovine serum and 1%
antibiotic (PSN). For experimental studies, the cells were allowed to grow to 80–90% confluence, harvested
followed by trypsinization and plated at desired density. Cells were then re-equilibrated for 24 h before any
treatment [1].
Cytotoxicity of silver nanoparticles

It is essential to determine the drug compatibility and dosage on normal cell lines before studying the
antiproliferation effect against any cancer cell lines. In the present study different concentration (1000 to
1.95 µg/ml) of silver nanoparticles were treated against both Vero cell line and MCF cell line. Both the
cells were cultured at a density of 1x106/well in Eagle’s minimal essential medium supplemented with 10%
fetal bovine serum and 1% antibiotic in 96-well plates and incubated at 37°C in a humidified atmosphere
supplemented with 5% CO2 for 48 hours. Then the cells were treated with different known concentrations of
silver nanoparticles (10 to 100 µg/ml) for 48 hrs. After incubation, the wells were washed with phosphate-
buffer saline (PBS), pH-7.4 and then 20µl/well (5mg/ml in PBS) of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-
diphenyl--tetrazolium bromide (MTT) was added in each wells. After 4 hours of incubation, 100 µl of
dimethyl sulfoxide (DMSO) was added and absorbance was determined at 450 nm using micro plate reader.
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The readings were used for the calculation of % cell viability [11]. The effect of the biologically synthesized
silver nanoparticles on the proliferation of Vero and MCF cell lines was expressed as the % cell viability,
using the following formula:
% cell viability = (A450 of treated cells / A450 of control cells) × 100%
Acridine orange/ethidium bromide dual staining
Acridine orange/ethidium bromide (AO/EB) dual staining was performed to detect the morphological
characteristics of apoptosis in silver nanoparticles treated cells MCF cell line. Treated and untreated cell
grown in 24 well plates (5 × 106 cells/mL) was stained with 10 μl of acridine orange and ethidium bromide
dye mix (100 μg/ml of acridine orange and ethidium bromide, separately prepared in PBS) [12]. The samples
were then examined and photographed on the FLoid™ Cell Imaging Station (Life Technologies).

The test strain, Aspergillus terreus was isolated from soil sample and identified as Aspergillus terreus using
ITS 1 and 2 partial sequencing. The sequence was analyzed using BLAST tool and submitted in GenBank
under the accession no KF032709.1.
The formation of silver nanoparticles was studied based on the initial change in the colour from yellow
to dark brown (Fig. 1). It is inferred that silver nanoparticles exhibit yellowish brown color in aqueous
solution due to excitation of surface plasmon vibrations in the metal nanoparticles [13, 14].

Fig. 1: Screening of AGNPs Fig. 2: UV Spectrum Analysis of AGNPs

Fig 3: TEM image of AGNPs Fig 4: IC50 value of MCF cell line treated with AGNPs
Fig 5: AO/EB staining of MCF cell line treated with AGNPs
175
The formation and stability of the reduced AgNPs in the fungal filtrate was monitored by using UV–vis
spectral analysis [15]. As illustrated in Fig 2, a strong and broad peak located at 435 nm was observed for
the AGNPs synthesized using the fungal extract. Observation of this peak, assigned to a surface plasmon, is
well-documented for silver nanoparticles with size ranging from 2 to 100 nm [16].
The TEM and EDS studies reveals that the AgNPs are spherical in shape with size ranging from 5 to 30
nm (Fig.3) and presence of elemental silver ions were also confirmed using EDS technique. The crysltalline
nature of AgNPs was studied using XRD spectrum, which showed peaks at 2θ values of 38.25°, 46.37°,
64.60° and 77.62°, corresponding to 111, 200, 220 and 311 planes for silver, respectively which agree well
with those reported for silver nanoparticles.
The toxicity study of biologically synthesized silver nanoparticles against Vero cell line and MCF
was studied and the IC50 value of 70 and 36 µg/ml were determined respectively (Fig 4). The apoptotic
morphological changes during the treatment of AgNPs against the MCF cell line was also observed using AO/
EB staining method (Fig 5), which also revealed viable (green colour), apoptotic (early-green fluorescence
with condensed chromatin and late-orange colour) and dead (red colour) cells. Contrary studies were also
investigated by various researchers who have also reported the ability of silver nanoparticles for the cytotoxic
activity various cancer cell lines (17-19).
Apoptosis is broadly considered as a distinctive mode of programmed cell death that eliminates
genetically determined cells. The induction of apoptosis is confirmed by two factors, (1) reduced and
shrunken cells and (2) DNA fragmentation. In this study, silver and gold nanoparticles treated cells showed
apoptotic features such as condensed nuclei, membrane blebbing and apoptotic bodies at 48 h and these
morphological changes were evident through AO/EB dual staining. Adding strengthen to the fact, silver and
gold nanoparticles treated MDA-MB-231 cells showed clear fragmented DNA ladders, suggesting that cell
death is due to apoptosis [20-21].
The above results revealed that that the biologically synthesized silver nanoparticles using Aspergillus
terreus strain shows significant anti proliferation activity against MCF cell lines, which gives way to explore
the potential of these components in the healthcare and pharmaceutical industries.
CONCLUSION
In this present study, AgNPs synthesized using fungal extract of Aspergillus terreus was investigated as
it is considered to be simple, less toxic and economically safe clinical research. Besides, the synthesized
nanoparticles were in the range of 5 to 20 nm it also exhibited impressive cytotoxicity property against MCF,
breast cancer cell line. The results of AO/EB staining also showed the presence of early and late apoptotic
cells which is a further evidence of apoptotic property. However, further extensive studies are required to
understand mechanism of apoptotic activity against the cancer cell line which may help in the future research.
REFERENCES
1. Bhattacharyya D, Singh S, Satnalika N, Khandelwal A and Jeon SH, Nanotechnology, big things from a
tiny world: a review. Int J u- and e- Serv, SciTechnol. 2009; 2(3):29–38.
2. Aboofazeli R, Carbon nanotubes: a promising approach for drug delivery. Iran. J. Pharm. 2010; 9: 1–3.
3. Orafai H., Kallinteri P, Garnett M, Huggins S, Hutcheon G and Pourcain C, Novel poly (glycerol-
adipate) polymers used for nanoparticle making: a study of surface free energy. Iran.J. Pharm. Res.
2008; 7: 11–19.
4. Nair LS and Laurencin CT, Silver nanoparticles: synthesis and therapeutic applications, J. Biomed.
Nanotechnol., 2007; 3: 301–316.
176
5. Choi O, Deng KK, Kim NJ, Ross L.Jr, Surampalli RY and Hu Z, The inhibitory effects of silver nanoparticles,
silver ions, and silver chloride colloids on microbial growth, Water Res., 2008; 42: 3066–3074.
6. Rai M, Yadav A and Gade A, Silver nanoparticles as a new generation of antimicrobials, Biotechnol.
Adv., 2009; 27: 76–83.
7. Ansary A and Al-Daihan S, On the toxicity of therapeutically used nanoparticles: An overview.
J. Toxicol. 2009; 1–9.
8. Tolaymat T, El Badawy A, Genaidy A, Scheckel K, Luxton T and Suidan M, An evidence-based
environmental perspective of manufactured silver nanoparticle in syntheses and applications: A
systematic review and critical appraisal of peer-reviewed scientific papers. Sci. Tot. Environ., 2010;
(408) 5:999–1006.
9. Fayaz M, Girilal M, Venkatesan R and Kalaichelvan PT, Biosynthesis of anisotropic gold nanoparticles
using Maduca longifolia extract and their potential in infrared absorption. Colloids and surfaces. B,
Biointerfaces, 2011; 88 (1): 287–91.
10. Fayaz M, Balaji K, Kalaichelvan PT and Venkatesan R, Fungal based synthesis of silver nanoparticles--an
effect of temperature on the size of particles. Colloids and surfaces. B, Biointerfaces, 2009; 74 (1) p. 123–6.
11. Mosmann T, Rapid colorimetric assay for cellular growth and survival: application to proliferation and
cytotoxicity assays. J Immunol Methods. 1983; 65(1-2):55–63.
12. Winnicka F, Bielawski K, Bielawska A and Milty W, Apoptosis-mediated cytotoxicity of ouabain,
digoxin and proscillaridin A in the estrogen independent MDA-MB-231 breast cancer cells. Arch.
Pharm. Res., 2007; 30: 1216–1224.
13. Basavaraja S, Balaji SD, Lagashetty A. Rajasab AH and Venkataraman A, Extracelluar biosynthesis of
silver nanoparticles using the fungus Fusarium semitectum. Mater. Res. Bull., 2008; 43; 1164–1170.
14. Ahmad A, Senapati S, Khan MI, Kumar R and Sastry M, Extracellular Biosynthesis of Monodisperse
Gold Nanoparticles by a Novel Extremophilic Actinomycete, Thermomonospora sp. Langmuir, 2003;
19(8) 3550.
15. Sastry M, Ahmad A, Khan MI and Kumar R, Biosynthesis of metal nanoparticles using fungi and
actinomycete. Current Science, 2003; 85; 162–170.
16. Karuppaiya P, Satheeshkumar E, Chao WT, Kao Y, Chen ECF and Tsay HS, Anti-metastatic activity of
biologically synthesized gold nanoparticles on human fibro sarcoma cell line HT-1080. Colloids Surf.
B, 2013;110; 163–170.
17. Fong J and Wood F, Nanocrystalline silver dressings in wound management: a review Int. J. Nanomedicine,
2006; 1: 441–449.
18. Gurunathan S, Jegadeesh R, Sri Nurestri AM, Priscilla AJ and Vikineswary S, Green synthesis of silver
nanoparticles using Ganoderma neo-japonicum Imazeki: a potential cytotoxic agent against breast
cancer cells. Int. J. Nanomedicine, 2013; 8: 4399–4413.
19. Franco JL, Posser T and Dunkley PR, Methyl mercury neurotoxicity is associated with inhibition of the
antioxidant enzyme glutathione peroxidase. Free Radical Bio. Med., 2009; 7: 449–457.
20. Jeyaraj M, Rajesh M, Arun R, Mubarak Ali D, Sathishkumar G, Sivanandhan G, Kapildev G,
Manickavasagam M, Premkumar K, Thajuddin N and Ganapathi A, An investigation on the cytotoxicity
and caspase-mediated apoptotic effect of biologically synthesized silver nanoparticles using Podophyllum
hexandrum on human cervical carcinoma cells. Colloids Surf. B, 2013; 102: 708–717.
21. Vivek R, Thangam R, Muthuchelian K, Gunasekaran P, Kaveri K and Kannan S, Green biosynthesis of
silver nanoparticles from Annona squamosa leaf extract and its in vitro cytotoxic effect on MCF-7 cells.
Process Biochem., 2012; 47: 2405–2410.
177
Larvicidal and Antimicrobial Potency of Silver
Nanoparticles Synthesized Using an Actinomycete,
Saccharopolyspora Erythraea
Prabha S. B. and Murugesan K.
CAS in Botany, University of Madras, Guindy campus, Chennai- 25, Tamilnadu, India.
e-Mail: pkprabha27@gmail.com, Mob: 9445319254
Abstract:
The actinomycetes are ubiquitous in nature. They are widely exploited for their antimicrobial activity.
However, it is less exploited in terms of production of nanoparticles. Thus the species of actinomycetes,
Saccharopolyspora erythraea is used in the synthesis of Silver Nano Particles (SNPs). The synthesized
nanoparticles are characterized using UV spectrophotometer, XRD, FTIR, FE-SEM and HR-TEM. The
surface plasma resonance was observed at 450nm which confirmed the formation of AgNP’s. The XRD
pattern confirmed the formation of nanocrystals. The FTIR analysis proved the reduction of silver salts into
respective nanoparticles. In FE-SEM with EDX, the average size of the nanoparticle was found to be 82.13 to
93.86nm. The HR-TEM analysis with SAED showed that the synthesized AgNP’s were polydispersed in 6 to
26 nm scale. The antimicrobial activity of the ethyl acetate extract of the isolate Saccharopolyspora erythraea
(AMP14) amended with that of the SNPs gave good results and showed maximum zone of inhibition against
human pathogens namely Bacillus subtilis, Klebsiella pneumoniae, Vibrio cholerae, Salmonella typhi and
Staphylococcus aureus. The ethyl acetate extract of the isolate Saccharopolyspora erythraea (AMP14) was
found to be less toxic when compared with that of the synthesized silver nanoparticles against the larvae of
Anopheles stephensi and Aedes aegypti.
Keywords: Saccharopolyspora erythraea, Silver nanoparticle, Antimicrobial activity, Larvicidal activity,
HR-TEM. Anopheles stephensi, Aedes aegypti.
INTRODUCTION
Actinomycetes are screened for its routine bioactive substances for its high commercial value of bioactive
compounds. Approximately two thirds of antibiotics and several medicinal important compounds have
been isolated from actinomycetes. Some of the secondary metabolites species of actinomycetes produce are
avermectins, tetranectin, faerifungin and macrotetrolides and flavonoids produced by the genera involved are
Micromonospora, Actinomadura, Actinoplanes, Micropolyspora, Nocardiopsis, Oerskonia, Thermonospora,
Streptoverticillium and Chainia were found to be toxic for the mosquitoes (Ando, 1983). The Gram positive
actinomycetes are primarily recognized as the potential source for antibiotics, potential degraders and
organism of academic curiosity. Actinomycetes are less exploited for production of nanoparticles, eventhough
they are well exploited for the production of high metabolite value and antibiotics. For the intracellular and
extracellular biosynthesis of nanoparticles, only limited reports are available for actinomycetes.
Synthesis of nanoparticles through green synthesis using plants or microorganisms could potentially
eliminate the problems like high chemical cost, environmental toxicity, energy consumption etc.,. Indeed,
over the past several years, plants, algae, fungi, bacteria, and viruses have been used as an energy-efficient
and nontoxic production of metallic nanoparticles. Silver nanoparticles shows very strong bactericidal
activity against Gram positive as well as Gram negative bacteria including multiresistant strains (7). Hence
there is a huge scientific progress in the study of biological application of ZnO and Ag and other metal
nanoparticles. Actinomycetes provide a valuable resource for novel products of industrial interest, including
antimicrobial agents. Silver nanoparticles have been used extensively in the food storages, textile coating, in
health industry and it contains number of environmental applications as anti bacterial and larvicidal agents.
Mosquitoes are the vectors of various dreadful diseases of mankind and one of the most medically
significant vectors transmitting pathogens and continue to have a devastating impact on human beings and
other animals. Among the 3492 species of mosquitoes recorded worldwide, more than a hundred species are
capable of transmitting various diseases to human and other vertebrates such as malaria, dengue, filariasis,
yellow fever, Japanese encephalitis and chikungunya.
Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus are the major urban vectors of malaria,
dengue and lymphatic filariasis respectively. About 90% of all malarial deaths in the world occur mostly in
Africa and South Sahara. Aedes aegypti is the principal vector of dengue fever, hemorrhagic dengue fever
and is reported to infect more than hundred million, every year in South East Asia to a lesser extent in more
than 110 countries in the tropics. incidence of dengue fever has increased in the last few years, and nearly
half of the world’s population is now at risk. Among 72 countries worldwide more than 1.3 billion people are
threatened by lymphatic filariasis, commonly known as elephantiasis. Over 120 million people are infected
currently, with about 40 million disfigured and incapacitated by the disease (WHO, 2012). Culex mosquitoes
are painful and persistent biters and are responsible for filariasis.
The most widely used conventional methods for mosquito control are the chemical control since it
produces immediate control and relatively inexpensive. Chemical control is generally carried out by the
indoor residual spraying of insecticides such as dichloro diphenyl tri-chloro ethane, hexa chlorocyclo
hexane, benzene hexa chloride, melathion and synthetic pyrothroid. The development of resistance against
these chemicals in various mosquito populations has been reported. One of the approaches for controlling
mosquito- borne diseases is the interruption of disease transmission by either killing or preventing mosquito
bite by using repellents or by causing larval mortality in a large scale at the breeding centers of the vector.
The control of mosquito larvae worldwide depends on continued application of organophosphates and insect
growth regulators.
In this study, an attempt was made to synthesize silver nanoparticles using actinomycete species,
Saccharopolyspora erythraea and its application against bacteria and mosquito larvae.
Saccharopolyspora erythraea
The species of actinomycetes was isolated from the soil sample and characterized for its physio-chemical
and biochemical characters for identification.
Screening of actinomycetes for antibacterial efficacy

The selected actinomycetes were screened for antibacterial efficacy by agar well diffusion method. The five
human pathogens namely Bacillus subtilis, Klebsiella pneumoniae, Vibrio cholerae, Salmonella typhi and
Staphylococcus aureus were obtained from Microbial Type Culture Collection (MTCC), Chandigarh, India.
Synthesis of Ag NPs
Five mL of this bacteria S. erythraea ethyl acetate extract was added to 95 mL of 1 mm aqueous silver nitrate
solution for the reduction of Ag+ ions. The effect of temperature on the synthesis rate of AgNPs was studied
by incubation at 37°C for 24 h. The AgNPs solution, thus obtained was purified by centrifugation twice at
179
10,000 g. for 15 min at 4°C. A control setup was also maintained without S. erythraea ethyl acetate extract
and color intensity of the extracts was measured.

UV- visible spectra were recorded as a function of the reaction time on UV- 160V, spectrophotometer,
Shimadzu, Japan. The studies on size, morphology and composite of the nanoparticles were performed by
means of HR TEM, FE SEM and EDX. The purified AgNPs were examined for the presence of biomolecules
using FTIR analysis. The spectrum obtained from the dried sample was recorded on FTIR spectrum in the
diffuse reflectange mode at a resolute on of 4 cm-1 in KBr pellets. Crystalline AgNPs were determined by
X-ray diffraction analysis. Briefly, the biosynthesized AgNPs were laid onto glass substrate on Phillips PW
1830 instrument operating at a voltage of 40 kv and current of 30 mA with Cu Kα 1 radiation.
Larvicidal bioassay
The larvae of A. stephensi and A. aegypti were collected from rice field and stagnant water areas of Kanchipuram
and identified in The Zonal Entomological Research Centre, Vellore, Tamil Nadu to start the colony. The larvae
were reared in the laboratory accordingly. One gram of ethyl acetate extract was first dissolved in 100 ml of
distilled water (stock solution). From the stock solution, 100 mg/L was prepared with dechlorinated tap water
for bioassay test of ethyl acetate extract. The larvicidal activity was assessed by the procedure of WHO (1996)
with modifications. For bioassay test, larvae were taken in five batches of 20 in 199 mL of water and 1.0 ml of
ethyl acetate extract concentration. The control was set up with dechlorinated tap water. The numbers of dead
larvae were counted after 24 h of exposure, and the percentage of mortality was reported from the average of
five replicates. The experimental media in which 100% mortality of larvae occurs were alone selected for dose–
response bioassay. Synthesized AgNPs toxicity test was performed by placing 20 mosquito larvae into 200 ml
of sterilized double distilled water with nanoparticles. The nanoparticle solutions were diluted using double
distilled water as a solvent according to the desired concentrations (10, 8, 6, 4 and 2 mg/l). Each test included
a set of control groups (silver nitrate and distilled water) with five replicates for each individual concentration.
Mortality was assessed after 24 h to determine the acute toxicities against fourth instar larvae of A. stephensi
and A. aegypti . Twenty mosquito larvae were placed into 250 ml glass beakers and set in an environmental
chamber at 25°C with a 16:8 h light/dark cycle. Each beaker containing the mosquito larvae distilled water
was spiked with solutions of AgNPs in order to achieve target nominal concentrations of 10, 8, 6, 4 and 2 mg/l
with a final volume of 200 ml. A negative control (silver nitrate) was used in all experiments and all conditions
were tested in five replicates. In order to compare the mortality of synthesized AgNPs to that of dissolved Ag
released the mosquito larvae were exposed to a range of dissolved Ag concentrations so as to cover the range
released from all doses of AgNPs. To avoid settling of particles especially at higher doses, all treatments were
sonicated for an additional 5 min prior to the addition of mosquito larvae. This additional sonication appeared
to significantly decrease the settling of particles.
Dose response bioassay

Based on the preliminary screening results, crude bacteria ethyl acetate extract of S. erythrae and synthesized
AgNPs were subjected to dose–response bioassay for larvicidal activity against the larvae of A. stephensi
and A. aegypti. Different concentrations ranging from 6.25 to 100.0 mg/l (aqueous extract) and 2.0 to 10.0
mg/l (synthesized AgNPs and silver nitrate) were prepared for larvicidal activity. The numbers of dead larvae
were counted after 24 h of exposure, and the percentage mortality was recorded from the average of five
replicates.
Data analysis
The average larval mortality data were subjected to probit analysis for calculating LC50, LC90 and other
statistics at 95 % confidential limits and chi-square values were calculated using the software. Results with
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p < 0.05 were considered to be statistically significant. Mean percent larval mortality data were subjected to
analysis of variance and compared with Duncan’s multiple range tests to determine any differences between
plant species and within species and concentrations (SPSS, 11.5). Prior to analysis, mortality in treatments
was corrected with that in controls using Abbott formula.
UV- vis analysis of Saccharopolyspora erythraea

Silver nanoparticles were synthesized using ethyl acetate extract of S. erythraea and within five seconds,
of incubation period, the colour change was observed by visual observation (Fig 1) Ethyl acetate extract
of S. erythraea without AgNO3 did not show any color change (Fig 2). The aqueous silver nitrate solution
turned to brown within five seconds and the intensity increased in direct proportion to incubation period.
The absorption spectrum of S. erythraea ethyl acetate extract at different wavelength range from 200 to
800 nm and the silver surface plasma resonance observed at 450 nm confirmed the formation of AgNPs.
Chemical synthesis resulted in the formation of brown colour which results from absorption by colloidal
silver nanoparticles in the visible (380- 450 nm) range of the electromagnetic spectrum. The colour formation
was mainly due to the surface plasmon resonance of deposited silver nanoparticles and silver nanoparticles
exhibit striking colors due to excitation of surface plasma on vibrations in the particles (1).
XRD pattern of synthesized silver nanoparticles

The XRD pattern compared with the standard confirmed spectrum of silver particles formed in the present
study were shown in (Fig 3) the form of nanocrystals as evidenced by the peaks at 2θ values of 27.71°,
32.16°, 46.15°, 54.70° and 57.68° corresponding to 210, 122, 231, 142, and 241 facets of the face centered
cubic crystal structure, which is a characteristic of nano crystallites, the prominent peaks corresponding to
210 brags reflection. X-ray diffraction (XRD) pattern show in tense Braggs reflection that can be indexed on
the basis of the fcc structure of silver (5).
FTIR analysis
FTIR analysis was performed to identify the biomolecules localized on the surface and responsible for the
reduction of silver salts into the respective nanoparticles. FTIR band intensities in different regions of the
spectrum for the synthesized Ag NPs were analyzed (Fig4) and the spectra showed the respective peaks and
functional groups of 2922 cm-1 (C-H, stretch, alkanes), 1753 cm-1 ( C=O, stretch carboxylic acid), 1633 cm-1
(N-H bend 1˚ amines), 1107 cm-1 ( C-N, stretch aliphatic amines). Silver nanoparticles were probably attached
to the nitrogen of amine and the amide groups and oxygen of carbonyl groups. The overall observations
confirmed the presence of protein in the sample of silver nanoparticles. It is reported earlier that proteins
could bind to nano particles either through free amine groups or cystiene residues (2).
HR- TEM analysis with SAED

HR-TEM images revealed that the synthesized silver nanoparticles are polydispersed in 6 to 26 nm scale
and they were spherical in shape with varying size (Fig 5). However, in contrast to many other reports of
metal-resistant bacteria, for which efflux of toxic ions is the main detoxification mechanism (3), majority
of the accumulated silver is deposited as seed like particles between the outer membrane and the plasma
membrane, the size of the deposits small (5–15 nm) compared to previous studies using P. stutzeri Ag259
(35–46 nm), which may be caused by different cell growth and metal incubation conditions.
FE- SEM analysis with EDX

The FE-SEM images of nanoparticles having prominently cubic structure and the magnified SEM image
confirmed that the silver nanocubes are in well resolved cubic structure with soft surface and with sharp
181
edges with an average size of 82.13 to 93.86 nm. The EDX attachment with the FE-SEM was known to
provide information on the chemical analysis of the fields that are being investigated or the composition at
specific locations (Fig 6). Representative profile of the spot EDX was obtained by focusing on AgNPs. In
this micrograph, silver nanoparticles in the size range 50-100 nm were observed (4).
Larvicidal activity of ethyl acetate extract of S. erythraea and synthesized silver

nanoparticles
In the present study, range of concentrations of S. erythraea (100, 50, 25, 12.5 and 6.5 mg/L) and synthesized
AgNPs (10, 8, 6, 4 and 2 mg/L) were tested against 4th instar of A. stephensi, and A. aegypti. The LC50 and
LC90 along with upper and lower confidence limit values and regression equations of A. stephensi, and A.
aegypti against ethyl acetate extract of S. erythraea and the synthesized Ag NPs are presented in (Table 1).
The ethyl acetate extract of S. erythrae were less toxic than the synthesized silver nanoparticles against
three tested mosquito species. The LC50 and LC90 values of ethyl acetate extract of S. erythrae treated against
fourth instar larvae of A. stephensi (LC50 = 32.99; LC90 = 94.07 mg/L) and A. aegypti (LC50 = 37.26; LC90 =
114.00 mg/L). While LC50 and LC90 values of synthesized AgNPs treated against fourth instars larvae of A.
stephensi (LC50 = 3.89; LC90 = 11.82 mg/L) an A. aegypti (LC50 = 4.02; LC90 = 12.64 mg/L) respectively. The
larvicidal potential of hexane, chloroform, ethyl acetate, acetone, methanol, and aqueous leaf extracts of N.
nucifera and synthesized silver nanoparticles using aqueous leaf extract against the fourth instar larvae of
A. subpictus and C. quinquefasciatus have been tested . These silver nanoparticles have been tested against
dengue and malarial vector. There is no data available on the efficacy of silver nanoparticles synthesized
using bacteria against mosquito larvae. The larvicidal potential of silver nanoparticles synthesized using
fungus C. lunatus A. aegypti and A. stephenesi has been tested .
Antimicrobial efficacy of Saccharopolyspora erythraea

Screening of the isolated actinomycetes amended with silver nitrate for their antibacterial activity against
five human bacterial pathogens was do ne using well diffusion method. Among all the isolates which are
amended with silver nitrate screened the maximum inhibition zone was observed in Staphylococcus aureus
compared with the other four human bacterial pathogens such as Bacillus subtilis, Klebsiella pneumoniae,
Vibrio cholerae, and Salmonella typhi.
Fig 1: Synthesis of Nanoparticles from the isolate AMP14
182
Fig 2: UV- VIS absorption spectra of silvernanoparticle synthesized frrom Saccharopolyspora erythraea at 1mM silver
nitrate.
Fig 3: XRD patterns of AgNPs synthesized using ethyl acetate extract of Saccharopolyspora erythraea.
183
Fig 4: FTIR spectrum of synthesized AgNPs using ethyl acetate extract of Saccharopolyspora erythraea.
Fig 5: Transmission electron microscopic image showing synthesized AgNPs from Saccharopolyspora erythraea.
Fig 6: FE-SEM and EDX image of AgNPs synthesized by Saccharopolyspora erythraea.
184
Table 1: Larvicidal activity of broth S. erythraea and synthesized AgNPs of S. erythraea against fourth instar larvae of
Anopheles stephensi and Aedes aegypti.
Extract / Species Concentration 24hr % LC50 ± SE (UCL– LC90 ± SE (UCL– 2 to the power
(mg/mL) Mortality* ± SD LCL) (mg/mL) LCL) (mg/mL) of r
Aqueous Aedes aegypti 100 92±1.30 37.26±1.58 114.00±18.36 0.984
50 72±2.06 (44.61-31.13) (130.42-98.17)
25 53±1.93
12.5 40±1.40
6.25 19±1.82
Aqueous Anopheles 100 97±1.28 32.99±3.18 94.07±12.38 0.972
stephensi 50 75±1.04 (40.52-26.85) (104.27-82.92)
25 59±3.04
12.5 42±2.56
6.25 23±1.88
AgNPs Aedes aegypti 10 100±0.00 4.02±0.19 12.64±0.44 0.992
8 82±1.60 (5.73-2.82) (16.49-9.31)
6 69±2.30
4 54±1.82
2 41±2.00
AgNPs Anopheles 10 100±0.00 3.89±0.22 11.82±0.99 0.991
stephensi 8 87±4.28 (7.59-1.99) (13.75-9.42)
6 73±2.08
4 52±0.82
2 46±2.56
ACKNOWLEDGEMENT
One of the authors SBP is thankful to the UGC- BSR for providing the Meritorious fellowship and to the
Director, CAS in Botany for the laboratory facilities.
REFERENCES
1. Kapoor,S., (1998), Preparation ,characterization and surface modification of silver particles.

Langmuir,14;1021-1025
185
2. Csaki, A., Moller, A. and Fritzsche, W., (2002). Gold nanoparticles as novel label for DNa diagnostics.
Expert Rev. Mol. Diag. 2(2); 187-193.
3. Gupta A, Matsui K, Lo JF and Silver S, (1999) Molecular basis for resistance to silver cations in
Salmonella. Nat Med 5:183–188. doi:10.1038/5545
4. Minaeian, S., Shahverdi, A. R., Nohi, A. S. and Shahverdi, H. R., 2008. Extracellular biosynthesis of
silver nanoparticles by some bacteria. J. Sci. I . A . U (JSIAU),Vol 17, No. 66, 2008.
5. S. Mandal, S. Phadtare and M. Sastry, Interfacing biology with nano particles. “Current Applied
Physics”, Vol. 5, No. 2; 2005. Pp. 118-127.
6. Mitsuiki, S., M. Sakai, Y. Moriyama, M. Goto and Furukawa, K., 2002. Purification and some properties
of Keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. Biosci. Biotechnol. Biochem. 66: 164-
167.
7. Shrivastava, S., T. Bera, A. Roy, G. Singh, P. Ramachandrarao and Dash, D., 2007. Characterization of
enhanced antibacterial effects of novel silver nanoparticles. Nanotechnol, 18:9-12.
8. Roe, D., B. Karandikar, N., Bonn – Savage, B. Gibbins and Roullet, J.B., 2008. Antimicrobial surface
functionalization of plastic catheters by silver nanoparticles. J. Antimicro. Chemo. 61: 869-76.
9. Zhang, L., Y. Jiang, M. Povey and York, D., 2007. Investigatin into the antimicrobial behavior of
suspension of ZnO nanoparticles (ZnO nanofluids). J. Nanoparti. Res. 9: 479-489.
186
Biomimetic Production and Cytotoxic Effect of Silver
Nanoparticles from Fungi
*S. Akila and Anima Nanda
Department of Biomedical Engineering, Sathyabama University, Rajiv Gandhi Salai, Chennai - 600119, India.
*e-mail: akila.hai@gmail.com, Mobile: 9176295082
Abstract:
Nanosized materials have been an important subject in basic and applied sciences because of their unique
optical, thermal, electrical, chemical, and physical properties that are due to a combination of the large
proportion of high-energy surface atoms compared to the bulk solid. AgNPs have been used extensively
as anti-bacterial agents in the health industry, food storage, textile coatings and a number of environmental
applications. The microbial-mediated biological synthesis of metallic nanoparticles has recently been
recognized as a promising source for mining nanomaterials. The biological approach to synthesize metal
nanoparticles is an important aspect of current nanotechnology research. The ever-increasing antibiotic
resistance in pathogenic and opportunistic microorganisms is a major threat to the health care industry.
The research work deals with the synthesis of silver nanoparticles from fungi Penicillium. Biosynthesized
silver nanoparticles were characterized by means of several analytical techniques including a UV-Visible
spectrophotometer, Fourier Transform Infrared Spectroscopy, X-ray diffraction pattern analysis, and Scanning
Electron Microscopy techniques. An evaluation of the antimicrobial activity of silver nanoparticles (AgNPs)
was carried out against clinically important pathogenic microorganisms. The metal nanoparticles were
also evaluated for their combined effects with antibiotics against the clinical pathogens. The antibacterial
activities of the antibiotics increased in the presence of the biologically synthesized AgNPs against the
clinically important pathogens.
Keywords: Biosynthesis of silver nanoparticles, Penicillium sps, Characterization, Cytotoxic activity
INTRODUCTION
Environmental pollution broadly refers to the presence of undesirable substances in the environment which
are harmful to man and other organisms. Microorganisms are ubiquitous in the environment that can be
beneficial or harmful or in apparent with regard to human measure or observation. The pollutants are
chemical, biological and physical in nature. Bacteria have developed resistance to all different classes of
antibiotics and its activity varies based on the climatic conditions. Hospital environments that require great
care in terms of the environmental monitoring is now critical [1]. The beneficial effects of microbes derive
from their metabolic activities in the environment, use in food production, biotechnological processes, for
the treatment and prevention of infectious diseases [2]. Many fungi also produce antibiotic substances, which
are now widely used to control diseases in human and animal populations.
Hospitals have become a breeding ground for antibiotic resistant bacteria, where meteorological
parameters play a very important role. Serious infections caused by bacteria that have become resistant to
commonly used antibiotics have become a major global healthcare problem in the 21st century. Fungi are
among the most successful organisms in their adaptations to different ecological conditions, mainly due to their
diverse reproductive capacity. Indoor fungal epidemiology involves numerous factors, including moisture,
ventilation, temperature, and organic matter present in building materials, but also outdoor meteorological
parameters, seasonal variations in climate, and presence of construction activities. The advancement in the
field of nanotechnology and nanoscience which involves with the use of metallic nanoparticles has increased
their application in the field of medicine, biotechnology, ceramics and food packing [3].
In the ancient times silver and copper were used to treat the bacterial infections [4]. Among, all metallic
nanoparticles, silver Nanoparticles posses high antibacterial activity and can be used for various medical
products, textile products, water filtration, coatings etc due to high thermal stability and less volatility[5,6].
Biological method for the synthesis of silver nanoparticle is the method of choice as it is simple and free
from toxic chemicals, easy to handle and ecofriendly. Silver nanoparticles can be synthesized from bacteria,
plants and fungi but fungi has the advantage over bacteria and plants because they are very tolerant towards
the metals, secretes large amount of enzymes, high capacity to bind with the cell wall and production of mass
is quite simple for the synthesis of silver Nano particles[7,8]. The research study deals with the extracellular
biosynthesis of sliver nanoparticles from Penicillium species, followed by characterization of nanoparticles
using UV-vis spectrophotometry, FTIR, XRD and SEM. These biologically synthesized nanoparticles have
been checked for its antibacterial activity against various bacterial pathogens and evaluate the synergistic
effect of these nanoparticles with different antibiotics.

Sample collection
The samples were collected from in and around hospital environment near Chennai Central, Chennai, by
exposing the Sabouraud’s dextrose agar media plate incorporated with antibiotic (50mg1) by settle plate
technique. The plates were exposed to air inside the hospital 5ft from the ground and 1km distance from the
hospital for 5 minutes just by keeping the media plates 2 m above the ground level to avoid dusting. The
plates were incubated at 25 ± 3°C temperature for 5-7 days.
Isolation of fungal culture

Fungal colonies developed in plates were counted for individual species and to get the total number CFUs.
Microscopic slides stained with lacto phenol cotton blue were prepared from each CFUs and observed
microscopically to identify them up to species level.
Microscopic and colony characterization

The fungal isolate of Penicillium species was observed by the author expertise using hand lens and the
colony morphology was recorded with respect to color, shape, size and nature of colony and also by using
laboratory manuals.
Synthesis of silver Nanoparticle

Penicillium species was utilized for the extracellular synthesis of silver nanoparticles. Fungal biomass
was grown aerobically in a liquid medium containing (g/L): KH2PO4 7.0; 2.0 K2HPO4 MgSO4. 7H2O 0.1;
(NH4)2SO4 1.0; yeast extract 0.6; glucose 10.0 at 25±30°C. After incubation, the biomass was filtered using
Whatman filter paper No.1 and extensively washed with distilled water to remove residual parts. The fresh
and clean biomass was taken into an Erlenmeyer flask, containing 100ml of deionized Milli-Q water. The
flask was incubated at 25°C in a shaker incubator at 140 rpm for 72 hours. The biomass was filtered again
with Whatmann filter paper No.1 and the cell free extract was used further. 1mM AgNO3 was prepared and
50ml was added to the cell-free extract and kept further in the incubator at 25°C, 140rpm for 72 hours in
dark condition.
188

The samples were observed for change of color and maximum absorbance was analyzed using UV-
spectrophotometer. 1ml of sample supernatant was taken after 72 hours and absorbance was measured by
using UV-visible spectrophotometer between 300-600nm. The powder form of the sample was subjected
to FTIR spectroscopy analysis. Two milligram of sample was taken and press it to form pellet. The sample
was placed into the sample holder and FTIR graph was taken. After the synthesis of silver nanoparticles
were further characterized by X-ray diffraction analyses which determine the crystalline nature of silver
nanoparticles. For XRD sample is prepared by the centrifugation of the silver nanoparticle solution, for 10
minutes at 15000 rpm. The pellet is then dried and makes it in to powder form and subjected for the XRD
analysis. SEM analysis is performed to determine the surface morphology and size of the nanoparticles. The
sample has been prepared by centrifugation and then dried into powder form and subjected to SEM analysis.
Evaluation of antibacterial Activity

Biologically synthesized silver nanoparticles were checked for its antibacterial activity by using disc
diffusion method. The antibacterial activity of silver nanoparticles from Penicillium sp. was tested against the
pathogenic bacteria such as Staphylococcus aureus, Bacillus cereus, Escherichia coli and Proteus vulgaris.
The combined formulation of silver nanoparticles with standard antibiotic disc Ampicillin and Tetracycline
were used to find out the synergistic activity against the above bacterial pathogens. The zone of inhibition
was measured after overnight incubation at 37°C [9].

The Penicillium species were used for the biosynthesis of silver nanoparticles (Fig 1). The change in the
color of solution in to the dark brown by the addition of Silver nitrate (AgNO3) results in the formation
of silver nanoparticles due to the reduction of Ag + to Ag0 (Fig 2). These reports have been shown many
researchers in the past [10-12]. After the formation of silver nanoparticles from the fungal filtrate which have
been further characterized by using UV-Vis spectrophotomer which shows the absorption peak at 416nm due
to surface Plasmon resonance[14,15] which reports the synthesis of silver Nanoparticles and peak is specific
for silver nanoparticles[13].
FTIR analysis were used to identify the molecules, proteins and functional groups involved in the
reduction of silver ions in to silver nanoparticles and stabilizes them as capping agent (Fig 3). The FTIR
analysis obtained for the Nanoparticles shows the absorption peaks located at 3419.5 cm-1(O-H stretch),
2920.2 cm-1(C-H stretch alkanes), 1640.2 cm-1 (NH stretch of amide), 1431.2 cm-1 (N-H(10) bend of
amide),1378.9cm-1 (NO2 aliphatic) ,1163.9 cm-1 (C-O stretch of carboxylic acid),1113.7cm-1 (C-N Stretch
of amines) and also some small peaks are there.
XRD is used to determine the crystalline metallic nature of silver Nanoparticles by using X ray
diffractometer (Fig 4). The XRD analysis shows that silver Nanoparticles from Penicillium species are face
centered cubic structures. Mohsen et al 2012[16], and also shows that silver nanoparticles are crystalline in
nature. These Nanoparticles have been further characterized by scanning electron microscopy (Fig 6) which
has been used to understand the surface topology and the size of silver nanoparticles and also shows silver
nanoparticles are spherical in shape with the diameter ranges between 35 nm and 60nm.
Invitro antibacterial activity of silver nanoparticles synthesized from Penicillium species were carried
out and also in combination with the Ampicillin and Tetracycline antibiotics available in the market against
clinically isolated pathogens viz., Staphylococcus aureus, Bacillus cereus, E. coli, and Proteus vulgaris were
satisfactory in the present study. The combined formulation of silver nanoparticles with different available
antibiotics in the market like Ampicillin (5mcg disc) and Tetracyclne (5mcg disc) showed remarkable results
against all the pathogens studied. Each disc is impregnated with 20 μg silver nanoparticle solution. The
189
antibacterial activity of Ampicillin and Tetracycline were amplified in the presence of Ag-NPs against the
bacterial strains. The highest increase in fold area was observed for Tetracycline and Ampicillin were against
E. coli at (1.4%) and (0.73%) respectively.
Silver Nanoparticles showed good antimicrobial activity alone. It was found that the silver nanoparticles
produced from Penicillium species enhanced the reaction rates of the antibiotics in a synergistic mode as well
as in its own way on these clinically isolated pathogens. Fig (6) and Fig (7) shows the graphical representation
of the combination effect of Ampicillin and Tetracycline with Ag-NPs against clinically isolated pathogens.
Fig 1: Growth of Penicillium sps in growth media and in distilled water
A) after addition of silver nitrate (B) before addition of silver nitrate

Fig 2: Biosynthesis of silver Nanoparticles
Fig 3: FTIR analysis of Silver Nanoparticles Fig 4: XRD pattern of silver Nanoparticles
190
Fig 5: Scanning electron microscope of silver Nanoparticles synthesized from Penicillium species
Fig 6: Graphical representation of the combination Ampicillin with Ag-Nps against the selected pathogens
Fig 7: Graphical representation of the combination Tetracycline with Ag-Nps against the selected pathogens
REFERENCES
1. Jose Nelson marins-Dinz, Rosangela Aparecisa Moraes Da Silva Monitoringof airborne fungus and
yeast species in a hospital unit, RevSaude Publica (2005), 39
2. Kenneth Todar, PhD., The Impact of Microbes on the Environment and Human Activities Todar’s Online
Textbook of Bacteriology ©, 2011.
191
3. Gajjar P, Pettee B, Britt DW, Huang W, Jhonson WP and Aderson J, Antimicrobial activities of
commercial Nanoparticles against an environmental soil microbe, Pseudomonas putidanKT2440.
Journal of Biological Engineering (2009) 3,9-22.
4. Moghimi S M, Nanomedicine: Prospective diagnostic & therapeutic potential. Asian pacific Biotech
News 9, (2005). 1072- 1077.
5. Rai M, Yadav A and Gade A, Silver Nanoparticles as a new generation of antimicrobials. Biotechnology
Advances. (2009). 27, 76-83.
6. Gong P, Li,X.X. He H.M., Wang K.M., Hu J.B., Tan W.H., Chun Z.S. and Yang X.H, Preperation and
antibacterial activity of Fe3o4@Ag Nanoparticles. Journal of Nanotechnology.18, (2007) 604-611.
7. Chen J.C., Lin Z.H. and Ma. X.X., Evidence of the production of silver Nanoparticles via pretreatment
of Phoma sp.32883 with silver nitrate. Letters in applied microbiology. (2003) 34, 105-108.
8. Dias I.C., Lacerda, Pimentel P.F., De Castro and Rosa C.A., Removal of heavy metal by an Aspergillus
terrus strain immobilized in a polyurethane matrix. Letters in applied microbiology (2002) 34, 46-50.
9. Bauer AW, Kirby M, Sherris JC and Truck M, Antibiotic susceptibility testing by a standardized single
disk method. Clinical Pathology (1996) 45,493-496.
10. Gardea- Torresedey JL, Gomez E,Jose–yacaman M, Parson JG, Peralta- Videa JR and Tioani H, Alfaalfa
Sprouts: A natural source for the synthesis of silver Nanoparticles. Langmuir.19, (2003)1357- 1361.
11. Gade Ak, Bonde PP, Ingle AP, Marcato P, Duran N and Rai MK, Exploitation of Aspergillus niger for
synthesis of silver Nanoparticles. Biobased Material Bioenergy, (2008). 2,243-7.
12. Ingle A, Rai M, Gade A and Bawaskar M, Fusarium solani: a novel biological agent for the extracellular
synthesis of silver Nanoparticles. Nanoparticle Research (2009). 11, 2079-2085.
13. Sastry M, Ahmad A, Khan MI and Kumar R, Biosynthesis of metal nanoparticles using fungi and
Actinomycetes. Current Science (2003). 85,162-170.
14. Mulvaney, P.Surface Plasmon Spectrocopy of Nanosized Metal particles. Langmuir (1996)12,788-800.
15. Ingle A, Gade A, Pierrat S,Sonnichsen C and Rai M, Mycosynthesis of Silver Nanoparticles using
fungus Fussarium acuminatum and its activity against some huaman pathogenic bacteria.current.
nanosience.4(2008), 141-144.
16. Mohsen Z, Aziz a A, H, Fatima, A.B, Mariana, N.S, Kamyar, S, Fatemah, J and Farah, F, Evidence based
green synthesis of Nan particles. VBRI press, doi; 10. 5185/amlett.Incnano. (2012) 353
192
Synthesis and Characterization of Andrographis
Paniculata Loaded Nanoparticles for the Development of
Antimicrobial Poly Cotton Fabrics
*R. Rajendran, K. Hemalatha, A. Manikandan and R. Radhai
PG & Research Department of Microbiology, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India.
*Corresponding author’s
email id: rrajendranmicro@yahoo.co.in
Abstract:
The present study was focused on the development of herbal based nanoparticle finishes on Polycotton fabric
for protective purpose. Andrographis paniculata was used to study the antimicrobial property. Methanolic
extract of this leaves showed the higher antimicrobial activity. MIC was performed with plate titre method
against gram positive (S. aureus, B. subtilus, MRSA) and gram negative microorganisms (E. coli, P. aeruginosa,
K. pneumoniae, A.baumannii, S. marcescens, P. vulgaris) respectively. Antifungal study was done with
Agar plug method against A. niger, T. reesei and P. notatum. Leaf extract was checked for the presence of
phytochemical components. GCMS and FTIR analysis were done with methanolic extract of A. paniculata.
Nanoparticles were prepared by using emulsion technique. Physical and chemical characterizations were
done for the synthesized nanoparticles. Average size of the nanoparticles was determined as 164.5 nm by
DLS analysis. Shape of the nanoparticles was determined by HRSEM analysis. Nanoparticles were coated
on the fabric by pad-dry cure method. Antibacterial activity of the coated fabric was checked using AATCC
147 and AATCC100. The antibacterial efficacy of the fabric was achived up to 35 washes of the coated
fabric. Antifungal activity was checked using AATCC 30, humidity jar method and soil burial test. Treated
fabric was showed higher antimicrobial activity. The physical parameters of the treated fabric showed good
results when comparing with the untreated fabric. As a future prospectus application of the nanoparticle
loaded fabric can be used as nose masks, head caps, aprons, curtains, etc.
Keywords: Antibacterial activity, Andrographis paniculata, AATCC and FTIR, Nanofinish.
1. INTRODUCTION
The growth of microorganisms in the textile materials cause innumerable problems such as unacceptable
odor, loss of strength in fabric and stains and moreover, affect the health of the wearer. It is therefore important
to impart antimicrobial effect on textile materials so as to protect the health of the wearer [1]. Antimicrobial
fabrics gained significant importance in recent years due to its wide range of application in various fields like
medical, technical, industrial, home furnishing and apparel categories [2]. The present study deals with the
development of anti microbial herbal nanoparticles finished textiles for protective purpose to improve the
functions of textile materials to medical protective purposes.
2. MATERIALS AND METHODS

The leaves of A.paniculata were collected and extracts prepared with different solvents like methanol,
petroleum ether, hexane, and acetone. Bacterial strains (E. coli, P. aeruginosa, K. pneumoniae, A. baumannii,
S. marcescens, P. vulgaris, S. aureus, B. subtilus and MRSA) and fungal strains (A. niger, P. notatum and T.
reesei) were obtained from the Department of Microbiology, PSG Institute of Medical Science, Coimbatore.
Antibacterial and antifungal study was done with well diffusion method [3] and agar plug method respectively
[4]
. Methanolic extract of this plant showed the higher antimicrobial activity. MIC was performed with plate
titre method against all the bacterial strains. Leaf extract was checked for the presence of phytochemical
components using standard methods [5]. Leaf extract was chemically characterized by GCMS (Hewlett
Packard GC-MS system) and FTIR (Bruker tensor 27, Germany) analysis. Nanoparticles loaded with leaf
extracts of A. paniculata were synthesized using micro-emulsion technique [6]. Synthesized nanoparticles
were physically characterized by DLS (Malvern zetasizer version 2.2) and HR SEM (FEI Quanta FEG 250)
analysis. Nanoparticles were coated on the polycotton fabric by pad-dry cure method [6]. Antibacterial
activity of the coated fabric was checked using AATCC 147 and AATCC100 [7]. Antifungal study was done
using AATCC 30, humidity jar method and soil burial test [8]. The physical parameters like air permeability,
stiffness, abrasion and tensile strength were analyzed in the Department of CDF, PSG College of arts and
science, Coimbatore.
3.1 Antimicrobial activity and Phytochemical analysis of leaf extract of A.paniculata

Antibacterial activity of A.paniculata was performed using different solvent extracts. The maximum zone
of inhibition against the entire test organism was observed in methanolic extract. Methanolic leaf extract
showed zone of inhibitions of 8 mm for A. baumannii, 4 mm for E .coli,7 mm for K .pneumoniae, 10 mm
for S. aureus, 7.5mm for B. subtilus and 8 mm for MRSA (table 1). Whereas 11mm inhibition showed by
Chloramphinicol against A. baumannii. Antifungal activity of methanolic leaf extract of A.paniculata was
checked using agar plug method [4]. The zone of inhibition of 2 mm for A. niger, 3 mm for P. notatum and
5mm for T. reesei were observed (Table 2). Ketoconazole inhibited the growth of T. reesei with 10mm of
zone. Acetone extract exhibited a moderate activity of 6mm for K .pneumoniae and 1mm for P. notatum.
Whereas other solvent extracts were not effective as evidenced in the methanolic extract. The increases in
inhibitory effects are may be as a result of the methanolic extraction [5]. MIC ranged between 40-80 µg/ml.
The qualitative analysis of the methanolic extract of A.paniculata was showed the presence of phytochemical
constituents such as carbohydrates, flavonoids, phenols, thiols, tannins and glycosides. Flavonoids, phenols
and tannins are proved to possess antimicrobial activity [3].
3.2 Synthesis and characterization of nanoparticles

Nanoparticles were prepared using Micro Emulsion procedure [6]. The Synthesized nanoparticles were
characterized by DLS analysis. The average particle size and zeta potential of the nanoparticles loaded with
herb extract was found to be 164.5 nm in diameter and 24.9mV respectively, as shown in figure (1.a,b). HR
SEM analysis showed the particle size ranged from 10-30 nm (fig 2.b). It could be inferred from the fig2.b
that the A. paniculata loaded nanoparticles were coated on to the polycotton fabric.
Fig 1: a- Average particle size and b- Zeta potential of the leaf extract of A. Paniculata loaded nanoparticles
FTIR analysis was done for the herbal extract and herbal extract loaded nanoparticles. Alcohols, phenols,
alkenes, aldehydes, alkenes, nitro compounds, alcohols, carboxylic acids, esters and ethers were present
in methanolic leaf extract of A. paniculata. Alcohols, phenols, alkanes, alkynes, esters, saturated aliphatic
aldehydes, nitro compounds, carboxylic acids and ethers were present in the spectra of herb extract loaded
nanoparticles. Spectra of nanoparticles showed a shift spectral of functional groups when compared with the
spectra of methanolic leaf extract (Fig 2.a). C–H stretch was shifted to 1458.18 cm-1 and O-H stretch shifted
194
from 3448.72, 3410.15 cm-1 to 3340.71 cm-1. These shifts confirmed that the Herbal extract was loaded inside
the polymeric matrix.
Fig 2: a - The FTIR spectrum of methanolic extract A. paniculata and Nanoparticles loaded with herb extract, b - HR
SEM image of polycotton fabric finished with Nanoparticles
The chemical composition of methanolic leaf extract and leaf extract loaded nanoparticles were
analyzed by GC-MS. The results revealed that the major compounds such as n-Pentadecanol, Dodecanoic
acid, Benzenedicarboxylic acid, 2(1h)-naphthalenone,9,12,15-octadecanoic acid, 1-(+)- Ascorbic acid
2,6-dihexane, andrographolide ,Eicosane(0.33%), 1-Heptanol,1-hexadecanol, 3,7,11,15- tetra, Phenazine,
Acridin-9-amine, 1H-Indazole, 6-methyl-1-phenyl, 5,6- Dimethyl-1,10-phenanthroline, Silicone oil,
Squalene, 1h-purin-6-amine, Vitamin E.
Fig 3: a - GC – MS analysis of metahnolic extract of A. paniculata and b- nanoparticles loaded with A. paniculata
The compounds like Phytol isomer, Dodecanoic acid, 17-pentatriacontene, n-hexadecanoic acid and
trimethyl 14- ethylene-14-pentadecne were present in both methanolic extract of A. paniculata (Fig 3.a) and
nanoparticles loaded with A.paniculata (Fig 3.b). Presence of dodecanoic acid and n-hexadecanoic acid were
responsible for the antimicrobial activity. So it could be conferred that the herb extract was loaded inside the
polymeric matrix.
3.3 Antimicrobial assessment of A.paniculata loaded nanoparticles

coated polycotton fabric.
The antimicrobial efficacy of the polycotton fabric was determined qualitatively by parallel streak method
(AATCC 147). S. aureus was inhibited with a zone of 15 mm, 18.5 mm for A. baumannii, 13 mm for
K. pneumonia, 9 mm for E. coli, 14 mm for MRSA, 9 mm for S. Marcescens,12 mm for P. vulgaris,19 mm
for P. aeruginosa and 10 mm for B. subtilis. The quantitative assessment was studied by bacterial reduction
method (AATCC 100). The percentage of reduction was calculated and given in Table 1. Maximum level of
195
reduction was obtained against all the test organisms. Wash durability was checked against E.coli, S.aureus
and MRSA. Durability of the treated fabric was achived until 35 wash cycles.
Table 1: Antibacterial activity of methanolic extract of A.paniculata and herb extract loaded nano particles finished
polycotton fabrics.
S.No Antibacterial activity (Zone of inhibition in mm)

Test organism S.aureus E. coli B. P. A. K. P. vulgaris MRSA S.
subtilus aeruginosa baumannii pneumoniae marcescens
1 Well Diffusion 10 4 7.5 2 8 7 3 8 2
(25mg/ml)
2 AATCC 147 15 9 10 19 18.5 13 12 14 9

3 AATCC 100(% 99.69% 99.20% 100% 100% 98.78% 100% 96.55% 100% 100%
reduction)
Antifungal activity of the nanoparticles treated fabric was assessed against A. niger, T. reesei and
P. notatum. A.niger showed no growth under the fabric (Table 2). P. notatum and T. reesei exhibited 6mm
and 13 mm zone of inhibitions respectively. Soil burial test and humidity jar test were done to assess the
antifungal activity of nanoparticles treated fabrics (AATCC 30). The results of soil burial test clearly
showed the damaged nature of the fibres in the control fabrics. On the other hand the fabrics treated with A.
paniculata loaded nanoparticles did not indicate any remarkable change in the fabrics even after two weeks
of soil burial. The results of humidity jar test revealed the presence of the growth of fungal strains in control
fabric and absence of growth in treated fabric was observed. The results of physical parameters of treated
fabrics like stiffness and abrasion resistance were increased from 2.5 to 2.8 cm and 93.54% to 97.22%
when compared to the untreated fabrics. It proved the presence of nanoparticles on the fabric samples.
Above results can be found that A. paniculata loaded nanoparticles on polycotton surface are necessary for
inhibition of the microbial growth.
Table 2: Antifungal activity of methanolic extract of A.paniculata and herb extract loaded nano particles finished
polycotton fabrics.
S. No Antifungal activity (Zone of inhibition in mm)

Test organism Penicillium notatum Aspergillus niger Trichoderma reesei
1 Well Diffusion(25mg/ml) 3 2 5
2 AATCC 30 6 No growth under the fabric 13
4. CONCLUSION
The results showed that the fabric coated with the A. paniculata leaf extract loaded nanoparticles inhibits the
growth of test organisms (bacterial and fungal strains).This study suggests that, Andrographis paniculata
extract coated on polycotton fabric can be used as nose mask and gloves in hospitals. This will provide the
better protection against the tested organisms.
5. REFERENCE
1. Chavan.R.B., ``Development in environment friendly functional finishes for cotton fabrics and
garmenrs”, NCM, December 2003,pp26
2. Helmut Mucha, Dirk Hofer, Sigrid Assfalg and Maximillian Swerev, Mlliand International, 8(5) 2002:
148.
196
3. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999; 12(4):564-82
4. Schlumbam A,Mauch F, Vogeli U and Boller T, Plant chitinases are potent inhibitors of fungal growth.
Nature 1986: 324: 365-367.
5. Obi, V.I. and Onuoha, C., Extraction and characterization methods of plants and plants products. In:
Biological and Agricultural Techniques. Ogbulie, J.N., Ojaiko, O.A(eds). Websmedia publishers,
Owerri. 2000: 271-286.
6. Rajendran, R., Radhai, R., Balakumar, C., Ahamed, H. M., Vigneswaran, C and Vaideki,K., ``Synthesis
and characterization of neem chitosan nanocomposites for development of antimicrobial cotton textiles’’.
Journal of Engineered Fibres and Fabrics,2012: 7, 136–141.
7. AATCC Test method 100- 200, Antibacterial activity assessment of Textile materials: Percentage
reduction method, AATCC technical manual,149-150.
8. AATCC Test method 30-2004. Antifungal activity, Assessment on Textile Materials: Mildew and Rot
Resistance of Textile Materials, AATCC Technical Manual,American Association of Textile Chemists
and Colorists, Research Triangle Park, NC 2007.
197
SECTION - II
NANO DRUG DELIVERY

SYSTEMS
Modified Release Multiparticulate
Delivery System for Tramadol HCl
Using Gelucire 43/01 as Rate
Retarding Material
Divya Priya S, Sruthi S, Hema R, Rajasekar K,

Ganesan V, Lakshamana Prabu S, Puratchikody A and Shanmugarathinam A
Department of Pharmaceutical Technology, BIT Campus, Anna University,

Tiruchirappalli, 620024, Tamilnadu.
* Corresponding Author
e-mail address: shanmugarathinam@gmail.com
Abstract:
Tramadol HCl is an opioid analgesic was formulated as granules by melt granulation technique in the
present research by using gelucire 43/01 as rate retarding material. Tramadol HCl is highly soluble in
water and has immediate release. Gelucire 43/01 has a tendency of retarding the drug release profile at
higher ratios. The formulated granules have been characterized and evaluated. The results were obtained
as percentage yield (73 – 85 %) and drug content (69 – 98 %). FT-IR studies show that there is no
interaction between drug and excipient. DSC analysis is used for thermal analysis of granules. The in vitro
release studies were done for 10 hr (interval of 1 hr) by using distilled water as dissolution medium. The
study shows that the drug release rate gets retarded SI unit by Gelucire 43/01.
INTRODUCTION
Oral dosage form can be classified into two categories such as a single unit and multiple unit dosage forms.
The single unit dosage form include matrix tablets or coated/uncoated tablet or capsules. The multiple unit
dosage form consist of pellets or a microencapsulated drug filled in a capsule or compressed into tablets,
each exhibiting different characteristics. The idea behind designing multiparticulate dosage forms is to build
up a reliable formulation which has all the advantages of a single unit formulation behavior owing to unit
variation [1, 2].
Tramadol HCl, an opioid analgesic used primarily to treat severe acute and chronic pain, both acute and
chronic. Gelucire 43/01 is a highly hydrophobic lipid with an HLB value of 1 and a melting point of 43°C.
The extreme hydrophobicity of Gelucire 43/01 provides release-retarding properties and floating behavior.
The advantages of Gelucires over polymers in the design of controlled drug delivery systems include low
melt viscosity, obviating the need for solvents; absence of toxic impurities; potential for biocompatibility
and biodegradability. Thus, the major objective of the present study was to design multiparticulate drug
delivery system by varying lipid ratios. In the present study, Tramadol HCl, Gelucire 43/01 lipid granules
were prepared by melt granulation technique and were evaluated.

Materials used
Tramadol HCl was a kind gift sample from Fourrts India Laboratories Pvt ltd., Chennai, and Gelucire 43/01
was supplied from Gattefosse India Pvt Ltd., Mumbai.
Preparation of Lipid granules by melt granulation technique
The formulation adopted here for the preparation of the Tramadol HCl granules was melt granulation
technique. The drug Tramadol HCl and Gelucire 43/01 is weighed accurately as shown in Table 1; Gelucire
43/01 was heated above its melting point. Drug was added into the molten Gelucire 43/01 and triturated
[3]. The resultant mixture was allowed to solidify at room temperature and stored in refrigerated condition
for 24 hr, later it was passed through the sieve number 25 and stored in refrigerated condition until further
used [4].
Table: 1 Composition of formulation in ratio
Sl.No. Gelucire 43/01 Tramadol HCl

F1 1 5.0
F2 1 7.5
F3 1 10.0
F4 1 12.5
CHARACTERIZATION AND EVALUATION

Percentage Yield
Granules dried at room temperature were weighed, and yield of granule preparation was calculated. The
percentage yield of each formulation was calculated by using the formula.
{Practical mass / Theoretical mass} × 100
FT-IR spectroscopic studies
Fourier Transform Infrared (FT-IR) Spectroscopy Infrared spectroscopy was used to predict possible drug
excipients interaction using an FTIR spectrometer (JASCO 6300) at 4000 – 400 cm-1[5]. The granules
of optimized batch were recorded using KBr mixing method for drug excipients interaction study. FTIR
spectra of the Gelucire 43/01 and optimized formulation were recorded using a Fourier transform infrared
spectrophotometer.
Differential Scanning Calorimetric analysis (DSC Analysis)
The sample was prepared by physical mixture of drug and excipients and the thermo gram of pure drug,
polymer and formulation was carried out using DSC. In this study, approximately 5 mg of sample were
placed on aluminium plates, sealed with aluminium lid and heated at a rate of 10° C/min over a temperature
range of 0 – 400° C Thermograms were recorded for Gelucire 43/01and the optimized formulation using a
differential scanning calorimeter (Perkin-Elmer, USA) [6].
Drug content
Drug content of the formulation was determined by dissolving 50 mg of granules in 10 ml of methanol,

and the solution was filtered through Whatman filter paper. The sample was analyzed for drug content by
UV spectrophotometrically at 271 nm after suitable dilutions.
202
IN VITRO RELEASE TEST

In vitro dissolution, studies were carried out using USP type II dissolution test apparatus (LABINDIA
DS8000). The paddle was rotated at 75 rpm, and the temperature was maintained at 37.5 ± 0.5°C and 900
ml of dissolution medium was used. The granules were placed in 900 ml of dissolution media (distilled
water). The samples were withdrawn at interval of 1 SI unit up to 10 h... The test solutions were analyzed
by UV spectrophotometrically by measuring absorbance at 271 nm [7, 8].

Granules were developed successfully prepared by melt granulation technique as lipid multiparticulate
drug delivery systems using Gelucire 43/01 as lipidic excipient. The percentage yield of the product is
good and ranged from the 75.13 to 83.45%. The highest yield is obtained for the formulation F4 83.45%
and the least is obtained for the formulation F1 75.13%. This shows that melt granulation technique is
well suitable for the formulation of granules of Tramadol HCl [9, 10]. The percentage yield was shown
in Table 2.
Table: 2 Percentage Yield

Formulation Yield(%)
F1 75.13
F2 78.24
F3 80.13
F4 83.45
FT-IR spectroscopic studies [11]
The FT IR of pure drug and physical mixtures of drug and polymer were studied. The study reveals that
there is no drug-excipient interaction [Figure1]. The characteristics absorption peaks of Tramadol HCl were
obtained at 3637.09, 3058.55, 3012.27,2859.92, 2601.50, 1924.61, 1819.70, 1740.12, 1604.48, 1579.41,
1436.71,1286.29,1081.87, 1002.90, 973.60, 781.03,700.05,466.69, 511.04 cm-1.
Figure 1: IR spectrum of Tramadol HCl, FT-IR analysis of the Gelucire 43/01, FT-IR analysis of the Formulation (F1)
203
DSC analysis
DSC thermograms are shown in [Figure 2] below. Endothermic peaks were observed at 185.110C due to
melting of drug. From this, it is observed that there is no interaction between drug and excipient [12].
Figure 2: DSC spectrum of Tramadol HCl, DSC spectrum of Gelucire 43/01, DSC spectrum of Formulation (F1)
Drug content
The amounts of drug present in the granules were determined. This result was mentioned in the Table 3. The
range of drug content is 69.44 (F4) to 98.12 (F1) %. The drug content found to be more for increasing the
release of the drug [13]
Table: 3 Drug content
Formulation Drug Content (%)

F1 98.12
F2 85.33
F3 80.75
F4 69.44
In vitro release study
There is a significant difference in release rate from the formulation prepared from different concentration
of Gelucire 43/01. This excipient significantly affected the drug release rate in the order of increase in the
concentration of Gelucire 43/01 [14]. The dissolution profiles of Tramadol HCl for all the concentration of
Gelucire 43/01 are shown in [Figure 3]. It shows that the release is faster in lower concentration of Gelucire
43/01 than the higher concentration thus the release of the drug is retarded at the higher concentration of
Gelucire 43/01 [15, 16].
204
Figure 3: Percentage cumulative drug release of different formulation
CONCLUSION
The granules were formulated with the rate retarding lipid excipient Gelucire 43/01. The prepared granules
were characterized by FT-IR, differential scanning calorimetry, drug content, in vitro release study. FT-
IR analysis showed that there was no interaction between the drug and the excipient. The differential
scanning calorimetry also showed the melting point of the granules formulation is almost similar with
the drug and the excipient. Each formulation with different Gelucire 43/01 concentration shows various
release profile. Thus the result shows that, if the concentration of the Gelucire 43/01 is increased to a
particular extent and the release rate of the drug is retarded.
ACKNOWLEDGEMENT
The authors are grateful to CENTRE-NFDD, Department of Pharmaceutical Technology, Anna University
(BIT campus), Tiruchirappalli for providing required facilities to carry out this research work.
REFERENCES
1. Kodiak Y, Irisawaa Y, Okimotoa K, Osawaa T and Yamashitab S, A new formulation for orally
disintegrating tablets using a suspension spray-coating method, Int. J. Pharm. 2009; 382: 80–87.
2. Srikonda VS, Janaki RN and Joseph A, Recent technological advances in oral drug delivery – a review,
Pharm. Sci. Technol. Today. 2000; 3: 138-145.
3. Shimpi S, Chauhan B., Mahadik K.R. and Paradkar A., Preparation and Evaluation of Diltiazem
Hydrochloride-Gelucire 43/01 Floating Granules Prepared by Melt Granulation, AAPS PharmSciTech
2004, 5 (3) Article 43, 1–6.
4. Solanki HK, Tarashankar Basuri, Thakkar JH and Chirag A. Patel, Recent Advances in Granulation
Technology. Volume 5, Issue 3, November – December 2010; Article-008 ISSN 0976 – 044X. 50-51.
5. Yu Cui, Formulation of Sustained-Release Matrix Tablet of Nifedipine, J. Kor. Pharm.Sci., 32, No. 2,
95-101 (2002).
6. Craig DQM and Reading M. Thermal Analysis of Pharmaceuticals, C.R.C. Press, Taylor & Francis
Group, London. Pp. v-viii (2007)
7.
Gibaldi M. and Feldman S. Establishment of sink conditions in dissolution rate determinations:
theoretical considerations and application to nondisintegrating dosage forms. J. Pharm. Sci. 1967, 56,
1238–1242
205
8. Srikonda VS, Janaki RN and Joseph A., Recent technological advances in oral drug delivery – a review.
Pharm. Sci. Technol. Today. 2000; 3: 138-145.
9. Simone S and Peter CS, Fast dispersible ibuprofen tablets, Eur. J. Pharm. Sci. 2002; 15: 295–305
10. Lachman L, Lieberman HA and Kanig JL, The theory and practice of industrial pharmacy, 3rd ed.
Bombay: Varghese publishing house; 1986. p. 30-250.
11. Sekar V and Chellan VR, Immediate release tablets of telmisartan using super disintegrant- formulation,
evaluation and stability studies, Chem Pharm Bull (Tokyo). 2008 April; 56(4): 575-7.
12. Ainaoui A and Vergnaud JM, Modelling the plasma drug level with oral controlled release forms with
lipidic Gelucire, Int. J. Pharm. 1998, 253, 155–162
13. Subrahmanyam CVS, Thimmasetty J, Shivanand KM and Vijayendra Swamy SM, Laboratory manual
of industrial pharmacy, Delhi: Vallabh Prakashan; 2006; 87-225.
14. Rama RN and Chowdary KPR, Improvement of dissolution rate and Bioavailability of Piroxicam with
pre gelatinized starch, Ind. J. Pharm. Sci. 2001; 63: 36-40.
15. Deshpande AA, Shah NH, Rhodes CT and Malick W, Development of a novel controlled-release
system for gastric retention, Pharm. Res. 1997, 14, 815–819.
16. Dennis AB, Farr SJ, Kellaway IW, Taylor G and Davidson R, In vivo evaluation of rapid release and
sustained release Gelucire capsule formulations, Int J Pharm. 1990; 65:85-100.
206
Extraction and Characterization
(Both Physico-Chemical and Analytical)
of Pectin Obtained from Dillenia Indica and
Abelmoschus Esculentus and Compatibility
Study with Glipizide for Application in Novel
Drug Delivery Systems
Sivasankar Mohanty, G. Krishna Mohan and M. Sunitha Reddy
Centre for Pharmaceutical Sciences, Institute of Science & Technology, Jawaharlal Nehru Technological
University, Hyderabad, 500085
e-mail: smohanty55@gmail.com, Mobile No: 7569310018
Abstract:
The present study was focused on extraction of pectin from Dillenia indica and Abelmoschus
esculentus and characterized for establishment of polymer in place of synthetic or semi-synthetic in
novel drug delivery systems. The natural mucilaginous substances were collected from Dillenia Indica
and Abelmoschus esculentus by Acetone precipitation method and were undergone characterization
like physico-chemical test, analytical study and compatibility study with glipizide. The physico-
chemical characterization were included solubility, ash value, extractive value, Moisture content &
the analytical study includes TGA, XRD, FTIR, DSC & HPTLC. The nature of pectin was confirmed
by phytochemical screening and with Thin Layer chromatography (TLC), and High Performance Thin
Layer chromatography (HPTLC) which indicated distinct fingerprints with methanol extract. The
outcome of results has come within the limits according to standard monograph. The compatibility
study represents there was no interaction between the drug (glipizide) and pectin. The TGA & XRD
showed that the mucilage is amorphous in nature and has one-step mass loss event. The mucilage
obtained from Dillenia indica & Abelmoschus esculentus can be used as a natural polymer for
formulating various drug delivery systems.
Keywords: Dillenia indica, Abelmoschus esculentus, Pectin, Glipizide, Mucilage’s, Natural polymers.
INTRODUCTION:
Dillenia indica (DI) is commonly known as Elephant-apple, Indian catmon in English, Ou tenga
in Assamese, Lisora in Hindi and Bahubar in Sanskrit [1]. It occurs in India, Siam, and Malaya. In
India, it grows in moist and evergreen forests of sub-Himalayan tract from Kumaon to Garhwal,
eastward to Assam and Bengal and southwards to Central and Southern India. The fruit, which
is made of ripened carpels and enclosed by greatly enlarged and thickened imbricating sepals, is
large, somewhat rounded or broadly ovoid, 12.5 to 15 centimeters in diameter, yellowish green in
color, tough and fibrous. The seeds are numerous and compressed, with a hairy margin. In North east,
traditionally the unripe fruits are used to make curries because of its sour taste and ripe fruits are making
pickles. Abelmoschus esculentus (AE) or okra or bhendi also known as ladies’s finger is an important
vegetable crop being native of tropical Africa [2]. Okra is a tall annual dicotyledonous plant related to
cotton and thought to be of African origin. It is still found growing wild along the river Nile in Egypt
as well as Ethiopia French colonialist carried okra to the new world soon after 1700. Now it is a widely
grown vegetable crop in the tropics and subtropics and also in the warmer temperate areas. Pectin occurs
as a coarse or fine, yellowish-white, odourless powder that has a mucilaginous taste. It is a high-molecular-
weight, carbohydrate-like plant constituent consisting primarily of chains of galacturonic acid units
linked as 1, 4-a-glucosides, with a molecular weight of 30 000–100 000. It comprises mainly esterified
D-galacturonic acid residues in a-(1–4) chain. Pectin is a predominately linear polymer of mainly α-(1-4)-
linked D-galacturonic acid residues interrupted by 1, 2-linked L-rhamnase residues. The acid groups along
the chain are largely esterified with methoxy groups in the natural product [3].
MATERIALS AND METHODS:

Chemicals and Reagents:
All the chemicals used were of analytical grade & laboratory reagents. Acetone purchased from loba
chemicals with 99.9% A.R.
Plant materials:
The fruits of D.indica were collected from Balasore, odisha, especially during the month of September-
November & Ladies finger collected from the local market of Hyderabad.
(1) Extraction of pectin:
At first the fruits were collected and were cut into small Pisces with knife & seeds were separated. The
chopped pieces of the fruits were kept in a beaker containing distilled water with ratio 1:1.5. The beaker
was then placed on a heating mantle with temp. 600°C for 5-6 hours. After about 6 hours the slurries
were strained through a Buchner funnel and the filtrate was kept in the refrigerator in a beaker for
overnight for sedimentation. The decanted filtrate was taken out of the refrigerator, and the supernatant
was poured into a clean and dry beaker of 1 L size. The supernatant was evaporated to 1/5th of its
volume by heating mantle at 600°C. The concentrated samples were washed with acetone and dried at
50 to 600°C in a hot air oven for 4 hours. On drying, the sample becomes hard and brownish in colour.
The powdered samples were passed through sieve no 120 & were stored in desiccators under sealed
conditions for further study [4, 5].
(2) Identification test of pectin:
After the extraction process, the pectin was confirmed by various identification tests like Stiff gel Test, Test
with 95% Ethanol, Test with Potassium Hydroxide (KOH), Iodine test &Test for Acidity described by H.K.
Sharma et al. [4].
(3) Physico-chemical characterization of DI & AE:
Solubility test: The pectin extracted from DI & AE was evaluated for solubility in Distilled water, Phosphate
buffer pH 6.8 and organic solvents in accordance with I.P. specifications [6, 7, 14].
Determination of swelling index of DI & AE:

Powdered mass equivalent to 1 ml volume was weighed and taken in a measuring cylinder and to that 9.0
ml of phosphate buffer (pH 6.8) and distilled water were separately added and allowed to swell for 24 hours.
The swelling index (S.I.) in the respective medium was then obtained from the following relation [8].
S.I = S2-S1/S1×100
208
Ash values:
It is used to determine quality and purity of a crude drug. It is also used to determine the contents of inorganic
radicals like phosphates, carbonates and silicates of sodium, potassium, magnesium, calcium, etc. So water
soluble ash, total ash, and acid insoluble ash were obtained by reported methods [6, 7, 9].
Determination of extractive values:

The extractive value used to get an idea about the nature of the chemical constituents presents in pectin.
Extracts were prepared with various solvents by standard methods. Percentage yield for extracts was obtained
[6, 7, 15].
Determination of moisture content:

Moisture content was determined as per I.P. specifications. 2 g of samples were transferred uniformly in
the weighed Petri dishes and then dried in hot air oven at 105°C until a constant weight was obtained. The
moisture content was determined as the ratio of weight of moisture loss to weight expressed as a percentage
[6, 7, 17].
(4) Analytical studies of pectin obtained from DI & AE:
TGA studies:
Thermal analysis of the sample was carried out using a Perkin Elmer, SII, Pyris Diamond, TG/DTA Instrument
with sample weighing about 10 mg and the programmed heating of the samples were done at a rate of 100°C/
min with temperature starting from 400°C to 500°C and the various plots were recorded [10].
XRD studies:
This technique was used to know the X-ray diffraction of sample obtained from DI & AE. In this technique,
a small collimated beam of nearly monochromatic X-ray were directed onto a small specimen in the form of
powder producing a pattern that is recorded with a counter-tube. The target material of the instrument was
Copper (Cu) and Nickel was used as a filter and a voltage of 35 kV and a current of 30 mA was used. The
diffraction was done at room temperature of 30°C [11].
HPTLC studies:
This technique was used to determine the purity or degree of complexity in the extract.
Chromatographic Conditions
Stationary phase: Pre-coated silica gel plates, Merck 60F254
Mobile phase: Chloroform: Methanol: Acetic Acid: Water (4:4:1:1)
Lamp: Deuterium
Wavelength: 254 nm
Application mode: CAMAG Automatic TLC Sampler III
Development mode: CAMAG Twin Trough Chamber
Scanner: CAMAG TLC Scanner 3 and WinCATS software [12, 16].
(5) Compatibility studies:
FTIR studies:
The FTIR-8400S spectrophotometer (Shimadzu, Japan) was used to obtain the infrared spectra of pure
drug (glipizide) and physical mixture containing drug & pectin. Samples were prepared in KBr (Potassium
Bromide) disks of 2mg sample in 200mg of KBr with the Scanning range was 400-4000cm-1 [18].
209
DSC studies:
The DSC thermograms of the pure drug & physical mixtures were obtained using a Perkin Elmer JADE DSC
system, to identify any interaction between the components of drug and pectin [18].

(1) Percentage yield of pectin from DI and AE
The percentage yield of pectin obtained from DI and AE are calculated by using the following equation
Percentage yield = Quantity of pectin obtained (g)/Quantity of fruit (g) ×100 shown in (Table-1)
Table 1: Percentage yield of pectin
Plants Batches Quantity of Fruit (g) Weight of pectineous Percentage

substances after drying (g) Yield (%)
DI DB1 1000 7.1 0.71
DB2 1000 7 0.70
DB3 1000 7.2 0.72
AE AB1 1000 6.8 0.68
AB2 1000 6.4 0.64
AB3 1000 6.6 0.66
(2) Identification test of pectin:
The pectin was identified by different test like Stiff gel Test, Test with 95% Ethanol, Test with Potassium
Hydroxide (KOH), Iodine test &Test for Acidity and shown in (Table-2)
Table 2: Identification Test of Pectin
Types of test Observation

Stiff gel Test A Sticky gel was formed after cooling.
Test with 95% Ethanol Sample formed gelatinous ppt.
Test with Potassium Hydroxide(KOH) A semi-gel or transparent was formed.
Iodine Test No change
Test for Acidity Sample turns blue litmus paper to red.
(3) Physico-chemical characterization of DI & AE:
The physico-chemical properties of pectin obtained from DI & AE was carried out and the results were
shown in (Table-3).All the results were come according to the standard limit.
Table 3: Physico-chemical characterization of pectin obtained from DI & AE.
Properties Pectin from DI Pectin from AE

Colour Brown Light brown
Solubility Insoluble In organic Insoluble In organic
solvent but forms colloidal solvent but forms colloidal
dispersion & good swelling dispersion & good swelling
property in distilled water & property in distilled water &
phosphate buffer pH 6.8. phosphate buffer pH 6.8.
Swelling Index phosphate buffer 680% 700%
pH 6.8
Distilled Water 600% 610%
0.1N HCl 490% 500%
210
pH 5.99 ± 0.837 6.07 ± 0.063

LOD 11 ± 0.03 12.6 ± 0.06
Ash Value Total Ash 6.8 ± 0.03 7.1 ± 0.06
Acid insoluble Ash 0.341 ± 0.02 0.451 ± 0.062
Water soluble Ash 2.56 ± 0.021 3.01 ± 0.041
Extractive value Pet ether 0.44 ± 0.09 0.31 ± 0.03
Ethyl Acetate 0.184 ± 0.02 0.192 ± 0.012
Chloroform 0.615 ± 0.113 0.914 ± 0.145
Acetone 2.91 ± 0.12 3.87 ± 0.11
DI = Dillenia Indica, AE = Abelmoschus Esculentus, HCl = Hydrochloric acid, LOD = Loss on drying
(4) Analytical studies of pectin obtained from DI & AE:

TGA Studies: The thermogram of pectin obtained from DI & AE as shown in Figure 1, shows that the
physical changes of material according to increase in temperature i.e. from 400°C to 500°C.
Figure 1a: TGA of Dillenia indica Figure 1b: TGA of Abelmoschus esculentus
XRD studies:
The XRD spectra of Dillenia shows that it is of the amorphous nature as there are no sharp peaks present in
the spectra as shown in Figure 2.
Figure 2a: XRD of Dillenia indica Figure 2b: XRD of Abelmoschus esculentus
HPTLC studies:
The pectin obtained from DI & AE was compared with pure pectin by RF values as shown in figure-3.There
was not so much difference in RF values. The RF value came for pure pectin was 0.75 but for DI & AE was
0.73 & 0.74 respectively.
Figure 3a: Pure Pectin Figure 3b: Pectin from DI Figure 3c: Pectin from AE
211
(5) Compatibility studies:

FTIR studies:
The IR spectrum of glipizide is shown in (Fig 4) and the following characteristic bands were Observed
3324.5(N-H stretching), 1690 (- C = O, Amide), 1649.9 (- C = O, Urea), 1531 (Ar- CH, stretching), 1444
(ArCH, bending), and 1333.1 and 1159.2 cm-1 (- SO2NH). The IR spectrum of physical mixture (Glipizide+
pectin) showed the presence of characteristic bands as that of glipizide shown in (Fig4). Thus, any change
in the structure of glipizide was ruled out, and it was concluded that there is no chemical incompatibility
between glipizide and pectin obtained from DI & AE.
Figure 4a: FTIR of pure drug (Glipizide)
Figure 4b: FTIR of Physical mixtures
DSC studies:
The DSC thermogram of glipizide (Fig 5) showed a short endothermic peak at 215.3ºC the physical mixtures
showed an endothermic peak of drug at 260°C indicating a slight change in terms of shifting towards the
higher temperature. It has been reported from the graphs that the quantity of material used affects the peak
shape and enthalpy. Thus, these minor changes in the melting endotherm in the drug could be due to the
mixing of the drug and excipients which lower the purity of each component in the mixture and may not
necessarily indicate potential incompatibility.
Figure 5a: DSC of Glipizide Figure 5b: DSC of Physical Mixture
CONCLUSION:
From the above studies, it can be concluded that pectin obtained from DI & AE proves to be a good natural
substances, and there was no interaction between Drug & Pectin confirmed by FT-IR & DSC study. Thus the
pectinous substances can be used as a very promising polymer in Several Drug delivery systems.
212
REFERENCES:
1. A
purba Talukdar, Nayan Talukdar, Satyendra Deka, Bhargab Jyoti and Sahariah, Dillenia Indica
(Outenga) as Anti-diabetic herb found in Assam, IJPSR, 2012, 3(8):2482-2486.
2. Nipaporn, Sengkhampar, Leonard and M.C. Sagis, Physico chemical properties of pectins from okra
(Abelmoschus esculentus), Food hydrocolloids, 2010(24), 35-41.
3. Paharia A., Yadav A.K. and Rai G., Eudragit-coated Pectin Microspheres of 5-Fluorouracil for Colon
Targeting, AAPS PharmSciTech, 2007, 8 (1), 46-57.
4. Shrivastava P. and Malviya R., Sources of pectin, extraction and its applications in the pharmaceutical
industry- An overview, Indian journal of product and resources, 2011, 2 (1), 10-15.
5. H.K. Sharma, Sunita, Lahkar and L.K. Nath, Extraction, characterization and compatibility study of
polysaccharides from Dillenia indica and Abelmoschus esculentus with Metformin hydrochloride for
development of drug delivery system, 2013, 5 (1), 275-283.
6. Khandelwal K.R., Practical Pharmacognosy, Nirali Prakashan, Pune, 2005, 1, 149- 156.
7. Mukherjee P.K., Quality control of Herbal Drugs, Development of Standardisation Parameters, Business
Horizons, New Delhi.1, 2007, 184-245.
8. Goud K., Desai H. and Kumar T.M., Preparation and evaluation of a novel buccal adhesive system,
AAPS PharmSciTech, 2004, 5 (3), 135-141.
9. Sivasankar Mohanty and Krishna Mohan G., Proximate Analysis and standardization of plant exudates:
Gum olibanum and Gum Dikamali. Int. J. Pharm. Sci. Rev. Res., 24(1), 172-176
10. Trishna Bal., Padala Narasimha, Murthy, Shubhranshu and Sengupta, Isolation and Analytical studies of
mucilage obtained from the seeds of Dillenia Indica (Family Dilleniaceae) by use of various Analytical
Techniques, Asian Journal of Pharmaceutical and Clinical Research, 2012, 5(3), 65-68.
11. Amit Kumar and Ghanshyam S., Extraction and characterization of pectin from apple pomace and its
evaluation as lipase (steapsin) inhibitor, Carbohydrate Polymers(ELSEVIER), 2010, 82, 454-459.
12.
Kavitha Jayapala Reddy, Krishna Mohan G., Switi and Gaikwad, Preliminary Phytochemical
Standardization of Tree Exudates from India: Gum Kondagogu and Gum Ghatti, Research Journal of
Pharmaceutical, Biological and Chemical Sciences, 2011, 2(4), 1023-1034.
13.
Igor Maria De Rosa, Jose Maria Kenny, Debora Puglia, Carlo Santulli and Fabrizio Sarasini,
Morphological, thermal and mechanical characterization of okra (Abelmoschus esculentus) fibres as
potential reinforcement in polymer composites, Composites Science and Technology (ELSEVIER),
2010, 70, 116-122.
14. Anonymous, The Indian Pharmacopoeia, Government of India, The Controller of Publications, New
Delhi, 1996, 4th ed, vol II, A-53, A-54, A-089.
15. Heinrich M., Fundamentals of Pharmacognosy and Phytotherapy, Production, Standardisation and
Quality control, Elsevier publication, London, 2004:144-159.
16. Stahl E., Thin layer Chromatography: A Laboratory Handbook, Springer Publication, New York, 2005,
2, 807-837.
17. Quadry JS., Pharmacognosy, Evaluation of Natural Products and significance of Pharmacopoeial
standards, CBS Publishers and Distributors Pvt Ltd, New Delhi, 2010, 16,32-39.
18. Hemanta Sharma, Siba Prasad Pradhan and Babita Sarangi, Preparation and in vitro evaluation of
Enteric Controlled release Pantoprazole loaded Microbeads using Mucoadhesive Substance from
Dillenia indica, International Journal of PharmTech Research, 2010, 2(1), 542-551.
213
Preparation and Characterization of Leflunomide Loaded
Nanosuspensions for Rheumatoid Arthritis
S. Lakshmana Prabu, S.P. Sharavanan, A. Shanmugarathinam, K. Ruckmani, A.

Bhuvaneswari, S. Aravindan and V. Manikandan
Dept. of Pharmaceutical Technology, Bharathidasan Institute of Technology, Anna University,

Tiruchirappalli – 620 024.
e-mail: slaxmanvel@gmail.com
Abstract:
Drugs with low aqueous solubility and high permeability (BCS Class II) present a high proportion of
all drugs coming out of high throughput screening. Colloidal nano carriers in their various forms have
the possibility of providing endless opportunities in the area of drug delivery. Drug delivery through
nanosuspensions formulation is found to be a promising approach in the enhancement of bioavailability of
low water soluble drugs. Nanosuspensions containing Leflunomide stabilized with PEG was formulated
by solvent evaporation method to develop a nanoparticulate delivery system for rheumatoid arthritis.
The prepared formulations were characterized for particle size, zeta potential, surface morphology,
dissolution and short-term stability. The optimized formulation had a mean particle size of 491 nm. The
AFM images showed spherical shape of the nanoparticles. Controlled drug release of Leflunomide from
nanosuspensions was observed during the dissolution study. The nanosuspensions were physically stable
under stress conditions. These study results designate a suitable procedure for the formulation of colloidal
Leflunomide loaded nanosuspensions with considerably enhanced in vitro dissolution rate, and thus
perhaps enhance the therapeutic effect for rheumatoid arthritis.
Keywords: Leflunomide, Nanosuspension, Solvent evaporation, Rheumatoid arthritis
INTRODUCTION
It has been reported that 75% of recent and current drug development candidates show low solubility in
water. Two important factors in the development of medicinal products are drug solubility and permeability,
as they determine the large extent of bioavailability of the drug substance. A conventional dosage form has
limited effectiveness, poor biodistribution and lack of selectivity. But these limitations can be managed by
controlled drug delivery systems [1, 2]. Formulating a poor water soluble drug has always been a challenging
problem confronted by the pharmaceutical scientist. Over the last few decades a considerable interest
has been shown in the field of nanotechnology, various nanostructured materials have been successfully
synthesized for biomedical applications [3-6]. Nanotechnology has the power not only to improve existing
technologies, but to dramatically enhance the effectiveness of new applications in all sectors. Nowadays
novel drug delivery systems use even particulate matters like Nanotechnological products as carriers of drug
and they have various advantages over the conventional drug delivery systems. Nanostructured products
exhibit distinctive physicochemical and biological properties. They can be used to target particular areas of
interest and are favorable for various biomedical applications [7-12]. There are over 120 different types of
arthritis and rheumatic diseases that affect joints, surrounding tissues and other connective tissues. Some
forms of arthritis can result in kidney disease, blindness, and premature death. Osteoarthritis, rheumatoid
arthritis and fibromyalgia are the most common types. Although rheumatism is one of the oldest known
diseases of the mankind this affects a large percentage of the population in the world. Rheumatoid arthritis
has been called the ‘King of Human Miseries’. Rheumatoid arthritis is the one of the challenging disorder
associated with an inflammatory condition. Rheumatoid arthritis (RA) is an inflammatory arthritis that affects
nearly 1% of the world’s adults. This inflammation results in pain and stiffness, and can lead to progressive
joint damage resulting in deformity and loss of function [13-17]. Formulation of polymeric nanosystems is
one of the most interesting approaches to achieve local controlled drug delivery. The objective of the present
investigation was to formulate controlled release Leflunomide (5-methyl-n-[4-trifluoromethyl] phenyl]-
isoxazolo-4-carboxamide) [18] nanosuspensions (LFN) by solvent evaporation technique to improve the
treatment for rheumatoid arthritis.
Experimental Materials and Methods

Aravind Laboratories, Chennai generously gifted leflunomide; ethanol and tween 80 were procured from
Loba Chemie Pvt., Ltd., Mumbai, India; potassium dihydrogen phosphate, sodium hydroxide and sodium
chloride were purchased from SD fine chemicals, India. Dialysis membrane was acquired from Sigma-
Aldrich, USA.
Formulation of Nanosuspensions
Nanosuspensions was prepared by solvent evaporation method. Leflunomide (drug) and PEG (polymer)
were dissolved in ethanol. Tween 80 (surfactant) was dissolved in water separately. Then the drug-polymer
solution was quickly injected into aqueous surfactant solution under stirring in a magnetic stirrer at 3000
rpm. The stirring was continued for five hr to remove the organic solvent. After complete solvent evaporation
the resultant LFN was packed in amber colored bottle for further evaluation studies. The formulation
composition is shown in Table 1.
Table 1: Physicochemical characterization results of Leflunomide loaded nanosuspensions
Drug: Mean Zeta Drug Sedimentation

Formulation
Polymer particle size potential content (24 hr)
code
ratio (nm) (mV) (%)
F1 1:1 352 2.1 98.90 No
F2 1:2 386 4.8 99.03 No
F3 1:3 491 3.5 99.11 No
F4 2:1 400 6.1 99.33 No
F5 3:1 502 4.2 99.67 No
Drug-Excipient Interaction Studies

FT-IR spectral data shows the compatibility between the drug and the chosen excipients. In the present study
1:1 ratio of physical mixtures of drug and excipient were prepared and analyzed by FT-IR (Perkin Elmer
Model 1600) and UV (Shimadzu) techniques [19].
Physicochemical Characterization
Physicochemical characterization viz., Particle size distribution was determined by Photon Correlation
Spectroscopy (PCS) using a Zetasizer 3000; Zeta potential by a Laser Doppler Anemometer connected with
Malvern Zetasizer and Atomic Force Microscope analysis using the TECNAI-10 (PHILIPS) operated at 70-
80kV.
Drug Content
The amount of drug present in the formulated nanosuspensions was determined spectrophotometrically by
using phosphate buffer solution (pH 7.4) at 258 nm.
215
In-Vitro Release Studies

In-vitro release studies were carried out using dialysis membrane by soaking in pH 7.4 phosphate buffer
(PBS 7.4) for 5 hours. Then, 1 mL aliquots were collected at different time intervals and an equal volume of
the same buffer was replaced to maintain a sinking condition. The samples were suitably diluted with pH 7.4
phosphate buffers and the concentration of Leflunomide was analyzed with UV-visible spectrophotometer
at 258 nm.
Stability Study
A short stability study (3 months) was performed by keeping the nanosuspension in tightly sealed amber
colored bottles and kept in a place at room temperature. Samples were analyzed at regular intervals for its
physicochemical parameters and drug content.
Result and Discussion

One of the major problems associated with poorly soluble drug is very low bioavailability. Nanocarriers, in
their various forms, have the possibility of providing endless opportunities in the area of drug delivery and
therefore are increasingly being investigated to harness their potential. Excipients are integral components
of almost all pharmaceutical dosage forms thus it is mandatory to detect any possible physical or chemical
interaction of the drug with the excipients to produce a stable product, effective, attractive, easy to administer
and safe.
Excipient compatibility studies were carried out by FT-IR and UV techniques to investigate chemical
interactions between drug and the excipients. Leflunomide contains chemical functional groups like
with wave numbers of 3438, 2921, 1641, 1461, 1101 cm-1 for NH stretching, CH stretching, C = O
amide bond (stretching), CH bending and C-O stretching respectively. These characteristic bands
were present in the IR spectra of nanosuspension formulation. The IR spectrum of nanosuspension is
shown in Figure 1.
Figure 1: IR spectra of Leflunomide loaded Nanosuspension
In UV technique, the UV spectrum of drug is super impossible with the spectra obtained with drug excipients
mixtures and there is no change in the λmax (258 nm) between the drug and drug excipients mixtures. From the
FT-IR and UV results (Data not shown) revealed that there is no chemical interaction between the drug and the
excipients used in the formulation. Particle size of the formulation has a direct impact in the release of the drug
and its bioavailability. For suspension based products, the particle size of droplets of the internal phase have an
impact on the stability of the suspension itself. The physicochemical characterization results of the formulations
were shown in Table 1. Based on the physicochemical characterization results, formulation F3 was selected
for further studies. The AFM results showed that the particles were of nanometer in size range with uniform,
spherical with a smooth surface. The AFM image is shown in Figure 2.
216
Figure 2: 3D surface topography of Leflunomide loaded nanosuspension
Based on the release study results formulation F3 showed maximum release in 24 hr; hence
the formulation F3 has been taken for comparative in vitro release studies with the marketed tablet
formulation. Comparative release study results revealed that 99.51% w/w was released in one hour for
market formulation whereas optimized formulation showed 96.56% w/w in 24 hr. The release results
revealed that LFN showed faster onset of action and controlled drug release pattern. This also proves
the advantage of a polymeric nanosuspension for controlled delivery of leflunomide to improve anti-
rheumatoid arthritis therapy. The comparative in vitro release result is shown in Figure 3.
Figure 3: Comparison of drug release profile of optimized Leflunomide loaded nanosuspension (LNS) and commercial
tablet
Stability testing of the prepared formulation was carried out at room temperature for three months.
No changes were found with respect to the initial analysis results of the physicochemical parameters like
appearance, sedimentation, particle size and drug content. Stability study results revealed that the prepared
formulation was stable under stress conditions.
CONCLUSION
Solubility and dissolution rate are desirable factors to improve bioavailability. Nanosuspension of poor
water soluble drugs can be prepared by solvent evaporation method with surfactant and hydrophilic
polymer. Nano-sized leflunomide dissolved more rapidly. Our results findings clearly indicated the
suitability of formulation procedure for preparation of nano-sized poorly water soluble drug Leflunomide.
The release study results demonstrated that the compound’s aqueous solubility was increased by converting
217
into nanosized product and the developed Leflunomide nanosuspension exhibited controlled drug release.
These nanosized formulations can be used as a valid therapeutic approach and alternate to the existing
commercially available immediate release tablet formulation for the treatment of rheumatoid arthritis.
REFERENCE
1. Wilczewska AZ, Niemirowicz K, Markiewicz KH and Car H, Nanoparticles as drug delivery systems,
Pharmacological Reports, 2012; 64; 1020.1037.
2. Nevozhay D, Kanska U, Budzynska R and Boratynski J, Current status of research on conjugates and
related drug delivery systems in the treatment of cancer and other diseases (Polish), Postepy HigMed
Dosw. 2007; 61: 350–360.
3. Bamrungsap S, Zhao Z, Chen T, Wang L, Li C, Fu T and Tan W, Nanotechnology in therapeutics: a focus
on nanoparticles as a drug delivery system, Nanomedicine, 2012; 7: 1253–1271.
4. Koo OM, Rubinstein I and Onyuksel H, Role of nanotechnology in targeted drug delivery and imaging:
a concise review, Nanomedicine, 2005; 1: 193-212.
5. Sahoo SK and Labhasetwar V, Nanotech approaches drug delivery and imaging, Drug Discov Today.
2003; 8: 1112-20.
6. Lobenberg R, Smart Materials: Applications of nanotechnology in drug delivery and drug targeting,
Proceedings of the international conference on MEMS, NANO and Smart Systems (ICMENS’03), 2003.
7. Arruebo M, Fernandez-Pacheco R, Ibarra MR and Santamaria J, Magnetic nanoparticle for drug delivery,
Nanotoday; 2007; 2(3): 22-32.
8. Mishra B, Patel BB and Tiwari S, Colloidal nano carriers: a review on formulation technology, types and
applications toward targeted drug delivery, Nanomedicine: Nanotechnology, Biology, and Medicine,
2010; 6: 9–24.
9. Kingsley JD, Dou H, Morehead J, Rabinow B, Gendelman HE and Destache CJ, Nanotechnology: a
focus on nanoparticles as a drug delivery system, J Neuroimmune Pharmacol, 2006; 1: 340-50.
10. Caruthers SD, Wickline SA and Lanza GM, Nanotechnological applications in medicine, Curr Opin
Biotechnol, 2007; 18: 26-30.
11. Soppimath KS, Aminabhavi TM, Kulkarni AR and Rudzinski WE, Biodegradable polymeric
nanoparticles as drug delivery devices, J Control Release 2001; 70: 1-20.
12. Bawarski WE, Chidlowsky E, Bharali DJ and Mousa SA, Emerging nanopharmaceuticals,
Nanomedicine 2008; 4: 273-82.
13. Sen S, Raja C, Biplab D, Ganesh T, Raghavendra HG and Debnath S, Analgesic and anti-inflammatory
herbs: a potential source of modern medicine, International Journal of Pharmaceutical Sciences and
Research, 2010; 1(11): 32-44.
14. Agnihotri S, Wakode S and Agnihotri A, An overview on anti-inflammatory properties and chemo-
profiles of plants used in traditional medicine, Indian Journal of Natural Products and Resources, 2010;
1(2): 150-167.
15. Chatterjee GK and Pal SP, Search for anti-inflammatory agents from Indian Medicinal Plants-A review,
Indian Drugs, 1984; 21: 413.
16. Theofilou P, Psychological Impact of Rheumatoid Arthritis on Patient’s Health - Related Quality of
Life, J Arthritis, 2012; 1(1): 1-2.
218
17. Victoria Ruffing RN and Bingham CO, Rheumatoid Arthritis Signs and Symptoms, http://www.
hopkinsarthritis.org/arthritis-info/rheumatoid-arthritis/ra-symptoms/
18. Martindale, The complete drug reference, 2002. 33rd ed. Sweetman, C.S. (Ed.), Pharmaceutical Press,
London, pp.
19. Lakshmana Prabu S, Shanawaz S and Dinesh Kumar C, Compatibility Studies Between Duloxetine
Hydrochloride and Tablet Excipients Using Thermal and Non-thermal Methods, J Pharm Research,
2008; 7(1): 20-23.
219
Diclofenac Sodium Nanosuspension: An Approach to
Improve Anti-Inflammatory Therapy
S. Lakshmana Prabu, S.P. Sharavanan, A. Shanmugarathinam, K. Ruckmani, S.

Aravindan, A. Bhuvaneswari and V. Manikandan
Dept. of Pharmaceutical Technology, Bharathidasan Institute of Technology,

Anna University, Tiruchirappalli – 620 024
e-mail address: slaxmanvel@gmail.com, Tel.: +91-9750550965
Abstract:
Recently drugs with low aqueous solubility and high permeability present a high proportion of all drugs.
Poorly water-soluble compounds are frequently abandoned early in discovery and are difficult to be developed
into drug products by using conventional formulation techniques. In this study, we present an approach of
nanosizing a drug/polymeric complex to increase both solubility and dissolution rate of poorly water soluble
drug diclofenac sodium. Polymeric nanosuspensions were prepared by nanoprecipitation method by using
biodegradable polymer loaded with diclofenac sodium (DS), with the aim to improve anti-inflammatory
therapy. The prepared formulation was evaluated for drug excipients compatibility study, polydispersity
index, particle size analysis, surface morphology, zeta potential, drug release profile and stability. These
study results indicate a suitable formulation procedure for preparation of nanosized poorly water soluble drug
with significantly improved in vitro dissolution rate, and thus possibly enhanced fast onset of therapeutic
drug effect.
Keywords: Diclofenac sodium, Nanosuspension, Nanoprecipitation, Dissolution
INTRODUCTION
Oral drug delivery is the preferred way of drug administration, since it avoids the pain and discomfort
associated with injections and is more attractive from a marketing and patient compliance perspective
[1]. Recent advances in synthetic, analytical and purification chemistry along with the development of
specialized tools such as high throughput screening, combinatorial chemistry and proteomics have lead to
a sharp influx of discovery compounds entering into the development. It is estimated that 40% of all newly
developed drug compounds are poorly soluble or “insoluble” in water and upto 50% of orally administered
drug compounds present formulation problems related to high lipophilicity [2]. The dissolution properties
of a drug and its release from a dosage form have a basic impact on its bioavailability. Poor aqueous
solubility is one of the major hurdles in the development of new compounds into oral dosage forms, since
dissolution is the first step in the absorption of the drugs to produce the therapeutic effect [3]. Delivering
the drug precisely and safely to its target site at the right period of time to have a controlled release and
achieve the maximum therapeutic effect remains a yardstick in the design and development of novel
drug delivery systems. Reduction of particles size by micronisation does not lead to sufficiently high
bioavailability. Consequently, the next step was taken to move from micronisation to nanonisation, by
means of producing drug nanoparticles.
In recent years, nanoparticle engineering process has been seen as a promising approach for the
enhancement of drug solubility [4-6]. Thus, nanoparticle technology could play a major role in the
successful development and marketability of poor water soluble drug compounds. Nanocarriers, on account
of their higher ratio of surface area to volume, show improved pharmacokinetics and biodistribution of
therapeutic agents and thus minimize toxicity by their preferential accumulation at the target site [7]. Use
of polymeric nanoformulations is one of the most interesting approach to achieve local controlled drug
delivery [8-10].
Nanosuspension is produced by various technologies such as precipitation, pearl milling and high-
pressure homogenization, either in water or in mixtures of water and water-miscible liquids or nonaqueous
media [11-15].
Diclofenac (DCF), 2-[(2,6-dichlorophenyl)amino] phenylacetic acid, is a potent nonsteroidal anti-
inflammatory drug (NSAID) with a very low aqueous solubility and gastrolesive actions. It is used in
inflammatory and painful conditions of rheumatic and nonrheumatic origin [16-18]. The aim of this
investigation was to formulate and characterize diclofenac sodium nanosuspension by nanoprecipitation
technique to improve anti-inflammatory therapy.

Diclofenac sodium was graciously gifted by Cipla Pharmaceuticals, Mumbai, India; ethanol, hydrochloric
acid, tween 80, benzalkonium chloride, hydroxyl propyl methyl cellulose were obtained from Loba Chemie
Pvt. Ltd., Mumbai, India; potassium dihydrogen phosphate, sodium hydroxide and sodium chloride were
purchased from SD fine chemicals, India. Dialysis membrane was procured from Sigma Aldrich, USA.
Formulation of Nanosuspension
Nanosuspension was prepared by precipitation technique. Eight different compositions of formulations were
prepared with various ratios of drug and polymer. Diclofenac sodium was dissolved in 2 mL of ethanol, and
the polymer hypromellose was dissolved in 2 mL of 1:1 ratio of water and ethanol mixture. Drug solution
was dispersed in the polymer solution at room temperature and sonicated for 5 minutes. The solution was
slowly injected with the help of the syringe into an aqueous phase (50 ml) containing various ratio of Tween
80 and 0.02 to 0.04% v/v of benzalkonium chloride which was kept in at room temperature. During an
addition of the drug solution the mixture was mixed by mechanical stirring at 4000 rpm for one hour and
then the dispersion was ultra sonicated for 10 minutes. Ethanol residues were left to evaporate under slow
mechanical stirring of the nanosuspension at room temperature for about 8 hours. Formulation composition
is shown in table 1.
Table 1: Composition of Diclofenac Nanosuspension formulations
Formulation Drug (mg) HPMC Surfactant Preservative Solvent Solvent

code (Tween 80) (Benzalkonium (Ethanol) (Water) (ml)
(% w/w) chloride) (ml)
(% w/w)
F1 200 200 2 0.04 2 20
F2 200 300 2 0.04 2 20
F3 200 400 2 0.04 2 20
F4 200 500 2 0.04 2 20
F5 200 600 2 0.04 2 20
F6 200 700 2 0.04 2 20
F7 200 800 2 0.04 2 20
F8 200 1000 2 0.04 2 20
221
Compatibility Studies
The compatibility studies have a considerable importance to choose appropriate excipients to produce
a product that is stable, efficacious, attractive, easy to administer and safe. The physical mixture
in 1:1 ratio of drug excipients maximizes the possibility of interaction and helps to determine the
incompatibilities. In the present study 1:1 ratio physical mixture was prepared and analyzed by FT-IR
and UV techniques [19].
Physicochemical characterization
Physicochemical characterization viz., Particle size distribution & Polydispersity Index were performed
by Photon Correlation Spectroscopy (PCS) using a Zetasizer 3000; Zeta potential by a laser Doppler
Anemometer connected with Malvern zetasizer and Transmission Electron Microscopic (TEM) Analysis
using the TECNAI-10(PHILIPS) operated at 70-80kV.
Drug Content
The amount of drug present in the formulation was determined spectrophotometrically by using phosphate
buffer solution at pH 7.4 at 276 nm.

In-vitro release studies were carried out by using dialysis membrane by soaking in phosphate buffer 7.4 for
5 hours. Sample of 1 mL was withdrawn from the dissolution setup at regular intervals for 5 hours, and an
equal volume of phosphate buffer (pH 7.4) was replaced to maintain a sink condition and anlaysed by UV
spectrophotometer at 276 nm.
Drug Release Kinetics

The model that best fits the release data was selected based on the correlation coefficient value in
various models. The model that gives highest R value was considered as best fit of release data.
Stability Study
The prepared nanosuspension was tightly sealed in amber colored bottles and kept in a place at room
temperature. At regular intervals, the stability samples was tested for its physicochemical parameters and
drug content. The stability study was carried out for a period of 3 months.
Results and Discussion

Low oral bioavailability of poorly water-soluble drugs poses a great challenge during drug development.
Various approaches have been developed to improve bioavailability by increasing the drug’s dissolution rate
and solubility.
Excipient compatibility studies were carried out by FT-IR and UV techniques to investigate
chemical interactions between drug and the excipients. Diclofenac sodium contains chemical functional
groups like carboxyl group, secondary amine group and C-Cl group, the corresponding wave numbers
are 2395.57, 3426.30 and 699.57 cm-1 respectively; these characteristic bands were present in all the
formulation composition. IR spectrum of nanosuspension is shown in Figure 1. In UV technique, the
UV spectrum of drug is super impossible with the spectra obtained with drug excipients mixtures and
there is no change in the λmax (276 nm) between the drug and drug excipients mixtures. From the FT-
IR and UV results revealed that there is no interaction between the drug and the excipients used in the
formulation.
222
Figure 1: IR spectrum of diclofenac sodium nanosuspension formulation
The best formulation was optimized based on the particle size and stability of the formulation. Since,
reduced particle size helps in the improvement of solubility of poorly soluble drug thereby increase in the
dissolution and bioavailability.
Table 2: Physicochemical characterization results of DNS formulations

Formulation Mean Particle Polydispersity Zeta potential
Drug content (%)
code size (nm) Index (PDI) (mV)
F1 522.0 0.511 -10.5 99.34
F2 527.5 0.521 -9.6 99.23
F3 546.9 0.574 -13.1 99.67
F4 549.2 0.645 -14.0 99.76
F5 560.1 0.538 -12.0 99.99
F6 564.3 0.456 -12.4 98.91
F7 570.0 0.234 -13.5 99.12
F8 600.4 0.367 -10.4 99.24
The polydispersity of the optimized formulation F3 was measured, and the value was found to be 0.574;
which indicates that the particles were broadly distributed. An increase or decrease in the particle size of the
drug in a formulation can affect its in vitro release and subsequently its bioavailability. For suspension based
products, the particle size of droplets of the internal phase has an impact on the stability of a suspension
itself. The mean particle size was found to be 546.9 nm. The physicochemical characterization results of the
DNS formulations were shown in Table 2. The TEM results showed that the particles were of nanometer in
size range with uniform, spherical with a smooth surface. The TEM image is shown in Figure 2.
Figure 2: TEM image of diclofenac nanosuspension F3
223
Based on the release study results formulation F3 showed maximum release in 5 hrs; hence the
formulation F3 has been taken for comparative in vitro release studies with the market formulation.
Comparative release study results revealed that 99.96% w/w and 79.74% w/w of drug release was
observed after 5 hours for Nanosuspension formulation and marketed product respectively. The release
results revealed that diclofenac sodium nanosuspension showed faster onset of action. This also
proves the advantage of a polymeric nanosuspension in delivering diclofenac sodium to improve anti-
inflammatory therapy. The comparative in vitro release result is shown in Figure 3.
Figure 3: Comparative in-vitro release profile of diclofenac nanosuspension and marketed tablet
The regression coefficient (R2) for the drug release kinetic studies were found to be 0.835, 0.9722,
0.9475, 0.9603 and 0.9885 for Zero order, the first order, Korsmeyer peppas, Higuchi plot and Hixon
crowell model respectively. From the release kinetics studies, it was observed that the drug release from the
optimized nanosuspension formulations (F3) obey the first order kinetics and shows that the drug release
was dependant of its concentration. The Higuchi plot confirmed that all formulations obeyed the Higuchi
equations that the drug released through diffusion from the polymeric matrix. The Korsmeyer peppas plot
also shows a good linearity. The n values of the formulation were found to be less than 0.5, confirmed
that the release was Fickian diffusion transport. The Hixon crowell plot found with good linearity for the
formulation. It confirmed that the formulation loses its weight with time either due to erosion or diffusion of
the drug from the system.
Stability testing of the prepared formulation was carried out at 25°C and 65% RH for 3 months. The
samples were taken and analyzed for their physicochemical parameters for 3 months. The physicochemical
parameters like appearance, particle size and drug content were found to be satisfacotry, and there is no change
with respected to the initial analysis results. Stability study results revealed that the prepared formulation
was stable in the stress condition.
CONCLUSION
Dissolution rate and solubility are two of several factors that affect oral bioavailability of poorly water-
soluble compounds. In this study an approach to prepare a suitable procedure for preparation of diclofenac
sodium nanosuspension with HPMC by nanoprecipitation technique was designed. The optimized
formulation shows increased dissolution rate and bioavailability. These nanosized particles with a very
large interfacial area can influence the transport and delivery properties of the incorporated drugs and
provide for site-specific targeting. The results clearly indicated the suitability of formulation procedure
for preparation of nanosized poorly water soluble drug with significant improvement of the in vitro
dissolution rate, and thus possibly improve oral bioavailability and to produce better therapeutic effect
than the existing conventional tablet.
224
REFERENCES
1. Fasano A, Innovative strategies for the oral delivery of drugs and peptides, TIBTECH, 1998; 16: 152-
157.
2. Lomabardino JG, Joseph GL and John AL, A guide to drug discovery: The role of the medicinal chemist
in drug discovery then and now. Nat Rev. Drug Disco, 2004; 3: 853–862.
3. Lipinslki C, Poor aqueous solubility an industry wide problem in drug discovery, Am Pharm Rev, 2002;
5: 82–85.
4. Wong SM, Kellaway IW and Murdan S, Enhancement of the dissolution rate and oral absorption of a
poorly water soluble drug by formation of surfactant-containing microparticles, Int J Pharm, 2006; 317:
61-68.
5. Rabinow BE, Nanosuspensions in drug delivery Nat Rev. Drug Disco. 2006; 3: 785–796.
6. Patravale VB, Date AA and Kulkarni RM, Nanosuspensions: a promising drug delivery strategy, J Pharm
Pharmacol, 2004; 56: 827–840.
7. Alexis F, Rhee J, Richie J, Radovic-Moreno A, Langer R and Farokhzad O, New frontiers in
nanotechnology for cancer treatment, Urol. Oncol, 26: 74-85, 2008.
8. Li VHK, Wood RW, Kreuter J, Harmia T and Robinson J.R., Ocular drug delivery of progesterone using
nanoparticles, J Microencapsul, 1986; 3: 213–218.
9. Marchal-Heussler L, Sirbat D, Hoffman M and Maincent P, Poly (epsilon-caprolactone) nano-capsules
in carteolol ophthalmic delivery, Pharm Res, 1993; 10: 386-90.
10. Calvo P, Vila-Jato JL and Alonso MJ, Evaluation of cationic polymer-coated nanocapsules as ocular
drug carriers, Int J Pharm, 1997; 153: 41–50.
11. Liversidge GG and Cundy KC, Particle size reduction for improvement of oral bioavailability of
hydrophobic drugs: Absolute oralbioavailability of nanocrystalline danazol in beagle dogs, Int J Pharm,
1995; 125(1): 91- 97.
12. Peters K, Leitzkeb S, Diederichs JE, Borner K, Hahn H, Muller R.H. et al., Preparation of a clofazimine
nanosuspension for intravenous use and evaluation of its therapeutic efficacy in murine Mycobacterium
aviuminfection, J Antimicro Chem, 2000; 45(1): 77–83.
13. Trotta M, Gallarate M, Pattarino F and Morel S, Emulsions containing water-miscible solvents for the
preparation of drug nanosuspensions partially, J Control Release, 2001, 76(1-2): 119-128.
14. Debuigne F, Cuisenaire J, Jeunieau L, Masereel B and Nagy J.B., Synthesis of nimesulide nanoparticles
in the Microemulsion epikuron/isopropyl myristate/water/n-butanol (or/isopropanol), J Colloid Interface
Sci, 2001; 243: 90–101.
15. Hecq J, Deleers M, Fanara D, Vranck H and Amighi K, Preparation and characterization of nanocrystals
for solubility and dissolution rate enhancement of nifedipine, Int J Pharm, 2005; 299: 167-177.
16. Martindale, The complete drug reference, 2002, 33rd ed. Sweetman, C.S. (Ed.), Pharmaceutical Press,
London, pp. 30–32.
17. Fini A, Fazio G and Feroci G, Solubility and solubilization properties of non-steroidal antiinflammatory
drugs, Int J Pharm, 1995; 126: 95–102.
18. Kockbek P, Baumgartner S and Kristl J, Preparation and evaluation of nanosuspensions for enhancing
the dissolution of poorly soluble drugs, Int J Pharm, 2006; 312: 179–186.
19. Lakshmana Prabu S, Shnanawaz S and Dinesh Kumar C, Compatibility Studies Between Duloxetine
Hydrochloride and Tablet Excipients Using Thermal and Non-thermal Methods, J Pharm Research,
2008; 7(1): 20-23.
225
Formulation and In Vitro - In Vivo Evaluation
of Wedelolactone Loaded Nanosuspension for
Hepatoprotective Activity in CCl4 Induced Significant
Hepatic Damage and Oxidative Stress Model
S. Brito Raj1, P. Sucharitha*1, T. Murali1, S. Wasim Raja2 and K. Bhaskar Reddy1
Department of Pharmaceutics, Sri Venkateswara College of Pharmacy, RVS Nagar, Chittoor - 517 127,
Andhrapradesh, India
Department of Pharmacy Practice, Sri Venkateswara College of Pharmacy, RVS Nagar, Chittoor - 517 127,
Andhrapradesh, India
e-mail: sucharitha89pharma@gmail.com, Mob: 09052879033
Abstract:
Nanosuspension have emerged as a promising strategy for the efficient delivery of hydrophobic drugs because
of their versatile features and unique advantages. The aim of present study was to develop Nanosuspension
of poorly water soluble drug wedelolactone by using a combination of solvent evaporation followed by high-
pressure homogenization and it was an optimized technique for the preparation of Nanosuspension, which
lead to better results like high efficiency, high drug content. Prepared Nanosuspension was evaluated for
drug content, SEM, particle size distribution, zeta potential, polydispersity, in-vitro drug release, and in-vivo
hepatoprotective activity by CCl4 induced significant hepatic damage and oxidative stress model. The results
Show that the prepared Nanosuspension persists good stability with 98.91% drug content; Droplet size
was found to be 115.9 nm with poly dispersive index 0.263 and -3.04 mV of zeta potential. This indicates
that nanosuspension is a stable formulation. SEM photography showed smooth surface and spherical in
shape. The drug release of Nanosuspension formulation is about 97.72 % which indicates the increase in
dissolution rate of wedelolactone compared to conventional dosage forms. The formulated wedelolactone
Nanosuspension having improved dissolution rate and bioavailability compared to conventional dosage
forms.
INTRODUCTION
Oral route has been the commonly adopted and most convenient route for the drug delivery. The formulation
of poorly water-soluble drugs has always been a challenging to pharmaceutical scientists and it is expected
to increase because approximately 40% or more of the new chemical entities being generated through drug
discovery programmes are poorly water-soluble.[1]
A pharmaceutical Nanosuspension is defined as “very finely dispersed solid drug particles in an aqueous
vehicle, stabilized by surfactants, for either oral and topical use or parenteral and pulmonary administration,
with reduced particle size, leading to an increased dissolution rate and therefore improved bioavailability”.
The diameter of the suspended particle is less than 1 μm in size (i.e. 0.1nm-1000 nm) [2, 3].
Techniques of Nano Suspensions Preparation:

Two techniques prepare Nanosuspensions. They are
i. Top-down technology

These are the mechanical communication process of larger drug particles, as in milling (jet mills and pear-
ball mills) and homogenization (high-pressure homogenizers).
ii. Bottom-up technology
In Bottom-up Technology the drug is dissolved in a solvent, which is then added to non-solvent to precipitate
the crystals. This technique is that during the precipitation procedure the growing of the drug crystals needs
to be controlled by addition of surfactant to avoid formation of Nano or Microparticles. The ‘Top Down
Technologies’ are the disintegration methods and are preferred over the precipitation methods.

The chemicals used for the following research includes methanol, Glacial acetic acid, Sodium lauryl sulphate,
CCL4, Olive oil, Distilled water, and all the chemicals are Spectrum grade.
Extraction Methodology of Eclipta Alba:
Extraction by using Soxhlet apparatus- methanolic extraction process:

The coarse powder of whole plant of Eclipta alba was extracted with methanol in herb: menstrum ratio of 1:6
by hot solvent extraction by heating with it for 12 hr. The methanolic extract was concentrated under reduced
pressure to obtain a dark green coloured sticky mass.
Screening of Phytochemical Constituents of Crude Extract:
Table 1: Screening of phytochemical constituents of crude extract
Test for alkaloids Dragendorff’s test Reddish brown precipitate with

Dragendorff’s reagent- presence of alkaloids.
Mayer’s test Cream colour precipitate with Mayer’s reagent
Test for carbohydrates Molisch’s test Purple to violet colour ring appears at the
junction-presence of carbohydrates.
Test for flavonoids Alkaline reagent test By adding few drops of sodium hydroxide
solution, intense yellow colour is formed-
presence of flavonoids.
Test for tannins Ferric chloride test By treating the extract with ferric chloride
solution, blue colour appears -hydrolysable
tannins are present & green colour appears if
condensed tannins are present.
Test for proteins Biuret test Violet colour indicates presence of proteins.
Test for steroids Sulphur powder test Sulphur sinks at the bottom-presence of steroids.
Test for starch Amylum Produce blue colour with weak aqueous iodine
solution.
Test for amino acids Ninhydrine test violet colour- Amino acid
Test for naphthoquinones Dam-Karrer test By adding potassium hydroxide solution blue
colour develops.
Test for cardiac glycoside Baljet’s test Orange colour is formed by treating with picric
acid.
Test for saponin Froth formation test Place 2ml solution of drug in water in a test tube
glycoside –froth is formed
Formulation of Nanosuspension Containing Isolated Wedalolactone

Rapid addition of a drug solution to anti-solvent leads to sudden supersaturation of the mixed solution and
227
generation of fine crystalline or amorphous solids. Precipitation of an amorphous material may be favoured
at high supersaturation when the solubility of the amorphous state is exceeded. This is the basic principles of
precipitation and homogenization. The combination of these techniques results in smaller particle size and
better stability in a shorter time.
Table 2: Composition of various formulations of Nanosuspension containing Wedelolactone
Sl.No. Drug Organic Phase Aqueous Phase Surfactant (Sodium

(wedelolactone) (Glacial Acetic Acid) (Water) Lauryl Suphate)
F1 40 mg 5 ml 10 ml 25 mg
F2 40 mg 5 ml 10 ml 20 mg
F3 40 mg 5 ml 10 ml 30 mg
F4 40 mg 5 ml 10 ml 5 mg
F5 40 mg 5 ml 10 ml 15 mg
F6 40 mg 5 ml 10 ml 10 mg
Evaluation of Nanosuspension
Nanosuspensions are characterized for appearance, colour, odour, assay, related impurities, particle size,
zeta potential, crystalline status, dissolution studies and in vivo studies. Among this, the most important
characterization techniques were discussed. [5]
Mean particle size and size distribution

Various parameters of nanosuspensions like saturation solubility, dissolution velocity, physical stability,
dissolution velocity, physical stability and biological performance depend on the mean particle size
and particle size distribution. Mean particle size and particle width (poly-dispersity index) can be
determined by Photon Correlation Spectroscopy (PCS), laser diffraction, and counter current multi-
sizer. Poly-dispersity index (PI) should be low for the long-term stability of the nanosuspensions.
To detect and quantify the microparticles that might have been generated during the production process,
laser diffractometry (LD) analysis should be carried out in addition to PCS analysis. Particles ranging from
0.05–80 μm and in certain instruments particle sizes up to 2000 μm can be measured by using LD. Particle
size analysis by the Coulter counter technique is essential (in addition to PCS and LD) for nanosuspensions
228
that are intended for intravenous administration. [6] Coulter counter is a more efficient and appropriate
technique than LD analysis as it gives the absolute number of particles per volume unit for different size
classes. It quantifies the contamination of nanosuspensions by micro particulate drugs.
Particle charge (Zeta Potential):

Zeta potential determines the stability of the Nanosuspension. Both the stabilizer and the drug govern the
zeta potential of a Nanosuspension. A zeta potential of minimum ± 30mV is required for electrostatically
stabilized Nanosuspension and ± 20mV is required in case of electrostatic and steric stabilization.
Scanning electron microscopy:

Scanning electron microscopy (SEM) was conducted to characterize the surface morphology of the
Nanosuspension.[5] The samples were mounted on alumina stubs using double adhesive tape, coated with
gold in HUS-5GB vacuum evaporator. Then the sample was observed in Hitachi S-3000N SEM at an
acceleration voltage of 10KV and a magnification of 5000X.
Saturation solubility and dissolution velocity:

Reduction in particle size results in increased dissolution pressure and hence the solubility. Change in surface
tension occurs as the solubility increases (due to particle size reduction) which lead to increased saturation
solubility. Different physiological solutions at different pH and different temperatures are used to carry out
the determination of the saturation solubility and dissolution velocity according to the methods reported in
the pharmacopoeia. Increase in saturation solubility can be explained by the Ostwald-Freundlich equation [5].
Determination of the dissolution velocity of nanosuspensions provides the information about the advantages of
Nanosuspension over conventional formulations, especially in sustained-release dosage forms.
The Ostwald-Freundlich equation is:
C(r) = C(∞) exp (2γM / rρRT) ------Equation (2)
Where C(r) and C (∞) are the solubilities of a particle of radius r and infinite size. γ, M, and ρ are
interfacial tension at the particle surface, the molecular weight of the solute, and the density of the particle,
respectively.[5]
Stability:
Nanosuspensions Stability depends on the particle size of the suspended particles. Decrease in the particle
size to the nano range increases the surface energy of the particles, and the tendency of the particles to
agglomerate increases. Therefore, the stabilizers are used to decrease the chances of Ostwald ripening and to
improve the stability of the suspension.
Methodology:
Nanosuspensions can be stored at different stress conditions like different temperature (15, 25, 35 45°C),
thermal cycling, and mechanical shaking and change in their mean particle size can be followed for
three months. Different concentrations of small molecule surfactants (like sodium lauryl sulphate (SLS)
and dowfax 2A1 (DF)) and polymeric stabilizer (like Hydroxypropyl methylcellulose (HPMC)) can be
evaluated to determine the effect of stabilizer type and micellar solubilized drug on Ostwald ripening. [7]
pH:
The pH of the Nanosuspension can be easily measured by using pH meter [8]
Osmolarity:
Practically, Osmolarity of Nanosuspension can be measured by using Osmometer [8]
229
Drug content:
Drug content of Nanosuspension formulation can be carried out by extracting the Nanosuspension in suitable
solvent mixture, like Methanol: THF (1:1) mixture, shaken well, and then centrifuged. The supernatants can
be separated and diluted with same solvent mixture, and the absorbance can be measured at suitable λmax.
The drug content then can be calculated using the calibration curve. [8]
In vivo studies:
The present study was conducted to compare the hepatoprotective activity of a marketed formulations Liv-
52 and nano suspension containing fraction of Eclipta alba against CCl4-induced hepatotoxicity in rats.
Requirements:
Animals:
Wister strain male albino rats having a weight range of 150-180 g were used for the experiment. The animals
were well housed in polypropylene cages under hygiene conditions and maintained at 28 °C temperature.
The animals were allowed to have food and water adlibitum. The Institutional Animals Ethics Committee
approved all the experimental protocols IAEC no (SVCP/1029/2014).
Chemicals and Drugs: Liv-52 syrup (The Himalaya Drug Company) and nano suspension containing
wedelolactone were used to evaluate the hepatoprotective activity, and carbon tetrachloride was used to
induce hepatotoxicity.
Acute toxicity studies:

According to the guideline 423 acute toxicity studies were performed for the isolated wedelolactone. Two
groups of albino rats such as group A and group B were selected containing 6 rats in each group. 1g/kg body
drug were given to each rat and observed for 24 hrs. After 24 hrs 8 rats survived and 4 rats died. Finally, the
dose was fixed to 40 mg/kg body after the acute toxicity studies.
Experimental Design
Animals are divided into five groups (n = 6). Considering human dose (HD) of
Liv-52 (HD-15 ml daily) and, the rat dose was calculated on the basis of weight of the rats.
Group I : Normal control vehicle i.e. distilled water,
Group II : Negative control CCl4 (1 ml/kg, i.p.)
Group III : Liv-52 (0.216 ml/kg, p.o.)
Group IV : isolated wedelolactone (40 mg / kg)
Group V : Nanosuspension treated (0.216 ml/kg)
Treatment duration was 13 days, and the dose of CCl4 (30% v/v in olive oil) is administered after every
24 hrs. CCl4 is given orally for 5 days to induce hepatotoxicity. On 6th day the dose is given to Group III, IV,
and V according to the weight of the rats. This dose is given orally for 7 days.
Animals were sacrificed on 13th day. Blood was collected, allowed to clot, and the serum is separated.
The liver was dissected out and used for biochemical and histo-pathological studies.
Activation and preparation of silica gel:

20 g of silica gel (60-100 mesh no.) powder were activated by using oven with 150°C for 1 hr., it was
hydrophobized by adding 100ml chloroform, and heated continuously at 150 °C in vacuum drying oven,
230
then it was cooled for few minute and suspended with 100 ml of methanol with stirring , the column was
prepared (5x54cm), and the silica gel suspension was impacted into column until settling , the final silica
gel length was 50 cm, addition 10ml from crud in column and setting the flow rate with methanol into
3ml /5min.
Preparation of mobile phase:

The mobile phase was prepared from two different organic solvent, This solvent mixture was which were
methanol and chloroform (70:30).known as the “eluent” or “mobile phase”, each component of the mixture
will exhibit a different interaction with stationary and mobile phases; this interaction will largely depend
on the chemical structure and geometry of the molecule concerned. The components of the mixture will be
carried down the column at variable rates separation, may be achieved.
Fractions collection:
The fractions were eluted after the sample was poured in the prepared column; each colored sample is collect
separately in a conical flask or any beaker. The collected samples are concentrated by using Rotary Flash
Evaporator, and the concentrated samples are subjecting for Chemical test, FTIR, and TLC to conform the
active component. The fraction was measured by spectrophotometer at UV at 351 nm.
Biochemical Estimation
The blood was obtained from all the animals by puncturing the retro-orbital plexus. The blood samples were
allowed to clot for 45 min at room temperature.[9] Serum was separated by centrifugation at 2500 rpm for 15
min at 30°C and utilized for the estimation of various biochemical parameters, namely, SGOT, SGPT, Total
Protein (Beacon diagnostic kit, Navsari) and Bilirubin (Bio lab diagnostic kit, Maharashtra).
SGOT = Activity (U/L) =∆OD/min. x 1768
SGPT= Activity (U/L) =∆OD/min. x 1768
Total Bilirubin in mg/dl = (Abs of Tt – Abs of Bt) x 26.31
Direct Bilirubin in mg/dl = (Abs of TD – Abs of BD) x 26.31
Histopathological Examination
All animals were sacrificed on the last day of the study; blood was collected, and liver was removed and
washed with saline. Liver pieces were preserved in 10% formalin for histopathological study. The sections
were approximately 4-6 micron in thickness. They were stained with hematoxylin and eosin and photographed.
The data were expressed as mean S.D.; for obtaining this data, biochemical and physiological parameters
were statistically analyzed using one-way ANOVA followed by Dunnett’s test. [10]

Identification of Wedelolactone from the Extract of Eclipta alba
Coumarin test: A mixture of Toluene: Acetone: Formic acid in the ratio of 1:2:1 was taken as
mobile phase was taken in a beaker, and the extract was spotted in the TLC plate was kept in the beaker.
Appearance of purple to violet color was indicated the coumarins (Wedelolactone) presence in Eclipta
alba plant extract.
The detection of the wedelolactone in the partially purified samples after concentrated the collected
fraction using TLC technique,the results showed a positive reaction for wedelolactone throughout
231
the appearance of purple color under UV light, and also by calculating the RF value which reached
into 0.56 cm as noted. This result was identical to several recent researches that mention by Harborne
(1973). [11]
Figure 1: TLC showing light violet spot indicating the presence of wedalolactone
Measure the absorption spectrum of wedelolactone (UV-visible light):

The tested wedelolactone shows emergence of one sharp peak at a wavelength of 351nm.
Evaluation of Nanosuspension:
SEM: Scanning electron microscopy (SEM):

Shape and surface morphology of the Nanosuspension prepared with optimized parameters was observed by
research microscope and scanning electron microscopy. The study revealed that most of the Nanosuspension
was fairly spherical in shape; the surface of the particle showed a characteristic smoothness and that the
particle size was in the nanometric range as depicted by SEM. Some of the particles were found to be in
clusters as shown in the Figure 2.
Figure 2: SEM-Surface Morphology of Nanosuspension
Optimization of formulation based on the effect of SLS concentration on Average particle size, PDI and Zeta
potential
The particle size analysis revealed that, the Nanosuspension was in the nanometer range. The size of the
nanoparticles was affected by the stirring time and the concentration of SLS. The size of the Nanosuspension
containing Eclipta alba was found to be between 115.9nm to 407.4 nm that were tabulated below.
The stability of the formulated Nanosuspension was evaluated by measuring the zeta potential of the
Nanosuspension by the Malvern particle size analyzer. The results are shown in Table 3 and Figure 3, 4.
Zeta potential of Eclipta alba loaded formulations was in the range of -1.84 ± 1.82 to -3.70 ± 2.28 mV and
Polydispersity index was found to be between 0.234±0.028 to 0.326±0.012.
232
Table 3: Effect of SLS on Particle size, PDI and Zeta Potential
Formulation SLS Mean Particle Poly Dispersibility Zeta Potential

concentration size (nm) Index (mV)
%
F1 25 260.2±3.9 0.326±0.012 -3.70 ± 2.28
F2 20 246.4±1.8 0.312±0.018 -3.33 ± 2.40
F3 30 224.6 ±4.1 0.535±0.024 -3.04 ± 1.84
F4 5 407.4±6.7 0.358±0.020 -1.84 ± 1.82
F5 15 311.6±1.4 0.244±0.018 -2.44 ± 2.80
F6 10 378.00±1.8 0.234±0.028 -2.85± 1.80
*Standard deviation (n=3)
Figure 3: Particle size distribution of Best optimized formulation F3
Figure 4: Zeta potential studies of best-optimized formulation F3
233
Drug content
The prepared formulations were analyzed for drug content. It was observed that the drug content in the
prepared Nanosuspension was satisfactory, and the drug was uniformly distributed in all the formulations.
The percentage drug content is highest for F3 formulation was about 98.91 ± 2.050 and lowest
In-vivo Studies:
Table 4: In-vivo studies showing the difference in biochemical parameters in infected and drug-treated liver
GROUP SGOT (IU/L) SGPT (IU/L) TOTAL BILURUBIN – LIVER WEIGHT

PROTEIN (g/dL) TOTAL (mg/dL) (g/100 b.w)
I – Control Group 21.54 ± 0.15 a 41.53 ± 0.16 a 6.34 ± 0.02 a 0.80 ± 0.005 a 5.01
II – Negative control 26.09 ± 0.80 46.47 ± 1.07 9.10 ± 0.47 1.98 ± 0.002 6.03
III – LIV 52 groups 22.98 ± 0.54 a 43.94 ± 0.54 a 7.77 ± 0.12 a 1.44 ± 0.002 a 5.48
IV – Extract treated 24.52 ± 0.39 b 45.58 ± 0.96 c 7.46 ± 0.02 a 1.24 ± 0.002 a 5.51
group
V – Nanosuspension 22.92 ± 0.83 a 42.55 ± 0.12 a 6.85 ± 0.05 a 0.94 ± 0.002 a 5.16
treated group
a = p < 0.001 (***); b = p < 0.01 (**);c = p < 0.05 (*); All the values are shown are mean ± SEM, n=6.

Figure 5: Serum SGOT level on 30th day Figure 6: Total protein level on 30th day
Figure 7: Serum SGPT level on 30th day Figure 8: Bilirubin level on 30th day
In this study, rats treated with a single dose of CCl4 developed significant hepatic damage and oxidative
stress, which was observed from a substantial increase in the activities of serum, SGOT, SGPT, total protein
234
and bilirubin. This is indicative of cellular leakage and loss of the functional integrity of the cell membrane
in liver.CCl4 is one of the most commonly used hepatotoxins experimental study of liver disease. The lipid
peroxidative degradation of biomembrane is one of the principle causes of hepatotoxicity of CCl4. This is
evident from an elevation in the serum marker analysis, namely AST, ALT, total proteins and bilirubin. The
formulations significantly reduced this serum enzyme in groups
The simultaneous administration of formulations and CCl4 produced significant recovery of the liver
damage induced by CCl4. The hepatotoxic effect of CCl4 is largely due to its active metabolite trichloromethyl
radical that binds to the macromolecule and induces peroxidative degradation of the membrane lipids of
an endoplasmic reticulum that is rich in polyunsaturated fatty acids. This leads to the formation of lipid
peroxide, which in turn produces a toxic aldehyde that causes damage to the liver. This was evident by an
increase in the level of lipid peroxidation in the CCl4 group, and there was a significant decrease in lipid
peroxidation in the groups treated with CCl4 and formulations.
The comparative histopathological study of the liver from different groups of rats corroborated the
hepatoprotective efficacy of formulations. Various pathological changes such as steatosis, centrilobular necrosis
and vacuolization observed in group II rats were prevented to a moderate extent in groups III, IV and V. All the
effects of formulations were comparable with Liv-52 as a positive control. The biochemical studies in albino
rats revealed that CCl4-induced hepatic injury was inhibited extract and Nanosuspension containing Eclipta
alba significantly. All the results can be compared with the standard drug Liv-52. The above results also state
that nanosuspensions containing Eclipta alba has shown significant hepatoprotective activity in comparison to
Liv-52. In support, the histopathological reports also revealed that there is a regenerative activity in the liver
cells.
The present study is to investigate the Nanosuspension containing Eclipta alba extracts against
the hepatotoxic activity. And results have shown that Nanosuspension shows very significant effect on
male Wistar albino rats induced by CCl4. When compared to Negative control group, Extract group and
Nanosuspension group shown Hepatoprotective effect based on SGOT, SGPT, Bilirubin and Total protein
data. When compared to extract group Nanosuspension group shows very significant Hepatoprotective
activity.
SUMMARY
Nanosuspension containing Eclipta alba was prepared and evaluates by Solvent Evaporation Method
followed by Homogenization. From the results it can be concluded as follows, i.e., Particle size
parameter shows that, particle size of Nanosuspension (407.4 to 224.6 nm) decreases with an increase
in the concentration of the SLS. Polydispersity index was within the range of 0.244 to 0.326, which
shows the prepared Nanosuspension formulation shows a homogeneous size distribution in all over
the formulation. The results of Zeta potential in formulation F3 shows -3.04 mV that shows that F3
formulation having high potential to conduct the surface charges with good stability, which confirms
that F3 Nanosuspension containing Eclipta alba shows better dispersion mechanism in the medium.
SEM clearly represents that the prepared Nanosuspension containing Eclipta alba were nearly spherical
in nature, the surface of the particle was smooth and that the particle size was in the nanometric range.
Prepared Nanosuspension is evaluated for the drug content. All the formulation shows better results i.e.
there is no any major difference between the formulations.
CONCLUSION:
It concludes that F3 is the best formulation among all the formulation. In-vivo studies that was carried for
the best formulation shows that, F3 Nanosuspension containing Eclipta alba shows maximum solubility and
bio-availability compared to marketed product LIV 52.
235
REFERENCES:
1. Dhiman S, Dharmila and Thakur GS, Nanosuspension: A recent approach for nano drug delivery system,
Int J Curr Pharm Res. 2010, 3(4); 96-101.
2. Jagdale DM, Kamble VA and Kadam VJ, Nanosuspension a novel drug delivery system, International
Journal of Pharma and Bio Sciences. 2010, 1(4); 352-360.
3. Chingunpituk J, Nanosuspension technology for drug delivery, Walailak J Sci & Tech. 2007, 4(2); 139-
153.
4. Elaine M, Liversidge Gary G and Liversidge M, Drug nanoparticles formulating poorly water soluble
compounds, Toxicologic Pathology, 2008, 36; 43-48.
5. Kawakami K, Modification of physicochemical characteristics of active pharmaceutical ingredients &
application of supersaturable dosage forms for improving bioavailability of poorly improving poorly
absorbed drugs, Advanced Drug Delivery Reviews, 2012, 3;168 -173.
6. Jacobs C, Kayser O and Muller R, Nanosuspensions as particulate drug formulations in therapy: rationale
for the development & what we can expect for the future, Advanced Drug Delivery Reviews, 2001, 47
(1); 3-19.
7. Burgess DJ, Gokhale R, Kumar S and Verma S, Physical stability of nanosuspensions: Investigation
of the role of stabilizers on Ostwald ripening, International Journal of Pharmaceutics, 2011, 406 (1-2);
145-152.
8. Patel M, Shah A and Patel KR, Nanosuspension: A novel approach for drug delivery system, JPSBR,
2011, 1(1), 1-10.
9. Manoj B and Aqueed UK, Protective role of Lawsonia alba Lam against CCl4 induced hepatic damage
in albino rats, Indian J Exp Biol. 2003, 41; 85-7.
10. Rui Yang, Renchao Gao, Fang Li, Haibing He and Xing Tang, The influence of lipid characteristics on
the formation, in vitro release, and in vivo absorption of protein loaded SLN prepared by the double
emulsion process, Drug Development and Industrial Pharmacy, 2011; 37(2): 139-148.
11. Harborne JB, Phytochemical methods, Chapman and Hall Ltd., London, 1973,
49-188.
236
Enhanced Oral Bioavailability of Naringenin Using
Polymeric Nanoparticles: Formulation, Characterization
and In Vivo Studies
P. Suseelaa, K. Premkumarb, S.D. Saraswathya
a
Molecular Pathology and Toxicology Laboratory, bCancer Genetics and Nanomedicine Laboratory
Department of Biomedical Science, School of Basic Medical Sciences, Bharathidasan University,
Tiruchirappalli 620024, Tamilnadu, India
e-mail: sd.saraswathy@gmail.com, Mobile: 9940181253
Abstract:
Flavonoids are natural products widely distributed in plant kingdom and currently consumed in large amounts
in the daily diet. Naringenin (NRG) is a flavonoid specific to citrus fruits and possesses anti-inflammatory,
anticarcinogenic, and antitumor effects. To improve the solubility and prolong its duration in body system,
the present study was designed to formulate, characterize and evaluate its pharmacokinetic profile in NRG
encapsulated polymeric nanoparticles (PNPs). NRG encapsulated PNPs were formulated by nanoprecipitation
method in ten different batches and named as NF1, NF2 ………..… NF10 respectively. The formulated
nanoparticles were characterized by Dynamic light scattering (DLS), Zeta potential (ζ), Scanning Electron
Microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). The in vitro drug encapsulation
efficiency, drug release and in vivo pharmacokinetic profile and toxicity studies were performed with the
formulated nanoparticles. Among ten different batches studied, the NF10 batch showed lowest mean particle
size and highest zeta potential was found to be 78 ± 12 nm and -34 ± 1.64 respectively. The characteristic
bands of NRG in FTIR were either shifted or reduced in the spectrum of PNPs. Scanning electron microscopy
of naringenin nanoparticles morphology revealed that spherical in shape. In vitro drug release, study showed
that PNPs was capable of releasing the drug in sustained manner. Enhanced pharmacokinetic parameters
were seen in PNPs as compared to free NRG. From our results, we thus conclude that PNPs prepared in
NF10 batch was the most effective formulation for enhanced oral bioavailability which can be used for
further studies to identify its biomedical applications.
Keywords: Naringenin, Oral drug delivery, Nanoparticles, Sustained release, Eudragit, Oral bioavailability
INTRODUCTION
Polymeric nanoparticles have attracted tremendous attention for their diverse biomedical applications
including optical materials, drug delivery systems and biomaterials [1-3]. In recent years, the approach of
utilizing the polymeric nanoparticles as a carrier system for delivery of drugs has gained much more interest
[4]. Oral drug delivery system has involved great attention in the pharmaceutical field to attain numerous
advantages over the conventional dosage forms. These embrace improved efficiency, reduced side effects
and improved bioavailability [5-6].
Naringenin (NRG, 4,’ 5, 7- trihydroxy flavanone), a naturally occurring flavonoid, and aglycone of
naringin, is widely present in citrus fruits, tomatoes, cherries, grapefruit and cocoa [7-11]. It is well known
for various biological actions, including antioxidant, anti- inflammatory and anticarcinogenic effects [12-
13]. Naringenin possesses tri hydroxyl groups in its aromatic rings which are accountable for its potent
antioxidant activity and widespread pharmacological property. Despite the wide spectrum of pharmacological
activities, the therapeutic effects of naringenin is limited due to its aqueous solubility and instability in
physiological medium [14]. These properties of naringenin result in poor bioavailability, poor permeability
and poor absorption, therefore developing strategies to overcome these difficulties and to enhance the oral
delivery system of naringenin are highly desirable.
In recent years, different strategies have been investigated to improve the dissolution and bioavailability
of naringenin [15-17]. These formulated nanoparticles that reported sustained release for only few hours,
thus improvement in bioavailability has been limited. It was expected that this system would improve
the water solubility of the drug, and hence improve its bioavailability by oral administration. All of these
reasons the study was necessary to formulate and characterize through mean particle size and zeta potential,
encapsulation efficiency, FTIR, morphology (SEM), in vitro drug release and in vivo toxicity studies.

Eudragit® E 100 (EE 100) was obtained from Evonik Industries, Mumbai, India. Naringenin (NRG) was
purchased from Sigma- Aldrich Chemicals, St. Louis, MO., Mumbai, India. While other fine chemicals, reagents
and solvents used for the experiments were of analytical grade, purchased from Merck Ltd., Mumbai, India. Milli
Q water was used in all experiments.
Formulation and Optimization of PNPs

Naringenin loaded with Eudragit® E 100 nanoparticles was prepared by using standard nanoprecipitation
method according to the procedure of Bilati et al., 2005 [18]. Briefly, Naringenin and Eudragit® E 100 at
ten different concentrations were dissolved in ethanol. These internal organic phase solutions were slowly
injected to external aqueous solution containing PVA, and the mixtures were then stirred at 1000rpm for
12hrs. Internal organic phase solutions are always composed of solvents, making the NRG and EE soluble
completely, and the external aqueous phase comprises aqueous solution, sometimes with polymeric stabilizer
in it. Rotary vacuum evaporation completely removed the ethanol at 40 °C water bath, and the remaining
fraction was washed with double- distilled water several times and lyophilized to obtain pure nanoparticles.
All the preparation was carried out in triplicate.
Size and zeta potential analysis of PNPs

Measurement of particle size and polydispersity index of the nanoparticles was performed by Photon
Correlation Spectroscopy (PCS) known as Dynamic Light Scattering using a Zetasizer® 3000 (Malvern
DTS1061). Each sample was appropriately diluted 10 fold with ultra purified water and measured at 25oCand
90° scattering angle, recorded for 180 s. Each value was measured in triplicate determination. The results are
showed as mean ± SD distribution. Surface charge of the nanoparticles were characterized with Zeta potential
(ζ) using a Zeta Sizer 4 (Malvern Instruments ltd., Malvern UK). The measurements were performed using
an aqueous dip cell in an automatic mode by placing diluted samples (with ultra-purified water) in the
capillary measurement cell and cell position was adjusted.
Determination of Encapsulation Efficiency

The encapsulation efficiency (EE) of NRG was determined by measuring the amount of NRG in the
supernatant after the twice centrifugation (12,000 X g for 25min at 4oC) using UV spectrophotometer (UV
1800, Shimadzu, Japan) with the detection wavelength of 271nm. The following equation were applied to
calculate the encapsulation efficiency (EE %) = [(Wo – Wt) / Wo] X 100. Wo and Wt are the weight of initial
NRG, and that of the total amount of NRG detected in the supernatant after twice centrifugation respectively.
Each sample was assayed in triplicate.
Fourier Transform Infrared Spectroscopy (FT-IR)

FT-IR spectra were obtained using a Perkin Elmer FT-IR spectrometer (Spectrum-RXI Perkin- Elmer). In
order to collect the spectra, a small amount of each material was used by compression in KBr tablets. The
238
IR spectra were obtained in the spectral region 400-4000 cm-1 using resolution 4 cm-1 and 10 co-added scans.
The spectrum of the KBr pellet was used as background. The spectra presented are baseline corrected and
normalized.
Morphological examination of PNPs

SEM was applied to examine the surfaces of the polymeric nanoparticles. The SEM specimens were
prepared by cutting cross sections of samples were sputter- coated, before being examined and observed
under field emission scanning electron microscopy (FE-SEM, JEOL, JSM6701F, Japan) at an accelerating
voltage of 15 kV.
In vitro release studies

The in vitro drug release studies were conducted in phosphate buffered saline (PBS; pH 7.4), stimulated gastric
fluid (SGF; pH 1.2) and stimulated intestinal fluid (SIF; pH 6.8). An appropriate formulated nanoparticles
was placed in a beaker containing 100ml of releasing media at 37oC with slow magnetic stirring under perfect
sink conditions and 1ml releasing media were withdrawn at each time point periodically and replaced with
the same volume of fresh releasing media and the amount of drug was quantified spectrophotometrically
(UV- 1800, Schimadzu, Japan) at 286 nm
In vivo Pharmacokinetic assessment

Animals and Dosing: All the animal study protocols were duly approved by the Institutional Animal Ethics
Committee, India. Male Wister strain rats weighing 200-250 g were maintained in an environmentally
controlled room 20°C ± 2°C and 60% ± 10% relative humidity on a 12-hour light/dark cycle. Water
and commercial laboratory complete food for rats were available ad libitum. They were acclimatized to
this environment for 7 days before receiving the experimental treatment. The animals were randomly
distributed into two groups each containing six animals. Different groups of animals received oral free NRG
(20 mg/ kg), and NRG encapsulated PNPs (20 mg/ kg). Blood samples (approximately 0.2 ml) were collected
at periodic intervals from the retro-orbital plexus. After each sampling, 1ml of dextrose-, normal saline was
administered orally to prevent changes in the central compartment volume and electrolytes. Plasma was
separated by centrifuging the blood samples at 10,000 rpm for 10min at 4 °C and kept at – 80 °C until
analyzed.
Determination of naringenin in rat plasma by HPLC

Naringenin in rat plasma was analyzed by HPLC using a mixture of acetonitrile and 2% acetic acid
in a volume ratio of 51:49 (v/v) as the mobile phase at a flow rate of 1.0 ml ⋅ minute−1. For the
measurement of plasma NRG, 50 μl of internal standard solution (hesperitin, 4 μg ⋅ ml−1 in ethanol)
was added to 0.2 ml plasma. After vortex mixing for 1 minute, 500 μl of anhydrous diethyl ether
was added and again vortex-mixed for 1 minute. After centrifugation at 10,000 rpm for 10 minutes,
the supernatant was withdrawn and evaporated under a light stream of nitrogen at room temperature.
The residue was dissolved in 100 μl of the mobile phase and again centrifuged at 10,000 rpm for 10
minutes. Twenty microliters of the supernatant were then injected for HPLC analysis. Quantification
was based on the peak area ratio R (ACUR/AEMO). The calibration curve obtained was R = 0.0023C +
0.0335 (r = 0.9998, n = 5), with a correlation coefficient 0.999. The pharmacokinetic parameters of each
formulation were calculated by the non-compartmental method. The area under the curve and the mean
residence time were determined by standard methods applying the linear trapezoidal rule.
In vivo toxicity studies

For the toxicity study, the rats were gavaged with free NRG and NRG encapsulated PNPs at a single dose of
20mg/ kg body weight. After 24h, rats were sacrificed, and the blood samples were collected for biochemical
239
analysis and different tissues samples for histopathological examination. The biochemical assays were
performed on fully automated clinical biochemistry analyzer (Beckmann Coulter AU480, Japan).
GraphPad Prism 5 software was used for statistical analysis (GraphPad Software Inc., San Diego, USA).
Data are expressed as mean ± s.d. Data were analyzed by one-way analysis of variance followed by the
Student–Newman–Keul multiple comparison tests. Differences were considered significant at P<0.05.

In this study, we formulated a new concept for synthesizing polymeric nanoparticles for oral drug delivery
using naringenin and Eudragit® E 100 polymers using nanoprecipitation method. Nanoprecipitation is
a simplistic and mild process technique for the preparation of nanoscale compounds, and it was one of
the most superior to other encapsulation methods [19]. During the particle synthesis, both internal phases
NRG and EE 100 are hydrophobic in nature interacts each other and form a suspension. The internal phase
suspension was introduced into external aqueous phase and produce interfacial tension to form stronger
outer surface with the help of PVA as a stabilizer. Nanoprecipitation was conducted through the precipitation
of a performed polymer from an organic solution which was previously solubilized, with the diffusion of
this organic solvent into the aqueous medium. In the aqueous phase, the polymers get insoluble, precipitating
immediately leading to the formation of nanoparticles [20].
Fig 1 shows the mean particle size distribution of the prepared PNPs. Nanoparticles size is a useful
parameter since its affects the drug loading and release. The magnitude of the zeta potential on the PNPs
was relatively high due to their stronger electrostatic repulsion that inhibits the particle aggregation [21]
that showed in Table 1. The high concentration of polymer and emulsion stabilizer does not affect the size
and charge of the PNPs. An increasing the concentration of EE and PVA had an antagonistic effects and
result in a decreased particle size. Also, the high proportion of PVA could offer sufficient stabilization to
nanoparticles formulation, and reduce their particle size and increase the stability [22].
Hydrodynamic process takes place during the particle preparation which leads to the higher encapsulation
efficiency of NRG that showed in Fig 2. When adjusting the proportion of stabilizer (PVA) in an aqueous
phase that influencing the encapsulation efficiency of NRG. The possible reason was the hydrophobic nature
of EE 100 interpenetrates into the hydrophilic nature of PVA during particle preparation and remained that
trapped to the matrix structure. The intermolecular interaction of nanoparticles was recognized by FTIR that
showed in Fig 3. The characteristic peak due to pure naringenin has appeared in the spectra of nanoparticles
without markable change in the position. It indicated that there was no chemical interaction between
naringenin and polymer. Polymeric nanoparticles morphology was showed in Fig 4. The particles had a
spherical morphology, and particles are smaller in size, among most of them are similar in dimensions.
The in vitro drug release study of PNPs was replicated in various drug-releasing medium showed in
Fig 5. It was obviously reported that faster and sustained release find out in SGF when compare to various
drug-releasing media such as SIF and PBS. In the first several minutes, an initial burst of 20% - 30% drug
was released from the surface of the nanoparticles. In the following period of the study, nearly 90% of the
drug was released from the nanoparticles. The release of NRG mainly depended upon the polymer and
stabilizer concentration, the release rate of NRG from nanoparticles also increased drastically. It was found
that the in vitro drug release of PNPs was best explained by zero order kinetics shows highest linearity. So
the sustained release pattern of NRG, which might contribute to the better pharmacological assessments,
resulted in enhanced bioactive efficacy. Main pharmacokinetics parameters in plasma following a single oral
administration of 20mg/ kg body weight in rats showed in Table 2. The pharmacokinetic parameters showed
significant differences between free NRG and PNPs in plasma that indicating improved oral bioavailability.
240
Naringenin is a bioactive flavanone involved in the inhibition of drug metabolism that exhibits various
biological activities. Biochemical analysis was carried out on serum samples taken from rats, 12 hours
and 24 hours after the single treatment of saline as a control, NRG alone as well as rats treated with NRG
encapsulated PNPs. The biochemical examination revealed that no major changes (Table 3). Electrolytes and
blood glucose levels were within normal ranges, In spite of unchanged urea, creatinine and uric acid levels
suggesting normal kidney function. Liver function tests were also within normal indicates no liver damage.
Together with the histopathological results that showed in Fig 6 suggest that oral administration of NRG
encapsulated PNPs was not associated with any adverse effects.
CONCLUSION
The naringenin encapsulated polymeric nanoparticles have been formulated by a facile nanoprecipitation
method at ten different formulations. The best formulation of NRG encapsulated PNPs has been optimized
by size, stability and encapsulation efficiency, and it was characterized by FTIR and SEM. The in vitro drug
release and the pharmacokinetic fate was investigated in PNPs as compared to free NRG. The overall findings
demonstrated that the NRG encapsulated polymeric nanoparticles have enhanced oral bioavailability, which
might be a promising novel approach for biomedical applications.
ACKNOWLEDGEMENT
Table 1: Arrangement of NRG encapsulated polymeric nanoparticles formulation and their response data for mean
particle size, poly dispersity index and zeta potential.
Nanoparticles NRG (mg) EE 100 (mg) PVA (mg) Mean particle Poly Dispersity Zeta Potential(mv)
Formulation Size (nm) Index (PDI)
(Ratio)
NF1 (1:2:1) 50 100 50 351 0.775 -25.3
NF2 (1:4:2) 50 200 100 285 0.429 -26.0
NF3 (1:6:3) 50 300 150 259 0.380 -28.6
NF4 (1:8:4) 50 400 200 215 0.314 -28.5
NF5 (1:10:5) 50 500 250 163 0.268 -29.3
NF6 (1:2:2) 50 100 100 319 0.181 -26.1
NF7 (1:4:4) 50 200 200 278 0.168 -28.4
NF8 (1:6:6) 50 300 300 226 0.157 -28.9
NF9 (1:8:8) 50 400 400 112 0.427 -30.7
NF10 (1:10:10) 50 500 500 83 0.153 -34.8
Figure 1: Size plot depicting the sizes of Figure 2: Encapsulation Efficiency of

different PNPs different PNPs formulations
241
Figure 3: Infrared spectrum of individual compounds NRG, EE 100, PVA and PNPs
Figure 4: SEM micrograph of formulated PNPs
Figure 4: SEM micrograph of formulated PNPs
Figure 5: In vitro release profile of PNPs in various releasing media (mean ± SD, n = 3).
242
Table 2: Main pharmacokinetics parameters following a single oral dose (20mg/kg) administration
Groups Dose Cmax Tmax MRT t1/2 AUC 0-∞

(mg/kg) (µg/ml) (h) (h) (h) (ng h/ml)
Free NRG 20 8.11 4.0 2.84 3.06 2710.48
PNPs 20 5.32 8.0 5.37 5.88 4529.05
AUC – area under the curve; Cmax – peak plasma concentration; Tmax – time to attain peak concentration;
MRT – mean residence time; t1/2 – terminal half life
Figure 6: In vivo toxicity study of free NRG (series- I) and PNPs (series- II) treated rats of different organs of kidney
(A), liver (B), duodenum (C) and stomach (D)
Table 3: Results for biochemical parameters of free NRG and PNPs treated rats at two different time intervals (12hr &
24hr) were compared with untreated controls.
Parameters (Units) after 12hr treatment after 24hr treatment

Control Free NRG PNPs Control Free NRG PNPs
Na (mEq/L)
+
132.7 ± 2.0 130.9 ± 1.5 140.2 ± 1.6 135.0 ± 1.2 135.8 ± 1.8 137.0 ± 1.7
K+ (mEq/L) 3.6 ± 0.9 4.1 ± 0.4 3.9 ± 0.5 4.8 ± 0.4 5.2 ± 0.3 5.5 ± 0.4
Cl- (mEq/L) 95.3 ± 3.1 98.7 ± 2.5 100.8 ± 1.8 97.0 ± 2.4 102.0 ± 1.7 103.0 ± 1.6
HCO3- (mEq/L) 22.6 ± 2.0 21.8 ± 1.8 23.7 ± 1.5 19.6 ± 1.8 20.1 ± 1.9 20.7 ± 1.5
Glu (mg/dl) 82.3 ± 8.7 79.2 ± 6.9 85.0 ± 5.1 71.1 ± 6.7 71.3 ± 5.5 73.5 ± 6.8
BUN (mg/dl) 21.4 ± 2.7 19.7 ± 3.5 22.6 ± 3.7 16.0 ± 2.9 18.1 ± 4.3 19.4 ± 3.5
Cre (mg/dl) 0.42 ± 0.5 0.45 ± 0.3 0.51 ± 0.3 0.36 ± 0.2 0.48 ± 0.1 0.39 ± 0.1
ALT (U/l) 37.9 ± 15.1 43.2 ± 16.8 40.1 ± 16.9 45.8 ± 19.3 48.6 ± 17.5 46.0 ± 20.1
AST (U/l) 112.5 ± 15.4 129.8 ± 16.1 117.6 ± 12.6 134.6 ± 19.8 138.7 ± 9.6 140.9 ± 12.4
ALP (U/l) 179.3 ± 20.6 185.4 ± 18.3 182.2 ± 22.7 182.0 ± 25.7 169.5 ± 24.1 170.1 ± 29.3
TBIL (mg/dl) 0.39 ± 0.2 0.35 ± 0.1 0.38 ± 0.1 0.36 ± 0.1 0.34 ± 0.1 0.34 ± 0.0
TP (gm/dl) 6.91 ± 1.2 6.58 ± 0.9 6.62 ± 0.7 6.44 ± 0.5 6.30 ± 0.3 6.37 ± 0.3
ALB (gm/dl) 3.14 ± 0.8 2.96 ± 0.5 3.22 ± 1.0 2.97 ± 0.5 3.04 ± 0.2 3.02 ± 0.3
UA (mg/dl) 2.6 ± 1.9 2.4 ± 1.9 3.0 ± 2.1 2.3 ± 2.4 2.7 ± 1.9 2.9 ± 2.0
Ca2+ (mg/dl) 8.8 ± 1.4 9.3 ± 1.0 9.2 ± 1.0 9.1 ± 1.2 8.9 ± 1.6 9.5 ± 1.0
Mg (mg/dl)
2+
2.0 ± 1.1 1.7 ± 1.3 2.2 ± 1.1 1.9 ± 0.9 2.1 ± 1.0 1.7 ± 1.2
PHOS (mg/dl) 4.98 ± 1.5 5.23 ± 1.9 5.51 ± 1.4 5.88 ± 1.6 5.90 ± 1.3 6.01 ± 1.3
Glu- Glucose, BUN- Blood Urea Nitrogen, Cre- Creatinine, ALP- Alkaline phosphatase, ALT- Alanine
transaminase, AST- Aspartate transaminase, TBIL- Total bilirubin, ALB- Albumin, TP- Total protein.
243
REFERENCES
1. A. Shrivastav, Y. Kim and R. Kim, Advances in the applications of polyhydroxyalkanote nanoparticles

for novel drug delivery system, BioMed Research International, pp. 1-12, 2013.
2. S. Naahidi, M. Jafari, F. Edalat, K. Raymond, A. Khademhosseini and P. Chen, Biocompatibility of
engineered nanoparticles for drug delivery, Journal of Controlled Release, pp. 182-194, 2012.
3. L. Li, H. Mohwald and G. Shchukin, Precipitation polymerization for fabrication of complex core-shell
hybrid particles and hollow structures, Chem Soc Rev, pp. 3628-3646, 2013.
4. S. Kalepu, M. Manthina and V. Padavala, Oral lipid-based drug delivery systems-an overview, Acta
Pharmaceutica Sinica B, pp. 361-372, 2013.
5. J. Salustio, P. Pontes, C. Conduto, I. Sanches, C. Carvalho, J. Arrais and M. Marques, Advanced
technologies for oral controlles release: cyclodextrins for oral controlled release, AAPS PharmSciTech,
pp. 1276-1291, 2011.
6. M. Ensign, R. Cone and J. Hanes, Oral drug delivery with polymeric nanoparticles: the gastrointestinal
mucus barriers, Advanced Drug Delivery Reviews, pp. 557-570, 2012.
7. A. Dembinski, Z. Warzecha, J. Konturek, P. Ceranowicz, M. Dembinski, W. Pawlik, B. Kusnierz Cabala
and W. Naskalski, Extract of grapefruit-seed reduces acute pancreatitis induced by ischemia/reperfusion
in rats: possible implication of tissue antioxidants, J. Physiol. Pharmacol, pp. 811-821, 2004.
8. G. Le Gall, S. DuPont, A. Mellon, L. Davis, J. Collins, E. Verhoeyen and J. Colquhoun, Characterization
and content of flavonoid glycosides in genetically modified tomato (Lycopersicon esculentum) fruits, J.
Agric. Food Chem, pp. 2438- 2446, 2003.
9. H. Wang, G. Nair, M. Strasburg, M. Booren and I. Gray, Antioxidant polyphenols from tart cherries
(Prunus cerasus), J. Agric. Food. Chem, pp. 840- 44, 1999.
10. I. Erlund, E. Meririnne, G. Alfthan and A. Aro, Plasma kinetics and urinary excretion of the flavonones
naringenin and hesperitin in humans after ingestion of orange juice and grapefruit juice, J. Nutr, pp.
235- 41, 2001.
11. T. Stark, S. Bareuther and T. Hofmann, Sensory-guided decomposition of roasted cocoa nibs (Theobroma
cacao) and structure determination of taste- active polyphenols, J. Agric. Food. Chem, pp. 5407- 5418,
2005.
12. G. Ekambaram, P. Rajendran, V. Magesh and D. Sakthisekaran, Naringenin reduces tumor size and
weight loss in N-methyl-Nnitro-N-nitrosoguanidine-induced gastric carcinogenesis in rats, Nutr Res,
pp. 106–112, 2008.
13. R. Lauro, F. De Simone, F. Sansone, P. Lannelli and P. Aquino, Preparations and release characteristics
of naringin and naringenin gastro-resistant microparticles by spray- drying, J. Drug Del. Sci. Tech. pp.
119-124, 2007.
244
Design and Characterisation of Baclofen Sustain Release
Tablet using Hydrophilic Matrix
R. Mohan Kumar1, M. Balakumaran2, M. Annapoorni3, P. Selvamani4,

N. Subramanian5 and K. Ruckmani6
Department of Pharmaceutical Technology, 6Author for Correspondence, Prof & Head,

1, 2, 3, 4, 5
Director – CENTRE, Department of Pharmaceutical Technology, BIT Campus, Anna University,

Tiruchirappalli-620024, Tamilnadu, India
e-mail: hodpharma@gmail.com, Mobile: +919842484568
Abstract:
The baclofen sustains release tablet formulated by using different types and levels of hydrophilic matrix
agent, including Hydroxy Propyl Methyl Cellulose 100LV, Microcrystalline Cellulose PH 102 and
Hydroxy Propyl Methyl Cellulose K-15M. These tablets contained 25 mg baclofen and prepared by wet
granulation method. Prior to compression, the prepared granules were evaluated for flow and compression
characteristics. The excipients used in the formulation were tested by FT-IR spectrophotometers that
indicate that there is no change in the physicochemical properties of the drug were not changed due to
using of the excipient. The Baclofen matrix tablet showed good mechanical properties like hardness and
friability. The Hydroxy Propyl Methyl Cellulose k15M and Microcrystalline Cellulose PH 102 shows
sustain release effect and good reproducibility and stability of release profile in ambient room condition,
suggesting that these are a good candidate for preparing sustain release baclofen hydrophilic matrix
formulation. These matrix tablets used as an antispastic agent or muscle relaxant. These formulations
minimize dose fluctuation and improve therapeutic response and patient suffering from spasticity and
chronic musculoskeletal condition.
Keywords: Baclofen, Matrix tablets, Sustained release, Hydroxy Propyl Methyl Cellulose 100LV and
K-15M
INTRODUCTION
Baclofen is the centrally acting muscle relaxant, indicated in long-term treatment of spasticity. Baclofen is
rapidly and extensively absorbed and eliminated. The half-life of the drug is 2.5 – 4 hrs in plasma. Many
report states that absorption of Baclofen is through facilitated intestinal transport.
The formulation of Baclofen sustain release tablet modifies the drug release in an attempt to minimize
those fluctuations and improves therapeutic response for patients suffering from spasticity and chronic
musculoskeletal conditions. When it injected chronically in an intrathecal space by implanted pumps, which
are very expensive are uncomfortable and sometimes lead to side effects. Therefore, in this study, Hydroxy
Propyl Methyl Cellulose 100 LV, Microcrystalline Cellulose PH 102 and Hydroxy Propyl Methyl Cellulose
K-15M in combination has been used as matrix materials in order to get the required release profile of
baclofen.[1-4]

Materials
Baclofen-USP was a gift sample from Sun Pharma; Hydroxy Propyl Methyl Cellulose 100LV Sun Pharma,
Microcrystalline Cellulose PH 102 and Hydroxy Propyl Methyl Cellulose K-15M Loba chemie PVT Ltd,
Mumbai, Mannitol, Starch, Magnesium stearate Loba chemical Pvt Ltd, Mumbai. All these chemicals and
reagent were of analytical grade. Distilled water was used for the evaluation study.
METHODS
Preparation of baclofen sustained release matrix tablets:
The composition of 25 mg of Baclofen tablet is given in table No.3, powder mixed was sieved through a No.
60 sieve, calculated amount (required to prepared 100 tablets batch) of the drug, polymer, (Hydroxy Propyl
Methyl Cellulose, Microcrystalline Cellulose PH 102, Mannitol) and lubricants (magnesium stearate) was
mixed thoroughly. Sufficient volume of specified granulating agent (PVP) was added slowly.
After enough cohesiveness, the mass was sieved portion wise through a No. 10 and 20 sieves. The
granule was dried at 50°C for 2 hours in a vacuum oven and kept in desiccators for 24 hours at room
temperature prior to compression all prepared granules evaluated for their flow properties and density.
Magnesium stearate was added as a lubricant and other excipients also added. These prepared granules were
compressed to give 250 mg baclofen hydrophilic matrix tablet. The entire compressed table stored in an
airtight container at room temperature for further study.
Formulation table no 1
S.No Ingrdients (Quantities in mg/Tablet)

Formulation No.
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11
1 Baclofen 25 25 25 25 25 25 25 25 25 25 25
2 HPMC100LV 187.5 175 162.5 150.0 – – – – 140.63 93.75 46.84
3 HPMC K 15M - - - - 187.5 175.0 162.5 155.0 46.87 93.75 140.63
4 Microcrystalline - 12.5 25.0 37.5 - 12.5 25 37.5 - 12.52 25.0
cellulose pH 112
5 Strach 35.5 22.66 11.84 - 35.5 22.66 11.84 - 35.5 22.66 11.84
6 Mannitol - 11.84 22.66 35.5 - 11.84 22.66 35.5 - 11.84 22.66
7 Magnesium Stearate 2 2 2 2 2 2 2 2 2 2 2
Evaluation of powder blends

The formulated powder blends were evaluated for compatibility, angle of repose, bulk density, at ed density,
percentage compressibility index and loss on drying.[3, 5]
Evaluation of tablets
The compressed tablets (formulations F1 to F11) were evaluated for hardness, percentage friability,
percentage weight variations and percentage drug content.[5, 6]
In vitro Release studies

In vitro dissolution studies were carried out using six stage dissolution rate test an aratus IP/BP/USP at
50 rpm. The dissolution medium consisted of simulated gastric fluid (pH 1.2 acid buffer, for first 2 h) and
followed by in simulated intestinal fluid
246
(pH 6.8 phosphate buffer) from 2 to 8 hours (900ml), maintained at 37±0.5C. Sample was withdrawn at
predetermined time intervals, and drug content was analyzed by UV-visible spectrophotometer at 266 nm
respectively compared with blank.[5, 7]

Baclofen is a water insoluble drug. Its poor inherent compressibility which possess a significance challenge
for developing sustained release matrix tablet with desirable drug release profile, cost effectiveness and
broader regulatory acceptance combination of HPMC 100 LV and HPMC K-15M. Was chosen as release
controlling polymer.
Compatibility study of baclofen by FT-IR

To identify drug Baclofen, FT- IR Spectrophotometric analysis was Carried Out by KBr disc method and
recorded the spectrum in the range of 2000cm1 and 600Cm-1.
The peak in a graph of pure drug and blends of drug in the polymer confirms that there is no any
interaction and hence the polymer is compatible with drug.[8] result shown in Table-2.
FT-IR peaks of various components
S.No. Name of Component Peaks Obtained (Weavenumber,cm-1)

1 Drug (Baclofen) 835.21,1018.45,1093,,1531.53,1627.97,1530.53,2648.35,2156.49
2 HPMC K-15M 835.21,1018.45,1093.67,1533.46,1627.97,1533.46,2648.35.
3 HPMC 100LV 835.21,1072,1136.11,1533.46,1627.97,2160.35,2652.21
Evaluation of physical and chemical parameters of formulated powder blends

Formulation of proper powder blend is the key factor in the production of tablet dosage form involving
sustained release of drug from matrix type particle. Physical parameters such as specific surface area, shape,
hardness, surface characteristics and size can significantly affect the rate of dissolution of drugs contained
in a complex system. The formulated powder blend of different formulations (F1 to F11) was evaluated for
angle of repose, true density, bulk density, compressibility index in Table-3. The results of angle of repose
(<30) indicated good flow properties of all the formulated powder blends. The compressibility index values
were recorded <15%, result in good to excellent flow properties in all formulation. Formulated powder blends
density; porosity and hardness are often interrelated properties and are likely to influence compressibility,
porosity, dissolution profile and properties of tablets made from it.
Physical Properties of the Prepared Granules, Using hydroxyl propyle methyle cellulose 100LV and K-15M,
microcrystalline cellulose, as Matrix-Forming Polymers
Formulations Angle of Repose (Ө) Poured Density Ta ed Density (g/ Hausner Factor Compressibility (%)
(g/cm3) cm3)
Pure drug — 0.241 0.611 2.535 25.48
F-1 23 0.282 0.325 1.152 13.23
F-2 25 0.301 0.351 1.166 14.24
F-3 20 0.291 0.332 1.141 12.34
F-4 20 0.501 0.581 1.159 13.76
F-5 22 0.491 0.552 1.124 11.05
F-6 22 0.481 0.541 1.124 11.09
F-7 27 0.322 0.402 1.248 19.90
F-8 30 0.401 0.471 1.174 14.86
F-9 30 0.422 0.483 1.144 12.62
F-10 20 0.350 0.421 1.203 16.86
247
Evaluation of formulated tablets
The tablets of different formulations (F1 to F11) were evaluated for various parameters viz., hardness, friability,
percentage weight variation and percentage drug content. The results of these parameters are given in Table-3, 4
Formulations Hardness (Kg/cm2 ) Friability (%) Thickness (mm) Weight variation Percent drug content
F-1 7.5 0.21 3.99 250 99.86
F-2 8.0 0.19 3.98 250 99.94
F-3 8.5 0.17 3.99 250.5 99.82
F-4 8.0 0.19 3.97 249 100.02
F-5 7.5 0.23 3.98 250.5 99.98
F-6 7.0 0.18 3.98 250 99.89
F-7 8.5 0.16 3.99 250 99.70
F-8 8.0 0.18 3.99 249.5 98.89
F-9 8.0 0.19 3.97 251.5 99.86
F-10 8.5 0.15 3.99 250 99.98
F-11 8.5 0.17 3.98 250 100.10

Results of the in vitro release studies of various formulations designed and manufactured are presented in
Table-3. A graphical representation of the data presented in the Table-3.The plot of cumulative percentage
In vitro drug release profile of Baclofen from 11 formulations F1 to F11 made with different concentration
and combination of hydroxypropyl methyl cellulose k-15m and hydroxypropyl methyl cellulose 100M in
simulated gastric fluid (pH 1.2 – acid buffer) (for first 2 h) followed by simulated intestinal fluid (pH 6.8-
phosphate buffer) for 2 to 8 h is shown in Fig-2. It is found that the cumulative percentage drug release of the
formulation, F1 to F4 is faster than remaining formulation, with formulation F11showing the slowest release.
So, it can be inferred that the proportion of HPMC (K15M) and (100M) is increased to release is retarded
and Drug: HPMC ratio is found to be optimum for comparable release profile with reference. Results were
shown in Table-5 and Fig-1
%of Drug Release

Time
Sr. No Medium Formulation NO.
(Min)
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11
1 30 13.21 14.46 15.28 18.54 12.48 13.21 13.78 15.21 12.48 13.97 14.21
2 60 22.29 24.38 26.19 32.48 21.32 20.47 22.41 28.38 20.36 21.46 24.38
3 120 42.66 39.28 42.34 54.76 34.28 35.28 33.64 41.56 39.41 42.17 41.76
pH1.2
4 180 59.71 51.42 54.72 76.37 49.56 47.56 49.31 54.46 55.71 58.49 55.21
5 240 78.81 67.54 69.84 98.83 61.74 56.67 57.27 66.72 66.34 69.79 63.86
7 300 99.92 80.67 83.58 73.78 64.46 66.43 78.27 74.64 74.93 75.78
8 360 99.72 100.26 82.76 70.81 72.69 98.25 82.78 85.19 83.76
9 420 99.21 75.78 79.25 9019 93.29 91.48
10 480 82.58 85.74 95.7 98.35 96.78
pH6.8
248
Stability studies on In vitro release

Since the in vitro release of formulations F10 of 25 mg label claimed were found to be desirable than the
other formulations, hence they were chosen for stability studies. Any ideal dosage form apart from other
dosage requirements should provide consistency of drug content and release throughout its self-life. The
SR Baclofen tablets 25 mg were stored at temperature of 40°C ± 2°C / 75 % RH ± 5 % RH and room
temperature. The products were stored for a period of 30 days in the above-mentioned conditions. The
product was analyzed at initial, 30 days of storage at 40°C ± 2°C / 75 % RH ± 5 % RH and room temperature.
After the storage period the formulation were evaluated for drug content and drug release profile in
pH1.2 (0.1N HCl), pH6.8 phosphate buffer and physiochemical parameters by method described.[9] And
results were shown in Table-6.
Time % of Drug Release Formulation No. F10
Sr. No Medium
(minutes) 40±2°C/75%RH±5% Room temperature
1 30 14.21 13.76
2 pH 1.2 60 24.38 21.39
3 120 41.76 41.21
4 180 55.21 57.49
5 240 63.86 68.78
6 300 75.48 75.93
pH 6.8
7 360 83.76 84.24
8 420 91.48 93.78
9 480 98.05 98.30
SWELLING CHARACTER OF POLYMER BATCH NO: F10

In order to assess the swelling character of the matrix polymer, the Pe a’s plot was drawn for formulation and
the values were calculated using. The slope values for the entire polymer were in the range of 0.5 – 0.9 that
the drug release may be controlled by swelling of the polymer 10, 11.Table No.7,8.
249
Time (minutes) Log time Cumulative % release Log cumulative % release

30 1.477 13.97 1.145
60 1.778 21.46 1.331
120 2.079 42.17 1.625
180 2.255 58.49 1.946
240 2.380 69.89 1.844
300 2.477 74.93 1.874
360 2.556 85.19 1.930
420 2.623 93.29 1.969
480 2.681 98.35 1.992
Formulation Regression value Slope
Label claimed 25 mg/Batch No. 10 0.9844 0.5231
KINETICS OF DRUG RELEASE

The order of drug release can be assessed by graphical treatment of drug release data. A plot of % drug
remaining versus time would be linear if the drug release follows zero order (i.e. Concentration independent
release).
A plot of log of % remaining drug versus time would be linear if the drug release follows first order (i.e.
concentration-dependent release).[12-14] see in Table-9,10.
Time (Time)1/2 Cumulative % Amount of % of drug Log % of drug 1/ 3

(minutes) release drug release remained remained  Mt 
1 − M 
30 5.477 13.47 3.492 86.03 1.934 0.950
60 7.745 21.46 5.365 78.54 1.895 0.921
120 10.954 42.17 10.542 57.83 1.762 0.830
180 13.416 58.49 14.621 41.51 1.618 0.740
240 15.491 69.89 17.473 30.11 1.478 0.661
300 17.320 74.93 18.732 25.07 1.399 0.620
360 18.973 85.19 21.297 14.81 1.170 0.511
420 20.493 93.29 23.328 6.71 .826 0.457
480 21.908 98.35 24.589 1.65 .217 0
Formulation Zero order R2 value First order R2 value Order of drug release
Baclofen 25 mg 0.9559 0.8963 Zero order
CONCLUSION:
In the present study, attempts were made to formulate 25 mg Sustained release which can provide effective
drug release for 8 hours. Wet granulation prepared sustained release matrix tablets of Baclofen. In vitro study
showed batch No F10 for 25 mg label claimed were well suited to be Sustained release formulation.
Batch No. F10 was found to obey Zero order drug release, governed by diffusion through swollen matrix
and erosion of the matrix, showing anomalous diffusion or nonfickian transport. Infra Red spectrum of the
tablet reveals that there is no interaction of the polymer and tablet matrix with the Baclofen. Further, in-vivo
and continuation of stability studies are recommended
250
REFERENCES:
1. K.D. Triphati, Essential of Medical pharmacology, 6th edition, 240, 250-51

2. Miller R.D., “Skeletal Muscle Relaxants,” in “Basic & Clinical Pharmacology: Seventh Edition,” by
Bertram G. Katzung, Published by A leton & Lange, 1998, 434-449.
3. Hamdy Abdelkader, Hesham salem, formulation of CR Baclofen Matrix Tablet, AAPS Pharmascitech
2007, 1-2
4. Vorapann mahaguna et al., Influence of hydroxypropyl methyl cellulose polymer on in vitro and in vivo
performance of CR tablet containing alprazolam, EJPB, 2003
5. United State Pharmacopoeia, 24th edition, 2000, 194-195
6. Liberman H.A. and Lachaman L.BS Joseph, Pharmaceutical dosage form tablets, 2nd edition, volume –
I, New york: Macel Decker. Inc; 1989, 356-9
7. Muhammad Khan sarfraz et al., The sustained release matrix tablet formulation of Naproxen by using
HPMC and Ethyl cellulose, IJP2006
8. Kalus Florey, Analytical profile of drug substance, Volume -14, 528-530
9. ICH Q 1A9 R2 Stability testing guidelines, Stability testing of new drug substance and products
10. Pe as N.A., Gurny R., Doelker E and Buri P., 198 Swell polymerize systems, J. membrane science.,
7: 241-253.
11. Bettini R., N.A. Pe as and P. Colombo, 1988, polymer relaxation in swellable matrixes contributes to
drug release, produce int. symptoms control. Release bioact. Mater., 25, 26-37.
12. D.M. Brahmankar and Sunil B. Jaiswal “Biopharmaceutics and Pharmacokinetics a Treatise, 2002
reprint, Vallabh prakashan, 335-337.
13. Kaushal A. et al..2001, “Regulatory requirements for oral controlled release drug delivery systems,”
pharmacy times, volume 33, april, 14 – 17
14. Gul Majed khan: “past and present status of controlled release matrixes”; Asia network for scientific in
permeation ISSN 1608-8689, vol 1 (5): 350-350, 2001.
251
Preparation and Evaluation of Miconazole Nitrate
Nanoemulsion using Tween 20 as Surfactant for Effective
Topical and Transdermal Delivery
P. Jaya Sree and Dr. C. Thirumal Azhagan
Department of Management Studies, Anna University, BIT Campus, Tiruchirapalli-620 024, India
e-mail: azhaganct@gmail.com
Abstract:
The present study has investigated the effective transdermal delivery of anti-fungal miconazole nitrate
(MN) using tween 20 nanoemulsion (NE). Ne’S were prepared by spontaneous emulsification technique
and formulated as gel using carbopol 934. Clove oil, tween 20 & ethanol were chosen as oil, surfactant &
cosurfactant respectively after optimization. The ratio of coded as F1 to F4 and their gel as F1(G) to F4(G).
Ne’S were characterized for transparency, drug content, compatibility, particle size, zeta potential, viscosity,
pH, transmission electron microscopic (TEM) analysis, in vitro drug release, release kinetics and stability
studies. In vitro skin permeation for NE gel and marketed preparation was conducted. Anti-fungal assay
was performed for F3(G) and compared with the marketed one. Transparent NE of narrow size distribution,
suitable zeta potential, pH and viscosity were obtained. TEM revealed the formation of discrete nanosized
(247.5nm - 511nm) droplets. Maximum drug loading and compatibility of the drug with excipients were
obtained. NE F3 had produced a maximum drug release of 94.8%±2.6% for 8h and the gel F3(G) produced
a skin permeation of 93.7%±2.1% in 12h, whereas the marketed gel exhibited 42.9%±1.8% permeation.
The release followed higuchi kinetics and fickian diffusion. Anti-fungal assay revealed higher % zone of
inhibition against Aspergillus niger and Candida albicans for F3(G) compared to marketed sample. The F3
was stable at room temperature, and the developed NE has attained the objective of topical & transdermal
delivery of miconazole nitrate.
Keywords: Nanoemulsion, Miconazole nitrate, Topical & transdermal delivery, Nanoemulsion gel, In vitro
characterization.
INTRODUCTION
Nanoemulsion (NE) offers advantages for topical and transdermal delivery of pharmaceutical agents
including controlled droplet size, lower concentration of surfactant and the ability to effectively dissolve
lipophilic drugs, enhanced skin permeation and extended release of lipophilic drugs. Moreover, they exert
good sensorial and physical properties such as complete dispersion on skin and skin hydration in topical and
transdermal products.. The drug Miconazole nitrate is fungicidal, used for topical and transdermal fungal
infections. It is available as gel in the market, and the development of nanoemulsion of this drug may
increases its effectiveness and allow for both topical and transdermal delivery.

Preparation of nanoemulsions
The homogeneous organic solution (S1) composed of clove oil in water–miscible solvent acetone 5ml and
methanol 5ml. The homogeneous aqueous phase (S2) was formed by water 30ml and Tween 20. The organic
phase was injected into the aqueous phase under magnetic stirring (1500 rpm). The o/w emulsion was
formed instantaneously leading to the formation of nanodroplets. The water miscible solvent was removed
by evaporation. The prepared nanoemulsion was stored in screw capped vials and kept at room temperature.
Preparation of NE Gel Formulation

The gel was prepared by dispersing 1g of carbopol 934 insufficient quantity of distilled water. After complete
dispersion, the solution was kept in the dark for 24 h for complete swelling. Then the miconazole nitrate
loaded nanoemulsion containing 5mg of drug was slowly added to the viscous solution of carbopol 934
under moderate magnetic stirring at 550 rpm for 10-15 min. The pH values were subsequently regulated
to 6-9 and then isopropyl alcohol, PEG 400, propylene glycol and triethanolamine were added to obtain a
homogeneous dispersion of gel.
Particle Size Analysis

Particle size or droplet size depends on the rate of the emulsification process but also for the volume and the
particle size distribution of produced emulsion. It is determined by using a zetasizer.
TEM and Zeta analysis

NE’S were subjected to Transmission Electron Microscopy (TEM) and zetasizer was used to find droplet
size distribution, zeta potential, pH and viscosity. The drug content was determined by UV-visible
spectrophotometer.
Dissolution studies for nanoemulsion & Gel formulations

The dissolution studies were conducted in a microdialysis setup. The nanoemulsion (20 ml) was suspended in
phosphate buffer of pH 7.4. The bulk solution was stirred continuously by a magnetic stirrer at 550 rpm and 5 ml
sample solution was drawn at regular intervals by replacing with an equal volume of the buffer. The quantity of
released drug was analyzed by means of a UV spectrophotometer. In the case of gel, 1 g of gel containing 100 mg
of drug was used for the study.
In vitro Permeation Studies

The in vitro permeation was performed using Franz diffusion cell. The cellulose acetate membrane was
clamped between the donor and receiver compartment of the cell. 10 ml of nanoemulsion was administered
253
in the donor compartment. The receptor compartment was filled with 24 ml of phosphate buffer pH 7.4. The
diffusion cell was thermo stated at 37°C and stirred at 600 rpm. Samples of 1ml aliquots were drawn from
the receiver compartment and replaced immediately with an equal volume of fresh phosphate buffer pH 7.4.
All the samples were analyzed by UV.
Antifungal Screening by Agar Well Method

Sabouraund agar plates were prepared aseptically to get the thickness of 5-6 mm; the plates were allowed
to solidify and inverted to prevent the condensate falling on the agar surface. The plates were dried at 37°C
before inoculation. The organisms were inoculated in the plates by dipping a sterile swab in the previously
standardized inoculums, removing the excess of inoculums by pressing and rotating the swab firmly against
the sides of the culture tube above the level of the liquid and finally streaking the swab all over the surface of
the medium 3 times, rotating the plates through an angle of 60°C after each application. Finally, the swab was
pressed round the edge of the agar surface. It was allowed to dry at room temperature with the lid closed. Then,
wells of about 3 mm diameter were punched using sterile core borer into the agar medium and filled with test
formulation, control (solution of drug in methanol) and nanoemulsion gel and marketed the gel. The plates were
kept in the refrigerator for one hour to facilitate uniform diffusion of the drug. Then the plates were incubated
for 18-24 h. Observation was made for zones of inhibition around the well and compared with that of standard.
100 mg of drug was present in all the formulations. All the formulations were tested for antifungal activity
against two organism’s Aspergillus niger and Candida albicans.
Thermodynamic Stability Studies

The studies were conducted for the optimized NE F3 by centrifugation, freeze-thaw cycle. The stability was
studied via clarity and phase separation observation at room temperature for three months.
RESULTS
The most important criteria for selection of all the nanoemulsion components are that all the excipients
should be pharmaceutically acceptable for topical application and transdermal delivery.
Optimization Criteria for Surfactant and Cosurfactant Ratio (Smix)
Optimization criteria for surfactant and cosurfactant ratio (Smix) in different volume ratios (1:0, 1:3, 1:2,
1:1, 2:1, 3:1, 4:1) results were given below.
Selection of Surfactant and Co-surfactant (Smix) Ratio
Tween 20 : Ethanol (Smix) Ratio formulations

Tween 20 Ethanol Physical state of nanoemulsion
1 0 Not clear
1 3 Transparent (low stability)
1 2 Light transparent
1 1 Transparent NE
2 1 Transparent NE
3 1 Transparent NE
4 1 Yellowish transparent
Transparent nanoemulsion was obtained for the ratio 1:1, 2:1 and 3:1 from which the 1:1 ratio has been
selected for further study. It has been reported that the 1:1 ratio of Smix is found to produce transparent
nanoemulsions
Construction of Calibration Curve
254
For this a calibration curve was constructed by selecting methanol as the primary solvent and Phosphate
buffer pH, 7.4 and the absorbance were measured at UV spectrophotometer at 230nm. The absorbances
obtained are given in table and calibration curve shown in figure.
Calibration Curve of Miconazole Nitrate at 230nm
S.No Concentration(μg/ml) Absorbance At 230nm

1. 4 0.160
2. 6 0.241
3. 8 0.315
4. 10 0.412
5. 12 0.494
6. 14 0.560
7. 16 0.618
Calibration Curve of Miconazole Nitrate at230nm
Particle Size for Miconazole Nitrate Nanoemulsion (F3)
In vitro Dissolution Studies for NE formulation
255
In vitro dissolution studies for Nanoemulsion Gel formulations.
Comparisons of Miconazole Nitrate Nanoemulsion Gel and Marketed Gel by Microbiological assay
256
DISCUSSION
The ratio of surfactant and cosurfactant (Smix), oil and Smix ratio were optimized as 1:1. Addition time and
rpm were optimized to 10 min & 1500 rpm respectively. Transparency of F3 formulation was more compared
to other formulations. The value of pH (5.92-6.83) and viscosity (0.8290-0.8729 cp) were obtained. The
results of the particle size analysis showed the formation of nanodroplets with narrow size distribution
having a suitable zeta potential. TEM studies showed the formation of discrete, spherical nanoglobules.
A maximum of 88.9%±1.84% of drug has got released from F3 in a period of 210 min, whereas the NE
gel (F3 (G)), showed 94.8%±2.06% release in 8h. The gel exhibited three-fold higher drug release than
the NE. In vitro permeation F3 (G) was found to be 93.7%±2.1% at 12h, whereas the commercial gel had
42.9%±1.8% of permeation. F3 (G) had two-fold higher permeation than the commercial formulation. The
drug release followed higuchi kinetics and was found to be fickian diffusion controlled according to the n
value (0.158) obtained from korsemeyer peppas model. A significant enhancement of antifungal activity
against Aspergillus niger 73% and Candida albicans 77% was elicited by NE gel F3 (G) compared with the
commercial one the activity of Miconazole nitrate in antifungal assay.
CONCLUSION
Overall the results the topical and transdermal delivery of antifungal drugs using clove oil nanoemulsion
can give better treatment compared to conventional gels. So the clove oil nanoemulsion may be used as a
potential carrier for topical and transdermal antifungal drugs.
REFERENCES
1. Bazigha K Abdul, Rasool Saeed A Khan et al., In vitro evaluation of miconazole mucoadhesive buccal
films, Int J Appl Pharm, 2010; 2:23-26.
2. Bouchemal K, Briancon S, Perrier E, Fessi H. et al., Nanoemulsion formulation using spontaneous
emulsification: solve.
257
Development and Characterization of Enrofloxacin Slns
and its Pharmacokinetics following Oral Administration in
Emu (Dromaius Novaehollandiae) Birds
P. Senthil Kumar1*, A. Arivuchelvan2, A. Jagadeeswaran3, N. Subramanian4, C. Senthil

Kumar5 and P. Mekala6
1
Veterinary College and Research Institute, Orathanadu-614625, Tamil Nadu, India
2, 3, 6
Veterinary College and Research Institute, Namakkal, Tamil Nadu, India
4
5
e-mail: 1p.senthilkumar@tanuvas.org.in, Phone: +91 94431 42359
Abstract:
The study was conducted to formulate the enrofloxacin SLNs and evaluate its pharmacokinetic (PK) behaviour
in emus. Enrofloxacin SLNs were prepared by a hot homogenization coupled with ultrasonication method
and characterized for further investigation in emus. PK of native enrofloxacin was studied after i.v. and oral
bolus administration at 10mg/kg in emus and it was compared with disposition kinetics of enrofloxacin SLNs.
Enrofloxacin and its metabolite ciprofloxacin in plasma were estimated using HPLC and PKs were calculated by a
noncompartmental analysis. The results demonstrated that the particle size, polydispersity index, zeta potential,
encapsulation efficiency and loading capacity of the SLNs were 154.72± 6.11nm, 0.42±0.11,-28.83±0.60mV,
59.66±3.22% and 6.13±0.32%, respectively. AFM and TEM images showed spherical to circular particles with
well defined periphery. In vitro drug study exhibited biphasic release pattern. Pharmacokinetic results showed
that the t1/2β, AUC0-∞, Vdarea/F, MRT and F were 3.107, 1.894, 1.594, 2.993 and 1.895 times enhanced, while CLB
and β were significantly decreased by 1.958 and 3.056 times compared to the values of native enrofloxacin. The
t1/2β and MRT of the metabolite were longer than those of parent substance. Based on the PK/PD indices, the
SLNs extended the enrofloxacin concentration upto 48 h.
Keywords: Enrofloxacin, Pharmacokinetics, SLNs, Tripalmitin, PK/PD integration.
INTRODUCTION
bactericidal activity against a range of clinically relevant Gram negative and Gram positive pathogens as
well as Mycoplasma and Chlamydiae. Enrofloxacin and its active metabolite ciprofloxacin possess high
bactericidal activity, killing the bacteria in a concentration dependent manner. The relative safety of
enrofloxacin, its low minimum inhibitory concentrations, broad spectrum of activity, long post-antibiotic
effect and good tolerance has encouraged their use in veterinary medicine ((Scheer 1987). Despite the
therapeutic potential of enrofloxacin, the very poor aqueous solubility of enrofloxacin leads to difficulty in
designs of pharmaceutical formulation and variations in bioavailability (Martinez et al., 2006)
Emu (Dromaius novaehollandiae) belongs to ratite group. Bacterial infections are important causes of
morbidity and mortality in domestic emu birds (Sales, 2007). Restraining is not easy and it causes stress in
emu birds. Hence, enrofloxacin with sustained release profile is highly convenient to utilize in emu birds. But,
all the oral enrofloxacin formulations are available as conventional, immediate-release form that necessitates
administration twice daily or daily for several days or weeks. Numerous efforts have been made to develop
alternative formulations of enrofloxacin to reduce frequency of administration.
The solid lipid nanoparticles (SLNs) introduced in 1991, are submicron-sized (50 to 1000 nm) carriers
composed of a lipid matrix stabilized by a surfactant. SLNs possess good tolerability, stability, scaling
up feasibility and the ability to incorporate hydrophilic/hydrophobic drugs (Muller et. al., 2000). The
incorporation of poorly soluble drugs into SLNs can enhance gastrointestinal solubilisation, absorption,
and bioavailability of drugs (Muller et al., 2002). Further, SLNs formulation has the ability to prolong or
sustain the release profile of the loaded molecules and hence reduce need for the repeated administration and
increase the therapeutic value of the treatment (Xie et al., 2011).
Hence, the present research was premeditated with the objectives of preparation and characterization
enrofloxacin SLNs; determination of pharmacokinetics of enrofloxacin SLNs following oral bolus
administration in domestic emu birds. This comprehensive study not only provides pharmacokinetic data of
enrofloxacin nanoparticle in domestic emus, for first time perpetually, but also lays down the pharmacokinetic
comparison between the native enrofloxacin and enrofloxacin nanoparticles. Based on these findings,
recommendations on the dosage of enrofloxacin SLNs are made.

Drugs and chemicals
Enrofloxacin, ciprofloxacin hydrochloride (Himedia Laboratories Pvt. Ltd., India), tripalmitin, span 80,
tween 80 and polyvinyl alcohol (Sigma Aldrich Chemicals Pvt. Ltd., USA) were utilized for the study.
Dialysis membrane procured from Himedia Laboratories Pvt. Ltd., India was used. For HPLC analysis,
HPLC grade acetonitrile, methanol, triethyl amine and phosphoric acid were purchased from Merck
Specialities Ltd., India. Water for HPLC obtained by Millipore water purification system was utilized. All
solvents and solutions for HPLC analysis were filtered through 0.2μ HNN nylon membrane filter (Nupore)
and degassed using sonicator. All other chemicals and solvents were analytical reagent grade and were used
without further purification.
Preparation of enrofloxacin SLNs

Enrofloxacin SLNs were prepared by hot homogenization followed by ultrasonication method. Enrofloxacin,
tripalmitin, span 80 were added at the ratio of 1:5:20 to get organic phase of preparation. The lipid content
in the organic phase was melted by heating at 70°C using magnetic stirrer with hot plate. The contents in
the organic phase were mixed properly by placing in the shaker (Spinix). An aqueous phase was prepared
by dissolving tween 80 and polyvinyl alcohol at the ratio of 20:20 by heating to the same temperature as
the organic phase. The hot aqueous phase was added to the organic phase under magnetic stirring (Remi,
Mumbai, India) at 1000rpm to form pre-emulsion. The hot pre-emulsion was then homogenised at 10,000
psi for 3 min using the high pressure homogenizer (Heidolph Electro, Germany) kept in a water bath
maintained at 70°C.
The hot emulsion so obtained was ultrasonicated (Sonics Vibra Cell, USA) using high-intensity (5/64’’
2 mm tip diameter) microprobe with amplitude 20% for 15min to form nanoemulsion. Then, the nanoemulsion
was run under magnetic stirring at 1000 rpm for 4 h to obtain enrofloxacin loaded tripalmitin SLNs.
All the batches were prepared in triplicate and the average size was measured.

Determination of particle size, polydispersity index and zeta potential
Particle size and polydispersity index (PDI) of enrofloxacin SLNs were measured by Photon Correlation
Spectroscopy (PCS) using zetasizer nanoZS with the Malvern PCS software version 6.20. The zeta potential
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was measured by electrophoretic light scattering (ELS) mode using zetasizer nanoZS. The particle charge of
enrofloxacin SLNs were quantified at 25°C. Each value was the average of three measurements.
Surface morphology
Surface morphology and shape of the enrofloxacin SLNs were examined using Transmission electron
microscope (Philips, Tecrai10, Dutch) and Atomic force microscopy (PARK XE-100).
Determination of loading capacity and encapsulation efficiency

To determine the entrapment of enrofloxacin in the SLNs, 0.1 mL of freshly prepared nanoemulsion was
taken and diluted with 9.9 mL chloroform. The obtained suspension was centrifuged for 45 min at 6,000 rpm.
The supernatant was separated and filtered through 0.2 µm filter. The filtrate was diluted using chloroform
and analysed at 273.8 nm using UV spectrophotometer. The SLNs formulated without enrofloxacin were
treated similarly and used as control for the measurements. The assay was repeated 3 times using different
preparations. Loading capacity and encapsulation efficiency were calculated as shown below:
Weight of enrofloxacin in SLNs

Loading capacity = × 100%
Weight of SLNs
Encapsulation efficiency = × 100%
Weeight of enrofloxacin added

In vitro release of enrofloxacin SLNs and native enrofloxacin was performed by dialysis bag diffusion
technique over a period of 120 h. Enrofloxacin nanosuspension equivalent to 5 mg of enrofloxacin was
filled in dialysis bag (Himedia Laboratory Pvt. Ltd, India). The receiver solution containing 100mL of
phosphate buffer with pH 6.7 was prepared and heated to 37°C under magnetic stirring at a speed of 100rpm.
The drug containing dialysis bag (Molecular weight 12 to 14 k.Da, pore size 2.4 nm) was dialysed against
receiver compartment. To determine the enrofloxaxin diffusion through the dialysis bag, 2mL samples were
withdrawn at regular intervals (0, 5, 10, 20, 30, 45, 60, 90 min, and 2, 4, 8, 12, 18, 24, 36, 72, 96 and
120 h.) from the receiver solution and same amount of fresh receiver solution was added to maintain the
volume constant. Enrofloxacin in the samples was measured spectrophotometrically at 273.8 nm using a
UV spectrophotometer (Systronics 2203 Smart, India). The control nanoparticles without enrofloxacin were
treated similarly and used as blanks for the measurements.
Pharmacokinetic study
Experimental design
The study was conducted in apparently healthy 8 emu birds (4 male + 4 female) aged 18 to 24 months with
a mean (±SE) body weight of 38.20±1.03 kg. The birds were under uniform conditions of housing (semi
intensive system) and feeding, according to the birds requirements. Birds were offered feed and water ad
libitum. Before the start of the experiment, the birds were examined clinically to rule out the possibility of
any disease. No antibiotics and anthelmintics were administered 2 months prior to the start of experiment.
The use of the birds and experimental design was approved by Institutional Animal Ethics Committee
(IAEC), TANUVAS, Chennai.
Administration of drugs and collection of blood samples

Trial I: Native enrofloxacin was administered i.v. (bolus dose) to the emu through the jugular vein. Two
millilitre blood samples were drawn by jugular venipuncture into heparinized tubes immediately before and
at 0.083, 0.167, 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, 8, 12, 18, 24 and 36 h after dosing.
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Trial II: After 2 weeks wash out period, the same birds were administered orally with the same dose of
native enrofloxacin directly using a thin plastic tube attached to a syringe. Then, 2 ml of blood samples were
drawn as same method at 0.25, 0.50, 0.75, 1.5, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48 and 60 h after dosing.
Trail III: With 2 weeks wash out period, the same birds were administered orally with enrofloxacin SLNs
directly using a thin plastic tube attached to a syringe. Blood samples (2 mL) were drawn by jugular
venipuncture into heparinized tubes at 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, 24, 36, 48, 60, 72, 84 and
96h after dosing. The birds were checked for observable signs of toxicity for up to 7 days after administration
of enrofloxacin SLNs.
The collected blood samples were centrifuged at 950xg for 20 min to separate the plasma. The plasma
samples were stored at -40°C until assay.
Drug assay
Determination of enrofloxacin and ciprofloxacin was performed by high performance liquid chromatography
(HPLC). The method developed by Kung et al., (1993) was followed.
The HPLC system comprised of LC-20 AD double plunger pump, Rheodyne manual loop injector with
a 20 μL loop, column oven CTO-10 AS vp, SPD-M20A diode array detector and a software LC Solution
for data analysis. The compound separation was achieved using a reverse phase C18 column (Hibar 250-4,
6 RP-18 endcapped, Particle size 5 μm, 4.6x250 mm, Merck, Darmstadt, Germany) as a stationary phase.
The column was protected with 2 to 8 mm Phenomenax guard column (KJO-4282). The mobile phase was
consisted a mixture of acetonitrile, methanol and water (containing 0.4% phosphoric acid (85%, v/v) and
adjusted to pH 3.0 using triethylamine in the ratio of 17:3:80 (v/v/v). The flow rate of mobile phase was 1
mL/min and samples were analysed for 10min at 40°C. The scan range of PDA was 220 to 400 nm, and the
detection wavelength was 278nm. The mean (±SE) retention times for ciprofloxacin and enrofloxacin were
5.65±0.003 min and 7.16±0.006 min, respectively.
The standard curves of enrofloxacin and ciprofloxacin were linear in the range of 0.01 to 10.0µg/mL.
The calibration curve for enrofloxacin was characterized by its regression coefficient (r2=0.999), slope
(19070) and intercept (13182), and was used to determine the analyte concentrations in the sample. The
calibration curve for ciprofloxacin was characterized by its regression coefficient (r2=0.998), slope (14777)
and intercept (6507.4), and was used to determine the analyte concentrations in the sample. Absence of
change in the retention time was considered the method found specific and selective. The mean absolute
recovery was within the range of 97.778 to 107.45% for plasma and the coefficient of variation (CV) was
2.129 to 7.676%. The intra-day and inter-day CV were within the limits (<10%) specified and hence the
method was suitable for assay of both enrofloxacin and ciprofloxacin in emu plasma. The limit of detection
and quantification were 0.01 and 0.025 µg/mL for enrofloxacin and 0.025 and 0.05 µg/mL for ciprofloxacin,
respectively.
The concentrations of enrofloxacin and ciprofloxacin in the plasma samples were determined by
substituting the respective peak areas/peak heights in the linear regression formula.
Pharmacokinetic analysis
Pharmacokinetic parameters were derived from concentration vs time curves obtained for each bird after
administration of native enrofloxacin and enrofloxacin SLNs. Non-compartmental pharmacokinetic analysis
was used to describe the pharmacokinetics of enrofloxacin and ciprofloxacin based on statistical moments
theory using pharmacokinetic software PK function (Usansky et al., 2011).
The elimination rate constant (β) was calculated from the log-linear portion of the elimination curve
using linear regression analysis. The elimination half-life (t1/2β) was calculated according t1/2β = ln2/β, where,
ln2-0.693. The area under the plasma concentration-time curve (AUC) and the area under the first moment
261
curve (AUMC) were calculated using the trapezoidal rule and extrapolated to infinity by means of the
elimination rate constant. The mean residence time (MRT = AUMC/AUC), total body clearance (CLB=Dose/
AUC), volume of distribution to steady state (Vdss= CLB x MRT) and apparent volume of distribution (Vdarea
=Dose/ β x AUC0-∞) were calculated after i.v. administration.
After oral administration, AUC, AUMC and MRT were calculated as above. Comparing the corresponding
oral and i.v. route of administration, the bioavailability (F) after oral administration was calculated as F=
AUC0-∞.(oral)/AUC0-∞ (i.v.)X100 ; mean absorption time as MAT = MRToral - MRTi.v.; total body clearance
as CLB=Dose x F/ AUC0-∞; apparent volume of distribution as Vdarea= Dose x F/ β x AUC0-∞ .
Pharmacokinetic/Pharmacodymanic (PK/PD) integration

The ratios Cmax/MIC and AUC/MIC were calculated for hypothetical MIC90 (0.05, 0.125, 0.25 and 0.5µg/
mL) values using the means of Cmax and AUC obtained in this study.
Statistical analysis of the data was performed by using SPSS 17.0 software. The results were expressed as
mean±SE. Harmonic mean was used with data not distributed normally. Test of significance such as t-test
and analysis of variance (one way ANOVA) were applied to find out difference between and among various
groups respectively (Snedecor and Cochran, 1989). Comparison of the means of different subgroups was
performed by Duncan‘s multiple range tests as described by Kramer (1957).
RESULTS
enrofloxacin SLNs are given in Table 1. AFM and TEM analysis showed that the enrofloxacin SLNs
were spherical and circular in shape (Fig.1 and 2). The particles were well dispersed with good particle
size distribution. The surfaces of the nanoparticle were smooth.
Table 1: Mean(±SD) particle size, PDI, zeta potential, EE and LC of selected enrofloxacin SLNs formulations
Particle size (nm) PDI Zeta potential (mV) EE (%) LC (%)

154.717± 6.149 0.422±0.109 -28.83±0.603 58.33±3.51 6.03±0.97
Figure 1: Atomic force microscopic and Transmission electron microscopic image of enrofloxacin SLNs
In vitro release of enrofloxacin from SLNs formulation and native enrofloxacin is illustrated in Fig.3.
The release curve of enrofloxacin SLNs exhibited a biphasic pattern. There was an initial burst release with
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about 39.23% drug released within the initial 24 h, followed by a slow and sustained release. The amount of
cumulated drug release over 96 h was 51.1%. In the native enrofloxacin, the release was 93.67% within 2 h
and reached 100% by 24 h.
Figure 2: In vitro release of native enrofloxacin and enrofloxacin SLNs (mean± SD, n=3).
The mean (±SE) plasma concentrations of enrofloxacin and its metabolite ciprofloxacin after
native enrofloxacin (i.v. and oral) and enrofloxacin SLNs (oral) administration are depicted graphically
in Fig.4. After i.v. administration of native enrofloxacin, enrofloxacin could be detected upto 18 h in one
bird while in seven birds the drug was detected upto 24 h. The hi ghest mean concentration was 14.756
µg/mL at 5 min and lowest was 0.054 µg/mL at 24 h. The mean (±SE) values of plasma concentration
of enrofloxacin following oral administration of native enrofloxacin rapidly increased from
0.591±0.073 µg/mL at 15min to 2.207±0.098 µg/mL within 1.5 h and then declined to 0.004±0.004 µg/mL
at 36 h. Detectable concentrations of enrofloxacin after oral administration of native enrofloxacin were
found upto 24h in seven birds while in one bird the drug was detected upto 36 h. The plasma concentration
of the active metabolite ciprofloxacin was observed from 15 min to 24 h for the both routes of i.v. and
oral administration of native enrofloxacin.
Figure 3: Semilogarithmic plot of mean plasma enrofloxacin and its metabolite ciprofloxacin concentration (µg/mL)
vs. time in emus (n = 8) following administration of native enrofloxacin and enrofloxacin SLNs (10 mg/kg).
The mean (±SE) plasma concentration of enrofloxacin after oral administration of enrofloxacin SLNs
was 0.964±0.074 μg/mL at 15min, reached a significantly higher peak concentration of 3.721±0.128 μg/mL
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at 1h, then decreased sharply to the same levels of native drug 2 h post-administration. Although the plasma
drug concentration decreased to 0.580±0.032 μg/mL at 6 h, the concentration was maintained over 0.012
μg/mL for up to 60h. Enrofloxacin could be detected in plasma up to 60 h in four birds and up to 48h in
four birds. The metabolite ciprofloxacin could be observed from 15 min to 48 h after the administration of
enrofloxacin SLNs orally.
Enrofloxacin showed AUC0-∞ of 20.085±3.493 µg.h/mL with large apparent volume of distribution
(3.921±1.005 L/kg) and slower elimination half-life (4.364±0.179 h) following i.v. administration (Table
2). After oral administration, enrofloxacin peak plasma concentration (Cmax) of 2.397±0.052 µg/mL was
achieved at (tmax) 2.167±0.279h with bioavailability of 79.941±7.147%. Whereas, for enrofloxacin SLNs,
the mean (±SE) peak plasma concentration (Cmax) was 3.815±0.059 μg/mL at 1.167±0.105 h. The t1/2β,
AUC0-∞, AUMC0-∞, MRT, MAT and Vdarea/F were 3.107, 1.894, 5.531, 9.730, 2.993 and 1.594 times
enhanced significantly (P≤0.01) than the values of native enrofloxacin. Total plasma body clearance (CLB)
and elimination rate constant (β) of drug was significantly (P≤0.01) decreased by 1.958 and 3.056 times,
respectively in enrofloxacin SLNs compared to native enrofloxacin. In SLNs groups, the bioavailability was
1.895 times higher than the bioavailability recorded for native enrofloxacin. The Cmax of enrofloxacin SLNs
was 1.499 folds higher than those obtained with the native enrofloxacin.
Table 2: Pharmacokinetic parameters of enrofloxacin after administration (10 mg/kg) of native enrofloxacin (i.v. and
p.o.) and enrofloxacin SLNs (p.o.) in emus
Variable Unit Routes of administration

Native Enrofloxacin Enrofloxacin SLNs
Intravenous Oral Oral
Enrofloxacin Ciprofloxacin Enrofloxacin Ciprofloxacin Enrofloxacin Ciprofloxacin
β. h-1 0.159±0.007 0.152±0.006 0.162±0.015 0.129±0.004 0.054±0.003 0.044±0.005
AUC0-t µg.h/mL 19.553 ±3.518 1.518±0.258 15.756 ±1.416 1.423±0.130 29.511±0.880 2.675±0.081
AUC0-∞ µg.h/mL 20.085 ±3.493 1.561±0.262 16.056 ±1.436 1.496±0.128 30.420±0.760 2.970±0.153
AUMC0-t µg.h /mL
2
90.670±19.068 10.591±2.058 102.756±16.766 10.575±1.106 538.882±36.290 46.035±1.673
AUMC0-∞ µg.h2/mL 104.619±19.920 11.889±2.058 109.083±17.395 12.892±1.063 603.401±27.268 75.657±10.032
MRT h. 5.105 ±0.216 7.454±0.223 6.616 ±0.475 8.625±0.173 19.807±0.590 25.463±3.288
MAT h. - - 1.511±0.475 - 14.702±0.590 -
Vd area/F L/kg - - 3.881±0.234 - 6.186±0.357 -
Vdarea L/kg 3.921 ±1.005 - 3.171 ±0.269 - 15.291±1.147 -
Vdss/F L/kg - - 4.168±0.191 - 6.520±0.196 -
CLB L/h.kg 0.629±0.164 8.256±2.385 0.507±0.003 6.897±0.509 0.3330±0.007 3.421±0.177
CLB/F L/h.kg - - 0.646±0.052 - - -
t1/2β h. 4.364±0.179 4.595±0.163 4.125±0.361 5.393±0.186 13.012±0.717 16..913±2.061
Cmax µg/mL - 0.197±0.029 2.397±0.052 0.169±008 3.815±0.059 0.358±0.011
tmax h. - 1.417±0.834 2.167±0.279 3.167±0.167 1.167±0.105 1.33±0.105
AF % - - 79.941±7.147 - 151.462±3.782 -
AUC0-t Cipro/ 7.764 9.031 9.063
AUC0-t Enro
After i.v. and oral administration of native enrofloxacin, the ciprofloxacin AUC0-t was 7.764% and
9.031% of enrofloxacin AUC0-t , respectively (Table 2). The ratio of AUC0-tcipro/ AUC0-tenro after oral
administration of enrofloxacin SLNs was 9.063%. The elimination half life (t1/2) and MRT of the metabolite
after administration of native enrofloxacin and enrofloxacin SLNs were longer than those of parent substance.
The clearance of the active metabolite recorded in this study was faster compared to the enrofloxacin.
The PK/PD integration parameters of Cmax/MIC and AUC0-24/MIC were calculated from the obtained PK
parameters is presented in Table 3.
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Table 3: PK/PD parameters of native enrofloxacin and enrofloxacin SLNs considering MICs of 0.05, 0.125, 0.25 and
0.5 µg/mL
Ratio MIC (µg/mL) Intravenous (Native Oral (Native enrofloxacin) Oral (Enrofloxacin SLNs)
enrofloxacin)
Cmax/MIC 0.05 295.11±44.52 47.94±1.04 76.29±1.18
0.125 118.04±17.81 19.17±0.42 30.52±0.47
0.25 59.02±8.90 9.59±0.21 15.26±0.24
0.5 29.51±4.45 4.79±0.10 7.63±0.12
AUC0-24/MIC 0.05 391.06±70.35 315.11±28.31 374.43±10.97
0.125 156.42±28.14 126.05±11.32 149.77±4.39
0.25 78.21±14.07 63.02±5.66 74.89±2.19
0.5 39.11±7.03 31.51±2.83 37.44±1.10
*For Cmax, a value of 14.755 µg/mL (mean peak plasma concentration at 5 min) was used for the calculation
DISCUSSION
Hot homogenization followed by ultrasonication technique applies high shear stress disrupting lipid
particles down to the submicron range. According to Schwarz et al., (1994) a sufficient high-energy input
was necessary to break down the droplets into the nanometer range. A high energy such as high production
temperature, high stirring rate, longer emulsification time and stronger ultrasound power were applied in
this study to obtain a finer dispersion of formulation. In the present study, the homogenization pressure
10,000 psi was applied for 3 min and followed by ultrasonication resulted the mean (± SD) particle size
of 154.717±6.149 nm with narrow size distribution. The result suggests that the hot homogenization and
ultrasonication method was a feasible and compatible method for preparing enrofloxacin SLNs.
(219°C–233°C), hence the stability and antibacterial activity of the enrofloxacin are not affected. According
to Luo et al., (2006), the size of nanoparticles ranges from 100 to 200 nm was favourable for better per oral
A narrow particle size distribution was an indication of nanoparticles stability and homogeneous
dispersion (Olbrich et al., 2002). PDI values ranging from 0 to 0.5 were considered to be monodisperse and
homogenous, but those of more than 0.5 indicated nonhomogenity and polydispersity (Zhang et. al., 2009;
Anton et. al., 2008). In the present study, the particle size distribution was monodisperse and homogenous as
formulation has less mean (±SE) PDI of 0.42±0.11.
Nanoparticle with zeta potential values greater than +25 mV or less than -25mV typically have high
degrees of stability due to electric repulsion between particles. Dispersions with a low zeta potential value
aggregates due to Van Der Waal inter-particle attraction (Muller et al., 2000). In this study, the mean (±SD)
zeta potential of -24.90±1.00 mV was recorded and it could provide proper stability to the enrofloxacin
SLNs. According to Schwarz and Mehnert (1997) and Zimmermann et al., (2000), the negative charge of
zeta potential was conferred by the lipids used in the SLNs. In agreement with this report, the tripalmitin
utilized in this study provided negative charge of zeta potential.
AFM images revealed spherical and circular in shape with the presence of some particle aggregates.
The presence of aggregates might be due to redistribution of particles after preparation. The images of AFM
and TEM represented that the particles were ranging from 100 to 200 nm and well dispersed with smooth
surfaces.
The enrofloxacin SLNs obtained in the present study had relatively medium drug entrapment efficiency
(59.67%). To get sufficient loading capacity, the drug should have sufficiently high solubility in the lipid
265
melt. (Bunjes et al., 2002). The percentage encapsulation efficiency data obtained in this study are consistent
with the findings of Xie et al (2011b).
In vitro release data obtained under sink conditions are consistent with drug release reported from
different SLNs by Ji et al (2011) and Xie et al., (2011b). The initial fast release (burst effect) could be
attributed to the presence of a small fraction of unentrapped drug or drug embedded near the SLNs surface.
Other factors contributing to a fast release were large surface area, high diffusion coefficient (small molecular
size), low matrix viscosity and short diffusion distance of the drug. The slow release was mainly due to
the low diffusion of drug molecules through the lipid matrix of the nanoparticles and hindering effects by
surrounding solid lipid shell (Muller et al., 2000; Mehnert and Mader 2001).
No overt signs of toxicity or abnormal behaviour were observed when enrofloxacin SLNs were
administered to emus through oral route. Published data regarding pharmacokinetics of drug loaded SLNs
in ratites and other domestic animals was limited. Hence, the results obtained in the present study are
interpreted by comparing pharmacokinetic results reported for laboratory animals administered with SLNs.
The plasma concentration of enrofloxacin after enrofloxacin SLNs administration in emus showed biphasic
release pattern. The initial fast (burst release) release of the drug could be due to desorption and diffusion of
enrofloxacin accumulated at the oil–water interface and in the outer shell of nanoparticles (Xie et al., 2008;
Muller et al.2000; Han et al.2009; Wang et al., 2011). The initial release should be sufficiently rapid to ensure
that the therapeutic drug levels are achieved in a timely manner in vivo. The subsequent slow release was
mainly due to the slow diffusion of drug molecules through the lipid matrix of the nanoparticles (Mehnert and
Mader 2001; Muller et al., 2000) which maintains the effective therapeutic drug concentrations for a longer
period. In the present study, the sustained release performance of enrofloxacin loaded SLNs provided the
plasma concentrations of enrofloxacin exceeding 0.012 μg/mL for 60h which was therapeutically effective
for many common pathogens (Prescott and Yielding 1990). The bi-exponential release of enrofloxacin from
SLNs observed in this study are in accordance with the reports of Xie et al., 2011b (ofloxacin loaded palmitic
acid SLNs in mice), Xie et al., 2011a (enrofloxacon loaded palmitic acid SLNs in mice), Kurtz et al., 1994
(doxorubicin loaded SLNs in rats) and Pandita et al., 2011 (paclitaxel loaded in SLNs in mice).
The plasma concentration of enrofloxacin recorded in this study was not consistent with the in vitro
release profile. The in vivo degradation of SLNs could be the main reason which is an important parameter
in determining drug release in vivo (Olbrich et al., 2002)
Significantly higher (p<0.01) AUC0–∞ and Cmax value with shorter (p<0.01) tmax was observed for
enrofloxacin SLNs compared to native enrofloxacin after administration at the same dose in emus. The
increased absorption of enrofloxacin loaded SLNs might have been contributed via six possible mechanisms.
First, the SLNs formulations entering into the GI tract stimulated secretions of bile salts (BS), phospholipids
(PL) and cholesterol, due to the presence of lipids in the formulation (Fleisher et al., ,1999; Dahan and
Hoffman 2008). The SLNs products along with the gastric shear movement formed a crude emulsion. It
promoted the solubilization of the coadministered lipophilic drug. Secondly, the SLNs are degraded by
lipase/co-lipase complex being anchored onto their surface. Triglycerides of SLNs are degraded into surface-
active monoglycerides, forming micelles. Drugs present in the degrading lipids may be entrapped in the
micelles. The micelles can interact with bile salts present in the gut, leading to the formation of mixed
micelles. Then, the lipids are absorbed via chylomicron formation primarily into lymphatic system, and
simultaneously the drug goes with the lipid which is called as ‘Trojan horse effect’ (Muller et al., 2000).
Thirdly, the cellular lining of the gastrointestinal tract is composed of absorptive enterocytes interspersed
with membraneous epithelial (M) cells. M cells that cover lymphoid aggregates, such as Peyar’s patches,
take up microparticles by a combination of endocytosis or transcytosis (Norris et al., 1998; Andrianov and
Payne 1998). Fourth, the permeability of gut wall was enhanced by lipids presence in the SLNs and thus
increased the drug absorption (Constantinides and Wasan 2007). Fifth, the activity of p-glycoprotein efflux
transporters in the GI wall was suppressed by lipids and surfactants and hence, increased the fraction of drug
266
absorbed (Dintaman and Silverman 1999; Nerurkar et al., 1996). Sixth, the lipids in the GI tract provoked
delay in gastric emptying which resulted in increased residence time of the coadministered lipophilic drug in
the small intestine. This enabled better dissolution of the drug at the absorptive site, and thereby improved
the absorption (Citters and Lin 1999).
The MRT for enrofloxacin SLNs was significantly increased compared to native enrofloxacin in this
study. According to Duchene and Ponchel (1997); Vasir et al., (2003), the adhesive properties of nanoparticles
with gastrointestinal tract wall increase their residence time in the gastrointestinal tract. Moreover, Xie et al.,
(2010b) explained that nanoparticles could protect the drug from chemical and enzymatic degradation and
gradually release drug from the lipid matrix into blood, resulting in a several-fold increase in MRT. Mehnert
and Mader (2001) reported that the drug transported as lipid vesicles remained intact for extended periods
and, thereby, resulted in prolonged release of the encapsulated drug.
The relative bioavailability obtained in this study (189.47%) is comparable to that reported by Suresh et
al., (2007) for lovastatin SLNs (173.0%). In concurrence with Xie et al., (2011a), the triglycerides utilized
in the SLNs formulation enhanced the lymph formation and simultaneously promoted the lymph flow rate.
The possible mechanisms for increased absorption of enrofloxacin SLNs discussed under AUC0-∞ could
be the reasons for higher bioavailability obtained in the present study. According to Suresh et al., (2007),
the increased relative bioavailability was due to transport of SLNs by intestinal lymph which avoided
first pass hepatic metabolism of drugs. Investigation of lymph at regular intervals for enrofloxacin could
have provided valuable information in this study and support suggestion of Suresh et al., (2007) regarding
lymphatic transport of SLNs.
The result after administration of enrofloxacin SLNs indicated longer t1/2 and prolonged clearance (CLB)
of enrofloxacin in emus. The increase in AUC and F reflect higher availability of enrofloxacin in SLNs
treated groups which is attributed to the low clearance of enrofloxacin in these birds.
The degree of metabolism varies considerably across species (Cox et al., 2004). In the present study,
ciprofloxacin AUC0-t was lower than 10% of enrofloxacin AUC0-t after administration of native enrofloxacin
and enrofloxacin SLNs. Similar results were obtained by De-Lucas et al., (2004) in ostrich for native
enrofloxacin. Helmick et al., (1997) reported that the plasma concentration of metabolite ciprofloxacin was
not consistent in emus. However, Anadon et al., (1995) observed a high hepatic conversion of enrofloxacin
to ciprofloxacin in the chicken. The ratio of AUC0-t cipro/AUC0-t enro recorded in this study indicated limited,
but rapid conversion of ciprofloxacin in the liver of emu birds after native enrofloxacin and enrofloxacin
SLNs administration.
The use of SLNs sustained the therapeutic concentrations up to 48 h, and enhanced the bioavailability
and volume of distribution besides appreciably increasing the AUC/MIC ratio. However, these derived
values do not take into account the contribution made by the active metabolite ciprofloxacin, and
therefore underestimate enrofloxacin efficacy. From these results, it is obvious that use of enrofloxacin
SLNs administration at 10 mg/kg every 48 h is able to produce an ideal clinical outcome against
pathogens susceptible to 0.125 µg/mL.
CONCLUSION
From the results of the executed experiments, it can be concluded that hot homogenization coupled with
ultrasonication method is suitable for producing SLNs with optimal particle size, shape, PDI, zeta potential,
drug loading and encapsulation efficiency. In vitro release of enrofloxacin SLNs exhibited biphasic pattern
with an initial burst release followed by sustained release over 96 h. Enrofloxacin SLNs were rapidly
absorbed after oral bolus administration and therapeutic concentrations in plasma were achieved for
extended period of time. SLNs significantly improved the bioavailability, t1/2β, AUC0-∞, Vdarea/F and MRT
while significantly decreased the CLB and elimination rate constant compared to native enrofloxacin. In
267
the present study, the SLNs played an important role in the drug delivery system and significantly changed
the in vivo pharmacokinetic behaviour of drug molecules. The results of pharmacokinetic parameters of
enrofloxacin SLNs strongly support the potential application of SLNs in emus as sustained delivery system
for enrofloxacin.
ACKNOWLEDGEMENT
Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai is gratefully acknowledged.
The authors wish to thank Dr. K. Rukumani, Professor and Head, Department of Pharmaceutical Technology,
Regional Centre- Anna University, Trichy for their support of this work.
Conflict of interest
The authors declare no conflicts of interest
REFERENCES
1. Andrianov AK and Payne LG, (1998), Polymeric carriers for oral uptake of microparticulates Adv Drug
Deliv Rev 34: 155-170
2. Anton N, Benoit JP and Saulnier P, (2008), Design and production of nanoparticles formulated from
nano-emulsion templates-a review J Control Release 128: 185–199
3. Bunjes H, Koch MHJ and Westesen K, (2002), Effect of surfactants on the crystallization and
polymorphism of lipid nanoparticles Prog Colloid Polym Sci 121: 7-10
4. Citters GWV and Lin HC, (1999), The ideal brake: a fifteen year progress report. Curr Gastroenterol
Rep 1: 404–409
5. Constantinides PP and Wasan KM, (2007), Lipid formulation strategies for enhancing intestinal transport
and absorption of p-glycoprotein (p-gp) substrate drugs: in vitro/in vivo case studies, J Pharm Sci 9:
235-248
6. Dahan A and Hoffman A, (2008), Rationalizing the selection of oral lipid based drug delivery systems
by an in vitro dynamic lipolysis model for improved oral bioavailability of poorly water soluble drugs,
J Control Release 129: 1-10
7. Dintaman JM and Silverman JA, (1999), Inhibition of pglycoprotein by D-α-tocopheryl polyethylene
glycol 1000 succinate (TPGS), Pharm Res 16: 1550-1556
8. Duchene D and Ponchel G, (1997), Bioadhesion of solid oral dosage forms, why and how? Eur J Pharm
Biopharm 44: 15–23
9. Fleisher D, Li C, Zhou Y, Pao LH and Karim A, (1999), Drug, meal and formulation interactions
influencing drug absorption after oral administration Clinical implications, Clin Pharmacokinet 36: 233-
254
10. Han C, Qi C and Zhao MBK, (2009), Hydrogenated castor oil nanoparticles as carriers for the subcutaneous
administration of tilmicosin: in vitro and in vivo studies. Journal of Veterinary Pharmacology and
Therapeutics 32: 116–123
11. Ji J, Hao S, Wu D, Huang R and Xu Y, (2011), Preparation, characterization and in vitro release of
chitosan nanoparticles loaded with gentamicin and salicylic acid Carbohydrate, Polymers 85: 803–808
12. Kramer CY, (1957), Extension of multiple range tests to group correlated adjusted means Biometrics,
13: 13-18 doi:102307/3001898 Access date: 16032014
268
13. Kurtz MB, Heath IB, Marrinan J, Dreikorn S, Onishi J and Douglas C, (1994), Morphological effects
of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan
synthase, Antimicrobial Agents and Chemotherapy 38: 1480-1489
14. Luo YF, Chen DW, Ren LX, Zhao XL and Qin J, (2006), Solid lipid nanoparticles for enhancing
vinpocetine‘s oral bioavailability, J Control Release 114:53–59
15. Martinez M, McDermott P and Walker R, (2006), Pharmacology of the fluoroquinolones: A perspective
for the use in domestic animals, The Veterinary Journal 172, 10–28
16. Mehnert W and Mader K, (2001), Solid lipid nanoparticles: Production, characterization and applications,
Advanced drug delivery reviews 47:165-196
17. Mitchell M, (2006), Enrofloxacin, Journal of Exotic Pet Medicine 15, 66-69
18. Muller RH, Mader K and Gohla S, (2000), Solid lipid nanoparticles (SLN) for controlled drug delivery-a
review of the state of the art, Eur J Pharm Biopharm 50: 161-177
19. Muller RH, Radtke M, Wissing SA, (2002), Solid lipid nanoparticles (SLN) and nanostructured lipid
carriers (NLC) in cosmetic and dermatological preparations, Adv Drug Deliv Rev 54: 131-55
20. Nerurkar MM, Burton PS and Borchardt RT, (1996), The use of surfactants to enhance the permeability
of peptides through caco-2 cells by inhibition of an apically polarized efflux system, Pharm Res 13:
528-534
21. Norris DA, Puri N and Sinko PJ, (1998), The effect of physical barriers and properties on the oral
absorption of particulates, Adv Drug Deliv Rev 34: 135-154
22. Olbrich C, Kayser O and Muller RH, (2002), Lipase degradation of Dynasan 114 and 116 solid lipid
nanoparticles (SLN) effect of surfactants, storage time andcrystallinity, Int J Pharm 237: 119-128
23. Pandey R, Sharma S and Khuller GK, (2005), Oral solid lipid nanoparticles based antituberclar
chemotherapy, Tuberculosis (edinp) 85: 415-421
24. Pandita D, Ahuja A and Lather V, (2011), Development of lipidbased nanoparticles for enhancing the
oral bioavailability of paclitaxel, AAPS PharmSciTech 12, 712–722
25. Prescott JF, Yielding KM, 1990, In vitro susceptibility of selection veterinary bacterial pathogens to
ciprofloxacin, Canadian J Vet Res 54: 195-197
26. Reddy LK, Sharma RK, Chuttani K, Mishra AM and Murthy RR, (2004), Etoposide-incorporated
Tripalmitin Nanoparticles With Different Surface Charge: Formulation, Characterization, Radiolabeling
and Biodistribution Studies, AAPS PharmSciTech 6:1-10
27. Scheer M, (1987), Studies on the antibacterial activity of Baytril, Veterinary Medical Reviews 2: 90-98
28. Schwarz C and Mehnert W, (1999), Solid lipid nanoparticles (SLN) for controlled drug delivery II Drug
incorporation and physicochemical characterization, J Microencapsul 16: 205-213
29. Schwarz C, Mehnert W and Muller R, (1994), Influence of production parameters of solid lipid
nanoparticles (SLN) on the suitability for intravenous injection. Eur J Pharm Biopharm 40: 24-30
30. Snedecor G W and Cochran WG, (1989), Statistical methods 8th Edn Iowa State University Press, Ames
Iowa, USA
31. Suresh G, Manjunath K, Venkateswarlu V and Satyanarayana V, (2007), Preparation, characterization
and in vitro evaluation of lovastatin solid lipid nanoparticles, AAPS Pharm Sci Tech 8: E1-E9
32. Usansky JI, Desai A and Tang-Liu D, (2011), PK Functions for Microsoft Excel Department of
Pharmacokinteics and Drug Metabolism Allergan, Irvine, CA 92606, USA
269
33. Vasir JK, Tambwekar K and Garg S, (2003), Bioadhesive microspheres as a controlled drug delivery
system, Int J Pharm 255: 13–32
34. Wang XF, Zhang SL and Zhu LY, (2011), Enhancement of antibacterial activity of tilmicosin against
Staphylococcus aureus by solid lipid nanoparticles in vitro and in vivo, Vet J 55: 672-680
35. Xie S, Zhu L, Dong Z, Wang X, Wang Y, Li X and Zhou W, (2011a), Preparation, characterization and
pharmacokinetics of enrofloxacin-loaded solid lipid nanoparticles: Influences of fatty acids, Colloids
and Surfaces B: Biointerfaces 83: 382–387
36. Xie SY, Wang SL, Zhao BK, Han C, Wang M and Zhou WZ, (2008), Effect of PLGA as a polymeric
emulsifer on preparation of hydrophilic proteinloaded solid lipid nanoparticles, Colloids Surf B:
Biointerfaces 67: 199–204
37. Xie Y, Zhu L, Dong Z, Wang X and Zhou WZ, (2011b), Preparation and evaluation of ofloxacin-loaded
palmitic acid solid lipid nanoparticles, International Journal Nanomedicine 5: 693–701
38. Yang T, Hussain A and Bai S, (2006), Positively charged polyethylenimines enhance nasal absorption of
the negatively charged drug, low molecular weight heparin, J Control Release 115: 289-297
39. Zhang J, Fan Y and Smith E, (2009), Experimental design for the optimization of lipid nanoparticles, J
Pharm Sci 98: 1813–1819
40. Zimmermann E, Muller RH and Mader K, (2000), Influence of different parameters on reconstitution of
lyophilized SLN. Int J Pharm 196: 211-223
270
Redox Environment Cleavable Polymeric Nanoparticles
for Drug Delivery
M. Gover Antoniraj, C. Senthil Kumar, Angeline Tisha and K.Ruckmani
Department of Pharmaceutical Technology, Bharathidasan Institute of Technology,

Anna University, Tiruchirappalli-620024
Abstract:
Chitosan and Methoxy polyethylene glycol (mPEG) have been broadly studied as delivery carrier for
drugs and various biomolecules such as DNA and siRNAs. Conjugation of chitosan with mPEG via
disulphide linkage was, therefore, anticipated to produce reduction sensitive drug delivery at disease
site. In this study, thiolated chitosan and mPEG-SH was synthesised and conjugated through disulphide
linkage CH-SS-mPEG by oxidation reaction. Synthesised polymer characterized by FTIR analysis. CH-
SS-mPEG nanoparticles (CNs) were prepared by ionic gelation method using Sodium tripolyphosphate
(STPP) as gelation agent. Size of the particles and surface charge value was evaluated for formulated
polymeric nanoparticles. An in-vitro drug release study was carried out for disulphide linked polymeric
nanoparticles with and without reducing agent glutathione (GSH). A drug release study shows that
the rapid release at reduction medium indicates glutathione induced drug release by the reduction of
disulphide. In conclusion, disulphide conjugated CH-SS-mPEG was successfully synthesized, and
prompt drug released in the presence high concentration of GSH.
Keywords: Polymeric nanoparticles, Redox-sensitive drug delivery, Ionic gelation and Glutathione (GSH).
1. INTRODUCTION
Designing an ideal drug delivery carrier an objective of present day has progressed significantly. Preliminary
research efforts were focused on formulating different drug delivery carriers to protect drug from degradation
and modify the drug release profiles. But now, the challenge has been to increase the effectiveness and
efficiency of drug delivery, and therefore reduce side effects, such that drug carriers are specially releasing
therapeutic agents at the required site. Nanoparticles drug delivery carriers able to deliver the drug at
targeted sites. Therapeutic efficiency of nanocarriers can enhance by the ability of rapidly distribute to
pathophysiological sites in the body and established effect of enhanced permeability and retention can
passively deliver the drug at disease sites.
However, rapid distribution of nanoparticles in pathophysiological sites the concentration of drug
within cancer cells is often inadequate due to the inefficient release of drug from the vehicle into
the cytoplasm, resulting in a requirement for higher drug dosage. Reason behind this, intracellular
environmental pH, temperature, glutathione and enzyme levels of cancer cells varied from normal
cells. To improving therapeutic efficiency promising method is to develop carrier systems that can
release the drug triggered by intracellular stimuli, such as pH, temperature, glutathione and enzyme.
Stimuli sensitive nanocarriers when reaching the targeted cancer cells, such carriers can be rapidly
localized intracellularly and subsequently activated by the stimuli to release the drug, hence inducing
required activity within tumor cells and leading to maximal therapeutic efficacy with reduced side
effects.
Reduction sensitive disulphide containing polymeric nanoparticles prepared and their reductive
degradation of disulfide bonds of polymers by intracellular glutathione (GSH) has been widely
investigated for responsive drug and gene delivery. GSH is a thiol-containing tripeptide and reduces
disulfide bonds in the cytoplasm. It has been revealed that the intracellular concentration of GSH (10
mM) is significantly greater than the level in the cell exterior (2 μM). In the present work, are we
aiming to synthesise disulfide-linked copolymer by the reaction thiolated chitosan with mPEG-thiol.
Polymeric nanoparticles formulated from synthesised copolymer, dextromethorphan used as model drug
to encapsulate into polymeric nanoparticles. Formulated disulfide bond containing drug delivery carrier
cleaved the shell material under GSH stimulus, subsequent in quick drug release with destruction of
polymeric nanoparticles.
2.1. Materials
Chitosan (degree of deacetylation, 75-85%) was obtained from Sigma-Aldrich. mPEG ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride and thioglycolic acid were purchased from Sigma-
Aldrich. Triethylamine and sodium hydroxide were purchased from Merck India. Tetrahydrofuran (THF)
were distilled under reduced pressure from magnesium sulfate and stored over molecular sieves (4A°). All
other commercially available chemicals were used as-received
2.2. Synthesis of thiolated chitosan:

Thiolated chitosan was synthesised by the reaction of chitosan with thioglycolic acid. Chitosan 50 mg
was dissolved in 25 mL of 1% acetic acid. To proceed the reaction thioglycolic acid 20mg (TGA) and
1.5 g of ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) was added to the CS
solution and the pH was adjusted to 5.0 with 1 M NaOH. The reaction mixture was stirred at room
temperature for 3 h. To remove the unbounded TGA and to isolate the thiolated chitosan, the reaction
mixture was dialyzed against acidic condition (molecular weight cut-off 12-14 kDa) over a period of 2
days in the dark.
2.3. Synthesis of mPEG-SH
mPEG-SH was synthesised by reaction of Dithiodipropionic chloride (5 mmol) with mPEG (4.8 g, one
mmol) in THF solution in the presence of triethylamine. Reaction mixture was maintained under nitrogen
condition for 75°C at 5 h. The desired mPEG-SH was isolated by filtration and purified by three times
precipitation with cold diethyl ether followed by vacuum drying.
2.4. Synthesis of CH-SS-mPEG
Disulfide bond linked CH-SS-mPEg was synthesised by addition of equal molar ratio of thiolated chitosan
with mPEG-thiol in distilled water by oxidation reaction. pH of the solution adjusted to 6.5 with the addition
of 1M NaOH solution. Formation of disulphide bond was enhanced by adding of the catalytic amount of
hydrogen peroxide to the above reaction mixture.
272
Figure 1: Synthesis of CH-SS-mPEG copolymer
2.5. Characterization of Synthesised polymer

FTIR studies were performed using spectrophotometer (JASCO 6300 series), FTIR spectral studies were
performed for thiolated chitosan, mPEG-SH and CH-SS-mPEG. Spectral scanning was done in the range
between 4000cm-1 and 400 cm-1.
2.6. Formulation of the CH-SS-mPEG nanoparticles

CH-SS-mPEG nanoparticles were obtained based on ionic gelation method TPP used as gelation agent.
Briefly, CH-SS-mPEG was dissolved in 1 % acetic acid to obtain a concentration of 2mg/ml and TPP
dissolved in double distilled water to get the solution of 1 mg/ml. The chitosan nanoparticles were formulated
upon addition of 10 ml TPP solution into 50 ml chitosan solution under mechanical stirring (1000 rpm) at
room temperature, and the pH was maintained 6.5. In that way, drug-loaded polymeric nanoparticles were
prepared by using a CH-SS-mPEG solution18.
2.7. Characterization of CH-SS-mPEG nanoparticles:

Drug loading of polymeric nanoparticles was made indirectly from the calculation of the unbound drug which
remained dissolved in the nanoparticles suspension medium. The drug-loaded polymeric nanoparticles were
centrifuged at 15,000rpm for 30 min. The amount of unbound drug (free drug) was measured in the clear
supernatant at 278 nm. The drug loading efficiency (LE) and entrapment efficiency (EE) of the nanoparticles
were calculated according to the following equations:
273
2.8. Measurement of Particle Size, Zeta Potential and Morphology:

Total drug-Free drug
Drug entrappment efficiency % = ×100
Total drug
Total drug-Free drug
Drug loading efficiency % = ×100
Mass of nanoparticlles
The particle size and the zeta potential of the polymeric nanoparticles were measured using zetasizer
(Ver. 7.02, Malvern Instruments Ltd). To analyse particle size, lyophilized drug-incorporated polymeric
nanoparticles of CH-SS-mPEG copolymer was reconstituted into deionized water. This sample solution
prepared was prepared at concentration of 1.0 mg/mL for the particle size measurement. Atomic force
microscopy observed the morphology and size distribution of polymeric nanoparticles.
2.9. In vitro drug release studies:

In vitro drug release studies were carried out for CH-SS-mPEG polymeric nanoparticles were placed into a
dialysis tubing (MWCO 12-14 kDa) with 10 mM of GSH or without, as control, and immersed in 20 ml of
PBS (pH 7.4). The systems were kept at 37°C under shaking (600 rpm). At predetermined time, intervals 1
ml of buffer solutions were withdrawn and replaced with 1 ml of fresh PBS. The amount of drug released
was evaluated by UV spectroscopy measurement.
3. RESULT AND DISCUSSION

Reduction sensitive disulphide bond containing CH-SS-mPEG copolymer was synthesised by three steps of
chemical reaction. First step of the synthesis of thiolated chitosan was characterized by FTIR spectroscopy
(Fig.2). Chitosan polymer structure containing OH, amine, amide and COC functional group proves by band
appears at3340, 1640 and 1100cm-1 respectively. Thiol substitution on a chitosan backbone confirmed by the
appearance peak at 2550 cm-1. In addition, the peak at 1605 cm-1 ascribed to the unreacted amino functional
decreased evidently, showing that the amino group of chitosan was partly substituted by the thioglycolic acid
molecules via the acylamino bond.
Figure 2: FTIR spectrum of a. chitosan, b. thiolated chitosan, c. mPEG, d. mPEG-SH, e. CH-SS-mPEG
274
mPEG and mPEG-thiol compounds containing peak at illustrates the structure of methoxy polyethylene
glycol. Substitution of a thiol group on mPEG was confirmed by peak formed at 2500cm-1. Oxidation of
thiol group synthesised CH-SS-mPEG copolymer. Band appears at 3343 cm-1 and 1100cm indicates the
functional group of chitosan and peak formed at 2880cm-1 (-CH) illustrates mPEG functional group.
Disappearance of peak at 2500 cm-1 demonstrates the disulphide bond formation by oxidation of thiolated
chitosan with mPEG-thiol(CH-SS-mPEG). Ionic gelation method prepared the anti-inflammatory drug
dextromethorphan encapsulated polymeric nanoparticles. The characteristics of a sequence of drug-loaded
polymeric nanoparticles, including drug loading, entrapment efficiency particle size and zeta potential were
summarized in Table 1.
Table 1: Drug loading efficiency, drug entrapment efficiency, and mean size of polymeric nanoparticles
Nanoparticles Feed ratio of CH-SS- Drug loading Drug entrapment Particle Zeta
mPEG/TPP(w/w) efficiency (%) efficiency (%) size (nm) potential(mV)
Empty NPs 5:1 - - 187 +14.7
Drug Loaded NPs 5:1 20.12% 72.84% 205 +16.8
Drug loading efficiency and entrapment efficiency was found to be 20.12% and 72.84% respectively.
Particle size of drug-loaded polymeric nanoparticles slightly larger than empty nanoparticles entrapment of
drug into polymeric carrier. AFM images reveal the spherical shape of polymeric nanoparticles and confirm
the size variation of empty and drug loaded nanoparticles.
Figure 3: AFM images of (a) empty polymeric nanoparticles and (b) drug loaded polymeric nanoparticles
To investigate the effect of the disulphide bond cleavability upon the drug release, we performed release
experiments with drug-loaded polymeric nanoparticles. Fig. 6 showed the release of dextromethorphan from
polymeric nanoparticles against PBS (pH 7.4) at 37°C in the presence and absence of GSH. Slow release of
drug from CH-SS-mPEG polymeric nanoparticles was detected in the absence of GSH. For illustration, only
36.81 % of drug was released in the first 12 h, and approximately 80.24 % released within 24 h. However, in
the same buffer solution with 10 mM GSH, similar to intracellular levels reported for GSH in cancer cells,
the release of drug was radically accelerated. For instance, about 60 % of drug was released in 3 h and near
85% was released in 6 h.
275
Figure 4: In vitro drug release studies of CH-SS-mPEg polymeric nanoparticles in the presence and absence of
glutathione
4. CONCLUSION
In this study, bioreducible copolymer CH-SS-mPEG have been successfully synthesised in order to build
redox-sensitive polymeric nanoparticles. Redox sensitive disulfide bond formation confirmed by FTIR
analysis. Dexamethasone used as model drug to encapsulate into polymeric nanoparticles formulated by ionic
gelation method. The polymeric nanoparticles exhibit 20% drug-loading and 72% entrapment efficiency.
These CH-SS-mPEG polymeric nanoparticles and drug-loaded polymeric nanoparticles are spherically
shaped, and size varied based on drug loading. In-vitro drug release studies directed with presence and
absence of glutathione indicates at reduction medium quickly released the drug matched with absence of
glutathione. Therefore, the polymeric nanoparticles are an attractive platform to achieve the fast reduction
sensitive drug delivery and enhanced therapeutic efficacy at required site.
ACKNOWLEDGEMENT
We acknowledge that funding for this work from the Department of Science and Technology –Nanomission
scheme (DST/SR/NM/NS-19/2009) INDIA.
5. REFERENCES
1. Priya Bawa, Viness Pillay, Yahya E Choonara and Lisa C du Toit, Stimuli-responsive polymers and their
applications in drug delivery, Biomed Mater., 2009:4(2) ; 022001
2. Haliza Katas, Nik Nur Shamiha Nik Dzulkefli and Shariza Sahudin, Synthesis of a New Potential
Conjugated TAT-Peptide-Chitosan Nanoparticles Carrier via Disulphide Linkage, Journal of
Nanomaterials 2012:(2012); 134607
3. Yu Shao, Wenzhe Huang, Changying Shi, Sean T Atkinson and Juntao Luo, Reversibly crosslinked
nanocarriers for on-demand drug delivery in cancer treatment, Ther Deliv. 2012:3(12);1409–1427
276
4. Na Song, Wenming Liua, Qin Tua, Rui Liua, Yanrong Zhanga and Jinyi Wang, Preparation and in vitro
properties of redox-responsive polymeric nanoparticles for paclitaxel delivery, Colloids and Surfaces B:
Biointerfaces 2011:87;454– 463
5. Rahul Nahire et al. Polymer Coated Echogenic Lipid Nanoparticles with Dual Release Triggers,
Biomacromolecules, 2013:14(3);841–853
6. Hanjoung Cho, Jungeun Bae, Vivek K. Garripelli, Joel M. Anderson, Ho-Wook Junb and Seongbong,
Redox-sensitive polymeric nanoparticles for drug delivery, Chem. Commun. 2012:48;6043–6045
7. 7. Ling-Yan Tang, Yu-Cai Wang, Yang Li, Jin-Zhi Du and Jun Wang, Shell-Detachable Micelles Based
on Disulfide-Linked Block Copolymer as Potential Carrier for Intracellular Drug Delivery. Bioconjugate
Chem., 2009:20; 1095–1099
8. Mary Caldorera-Moore, Nathalie Guimard, Li Shi and Krishnendu Roy, Designer nanoparticles:
Incorporating size, shape, and triggered release into nanoscale drug carriers, Expert Opin Drug Deliv.,
2010:7(4); 479–495
9. Yangyun Wang, Guolin Wu, Xiaomeng Li, Jiatong Chen, Yinong Wang and Jianbiao Ma, Temperature-
triggered redox-degradable poly (ether urethane) nanoparticles for controlled drug delivery. J. Mater.
Chem., 2012:22;25217–25226
10. Hui Liu and Changyou Gao, Preparation and properties of ionically cross-linked chitosan nanoparticles,
Polym. Adv. Technol., 2009:20; 613–619
11. Jing Li, Meirong Huo, Jing Wang, Jianping Zhou, Jumah M. Mohammada, Yinlong Zhang, Qinnv Zhu,
Ayman Y. Waddad and Qiang Zhang, Biomaterials 2012:33; 2310-2320
12. Mohammad Reza Saboktakin, Roya M. Tabatabaie, Abel Maharramov and Mohammad Ali Ramazanov,
Synthesis and Characterization of Biodegradable Thiolated Chitosan Nanoparticles as Targeted Drug
Delivery System, J Nanomedic Nanotechnol. 2011:4;S4
13. Min Suk Shim andYoung Jik Kwon, Stimuli-responsive polymers and nanomaterials for gene delivery
and imaging applications, Advanced Drug Delivery Reviews.2012:64(11); 1046-1059
14. Xiangang Huang, Xulin Jiang, Qizhi Yang, Yanfeng Chu, Guangyan Zhang, Bin Yang and Renxi Zhuo,
Triple-stimuli (pH/thermo/reduction) sensitive copolymers for intracellular drug delivery, J. Mater.
Chem. B, 2013:1;1860–1868
15. Fenghua Meng, Wim E. Hennink and Zhiyuan Zhong, Reduction-sensitive polymers and bioconjugates
for biomedical applications. Biomaterials 2009: 30; 2180–2198
16. Marta Roldo, Margit Hornof, Paolo Calicetic and Andreas Bernkop-Schnurch, Mucoadhesive thiolated
chitosans as platforms for oral controlled drug delivery: synthesis and in vitro evaluation. European
Journal of Pharmaceutics and Biopharmaceutics 2004:57;115–121
17. Hui-Yun Wen, Hai-Qing Dong, Wen-juan Xie, Yong-Yong Li, Kang Wang, Giovanni M. Pauletti and
Dong-Lu Shi, Rapidly Disassembling Nanomicelles with Disulfide-linked PEG Shells for Glutathione-
mediated Intracellular Drug Delivery, Chem. Commun., 2011:47;3550-3552
18. Gordon A. Morrisa, Jonathan Castile, Alan Smith, Gary G. Adamsa and Stephen E. Harding, The effect
of prolonged storage at different temperatures on the particle size distribution of tripolyphosphate
(TPP) – chitosan nanoparticles. Carbohydrate Polymers 2011:84; 1430–1434
277
Preparation of Prednisolone Loaded CaCO3
Microparticles for Sustained Release
C. Prabu, S. Latha, P. Selvamani
Department of Pharmaceutical Technology & Centre for Excellence in Nanobio Translational Research, Anna
University, Bharathidasan Institute of Technology Campus, Tiruchirappalli 620024, Tamil Nadu, India
e-mail: lathasuba2010@gmail.com, Phone: +919842598097, Fax: +914312407333
Abstract:
The system was developed for the inclusion of poorly water soluble prednisolone drug into the porous CaCO3
microparticles. Porous CaCO3/PSS [sodium poly (styrene sulfonate)] doped microparticles were prepared
by using bio-mimetic mineralization method. Prednisolone is a corticosteroid used to treat inflammatory
and autoimmune diseases conditions was absorbed onto the porous of the CaCO3 by solvent evaporation
technique. Encapsulation of polyelectrolytes on microparticles via layer by layer technique was the promising
approach for the development of sustained delivery using the polyelectrolytes protamine sulfate (PRO) and
PSS. Absorption and release of drug from the microparticles were estimated using ultraviolet spectroscopy.
Particle size of the formulation was around 5 microns and SEM analysis shows that the particles were
spherical in shape, from FTIR results indicates that no interaction between the drug and excipients. Zeta
potential analysis confirms the layer by layer coating of the polyelectrolytes, and the release studies were
carried out in the pH 7.4. Release shows upto 48 hours. The release results was in a sustained manner.
Keywords: Bio-mimetic mineralization method, Microparticles, Layer by Layer technique, Sodium poly
(styrene sulfonate).
1. INTRODUCTION
Many micro- and nanoparticles mostly organic [1] and some inorganic [1, 2] have been studied for the use
in drug delivery systems (DDS), Calcium carbonate (CaCO3), calcium phosphate (Ca(H2PO4)2), tricalcium
phosphate (Ca3(PO4)2) and hydroxyapatite (Ca5(PO4)3OH) have been used in DDS [4, 5]. Above all, CaCO3
was reported to be useful as an intranasal carrier of insulin and hydrophilic compounds, because of its easy
production and slow biodegradability [6–8]. In these reports, however, drugs or bioactive proteins were
adsorbed on the surface of solid particles or porous CaCO3 material. In these cases, the binding of the
adsorbed drugs to CaCO3 was not strong, which may result in insufficient sustained release or targeting.
Previous studies have demonstrated that the presence of the pure polystyrene sulfonate. In this case the
CaCO3 particles have the same size (diameter:2mm) and the same crystal structures that are vaterite. The
main results from that work argued that the polymeric additives could be used as a tool to control the shape
and size of the calcium carbonate particles and the mineralogy of the unstable polymorphs. Porous inorganic
materials are emerging as a new category of host/guest drug delivery systems due to some interesting
features of biological stability and sustained release property. Layer-by-layer (LbL) self-assembly technique
has been a powerful tool for the micro-encapsulation [9-11], where polyelectrolyte multilayer films were
elaborated on various particles through alternating deposition of oppositely charged polyelectrolytes mainly
due to the electrostatic attraction [12]. This micro-encapsulation via LbL showed potential applications in
biochemistry, pharmacy, controlled release, cosmetic and catalyst by several approaches [11, 13]. The first
approach was directly using proteins, e.g., bovine serum albumin (BSA) [14], glucose oxidase (GOD) [15],
and urease [16] and superoxide dismutase (SOD) [17], as the depositing species to prepare bioactive core-
shell particles. Prednisolone is a glucocorticoid used for various the therapies of autoimmune conditions
that were rapidly eliminated and had a short plasma elimination half-life of 1 to 2 hours. Despite all efforts
towards its control and prevention, rheumatoid arthritis remains a global health problem affecting all ages.
Although a decline in its prevalence has been witnessed in some developed countries, the level of this decline
has been exaggerated.
2. EXPERIMENTAL SECTION
2.1. MATERIALS AND METHODS
Prednisolone was a gift sample from forruts india Pvt. Ltd, Calcium chloride, sodium carbonate, Ethylene
diamine tetra- acetic acid disodium salt (ETDA salt) from Loba Chemie Mumbai, Protamine sulfate) (PRO)
and Poly(sodium 4-styrene sulfonate) (PSS) from Sigma Aldrich.
2.2. Preparation of drug loaded CaCO3 microparticles by LBL technique

2.2.1. Preparation of CaCO3 microparticles
The CaCO3 hybrid microparticles were prepared by mixing 0.2 M, Na2CO3 of in 100ml deionized water
containing 400 mg of Poly (sodium 4-styrenesulfonate). Meanwhile 0.2 M CaCl2 was dissolved in 100 ml of
deionized water and rapidly poured into the Na2CO3 mixture solution and stirred for 30 minutes and CaCO3
microparticles were separated by filtration and repeatedly washed with deionized water and acetone. Then
the filtered microparticles were dried at vacuum oven for 4 hours at 60°C.
2.2.2. Drug loading on CaCO3 microparticles

250 mg of dried CaCO3 microparticles were soaked in 5 ml of Prednisolone solution of 50 mg/ml in closed
batch to prevent the evaporation of the solvent. The suspension was brought to equilibrium under gentle
stirring for 24 hours. Particles were collected by centrifugation and dried at 50˚C to remove the excess of
solvent.
2.2.3. Polyelectrolyte multilayer deposition on drug loading on CaCO3 microparticles

Polyelectrolyte microparticles were prepared by deposition of bilayers of PRO/PSS both on to the drug
loaded CaCO3 microparticles. The absorption of polyelectrolytes was made from their stock solution (2mg/
ml) in 0.5 M, NaCl with pH adjusted to 6.5. Each absorption cycle included mild agitation of the suspension
of CaCO3 microparticles 150 mg in polyelectrolyte on mild agitation for 15 minutes, centrifugation and triple
washing of the precipitate in 0.001 M, NaCl to remove the excess unbound electrolyte. Alternating PRO and
PSS layers were deposited in the identical way until the desired layer number was achieved.
2.3. Evaluation studies
2.3.1. Particles Size Analysis
Measurement of Particle size and PI of microparticles was performed by using photon correlation spectroscopy
(PCS) (Malvern master sizer 2000 Ver.5.54). Microparticles were diluted with 1mL of deionized water to
eliminate the effect of viscosity caused by the ingredients. Particle size and PI were obtained as the average
of three measurements at 25°C [19].
2.3.2. Zeta potential analysis

The zeta potential of the formulation was diluted using deionized water and analyzed using laser light
scattering Malvern Zeta Sizer 1000HS (Malvern instruments) at 25°C [19].
279
2.3.3. Scanning electron microscopic (SEM) studies

The morphology of CaCO3 particles with a thin layer of palladium gold alloy and studied by scanning
electron microscopy (Instrument model TS5136MM) at the magnification range of 10000x.[20].
2.3.4. Fourier transforms infrared spectroscopic (FTIR) studies

The FTIR spectra were recorded with an Equnox 55 Fourier-transform infrared spectrometer (Bruker,
Germany) by a direct transmission, The CaCO3 particles, drug prednisolone and drug loaded CaCO3
particles was placed on a crystal sample holder and scanning from 4000 to 400 cm−1 at a resolution of 2
cm−1 [18].
2.3.5. X- ray diffraction studies

X- ray diffraction (XRD) patterns of the CaCO3-PSS microparticles were recorded on a Rigaku Ultima III,
USA powder diffraction system using Cu Kα radiation of 0.15406 nm wavelength at a scanning rate of 0.02
◦/s in the 2θ range of 10-80 ◦ [21.22].
2.3.6. Encapsulation efficiency and Loading capacity

Percent drug loading
The percentage of drug loading was estimated by using the formula
Percent drug loading = Total prednisolone-free prednisolone / Total particle weight X 100
Where, total prednisolone is the initial weight of the drug dissolved in the solution and free drug is
the weight of the drug in the aqueous media measured immediately after preparing the drug loaded CaCO3
particles by using the absorbance of UV spectroscopy, total particle weight is the total amount of CaCO3
particles used for the formulation.
Percentage Encapsulation Efficiency
Percent Encapsulation Efficiency = Total prednisolone-free prednisolone / Total prednisolone X 100
Where, total prednisolone is the initial weight of the drug dissolved in the solution, and free drug is
the weight of the drug in the aqueous media measured immediately after preparing the drug loaded CaCO3
particles by using the absorbance of UV spectroscopy [23].
2.3.7. In vitro release studies

In vitro release studies of the drug have been carried out in triplicate by placing drug loaded CaCO3 particles
in definite volume of releasing medium at 37°C temperature in the intestinal pH conditions using 7.4 pH
phosphate buffers, respectively under gentle stirring. The amount of drug released in 7.4 pH buffer solutions
was measured by UV spectrophotometer, at the wavelength of 244 nm.The drug release was measured after
a fixed interval of time and release dynamics were studied.
2.3.8. Stability studies
The formulated microparticles were subjected to stability studies in amber and transparent airtight glass
containers to assess the stability of the prepared formulation with respect to drug content and drug release
characteristics after storing the multiple-units of the formulation in drug stability testing chamber (Model:
WIL-195, Wadegati Labe quip Pvt. Ltd., India) whilst, stress conditions like temperature was maintained at
25°C and 65% relative humidity to represent temperate conditions for 60 days [24].
280
2.4 Results and Discussion

2.4.1. Particle Size Analysis of CaCO3
Porous calcium carbonate particles were prepared by using biomineralization technique. Particles were
relatively in the narrow size collection of about 5µm (fig. 1a). PSS plays a very important role in the
prevention of aggregation and broad size range. And the zeta potential for the microparticles potential is
-18.3 mV (fig. 1b).
Figure 1a: Particles size of CaCO3 – PSS microparticles Figure 1b: Zeta potential of CaCO3 – PSS microparticles
2.4.2. Zeta potential analysis

Sodium poly (styrene sulfonate) and Protamine sulphate were alternately deposited on CaCO3 microparticles
by layer by layer technique. This encapsulation process was followed by zeta potential, (fig. 2a). and the zeta
potential is -18.3 mV. And for drug loaded microparticles it was -21.5. And for final polyelectrolytes loaded
microparticles particle zeta potential was -11.5 (fig. 2b).
Figure 2a: Variation of zeta potential after addition of Figure 2b: Zeta potential of final CaCO3 – PSS
alternative polyelectrolyte over CaCO3 – PSS microparticles microparticles

2.4.3. Scanning Electron Microscopy

Scanning Electron Microscopy images of Porous Calcium carbonate particle prepared using PSS were
mostly spherical in shape (Fig. 3). Particles with PSS will have no structural change during the storage for
281
months. Negatively charged PSS on the microparticles were responsible for the Pore channel and prevented
recrystallization.
Figure 3: SEM Images of CaCO3 microparticles
2.4.4. FT-IR Spectrum for CaCO3 microspheres

Peak for CaCO3 bands located at 1501cm-1 CO stretching, 876 cm-1 aromatic peak together 2925 cm-1 for the
asymmetric C-H stretching mode, indicate the presence of carbonate ions in calcium carbonate. The characteristic
peak of prednisolone was showed at 3813 cm-1 is due to OH involved in intermolecular association, 1710 cm-1 and
1653 cm-1 due to carbonyl stretching groups.1609 cm-1 and 1443 cm-1 for C=O and COOH bending group. On
a comparison with the FTIR of CaCO3 microparticles, pure prednisolone and drug loaded CaCO3 microparticles
shows all the absorbance band of the CaCO3 and drug without any further peaks indicates no interaction and the
drug loaded CaCO3 microparticles (fig. 4).
Figure 4: FT-IR spectrums of a - Calcium carbonate, b - Prednisolone, c - Prednisolone loaded calcium carbonate
2.4.5. X-ray diffraction studies

In the XRD pattern shows that the CaCO3/PSS has dominant phase of veterite at 21, 25, 27, 29, 39, 44, 50, 56. along
with the trace of calcite 23, 29, 36, 47 (fig. 5). The broadening peaks shows the presence of tiny particles, generally
particles on storage in water will be changed into calcite on storage in water, presence of PSS on the surface prevent
the calcite formation.
282
Figure 5: XRD pattern of CaCO3 – PSS microparticles
2.4.6. Encapsulation efficiency and loading capacity

The encapsulation efficiency and loading capacity was found to be 66% and 18.6% respectively.
2.4.7. In-vitro Drug release studies

Release has been carried for the formulation in dialysis bag with 2 ml of the formulation in the phosphate buffer
with the pH of 7.4 samples were taken at the fixed times intervals and release of the drug extended upto 48 hours in
the sustained manner(fig. 6). The drug release behavior obeys a diffusion mechanism.
Figure 6: Cumulative drug release for drug loaded CaCO3 microparticles
2.4.8. Stability studies
The prepared microparticles do not show any considers able changes in drug content and drug release on
storage for 60 days maintained at 25°C and 65%(Table. 1).
Table 1: Storage stability studies of prednisolone microparticles
S. No Time (Days) Drug Content (%) Cumulative Drug Release

1 0 94.18 96.45%
2 15 93.64 96.16%
3 30 93.08 95.56%
4 45 92.54 94.71%
5 60 92.16 93.39%
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CONCLUSION
In the present work an approach to the encapsulation of prednisolone microparticles using polyelectrolyte.
It utilizes the ability of CaCO3 microparticles formed by mixing of calcium chloride and sodium carbonate
solution to get porous nature to capture drug with efficiency as a result of both physical and chemical
absorption. The employment of the CaCO3 microparticles as the template for the LBL assembly of a
multilayered polyelectrolyte shell, results in the release of the drug. It is not only results in the higher filling
of the drug and to minimize the immobilization of the drug. This application results in higher content of
encapsulation as well as their sustained release or delivery.
REFERENCES
1. Majeti N and Kumar VR, Nano and microparticles as controlled drug delivery devices, J. Pharm. Pharm.
Sci. 2000; 3: 234–258.
2. Akerman ME, Chan WC, Laakkonen P, Bhatia SN and Ruoslahti E, Nanocrystal targeting in vivo, Proc.
Natl. Acad. Sci. U. S. A. 2002; 991: 2617– 12621.
3. Itokazu M, Sugiyama T, Ohno T, Wada E and Katagiri Y, Development of porous apatite ceramic for
local delivery of chemotherapeutic agents, J. Biomed. Mater. Res.1998; 39: 536– 538.
4. Paul W and Sharma CP, Ceramic drug delivery: a perspective. J. Biomater. Appl. 2003; 17 : 253–264.
5. Haruta S, Hanafusa T, Fukase H, Miyajima H and Oki T, An effective absorption behavior of insulin for
diabetic treatment following intranasal delivery using porous spherical calcium carbonate in monkeys
and healthy human volunteers, Diabetes Technol. Ther.2003; 5: 1– 9.
6. Ishikawa F, Murano M, Hiraishi M, Yamaguchi T, Tamai I and Tsuji A, Insoluble powder formulation as
an effective nasal drug delivery system, Pharm. Res. 2002;19: 1097– 1104.
7. Yanagawa A, Kudo T and Mizushima Y, A novel particle carrier for transnasal peptide absorption. Jpn.
J. Clin. Pharmacol. Ther.1995; 19: 127– 128.
8. Jada A and Verraes A, Preparation and microelectrophoresis characterisation of calcium carbonate
particles in the presence of anionic polyelectrolyte, Colloids and Surfaces A: Physicochem. Eng.
Aspects. 2003; 219: 7-15.
9. Caruso F, Nanoengineering of particle surfaces. Adv. Mater. 2001; 13, 11–22.
10. Peyratout CS and Dahne L, Tailor-made polyelectrolyte microcapsules: from multilayers to smart
containers, Angew. Chem. Int. Ed. 2004; 43 : 3762–3783.
11. Decher G and Schlenoff J.B.(Eds.). Multilayer Thin Films: Sequential Assembly of Nanocomposite
Material. Wiley-VHC, Weinheim; 2003.
12. Sun QL, Tong Z, Wang CY, Ren BY, Liu XX and Zeng F, Charge density threshold for LbL self-assembly
and small molecule diffusion in polyelectrolyte multilayer films, Polymer. 2005; 46 : 4958–4966.
13. Liang ZP, Wang CY, Sun QL and Tong Z, Novel microcapsule fabricated by LbL nano self-assembly.
Prog. Chem. 2004;16 : 485–491.
14. Caruso F and Mohwald H, Protein multilayer formation on colloids through a stepwise self-assembly
technique. J. Am. Chem. Soc. 1999; 121:6039–6046.
15. Schuler C and Caruso F, Preparation of enzyme multilayers on colloids for biocatalysis, Macromol.
Rapid Commun. 199; 21 : 750–753.
284
16. Liang ZP, Wang CY, Tong Z, Ye WH and Ye SQ, Bio-catalytic nanoparticles with urease immobilized in
multilayer assembled through layer-by-layer technique. React. Funct. Polym. 2005; 63 : 85–94.
17. He CY, Liang ZP, Wang CY, Liu XX and Tong Z, Immobilization of superoxide dismutase by layer-by-
layer assembly on surface of PS colloid particles and their bioactivity, Chem. J. Chin. U. (Chin.). 2005;
26 : 88–92.
18. Wang C, He C, Tong Z, Liu Z, Ren B and Zeng F, Combination of absorption by porous CaCO3
microparticles and encapsulation by polyelectrolyte multilayer films for sustained drug delivery, Int. J.
Pharm. 2006; 308 : 160-167.
19. Ghassabian S, Ehtezazi T, Forutan M and Mortazavi SA, Dexamethasone loaded magnetic albumin
microspheres preparation and in vitro release, Int. J. Pharm.1996; 130 : 49-66.
20. Jin Y, Liu W, Wang J, Fang J and Gao H, (Protamine/dextran sulfate) 6 microcapsules templated on
biocompatible calcium carbonate microspheres, Colloid Surf. A. 2009; 342 : 40-45.
21. Volodkin DV, Larionova NI and Sukhorukov GB, Protein Encapsulation via Porous CaCO3 Microparticles
Templating, Biomacromolecules, 2004; 5 : 1962-1972.
22. Volodkin DV, Petrov AI, Prevt M and Sukhorukov GB, Matrix Polyelectrolyte Microcapsules: New
System for Macromolecule Encapsulation, Langmuir. 2004; 20 : 3398-3406.
23. L.N. Ramana, S. Sethuraman, U. Ranga and U.M. Krishnan, Development of a liposomal nanodelivery
system for nevirapine, J. Biomed. Sci. 2010; 17 : 1-9.
24. S. Latha, P. Selvamani, C. SenthilKumar, P. Sharavanan, G. Suganya, V.S. Beniwal and P.R. Rao,
Formulation development and evaluation of metronidazole magnetic nanosuspension as a magnetic-
targeted and polymeric-controlled drug delivery system, J. Magn. Magn. Mater. 2009; 321:1580-1585.
285
Quercetin Loaded Chitosan/PEG Nanoparticles:
Formulation and In Vitro Characterization
Saravanakumar P, Vinoth J, Chandrasekar P and Subramanian N*
Laboratory for Lipid Based Systems, Department of Pharmaceutical Technology, Bharathidasan Institute of
Technology, Anna University, Tiruchirappalli -24
e-mail: natesansubbu@gmail.com
Abstract:
The present work address investigation on the development and characterization of quercetin encapsulated
nanoparticles made of chitosan and also chitosan modified with polyethylene glycols. Quercetin loaded
polyethylene glycols modified chitosan nanoparticles were prepared by ionic gelation of tripolyphosphate
and chitosan. The optimum encapsulation efficiency (92.66 ± 2.67) was obtained with chitosan concentration
of 1mg/mL, chitosan-to-tripolyphosphate mass ratio of two and quercetin concentration of 1mg/mL. The
average nanoparticles size was about 294 nm and polydispersity index is 0.338 while the zeta potential
values are positive. Transmission electron microscope imaging showed a spherical, smooth and almost
homogenous structure for quercetin loaded chitosan nanoparticles but polyethylene glycols modified
chitosan nanoparticles shape slightly modified and size of the particles were increased. Fourier transform
infrared spectroscopy confirmed the linkage of hydroxyl group of polyethylene glycols with amino groups of
chitosan in the nanoparticles. The in vitro release of nanoparticles showed an initial burst release of quercetin
(45%) and followed by controlled drug release over the period of 12 h. It is concluded that, the developed
quercetin loaded polyethylene glycols modified chitosan nanoparticles might be used as vehicles for the
improved solubility and prolonged delivery of quercetin.
Keywords: Quercetin, Chitosan, Poly ethylene glycol and Ionic gelation
1. INTRODUCTION
Polyphenols are “bioactive compounds” that gained a lot of importance due a variety of biological effects
relevance to numerous area of health care. It has been chosen as a drug molecule(s) and gained attention
in the area of novel drug delivery systems because of their disease-preventing property and therapeutic
expediency in multiple biological effects. Among the polyphenols, quercetin (QUR) is the most attracted
and well beneficial molecule [1-2].
QUR (3, 5, 7, 3 and 4 – pentahydroxyl flavanone, Fig.1) is present in foods and herbals and showed
dilation of coronary arteries, decreasing of blood lipid level, anti-inflammation property, anti-aging
effects, prevention of cataract, lowering of the intra ocular pressure and anti-tumour activities. But its
clinical use is limited due its poor water solubility, intestinal degradation by bacteria and quick first pass
metabolism (3).
Figure 1: The structure of Quercetin
Chitosan (CS) improves the biological and pharmacokinetic property of the incorporated drugs due to
its mucous adhesive, non-toxic, biocompatible and biodegradable properties. The presence of poly ethylene
glycol (PEG), amphiphilic polymer, on the surface of nanoparticles (NPs) reduce the steric hindrance and
makes the NPs more hydrophilic and prevents binding with blood proteins and hence encapsulated drug in
the nanoparticles survive in the blood circulation for longer period of time. CS NPs can be prepared by the
ionic gelation method using tripolyphosphate (TPP) as a cross linking agent [4]. The blends of PEG with CS
NPs has been prepared by this method utilized as a therapeutic drug-delivery system for promoting tissue
repair and regeneration through controlled and sustained release of loaded drugs [5]. In the present study
QUR loaded NPs with CS and PEG modified CS were developed and characterized with the aim to obtain
improved solubility and prolonged delivery of QUR.
2. EXPERIMENTAL
2.1. Materials
CS was received as gift sample from Cognis, Mumbai, India. Quercetin dihydrate was purchased from Sigma-
Aldrich, St. Louis. PEG 2000, PEG 4000, PEG 6000 and other reagents and solvents were of analytical
grade. The solutions were prepared using double distilled water.
2.2. Methods
2.2.1. Preparation of Quercetin -loaded chitosan and PEG modified chitosan NPs.

CS NPs were prepared by the ionic gelation using CS and TPP [6]. CS was dissolved in 20 mL of acetic
aqueous solution. TPP was dissolved in double distilled water to get 1mg/mL aqueous solution. Different
concentration of 2.5, 5, 7.5 mg/mL PEG solution of various molecular weights (PEG 2000, PEG 4000 and
PEG 6000) was prepared in TPP solution. QUR (20mg) was added into CS solution for the formulation of
QUR loaded NPs. Chitosan NPs are formed by drop wise addition of TPP (1mg/mL) aqueous solution into
chitosan solution under magnetic stirring at room temperature. PEG-modified chitosan NPs were formed
spontaneously upon incorporation of TPP containing PEG solution drop by drop into the acetic aqueous
solution of chitosan under magnetic stirring at room temperature. The formed opalescent suspensions were
subjected to centrifugation at 15,000 rpm, drug loaded NPs and drug free NPs were collected and dried under
vacuum.
290
3. CHARACTERISATION
3.1. Particle size and zeta potential
The mean particle size, polydispersity index and zeta potential of the formulations were determined by using
Zetasizer (Malvern Instruments Ltd., UK).
3.2 Drug content
The QUR content in the developed NPs was determined by dissolving accurately weighed NPs in acetic acid
solution and then diluted with freshly prepared Phosphate Buffered Saline (PBS). The absorbance of the
solution was measured at 370 nm for QUR by using UV–Vis spectrophotometer.
3.3. Transmission electron microscopy (TEM)

The morphology of the QUR loaded CS NPs and PEG modified CS NPs was examined by a TEM (Philips
EM430 Transmission electron microscopy, USA). NPs were fixed on copper grids with 0.5% (w/v)
phosphotungstic acid staining for TEM study.
3.4. Fourier-transformed infrared spectroscopy (FTIR)

FTIR spectra of CS, PEG 2000, QUR and QUR loaded NPs formulations dispersed in potassium bromide
pellets were recorded from 4000 to 400 cm−1 using FTIR spectrometer (Affinity 1, Shimadzu, Japan) with
32 scans and resolution of 4 cm−1.
3.5. Differential scanning calorimetry (DSC)

The thermal properties of the pure polymers (CS and PEG 2000), QUR and formulated NPs were
characterized by Differential scanning calorimeter (TA Instruments, USA) using purified nitrogen as purge
gas at a pressure of 20 psi.
3.6. In vitro drug release

Drug loaded NPs formulations (10mg) were dispersed in 2 mL PBS, transferred to dialysis bag (12-14 kDa
cut-off, Himedia Labs, India) and it is suspended in a beaker containing 40mL of dissolution medium PBS
(pH 7.4) under magnetic stirring at 600rpm in room temperature. At the specific time intervals, aliquots of
samples were withdrawn and replaced the same amount of fresh PBS to maintain the sink condition. The
aliquots were analysed for the concentration of QUR released by UV- spectrophotometer at 370 nm.

QUR loaded CS and PEG modified CS NPs were obtained by ionic gelation method where mixture of two
aqueous phases involved, one is the CS solution and another is a polyanion TPP solution/TPP with PEG(s)
solution at room temperature. The composition of NPs is shown in Table 1. The particle size QUR loaded
CS NPs is 14 nm and PEG modified CS NPs were in the range of 294 - 814 nm (Fig. 2). The polydispersity
index of the developed NPs were in the range of 0.33 – 0.68. The particle size and polydispersity index of
the formulation was increased with increased concentration and molecular mass of PEG (Table 1). It might
confirm that PEG and QUR are bound or trapped in CS-TPP matrix.
It has been previously reported that the incorporation of PEG in the formulations, might forms the
intermolecular hydrogen bonding between the amino hydrogen of CS and the oxygen atom of PEG,
thus forming a CS-PEG semi-interpenetrating network due to its electropositive and electronegative
effects respectively [7]. Consequently, increase in the concentration of PEG leads to an increase the
size and decrease the positive charge of the NPs. Thus the possibilities of ionic interaction between the
291
TPP and CS are reduced. Fig. 3 demonstrated changes in the zeta potential of NPs before and after PEG
modification.
Table 1: Particle size and Polydispersity index of the formulations
Formulation code PEG (Mol. Wt) PEG concentration (%) Average Particle size (nm) Polydispersity Index
FQ - - 14±1.12 0.231
FQ1 2000 0.25 294±2.32 0.338
FQ2 0.5 328±1.25 0.312
FQ3 0.75 494±1.35 0.418
FQ4 4000 0.25 527±1.41 0.507
FQ5 0.5 612±1.28 0.260
FQ6 0.75 660±2.86 0.559
FQ7 6000 0.25 713±1.23 0.557
FQ8 0.5 783±2.31 0.654
FQ9 0.75 814±5.06 0.689
Figure 2: Particle size and its distribution of the chitosan NPs and PEG-chitosan NPs.
Figure 3: Zeta potential distribution of the Chitosan nanoparticles and PEG-Chitosan nanoparticles.
The drug loading and encapsulation efficiency (EE) of developed nanoparticle formulations are given
in Table 2. The EE of QUR decreases with increase in the concentration of PEG. QUR loading of the
formulation is decreases when increases the PEG concentration and the molecular weight of PEG added with
the TPP solution.
292
Table 2: Drug loading and encapsulation efficiency of the formulations
Formulation code % QUR Loading EE of QUR

FQ 6.38±0.18 92.66±2.67
FQ1 8.11±0.07 91.52±0.89
FQ2 5.25±0.04 86.35±0.66
FQ3 4.56±0.07 80.23±1.39
FQ4 8.07±0.14 88.85±1.55
FQ5 8.05±0.15 81.68±1.62
FQ6 6.90±0.08 62.41±0.72
FQ7 7.98±0.11 89.18±1.27
FQ8 5.04±0.11 79.63±1.78
FQ9 4.23±0.14 61.66±2.11
Mean ± SD of three determinations.

FT-IR spectrum of QUR, PEG 2000, CS and NPs are shown in Fig. 4. The characteristic peak
of QUR C = C stretching of aromatic ring was observed at 1517 cm−1 and presence of strong O-H
indicated at 3295 cm-1. Strong C-O-C stretching and the N-H primary amine of CS confirms the by the
peaks at 1243 cm-1 and 1612 cm-1 respectively. PEG confirmed by the O-H stretch at 3459 cm-1 and the
characteristic C–O–C stretching vibration of the repeated -OCH2CH2- units of the PEG backbone at
1170 cm−1.
The peak at 3421 cm-1 of FT–IR spectrum of formulation indicates the hydrogen bond formed between
the polymer and drug. The peak at 1747 cm-1 confirms the presence of C-O-C stretching of the repeated
-OCH2CH2- units of the PEG backbone of pure PEG. This indicates that the PEG (s) incorporated in the CS
NPs formulation.
Figure 4: FTIR spectra of Chitosan/PEG Quercetin Nanoparticles (A), Quercetin (B), PEG (C) and Chitosan (D)
The surface morphology of CS NPs and QUR loaded PEG-CS NPs formulations were characterised
using TEM. The Fig. 5A and 5B shows that the CS NPs are smaller in size compare to the PEG modified
CS NPs.
293
Figure 5: TEM images of (A) Chitosan QUR Nanoparticles and (B) Chitosan/ PEG QUR Nanoparticles
DSC studies were performed to understand the nature and interaction of the encapsulated QUR in the polymer
matrix. DSC analysis was performed on CS, PEG 2000, QUR, PEG/CS NPs and FQ1 and the thermograms
are not shown. QUR showed endothermic peak at 250°C indicating the melting point of the compound. The
peak corresponding to the melting of QUR was not prominent in the thermogram of nanoparticle formulation
which could be due to the dispersion of QUR in the NPs. The DSC curves of drug free NPs and CS are almost
identical, indicating no influence of any residual solvent in the NPs on their thermal properties.
The drug release profile of CS-PEG NPs was established by dialysis bag method. Fig. 6A & 6B shows
the cumulative release of the QUR from CS-PEG NPs with different molecular mass PEG with the fixed CS
content (20mg). The cumulative amount of drug release observed from the developed formulations are in the
following order pure QUR < CS NPs < CS-PEG NPs. This indicates the increase in the solubility of QUR
when encapsulated in the polymeric systems. The polymeric NPs system showed initial rapid releases of the
QUR and sustained release up to 12 hours. This rapid release was indicated that some of the amount of QUR
is adsorbed on the NPs surface and after that release medium diffused into the NPs and released the QUR.
Figure 6 (A) and (B): In vitro release of Quercetin from pure Quercetin, Chitosan Quercetin Nanoparticles, Chitosan/
PEG Quercetin Nanoparticles
294
5. CONCLUSION
QUR loaded CS and PEG modified CS NPs were prepared by the ionic gelation method. The particle size
of the formulation was increased when increasing the concentration of PEG and it indicated the presence of
PEG and QUR in the CS-TPP complex. Entrapment of QUR in the polymer matrix is also confirmed with
FTIR spectrum and DSC thermograms. The low cumulative amount of QUR released from the CS NPs
than modified with PEG. CS NPs made with higher molecular weight PEG showed higher amount of QUR
release than the CS NPs made with lower molecular weight PEG. The developed CS-PEG NPs might be used
as vehicles for the improved solubility and prolonged delivery of QUR.
6. REFERENCES
1. Mikstacka R,Rimando AM,Ignatowicz E. Antioxidant effect of trans-reveratrol, pterostilbene, quercetin

and their combinations in human erythrocytes in vitro.Plant Foods Hum Nutr.2010 Mar;65(1):57-63.
2. Kleinedler JJ, Foley JD, Alexander JS, Roerig SC, Hebert VY, Dugas TR. Synergistic effect of resveratrol
and quercetin released from drug-eluting polymer coatings for endovascular devices. J Biomed Mater
Res B Appl Biomater. 2011 Nov;99(2):266-75.
3. Gao L, Liu G, Wang X, Liu F,Xu Y,Ma J.Preparation of chemically stable quercetin formulation using
nanosuspension technology. Int J Pharm. 2011 Feb 14;404(1-2):231-7.
4. Desai KG,Park HJ. Encapsulation of vitamin C in tripolyphosphate cross-linked chitosan microspheres
by spray drying. J Microencapsul.2005 Mar;22(2):179-92.
5. Huang M, Lieu L, Zhang G,Yuan G, Fang Y. Preparation of chitosan derivative with polyethylene glycol
side chains for porous structure without specific processing technique. Int J Biol Macromol.2006 May
30;38(3-5).
6. Calvo P, Remunan-Lopez C, Vila-Jato J L and Alonso M J. Novel hydrophilic chitosan-polyethylene
oxide nanoparticles as protein carriers. J Appl Polym Sci. 1997 Jan 63(1):125-132.
7. Wu Y, Yang W, Wang C, Hu J,Fu S. Chitosan nanoparticles as novel delivery system for ammonium
glycyrrhizinate. Int J Pharm. 2005 May 13;295(1-2):235-45.
295
Development and Evaluation of Artemisinin Magnetic
Nanosponges for Targeting Breast Cancer
S.P. Sharavanan, P. Chandrasekar, K. Sanjai, Padma @ Rajam,

R. Suriyakanth and N. Subramanian1
Laboratory for Lipid Based Systems, Department of Pharmaceutical Technology,

Anna University, BIT Campus, Tiruchirappalli-620024
1
natesansubbu@gmail.com
Abstract:
The most of cancer drugs have inability to accumulate selectively in the cancerous cells or tissues. Large
quantities of doses have to be administered to get the required therapeutic concentration to the target
site. As a result, many negative side effects may be caused by cytotoxic drugs, since the healthy cells get
exposed to higher concentrations of drugs. In the present after study we had made an attempt to resolve
the problems by selectively and quantitatively accumulating the drug to the target breast cancer tissues
using magnetic nanosponges with external magnetic field. Magnetic nanosponges containing artemisinin
was formulated and characterized for their drug-excipient compatibility, surface morphology, particle size
and size distribution, in-vitro drug release and evaluated for its in-vitro targeting potential. The FT-IR
studies confirmed the absence of chemical interaction between the drug and excipients in the formulation.
The average particle size was found to be 150 nm. The drug release at 8 hr was observed to be 52%, 65%
and 94% for formulations F1, F2 and F3 respectively. It was also observed that the drug release from the
magnetic nanosponges was slower in and controlled pattern. From the obtained results, we conclude that the
developed magnetic nanosponges may exhibit might be useful in in-vivo targeting of artemisinin to breast
cancer tissues and will provide better breast cancer activity.
Keywords: Artemisinin, Breast cancer, Nanosponges, Controlled release.
INTRODUCTION
Cancer is the uncontrolled growth of abnormal cells in the body [1]. Cancer is the second leading cause
for death worldwide and hence better drug delivery methods are innate demand for cancer treatment in the
world market. Breast cancer caused 458,503 deaths w o r l d w i d e in 2008 which account 13.7% of cancer
deaths in women [3, 4]. According to American cancer society, about 1.3 million women are diagnosed
with breast cancer annually worldwide. In India about 191,000 women were affected by breast cancer
out of which 104,000 women die each year [2]. The available treatments for breast cancer are surgery,
radiation therapy, chemotherapy, hormone therapy and targeted therapy. In conventional chemotherapy,
chemotherapeutic agents exhibits poor specificity in reaching tumor tissue and they are often restricted by
dose-limiting toxicity. Large quantities of doses have to be administered to get the required therapeutic
concentration to a target site. So the healthy cells also get chance a to expose in higher concentrations of
drugs. As a result, many negative side effects may be caused by cytotoxic cancer drugs. The limitations
in conventional chemotherapy can be surmounted by developing a controlled release nanoparticulate
system with efficient targeted drug delivery potential. Biodegradable polymeric nanoparticles, hydrogels,
micelles, liposomes, dendrimers, nanoshells, nanotubes, nanomaterials, polymersomes, nucleic-acid-based
nanoparticles, magnetic nanoparticles, polynucleotide nanoparticles and virus nanoparticles are some classes
of materials have been developed for targeting nanoparticles to the cancer target site [5, 6].
The non-specificity and side effects of most chemotherapeutic agents is overcome by magnetic drug
targeting. The accumulation of magnetic nanoparticles at target site are determined by particle size, surface
characteristic, field strength and geometry of particles and depth of the target tissue, vascular supply and
rate of blood flow etc [8]. These systems offers enhanced drug bioavailability, efficient biodistribution of
drug, improved timed release of drug molecules, precise drug targeting to specific site, avoidance of RES
clearance, and reduction in the concentration of free drug in blood.
Magnetic nanosponges are nano particulate systems having a particle size of <200 nm. In which
drug(s) and ferromagnetic particles are encapsulated with biocompatible polymer. It can be easily targeted
to the tumor site by the external application of magnetic field. On application of external magnetic field, the
sponges come into contact with a tumor cell and attach to the surface or engulfed into the cell, where they
release the encapsulated drugs in a predetermined rate. The mechanism of drug release may be surface or
bulk erosion, diffusion or swelling followed by diffusion, in a time or condition-dependent manner [7].
Artemisinin is a water insoluble anti-malarial drug recently screened for anti-cancer activity, anti-
proliferative activity and anti-angiogenic activity [9]. It reacts with iron to form free radicals which can
kill cells. Cancer cells require and uptake a large amount of iron to proliferate. They are more susceptible
to the cytotoxic effect of artemisinin than normal cells. The objective of present study is to formulate and
characterize polymeric artemisinin magnetic nanosponges for the effective targeting of drug to the breast
cancer tissues. The drug loaded magnetic nanosponges were characterized for its surface morphology, particle
size, polydispersity index, swelling index, and evaluated for its drug loading, encapsulation efficiency, in-
vitro drug release and in-vitro targeting potential.
EXPERIMENTAL METHODS
Materials
Artemisinin was graciously gifted by Fourrts India Laboratories Pvt. Ltd., Chennai, Gelatin and Ammonia
was purchased from S.D Fine Chemicals, Mumbai, Ferrous Chloride tetrahydrate was procured from
Loba Chemie, Mumbai, Gluteraldehyde was obtained from Marine Chemicals, Cochin, Sodium hydroxide
purchased from Sigma-Aldrich, USA and Potassium di-hydrogen phosphate from Hi-Media, Mumbai. All
other reagents used were analytical grade.
Drug-Excipient Compatibility Studies

The compatibility study was performed using Fourier Transform Infrared Spectrometer (FT-IR). The
physical mixtures of drug and excipients(1:1), Artemisinin, gelatin, ferric chloride tetrahydrate,
gluteraldehyde were individually mixed with potassium bromide (1:10) and compressed under 10 tones
pressure in a hydraulic press to form a transparent KBr pellets and then the pellets were scanned in IR
spectrophotometer in the wave number range of 4000 cm-1 to 400 cm-1. The formulation was also studied
in the same manner.
Preparation of Magnetic Nanosponges

Magnetic Nanosponges with gelatin were fabricated by co-precipitation method in which iron oxide
nanoparticles were deposited directly into the gelatin. Briefly, gelatin was dissolved in the water at 40 °C
and to this ferrous chloride tetrahydrate was added to form the hybrid sols at 600C. To the above solution
artemisinin dissolved in ethanol was injected drop by drop and then the hybrid sols were rapidly cooled
to form gel. To the above gel, aqueous ammonium hydroxide solution was added to start the iron
oxide formation process. The gel immediately becomes black indicating the formation of magnetic
nanoparticles. Then to the formed nanosponges, gluteraldehyde was added under stirring at 3000 rpm.
Finally, the solution was lyophilized to collect magnetic nanosponges.
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Encapsulation Efficiency and Drug Loading Capacity

The encapsulation efficiency and loading capacity of nanoparticles were determined by separation of
magnetic nanosponges from the medium containing free artemisinin by centrifugation at 12,000 rpm for
30min. The amount of free artemisinin in the supernatant was measured by UV-1700 spectrophotometer
(shimadzu, Japan) at 289nm.
Swelling Ratio
The weights of dry magnetic nanosponges were determined after lyophilization. The magnetic nanosponges
were then dispersed in phosphate buffer and allowed to swell for 2 days. It was then centrifuged and the
weight of wet magnetic sponge was calculated.
Particle Size Distribution and Surface Morphology

The mean particle size and its distribution of the sample were determined by using Particle size
analyzer (Malvern Instruments, UK). The surface morphology of the prepared magnetic nanosponges in
clusters was observed in compound microscope at 100 x magnification.
In-Vitro Drug Release

The drug release from the magnetic nanosponge was determined by taking 10 mg of formulation (F-1 to F-3)
in a dialysis membrane bag placed in 50 ml of phosphate buffer solution pH 7.4 at 37°C under magnetic
stirring (250 rpm). After 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7 and 8 hr time intervals, 2 mL of the sample was withdrawn
from the release medium and simultaneously replaced with the same to maintain sink condition [9].
Drug Release Kinetics Study

In order to understand the mechanism of drug release from the nanosponge, release kinetics of formulations
(F1 to F3) was determined by fitting the drug release data with mathematical model equations of zero
order, first order, higuchi, korsmeyer-peppas and hixon crowell.

I.R. spectrum of artemisinin, magnetite, gelatin and magnetic nanosponges of artemisinin were recorded to
identify the possible interactions between active constituent and excipients. The major functional groups
of the drug at wave numbers of 3798.02 cm-1, 3455.56 cm-1, 2579.63 cm-1, 1197.40 cm-1 and of gelatin at
3789.17 cm-1, 3446.20 cm-1, 1636.20 cm-1 were observed in the spectra of the formulation as shown in Fig
1. This shows the compatibility of the drug with excipients during formulation and the absence of chemical
interaction between them.
Figure 1: FT-IR spectra of artemisinin magnetic nanosponges
The compositions for the preparation of artemisinin magnetic nanosponges were shown in Table 1.
During the preparation of magnetic nanosponges, orange porous gel was formed during refrigeration of
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solution containing gelatin and ferrous chloride. There were fine pores with very low particle size in the
formed gel. On addition of ammonium hydroxide solution, the gels immediately turned black indicating
magnetic sponge formation process. The ammonium hydroxide permeates for two days into the gel to form
magnetic nanosponges. Slow permeation facilitates the nano sized particle formation. When sponges were
cross linked with gluteraldehyde, the color changes into brownish black and then lyophilized to get magnetic
nanosponges. The encapsulation efficiency and drug loading capacity of the prepared artemisinin nanosponge
formulation F1, F2 and F3 were found to be 89%, 81%, 73% and 38 %, 33% and 29% respectively. This shows
the decrease in drug loading with increase in concentration of polymer, gelatin. Compared to conventional
coating techniques the drug loading was found to be higher in magnetic nanosponges [8].
Table 1: Compositions of artemisinin magnetic nanosponges
Formulation Artemisinin (mg) Gelatin (g) Ferric chloride Gluteraldehyde (mL) Water (mL)
tetra hydrate (g)
F1 200 2 2 20 100
F2 200 3 2 20 100
F3 200 4 2 20 100
The swelling index of the formulation F1 was observed as 9.1 mL, F2 as 8.8 mL and F3 as 8.4 mL.
This shows that as the concentration of gelatin increases the swelling index decreases due to the decrease in
surface area and contact area. The size of magnetic nanosponges (F1, F2, and F3) was found to be 93 nm,
123 nm and 143 nm and PDI of less than 0.1. Because of its small size, it might show a superparamagnetism.
The gradual increase in particle size was observed for the formulations F-1, F-2 and F-3, which indicates
that increase in polymer concentration increases the size of the nanoparticles. So, it was clearly concluded
that concentration of polymer affects the particle size o f the nanoparticles. The PDI values depict the
monodispersity of the prepared formulations.
Figure 2: Surface morphology of magnetic nanosponges at low and high gelatin concentration
The surface morphology of the swelled magnetic nanosponges was examined under compound
microscope at 100x magnification (Fig 2). The particles were irregular in shape and seen in clusters. The
size of the sponges remains uniform throughout the image. The targeting potential of nanosponges has
been demonstrated by placing the formulation in the magnetic stirrer in presence and absence of magnetic
stirring (rpm). The suspended magnetic nanosponges get attracted towards the center of magnetic stirrer
when the rpm of the magnetic stirrer is raised (Fig 3).
Figure 3: In-vitro targeting potential of magnetic nanosponges at 0 rpm and at 2120 rpm in magnetic stirrer
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Figure 4: In-vitro drug release of magnetic nanosponges formulations
The drug release at 8 hr was observed to be 52%, 65% and 94% for formulations F1, F2 and F3 (Fig 4)
respectively. Generally, the increase in the concentration of gelatin shows increase in the particle size and
decrease in the release rate. But for the prepared magnetic nanosponges the increase in gelatin concentration
increases the particle size with increase in release rates. This pattern may be due to aggregation of the small
particles on application of external magnetic field over the larger particles leading to increase in the size.
From the release kinetics studies, it was observed that the r2 values for all the formulations (Table 2) were
0.9 for first order, higuchi, korsemyer peppas and hixon crowell except zero order equation. The drug
release from the formulations (F-1 to F-3) obey the first order kinetics and drug from the nanosponges
released by diffusion and erosion mechanism and follows non- Fickian diffusion or anomalous transport.
Table 2: Drug release kinetic model fitting data of the magnetic nanosponges formulations
Formulation Regression Coefficient (r2)

Zero order First order Higuchi Korsemeyer Peppas Hixson Crowell
F1 0.894 0.954 0.911 0.982 0.937
F2 0.853 0.960 0.913 0.984 0.931
F3 0.890 0.908 0.988 0.990 0.965
CONCLUSION
The artemisinin magnetic nanosponges were prepared by co-precipitation method by varying the concentration
of the gelatin and characterized for the effective targeting of breast cancer. The FT-IR compatibility studies
showed the absence of chemical interaction between the drug and excipients. The impacts of polymer
concentration on drug loading and in vitro drug release were determined. The particle size of the magnetic
nanosponge was found to be below 150 nm which is found to be suitable for the targeting to the cancer
cells. The kinetic studies revealed that all the formulations follows drug release through diffusion
and anomalous transport. Hence, we postulate that the developed artemisinin-gelatin magnetic nanosponges
possess desirable in-vitro drug targeting potential guided through external magnetic field and the formulation
may be further developed and screened for its in-vivo breast cancer activity.
REFERENCES
1. Cancer dictionary [Online], 2008 Jun 12 [Cited: 2009 Nov 09], Available from: URL: http://www.
cancer.gov/dictionary/?expand=C
2. Breast cancer: Statistics on incidence, survival and screening [Online], 2010 Jan 12 [Cited: 2010 Jan 20],
Available from: URL: http://www.imaginis.com/breast-health/breast-cancer-statistics-on-incidence-
survival-and-screening-2
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3. World Cancer Report, International Agency for Research on Cancer, 2008, Retrieved 2011-02-26.
4. Male Breast Cancer Treatment, National Cancer Institute, 2011, Retrieved 2011-02-26.
5. Understanding breast cancer [Online], 2009 Aug 12 [Cited: 2009 Nov 09]. Available from: URL: http://
www.breastcancer.org/symptoms/understand_bc/what_is_bc.jsp
6. Emerich DF and Thanos CG, The pinpoint promise of nanoparticle-based drug delivery and molecular
diagnosis, Biomol Eng 2006; 23(4):171-84.
7. Langer R, New methods of drug delivery, Science 1990; 249(4976):1527-33.
8. Panyam J and Labhasetwar V, Biodegradable nanoparticles for drug and gene delivery to cells and
tissue, Adv Drug Delivery Rev 2003; 55(3):329-47.
9. Lai H and Singh NP, Oral artemisinin prevents and delays the development of 7, 12- dimethylbenz[a]
anthracene (DMBA)-induced breast cancer in the rat, Cancer Letters 2006; 231(1):43-8.
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Cancer Targeting with Artesunate Magnetic Nanoparticles
Encapsulated with Thermo-Responsive Polymers
Vinoth J., Sharavanan S.P., Senthil Kumar C., Dhinakaran P. and Subramanian N.*
Laboratory for Lipid Based Systems, Department of Pharmaceutical Technology, Anna University,
BIT Campus, Tiruchirappalli- 620 024
Abstract:
In the present study, the artesunate magnetic nanoparticles were successfully formulated using thermo
responsive polymer like poloxamer (S) and evaluated for its in vitro characteristics to assess the systems for
the efficient targeting in to the cancer tissues. The magnetite was prepared by simple precipitation method.
The thermoresponsive polymeric magnetic nanoparticle formulations have been formulated by encapsulating
varying concentration of poloxamer 188 & 407 with chitosan (2:1) along with Artesunate on the surface of the
blank magnetic nanoparticles by ionic-gelation method . The FTIR studies confirmed the compatibility of drug
and excipients in the formulation. The synthesized magnetite and optimized Artesunate magnetic nanoparticle
formulation has 347 nm average particle size with -13.7mV zeta potential and 424 nm average particle size with
-12.0 mV zeta potential respectively. The magnetic susceptibility of the magnetite and optimized formulation
was found to be 70×10-6 and 23×10-6, respectively which showed super paramagnetism. The increase in
polymer concentration decreases the encapsulation efficiency and loading capacity of artesunate in the
formulation. The in-vitro drug release was found to be higher (64% in 36 h) in hyperthermia induced condition
(43ºC) compared to normal body temperature (34% in 36 h) conditions. Kinetic studies revealed that all the
formulations follows drug release through diffusion and anomalous transport. From the results, it was observed
that the developed artesunate loaded thermo responsive polymeric magnetic nanoparticles possessing shown
desirable in-vitro characteristic for drug targeting and delivery to cancer cells guided through external magnetic
field and it has to be screened for its in-vivo breast cancer activity.
Keywords: Artesunate, Cancer, Magnetic nanoparticles and Poloxamer.
INTRODUCTION
Cancer is one of the fatal diseases, in which abnormal cells divide without control and invade the nearby
tissues that spreads to other parts of the body through the blood and lymph systems. It is a major cause of
death worldwide [1]. The various treatment modalities available for cancer treatment therapies are surgery,
chemotherapy, radiation therapy, targeted therapy, immunotherapy, hyperthermia, photodynamic therapy
and laser therapy. The currently available treatments are inefficient in treating cancer as they failed to avoid
cancer recurrence [2]. The major therapy method, cancer chemotherapeutics are also not producing promising
effect due to non-site specificity and the inability to accumulate in cancer tissues at required therapeutic
concentration. This requires a large dose of drug to achieve therapeutic concentration at a diseased site,
which also brings cytotoxicity to normal healthy cells. This situation is particularly critical in case of drugs
having very low therapeutic indices [3].
Artesunate is a water soluble semi-synthetic derivative of artemisinin, has reported to have remarkable
anticancer activity, anti-proliferative activity and anti-antigenic activity [4] in addition to its antimalarial
activity.
Biodegradable magnetic nanoparticles are ideal carriers for targeted delivery of potent cytotoxic
drugs, which are biocompatible, biodegradable, stable and protect drug in physiological condition. The
temperature-responsive polymer encapsulated on the magnetic nanoparticles is designed in such a way;
it tends to shrink/breakup to release the drug at the hyperthermia condition (43°C) effect with the use of
external magnetic field but not in physiological temperature. On successful targeting, the hyperthermia
produced by the magnetic core under external alternative magnetic field, which disturbs the integrity of
drug containing thermo responsive polymeric matrix to release the drug in the controlled pattern by On-
off switch mechanism. The rate and extent of drug release are customized by controlling the temperature
and polymeric composition.
The objective of this study is to develop novel biodegradable thermo responsive nanoparticulate system
containing combination of thermo responsive polymers, (poloxamer 188 & 407 and chitosan) and non-
thermo sensitive polymer chitosan and characterize the thermo-responsive polymer encapsulated magnetic
nanoparticles for selective delivery of artesunate to the target cancerous site and enhance its cellular
transfection.
Thermo responsive nanoparticulate system was developed by mixing various ratios of poloxamers (188
& 407) with chitosan in specific concentration (2:1) by ionic-gelation method and the effect of poloxamer
ratio in the thermoresponsive drug release was studied using methyl orange as model drug. Further, developed
Polymer Encapsulated Artesunate Magnetic Nanoparticles (PEAMNP) is evaluated for particle size,
polydispersity index, zeta potential, surface morphology, magnetic susceptibility, encapsulation efficiency,
loading capacity, drug content, in vitro targeting potential, and in vitro drug release.
Drug-Excipient Compatibility Studies
As a part of preformulation studies, the drug and poloxamer were mixed in 1:1 ratio and stored at 50ºC
temperature for a period of 30 days and the compatibility study was performed using Fourier transform
infrared spectroscopy (FT-IR).
Preparation of Magnetite
Magnetite (Fe3O4) was prepared in nano size using a simple precipitation method. Ferrous chloride tetra
hydrate solution (0.5% w/v) was continuously stirred at 1200 rpm using a mechanical stirrer maintained at
80ºC. Ammonia solution (7M) was added drop wise into ferrous chloride solution until the colour change
was observed from orange colour to a stable black precipitation of iron oxide, washed with distilled water
and dried.
Preparation and evaluation of Poloxamer 188 and 407 for Thermo Responsive Drug
Release Effect
Accurately weighed chitosan was dissolved in 1% acetic acid solution. Then the magnetite, specific ratio
of poloxamer and methyl orange was added as shown in Table 1 under constant mechanical stirring at
2000 rpm at 35ºC. The 0.5% TPP solution was added drop wise for cross-linking to get thermo responsive
polymer encapsulated methyl orange magnetic nanoparticles. Tween 80 was used as stabilizer. The prepared
nanoparticles were evaluated for temperature responsive drug release at physiological temperature (37ºC)
and hyperthermia temperature (43ºC) by suspending nanoparticles in phosphate buffer pH 7.4 at both
temperatures. The release of methyl orange was measured using UV-spectrophotometer at 464 nm.
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Table 1: Composition of thermo-responsive polymer system containing methyl orange
Formulation Chitosan Poloxamer Magnetite Methyl Poloxamer

(mg) 188 (mg) 407 (mg) (mg) Orange (ml) ratio (188:407)
RF1 50 50 50 100 3 (1:1)
RF2 50 33 66 100 3 (1:2)
RF3 50 66 33 100 3 (2:1)
RF4 50 25 75 100 3 (1:3)
RF5 50 75 25 100 3 (3:1)
RF6 50 60 40 100 3 (1.5:1)
RF7 50 40 60 100 3 (1:1.5)
RF8 50 0 100 100 3 (0:1)
RF9 50 100 0 100 3 (1:0)
Preparation of PEAMNP
The polymer encapsulated artesunate magnetic nanoparticles were prepared by adopting the procedure
described for the preparation of methyl orange magnetic nanoparticles.
Surface Morphology, Particle Size Distribution, Polydispersity Index and Zeta potential
The surface morphology of the prepared magnetite and artesunate magnetic nanoparticles were examined
under scanning electron microscope (SEM, Tescan). The mean particle size and its distribution,
polydispersity index and zeta potential of the formulations were determined by using Zetasizer (Malvern
instruments, UK).
Magnetic Susceptibility and Targeting Potential

Magnetic susceptibility of the magnetic nanoparticles was determined using Fugro magnetic susceptibility
meter. The thermo responsive PEAMNP were dispersed in phosphate buffer solution (pH-7.4) in glass
container. Targeting potential was demonstrated by applying an external magnetic field (permanent magnet)
to sides of the glass container. The dispersant was stirred with a glass rod and the time taken by the magnetic
nanoparticles for complete accumulation towards magnet was noted.
Encapsulation Efficiency and Drug Loading

The encapsulation efficiency and loading capacity of nanoparticles were determined by separation of
magnetic nanoparticles from the medium containing free artesunate by centrifugation at 12,000 rpm for
30 min. The amount of free artesunate in the supernatant was measured by UV- 1700 spectrophotometer
(shimadzu, Japan) at 289nm
In vitro Drug Release

Accurately weighed 10mg of formulation (F1) dispersed in 2 ml of phosphate buffer solution pH 7.4 was
taken in a dialysis membrane and placed in 50 ml of phosphate buffer solution pH 7.4. The system was kept
at 37°C or 43°C with continuous constant magnetic stirring (100 rpm). At 30 min, 60 min, 90 min, 120 min
upto 2880 min time interval, 5 ml of the sample was withdrawn and simultaneously 5ml of phosphate buffer
solution pH 7.4 was replaced to maintain sink condition. The amount of artesunate in the release medium
was evaluated after appropriate dilution of sample with 0.1M NaOH solution, heated at 50°C for 30 min.
Then, it was cooled to room temperature and absorbance was measured using UV-1700 spectrophotometer
(shimadzu, Japan) at 289nm.
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Drug Release Kinetic Study

Release kinetic of formulation was determined by fitting the drug release date with the zero order, first order,
Higuchi equation, Korsmeyer-peppas equation and Hixson Crowell mathematical model equations.

Compatibility studies were carried out to study the possible interaction between active constituent and
excipient by FTIR spectroscopy. I.R. spectrum for artesunate, acetic acid soluble chitosan, magnetite,
poloxamer, chitosan, poloxamer, magnetic nanoparticles of artesunate formulation was recorded. Artesunate
was identified by its significant strong absorption band for C-O stretching at 1262 cm-1 and other multiple
C-O stretching bands upto 1262 cm-1, C=O stretching was given strong absorption band at 1561 cm-1, C-H
bending showed by bands at 1453 cm-1, 1368 cm-1 and 1262 cm-1. Absorption band for O-H stretching was
found at 3270 cm-1. The I.R spectrum shows an absorption band at 1779 cm-1 for primary amines N-H bending
vibration. C-H shows its characteristic band at 1380 cm-1. C-O stretching showed absorption band at 1198
cm-1. The I.R. spectrum for magnetite shows its characteristics absorption band for Fe-O at 643 cm-1. The I.R.
spectrum for poloxamer shows its characteristic absorption band at 3533 cm-1 for N-H stretching vibration,
O-H stretching vibration shows characteristic band at 2893, 2739 cm-1 respectively. The blank formulation
of I.R spectrum shows an absorption band at 1645 cm-1 for primary amines N-H bending vibration, C-H
shows its characteristic band at 1390 cm-1. C-O stretching gave absorption band at 1205 cm-1,then magnetite
shows its characteristics absorption band for Fe-O at 568 cm-1, The I.R. spectrum for poloxamer show its
characteristic absorption band at 3652 cm-1 for N-H stretching vibration, O-H stretching vibration shows
characteristic bond at 2358 cm-1respectively. The I.R. spectrum of formulation (F1) as shown in Fig.1(E)
has the characteristic peaks of artesunate at 1262 cm-1, 3270 cm-1, and 983 cm-1 and the characteristic of
poloxamer, chitosan and magnetite. The results of this study indicate that there is no interaction between the
drug and excipients.
Figure 1: FTIR spectrums
The release of methyl orange from the thermo responsive system was observed at 37°C and 42°C.
The data obtained are shown in Fig.2. From this study, the poloxamer 188 and poloxamer 407 in the ratio
1:1.5 was found to be a efficient ratio of the tested thermo responsive polymer system, which released
higher amount of drug at hyperthermial temperature, whereas very low drug release at normal physiological
temperature.
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Figure 2: Effect of poloxamer ratio in temperature responsive drug release
The artesunate magnetic nanoparticles were formulated on the basis of thermoresponsive behavior
of the various prepared systems. According to the model drug release from the polymeric system under
hyperthermia, the four formulations were prepared in varying the concentration of poloxamers. The
composition of PEAMNP was given in Table 2.
Table 2: Composition of thermo responsive Artesunate loaded poloxamer magnetic nanoparticles
Formulation Chitosan (mg) Ratio (1:1.5) Magnetite (mg) Drug (mg)

Poloxamer (188) (mg) Poloxamer (407) (mg)
F1 50 20 30 100 50
F2 50 40 60 100 50
F3 50 60 90 100 50
F4 50 80 120 100 50
The surface morphology of the prepared magnetite and Artesunate magnetic nanoparticles were
characterized using scanning electron microscope (SEM). The photomicrographs are shown in Fig 3 & 4.
The prepared magnetic particles were in the size range of 347-567nm. The surface morphology was nearly
uniform in all particles; appeared in almost spherical shape and some are appeared as cluster due to their
unique ionic property of magnetite. The artesunate loaded magnetic nanoparticles were in the size range of
424nm. The particles were appeared as cluster due to ionic property of magnetite present in the particles.
Figure 3: SEM image of magnetite Figure 4: SEM image of formuation F-1
The average particle size and polydispersity index values of formulation shown in Table 3. The size of
magnetite was found to be 347 nm. The gradual increase in particle size was observed for the formulations
F-1, F-2, F-3 and F-4 with increase in polymer concentration of the formulations. The magnitude of the
potential values gives in indication about the stability of the formulation. The zeta potential values for
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magnetite and formulation are shown in the Table 3. The presence of poloxamer in the nanoparticle itself
contributes towards the stability of the formulations. The Magnetic susceptibility results (Table 3) indicate
that magnetite, blank formulations and artesunate loaded magnetic nanoparticles were having super
paramagnetic property, which is essential for efficient targeting to disease site by external magnetic field
and to prevent accumulation and blockage of micro capillaries by magnetic nanoparticles when the magnetic
field was absent.
Table 3: Evaluation results of magnetite and thermo responsive PEAMNP
S.No. Formulation Average particle size (nm) Polydispersity index Zeta potential Magnetic Susceptibility
1 Magnetite 347 0.671 -13.7 70×10-6
2 F-1 424 0.459 -12.7 23×10-6
3 F-2 479 0.531 -15.5 29×10-6
4 F-3 554 0.512 -18.9 17×10-6
5 F-4 567 0.371 -12.0 76×10-6
The suspended magnetic nanoparticles gets attracted and completely accumulated towards the external
magnet placed on the side of container within 5 minutes (Fig. 5). This proved the targeting potential of the
formulated thermo responsive PEAMNP.
Figure 5: Magnetic targeting efficiency of magnetite
The formulation of thermoresponsive magnetic nanoparticles was possible within some moderate
concentartion of poloxamer and TPP. The increase in concentration of poloxamer in nanoparticles formulation
led to decrease in encapsulation efficiency of artesunate that shown in Table 4. The maximum encapsulation
efficiency (62%) was achieved with very low concentartion of poloxamer 188 (20 mg) and poloxamer 407
(40 mg). The loading capacity of artesunate was related to the concentration of poloxamer and that the
increase in polymer concentration lead to decrease the drug loading capacity. The maximum drug loading
capacity (13.7%) was abserved in formulation F1.
Table 4: Encapsulation efficiency and loading capacity of Artesunate in thermo responsive PEMNP
S.No. Formulation Encapsulation Loading

Efficiency (%) Capacity (%)
1 F-1 62.3 13.7
2 F-2 58.4 10.6
3 F-3 39.1 9.2
4 F-4 31.7 7.1
Percentage of artesunate released from magnetic nanoparticles containing thermoresponsive polymer

and chitosan (F1-F4) in body temperature (37ºC), and hyperthermic temperature (43°C) (Fig 6). Formulation
F1 exerts maximum drug release with 64% in 36 hours. Whereas, the drug release at normal temperature was
found to be less. The artesunate loaded poloxamer magnetic nanoparticles exerts better release in lowest
polymeric concentration.
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Figure 6: Drug release graph for PEAMNP with normal temperature and induced temperature.
Release kinetics of optimized formulation F1 were determined by fitting the drug released data with the
zero order, first order, Higuchi equation, Korsmeyer peppas equation and Hixon Crowell equation. The result
were given in the Table.5.
Table 5: Release kinetics for Normal and Temperature-Induced Formulation-F1
S.No Kinetics equation Regression coefficient (R2)

Normal Temperature Hypertermia Temperature
1 Zero order kinetics 0.781 0.739

2 First order kinetics 0.921 0.885
3 Higuchi kinetics 0.781 0.739
4 Korsmeyer-peppas equation 0.595 0.589
5 Hixon Crowell equation 0.880 0.840
From the release kinetics studies, it was observed that the drug release from the formulation at (37°C and
43°C) obey the first order kinetics and the drug release may be either due to erosion or diffusion of drug from
the system. So, from the drug kinetics studies of thermoresponsive magnetic nanoparticles of artesunate, it
was revealed both normal and temperature induced formulations perform in the same pattern.
CONCLUSION
The main objective of the study was to formulate thermo responsive magnetic nanoparticles for site
specific and targeted delivery of artesunate to breast cancer cells and to avoid the death of normal
cells. The FT-IR studies revealed that the drug and the selected excipient were compatible. Magnetite
was synthesized by simple precipitation method. Magnetite was characterized for its particle size and
targeting potential. The thermo responsive magnetic nanoparticles containing lower concentration
of polymer (1:1.5) ratio showed higher amount of drug release (64%), encapsulation efficiency and
loading capacity. The drug release was found to be higher with temperature induced condition of (43°C)
compared to normal temperature (37°C). Formulation F1 might be suitable for further in-vivo study due
to its small size (373 nm), satisfactory zeta potential (-15.5mV), high magnetic susceptibility (140×10-
6
), high encapsulation efficiency (64%), drug loading (13.3%) and adequate controlled drug release
(64% at 36 hours). Hence, from the obtained results we conclude that the developed artesunate loaded
thermo responsive polymer magnetic nanoparticles can be used as a potential nanocarriers system for the
targeted drug delivery to breast cancer therapy.
308
REFERENCES
1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013 Jan;63(1):11-30.
2. Forrest LF, White M1, Rubin G2, Adams J1. The role of patient, tumour and system factors in
socioeconomic inequalities in lung cancer treatment: population-based study. Br J Cancer. 2014 Jul
29;111(3):608-18.
3. HYPERLINK “http://www.ncbi.nlm.nih.gov/pubmed?term=Steichen%20SD%5BAuthor%5D&c
author=true&cauthor_uid=23262059”Steichen SD, HYPERLINK “http://www.ncbi.nlm.nih.gov/
pubmed?term=Caldorera-Moore%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23262059”
Caldorera - Moore M, HYPERLINK “http://www.ncbi.nlm.nih.gov/pubmed?term=Peppas%20NA%5B
Author%5D&cauthor=true&cauthor_uid=23262059” Peppas NA. A review of current nanoparticle and
targeting moieties for the delivery of cancer therapeutics. Eur J Pharm Sci. 2012 Dec 20;48(3):416-427.
4. Li-Weber M. Targeting apoptosis pathways in cancer by Chinese medicine. Cancer Lett. 2013 May
28;332(2):304-12.
309
Formulation and In Vitro Evaluation of Controlled Release
Tablets of Norfloxacin using Natural Polymers
V. Ganesan1, S.R. Senthilkumar2*
Department of Pharmaceutics, The Erode College of Pharmacy, Veppampalyam,

1
Erode- 638112, Tamil Nadu, India

2
Department of Pharmaceutics, S.B. College of Pharmacy, Annaikuttam, Sivakasi-626130,
Tami Nadu, India
*Corresponding Author
e-mail: 2srsmpharm@gmail.com
Abstract:
The aim of study was to prepare controlled release tablets of norfloxacin using xanthan and guar gum
as natural polymers. Tablets were formulated by direct compression technology employing the polymers
in different concentrations (5, 10, 15 and 20% w/w). The prepared batches were evaluated for hardness,
friability, thickness, weight variation, drug content & swelling index and subjected to in vitro drug release
studies. The pre compression studies results shows that the granules have good flow property. The results of
hardness and friability revealed that all the formulations were having good mechanical strength. In vitro drug
release data were fitted for studying the mechanism of drug release. Swelling index was reported to increase
with both increase in the concentration of gum and the time duration. The in vitro drug release decreased
from 93.56 to 77.92% (F1 to F4) with the increase in polymer (xanthan gum) concentration. Drug release
decreased from 76.43 to 64.89% (F5 to F8) with the increase in polymer (guar gum) concentration.
Keywords: Drug release, Friability, Guar gum, Norfloxacin, Xanthan gum.
1. INTRODUCTION
Oral drug delivery system have the advantages that these are easy to administer, ease of manufacturing and
higher patient compliance. In oral drug delivery systems certain modifications were done to achieve certain
specific objectives, the most profound need of which was to maintain constant drug plasma concentrations
for a certain period of time to reduce the dosage frequency which was achieved by controlled release drug
delivery systems [1]. Despite of the advancement in other drug delivery systems, oral sustain/controlled
release drug delivery systems is dominating the market and have an increased safety and patient compliance
[2]. To control the release of drug from a controlled release dosage forms, polymers are used, which release
the drug in a slow and nearly constant manner to obtain nearly constant peak plasma level. The drug is
release from such type of controlled release matrices occurs by diffusion or degradation [1, 3].
Norfloxacin is a synthetic chemotherapeutic broadspectrum antibacterial agent for oral administration.
Norfloxacin, a fluoroquinolone, is 1-ethyl-6-fluoro-1, 4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline
carboxylic acid. Its empirical formula is C16H18FN3O3 and the structural formula is:
Norfloxacin is a white to pale yellow crystalline powder with a molecular weight of 319.34 and a melting
point of about 221°C. It is freely soluble in glacial acetic acid, and very slightly soluble in ethanol, methanol
and water. Norfloxacin, a fluoroquinolone, differs from non-fluorinated quinolones by having a fluorine atom
at the 6th position and a piperazine moiety at the 7th position.

Norfloxacin (Suchem bulk drug manufacturer, Ahmedabad) was received as a gift sample. Xanthan
gum and guar gum (Dabur India Limited, India), Lactose (Paxmy speciality chemicals, Chennai), Talc
(Merck Limited, Mumbai), Magnesium stearate (Loba chemical pvt ltd Mumbai), PVP (poly vinyl
pyrrolidine) K-30, Iso propyl alcohol, (S.D fine chem. Pvt limited) were commercially procured and
used for this study.
2.1. Preparation of Norfloxacin Tablets

The tablets were prepared by wet granulation technique. Norfloxacin controlled release tablets were prepared
with xanthan gum, guar gum and other additives that are listed in the Table 1.norfloxacin, guar gum or
xanthan gum and lactose were mixed together. Isopropyl alcohol in which PVP K-30, previously dissolved
was added to the above powder mixture and mixed well to form a coherent mass. Then the coherent mass
was passed through sieve no. 16 and the granules were dried at 40° ± 2°C for 2 hours. Dried granules
were passed through sieve no. 20. After the granules were evaluated, magnesium stearate was added to the
granules. Then the lubricated granules were compressed into tablets weighing 400 mg using 12 mm round
flat faced punches in a rotary tablet press (Rimek mini press-1, Model RSB-4, Karnavathi Engineering,
Ahmedabad) to a hardness of 5, 6 and 7 kg/cm2. The compressed tablets were dedusted and evaluated for
various tablet properties [7].
Table 1: Composition of norfloxacin tablets
Ingredients F1 F2 F3 F4 F5 F6 F7 F8
Norfloxacin 200 200 200 200 200 200 200 200
Xanthan gum 20 40 60 80 - - - -
Guar gum - - - - 20 40 60 80
Lactose 170 150 130 110 170 150 130 110
PVP K-30 02 02 02 02 02 02 02 02
Isopropyl alcohol(IPA) Qs Qs Qs Qs Qs Qs Qs Qs
Talc 04 04 04 04 04 04 04 04
Magnesium stearate 04 04 04 04 04 04 04 04
Total 400 400 400 400 400 400 400 400
2.2. FT-IR studies
It was used to study the interactions between the drug and polymer. The drug and polymer must be compatible
with one another to produce a stable product. Drug and polymer interactions were studied by using FTIR
(Shimadzu, Japan model – 8400S) as per the method. IR spectral analysis of pure norfloxacin, guar gum ,
xanthan gum, norfloxacin with xanthan gum, norfloxacin with guar gum were carried out. The peak and
patterns produced by the pure drug were compared with combination of pure drug and polymer [10].
2.3. Measurement of Flow Properties

Flow properties of the dispersion granules were determined by measuring the Carr’s compressibility index
(CI). Bulk density was calculated by measuring the volume of 5g of granules in a 50 ml measuring cylinder.
The cylinder was tapped 100 times manually through a fixed height of 10 cm from the surface, till there was
311
no further reduction in volume of the granules. Tapped density was calculated using the volume obtained
after tapping [2].
Compressibility Index
[Tapped Density – Bulk Density/ Tapped Density] × 100
Optimized dispersion granules were also characterized for angle of repose; it was determined by allowing
the dispersion granules to flow through a glass funnel of internal diameter 10 mm on the horizontal surface.
The height (h) of the heap formed was measured, and the radius (r) of the cone base was also determined.
The angle of repose(θ) was calculated from the following formula
tan θ = h/r
θ = tan- 1 h/r
The tablets were compressed using prepared granules with the help of the compression machine and
evaluated for post compression parameters[4].
2.4. Evaluation of Tablets
Various standards have been set in the various pharmacopoeias regarding the quality of pharmaceutical
tablets. The following standards or quality control tests were carried out on compressed tablets. The
formulated tablets were evaluated for the following physicochemical parameters [4 – 6].
Physical tests for the prepared tablets

Ten tablets from each formulation were taken for measurement of average weight. Hardness of the norfloxacin
tablets were evaluated by using hardness tester (Pfizer) and mass determination was performed for twenty
Bulk density tablets from each batch and uniformity of weights were calculated. Friability of the norfloxacin
tablets were determined by first weighing 10 tablets after dedusting and placing in a friability tester (Roche
friabilator, Pharmalabs, Ahmedabad, India),which was rotated for 4 min at 25 rpm. After dedusting, the total
remaining weight of the tablets were recorded and the percent friability was calculated. The drug content of
the prepared tablets of each batch was determined in triplicate [5-8].
Swelling Index
The percentage water uptake of the formulations (F1-F8) were calculated (Table 5). The results show swelling
index increases through raising polymer concentration as well as time duration. Through raising polymer
concentration from (F1-F8), swelling index after12hr was increased from 35.12 to 67.90% and by increasing
time duration from 2 to 12hr in formulation F8. Swelling of the polymer stands crucial for the development
of sound matrix for retarding the release of drug from the formulation [8].
Dissolution rate studies

Dissolution is the process by which a solid solute enters a solution. In vitro drug release of the samples
were carried out using USP – type I dissolution apparatus [paddle type]. The dissolution medium used
was 900 ml of buffer with pH – 7.4 were placed into the dissolution flask maintaining the temperature of
37± 0.5° C. One norfloxacin tablet was placed in each flask of dissolution apparatus. The apparatus was
allowed to run for 10hrs. The sample measuring 10 ml was withdrawn after every 30 minutes interval
up to 8 hrs using 10 ml pipette. The fresh dissolution medium [37°C] was replaced every time with the
same quantity of the sample. Collected samples were suitability diluted with pH 7.4 and analyzed in UV
double beam spectrophotometer at 278 nm using pH 7.4 as blank. The % drug release was calculated and
its graphically illustrated in fig 1 & 2 [9].
312
Table 2: Pre compression studies
Formulation Parameters
code Angle of repose(θ) Bulk density Tapped density Carr’x Hausner ratio
(gm/mL) (gm/mL) index(%)
F1 23.34 ±0.63 0.576 ±0.08 0.643 ±0.15 10.41 1.11 ±0.35
F2 21.57 ±0.42 0.589 ±0.09 0.654 ±0.27 9.93 1.11 ±0.83
F3 21.52 ±0.27 0.532 ±0.03 0.683 ±0.05 22.10 1.28 ±0.04
F4 22.23 ±0.95 0.578 ±0.09 0.689 ±0.31 16.11 1.19 ±0.13
F5 25.12 ±0.76 0.569 ±0.01 0.721 ±0.05 21.08 1.26 ±0.17
F6 24.89 ±0.64 0.589 ±0.09 0.734 ±0.01 19.75 1.24 ±0.06
F7 23.13 ±0.54 0.592 ±0.07 0.743 ±0.05 20.32 1.25 ±0.86
F8 26.27 ±0.44 0.657 ±0.05 0.764 ±0.06 16.28 1.16 ±0.63
Table 3: Post compression studies
Formulation Hardness Friability (%) Thickness (mm) Weight variation Content

code (kg/cm2) (g) uniformity(%)
F1 6.4±0.23 0.34 4.25± 0.01 398.02±1.4 99.91±0.31
F2 6.8± 0.15 0.31 4.32 ±0.05 397.02±2.3 99.32 ±0.17
F3 6.7±0.34 0.28 4.19± 0.12 395.02±1.9 98.91±0.61
F4 6.9±0.27 0.30 4.21± 0.18 402.02±3.3 98.67±0.56
F5 7.2±0.29 0.27 4.26± 0.08 396.02±1.4 99.34±0.42
F6 6.8±0.65 0.37 4.31± 0.34 398.02±3.9 97.34±0.28
F7 7.0±0.25 0.25 4.45± 0.26 399.02±2.5 99.38±0.91
F8 6.9±0.58 0.33 4.23± 0.15 398.02±1.8 99.25±0.64
Table 4: In vitro release study
S.No Time Formulation Code

F1 F2 F3 F4 F5 F6 F7 F8
1 30 11.89 10.12 12.23 10.89 13.41 11.16 10.17 3.91
2 60 15.87 14.85 13.62 15.97 31.72 23.82 30.68 8.62
3 90 18.92 18.98 17.45 21.86 30.61 26.58 31.79 11.07
4 120 22.45 22.13 20.86 23.74 36.49 31.32 35.97 15.09
5 150 27.12 24.87 22.97 30.56 39.25 36.45 36.66 20.38
6 180 31.23 27.58 24.76 31.84 41.71 39.27 38.72 24.99
7 210 38.90 31.42 28.31 36.54 45.26 43.67 45.38 35.50
8 240 45.34 34.27 30.76 38.81 46.59 45.82 46.56 38.97
9 270 49.58 39.28 34.68 41.78 51.47 48.23 52.15 39.50
10 300 52.71 45.90 40.03 43.83 56.81 57.20 58.32 42.74
11 330 57.23 51.25 44.70 45.84. 58.25 61.29 60.21 46.36
12 360 65.78 58.36 48.93 48.25 59.02 63.81 62.99 50.19
13 390 74.67 62.95 56.73 51.33 63.80 65.75 67.70 51.36
14 420 79.59 67.47 59.85 54.87 67.57 67.62 58.72 56.43
15 450 83.86 72.58 64.92 64.90 68.13 69.33 58.42 59.87
16 480 86.75 76.56 67.84 70.49 73.72 71.86 63.91 62.32
17 510 93.56 82.41 76.42 77.92 75.85 76.43 65.34 64.89
313
Figure 1: Effect of drug release with xanthan gum Figure 2: Effect of drug release with guar gum
Table 5: Swelling index of various formulations
Formulation code Swelling index

2h 4 8 12
F1 20.08 36.32 46.23 52.46
F2 23.89 39.35 45.87 56.78
F3 22.56 41.74 47.32 59.32
F4 23.92 43.26 51.56 60.41
F5 25.67 42.18 54.89 62.19
F6 28.83 46.32 53.25 63.67
F7 32.47 49.74 57.85 66.74
F8 35.12 50.75 60.14 67.90
The granules of all the formulations were evaluated for angle of repose, bulk density, tapped density,
compressibility index and Hausner ratio. The angle of repose was found to be 22.23 ±0.95 to 26.27 ±0.44.
It indicates that granules have a good flow property. The bulk density and tapped density was found to be
in the range of 0.532 ±0.03 to 0.657 ± 0.05 g/cm3 and 0.643 ±0.15 to 0.764 ±0.06g/cm3 respectively. The
compressibility and Hausner ratio was found to be 9.93 to 22.10 and 1.11 ±0.35to 1.28 ±0.04 indicating good
flow character of the granules (Table 2). All the results were within the prescribed limits.
The hardness of the tablets for all the formulations were in the range of 5-7 kg/cm2. The uniformity
weights of twenty tablets of all the formulations were within 5% deviation. The friability of all the formulation
was less than 1%. Drug content of all the formulations were found to be in the range of 96 to 99 % (Table 3).
All the results were within the prescribed limits.
The FT-IR studies showed that N-H stretching, C-H stretching, C-O stretching, C-H bending, O-H
deformation, C-H out of plane bending of pure norfloxacin and norfloxacin with guar gum and norfloxacin
with xanthan gum were almost in the same region of wave number. It showed that there was no significant
interaction between the drug and polymer and they are compatible with each other.
The results of the in vitro release study for all the formulations are shown in table 4. At the end of
10 hours the cumulative percentage drug release for the formulations F1 to F8 was found to be 93.56,
82.41, 76.42, 77.92, 75.85, 76.43, 65.34 and 64.89 respectively. Among the eight formulations, F-8 showed
prolonged drug release. An increase in the compression force increases the hardness and the apparent density
of the tablet, thereby reducing the matrix porosity in the tablet. The release rate decreases with increase in
compression force. The drug release was found to be faster at lower compression force than at higher ones.
The controlled drug release may also be due to increased proportion of polymer.
314
CONCLUSION
Controlled release tablet containing norfloxacin can be prepared successfully by using direct compression
method. From the above observations it is concluded that slow and controlled release of norfloxacin over
a period of 12 hours was obtained from formulation (F1-F8). Use of natural hydrophilic polymer like guar
gum was successful in the formation of matrix and at the same time it is effective in retarding the drug release
compared to xanthan gum. Among all the formulation, F-8 shows that 64.9% of drug release at the end of
12 hours. The cumulative percentage drug was decreased by increase in polymer concentration. Thus, the
proposed formulation F-8 can be successfully used. It can be concluded that the release of norfloxacin can
be effectively controlled by using polymer like Guar gum.
REFERENCES
1. Shah SU, Shah KU, Rehman A and Khan GM. Investigating the in vitro drug release kinetics from
controlled release Diclofenac Potassium-Ethocel matrix tablets and the influence of Co-excipients on
drug release patterns. Pak J Pharm Sci. 2011; 24: 183-192.
2. Lakshmana PS, Shirwaikar AA, Shirwaikar A, Ravikumar G, Kumar A, et al. Formulation and evaluation
of oral sustained release of Diltiazem Hydrochloride using rosin as matrix forming material. Ars Pharm,
2009; 50: 32-42.
3. Cleary GW. Transdermal Drug Delivery, Cosm Toilet. 1991; 106: 97-107.
4. Pharmacopoeia of India, Ministry of Health and Family Welfare, New Delhi; Govt. of India Controller
of Publications; 1996.
5. Manikandan M, Kannan K, Selvamuthukumar S and Manavalan R. Formulation development and
evaluation of Emtricitabine and TenofovirDisproxilFumarate Tablets. Int J Drug Dev& Res. 2012; 4(1):
247-256.
6. Kannan K, Ramya Krishna, Manikandan M, Selvamuthukumar S and Manavalan R. Development and
Evaluation of Valsartan Film Coated Tablets, J Pharm Sci& Res.2012; 4(6):p. 1866 - 1871.
7. Rawlins EA. Tablets and Capsules, In: Bentleys, Text book of Pharmaceutics. New Delhi: All India
Traveller Publishers. 2006. p. 234-310.
8. Swati Jagdale, Mahesh Gattani, DhavalBhavsar, BhanudasKuchekar and AniruddhaChabukswar.
Formulation and evaluation of chewable tablet of levamisole, International, J Res Pharm Sci. 2010;
1(3): 282-289.
9. Rajalakshmi G, Vamsi CH,Balachandar R and Damodharan N. Formulation and evaluation of diclofenac
potassium effervescent tablets, Int J Pharm & Bio Res. 2011; 2(4): 237-243.
10. Pathra CH.N, BhanojiRao MK, Yadav KS and Prakash K. Influence of some cellulose ethers on
the release of propranolol hydrochloride from guar gum matrix tablets, Ind J Pharm Sci. 2004; 66:
636 – 641.
315
Exploration of Quail’s Egg Lecithin in Development and
Evaluation of Novel Celecoxib Loaded Lecithin Organogel
S. Balaguru, Ramya Devi D and B.N. Vedha Hari
Department of Pharmaceutical Technology, School of Chemical and Biotechnology, SASTRA University,

Thanjavur-613401, Tamil Nadu, India.
Corresponding e-mail: vedhahari@scbt.sastra.edu
Abstract:
The main purpose of this research work is to isolate and explore lecithin from Quail’s egg and development
of Pluronic lecithin organogel incorporated with Celecoxib drug as topical drug delivery systems. Lecithin
Organogel and combination of Pluronic lecithin organogel is a semi-solid gelled matrix composed of lecithin,
Pluronic F-127 polymer, n-Hexane and water as a polar solvent which are thermodynamically stable, and
biocompatible in nature. Celecoxib drug loaded in this organogel is a Sulphonamide Non-Steroidal Anti-
Inflammatory drug widely prescribed for rheumatoid arthritis and pain management. Firstly lecithin
macromolecule was successfully isolated from quails egg as an novel source and using this Quails egg lecithin,
Celecoxib incorporated Pluronic lecithin organogel was developed and it was explored, characterised by well
established techniques and formulated organogel formulations were subjected to evalvations like Microscopic
studies, FTIR, DSC, and dissolution release studies. Microscopic images showed net like structure formation
that confirms the Organogel formation. It was also elegant in appearance and non irritant to skin; instrumental
analysis such as FTIR shows the compatibility and stability of the drug with Quail egg lecithin and DSC-TGA
showed change in melting point of the compound that indicates the solid state modification of the compound.
The in-vitro drug release study with dialysis membrane and ex-vivo skin permeation study with goat skin
up to 6 hrs showed that Celecoxib drug from quail egg Pluronic lecithin organogel released in a sustained
manner. From the above studies, we could observe that lecithin isolated from Quail’s egg was compatible in
formulation both physically and chemically as a carrier for drug delivery.
Keywords: Lecithin, Organogel, Quails, In vitro, Ex-vivo skin permeation studies.
INTRODUCTION
Drug delivery system is the delivery of various drugs to the targeted site using numerous carriers, through
oral, parental, rectal, transdermal routes etc. All among these transdermal routes of delivery has gained
much importance and demands because of their highlighted properties which include creams, pastes, gels,
plastizers etc [1]. Gels may be defined as an intermediate state of matter, comprised of two components, one
of which is a solid act as the gelator molecule form a three-dimensional networked structure, which helps
in immobilising the loaded drug by the addition of either polar or non polar solvent component to them [2].
Based upon the solvent concentration gels get differentiate into hydrogel or Organogel.
In general, Organogels are semi-solid systems in which a three-dimensional network of gelator molecules
are aggregates immobilise an organic liquid continuous phase, typically a non-polar solvent [3]. They consist
of an organogelators (lecithin and Pluronic F-127 polymer) compounds in them form a cross-linked structure
either by physical or chemical interactions, thereby immobilising the organic phase within the network. Molecular
interactions such as hydrogen bonding, dipolar interactions are responsible for the Organogel structure [4].
Organogel is novel formulation techniques which has attracted the importance in topical route of delivery of drugs,
over the availability of other traditional topical formulations [5-6]. Advantages of using lecithin in preparing gel are
that they are zwitter ionic phospholipids, biocompatible nature, and doesn’t harm the stratum cranium of the skin
gives better penetration of drug. And Pluronic F-127 is a copolymer of polyoxyethylene and polyoxypropylens,
which are also called as poloxamer with a major compound of ethylene oxide makes it hydrophilicity. They are
freely soluble in cold water and at low temperature (4-5°C) they exhibit more as solution form and turns into a
gel when reaches body temperature. Pluronic polymers are non-toxic, biocompatible and act as good penetration
enhancer without arming the skin layers. These properties made the use of Pluronic polymer in preparation of
organogel loaded with various medicaments [7].
Main highlight of preferring Organogel in a topical formulation is that drugs of both hydrophilic and
lipophilic nature can load in them, and they have a good viscoelastic, thermally stable properties and are used
as matrix delivery of drugs [8]. They are mainly used in targeted drug delivery especially bioactive agents,
NSAID drugs and other components. And Celecoxib drug selected here is a sulfonamide class selective
NSAID drug of COX-2 enzyme inhibitor prescribed for arthritis, muscle cramps and pain [9]. This makes
the organogel formulation as a proposed lipid based carrier system for the delivery of various medicaments
through topical route worldwide [10].

Materials
Quail eggs were purchased from Aishwarya quail farm Pondicherry for lecithin isolation. Celecoxib drug was
obtained as gift sample from Glukem Pharmaceuticals Company, Hyderabad, India. And all the chemicals,
reagents used in this process were of analytical grades.
Methodology
Pluronic lecithin organogel preparation
Macromolecule compound lecithin from quail egg were isolated using singleton gray procedure by chemical
extraction method, and it was stored in the deep freezer at (-20°C) until use. PLO formation takes place by
two steps, preparation of the organic phase and the aqueous phase separately [11]. Organic phase is prepared
my mixing calculated amount of lecithin in n-hexane organic solvent, and Celecoxib is dissolved few drops
of methanol are allowed to stand overnight for complete dissolution. And aqueous phase is prepared by
adding Pluronic F127 polymer in cold water and seen that complete dissolution takes place [12].
Later addition of aqueous phase to lecithin organic phase under controlled stirring leads in the formation
of three dimensional networks of reverse micelles due to the interaction of hydrophilic molecules of lecithin
with the polar phase confirms the Organogel formation [13]. Using this procedure eight formulation of
organogel was prepared with different ratios of Pluronic polymer to lecithin concentrations and it are
tabulated in Table 2.
Table 1: Formulation variables of organogel developed using the lecithina, pluronic and combination of lecithin-
pluronic
S.No Ingredients F1 F2 F3 F4 F5 F6 F7 F8
1 Lecithin(mg) 500 750 1000 ----- ----- ----- 250 500
2 Pluronic F-127(% ----- ----- ----- 20 25 30 20 20
W/V)
3 Drug(mg) 10 10 10 10 10 10 10 10
4 n-Hexane(ml) 2.5 3.25 5 1 1 1 1.5 2
5 Water(µl) 81 81 81 3ml 3ml 3ml 81 81
6 Rose oil(µl) 50 50 50 50 50 50 50 50
7 Methanol(ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
317
Evaluation Studies for organogel

Organoleptic properties of organogel
The formulated PLO and LO were subjected to various physical parameters like pH, appearance, colour,
grittiness, greasiness, stickiness, ease of application and skin irritation studies. This skin irritation check of
the formulations was done on two human volunteers by applying small volume of gel on 5 square cm area
of the dorsal surface of the skin [14].
Microscopic Evaluations
The photo microscopic images were carried out for the formulated PLO and quail LO organogel incorporated
with drug. Images were taken with high resolution camera attached with the microscope at different
magnifications using 5X, 10X, 40X lens. Glass slides were properly clean, dried and small quantity of
sample was placed and kept for evaluations, light rays were focussed and images were captured. Primarily
gel formation was confirmed with the net like structure obtained.
Fourier Transform Infrared Spectroscopy (FT-IR)

Fourier Transform Infrared Spectroscopy was performed to find interactions and compatibility between the
drug and the excipients used. Analysis was performed for crude lecithin, pure drug, and drug loaded LO,
PO, PLO using ATR techniques, which was based on the aspect of measuring the intensity of internally
reflected beams from the samples. Analysis was carried out by placing 0.5 ml of liquid sample over KBr
material made into a thin film and it was placed in between the two IR transparent windows for total internal
reflections and all the samples were scanned in the wave number range of 4000cm-1 to 400cm-1 and the
output spectra obtained were recorded [15].
Differential Scanning Calorimetry Analysis (DSC)

DSC-TGA analysis was performed simultaneously to find the exothermic and endothermic changes taking
place in the sample with respect to rising in temperature. DSC gives information like glass transition
temperature, recristallization and melting point of the sample; whereas TGA helps in finding the weight loss of
the sample with respect to time and temperature increase. They also help in determining the thermal stability
and degradation nature of the sample as a function of heat. This process was carried out for crude lecithin,
pure drug, and drug loaded PLO and LO. It was carried out by placing 3-8 mg of sample in alumina pan under
nitrogen (N2) atmosphere as a carrier gas with flow rate of 30 ml/min on a scanning rate of 10°C/min to 500°C/
min. Thermograms of sample were obtained with distant peaks with respect to temperature [16].
In- vitro dissolution study

To find the drug release profile of formulated organogels dissolution studies was carried out using USP
XXII type I rotating basket type dissolution test apparatus using dialysis membrane at 37± 0.2°C at a speed
of 50 rotations per minute. The membrane was tied with a tube of 10mm diameter and a known quantity of
formulations were placed in the tube and it was kept inside the basket with a compendial dissolution media
of 100ml distilled water and 1% Sodium Lauryl Sulphate (SLS). At regular time intervals 5 ml of samples
were taken, and it was replaced with a fresh media. Later all the aliquots of samples were analyzed using UV/
Visible spectrophotometer at 254 nm using lecithin solution or dissolution media as blank.
Ex-Vivo Skin permeation study

The Ex-Vivo study was performed with the help of Keshary Chein type diffusion cell with the help of
goat skin received from Thanjavur local market. This system comprised of donor and a recipient cell with
a capacity of 5 ml and 20 ml respectively. 20 ml of compendial media 1% SLS was filled in the receptor
compartment and the setup over with the completely cleaned, hair removed and defatted goat skin layer
318
in between the donor and the recipient chamber in order the mimic the human transdermal skin condition.
Setup was placed over magnetic stirrer, and it was maintained at 37°C. 50µg quantity of formulation was
placed onto the surface of the skin and process was continued for 6 hours. Samples were withdrawn at
regular intervals of time, and equal volume of fresh media was replaced simultaneously. All the aliquots
were analysed in UV-Vis spectrophotometer and permeability coefficient was obtained from cumulative
percentage of drug permeated through skin vs time.
Stability studies
The formulated quail LO and PLO were stored at room temperature of 25°C and were observed at regular
intervals for any destability nature like colour, pH, odour, microbial contamination etc for a period of 1
month [17].

Organoleptic properties of organogel
Through the visual observations various physical parameters like colour, grittiness, skin irritation, and stickiness
were evaluated and sorted out in the table. Yellowish colour of the LO organogel was due to the presence
of fatty compound phospholipid in the formulations. Observations show that the formulated organogel does
not show any irritation sensation on the surface of the skin and suitable in nature. This contributes that the
formulated organogel are pharmaceutical acceptance and biocompatible in nature (table 2).
Table 2: Organoleptic properties of formulated organogel developed using the lecithina, pluronic and combination of
lecithin-pluronic
S.No Formulations Colour Greasiness Ease of Application Stickiness Skin Irritation
1 F1 Yellow No Yes Yes No
4 F4 Off White No Yes Yes No
7 F7 Off yellow No Yes Yes No
8 F8 Off yellow No Yes Yes No
Microscopic Evaluation
The photon microscopy images of the formulated PLO and LO were shown in figure 2. The thread or net
like structure formed in the formulations confirms the formation of organogel, due to the interactions of
hydrophilic head portion of lecithin molecules with a polar solvent addition. Three dimensional net like
reverse micelles in the formulation starts to disappear and clumps together as time passes, due to evaporation
of methanol and n- hexane organic solvents in them. Microscopic images of the proposed formulations were
shown in figure 1.
319
Figure 1: Microscopic images of Formulated Organogel-(2a, 2b, 2c, 2d, 2e – lecithin organogel (LO), Pluronic
organogel (PO) and Pluronic-lecithin organpgel (PLO) with plain and drug loaded ) respectively
FT-IR Spectroscopic Analysis

FTIR spectra were recorded for the pure drug, crude lecithin and selected formulations of LO, PO and PLO
were shown in figure 2&3 respectively. All the Spectral shifts of the proposed formulations with pure drug
were compared. Chemical shift peaks of the pure drug in the range of 2927.79, 1446.64, 1347.86, 1297.51 and
1103 cm-1 shows the presence of alkyl (C-H), aromatic(C=C), alkane (C-H), amine (C-N) and alkyl halide
functional group present in them respectively. It was observed that in the selected formulations of (F1, F4 and
F7) peaks appears to be similar in the same range with minor deviations of 2924.49, 1458.27, 1352.89, 1298.61,
and 1102.9 cm-1 respectively proves that there is no interaction between the drug and the excipients used in the
formulation preparations and finds that there is no compatibility between the molecules.
Figure 2: FTIR spectra of pure celecoxib drug
320
Figure 3: Combined FT-IR spectra of prepared formulations of lecithin organogel (LO), Pluronic organogel (PO) and
Pluronic-lecithin organpgel (PLO)
TG-DTA Analysis
The TG-DTA analysis for crude lecithin, pure drug, and selected formulations of PO, LO, PLO (F1, F4,
F7) was performed and output thermogram were recorded which elaborates the thermal properties of the
sample.56 DSC spectral peaks of the pure Celecoxib at 162.39°C and 320.65°C shows the melting point and
decomposition point of the drug which was confirmed from the TGA thermogram. Spectral datas of curd
lecithin and peaks at 75.24°C and 305.53°C shows the melting point and complete decomposition point of
the sample respectively. The DSC spectral peak data of LO, PO and PLO of selected formulation (F1, F4, F7)
shows peak at 57.27°C, 67.13°C and 59.80°C, which indicates the melting point of Pluronic F-127 polymer
and initial melting of lecithin fractions in the formulations respectively. The melting point of the drug in the
formulations seems to be decreasing from 162.39°C to 139.11°C, 137°C and 147.63°C correspondingly in
the formulations indicate the disturbance in the crystalline nature of the drug. Thermograms of pure drug,
crude lecithin, and proposed formulations of PLO, PO, LO where shown in figure4, 5&6 respectively.
Figure 4: Thermogravimetry-Diffrential thermal analysis (TG-DTA) thermogram of pure Drug
321
Figure 5: Thermogravimetry-Diffrential thermal analysis (TG-DTA) thermogram of Thermogram of Crude

lecithin
Figure 6: Thermogravimetry-Diffrential thermal analysis (TG-DTA) thermogram of Thermogram of formulated

Organogel; lecithin organogel (LO), Pluronic organogel (PO) and Pluronic-lecithin organpgel (PLO)
In Vitro drug release study

The comparative percentage release profile of all the eight formulations in both 1% SLS media and
phosphate buffer (pH – 6.8) were in shown in figure 7&8 respectively. The percentages of drug released
were calculated using the absorbance value obtained from a sample taken in regular intervals of time and
analysed in UV-Vis spectrometer. It was observed that the release rate of drug from all the formulations
after 6 hours study was found to be in the range of 65 -90 %. The formulation with low concentration of
lecithin (F1) and Pluronic (F4) with n-hexane organic solvent showed maximum release than compared
to increase in concentration of polymers, this is due to the lipophilic nature of lecithin and higher gelling
property of pluronic F-127 polymer. It was observed that organogel in combination with Pluronic and
lecithin (F7) also a showed better release than the other two formulations. At the end of 6 hour’s study,
results showed that the majority of the formulations exhibits more than 60% of drug release showed that
the release is to be in a sustained manner.
322
Figure 7: Comparative in-vitro % drug release profile of organogel in 1% SLS media
Figure 8: Comparative in-vitro % drug release profile of organogel in phosphate buffer (pH6.8)
Ex-Vivo Skin permeation Study

The percentage drug release of the optimized formulation (F1 and F7) through goat skin were calculated
using 1% SLS as compendial media. Release data revels that PLO shows higher release than compared to
lecithin organogel this is due to combinable effect of lecithin as natural penetration enhancer which alters
the lipid bilayer of the skin and the effect of Pluronic F-127 polymer that posse’s amphiphilic nature, good
solubilising property. This shows that Pluronic in combination gives a better release of drug.
Figure 9: Comparative Ex –vivo % drug release profile of Lecithin organogel and Pluronic-lecithin organpgel
323
Stability Studies
The optimized organogel was stored at room temperature of 25°C were found stable without any change
in colour and integrity of the formulations. And was free from microbial contamination this is due to
the presence of organic solvent, which indicates that there is no undesirable change in the formulated
organogel.
CONCLUSION
The macromolecular compound lecithin from the quail’s egg was isolated successfully, and it was explored,
characterised using well established techniques both quantatively and qualitatively by TLC, GC-MS, NMR
spectroscopy and FTIR spectroscopy. The extracted semi-solid mass lecithin seems to have compound of
phospholipid and fatty acids esters which was proved by TLC plate method using soya lecithin as standard.
NMR spectroscopy reveals the presence of carbon and hydrogen containing compounds in lecithin that
forms bonding between molecules during gel formation mechanism. The biocompatible and optimized
formulations of Lecithin Organogel, Pluronic Organogel and combination of Pluronic Lecithin Organogel
were prepared using the extracted quail’s lecithin and Pluronic F-127 polymer. Microscopic images show
the net like micelles formation that confirms the formation of organogels. The in-vitro dissolution study and
ex-vivo skin permeation study justifies the use of lecithin as a lipid based carrier molecule in topical delivery
of drugs. Also lecithin in combination with Pluronic F-127 polymer showed a slight increased release of drug
by following sustained release mechanism.
ACKNOWLEDGEMENT
Authors are thankful to the management of SASTRA University for providing the infrastructure facilities
and support.
REFERENCE
1. Srikonda Venkateswara Sastry and Janaki Ram Nyshadham, Recent technological advances in oral drug
delivery- a review, Pharmaceutical Science and Technology Today, 2000; 3(4): 113-150.
2. Almdal, Towards a phenomenological definition of the term ‘gel’, Journal of Pharmaceutics, 1993; 1:
5-17.
3. Sarath G and Chandra Reddy, Organogels - A Review, International Journal of Pharmacy & Technology,
2010; 2(4): 584- 602.
4. Schurtenberger P and Scartazzini R, Structural and dynamic properties of polymer like reverse micelles,
Jou Phys Chem., 1990; 94: 3695-3701.
5. Costas Kaparissides, Sofia Alexandridou, Katerina Kotti and Sotira Chaitidou, Recent Advances in
Novel Drug Delivery Systems, Azojono - Journ of nanotechnology online, 2010; 3:5-12.
6. Parthasarathi, Bhattacharjee and Sandeep Kumar, An overview of Novel Drug Delivery Systems
(NDDS), ISSN: 0974-6943.
7. Rajiv Kumar and Om Prakash Katare, Lecithin Organogels as a Potential Phospholipid-Structured
System for Topical Drug Delivery: A Review. AAPS Pharm Sci Tech. 2006; 6 (2): 1-12.
8. Willimann H, Walde R and Luisi P.L., Lecithin organogels as matrix for transdermal transport of drugs,
Journal of Pharmaceutical Science, 1991; 81: 871-874.
9. Hadgraft J, Plessis J and Goosen C., The selection of non steroidal anti-inflammatory agents for dermal
delivery, International Journal of pharmacy, 2000; 207: 31–37.
324
10. Prausnitz M.R, Mitragotri S and Langer R., Current status and future potential of transdermal drug
delivery, Journal of Drug Delivery and Formulations, 2004; 3:115-124.
11. Sahoo SS, Bhattacharya C, Sagiri S.S, Jain K, Pal K, Ray S.S and Nayak B, Organogels: Properties and
Applications in drug delivery, Designed monomers and polymers, 2011; (4): 95–108.
12. Varsha Agrawal, Vandana Gupta, Suman Ramteke and Piyush Trivedi, Preparation and Evaluation of
Tubular Micelles of Pluronic Lecithin Organogel, AAPS Pharm Sci Tech., 2010; 11(4): 1718–1725.
13. Mandal Surjyanarayan, Mandal Snigdha S and Sawant Krutika K, Lecithin Stabilized Organogel:
Design and Development for Topical Application of Clobetasol Propionate, International journal of
Pharmaceutical Technology, 2010; 2(2): 1133-1138.
14. Swapnil P, Thorat. Formulation and in-vitro evaluation of lecithin (soya and egg) based Aceclofenac
organogels, Journal of Pharmacy research, 2010; 3(6):1438-1441
15. Dantu AS, Ramya Devi D and Vedha Hari BN, Enhancement of Solubility of Nimesulide in the Presence
of Polymer with Milling Technique, J Pharm Sci & Res. 2012; 4(9):1907-1914
16. Ibrahim S.F., Thermal Analysis and Characterization of Some Cellulosic Fabrics Dyed by a New Natural
Dye. International journal of Chemistry, 2011; 2 (3): 1-10.
17. Zhongqi He and Jingdong Mao, Characterization of Plant-Derived Water Extractable Organic Matter by
Multiple Spectroscopic Techniques, Biol Fertil Soils, 2009; 45:609–616
325
Development of Response Surface Methodology for
Tenofovir Disoproxil Fumarate Microparticles using
Solvent Evaporation Method
Fathima A, Vedha Hari BN and Ramya Devi D*
Department of Pharmacy, School of Chemical and Biotechnology, SASTRA University, Thanjavur- 613 401,
Tamil Nadu, India
Corresponding e-mail: *ramya@scbt.sastra.edu
Abstract:
Tenofovir disoproxil fumarate is an antiviral agent specifically inhibiting the nucleotide reverse transcriptase
enzyme of human immune deficiency virus subsequently being used for management of its infections. Its
trivial and substandard properties are low bioavailability (25%) and poor solubility in water. To boost up
the probable in-vivo availability of the drug, a continued release dosage form is developed using response
surface methodology (RSM) and the release kinetics is studied. The desired microparticles are prepared
by emulsion solvent evaporation process with ethyl cellulose as release retarding agent at different ratios
of drug with respect to the polymer such as 1:1.5, 0.75:2.5, 0.75:2, 1:2.5, 1:2, 0.75:1.5, 0.5:2, 0.5:1.5 by
using 32 factorial design (RSM). Various characterization studies are performed for all the nine formulations
TEF-1, TEF-2, TEF-3, TEF-4, TEF-5, TEF-6, TEF-7, TEF-8, and TEF-9. The particle size (27.73 µm - 56.6
µm), drug content (23.5 - 50.18%), drug entrapment efficiency (11.75 - 37.63%) and in-vitro drug release
(52.28 - 86.34%) are found to show satisfactory results. The stability of the drug in the formulation is proved
by the infrared spectra and diffraction scanning calorimetry. The morphology of the samples is viewed in
optical microscope that showed irregular shaped microparticles with smooth surface. TEF-6 has maximum
entrapment efficiency and significant drug release than the other formulations. The records obtained from
in-vitro dissolution studies are en-suited to diverse kinetic models like Higuchi, Peppas, Hixon, zero order
and 1st order kinetics, and the release is found to be diffusion controlled and the statistical evaluation shows
significant (P<0.01).
Keywords: Microparticles, Tenofovir disoproxil fumarate, Ethyl cellulose, Factorial design.
INTRODUCTION
Tenofovir disoproxil fumarate is an antiviral therapeutic agent, which prevents human immune deficiency
virus (HIV) cells from multiplying in human body, but is not a cure for HIV (or) AIDS [1-3]. Tenofovir
DF has low bioavailability (25%) and poor solubility in water and freely soluble in methanol, very slightly
soluble in dichloromethane. Tenofovir DF used in the combination with other drugs to treat HIV infections,
and it belongs to a class of nucleoside reverse transcriptase inhibitors (NRTIs). It helps to block a protein
and result in reducing the amount of virus in human body and also reduce to getting sick from Aids-related
illness.
Microparticles are small spherical particles within the range of 1 - 1000 µm and for delivery of
macromolecules by various routes of administration and control the release of drugs over a period ranging
from few hours to months, because of effective protections of encapsulated drug against degradation
[4-7]. It is prepared for prolonged or controlled release, and it is used for improving the bioavailability of
the drug and also to target the specific site in the body [8]. Microparticles also have certain intrinsic worth
like restricting rise and fall within therapeutic range, plummeting side effects, falling dosing frequency and
ultimately elevating the patient compliance [9-11].
In present work ethyl cellulose (EC) is selected as the retardant material for Tenofovir disoproxil
fumarate microparticles. Ethyl cellulose is a hydrophobic polymer and capable of releasing the drug in
sustained manner [12-13]. EC is used as encapsulating material is extensively studied by many researchers
for controlled release of microparticles [14-15]. The main aspire of the current effort is to develop and test the
oral controlled delivery system of Tenofovir with ethyl cellulose by means of emulsion solvent evaporation
method and optimizing the formulation by statistical tool (RSM) with high entrapment efficiency, reducing
dosing frequency and sustained release.
MATERIALS AND METHODOLOGY

MATERIALS
Tenofovir disoproxil fumarate was obtained as a donation sample from (Matrix, Hyderabad, India), ethyl
cellulose (Loba Chemie Pvt. Ltd., Mumbai, India) dichloromethane (Fischer Chemie Ltd., Chennai, India),
tween 80 (Loba Chemie Pvt. Ltd., Mumbai, India) were obtained from commercial sources. All the other
materials used in the study were of analytical grade.
METHODOLOGY
Formulation of microparticles
The microparticle trial formulations were primed by emulsion solvent evaporation technique. The main
investigation of the work is based on the changes made in polymer: drug ratios to check its role in a variety of
dealing factors on the microparticle uniqueness [9-11]. Weighed amount of Tenofovir DF and ethyl cellulose
(1:1.5, 0.75:2.5, 0.75:2, 1:2.5, 1:2, 0.75:1.5, 0.5:2, and 0.5:1.5) were added to 10 ml solution containing a
combination of dichloromethane with methanol in 1:1 ratio. The organic solution was then slowly added to
water containing tween 80 (0.3% w/v) as surfactant with constant stirring for 1h. The resulting microparticles
were separated by filtration and over a period of air dried at 12 h.
CHARACTERIZATION OF MICROPARTICLES
Estimation of drug content
The drug content of the microparticles was determined spectrophotometrically with UV – Visible
spectrophotometer (Perkin Elmer, USA). A definite quantity of microparticles say 10mg equivalent to
the drug was introduced into a 100ml standard measuring flask containing 5ml of methanol to dissolve
and then added with phosphate buffer pH 7.4 to get the volume up to score line. After filtration and
respective dilutions, the sample was analyzed to estimate the amount of drug encapsulated contained by
the microparticles [16-17].
Percentage yield
The total amount of microparticles was weighted and the percentage yield calculated depending upon the
drug and the polymer.
% Yield = (Practical Yield / Theoretical Yield) × 100
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Effect of Drug entrapment

Precise measure of microparticles were weighed, compacted into fine powder and mixed with 10ml
of phosphate buffer pH 7.4. The consequential solution was exposed for bath sonication waves for 30
minutes [17] and then cleared through whatmann filter paper. The filtrate was subjected to analysis
by spectrophotometric method using UV – Visible spectrophotometer (Perkin Elmer, USA) by setting
wavelength maximum at 230.4nm. The entrapment potency of the formulation was resolute by using the
formula,
Drug entrapment efficiency = (Experimental drug content / Theoretical drug content) × 100
Particle Size
The size of the microparticles was observed by optical microscopy method, with the help of a compound
light microscope (Khera instruments, Pvt Ltd, New Delhi) and calibrated eyepiece micrometers. Small
amount of microparticles were placed on clean dried glass slide (5cm × 1cm) having thin film and oil layer
(glycerin), placed on platform and focused with 10X and 100 particles were measured and the results were
determined and tabulated [16].
FT – IR spectroscopy analysis
A small quantity of the Tenofovir DF microparticles was ground along with dry salt of IR grade potassium
bromide. The mechanical die press was used to crush a powder mixture to convert into a pellet along which
the IR beam was passed across to determine the interaction of drug and excipients. The unique grade of KBr
was used for the study to eradicate the scattering effects of non-uniform large crystals and avoid irrelevant
bands in the IR spectrum. The interaction of drug with excipients can be determined from the IR graph peaks
of formulation compared with the pure drug [17-19].
In-vitro drug release studies

An accurately weighed (10 mg) Tenofovir DF microparticles were dropped in 200ml of phosphate buffer
pH 7.4, maintained at a temperature of 37°C ± 0.2°C and stirred at a speed of 75 rpm using USP - XXVII
dissolution apparatus type I (basket) [17]. The time points for collection of aliquot samples were at every
one-hour interval, and each instance the samples were collected out, the baskets were added with equivalent
volume of warm fresh medium. The so withdrawn solutions were filtered and analyzed using UV-visible
spectrophotometer (Perkin Elmer, USA) fixing the wavelength maximum at 230.4nm, against phosphate
buffer pH 7.4 as a blank.
Differential scanning calorimetric (DSC) analysis:

The physicochemical compatibilities of the optimized formulations were checked by obtaining the DSC
thermograms from a differential scanning calorimetry (Perkin Elmer, USA) for the powdered microparticles
containing the drug and comparing with the blank microparticles. The instrument was equipped with a
thermal analysis data station system, computer and plotter interface. The system was previously calibrated
using standard sample of indium and the materials (2mg) were exposed for analysis at the range of 30°C –
220°C [17-19].
XRD analysis
XRD analysis was performed for the pure drug and the microparticles containing drug, using Rigaku ultima
III with copper target being run at room temperature. The other instrumental setup includes 40 Kv voltages
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and 30 mA current and examine rate of 0.05°C / min. The materials were located on to sample holder of the
diffractometer and scanned over a range of 2θ values from 3° to 60° [20-22].
Experimental analysis and statistical design

A 32 factorial design was employed to design sustained microparticles for Tenofovir DF. This design
is suitable for response surface of quadratic equations and the two independent variable was Tenofovir
DF (X1) and Ethyl cellulose (X2) [23-25]. The actual and coded levels of the design were represented in
(table 1). The lower, intermediate and higher levels of each factor coded as 0, +1, and -1 respectively. The
selected dependent variables were drug entrapment efficiency (Y1) and % drug release (Y2) [26-28].
Table 1: Actual values and coded values
Factors Actual values Coded values

Low level Medium level High level Low level Medium level High level
Tenofovir DF (X1) 50 75 100 -1 0 1
Ethyl cellulose (X2) 150 200 250 -1 0 1

Drug content
The proportion of drug present in the different microparticles of Tenofovir DF ranged from 79.35 ± 1.11
to 103.77±1.29 % (table 2). Higher percentage of drug loading was obtained by increasing the amount
of Tenofovir DF with respect to ethyl cellulose polymer. An analogous behavior was also observed by
researchers Sudhamani T et. al., and Deshmukh VN et. al. [4, 29].
Percentage yield
The percentage yield of all the formulation was ranging from 23.5 to 50.18% respectively (table 2). This
higher percentage yields indicates that this method shall be very useful for adoption in the formulation of
Tenofovir microparticles.
Drug entrapment effect

The outcome of dissimilarity in drug encapsulation potency with respect to change in polymer: Tenofovir
disoproxil fumarate concentration was shown in table 2. The percentages of encapsulation of all the
formulation were in the range of 11.75 – 37.63%. This suggests that the higher level of encapsulation of drug
in polymer can be achieved by simply modifying the proportion of polymer with respect to drug. The same
observation was reported by Sudhamani T et. al. [4].
Particle size
The size range of Tenofovir DF loaded microparticles has been analyzed with help of optical microscopy.
All the formulation of Tenofovir DF microparticles shows uniform size division. The dimensions of
Tenofovir microparticles were found to be within the range of 27.63 ± 1.11 to 56.19 ± 2.10µm. While the
ratio of polymer and drug was modified, the microparticles size was also found to be changing respectively.
Lakshmana Prabu S et. al. have also attributed that increase in the amount of polymer shall also increase to
the microparticle size [30].
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Figure 1: Optical microscopic images of Tenofovir disoproxil fumarate and ethyl cellulose microparticles a) TEF4 at
(40X) b) TEF5 at (40X) c) TEF6 at (40X) d) Plain microparticles (without drug) at (40X)
Table 2: Comparative study of various physical parameters for microparticles containing Tenofovir disoproxil fumarate.
S.No. Formulation Drug:polymer Particle size Drug content % yield Drug EE (%) % Drug
code ratio (µm) (%) release
1 TEF1 1:1.5 35.14±2.42 89.19±0.68 35.2 35.2 84.13±0.99
2 TEF2 0.75:2.5 55.5±2.53 93.42±0.73 46.76 35.07 77.58±0.81
3 TEF3 0.75:2 33.4±0.77 94.56±0.68 35.33 35.33 80.23±1.00
4 TEF4 1:2.5 33±1.04 96.27±0.87 26 26 75.49±0.84
5 TEF5 0.5:2.5 37.59±2.03 92.54±0.70 46.66 23.33 63.72±0.86
6 TEF6 1:2 56.19±2.10 103.77±1.29 50.18 37.63 86.23±0.77
7 TEF7 0.75:1.5 36.91±1.35 83.41±0.94 41.77 31.33 65.53±0.71
8 TEF8 0.5:2 32.32±1.13 86.64±0.70 26.8 13.4 74.27±0.86
9 TEF9 0.5:1.5 27.63±1.11 79.35±0.75 23.5 11.75 52.41±0.85
In vitro drug release

Microparticles of all the formulations had slow drug release approximately 25% within 1 hour. Then the
release is sustained over 8 hours, depending upon the polymer: drug ratio. By the end of 8th hour, the
percentage of drug release is found to be 52.35±0.95 to 86.59±0.70% (table 2). The formulation TEF6
containing 1:2 ratio of drug: polymer have shown elevated level of drug liberated by the end of time 8th
hour as compared to other formulations (fig 2). This may be due to superior drug loading, high potency
for encapsulation of the drug within polymer and finally enlarged size of particles as compared to other
formulations. The mechanism of drug release from each formulation was attributed by treating the percentage
drug release results obtained from dissolution studies in various kinetics models and the R2 values generated
by the respective graphs were 0.9746 for zero order, 0.8513 for first order, 0.9814 for Higuchi, 0.9699 for
Korsmeyer and 0.9727 for Hixon Crowell kinetics, estimated for the optimized formulation (TEF6) and
remaining showed in table 3.
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Figure 2: In vitro release of Tenofovir DF microparticles.
Table 3: Release kinetics of drug release from Tenofovir DF microparticles.
Ratio TEF1 TEF2 TEF3 TEF4 TEF5 TEF6 TEF7 TEF8 TEF9
r2
Zero order 0.9775 0.9755 0.9337 0.9738 0.9666 0.9746 0.9834 0.9461 0.9888
First order 0.8513 0.8513 0.8513 0.8513 0.8513 0.8513 0.8513 0.8513 0.8513
Higuchi 0.9992 0.9978 0.9936 0.9988 0.9951 0.9814 0.9953 0.9926 0.9953
Korsmeyer 0.905 0.9397 0.9857 0.9286 0.9817 0.9699 0.9378 0.8553 0.9531
Hixon 0.9252 0.9511 0.9733 0.9351 0.9694 0.9608 0.9727 0.9114 0.9745
FT - IR spectroscopy analysis
FTIR spectra of pure drug and microparticles of Tenofovir DF shown its principal peaks of C-N stretch at
1215.2 cm-1, C-H stretch at 2925.10 cm-1 and N-H stretch at 1643.43cm-1 revealed in figure 3. The IR
graph of microsphere formulations presented each and every characteristic peak of pure drug indicating no
interaction between the drug and polymer.
Figure 3: FTIR study of (a) Tenofovir DF (pure drug), (b) Microparticle formulation (TEF6)
Differential scanning calorimetric (DSC) analysis

The thermo grams of pure sample of Tenofovir DF and its optimized formulation of microparticles were
showed in the figure 4. In the case of Tenofovir, a pointed endothermic crest peak was observed at 116.80°C
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and 182.13°C which corresponds to the melting of crystalline Tenofovir. The peak of the drug did not appear
in the thermo gram of the formulation (ethyl cellulose loaded Tenofovir microparticles). It indicates that the
drug was uniformly dispersed at the molecular level in the polymeric matrix.
Figure 4: DSC study of (a) Tenofovir DF (pure drug), (b) Microparticle formulation (TEF6)
X – Ray diffraction (XRD) analysis

The XRD graph of pure sample of Tenofovir DF and microparticles were displayed in Fig 5. The frequent
divergent peaks seen in pure Tenofovir DF have confirmed the crystalline nature of the drug, where the
prominent peak was obtained at 2θ of 24°. Ethyl cellulose microparticles containing Tenofovir DF reveals
that the pure drug intensity peaks were sharp, but when it is incorporated into the polymeric matrix probably
the crystallinity decreased, because the drug peaks showed a loss of sharpness due to the presence and
masking effect of polymer ethyl cellulose. The drug was crystalline nature and powder XRD pattern with
different peaks appearing at 8° – 31.30° 2θ values, but in case of formulation no intense peak were observed
between this 2θ value range, indicating semi-crystalline or amorphous nature of drug after encapsulation into
the polymer matrix.
Figure 5: XRD study of (a) Tenofovir DF (pure drug), (b) Microparticle formulation (TEF6)
Experimental analysis and statistical design

The data was analyzed and recorded using analysis of variance and the evaluation of polynomial equation,
and individual factor was carried out by F-test. The nine different formulations of two different variables
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(X1, X2) and its related responses (Y1, Y2) were tabulated in the table 4. The selected response variables were
the percentage drug entrapment efficiency (Y1) and percentage drug release (Y2). The data was fitted with
various models of statistical tools like linear, 2F1, quadratic and cubic for the selected two responses using
design expert software (Design 8 ExpertTM), it helps to choose the good fit model for better analysis. The
good fit of the model was evaluated using ANOVA [23].
Table 4: Design and response summary data for Tenofovir DF microparticles with ethyl cellulose
Formulation code Factors Response

Tenofovir DF (X1) Ethyl cellulose (X2) Drug EE (%)(Y1) % Drug release (Y2)
TEF1 1 -1 35.2 84.13
TEF2 0 1 35.07 77.58
TEF3 0 0 35.33 80.23
TEF4 1 1 26 75.49
TEF5 -1 1 23.33 63.72
TEF6 1 0 37.63 86.23
TEF7 0 -1 31.33 65.53
TEF8 -1 0 13.4 74.27
TEF9 -1 -1 11.75 52.41
R2 (multiple correlation coefficients), adjusted R2 (adjusted multiple correlation coefficient) and PREES
(predicted residual sum of square) provided by Design – Expert software were used as factors for selection
of adequate models. With the above data quadratic model was chosen as a good fit model due to its small
predicted residual sum of squares (PRESS), it gives the quantity of robust of the model to the points in
experiments. For the better data point fit the PRESS must be small.
The quadratic model generated by the design is to form,
Y = β0 + β1 X1 + β2 X2 + β3 X1X2 + β4 X12 + β5X22
Where β0 is an intercept and β1- β5 are coefficient of respective factors and their interaction response are
following, Response: Drug entrapment efficiency (Y1) Values of “Prob > F” less than 0.0500 indicate model
terms are significant. In this case X1, X2, X1X2 is significant model terms (table 5) Final Equation in Terms
of Coded Factors:
Drug EE (Y1) = +37.06 + 8.01 X1 +1.02 X2 – 5.20 X1X2 – 11.27 X12 – 2.43X22
Response: Drug release (Y2) Values of “Prob > F” less than 0.0500 indicate model terms are significant.
In this case X1, X2 is significant model terms (table 6).
Final Equation in Terms of Coded Factors:
Drug release (Y2) = + 80.59 + 9.24 X1 + 2.45 X2 – 4.99 X1X2 – 1.25 X12 – 9.95 X22
Table 5 and 6 shows all the responses and their standardized main effects in the type of quadratic
equation. Constructive and destructive symbols in the head of coefficient in quadratic model specifies a
synergistic and antagonistic effect of the factor, by omitting the non-significant terms the significant models
were retained. The coefficient of X1, X2 and X1X2 were significant (P <0.05). Increasing the concentration
of ethyl cellulose resulted in reduction of drug release. However, its interaction terms had a retardation
influence on the release of Tenofovir DF. The similar observation was reported by Swati C et. al. and Pande
AV et. al. [27-28]
333
Table 5: ANOVA for Tenofovir DF drug entrapment efficiency
Source SS DF MS F P Model significant/ non-

significant relative noise
Model 995.90 5 199.18 34.70 <0.0001 Significance
Drug EE 384.80 1 384.80 67.03 <0.0001 Significance
% drug release 6.24 1 6.24 1.09 0.3317 Significance
Residual 40.19 3 13.40
Core total 1036.09 12
Drug EE - Drug entrapment efficiency, DF - degree of freedom, SS - sum of square, MS - mean sum of
square, F- Fischer’s ratio.
Table 6: ANOVA for Tenofovir DF percentage drug release
Source SS DF MS F P Model significant/ non-

significant relative noise
Model 1003.57 5 2007.71 18.74 0.0006 Significance
Drug EE 512.45 1 512.45 47.84 0.0002 Significance
% drug release 36.11 1 36.11 3.37 0.1090 Significance
Residual 74.98 7 10.71
Core total 1078.56 12
Drug EE - Drug entrapment efficiency, DF - degree of freedom, SS - sum of square, MS - mean sum of
square, F- Fischer’s ratio.
3D response surface and contour plot showing the effect of Tenofovir DF and ethyl cellulose on
drug entrapment efficiency and percentage drug release had shown in figure 6 & 7. Drug release was
found to be in sustained manner and a regular fashion as concentration of polymer was increased. The
optimized formulation was evaluated for drug entrapment efficiency and percentage drug release, table
7 enlisted the value of the observed and predicted response for the optimized formulation of Tenofovir
disoproxil fumarate. The data clearly indicate that the dependent variables were strongly dependent on the
independent variables.
Figure 6: (a) 3D Response surface plot and (b) Contour plot showing the effect of the Tenofovir DF and Ethyl cellulose
on drug EE
334
Figure 7: (a) 3D Response surface plot and (b) Contour plot showing the effect of the Tenofovir DF and Ethyl cellulose
on percentage drug release
Response Predicted Observed Residuals

Drug EE 37.060 37.630 0.57
% drug release 88.582 86.230 -2.352
Drug EE - (Drug entrapment efficiency)

Residual = observed value – predicted value
Table 7: Predicted and observed response of the optimized formulation for Tenofovir DF microparticles
Response Predicted Observed Residuals

Drug EE 37.060 37.630 0.57
% drug release 88.582 86.230 -2.352
Drug EE - (Drug entrapment efficiency)

Residual = observed value – predicted value
CONCLUSION
Sustained release microparticles of water insoluble drug using hydrophobic polymer can be successfully
prepared by emulsion solvent evaporation method with fine yield of production, elevated level of drug
content and improved potency of drug encapsulation. The formulations proved to show, soft surface and also
narrow range of size distribution. Tenofovir DF microsphere formulation demonstrated a typical sustained
release up to 8 hrs. The application of 32 factorial designs was a successful tool for optimization of Tenofovir
DF microparticles. The data so obtained clearly ensure that the size, content and encapsulation variables of
the microparticles undoubtedly depend on the drug and polymer concentration variables. The microparticles
certain the extended release of the drug that ultimately can reduce total dose administration and possible side
effects.
ACKNOWLEDGMENT:
The authors are grateful to the management of SASTRA University for providing the necessary infrastructure
and support to complete this work successfully.
335
REFERENCES
1. Patrick Marcellin MD, Jenny Heathcoat E, Maria Butti MD. et. al., Tenofovir Disoproxil Fumarate
versus Adefovir Dipivoxil for chronic hepatitis B. N Engl J Med 2008; 359: 2442-2455
2. Evanas, Melony Floyd and Marton Honeywell, Tenofovir: The first nucleotide analog for HIV-1, Drug
forecast. 2002; 27(7): 359 – 361.
3. Shu - Shan Zhao, Lan – Hua Tang, Xia – Hong Dai et. al., Comparison of the efficacy of Tenofovir and
Adefovir in the treatment of chronic hepatitis B: A systemic review, Virology journal, 2011; 8(111):
2 – 9.
4. Sudhamani T, Naveen Kumar Reddy K, Ravikumar VR, Revathi R, Ganesan V, et. al. preparation and
evaluation of ethyl cellulose microparticles of ibuprofen for sustained drug delivery, Int J pharma Res
and dev., 2010; 1:119-125.
5. Dhanasingh S and Nallaperumal SK, Chitosan/casein microparticles: preparation, characterization and
drug release studies, International Journal of Engineering and Applied Sciences, 2010; 6(4): 234 -238
6. Morimoto Y and Fujimoto S, Albumin microspheres as drug carrier, CRC Critical Reviews in Therapeutic
Drug Carrier Systems, 1985; 1(2) 19-63.
7. Gupta P and Fung C, Targeted delivery of low dose doxorubicin hydrochloride administered via magnetic
albumin microparticles in rats, J microencapsul, 1990; 7(1): 85-94.
8. Venkatesan P, Muralidharan C, Manavalan R and Valliyappan K, Selection of better method for the
preparation of microparticles by applying analytic hierarchy process, J Pharm Sci & Res. 2009; 1 (3):
64-78.
9. Patrick B deasy, Microencapsulation and related drug process, Marcel Dekkar, New York, 1984; 20:
01-159.
10. Fedreic Lagarce, Pascal Renaud, Nathalie Faisant, Guillaume Nicolas, Annie Cailleux, Joel Richard,
Philipp menei and Jean- pierre benoit, Eur J Pharm Biopharm, 2004; 59: 449-459.
11. Ambati Brahma Reddy, Karunanidhi M, Sudheer karna k and Samyuktha Rani B, Effect of SLS on ethyl
cellulose containing Cyclobenzaprine HCl floating microparticles, Research journal of pharmaceutical,
biological and chemical sciences, 2010; 1(4): 898-908.
12. Al-Omron MF, Al-suwayeh SA, El-helw AM saleh SI, Taste masking of Diclofenac sodium using
microencapsulation, J microencapsul, 2002; 19:45-52.
13. Gander B, Johnsan P, Nam-Tram H and Merkle HP, Thermodynamics approach to protein
microencapsulation into poly (DL-lactide) by spray drying, Int J Pharm 1996; 129: 51-61.
14. Rao B.S and Murthy K.V., Studies on Rifampicin release from ethyl cellulose coated nonpareil beads,
Int J Pharm, 2002; 231:97-106.
15. Yamada T, Onishi H and Machida Y, Sustained release Ketoprofen microparticles with ethyl cellulose
and carboxymethyl-cellulose, J Control Rel. 2001; 75:271-282.
16. Venkatesan P, Manavalan R, Valliappan K, et. al. microencapsulation: A vital technique in novel drug
delivery system, J Pharm Sci & Res, 2009; 1(4):26-35.
17. Vedha HB, Brahma RA and Samyuktha RB, Floating drug delivery of Nevirapine as gastroretentive
system, J Young Pharm, 2010; 2(4): 350-355
336
18. Luypaert J, Zhang MH and Massart DL, Feasibility study for the use of near infrared spectroscopy in the
qualitative and quantitative analysis of Green tea Camellia sciences (L), Analytica Chimica Acta 2003;
478 (2): 303-312.
19. Roy S, Panpalia SG, Nandy BC, Rai VK et. al. Effect of method of preparation on Chitosan microparticles
of Mefenamic acid, Int J Pharm Sci Drug Res., 2009; 1(1): 36 – 42.
20. China Gangadhar B, Shyam Sundar R, Vimal Kumar Varma et. al., Formulation and evaluation of
Indomethacin microparticles using natural and synthetic polymers as controlled release dosage forms,
Int J drug discovery, 2010; 2(1): 8 – 16.
21. Lalduhsanga Pachuau and Bhaskar Mazumder, A study on the effects of different surfactants on ethyl
cellulose microparticles, Int J Pharm Tech Res., 2009;1(4): 966 – 971.
22. Pachuau L, Sarkar S and Mazumder B, Formulation and Evaluation of matrix microspheres for
simultaneous delivery of Salbutamol Sulphate and Theophylline, Trop J Pharm Res., 2008; 7(2): 995 –
1002.
23. Hakan Eroglu, Reha Alpar and Leven toner, Chitosan in steroid delivery: formulation of microspheres
by factorial design and evaluation of in vitro release parameters, FABAD J Pharm Sci. 2008; 33: 144 –
150.
24. Chudiwal PD, Pawar PL, Nagaras MA and Mandlik SK, Statistical evaluation and optimization of
influence of viscosity and content of polymer on floating microspheres of Clarithromycin, Int J Pharm
Res. 2009; 1(4): 1366 – 1372.
25. Shaji and Shinde Amol, Formulation and optimization of floating pulsatile Aceclofenac microspheres
using Response surface methodology, Int Res J Pharm. 2012; 3(1): 166 – 169.
26. Gajanan Deshmukh, Deepti Ruikar and Seth A.K., Formulation and statistical optimization of Verapamil
Hydrochloride floating drug delivery system by Response surface methodology, Int J Pharm Sci. 2011;
2(3): 26 – 42.
27. Swati C, Jagdale and Swapnil, Effect of polymer and gas forming agent on floating drug delivery
of Tramadol HCl using Response surface methodology: in vitro and in vivo evaluation, Int J Pharm
Applications, 2011; 3(3): 181 – 194.
28. Pande AV, Nimbalkar and Dhoka MV, Floating microspheres of Cefpodoxime Proxetil: formulation and
optimization by factorial design, Int J Res Pharm & Chem. 2011; 1(3): 448 – 457.
29. Deshmukh VN, Jadhav JK, Masirkar VJ et.al., Formulation, optimization and evaluation of controlled
release alginate microspheres using synergy gum blends, Research J Pharm and Tech., 2009; 2(2): 324
– 327.
30. wLakshmana Prabu S, Shirwaikar AA, et. al., Formulation and evaluation of sustained release
microspheres of Rosin containing Aceclofenac, ARS Pharm, 2009; 50(2): 51 – 62.
337
Formulation and Evaluation of Mouth Dissolving Film
Leflunomide for Rheumatoid Arthritis
S. Lakshmana Prabu*, A. Shanmugarathinam, S. P. Sharavanan, M. Elakkiya,

V. Manikandan, A. Bhuvaneswari and S. Aravindan
Dept. of Pharmaceutical Technology, Bharathidasan Institute of Technology,

Anna University, Tiruchirappalli – 620 024
e-mail: *slaxmanvel@gmail.com
Abstract:
The present work aimed to prepare mouth dissolving films of leflunomide with the purpose of developing a
dosage form for a very quick onset of action, which is very convenient for administration, without the problem
of swallowing and using water. The films of leflunomide were prepared by using polymers such as PVA and
HPMC using PEG as plasticizer, by solvent casting method. Drug excipient compatibility was studied by
FTIR and UV techniques. The formulated mouth dissolving films were evaluated for physical characteristics
such as uniformity of weight, thickness, folding endurance, drug content uniformity, surface pH, in-vitro drug
release tests and stability study. Mouth dissolving film of leflunomide containing PVA as polymer showed
99.18 % drug release at 30 min. Stability studies revealed that optimized formulation was stable.
Keywords: Leflunomide, Mouth dissolving film, Solvent casting, Rheumatoid arthritis
INTRODUCTION
During 1970 fast dissolving drug delivery system was introduced as an alternative to oral solid dosage forms
for paediatric and geriatric patients who experienced difficulties to swallow the medicines in the form of
tablets and capsules [1, 2]. Oral route is the most preferred route by medicinal practitioners and manufacturer
due to high acceptability of patients. About 60% of all dosage forms available are the oral solid dosage
form. Based on the technological concept of transdermal patch mouth dissolving films for the oral delivery
of the drugs were developed. Mouth dissolving film is an ideal route for fast dissolving delivery systems
which satisfies the various needs such as easy handling by the patients, simple and convenient packaging,
avoiding of unpleasant taste and an attractive marketing by the pharmaceutical manufacturers. Recently
mouth dissolving films approach is increase among the consumer due to easy administration without water,
rapid dissolution and on set of action by absorbing the drug directly into the systemic circulation [3-8].
Rheumatoid arthritis (RA)is an inflammatory arthritis that affects nearly 1% of the world’s adults. It is characterized
by symmetric polyarticular inflammation of the synovium. Typically of the small joints of the wrists and feet [9-
10]. The present investigation was to formulate mouth dissolving film leflunomide (5-methyl-n-[4-trifluoromethyl]
phenyl]-isoxazolo-4-carboxamide) by solvent casting method to improve the treatment of rheumatoid arthritis.

Leflunomide was gifted by Aravind Laboratories, Chennai; PVA, HPMC, ethanol and tween 80 were obtained from
Loba Chemie Pvt., Ltd., Mumbai, India; ethanol, methanol, acetone, PEG 40, potassium dihydrogen phosphate,
sodium hydroxide and sodium chloride were purchased from SD fine chemicals, India.
Formulation of Mouth Dissolving Film

Mouth dissolving film was prepared by solvent casting method. Water soluble hydrocolloids dissolved in
water to form homogeneous viscous solution. Other ingredients including active agent dissolved in small
proportion of aqueous solvent. Both mixtures are mixed to form homogenous viscous solution using magnetic
stirrer and degassed under vacuum. Bubble free solution is coated on non-treated casting film and dried in
hot air oven at 40 - 50°C. Film was cut into desired shape and size and wrapped in aluminum foil. Various
formulations composition was shown in table 1.
Table 1: Composition of the mouth dissolving film leflunomide
S. No Ingredients Formulation code

F1 F2 F3 F4 F5
1 PVA (mg) 500 300 - 300 200
2 HPMC E5 (mg) - - 300 - -
3 Leflunomide (mg) 210 120 120 120 120
4 Tartaric acid (mg) 50 50 50 50 50
5 Glucose (mg) 60 60 60 60 60
6 PEG 400 (ml) 0.5 0.3 0.3 0.3 0.3
7 Tween 80 (mg) 50 50 50 50 50
8 Methanol (ml) - 1.5 1.5 1 -
9 Ethanol (ml) - 1.5 - 1 1
10 Acetone (ml) - - 1.5 - -
11 Water (ml) Q.S Q.S Q.S Q.S Q.S
Drug Exicipient-Interaction Studies

The drug excipients mixture in the ratio 1:1 can detect the incompatibility among the chosen excipients.
In the present study 1:1 ratio of physical mixtures were prepared and analyzed by FT-IR and UV
techniques [11].
Physicochemical characterization
The prepared films were characterized physicochemically for visual inspection (homogeneity, colour,
transparency and surface), thickness, folding endurance, weight variation and surface pH.
Disintegration time
The disintegration time was measured using modified disintegration method. For this purpose a petri dish
was filled with 10 ml of water. The film was carefully put in the centre of petri dish. The time taken for the
film to completely disintegrate into fine particles is noted. Measurements were performed five times for each
formulation.
Drug Content
The amount of drug present in the formulation was determined spectrophotometrically by using phosphate
buffer solution at pH 6.8 at 258 nm.

In vitro dissolution studies were carried out using USP basket type apparatus. Phosphate buffer (pH 6.8,
500 ml) was used as dissolution medium at 100 rpm speed. At periodic time interval 5 ml samples were
withdrawn and replaced with the equal quantity of fresh dissolution medium. Samples were filtered through
339
Whatman filter paper, and analyzed spectrophotometrically at 258 nm. The in vitro dissolution testing studies
were performed in triplicate for all the batches.

Oral mucosa in oral buccal cavity have a rich vascularization and offers higher permeability to many drugs
and drug absorbed in to the reticulated vein then drained into the systemic circulation to produce the onset
of action.
Excipient compatibility studies were carried out by FT-IR and UV techniques to investigate chemical
interactions between drug and the excipients. Leflunomide contains chemical functional groups like with
wave numbers of 3438, 2921, 1641, 1461, 1101 cm-1 for NH stretching, CH stretching, C=O amide bond
(stretching), CH bending and C-O stretching respectively. These characteristic bands were present in the IR
spectra of nanosuspension formulation. The IR spectrum of mouth dissolving film composition is shown in
Figure 1.
Figure 1: IR spectrum of mouth dissolving film composition
Various physicochemical characterization of the mouth dissolving film leflunomide was performed. The
result showed that all the film formulations are transparent one with smooth surface with good folding
endurance. Films were having thickness adequate for handling and use. The weight variation and drug content
results showed that the film was uniform and drug was also uniformly distributed in the films. Surface pH of
the films was found to be between 6.59 and 6.95, there is no significant difference was found in surface pH
of different films. The film surface pH either acidic or alkaline can irritate the buccal mucosa. The surface
pH of the film found to be close to neutral in all the formulations; hence the formulated film may have less
potential to irritate and fairly comfortable with the buccal mucosa. The physicochemical characterization of
mouth dissolving film leflunomide is given in table 2.
Table 2: Physicochemical characterization of mouth dissolving film leflunomide
S. No Formulation Folding Thickness Weight of the Drug content Surface pH Disintegration

endurance (mm) film (mg) uniformity (sec)
1 F1 >350 0.229±0.012 135.56 98.5% 6.93 120
2 F2 >250 0.144±0.010 110.02 100.03% 6.85 60
3 F3 >300 0.211±0.034 106.12 99.23% 6.72 80
4 F4 >250 0.181±0.002 107.22 100.17% 6.59 100
5 F5 >250 0.154±0.003 107.03 100.43% 6.85 40
340
In vitro release studies are carried out under physiological conditions and interpretation of the
dissolution data with respect to the in vivo performance of a drug product. Based on the release study results
formulation F5 showed maximum release in 30 min. The release results revealed that mouth dissolving
film leflunomide can provide faster onset of action to improve anti-rheumatoid arthritis therapy. The
comparative in vitro release studies of formulation F1 to F5 is shown in Figure 2.
Figure 2: Comparative in vitro release studies of formulation F1 to F5
CONCLUSION
Solubility and dissolution rate are desirable factors to improve bioavailability. Leflunomide
Mouth dissolving film Leflunomide poorly water soluble drug was prepared by solvent casting method with
surfactant and hydrophilic polymer. Mouth dissolving film disintegrate within one minute. Our research
results indicate the suitability of the formulation procedure for the preparation of mouth dissolving film
leflunomide poorly water soluble drug. The in vitro release study demonstrated that the aqueous solubility
of compound was increased when administered as mouth dissolving film. These mouth dissolving film
formulations can be used as an alternative approach to improve the treatment of rheumatoid arthritis.
REFERENCE
1. Siddiqui MDN, Garg G and Sharma PK, A Short Review on “A Novel Approach in Oral Fast Dissolving
Drug Delivery System and Their Patents”. Advances in Biological Research. 2011; 5 (6): 291-303.
2. Saini S, Nanda A and Dhari J, Formulation, Development & Evaluation of Oral Fast Dissolving Anti-
Allergic Film of Levocetrizine Dihydrochloride. J. Pharm. Sci. & Res. Vol.3(7), 2011,1322-1325.
3. Panchal MS, Patel H, Bagada A and Vadalia KR, Formulation and Evaluation of Mouth Dissolving Film
of Ropinirole Hydrochloride by Using Pullulan Polymers. Int J Pharm Res All Sci. 2012; 1: 60-72.
4. Nagar M, Nagar M and Chopra V, Formulation and evaluation of mouth dissolving film of antipsychotic
drug Aripiprazole. Der Pharmacia Lettre, 2012, 4 (4):1221-1227.
5. Tomar A, Sharma K, Chauhan NS, Mittal A and Bajaj U, Formulation and Evaluation of Fast Dissolving
Oral Film of Dicyclomine as potential route of Buccal Delivery. Int J Drug Dev & Res. 2012; 4 (2):
408-417.
341
6. Dixit RP and Puthli SP, Oral strip technology: Overview and future potential. J of Cont Release 2009;
139: 94–107.
7. Bradoo R, Fast Dissolving Drug Delivery Systems. JAMA 2001; 4 (10): 27-31.
8. Sabar MH, Formulation and in-vitro evaluation of fast dissolving film containing amlodipine besylate
solid dispersion. Int J Pharm & Pharm Sci. 2013; 5(4): 419-428.
9. Theofilou P, Psychological Impact of Rheumatoid Arthritis on Patient’s Health - Related Quality of Life.
J Arthritis. 2012; 1(1): 1-2.
10. Victoria Ruffing RN and Bingham CO, Rheumatoid Arthritis Signs and Symptoms.http://www.
hopkinsarthritis.org/arthritis-info/rheumatoid-arthritis/ra-symptoms/
11. Lakshmana Prabu S, Shnanawaz S and Dinesh Kumar C, Compatibility Studies Between Duloxetine
Hydrochloride and Tablet Excipients Using Thermal and Non-thermal Methods. J Pharm Research,
2008; 7(1): 20-23.
342
Development and Sustainable Release Evaluation of
Enrofloxacin Solid Lipid Nanopartilces
P. Senthil Kumar1,*, A. Arivuchelvan2, A. Jagadeeswaran3, N. Subramanian4, C. Senthil

Kumar5 and P. Mekala6
Department of Veterinary Pharmacology and Toxicology, Veterinary College and Research Institute,
Namakkal, Tamil Nadu
1
Veterinary College and Research Institute, Orathanadu-614625, Tamil Nadu, India.
2, 3, 6
Veterinary College and Research Institute, Namakkal, Tamil Nadu, India.
4
5
e-mail: p.senthilkumar@tanuvas.org.in, Phone: +91 94431 42359
Abstract:
The study was conducted to formulate the enrofloxacin solid lipid nanoparticles (SLNs) with sustained
release profile and improved pharmacological activity. The enrofloxacin SLNs were prepared using
tripalmitin as lipid carrier, tween 80 and span 80 as surfactants and poly vinyl alcohol (PVA) as a stabilizer
by a hot homogenization coupled with ultrasonication method. The formulation were characterized for
particle size, polydispersity index, zeta potential (using dynamic light scattering), shape (using atomic
force microscopy), drug encapsulation efficiency (using by dialysis and ultracentrifugation methods),
and in vitro drug release (using by dialysis). The prepared SLNs were analyzed by FT-IR spectroscopy
to confirm the cross-linking reaction between drug, lipid and surfactants. The results demonstrated
that encapsulation efficiency, loading capacity, diameter, polydispersity index and zeta potential of the
nanoparticles were 59.66±3.22%, 6.13±0.32%, 149.4±2.50 nm, 0.314±0.004, and -24.90±1.00 mV. The
prepared nanoparticle was spherical in shape with smooth surface. In vitro release study in phosphate
buffer saline (pH 6.7) showed an initial burst release and followed by a slow and sustained drug release.
FT-IR study showed no interaction between the drug, polymer and excipients in the present study. From
the results, it can be concluded that the enrofloxacin-loaded tripalmitin SLN proved promising formulation
for sustained release.
Keywords: Enrofloxacin, Solid lipid nanoparticle, Tripalmitin, In vitro Release
INTRODUCTION
bactericidal activity against a range of clinically relevant Gram negative and Gram positive pathogens
as well as Mycoplasma and Chlamydiae. Enrofloxacin and its active metabolite ciprofloxacin possess
high bactericidal activity, killing the bacteria in a concentration dependent manner. The relative safety of
enrofloxacin, its low minimum inhibitory concentrations, broad spectrum of activity, long post-antibiotic
effect and good tolerance has encouraged their use in veterinary medicine (Scheer, 1987).
Despite the therapeutic potential of enrofloxacin, the very poor aqueous solubility of enrofloxacin leads
to difficulty in the designing of pharmaceutical formulation and results variations in bioavailability. In
addition, all the oral enrofloxacin formulations are available as conventional, immediate-release form that
necessitates administration twice daily or daily for several days or weeks. Need to administer of enrofloxain
twice daily or daily causes stress in animals (Martinez et al., 2006). Numerous efforts have been made to
develop alternative formulations of enrofloxacin to reduce frequency of administration.
Nanoparticle-based drug delivery systems have considerable potential in improving the bioavailability
of the drug and as well reducing the dosing frequency. Lipid nanoparticles have received great attention
as carriers in recent years (Schawarz and Mehnert, 1999). Solid lipid nanoparticles (SLNs) possess good
tolerability, stability, scaling up feasibility and the ability to incorporate hydrophilic/hydrophobic drugs
(Muller et. al., 2000). The incorporation of poorly soluble drugs into SLNs can enhance gastrointestinal
solubilisation, absorption, and bioavailability of drugs (Muller et al., 2006). Further, nanoparticle
formulation has the ability to prolong, extend or sustain the release profile of the loaded molecules and
hence reduce need for the repeated administration and increase the therapeutic value of the treatment (Xie
et al., 2011).
Hence, the objective of the present study is to prepare and evaluate the enrofloxacin- loaded tripalmitin
SLNs for sustained oral delivery by using a hot homogenization technique coupled with ultrasonication
method.

Materials
Enrofloxacin and dialysis membrane was procured from Himedia Laboratories Pvt.Ltd., India. Tripalmitin
(Glyceryl palmitate), Span 80 (Polysorbate), Tween 80 (Sorbitate monooleate) and Polyvinyl pyrolidone
K30 were purchased from Sigma Aldrich Chemicals Pvt. Ltd., India. All other chemicals and solvents were
analytical reagent grade and were used without further purification.
Preparation of Enrofloxacin Solid Lipid Nanoparticles

Enrofloxacin SLN were prepared by hot homogenization followed by ultrasonication method.
Enrofloxacin, tripalmitin, span 80 were added at the ratio of 1:5:20 to get organic phase of preparation.
The lipid content in the organic phase was heated at 70°C using magnetic stirrer with hot plate until
it melted. The contents in the organic phase were mixed properly by placing in the shaker (Spinix).
An aqueous phase was prepared by dissolving hydrophilic surfactant tween 80 and polyvinyl alcohol
at the ratio of 20:20 by heating to the same temperature as the organic phase. The hot aqueous phase
was added to the organic phase under magnetic stirring at 1000rpm to form pre-emulsion. The hot pre-
emulsion was then homogenised at 10,000psi for 3min using the high pressure homogenizer kept in a
water bath maintained at 70°C.
The hot emulsion so obtained was ultrasonicated (Sonics Vibra Cell, USA) using high-intensity
(5/64’’ 2mm tip diameter) microprobe with amplitude 20% for 15min to form nanoemulsion. Then,
the nanoemulsion was run under magnetic stirring at 1000rpm for 4h to obtain enrofloxacin loaded
tripalmitin SLNs.
All the batches were prepared in triplicate and the average size was measured.

Determination of particle size, polydispersity index and zeta potential
Particle size and polydispersity index of enrofloxacin SLNs were measured by Photon Correlation
Spectroscopy (PCS) using zetasizer nanoZS with the Malvern PCS software version 6.20. The zeta potential
or the charge on the surface of colloidal particles in a liquid enrofloxacin nano suspension was measured
344
by electrophoretic light scattering (ELS) mode using zetasizer nanoZS. Each value was the average of three
measurements.
Surface morphology
Surface morphology and shape of the enrofloxacin SLNs were examined using Atomic force microscopy
(PARK XE-100).
Determination of loading capacity and encapsulation efficiency

To determine the entrapment of enrofloxacin in the SLNs, 0.1 ml of freshly prepared nanoemulsion
was taken and diluted with 9.9 ml chloroform. The obtained suspension was centrifuged for 45min
at 6,000rpm. The supernatant was separated and filtered through 0.2µm filter. The filtrate was diluted
using chloroform and analysed at 273.8nm using UV spectrophotometer. The SLNs formulated without
enrofloxacin were treated similarly and used as control for the measurements. The assay was repeated
3 times using different preparations. Loading capacity and encapsulation efficiency were calculated as
shown below:

Loading capacity = ×100
Weight of SLNs
Encapsulation efficiency = ×100
Weight of enrofloxacin added

In vitro release of enrofloxacin SLNs and native enrofloxacin was performed by dialysis bag diffusion
technique over a period of 120h. Enrofloxacin nanosuspension equivalent to 5mg of enrofloxacin was filled
in dialysis bag (Himedia Laboratory Pvt. Ltd, India). The receiver solution containing 100 mL of phosphate
buffer with pH 6.7 was prepared and heated to 37°C under magnetic stirring at a speed of 100 rpm. The drug
containing dialysis bag (Molecular weight 12 to 14k.Da, pore size 2.4nm) was dialysed against receiver
compartment. To determine the enrofloxaxin diffused through the dialysis bag, 2mL samples were withdrawn
at regular intervals (0, 5, 10, 20, 30, 45, 60, 90min, and 2, 4, 8, 12, 18, 24, 36, 72, 96 and 120h.) from the
receiver solution and same amount of fresh receiver solution was added to maintain the volume constant.
Enrofloxacin in the samples was measured spectrophotometrically at 273.8nm using a UV spectrophotometer
(Systronics 2203 Smart, India). The control nanoparticles without enrofloxacin were treated similarly and
used as blanks for the measurements.
Compatibility studies using FT-IR Spectroscopic analysis

Fourier Transform-Infra Red (FT-IR) spectral measurement for pure enrofloxacin, tripalmitin, span 80,
tween 80, polyvinyl alcohol and formulation were analysed separately and then correlated for compatibility.
In the present study, potassium bromide (KBr) pellet method was employed. A small drop of sample was
placed on one of the KBr plates. The second KBr plate was placed on the top and made a quarter turn to
obtain an even film. Then, the plates were kept on the sample holder to run a spectrum.
The data obtained on loading capacity, encapsulation efficiency, particle size, PDI and zeta potential were
analyzed using a Statistical Package for Social Sciences (SPSS version 11.0) software. All values are
expressed as their mean±S.D.
345
RESULTS
enrofloxacin SLNs are given in Table 1 and the size distribution graph is depicted in Fig. 1.
Table 1: Physico-chemical characteristics of enrofloxacin loaded SLN (mean±SD, n=3)
Particle Size (nm) PDI Zeta Potential Encapsulation Loading Capacity

(mV) Efficiency (%) (%)
149.4±2.50 0.314± 0.004 -24.90±1.00 59.66±3.22 6.13±0.32
Fig. 1: Particle size distribution and Zeta potential
Surface morphology
AFM and TEM analysis showed that the enrofloxacin SLNs were spherical and circular in shape (Fig. 2).
The particles were well dispersed with good particle size distribution. The surfaces of the nanoparticle were
smooth.
Fig.2: Atomic force microscopic 3D image of enrofloxacin SLN

In vitro release of enrofloxacin from SLNs formulation and native enrofloxacin is given in Table 2 and the
same is illustrated in Fig.3. The release curve of enrofloxacin SLNs exhibited a biphasic pattern. There
was an initial burst release with about 39.23% drug released within the initial 24h, followed by a slow and
sustained release. The amount of cumulated drug release over 96h was 51.1%. In the native enrofloxacin, the
release was 93.67% within 2h and reached 100% by 24h.
Table 2: In vitro release of native enrofloxacin and enrofloxacin SLN

Time (h) Cumulative Release (%)
Native Enrofloxacin (Mean ±SD) EnrofloxacinSLN (Mean ±SD)
0 0 0
0.08 83.03±1.15 5.07±0.60
0.17 83.73±0.97 5.53±0.21
346
0.3 84.57±0.51 6.10±0.30

0.5 85.57±1.03 7.37±0.21
0.75 87.83±0.90 10.33±0.61
1 90.33±0.95 12.03±0.59
1.5 92.10±0.72 15.07±0.32
2 93.67±0.59 18.03±0.46
8 95.87±0.71 29.07±0.95
12 96.83±0.87 34.13±0.76
18 98.53±0.25 37.00±0.96
24 99.83±0.15 39.23±1.03
36 - 43.07±0.85
72 - 49.70±1.31
96 - 51.10±1.59
Fig. 3: In vitro release of native enrofloxacin and enrofloxacin SLNs (mean± SD, n=3).
Compatibility studies using FT-IR spectroscopic analysis

The FT-IR spectra of drug, tripalmitin, span 80, tween 80 polyvinyl alcohol and formulation were exhibited
the peaks of specific functional groups at their respective frequencies as presented in Fig. 4.
Fig.4: Spectra of Span 80 (a), Tween 80 (b), PVP K30 (c), Tripalmitin (d), Enrofloxacin(e) and formulation (f)
347
DISCUSSION
Homogenization followed by ultrasonication technique applies high shear stress disrupting lipid particles
down to the submicron range. According to Schwarz and Mehnert (1997), a sufficient high-energy input
was necessary to break down the droplets into the nanometer range. A high energy such as high production
temperature, high stirring rate, longer emulsification time and stronger ultrasound power were applied in this
study to obtain a finer dispersion of formulation. In the present study, the homogenization pressure 10,000
psi was applied for 3 min and followed by ultrasonication resulted the mean (± SD) particle size of 154.717
± 6.149nm with narrow size distribution. The result suggests that the hot homogenization and ultrasonication
method was a feasible and compatible method for preparing enrofloxacin loaded tripalmitin SLNs.
(219°C–233°C), hence the stability and antibacterial activity of the enrofloxacin are not affected. According
to Luo et al. (2006), the size of nanoparticles ranges from 100 to 200nm was favourable for better per oral
A narrow particle size distribution was an indication of nanoparticles stability and homogeneous
dispersion (Olbrich et al. 2002). PDI values ranging from 0 to 0.5 were considered to be monodisperse and
homogenous, but those of more than 0.5 indicated nonhomogenity and polydispersity (Zhang et. al. 2009;
Anton et. Al. 2008). In the present study, the particle size distribution was monodisperse and homogenous as
formulation has less mean (±SE) PDI of 0.42±0.11.
Nanoparticle with zeta potential values greater than +25 mV or less than -25mV typically have high
degrees of stability due to electric repulsion between particles. Dispersions with a low zeta potential value
aggregates due to Van Der Waal inter-particle attraction (Muller et al. 2000). In this study, the mean (±SD)
zeta potential of -24.90±1.00mV was recorded and it could provide proper stability to the enrofloxacin
SLNs. According to Schwarz and Mehnert (1997) and Zimmermann et al. (2000), the negative charge of zeta
potential was conferred by the lipids used in the SLNs. In agreement with this report, the tripalmitin utilized
in this study provided negative charge of zeta potential.
AFM images revealed spherical and circular in shape with the presence of some particle aggregates.
The presence of aggregates might be due to redistribution of particles after preparation. The images of AFM
and TEM represented that the particles were ranging from 100 to 200nm and well dispersed with smooth
surfaces.
The enrofloxacin SLNs obtained in the present study had relatively medium drug entrapment efficiency
(59.67%). To get sufficient loading capacity, the drug should have sufficiently high solubility in the lipid
melt. (Bunjes et al. 2002). The percentage encapsulation efficiency data obtained in this study are consistent
with the findings of Xie et al. (2011b).
In vitro release data obtained under sink conditions are consistent with drug release reported from
different SLNs by Ji et al. (2011) and Xie et al. (2011b). The initial fast release (burst effect) could be
attributed to the presence of a small fraction of unentrapped drug or drug embedded near the SLNs surface.
Other factors contributing to a fast release were large surface area, high diffusion coefficient (small molecular
size), low matrix viscosity and short diffusion distance of the drug. The slow release was mainly due to
the low diffusion of drug molecules through the lipid matrix of the nanoparticles and hindering effects by
surrounding solid lipid shell (Muller et al. 2000; Mehnert and Mader 2001).
The FT-IR spectra of drug, excipients, polymer and formulation were exhibited all the characteristics
peaks as depicted in Fig 4. From the IR spectra, it was clear that functionalities of drug have remained
unchanged, including intensities of peak. This suggested that during the process of formulations, excipients
and polymer have not reacted with the drug to give rise to reactant products. So it was only physical mixture
and there was no interaction between them which is on favour to proceed for formulations.
348
CONCLUSION
Enrofloxacin was successfully incorporated into tripalmitin-SLNs by a hot homogenization coupled with
ultrasonication method. The physico-chemical study of enrofloxacin loaded tripalmitin SLNs showed
desired particle size, PDI, zeta potential, loading capacity and encapsulation efficiency. The prepared SLNs
had a sustained release effect in the in vitro release study. FT-IR study concluded that no interaction occurred
between the drug excipients and polymer used in this study. From this study, it can be concluded that, the
nanoparticle would be an effective vehicle for oral delivery of enrofloxacin to improve its pharmacological
activity.
ACKNOWLEDGEMENT
Authors are thankful to the Dr. K. Rukumani, Professor and Head and staff of the Dept. of Pharmaceutical
Technology, Anna University, Regional Office, Trichirappalli-620 024 for providing facilities and technical
guidance for the present study.
REFERENCES
1. Anton, N, Benoit, J.P and Saulnier, P, 2008. Design and production of nanoparticles ormulated from
nano-emulsion templates-a review. J. Control Release 128, 185–199.
2. Martinez M, McDermott P and Walker R. 2006. Pharmacology of the fluoroquinolones: A perspective
for the use in domestic animals. The Veterinary Journal, 172: 10-28.
3. Bunjes H, Drechsler M, Koch M H J and Westesen K, 2001. Incorporation of the model drug
Ubidecarenone into the solid lipid nanoparticles. Pharmaceutical Research, 18: 287-293.
4. Kharia A A, Singhai A K and Verma R, 2012. Formulation and evaluation of polymeric nanoparticles of an
antiviral drug for gastroretention. International Journal of Pharmaceutical Sciences and Nanotechnology,
4(4):1557-1562.
5. Muller R H, Runge S, Ravelli V, Mahnert W, Thunemaan A F and Souto, E B. 2006. Oral bioavailability
of cyclosporine: Solid lipid nanoparticle (SLN) versus drug nanocrystals. International Journal of
Pharmaceutics, 317: 82-89.
6. Luo Y F, Chen D W, Ren L X, Zhao X L and Qin J, 2006. Solid lipid nanoparticles for enhancing
vinpocetine’s oral bioavailability. Journal of Controlled Release, 114: 53–59.
7. Martinez M, McDermott P and Walker R, 2006. Pharmacology of the fluoroquinolones: A perspective
for the use in domestic animals. The Veterinary Journal, 172: 10-28.
8. Mehnert, W. and Mader, K, (2001) Solid lipid nanoparticles: production, characterization and
applications. Advanced Drug Delivery Reviews, 47: 165–196.
9. Muller R H, Runge S, Ravelli V, Mahnert W, Thunemaan A F and Souto, E B. 2006, Oral bioavailability
of cyclosporine: Solid lipid nanoparticle (SLN) versus drug nanocrystals. International Journal of
Pharmaceutics, 317: 82-89.
10. Muller R H, Mader K and Gohla S, 2000. Solid lipid nanoparticles (SLN) for controlled drug delivery –
a review of the state of the art. European Journal of Pharmeutics Biopharmeutics, 50:161–177.
11. Anton, N., Benoit, J.P., Saulnier, P., 2008. Design and production of nanoparticles ormulated from nano-
emulsion templates-a review. J. Control Release 128, 185–199.
12. Scheer M. 1987. Studies on the antibacterial activity of Baytril. Veterinary Medical Reviews, 2: 104-118.
349
13. Schwarz C and Mehnert W. 1999. Solid lipid nanoparticles (SLN) for controlled drug delivery. II. Drug
incorporation and physicochemical characterization, Journal of Microencapsulation, 16: 205-213.
14. Statistical Package for the Social Sciences. 1999. Computer software 11.00 SPSS Inc., Chicago,
Ilinois-60606, USA.
15. Westesen K, Bunjes H and Koch M H J, 1997. Physicochemical characterization of lipid nanoparticles
and evaluation of their drug loading capacity and sustained release potential. Journal of Controlled
Release, 48:223-236.
16. Mehnert, W. & Mader, K. 2001. Solid lipid nanoparticles: production, characterization and applications.
Advanced Drug Delivery Reviews, 47: 165–196.
17. Xie S, Zhu L, Dong Z, Wang X, Wang Y, Li X and Zhou W, 2011. Preparation, characterization and
pharmacokinetics of enrofloxacin-loaded solid lipid nanoparticles: Influences of fatty acids, Colloids
and Surfaces B: Biointerfaces, 83: 382–387.
18. Xie Y, Zhu L Dong Z, Wang X and Zhou W Z, 2011. Preparation and evaluation of ofloxacin-loaded
palmitic acid solid lipid nanoparticles. International Journal Nanomedicine, 5: 693–701.
19. Olbrich C, Kayser O, Muller RH (2002) Lipase degradation of Dynasan 114 and 116 solid lipid
nanoparticles (SLN) effect of surfactants, storage time andcrystallinity. Int J Pharm 237: 119-128.
20. Zhang J, Fan Y, Smith E. 2009. Experimental design for the optimization of lipid nanoparticles. J Pharm
Sci 98: 1813–1819.
21. Zimmermann E, Muller RH, Mader K. 2000. Influence of different parameters on reconstitution of
lyophilized SLN. Int J Pharm 196: 211-223.
350
Formulation and Evaluation of Stavudine
Loaded Niosomes and Quantitative Estimation
by HPLC
S. Latha, P. Selvamani, R. Bharathi , R. Benaseer Begam,

T. Keerthana, M. Ponmalar and K. Hari Gopala Reddy
Department of Pharmaceutical Technology &Centre for Excellence in Nanobio Translational Research,

Anna University, Bharathidasan Institute of Technology Campus,
Tiruchirappalli 620024, Tamil Nadu, India
e-mail: lathasuba2010@gmail.com, Phone: +919842598097, Fax: +914312407333
Abstract:
Stavudine is the commonly used antiretroviral drug, but it is limited by toxicity and high dosing needs.
Alternative drug delivery like niosomes has been proposed to overcome these drawbacks. Ether injection
method prepares stavudine niosomes, and the formulated Niosomes were characterized by parameters like
surface morphology, particle size analysis, zeta potential, polydispersity index, invitro studies and stability
studies. The results indicate that sonicated niosomes of tween-80 were in the size range of 1-100µ and
sonicated niosomes formulated with span-60 had a mean diameter of 95.98 nm and 75.41 nm. Encapsulation
efficiency of F4 formulation was found to be 74.73% of drugs entrapment and F8 formulation having 68.93%
of drug entrapment. Stavudine niosomes of F4 formulation with span-60 entrapped high amounts of drug
and enhanced drug release for a longer time (88.72% over 12 hrs). The mechanism of release from span-60
formulation was the Fickian type whereas F8 formulation of tween-80 shows 90.22% of drug release over a
period of 12 hrs with non-Fickian type of diffusion and both the formulations were obeyed zero-order release
kinetics.
INTRODUCTION
Novel Drug Delivery System (NDDS) are developed to overcome the limitations of conventional drug
delivery. To prevent harmful side effects and increase drug bioavailability drug carriers are used. The carriers
may be natural or synthetic polymers, microcapsules, lipoproteins, liposomes and micelles. Stavudine is
widely used for the treatment of AIDS and related conditions, either alone or in combination with other
antiviral agents. This virustatic drug has a low half-life due to considerable first pass metabolism, thus
necessitating frequent administration of large doses (30-40 mg twice daily) to maintain therapeutic
drug level. Niosomes of Stavudine provides the prolonged release of a single dose, thereby minimizing
the frequent administration and hence total dose required to elicit pharmacological activities, thereby
reducing the side effects. Niosomes are vesicular system similar to liposomes that can be used as carriers
of amphiphilic and lipophilic drugs since it is less toxic and improve the therapeutic index of Drugs [1, 2].
It may act as a depot, releasing the drug in a controlled manner. Also, niosomes are osmotically active and
increase the stability of entrapped Drug [3]. Niosomes are widely used carrier since liposomes exhibit certain
disadvantages as they are expensive and have phospholipids that are chemically unstable because of their
predisposition to oxidative degradation. Niosomes are prepared from uncharged single chain surfactant and
cholesterol whereas liposomes are prepared from double chain phospholipids [4]. The various methods for
the preparation of niosomes are ether injection method, Hand shaking method, sonication, micro fluidization,
multiple membrane extrusion methods, reverse phase evaporation technique, the bubble method and also
from proniosomes. Of these ether injections, method is widely used [5, 6].
S.NO Chemicals Manufacturing company Grade

1 Surfactants span 60, tween 80 Loba chemie laboratory reagents and fine AR
chemicals, mumbai
2 Cholesterol Loba chemie laboratory reagents and fine AR
chemicals, mumbai
3 Di acetyl phosphate Loba chemie laboratory reagents and fine AR
chemicals, mumbai
4 Phosphate buffer saline (PBS) SD fine chem-limited, mumbai AR
5 Diethyl ether Sigma alrich chemicals-Pvt Ltd, bangalore AR
6 Methanol SD fine chem-limited, mumbai HPLC
7 Chloroform SD fine chem-limited, mumbai AR
8 Acetonitrile Sigma alrich chemicals-Pvt Ltd, bangalore HPLC
Ether injection method
Niosomes containing Stavudine [7, 8, 9] was prepared by modified ether injection technique using non-ionic
surfactant (span-60, span-20) and cholesterol at different concentrations. Diacetyl phosphate, cholesterol and
surfactant were dissolved in 6ml diethyl ether and Stavudine is dissolved in methanol. These two solutions
were mixed and was slowly injected using a micro syringe at a rate of 1ml/sec. The solution was stirred
continuously on magnetic stirrer and temperature was maintained at 60-65 °C. As the lipid solution was
injected slowly into an aqueous phase, the difference in temperature between phases cause rapid vaporization
of ether, resulting in spontaneous vesiculation and formation of niosomes. The niosomes vesicles formed
were stored at refrigerated condition.
Formulation Surfactant Drug : Surfactant: Weight taken(mg)

Cholesterol Drug Surfactant Cholesterol
F1 1:1:1 30 30 30
F2 1:1.5:1.5 30 45 45
Span 60
F3 1:2:2 30 60 60
F4 1:2:2.5 30 75 75
F5 1:1:1 30 30 30
F6 1:1.5:1.5 30 45 45
Tween 80
F7 1:2:2 30 60 60
F8 1:2:2.5 30 75 75
352

Preformulation Studies
Compatibility Studies
FT-IR spectrum of physical mixture

The region 3375 cm-1 is a stretching region of the functional group N-H, At 2079 cm-1 there is a sharp
absorption peak that represent C-H stretching in (CH3). The region 1637 cm-1 shows a sharp peak which
confirms as C=O stretching of an aromatic structure, hence the physical mixture of Stavudine was compatible
with the ingredients.
CHARACTERISATION PARAMETERS
Surface Morphology
TEM image of Sonicated F4 Formulation

TEM images of niosomes prepared by ether injection method were spherical and smooth surface with
unilamellar in structure. The sonicated span-60 vesicles were nano-size with a mean diameter of 95.98 nm
and 75.41 nm respectively.
353
Particle Size Analysis
Particle Size of Sonicated F4 Formulation
Particle size of Sonicated F7 Formulation
354
Particle size of Sonicated F8 Formulation

The sonicated F3 and F4 formulations were in nano-range of particle size “95.98 and 79.4” and the sonicated F7,
and F8 formulation shows micron-size of particles, i.e., Vesicles of tween-80 formulations were larger than span-60
formulation. This suggests that when the hydrophilicity of the surfactant increases the vesicle size also increases.
Zeta Potential
Zeta potential of F8 Formulation

Few ml of the formulated suspension diluted with nano pure water and the zeta potential was measured. The magnitude
of the zeta potential values gives an indication about the stability of the formulation. The limit for the zeta potential value
of the particle in the formulated suspension was above +30 below -30. The both formulation shows an accepted value
for good stability. The both F4 and F8 formulation were stable up to 28 days due to the addition of diacetyl phosphate
(DCP). It acts as a stabilizer and prevents the aggregation by inducing charge to the particle. Particle and the Zeta
potential value for the formulation F8 was -5.67 mV, shows that the formulation was stable.
ENTRAPMENT EFFICIENCY
Percentage of Entrapment Efficiency

The Entrapment Efficiency was determined by separating the unentrapped drug using Centrifugation. Entrapment
efficiency for niosomes prepared with tween 20 was lesser than that with span-60. This is due to tween-80 contains
longer saturated alkyl chain; hence lesser drug will be entrapped. The entrapment efficiency of span-60 was higher
than tween-80. The niosomal formulation having low cholesterol content was found to cause low entrapment
efficiency, which might be because of leakage of the vesicles. These results can be explained by the fact that an
increase in cholesterol content resulted in micro-viscosity of the membrane indicating more rigidity of the bilayers.
S.No Entrapment efficiency %

F1 23.65
F2 52.76
F3 68.93
F4 74.73
F5 60.77
F6 31.57
F7 47.21
F8 68.93
355
Drug Release
Comparative Drug release of all Formulations

The tween-80 with cholesterol follows zero order kinetics and the Higuchi plot correlation coefficient confirms
the drug release was proportional to the square root of time indicating the Stavudine release from niosomes
was diffusion controlled. The n value from the Korsmeyer-peppas model for Stavudine niosomal formulation
was between 0.462 and 0.577 which confirms the F4 formulation of span-60 follows Fickian type diffusion,
whereas tween-80 formulations follows an anomalous diffusion mechanism with erosion. Release profile
Hixson-crowel model further confirm that drug release from niosomes followed anomalous diffusion.
STABILITY STUDIES
Assay by HPLC
HPLC peak of F4 formulation
No. R.T. Ht. Area Ht. % Area % Pk Ty Area/Ht
1. 1.46 0 7865 0.0000 0.1615 BB 0.103
2. 4.17 20203 3158964 100.0000 99.8385 BB 0.164

2e+04 3158964
SUMMARY AND CONCLUSION

Stavudine niosomes were successfully formulated with span and tween surfactants by ether injection method.
The sonicated F3 and F4 formulations were in nanoparticles, i.e., and vesicles of tween-80 formulations
356
were larger than span-60 formulation. This suggests that when the hydrophilicity of the surfactant increases
the vesicle size also increases. Entrapment efficiency for niosomes prepared with tween-80 was lesser
than that with span-60. This is due to tween-80 contains longer saturated alkyl chain. Hence, lesser drug
will be entrapped. The length of the alkyl chain influence the hydrophilic-lipophilic balance(HLB) value
of the surfactant and the lower the HLB value of surfactant the lower will be entrapment efficiency. The
entrapment efficiency of span-60 was higher than tween-80. When comparing the release of tween-80 with
span-60 formulations, the unsaturation in tween-80 was responsible for the higher rate of release. This is in
accordance with the concept that the unsaturation in the chain increases chain fluidity and permeability. The
best formulation F4 shows 88.72% of drug release in 12 hrs and F8 was found to give a cumulative release
of 90.22% over a period of 12hrs. The invitro release data was applied to various kinetic models to predict
the drug release mechanism. The F4 formulation shows Fickian diffusion type of drug release mechanism.
F8 formulation of tween-80 shows non-fickian type of diffusion and both the formulations were obeyed zero-
order release kinetics. HPLC determined the amount of drug present in niosomes.
REFERENCE
1. Azmin M.N., Florence A.T., Haandjani-Vila R.M., Stuart J.F.B., Vanlerberghe G. and Whittaker J.S.,
The effect of non-ionic surfactant vesicle (niosome) entrapment on the absorption and distribution of
methotrexate in mice. J. Pharm. Pharmcol.1985; 37: 237-242.
2. Sheena I.P., Singh U.V., Kamath R., Uma Devi P. and Udupa N., Niosomal withaferin A, with better
tumor efficiency. Indian J. Pharm. Sci. 1998; 60(1):45-48.
3. McCormack B. and Gregordias G., Drugs-in-cyclodextrins-in-liposomes: an approach to controlling the
fate of water insoluble drugs in vivo. Int. J. Pharm. 1998; 162: 59-69.
4. Baillie A.J., Florence A.T., Hume L.R, Rogerson A., and Muirhead G.T., The preparation and properties
of niosomes non-ionic surfactant vesicles. J. Pharm Pharmcol. 1985; 37(12):863-868.
5. Chandraprakash K.S., Udupa N., Uma Devi P. and Pillai G.K., Pharmacokinetic evaluation of surfactant
vesicles containing methotrexate in tumor-bearing mice. Int. J. Pharma. 1990; R1-R3: 61.
6. Rogerson A., Cummings J., Willmott N. and Florence A.T., The distribution of doxorubicin in mice
following administration in niosomes. J Pharm Pharmacol. 1998; 40(5): 337-342.
7. Hu C. and Rhodes D.G., Proniosomes: a novel drug carrier preparation. Int. J. Pharm. 1999;185:23-35.
8. B.L. Silver Ed., The Physical Chemistry of Membranes, Alan & Unwin and Soloman Press. New York,
USA.1985; p.209-230.
9. Stafford S., Baillie A.J. and Florence A.T., Drug effects on the size of the chemically defined non-ionic
surfactant vesicles. J. Pharm.Pharmcol. 1998; 40(suppl.): 26p.
10. Targeted and Controlled Drug Delivery novel carrier system-S.P. Vyas and R.K. Khar.
11. Ruth Duncan Niosomes containing N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin
(PK1): effect of the method of preparation and choice of surfactant on niosome characteristics and a
preliminary study of body distribution, International Journal of Pharmaceutics, Volume 155, Issue 1,12
September 1997, Pages 7-17
12. Kandasamy Ruckmani Formulation and Optimization of Zidovudine Niosomes, American Association
of Pharmaceutical Sciences Pharmasci Tech, July 16, 2010.
357
Optimization and Formulation of Drug Loaded Magnetic
Microcapsules
C. Prabu, S. Latha and P. Selvamani
Department of Pharmaceutical Technology & Centre for Excellence in Nanobio Translational Research, Anna
University, Bharathidasan Institute of Technology Campus, Tiruchirappalli 620024, Tamil Nadu, India
e-mail:lathasuba2010@gmail.com, Phone: +919842598097, Fax: +914312407333
Abstract:
A method was developed for the preparation of magnetic nanoparticles using oleic acid as stabilizer with
Pluronic F-127 and was drug loaded to the nanoparticles. Then magneticnanoparticles were loaded on to
the prepared porous calcium carbonate microparticles. Formulation was optimized by using Box Bennken
experimental design using the factors concentration of Pluronic and Oleic acid. Drug used to get response
asmaximize encapsulation efficiency, drug loading and to minimize burst release. The optimized formulation
gives exceptional response then characterization by using particle size analysis, XRD, encapsulation
efficiency, drug loading and in-vitro drug release and its release upto 36 hours. Thus the prepared formulation
is suitable for sustained release.
Keywords: Magnetic nanoparticles, Optimization, Encapsulation efficiency, Drug loading.
INTRODUCTION
Hollow capsules have attracted great interest in recent years for their potential applications in medicine,
pharmaceuticals, artificial cells or viruses, cosmetics, food and catalysts [1–5]. Polyelectrolyte microcapsules
are produced by stepwise adsorption of oppositely charged polymers onto the surface of colloidal particles
followed by core dissolution, which is called LbL (Layer by Layer) technique [6–11]. Using this approach, a
variety of materials, including charged and uncharged species, have been successfully assembled into nanoscale
multilayered structures. The most used polymers are commercially available synthetic polyelectrolytes
(PEs) such as PSS, PAH, PDADMAC and shells based on other synthetic polyelectrolytes such as poly
(ethyleneimine) or Nafion have been synthesized. Particularly, some naturally occurring polyelectrolytes,
such as polysaccharides are eye-catching. Recently, biocompatible polyelectrolytes of carrageenan [12],
dextran sulfate (DXS) [13], chitosan/chitosan sulfate [14], sodium alginate, and carboxymethyl cellulose
[15] were reported. The CaCO3 microparticles prepared by this method have a large number of nanopores,
which provide a capillary force to load substances regardless of their charging properties or hydrophilicity
[16]. Shortly, they prepared capsules that showed a unique feature of charge-controlled permeation for
electrolytes [17]. It is a highly efficient method and can be easily scaled up [18].were all negatively Charged
and nonbiodegradable. Surface charge of polyelectrolyte capsules is an important parameter that exhibits
essential influence on their properties and functionalities [19, 20]. The surface charge of microcapsules
fabricated by LbL protocol employing electrostatic interaction was determined by polyelectrolyte in the
outer layer and can be altered by further assembly procedure after the capsules were already formed [20].
Moreover, good biocompatibility and biodegradability are the essential prerequisites for capsule application
in biomedical fields [21] [A facile pathway to prepare] Response surface methodology (RSM) as a tool of
designing of experimentsis widely practiced approach in the development and optimization of drug delivery
devices [22–27]. The technique requires minimum experimentation and time, thus proving to be farm or
eeffective and cost effective than the conventional methods of formulating dosage forms.
Preparation of calcium carbonate microparticles

Porous CaCO3 microparticles were prepared by rapid mixing of an equimolar concentration of CaCl2.2H2O
and Na2CO3 aqueous solutions in equal volume. Typically, 0.33M CaCl2 was rapidly poured into an equal
volume of 0.33M Na2CO3 solution (containing 4 g/L Polystyrene sulfonate sodium) at room temperature.
After vigorous agitation with aultrasonication, the precipitate was filtered off, thoroughly washed with pure
water and dried in air.
Synthesis of Drug loaded Magnetic Nanoparticles

Stable Fe3O4 ferrofluid was synthesized by co-precipitation method using Fe (II) and Fe (III) salts. A solution
of FeCl3.6H2O and FeCl2.4H2O in 2:1 ratio was then quickly charged and mixed with 15 ml of aqueous
ammonia solution (25%) with vigorous stirring using a mechanical stirrer, under nitrogen atmosphere
followed by adding excess aqueous ammonia solution into the mixture slowly while stirring against air until
the pH value of the solution reached 11 & black precipitate was obtained. To the Fe3O4 ferro fluid of 10 ml
was added with of Oleic acid and the temperature was raised to 90 °C then stirred for 60 minutes. Then the
mixture was neutralized while cooling and then the magnetic particles were prepared. Further, Pluronic
F-127 was added and stirred for 12 hours to this was added with drug and the reaction mixture stirred for 12
hours.
2FeCl3+FeCl2+8NH3+4H2O Fe3O4 +8NH4Cl
Loading of Fe3O4 Nanoparticles into porous CaCO3

Before loading, CaCO3 particles were washed with acetone and dried in a vacuum oven at 50°C. 200 mg of
dried CaCO3 particles was soaked in 1ml of Fe-complex of 5 mg /ml in ethanol in closed batch to prevent
the evaporation of liquid. Incubate the loaded CaCO3 particles for 24 hours in shaker. Fe3O4 loaded CaCO3
particles were separated by using a centrifuge at 2500 rpm and remove the unloaded Fe3O4 nanoparticles.
The particles were collected and dried in a vacuum oven at 50°C.
Multilayer Fabrication of Fe3O4 Loaded CaCO3 particles by Layer by Layer Technique

Polycation protamine sulfate sodium poly (styrene sulfonate) and polyanion will be deposited on Fe-loaded
CaCO3 particles by layer by layer self-assembly in water.The first layer wasf ormed by the addition of
5ml of 1mg/ml (0.1%w/v) aqueous protamine sulfate solution containing 0.5M NaCl into 0.05gm of Fe-
loaded CaCO3 particles. The mixture was the nincubated for 15 minutes under gentle shaking. The excess
of polyelectrolyte will be removed by three repeated refined circles of centrifugation at 4000 rpm for 4
minutes washing/dispersions in water. The following sodium poly (styrene sulfonate)layer will be deposited
using the same procedure with 5ml of 1mg/ml (0.1w/v) solution with 0.5M NaCl. Subsequently alternate
polyelectrolyte layers will be deposited in the identical way until five layers was coated. CaCO3-PSS
microparticles were incubated in 0.2M of EDTA solution pH7.0 for 30 min in order to dissolve the CaCO3-
PSS core. And then centrifuged to get magnetic hollow micro capsules.
Optimization for the formulation was carried out using Box Bennken experimental design
Optimization was performingusingthedesirability Box – Behnken design was applied in this study to optimize
the formulation. Function to obtain the constraints X1, X2, X3 used to maximize Encapsulation Efficiency, drug
loading and to minimize burst release. In order to obtain a formulation having a higher RSM optimization was
used to determine the level of the factors. Response surface optimization is more advantages than the traditional
single parameter optimization in save time, space and raw material. The data was analyzed by multiple regression
analysis using version 7.0. Use of the Response Surface Methodology (RSM) has been proved to be a useful
tool in the development and optimization of sustained release formulations. Different steps involved in response
surface methodology include experimental design, regression analysis, constraint optimization and validation.
Response surface methodology (RSM) is an advanced tool, commonly applied, involves three factorial designs
359
giving number of input (independent) factors and their corresponding relationship between one or more measured
dependent responses. RSM is advantageous over conventional methods available, and it includes fewer numbers
of experiments. It is suitable for multiple factor experiments and searches for common relationship between
various factors towards finding the most suitable conditions for the processes. In this method, linear or quadratic
effects of experimental variables construct contour plots and a model equation fitting the experimental data. This
facilitates the determination of optimum value of factors under investigation and prediction of response under
optimized condition.
Factor X1 - Pluronic, Factor X2 - Oleic acid, Factor X3 – Prednisolone
Table.1 .A three factor, three level Box-Behnken design was applied for the optimization procedure.
IndepententVaribles Low Middle High

Factor X1 400 600 800
Factor X2 1.4 1.6 1.8
Factor X3 300 450 600
Factor X1 - Pluronic, Factor X2 - Oleic acid, Factor X3 -Drug

Dependent Variables Response 1 = Encapsulation Efficiency, Response 2 = Drug Loading, Response 3
= Burst Release.
Preparation of sample
Based on the experimental design generated factor combination resulted in different responses. There were
a total seventeen different runs for optimizing the three individual parameters in the current Box- Behnken
design. Consequently to the data generated and the responses for all the formulation were carried out.
Table.2: Variables and Observed Responses in Box-Behnken Design
Run Factor 1 A– Factor 2 B- Oleic Factor 3 Response 1 Response Response 3

Pluronic mg acid Ml C-Drug Mg EE % 2DL % BR %
1 0 1 -1 76.3 16.3 9.2
2 0 0 0 84.2 24.3 5.8
3 -1 0 -1 67.8 10.3 4.3
4 0 0 0 84.2 24.3 5.8
5 0 -1 1 74.6 15.3 9.4
6 1 0 -1 73.9 17.8 3.7
7 0 0 0 84.2 24.3 5.8
8 -1 1 0 73.5 15.9 5.8
9 -1 -1 0 69.5 16.7 2.9
10 -1 0 1 75.5 13.6 5.6
11 0 -1 -1 60.3 15.7 3.9
12 1 0 1 90.3 31.5 9.3
13 1 -1 0 75.3 17.8 3.8
14 1 1 0 90.35 30.4 5.9
15 0 1 1 69.3 12.3 8.6
16 0 0 0 84.2 24.3 5.8
17 0 0 0 84.2 24.3 5.8
Particles Size Analysis

Measurement of Particle sizeand PI of microparticles was performed by using photon correlation
spectroscopy (PCS) (Malvern Sizer 1000HS). Microparticles were diluted with 1mL of deionized water
360
to eliminate the effect of viscosity caused by the ingredients. Particle size was obtained as the average of
three measurements at 25°C [28].
X- ray diffraction studies

X- ray diffraction (XRD) patterns of the CaCO3-PSS microparticles were recorded on a RigakuUltima III,
USA powder diffraction system using Cu Kα radiation of 0.15406 nm wavelength at a scanning rate of 0.02
°/s in the 2Ө range of 10-80° [29,30].
Encapsulation efficiency and Loading capacity
Percent drug loading

The percentage of drug loading was estimated by using the formula Percent drug loading = Total Drug -free
Drug / Total particle weight X 100 Where, total prednisolone is the initial weight of the drug dissolved in the
solution and free Drugg is the weight of the drug in the aqueous media measured immediately after preparing
the drug loaded CaCO3 particles by using the absorbance of UV spectroscopy, total particle weight is the
total amount of CaCO3 particles used for the formulation.
Percentage Encapsulation Efficiency

Percent Encapsulation Efficiency = Total Drug -free drug / Total drug X 100 Where, total prednisolone is
the initial weight of the drug dissolved in the solution and free Drug is the weight of the drug in the aqueous
media measured immediately after preparing the drug loaded CaCO3 particles by using the absorbance of
UV spectroscopy [31].
In-vitro release studies

In-vitro release studies of the drug have been carried out in triplicate by placing drug loaded CaCO3 particles
in definite volume of releasing medium at 37 °C temperature in the intestinal pH conditions using 7.4 pH
phosphate buffers, respectively under gentle stirring. The amount of drug released in 7.4 pH buffer solutions
was measured by UV spectrophotometer, at the wavelength of 244nm.The drug release was measured after
a fixed interval of time and release dynamics were studied [32].

Response Surface Methodology graphs for the effects of Pluronic, Oleic acid and Drug on enhancement of
Encapsulation Efficiency, Drug Loading and Minimizing Burst Release.
Fig. 1: Effect of oleic acid and pluronic in burst release
361
Fig. 1: represents the when concentration of the oleic acid is increased the burst release also increased
where as the con pluronic shows the decreased when the con of the pluronic was also high. at the same time
where factor X3(drug) was kept constant.
Fig. 2: Effect of oleic acid and pluronic in drug loading
When concentration of the oleic acid is increased the drug loading decreased where as the con pluronic
is increased in an average shows the increase in drug loading.
Fig. 3: Effect of oleic acid and pluronic in Encapsulation efficiency,
when concentration of the oleic acid is increased the EE increased when it ia at optimum level. As same
as the as oleic acid pluronic also has the same effects in EE.
Effect of Formulation parameters on drug loading

The Perturbation plot shows the effect of all factors (pluronic, Oleic acid and prednisolone) on the drug
loading. Pluronic and drug have increased the drug loading when their levels increased where as oleic acid
exhibit negative effect.
Effects of pair of factors such as A, B; A, C and B, C on drug release have been displayed in Figure
while keeping other factors fixed at central level. The contour plot and response surface plots clearly exhibit
retarding effect of factor (A) on drug release at any level of B, C on drug release. The factors that control EE
are found to be controlled drug loading.
362
The effect of pluronic (A) and concentration of oleic acid (B) and their interaction on drugrelease (Y) at
fixed level of emulsifier are given in figure. It was showed that at a lower level of Pluronic (A), percentage
of drug loading was decreased from 15.7 to 10.3% when the amount of oleic acid (B) increases similarly at
high level of pluronic (A), and % drug loading decreased to17.8%.
Lower level of pluronic (A), an increase in Drug (C) concentration from low to high level resulted in an
increase in the percentage of drug loading from 15.6.72 to 30.3.
Effect of oleic acid (B) and Drug (C) and their interaction on drugrelease (Y) at fixed level of pluronic
weregiven in showed that at lower level of oleic acid, the percentage of drug loading increases from 13.6 to
15.3%.
Table.3: Predicted and Experimental Values of the Responses at Optimum Conditions
Batch X1 X2 X3 Y1 Y2 Y3 Predicted error

mg Ml mg Predicted Experimental Predicted Experimental Predicted Experimental Y1 Y2 Y3
1 600 1.6 450 84.2 83.5 24.3 23.7 5.8 8.6 -0.007 -0.006 0.028
2 600 1.6 450 84.2 80.6 24.3 25.2 5.8 6.1 -0.036 0.009 0.003
3 600 1.6 450 84.2 82.2 24.3 22.6 5.8 9.7 -0.020 -0.017 0.039
Table.4: Optimized valuesfor prepared drug loaded magnetic microcapsules.
Batch X1 X2 X3 Y1 Y2 Y3 Desirability
Mg ml mg Predicted Experimental Predicted Experimental Predicted Experimental Y1 Y2 Y3
1 600 1.6 450 84.2 82.2 23.8 23.7 5.8 8.1 -0.021 -0.010 0.07
From the response surface and perturbation plots it is obvious that pluronic concentration had a significant
effect on as compared to other variables. The studies of 3D surface plot also reveal the optimal values. The
experimental data of three batches prepared within optimum range were very close to the predicted values
with low percentage error suggesting that the optimized formulation was reliable.
Particle Size Analysis of drug loaded magnetic nanoparticles.

Drug loaded magnetic nanoparticles were prepared. They in the size range of 128 nm (fig. 4.) Oleic acid as
the stabilizers plays the important role in the stability of magnetic nanoparticles. And pluronic used does not
increase the size of the prepared nanoparticles.
Fig. 4: Particle size analysis for drug loaded Fe3O4 Loaded nano particles
363
X-ray diffraction studies

The characteristic diffraction peak were observed at, it could be indexed to the typical calcite structure of
CaCO3 particles, however, the characteristic diffraction peak of Fe3O4 nanoparticles were not found, and this
phenomenon can be interpreted that the strong characteristic diffraction peak of CaCO3 covered up the weak
diffraction peak of Fe3O4. In contrast characteristic diffraction peaks of CaCO3 were not fully detected which
implies that the CaCO3 particles with Fe3O4are remained.
Encapsulation efficiency and loading capacity

The encapsulation efficiency and loading capacity was found to be 82.2 % and 23.7% respectively.
In-vitro Drug release studies

Release has been carried for the formulation in dialysis bag with 2ml of the formulation in the phosphate
buffer with the pH of 7.4 samples were taken at the fixed times intervals and release of the drug extended
upto 36 hours in the sustained manner(fig. 5.) The drug release behavior obeys a diffusion mechanism.
Fig. 5: Cumulative drug release for Fe3O4 Loaded CaCO3 microcapsules
CONCLUSION
In this work preparation of magnetic nanoparticles was prepared and oleic acid as stabilizer with Pluronic F-127
and drug was loaded to the nanoparticles. Formulation was optimized by using Box Bennken experimental
design using the factors concentration of Pluronic, Oleic acid,Drug used to get response asmaximize
Encapsulation Efficiency, drug loading and to minimize burst release. And the optimized formulation gives
exceptional response then characterization by using particle size analysis, XRD, encapsulation efficiency,
drug loading and in-vitro drug release and its release upto 36 hours. Thus the prepared formulation is suitable
for sustained release.
REFERENCE
1. Majeti N and Kumar VR, Nano and microparticles as controlled drug delivery devices. J. Pharm. Pharm.
Sci. 2000; 3: 234–258.
2. Brannonpeppas L, Controlled release in the foods and cosmetics industries. In: Polymer delivery
systems. ACS Sym Ser. 1993;520: 42–52.
364
3. Wong MS, Cha JN, Choi KS, Deming TJ and Stuchy GD, Assembly of nanoparticles into hollow spheres
using block copolypeptides. NanoLett. 2002;2: 583–587.
4. Jiang P, Bertone JF and Colvin VL, A lost-wax approach to monodisperse colloids and their crystals.
Science 2001;291: 453–457.
5. Zhong ZY, Yin YD, Gates B and Xia YN, Preparation of mesoscale hollow spheres of TiO2 and SnO2
by templating against crystalline arrays of polystyrene beads. Adv Mater 2000;12:206–209.
6. Shchukin DG, Patel AA, Sukhorukov GB and Lvov YM, Nanoassembly of biodegradable microcapsules
for DNA encasing. J Am Chem Soc. 2004;126: 3374–3375.
7. Decher G, Fuzzy nanoassemblies: toward layered polymeric multicomposites. Science. 1997; 277: 1232.
8. Wang C, Ye S, Dai L, Liu X and Tong Z, Enhanced resistance of polyelectrolytemultilayer microcapsules
to pepsin erosion and release properties of encapsulated indomethacin. Biomacromolecules. 2007; 8:
1739.
9. Quinn A, Tjipto E, Yu A, Gengenbach TR and Caruso F, Polyelectrolyte blend multilayer films: surface
morphology,wettability, and protein adsorption characteristics. Langmuir. 2000; 23: 4944.
10. Li Z, Lee D, Rubner MF and Cohen RE, Layer-by-layer assembled janus microcapsules. Macromolecules.
2005; 38: 7876.
11. Glinel K, Déjugnat C, Prevot M, Scholer B, Schonhoff M and Klitzingd RV, Responsive polyelectrolyte
multilayers. Colloids Surf. A: Physicochem. Eng. Aspects. 2007; 303: 3.
12. Wang ZJ, Qian L, Wang XL, Yang F and Yang XR, Construction of hollow DNA/PLL microcapsule as
a dual carrier for controlled delivery of DNA and drug. Colloids Surf. A: Physicochem. Eng. Aspects.
2008; 326: 29.
13. Bartkowiak A and Hunkeler D, Carrageenan–oligochitosan microcapsules: optimization of the formation
process. Colloids Surf. B: Biointerfaces. 2001; 21: 285.
14. Georgieva R, Moya S, Hin M, Mitlöhner R, Donath E, Kiesewetter H, Möhwald H and Pumler HB,
Permeation of macromolecules into polyelectrolyte microcapsules. Biomacromolecules. 2002; 3: 517.
15. Berth G, Voigt A, Dautzenberg H, Donath E and Möhwald H, Polyelectrolyte complexes and layer-by-
layer capsules from chitosan/chitosan sulfate, Biomacromolecules. 2002; 3: 579.
16. Qiu XP, Leporatti S, Donath E and Möhwald H, Studies on the drug release properties of polysaccharide
multilayers encapsulated ibuprofen microparticles, Langmuir. 2001;7: 5375.
17. Wang CY, He CY, Tong Z, Liu XX, Ren BY and Zeng F, Combination of adsorption by porous CaCO3
microparticles and encapsulation by polyelectrolyte multilayer films for sustained
18. drug delivery, Int. J. Pharm. 2006; 308: 60-16.
19. Wang F, Tong WJ, Li J and Gao CY, In situ coacervated microcapsules with filled polyelectrolytes and
charge-controlled permeation for dye molecules, Macromol. Chem. Phys. 2008; 209: 957–966.
20. Wang F, Feng J,Tong W and Gao CY, A facile pathway to fabricate microcapsules by in situ polyelectrolyte
coacervation on poly(styrene sulfonate)-doped CaCO3 particles, J. Mater. Chem. 2007; 17: 670–676.
21. Peyratout CS and Dahne L, Tailor-made polyelectrolyte microcapsules: From multilayers to smart
containers, Angew. Chem. Int. Ed. 2004; 43: 3762– 3783.
22. Wang KW, He Q, Yan XH, Cui Y, Qi W, Duan L and Li JB, Encapsulated photosensitive drugs by
biodegradable microcapsules to incapacitate cancer cells, J. Mater. Chem. 2007; 17: 4018–4021.
365
23. De Geest BG, Vandenbroucke RE, Guenther AM, SukhorukovGB,Hennink WE, Sanders NN,
Demeester J and De Smedt SC, Intracellularly degradable polyelectrolyte microcapsules, Adv. Mater.
2006; 18:1005–1009.
24. Lewis GA, Mathieu D and Phan-Tan-Luu R, Pharmaceutical Experimental Design, Marcel Dekker,
New York, NY, USA, 1961.
25. Singh B and Ahuja N, Book Review on Pharmaceutical Experimental Design, 1999.
26. Nelson KG and Wang LY, Journal of Pharmaceutical Sciences. 1978; 67: 86–89.
27. Singh B, Dahiya M, Saharan V and Nahuja N, Critical Reviews in Therapeutic Drug Carrier Systems.
2005; 22: 215–293.
28. Singh B, Mehta G, Kumar R, Bhatia A, Ahuja N and Katare OP, Current Drug Delivery. 2005;2: 143–
153.
29. Aberturas MR, Molpeceres J, Guzm M and Garc F, Journal of Microencapsulation. 2002; 19: 61–72.
30. Ghassabian S, Ehtezazi T, Forutan M and Mortazavi SA, Dexamethasone loaded magnetic albumin
microspheres preparation and in-vitro release, Int. J. Pharm.1996; 130: 49-66.
31. Volodkin DV, Larionova NI and Sukhorukov GB, Protein Encapsulation via Porous CaCO3 Microparticles
Templating, Biomacromolecules. 2004; 5: 1962-1972.
32. Volodkin DV, Petrov AI, Prevt M and Sukhorukov GB, Matrix Polyelectrolyte Microcapsules: New
System for Macromolecule Encapsulation, Langmuir. 2004; 20: 3398-3406.
33. L.N. Ramana, S. Sethuraman, U. Ranga and U. M. Krishnan, Development of a liposomal nanodelivery
system for nevirapine, J. Biomed. Sci. 2010; 17: 1-9.
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Formulation and Characterization of Carisoprodol
Magnetic Nano-Emulsion for Rheumatoid Arthritis
A. Stephen Prawin Kumar, G. Phonnavan, G. VinothKumar, M. Neelumathi,

P. Selvamani and S. Latha
Department of Pharmaceutical Technology & Centre for Excellence in Nanobio Translational Research,
Anna University, Bharathidasan Institute of Technology Campus, Tiruchirappalli 620024, Tamil Nadu, India
e-mail:lathasuba2010@gmail.com, Phone: +919842598097, Fax: +914312407333
Abstract:
The aim of the present study is to formulate magnetic nano-emulsion using carisoprodol for treating
Rheumatoid Arthritis; this was intended in the sense to improve the bioavailability and half-life
and for targeting the inflammation using magnetite. Nutmeg oil, span-80, magnetite, tween-20
were used to formulate magnetic nano-emulsion. The method used for the formulation of magnetic
nano-emulsion is spontaneous emulsification method. The emulsification was achieved effectively
by using ultra sonicator. The globule size of the magnetic nano-emulsion is 256 nm. The In-vitro
release was determined, and they achieved the release of about 0.939 respectively, and the release
kinetics was performed.
INTRODUCTION
Carisoprodol acts as a sedative and skeletal muscle relaxant. Rather than acting directly on skeletal
muscle, carisoprodol interrupts neuronal communication within the reticular formation and spinal
cord, resulting in sedation and alteration in pain perception. Nano-emulsion is a group of dispersed
particles used for pharmaceutical, biomedical aids and biomedical aids and vehicles that show
great promise for the future of cosmetics, diagnostics, drug therapies, and biotechnologies. Nano-
emulsion can be defined as oil-in-water (O/W) emulsions with mean droplet diameters ranging
from 50 to 1000 nm. Usually, the average droplet size is between 100 and 500 nm. The particle
can exist as water–in–oil and oil in water forms, where the core of the particle is either water or
oil, respectively. Usually, Submicron emulsions SME contain 10-20 % oil stabilized with 0.5 to
2 % egg or soya bean lecithin. Nano-emulsions are made from surfactants approved for human
consumption and common food substances that are ‘Generally recognized as safe’ (GRAS) by
the FDA [1]. Magnetic targeting of therapeutic agents to specific sites in the body enjoys certain
advantages over other drug delivery methods since the risk of toxicity is reduced, and effectiveness
of treatment is increased.
Spontaneous Emulsification Method

Nano-emulsions consist of fine oil-in-water dispersions, having droplets covering the size
range of 100-600 nm. In the present work, nano-emulsions were prepared using the spontaneous
emulsification mechanism which occurs when an organic phase and an aqueous are mixed. The
organic phase is a homogeneous solution of oil, lipophilic surfactant and water. An experimental
study of nano-emulsion process optimisation based on the required size distribution was performed
in relation with the type of oil, surfactant and the water-miscible solvent. The results showed that
the composition of the initial organic phase was great importance for the spontaneous emulsification
process, and so for the physicochemical properties of the obtained emulsions. First, oil viscosity
and Hydrophilic Lipophilic Balance (HLB) surfactants were changed, nutmeg, the most viscous oil,
gave the smallest droplets size (171±2nm), HLB required for the resulting oil-in-water emulsion
was superior to 8. Second, the effect of water-solvent miscibility on the emulsification process
was studied by decreasing acetone proportion in the organic phase. The solvent-acetone proportion
leading to a fine nano-emulsion was fixed at 15/85% (v/v) with EtAc-acetone and 30/70% (v/v)
with Methyl Ethyl Ketone-acetone mixture. The choice pf solvents, physical characteristics, the
auto-ignition temperature and flash point were compared. This phase of emulsion optimisation
represents an important step in the process of polymeric magnetic nano-emulsion preparation with
spontaneous emulsification technique [2].
List of chemicals and materials used

Chemicals
Carisoprodol, Tween 20, Span 80, Nutmeg Oil, Iron (II, III) Oxide.
Equipments
Magnetic Stirrer, Ultrasonicator, UV spectrophotometer, Electronic Balance, Light Microscope, Transmission
Electron Microscope, Stability Chamber, Transmission Electron Microscope, Refrigerator, Cyclomixer
EXPERIMENTAL WORKS:
FORMULATION OF CARISOPRODOL MAGNETIC NANO EMULSION:
Carisoprodol (4mg) was thoroughly dissolved in Nutmeg oil (3ml). Span 80 (2ml) is added to the drug and
oil mixture as emulsifier, and this constitutes the oil phase. Tween 20 (2.5ml) was added to the ultrapure
water. Then magnetite was added to it and dissolved, this constitutes the water phase. The oil phase is added
drop by drop to the water phase and stirred with the aid of mechanical stirrer for 6 hours. This will result
in the formation of a primary emulsion. Then for reducing the droplet size it was sonicated for 10min with
pulse ON OFF for 10:3 sec. Then it was made up the required volume with ultrapure water and stored in a
light-resistant container.
EVALUATION OF MAGNETIC NANO EMULSION

Dye Solubility Test
A coloured dye soluble in any one of the component i.e. in water or oil was added to the emulsion. If the
colour spreads throughout the emulsion, the phase in which the dye is soluble is the continuous phase. Using
the solubility of the dye the formulation type can be determined i.e. O/W or W/O emulsion [3].
●● Water soluble dye will dissolve in the aqueous phase.
●● Oil-soluble dye will dissolve in the oil phase.
Phase Dilution Test

Phase dilution test was performed in order to determine the continuous phase or type of emulsion.
Here few ml of magnetic nanoemulsion is diluted with water, and it was visually observed and
confirmed if there was any change in the homogeneous phase. This test clearly indicates the type of
emulsion to which it belongs [1].
368
●● O/W emulsion can be diluted with water.

●● W/O emulsion can be diluted with oil.
Viscosity
Viscometer equipped with a capillary tube. 10ml of magnetic nanoemulsion was poured into the filling tube
and transformed into the capillary tube by gentle suction. The time was recorded for the flow of liquid from
the upper to lower a mark in the capillary tube. The analysis was performed in triplicate, and mean value was
taken. It was generally expressed in centi Poise (cP).
Density
It was determined with the help of specific gravity bottle. The magnetic nanoemulsion was filled up to the
neck mark, and its weight was taken. Similarly, it was done using the water and weighed. The Density of the
magnetic nanoemulsion is compared with that of water. It was generally expressed as Kg/m3.
Globule size analysis

The mean particle size analysis was measured at 25°C by Photon correlation spectroscopy (PCS) using
a Malvern Nanosizer/Zetasizer nano-ZS. All analysis was done in triplicate, and each 15µL sample was
diluted in 10 ml of ultrapure water and analysed.
Surface Morphology
The microscopical nature was examined using a Transmission Electron Microscope (TEM). The TEM was
performed in order to characterize the structure of the globules of the formulated magnetic nano-emulsion
[4].
Magnetic Susceptibility
It is the extent to which it is susceptible to induced magnetization by an external field. Before doing the
experiment, it was calibrated to zero. Magnetic composite is superparamagnetic in nature as observed in the
magnetic susceptibility.
In-vitro drug release study

In vitro drug release study of carisoprodol MNE as performed in USP model Type II dissolution apparatus
(paddle) at 37.5±0.5°C with an agitation speed of 50 rpm. One ml of MNE was placed in a dialysis bag (HI-
media 2.4nm pore size), then sealed at both the ends and placed at the bottom of the vessel. The dissolution
medium used was phosphate buffer saline pH 7.2 as the dissolution medium at designed intervals for 10
h, and the absorbance was measured by UV-visible spectrophotometer at 256 nm. The concentration of
Carisoprodol was determined from the calibration graph, and the percentage of drug release was calculated
by respective concentration of standard calibration curve with the suitable retention factor (Rf).

The formulated Carisoprodol magnetic nanoemulsion was evaluated for the following parameters, and the
following results was obtained.
Dye solubility test

The dye solubility test was performed, and the dye used was crystal blue, which clearly spreads throughout
the entire emulsion droplet and thus it indicate that the water is the continuous phase, and the formulated
emulsion are indeed O/W emulsion.
369
Phase dilution test

The phase dilution test was performed, and it includes complete solubility for the emulsion in the continuous
phase (water). Therefore, it is referred that it is an O/W type emulsion.
Viscosity
The viscosity was determined by using Ostwald’s viscometer for the formulation and was found to be
-0.847 cP
Density
The density of the Carisoprodol magnetic nano-emulsion are determined by specific gravity bottle method
and the density of the magnetic nano-emulsion formulation was found to be -0.994 kg/m3
Globule size distribution

Photon correlation spectroscopy found the particle size. The particle size of the globules was measured by
Zetasizer ZS-90, and the droplet size of the formulation was found to be 256 nm.
Table 1. Globule size analysis
Sample Globule Size (Avg diameter in nm)

CMNE 256
Fig. 1: Globule Size Distribution
Z-average (d.µm): 0.256 µm

Intercept: 0.398 µm
Result quality: Good
370
SUMMARY
Table 2:
Data Value (µm)

MV 0.001
MN 0.398
MA 0.194
SD 0.0134
Surface Morphology
The microscopical nature of CMNE was examined using a Transmission Electron Microscope. The TEM
picture reveals the spherical nature of the oil globules present in the formulations.
Fig. 2: TEM picture for CMNE
Magnetic susceptibility
The magnetic susceptibility of the formulated CMNE was measured using a magnetic susceptibility meter
and it gives extent to which formulation is susceptible to induce a magnetic field by an external magnetic
field. The magnetic susceptibility of the formulation CMNE was found to be 35×10-5.
In-vitro studies
Calibration curve
5mg of carisoprodol was accurately weighed and dissolved in 10ml of phosphate buffer solution pH 7.2 and
shaken to dissolve them make up to 10ml in a standard flask to produce the concentration of 50µg/ml. Label
this solution as primary stock solution. Then withdrawn 1ml of primary stock solution and diluted to 10ml
in a standard flask to produce the concentration of 10µg/ml. This solution is considered as secondary stock
solution.
371
Preparation of working stock solution

From the secondary stock solution withdrawn 0.4ml,0.3ml,0.1ml and 1ml, and then make up the volume to
10ml with phosphate buffer solution 7.2, in order to produce the concentration ranging from 2µg/ml,4µg/
ml,6µg/ml,8µg/ml and 10µg)ml. the resulted working standard solution was measured the absorbance in
uv-Spectrophotometrically at 256nm
The drug release data obtained for carisoprodol was plotted according to different modes of data
treatment. They are
Plot 1: percentage drug to be released vs. time according to zero order kinetics.
Plot 2: Log percentage drug to be released vs. time according to first order kinetic
Table 3: Drug release kinetic studies
Time % Drug % Drug to Log % Drug Log % Log T Root T (D)1/3 % Drug
Released be released to be released Drug to be Released
released
5 7.24 92.76 0.8597 1.9673 0.6989 2.2360 4.5267 7.24
10 10.32 89.68 1.0136 1.9526 1.0000 3.1622 4.4760 10.32
15 14.07 85.93 1.1482 1.9341 1.1760 3.8729 4.4128 14.07
20 18.66 81.34 1.2709 1.9103 1.3010 4.4721 4.3327 18.66
25 21.31 78.69 1.3285 1.8959 1.3979 5.0000 4.2852 21.31
30 24.13 75.87 1.3825 1.8800 1.4771 5.4772 4.2334 24.13
35 30.23 69.77 1.4804 1.8436 1.5440 5.9160 4.1167 30.23
40 30.59 61.41 1.5864 1.7882 1.6020 6.3245 3.9452 30.59
45 43.18 56.82 1.6352 1.7545 1.6532 6.7082 3.8444 43.18
50 52.92 47.08 1.7236 1.6728 1.6989 7.0710 3.6108 52.92
55 65.03 34.97 1.8131 1.5436 1.7403 7.4161 3.2701 65.03
60 74.33 25.67 1.8711 1.4094 1.7781 7.7496 2.9499 74.33
65 81.68 18.32 1.9121 1.2629 1.8129 8.0622 2.6361 81.68
Table 4: UV Absorbance @ 256 nm
Concentration Absorbance
2 0.1544
4 0.2903
6 0.4217
8 0.5601
10 0.7090
372
Fig. 3: Calibration curve
Fig. 4: % Drug release Plot
Fig. 5: Higuchi Plot
373
Fig. 6: Korsmeyer – Pepper Plot
Fig. 7: Hixon Crowell plot
Plot 3: percentage drug released vs. square root of time according to Higuchi classical diffusion Equation.
Plot 4: Log percentage drug released vs. log time according to Korsmeyer-peppas equation. This is
an exponential equation applicable to swelling polymer. In this system, the drug will be diffused after a
considerable swelling of the polymer.
Plot 5: Cube root or percentage drug to be released vs. time according to Hixon Crowell equation,
applicable to any tablet that loses weight with time either due to erosion or dissolution.
Further, to verify whether the data satisfies the above relationship, the plots were judged by linear
regression coefficient (r2).
The data’s obtained were plotted as a graph as a according to zero order rate kinetics, percentage drug
remaining along Y-axis and time (t) along X-axis. The linear regression value was calculated and was found
to be 0.952 respectively.
The data’s obtained were plotted as a graph as a according to first order rate kinetics, log percentage drug
remaining along Y-axis and time (t) along X-axis. The linear regression value was calculated and was found
to be 0.845 respectively.
374
The data’s obtained were plotted as a graph as a according to zero Higuchi equation, percentage drug
release along Y-axis and time (t) along X-axis and square root of time (t) along X-axis. The linear regression
value was calculated and was found to be 0.890 respectively.
The data’s obtained were plotted as a graph as a according to Hixon Crowell cube root equation, cube
root of percentage drug release along Y-axis and time (t) along X-axis. The linear regression value was
calculated and was found to be 0.888 respectively.
From the above kinetic studies, it was observed and referred that the release of the drug from the magnetic
nano-emulsion follows Zero order release and Hixon Crowell equation respectively.
CONCLUSION
The main objective of the study was formulation and evaluation of MNC containing nutmeg oil, tween
20, span 80 and nano pure water has been developed. The parameters evaluated confirm that formulation
parameters are well within the acceptable range. The phase dilution studies and the dye solubility test
confirmed that the prepared emulsion is O/W nature. The TEM reveals about the microscopical nature of
CMNE i.e. the globules were almost spherical in nature. The globule size indicates that the prepared MNC
was in the nanometre range. Also density, viscosity, magnetic susceptibility, was done for the formulated
magnetic nanoemulsion, and their result was well within the accepted range for a magnetic nano-emulsion
formulation. From the in-vitro studies, it can be concluded that the formulated magnetic nano-emulsion
formulations have great potential with better pharmaceutical and therapeutic activities. Also, the Carisoprodol
magnetic nanoemulsion is a better module for targeted drug therapy for Rheumatoid Arthritis. Since the
obtained results promised Pharmacokinetics, preclinical studies are to be undertaken and increase the half-
life period and bioavailability of the given Carisoprodol Drug.
REFERENCES
1. Swaminathana S, Pastero L, Serpe L, Trotta F, Vavia P, Aquilano D, Trotta M, Zara G and Cavalli
R, Cyclodextrin-based nanos[pmges encapsulating camptothecin: physicochemical characterization,
stability and cytotoxicity. Eur J pharm Biopharm. 2010 Feb; 74(2):193-201.
2. Tripathi K.D, Essentials for medical pharmacology. 5th ed. Newdelhi: Jaypee brothers; 2004
3. Li DC, Zhong XK, Zeng ZP, Jiang JG, Li L, Zhao MM, Yang XQ, Chen J, Zhang BS, Zhao QZ, Xie
MY, Xiong H, Deng ZY, Zhang XM, Xu SY and Gao YXJ, Control Release. 2009 Sepl; 138(2): 103-12.
4. Williams AC and Barry BW, Adv. Drug Deliv. Rev. 2004; 56:603. doi: w10.1016/j.addr.2003.10.025.
[PubMed]
375
Lipid Nanocarriers of Methotrexate in the Topical
Treatment of Psoriasis
Vanaja.K*1, Shailesh Biradar2, Narendra C3, Shobha Rani RH4 and Paradkar AV5.
*1, 4Department of Pharmaceutics, Al-Ameen College of Pharmacy, Hosur road, Bangalore, India.
Department of Pharmaceutics, BharatiVidyapeethUniversity,Poona College of Pharmacy,Pune, India.
2, 5
1, 3
Visveswarapura Institute of Pharmaceutical Sciences, Bangalore, India.
1
Visiting Scientist, Eberhard KarlsUniversitat, Tuebingen, Germany
e-mail: vanaja_ceutics@yahoo.com
Abstract:
Deformable liposomes are examined to be one of promising carriers in the topical treatment of psoriasis
due to their ability to enhance drug permeation and deposition. Deformable liposomal gel of MTX was
formulated using Carbopol P940 as the gelling agent, characterized and rheologically tested. Rheological
evaluation revealed that a significant difference existed between the viscoelastic properties of liposomal gel
systems. Higher gel strength with prevailing elastic component was observed in 1.0% Carbopol gel whereas
in other gels viscous component was exhibited. Liposome loaded gel (dispersed in 1.0% carbopol) was
found to be more suitable as it had optimum viscoelastic properties, good spreadability and extrudability
in comparison with other strengths. With confirmation of safety (with primary skin irritation test), in vitro
release was carried across human cadaver skin. Retarded drug release from higher cross-linked Carbopol gel
(1%) was observed, indicating diffusion to be the principle drug release mechanism.
INTRODUCTION
Psoriasis is a very common skin disorder affecting 1% to 2% of any given population, which is chronic,
genetically influenced, immunologically based inflammatory disease of the skin [1]. A variety of treatments
are commonly used to reduce the severity of symptoms and lessen their impact on the patient’s quality of life.
Topical therapy is useful for symptomatic relief and several topical preparations for treating psoriasis have
been developed but are often less effective probably because of poor penetration, cosmetically unacceptable,
inconvenient for long term use or associated with significant toxicity.
Since the mid-1950s, methotrexate (MTX) has become the gold standard by which other systemic
psoriasis medications are measured [2].
Attempts to deliver methotrexate topically to the psoriasis lesions have been tried. Despite the use of
various methods, MTX does not cross the intact cutaneous skin barrier. A major problem is that methotrexate
is hydro soluble and has a high molecular weight, and is in dissociated form at physiological pH; its capacity
for passive diffusion is limited [3-5].
Advancement in liposome technology has led to the introduction of ultradeformable/deformable,
flexible or ultraflexible liposomes which incorporate ‘edge activators’/surfactant molecules which make
them flexible and improve vesicle stability [6-8].These have been reported to improve drug uptake by the
skin in disorders such as psoriasis and dermatitis [6-8].
However, major disadvantage or limitation of liposome application for topical delivery is the liquid nature of
preparation. Suitable viscosity and topical application ability of liposome can be achieved by use of appropriate
vehicles. Polyacrylic acid polymer based hydrogels are widely used in the pharmaceutical field and are vehicle
of choice in cosmetics topical preparation due to their excellent bioadhesive properties, rendering prolonged
retention of formulation at the site of action[9]. A vehicle for incorporation of liposomes should provide adequate
pH value, stability and rheological characteristics like desired consistency and spreading at site of application.
Thus, the purpose of the present study was to formulate ultradeformable liposomes containing
Methotrexate, characterize and evaluate the rheological properties of gels.

Methotrexate was obtained from Biochem Pharmaceuticals, Mumbai, India. Soy Phosphotidyl Choline,
Cholesterol, Sodium Cholate were obtained from Himedia Laboratories, Mumbai, India. Propylene glycol
and glycerine were purchased from SRS scientific, Bangalore, India. Carbopol P 940 was a gift sample
from Karnataka Antibiotics Private Limited, India. Chloroform and methanol were obtained from Qualigens,
India. Double distilled water (Millipore) was used throughout.
Preparation of liposomes
Briefly, lipid phase comprised soya phosphotidylcholine (SPC), cholesterol and surfactant (sodium cholate) was
dissolved in CHCl3:MeOH (2:1 v/v) mixture in a dry round bottom flask. Organic solvents were removed by a
vacuum evaporator (Rotavapor-R, W.Büchi, FlawilSchweiz) above the lipid transition temperature to obtain a
uniform, thin lipid film on the wall of the flask. The deposited lipid film was hydrated with phosphate buffered
saline (pH 7.4) containing methotrexate, for 60 min at room temperature with continuous rotation for 1 hr.
Small unilamellar (SUV) liposomes were obtained by subjecting the dispersions to probe sonication (Ultrosonic
Processor, UP200S, Hielscher Ultrasound Technology) for 2 to 6 mins using cooling pads. Finally the liposomal
dispersions prepared were stored at room temperature for 2 hrs to anneal any structural defects.
Chromatographic Determination
Methotrexate concentration was quantified by HPLC using the method reported earlier with slight
modifications, at 302 nm using Shimadzu LV AT-10VP liquid chromatograph equipped with a variable UV
detector and a computer integrating apparatus [11]. The column used was Phenomenex Luna 5u C18 (2)
100 A, 250 x 4.60 mm. The mobile phase consisted of a mixture of acetonitrile and phosphate citrate buffer
(90:10) pH 6 at a flow rate of 1ml/min at ambient temperature with retention time (R.T.) of 7.2 ± 0.3 min.
Liposome Characterization
percent drug entrapment was determined after separation of free and entrapped drug. 5 ml of liposome
suspension was subjected to ultracentrifugation (Beckman L7 ultracentrifuge, Beckmann Coulter GmbH)
for 50 mins at 40,000 RPM at 6degreesC. Methotrexate content was determined indirectly from the free
concentration in the supernatant after ultracentrifugation using HPLC.
Amount of drug entrapped
PDE = × 100
Total amount of drug
Particle size (Z-average mean size and polydispersity index) and ζ - potential were determined by dynamic
light scattering (DLS) and Laser Doppler Electrophoresis (LDE), respectively, using a Nanosizer (Nano-ZS,
Nanoseries, Malvern Instruments, UK) after appropriate dilution of liposome preparation. Six independent
measurements were performed in each case. Mass distribution of particle size with polydispersity index as
well as the electrophoretic mobility was obtained. Sizes are reported as the Z-average mean diameter for
the hydrodynamic diameter (nm) along with polydispersity index. Zeta potential (mV) was calculated from
electrophoretic mobility in the electric field.
377
Preparation and evaluation of gels

Varying amount of Carbopol 940 P was weighed and dispersed in small quantity of distilled water to prepare
an aqueous dispersion. Dispersion was allowed to hydrate for 4-5 hrs. Propylene glycol and glycerin were
added and uniformly dispersed. The mixture was stirred until thickening occurred and then neutralized by
drop-wise addition of triethanolamine, until a transparent gel appeared. Quantity of triethanolamine was
adjusted to achieve pH 6. Final weight was adjusted with distilled water. Carbopol concentration in the
systems varied from 0.25% to 1% (i.e., 0.25 % w/w, 0.5% w/w, 0.75% w/w, 1 % w/w).
Liposomes containing methotrexate (previously separated from unentrapped drug) was mixed into
various concentrations of carbopol gel base with a mechanical stirrer at 500 rpm for 5-10 minutes. The
final gelscontained 0.25% of MTX encapsulated in ultradeformable liposomes. Blank gel(varying carbopol
concentration) was also prepared in the same manner without the inclusion of MTX in liposomes. Content
uniformity was determined by analyzing drug concentration (MTX) in gel samples by HPLC at 302 nm.
Extrudability and spreadability:

Weight in grams that was required for extruding 0.5 cm of ribbon in 10 seconds was determined. Extrusion
pressure in grams was recorded. Spreadability was measured with appropriate amount of gel being placed
between two slides and 1000g weight applied for 5 minutes to press the sample to a uniform thickness.
Required amount of weight was added to the pan and time in seconds required to separate two slides was
taken as a measure of spreadability [15].
Rheological evaluation of gels

Rheological studies were performed on a controlled stress rheometer (ViscotechRheometer, Rheologica
Instruments AB, Lund, Sweden). Measurements were performed in a cone and plate geometry with a
diameter of 25mm and cone angle 1.0°. Data obtained was analysed with Stress RheoLogic Basic software,
version 5.0[15-17].
In vitro permeation studies

In vitro study was carried out using Franz diffusion cell across human cadaver skin at 35 ± 2°C. The receptor
phase (PBS pH 7.4) was constantly stirred by magnetic stirrer at 100 rpm. The effective permeation area of
the diffusion cell and receptor cell volume was 2.5 cm2 and 75 ml, respectively. The deformable liposomal
MTX gel formulations (dispersed in varying concentrations of carbopol gel base) containing the final
concentration of 0.25% w/w of MTX was used for the in vitro release studies. Liposomal MTX gel containing
MTX equivalent to 2.5mg was applied to the surface of the cadaver skin. Samples were withdrawn through
the sampling port of the diffusion cell at predetermined time intervals over 24 hr and analyzed using HPLC
at 302 nm.
Primary skin irritation test

The irritancy of MTX liposomal gel; 0.25% w/w MTX was determined in male Wistar rats (150-200 gms).
The animals were housed in an air-conditioned room (20oC) and hair of the back was trimmed short, 24
hr before the beginning of the test. The animals were divided into two group of six each. First group of
rat received optimized liposomal formulation and second group received the marketed formulation. Two
squares were drawn on each side of the back of each rat, and 0.5 gms of each formulation were applied on
each square. The treated animal was protected by using nylon mesh, supported by the plastic squares having
small pores, kept above the treated area. After exposure for 72 hr, the test substance was removed and
exposed skin was scored for the formation of edema (graded 0–4) and erythema (graded 0–4) as per Draize
Evaluation of Dermal Reactions. (Ethical clearance was obtained from Institutional ethical committee, Al-
Ameen College of Pharmacy, approved by CPCSEA) [20].
378
RESULTS
Table 1: Characterization of ultradeformable liposomes of MTX
Formulation PDE* ± S.D Z-average PI* Zeta potential* Deformability* (corresponding

code size* (nm) (mV) blank vesicles)
MTX – SC- 1 51.589 ± 0.698 112.4 0.160 -18.84 84.4 % (Blank MTX-SC1: 86.7%)
MTX – SC- 2 52.051 ± 0.654 101.3 0.1283 -18.52 86.5 % (Blank MTX-SC 2: 87.9%)
MTX – SC- 3 51.236 ±0.529 97.00 0.125 -18.6 86.2 % (Blank MTX-SC 3: 85.3%)
*- Mean of three trials (n = 3); PDE ± S.D; Percent drug entrapment ± standard deviation; PI: Poly dispersity
index (n= 3)
Table 2: Stress sweep for liposome loaded gels
Gel Samples Stress sweep

(% of LVR (Pa) G’ (Pa) at 50 Pa
Carbopol)) LLG LLG
0.10 % NA NA
0.25 % 0.10-2.6 9.32
0.50 % 0.10-3.0 9.63
0.75 % 0.15-12.0 20.00
1.00 % 0.90-26.0 21.4
LVR: Linear Viscosity Range

G’ (Pa): Elastic modulus (Pascal)
LLG: Liposome loaded gel
A-0.25% carbopol; B- 0.5% carbopol; C- 0.75% carbopol; D- 1% carbopol.

G’ (Pa): Elastic modulus (Pascal) shown as –
G” (Pa): Viscous modulus (Pascal) shown as --
Fig 1: Effect of frequency sweep on G’ and G’’ for different loaded gels
379
Fig 2: Effect of frequency sweep on phase degree for different loaded gels
Fig 3: Effect of frequency sweep on complex viscosity for different loaded gels
Fig 4: Permeation profile of liposomal MTX gel from varying concentration of carbopol gel base
Preparation and characterization of deformable liposomes

To add to the “therapeutic ladder” of psoriasis treatment, ultra deformable liposomes of methotrexate (MTX)
were designed and formulated with biocompatible ingredients such as lipid, sodium cholate and cholesterol.
380
It was observed that higher amount of lipid and lower amount of sodium cholate resulted in higher percent
drug entrapment (PDE). The possible explanation could be due to the fact that MTX being a polar molecule
gets encapsulated into the aqueous core requiring higher amount of lipid to form the lipid lamellae. As
reported, incorporation of the right proportion of membrane softer (surfactant) plays an important role in
ultradeformable liposomes [21]. Higher amount of surfactant leads to aggregate disintegration, typically in
form of amphiphats, reducing the entrapment efficiency of the drug.
The Z-average size (nm) corresponding to the hydrodynamic radius was determined using Malvernizer.
All the vesicles formed were unilamellar with Z-average < 120 nm. The Z-average size (n =3) of MTX-SC1,
MTX-SC2, MTX-SC3 was 112.4, 101.3 and 97.00 nm respectively. The Polydispersity index (PI), which
measures the level of homogeneity of particle sizes revealed that the particle size distribution was unimodal
with PI (which is the width of the particle size distribution curve) of 0.160, 0.128, 0.125 for MTX-SC1,
MTX-SC2, MTX-SC3 respectively. The mean zeta potential values of MTX liposomes containing sodium
cholate as the surfactant were -18.84, -18.52 and -18.6 mV for MTX-SC 1, MTX-SC 2 and MTX-SC 3
respectively (Table 1).
The deformability test of blank liposomes and MTX liposomes formulations was measured by comparing
the volumes before and after extrusion through sheets of 50 nm polycarbonate membrane. Without MTX, the
percentages of extruded volume for Blank SC – OS1, Blank SC – OS2, Blank SC – OS 3 were 86.7%, 87.9%
and 85.3% respectively. Percentages of extruded volume of formulations with MTX i.e., MTX-SC1, MTX-
SC2, MTX-SC3 was 84.4%, 86.5% and 86.2% respectively. Microscopical examination revealed that the
liposomes were unilamellar vesicles. In agreement with the light scattering size measurements, liposomes
presented a size in the range of 95-115 nm.
Rheological evaluation of gels

Rheological property is one of the fundamental properties for any gel system as it provides structural
information about the gel system. Due to their hydrophilic nature and bioadhesive characteristics, crosslinked
poly (acrylic acid) polymers such as Carbopol® polymers are among the most widely used excipients for
thickening in topical vehicles [22].
Carbopol P gelling agents have been reported to have a disrupted gel structure which reforms very quickly
with no structure break down occuring under the shear conditions. Propylene glycol (PG) and glycerine
were incorporated to the liposomal gel system as co-solvent systems. Glycerin was shown to provide good
skin tolerability probably due to its good humectant property absorbing moisture from air, thus preventing
skin dehydration. Its effectiveness in reducing skin dryness implied the dominance of moisturizing property
which is an added advantage for psoriatic patients.The pH of the liposomal gels ranged from 6.0 to 6.5 [23].
Liposome suspension was uniformly dispersed throughout the gel base indicating good homogeneity; it
was also observed that incorporation of liposomes into the gel base reduced air entrapment.
Rheological studies
Stress sweep was carried out to determine the LVR, where G’ is independent of applied stress. LVR has great
significance for frequency sweep as it has to be carried out in rheological ground state of the material i.e.
there must not be any breakdown of the structure. G’ is measure of energy stored and recovered per cycle
of deformation and reflects the elastic component of a viscoelastic material. G’’ is a measure of the energy
lost per cycle and reflects the viscous component. When G’’ dominates over G’, the sample is predominantly
viscous and tends to be elastic when G’ is higher than G’’[24]. Both LVR and G’ were lower following the
addition of liposome in all gel samples. Stress sweep data indicated that 1.0% gel has highest gel strength
both before and after addition of liposomal preparation.
A frequency sweep provides a fingerprint of viscoelastic systems under non-destructive conditions
when performed with LVR. The trend of G’ and G’’ over applied frequency provided information about
381
microstructure or particle- particle interaction in the gel system (oscillation frequency sweep) [25-27].
Phase degree (δ) which is a good indicator of viscoelastic nature of system, is a measure of the lag in
sine response after an oscillatory frequency has been applied to the sample. For perfectly elastic system, δ
is close to 0° and perfect viscous system has δ close to 90°. The δ ≥45°indicates viscous nature of system
while δ ≤ 45°, represents elastic nature [28]. The phase degree of all gels was within the viscoelastic region.
The loss tangent is a dimensionless parameter, which is ratio of G’’ to G’. It has been reported that
[29] the system with tan δ<1 has predominant elastic behavior and system with tan δ>1 has a prevailing
viscous behavior. All gels exhibited very low loss tangent over applied frequency, indicating an elastic
behavior (Fig 3).
Among all liposome loaded gels, 1.0% gel had presented lowest phase degree (close to 1) and loss tangent
(below 0.1) (Fig 2and 3). Due to the presence of plateau region over entire frequency region, with higher
elastic behavior, retention of elasticity was observed even after addition of liposomal formulation. 1.0% gel
was most suitable delivery system for topical administration as; it is recommended that topical vehicle must
be elastic in nature with high spreadability [29-32]. This gel possessed the ability to withstand relatively
high shear without serious loss of consistency both before and after addition of liposomal formulation.
In vitro drug release

In vitro permeation studies give us valuable information about the behavior of the delivery system in vivo.
Physiological buffer, PBS (pH 7.4) was used as the receptor fluid in Franz diffusion cells to investigate the potential
of ultradeformable liposomes to enhance drug transport. In order to examine the drug permeation pattern, in vitro
permeation study was carried out using Franz diffusion cell across human cadaver skin. Permeation profile of
liposomal MTX gel with different concentration of Carbopol is shown in fig4. The values of transdermal flux for
all the gel formulations was found to be between 33.582 mcg/cm2/hr to 50.746 mcg/cm2/hr with MTX-loaded gel
(1% Carbopol gel base) showed a controlled and sustained release of the drug.
Primary skin irritation test

After 24 and 72 hours of application of liposomal MTX gel and Plain MTX gel, the test sites were examined
for dermal reactions in accordance with the Federal Hazardous Substance Act (FHSA)– recommended Draize
scoring. The Primary Irritation Index (P.I.I.) of the test article was calculated following test completion. The
Primary Irritation Index of the formulations was found to be 0.00; indicating the absence of irritation on the
skin of the rats.
Under the conditions of this test, the formulations (liposomal MTX gel and plain MTX gel) were not
primary skin irritants. The Primary Irritation Index was less than 5.00, which indicated the safety of both the
formulations.
CONCLUSION
Most stable liposome loaded gel was 1.0% with stable phase degree, higher G’ and LVR. Based on rheological
studies, 1.0% gel was identified as the suitable one as it presented higher G’ and LVR with applied stress
both with and without liposome dispersion. G’ was higher than G’’ in all applied stress range both with and
without liposome dispersion. Phase degree was within elastic region in case of stress as well as frequency
sweep. Higher elasticity which is a measure of higher cross-linking, therefore gives better consistency and
spreading was observed for 1.0% gel among all evaluated gels. Higher cross-linking, in turn can provide the
higher bioadhesion and higher residence at the site of topical application. More retarded drug release was
anticipated which was also confirmed from in vitro studies which also revealed that diffusion is the principle
drug release mechanism from higher cross linked carbopol gel system [33-34].
382
ACKNOWLEDGEMENT
We would like to acknowledge Indian Council for Medical Research (ICMR) for providing Senior Research
Fellowship. We also thank Principal and Management of Al-Ameen College of Pharmacy, India for all the
support rendered to carry out the work. We are very grateful to the Principal, BharatiVidyapeeth University,
Poona College of Pharmacy, for their support rendered for the rheological studies.
REFERENCES
1. Menter, A.M., Krueger, G.C., Feldman, S.R. and Weinstein, G.D., Psoriasis treatment 2003 at the new
millennium: Position paper on behalf of the authors. J. Am. Acad. Dermatol2003, 49(2), S39-43.
2. Frank, C.S. and Alan, M.M., Methotrexate and psoriasis in the era of new biologic agents. J. Am. Acad.
Dermato 2004, 50(2), 301-9.
3. Alvarez-Figueroa, M.J. and Blanco-Mendez, J., Transdermal delivery of methotrexate: iontophoretic
delivery from hydrogels and passive delivery from microemulsions. Int. J. Pharm 2001, 215, 57-65.
4. Trotta, M., Peira, E., Carlotti, M.E. and Gallarate, M., Deformable liposomes for dermal administration
of methotrexate. Int. J. Pharm 2004, 270, 119-5.
5. Trotta, M., Pattarino, F. and Gasco, F.R., Influence of counterions on the skin permeation of methotrexate
from water-oil microemulsions. Pharm Acta Hel 2006, 71, 135-40.
6. Barry, B.W., Novel mechanisms and devices to enable successful transdermal drug delivery. Eur J Pharm
Sci 2001,14, 101-114.
7. Cevc, G. and Gabriele, B., New, highly efficient formulation of diclofenac for the topical, transdermal
administration in ultradeformable drug carrier, Transfersomes. Biochim. Biophys. Acta 2001, 1514,
191-205.
8. Maghraby, G. M. M. El., Williams, A.C. and Barry, B.W., Interactions of surfactants (edge activators)
and skin penetration enhancers with liposomes, Int. J. Pharm 2004, 276, 143-161.
9. Knuth, K., Amiji, M. and Robinson, J.R., Hydrogel delivery system for vaginal and oral applications:
formulations and biological consideration. Adv. Drug Deliv Rev 1993, 11, 137– 167.
10. Robert, G., Rileya, J. D., Smart, J, T., Peter, W. D., Frank, H. J., Alf, D., Grant, K. and William, R. W., An
investigation of mucus/polymer rheological synergism using synthesized and characterisedpoly(acrylic
acid)s. Int. J. Pharm 2001, 217, 287-100.
11. Ioffe, V., Kalendarev, T., Rubinstein, I., Zabetski, R. and Zupkovitz, G., Reversed-phase high-performance
liquid chromatography of polar lipids II. Procedure for a phospholipid derivative of methotrexate. J.
Chrom A 2004, 1031, 249-258.
12. Majid, T., Naser, T., Mahmoud, R.J. and Saeid, D., Enhancement of follicular delivery of finasteride by
liposomes and niosomes 1. In vitro permeation and in vivo deposition studies using hamster flank and
ear models. Int. J. Pharm 2006, 323,1–10.
13. Samah, A., Michael, Laue., Claus-M, L., Udo, B. and Carsten, E., Assessing transferrin modification of
liposomes by atomic force microscopy and transmission electron microscopy. Eur. J. Pharm. Biopharm
2005, 60, 295–303.
14. Yun-Kyoung, Song and Chong-Kook, Kim., Topical delivery of low-molecular-weight heparin with
surface-charged flexible liposomes. Biomaterials 2006, 27 , 271– 80.
15. Panigrahi, L., John. T., Shariff, A. and Hiremath, S.R.R., Formulation and evaluation of lincomycin
HCL gels. Ind. J. Pharm. Sci 1997, Nov-Dec, 331-333.
383
16. Giulia, B., Marco, C., Monica, M.F., Giovanni and F.P., Rheological, adhesive and release characterization
of semisolid carbopol/tetraglycol systems. Int. J. Pharm 2006, 307, 129-140.
17. Giulia, B., Sante, M. and Giovanni, F.P., Rheological, mucoadhesive and release properties of Carbopol
gels in hydrophilic cosolvents. Int. J. Pharm 2006, 282, 115–130.
18. Narendra, C., Srinath, M.S. and Prakash, R.B., Development of three layered buccal compact containing
metoprololtartarate by statistical optimization technique. Int. J. Pharm 2005, 304, 102–114.
19. Nounou, M.M., El-Khordagni, L.K., Kalafallah, N.A. and Khalil, S.A., In vitro release of hydrophillic
and hydrophobic drugs from liposomal dispersions and gels, Acta. Pharm 2006, 56, 311-324.
20. Draize, J.H., Dermal Toxicity. In: Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics.
The Association of Food and Drug Officials of the United States, Bureau of Food and Drugs, Austin
1959, TX, 46-59.
21. Cevc, G., Lipid vesicles and other colloids as drug carriers on the skin. Adv. Drug Del. Rev 2004, 56,
675-711.
22. La¨ýla, B., Jean, L. G., Elias, F. and Amélie, B., Influence of methyl-β-cyclodextrin and liposomes on
rheological properties of Carbopol® 974P NF gels. Int. J. Pharm2003, 254, 59–64.
23. Keat, T.C., Lai, W,C. and Paul, W. S., Heng.Formulation of Hydrophilic Non-Aqueous Gel: Drug Stability
in Different Solvents and Rheological Behavior of Gel Matrices. Pharm. Res 2008, 25(1), 207-217
24. Varma, S. R. and Christy, M. W., Rheology of microcrystalline cellulose and sodiumcarboxymethyl
cellulose hydrogels using a controlled stress rheometer: part II. Int. J. Pharm 2005, 292, 63-73.
25. Lippacher, A., Muller, R.H. and Mader, K., Liquid and semisolid SLNdispersions for topical application:
rheological characterization. Eur. J. Pharm. Biopharm 2004, 58, 561-567.
26. Kim, J., Song, J. and Park, S., Rheological properties and microstructures of Carbopol gel network
system. Coll. Polym. Sci 2003, 281, 614-623.
27. Craig, D. Q. M., Tamburic, S., Buckton, G. and Michael N., An investigation into the structure and
properties of Carbopol 934 gels using dielectric spectroscopy and oscillatory rheometry. J. Cont. Rel
1994, 30, 213-223.
28. Wesis, J. and McClements, D. J., Influence of ostwald ripening on rheology of oil-in-water emulsions
containing electrostatically stabilized droplets. Langmuir 2000, 16, 2145-2150.
29. Korhenen, M.Rheological properties of pharmaceutical creams containing sorbitan fatty acid ester
surfactants. Academic Dissertation, University of Helsinki 2004, Finland.
30. Ikeda, S. and Nishinari, K., “Weak-Gel”-type rheological properties of aqueous dispersions of
nonaggregated k-carrageenan helices. J. Agric. Food Chem 2001, 49, 4436 – 4441.
31. English, R.J., Laurer, J.H., Spontak, R.J. and Khan, S.A., Hydrophobically modified associative polymer
solutions: rheology and microstructure in the presence of nonionic surfactants. Ind. Eng. Chem. Res
2002, 41, 6425–6435.
32. Mirka, K., Heikki, N., Juha, K. and Jouko, Y., Determination of optimal combination of surfactants in
creams using rheology measurements. Int. J. Pharm 2004, 19, 143–151.
33. Honeywell-Nguyen, L,P. and Bouwstra, J.A., The in vitro transport of pergolide from surfactant-based
elastic vesicles through human skin: a suggested mechanism of action. J. Control. Rel 2003, 86, 145-156.
34. Vanaja, K., ShobhaRani, R.H. and Sacchidananda,S., Formulation and clinical evaluation of
ultradeformable liposomes in the topical treatment of psoriasis.Clin. Res. Reg. Aff 2008, 25(1),41–52.
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SECTION - III
BIOTECHNOLOGY
Comparison of Cell Surface Glycosylation on
Apoptotic Cells
Keerthivasan Ambigapathy1 and Deepak Balaji Thimiri Govinda Raj2
Singapore-MIT Alliance, National University of Singapore-117576

1, 2
1
Syngene International Ltd, Biocon Park, Plot 2&3, Estate - Phase-IV Bommasandra-Jigani
Link Road, Bangalore-560 099
2
EMBL Grenoble out-station 6, rue Jules Horowitz BP 181 38042
Grenoble Cedex 9, France
e-mail: keerthivasan.a@gmail.com1, tdeepak@embl.fr2 equally contributed
Abstract:
The Chinese Hamster Ovary (CHO) cell line has been well established as a commercial recombinant protein
production system and has been extensively studied. Recombinant gene targeted (GT) sub-clones of CHO
IFNγ have been established in our laboratory by targeting genes that are actively involved during apoptosis
or programmed cell death. These genes include Fadd, Faim, Alg-2 and Requiem and the four sub-clones
have been termed as CHO GT cells. As expected, it was observed that the GT cell lines had increased
lifespan in batch and fed-batch cultures when compared with parental CHO IFNγ. In addition the GT cell
lines produced IFNγ with increased branch and sialylation of the N-glycans. This has led us to hypothesize
that the processes of apoptosis and N-glycosylation might be linked at the molecular level and delayed
apoptosis might lead to increased glycosylation. To analyze the glycosylation and sialylation patterns and
correlate it to apoptosis with respect to GT cells, we have optimized a lectin-based flow cytometry assay to
profile glycosylation pattern on the cell surface at distinct stages of the cell culture. We have also attempted
to analyze a batch culture time course, to compare the IFNγ glycosylation patterns on parental CHO IFNγ
and CHO GT Requiem cell lines using the lectin based assay.
Keywords: Lectin, Flow cytometry, CHO, Requiem, Apoptosis, Sialylation.
1. INTRODUCTION
The recombinant protein production technology has been a boon to mankind with the potential to
produce large amounts of therapeutic life-saving proteins which would otherwise be virtually impossible.
The two solutions available for large scale productions include using bacterial expression systems or
mammalian systems. Though bacterial systems can produce large amounts of protein, the proteins are
distinctly different from the human version as they are devoid of post-translational processing such as
glycosylation. The mammalian system on the other hand is slow-growing but produces an identical and
mostly similar version of the human counterpart. This has led to the adoption of the mammalian systems
to produce therapeutic proteins, where Chinese Hamster Ovary (CHO) cell line has been predominantly
used. However, even fed-batch cultures of CHO cells cannot prevent apoptosis that occur during the late
stages of culture resulting in loss of productivity. To investigate if it was possible to delay this apoptosis
in culture, transcriptional profiling of key cellular genes during the various phases of fed-batch cell culture
was carried out in our laboratory. It was observed that four key genes implicated in the apoptotic cascade
are highly modulated during the culture [1]. These include the Fadd and Requiem genes which are pro-
apoptotic and show increased expression. On the other hand, the Faim and Alg-2 are anti-apoptotic and
show decreased expression. On the basis of these observations, four cell lines were established in our
laboratory by overexpressing the Faim gene and the dominant negative mutant of Fadd gene and knocking
down the Requiem and Alg-2 genes. These cell lines were named as GT (Gene Targeted) cell lines and
as expected, these cell lines showed increased lifespan in culture and delayed albeit existent apoptosis.
This in turn led to higher levels of recombinant protein (Interferon γ) production. It was interesting that
the delayed apoptosis was accompanied by an increase in N-linked glycosylation. It was observed that
the amount of fully (tetra-), tri-and bi-sialylated interferon γ increased in the GT cells. This made us
hypothesize that the apoptosis and N-glycosylation pathways might be interlinked. It might be interesting
to study the molecular cross-talk between the two pathways and to decouple them. Indirect proof of
concept to our hypothesis comes from contemporary cancer research where loss of apoptosis in cancer
cells is correlated to the acquisition of the metastatic phenotype by the loss of contact with the Extra-
Cellular Matrix (ECM) [2]. Thus cell surface glycosylation profiling might allow us to correlate apoptosis
and N-linked glycosylation.
Recently lectin-based flow cytometric methods have been reportedly used to profile cell surface
glycosylation on U937 human monocytes [3]. The authors demonstrate a marked decrease in sialylation
with onset of apoptosis using a trivariate analysis by combining a lectin assay with the bivariate
Annexin V (AxV) – Propidium Iodide (PI) flow cytometry assay conventionally used to quantify cell
viability and apoptosis [4]. Lectins are the plant version of antibodies and show high specificity to
bind carbohydrate moieties on animal cells. Lectins are extremely toxic to animal cells and induce
potent cytotoxicity by necrosis and sometimes apoptosis. We have attempted to optimize the above
mentioned lectin-based trivariate flow cytometry for CHO cells. We chose Wheat Germ Agglutinin
(WGA), Succinylated Wheat Germ Agglutinin (sWGA) and Maackia amurensis Agglutinin (MAA) for
our analysis. Succinylated WGA binds N-acetylglucosamine; MAA binds sialic acid attached in α2→3
linkage while WGA binds both sialic acid and N-acetylglucosamine.
Thus by using this assay we can profile the glycosylation changes that happen in CHO cells during
different phases of the culture. We have optimized the lectin-based trivariate flow cytometric assay and
have compared the batch time course glycosylation profiles of the CHO IFNγ parental line and the CHO
GT Requiem cell line. The optimized parameters include the lectin concentration, choice of fluorophore
to detect the lectin and the flow cytometer equipment settings.

2.1. Cell Line
CHO IFNγ is a Chinese Hamster Ovary Cell line that produces recombinant human interferon gamma
which grows in suspension. CHO IFNγ was originally derived from DHFR- DuKx cells. CHO IFNγ
had been cotransfected with genes for DHFR and human interferon-γ [5]. CHO Gene Targeted Knock
down (GTKD) Requiem cell line was generated by transfecting CHO IFNγ cells with the Requiem siRNA
plasmid [1]. CHO IFNγ and GT cell lines were maintained in serum-free HyQ CHO MPS media (Hyclone,
Logan, UT) supplemented with 20 mM glucose, 4 mM glutamine and 0.25 μM methotrexate (Sigma, St.
Louis, MO).
2.2. Reagents
Biotinylated lectins WGA (Cat# B-1025), MAA (Cat# B-1265) and succinylated WGA (Cat# B-1025S)
were obtained from Vector Laboratories (Burlingame, CA, USA). Annexin V (human) (recombinant)
(FITC) kit was purchased from Bender MedSystem, Austria. The above kit (Cat# BMS306FI/100T)
was stored in freeze at -20°C. Streptavidin-Allophcocyanin (APC) cross linked, conjugate (Cat# S868)
388
and Streptavidin-Alexa Fluor 647 conjugate (Cat# S21374) were purchased from Molecular Probes
(Invitrogen). Lectin buffer were prepared using HEPES (10 mM), Sodium chloride (0.15 mM), Sodium
azide (0.08%) and Calcium chloride (0.1 mM, only for WGA and sWGA lectins) and fluorophore (APC
and AF647) buffer were prepared using Sodium phosphate (0.1 M), Sodium Chloride (0.1 M) and Sodium
azide (2 mM). The fine chemicals were obtained from Sigma Chemical Co.
2.3. Equipments
Haemocytometer, BD FACScalibur, MAC PC with BD CellQuest Pro software.
2.4. Biotinylated Lectin-Streptavidin Fluorophore Conjugation
Since streptavidin has 4 biotin binding sites, the amounts of biotin and Streptavidin-APC or Streptavidin-
AF647 were calculated using the molar ratio of 4:1. For lectin concentrations of 1, 5, 10, 25 μg/ml cell
suspensions, corresponding Streptavidin-APC or Streptavidin-AF647 were added and diluted in lectin
buffer to the required working volume. The conjugation was performed at room temperature for 15
minutes in the dark.
2.5. Annexin V and Propidium Lodide Dual-Labeling

Flow Cytometry
Using the manufacturer’s protocol (Bender Medsystems), apoptotic cells were detected using
Annexin V-FITC and Propidium iodide [6]. The above solution was analyzed on a Becton Dickinson
FACScalibur. 10,000 events per sample were recorded. Data were processed with BD Cell Quest Pro
software.
2.6. Lectin, Annexin V and Propidium Iodide Triple-Labeling

Flow Cytometry
CHO IFNγ GT cells (1E6) were centrifuged at 1000 rpm for 5 min. Supernatant was removed and resuspended
in ice-cold PBS. Cells were then resuspended in lectin-buffer containing Lectin-Biotin-Streptavidin-APC
conjugate and incubated at room temperature for 15 min in the dark. After incubation, cells were rinsed
with ice-cold PBS-1% BSA solution. After rinsing, cells were resuspended in binding buffer containing
Annexin V-FITC. The above solution was incubated for 10 min at room temperature in the dark. Cells were
then washed using ice-cold PBS 1% BSA solution and resuspended in binding buffer containing Propidium
iodide. The cells were then analyzed on the FACScalibur. The data were processed with BD CellQuest Pro
software.

3.1. Batch Growth Kinetics of CHO IFNγ vs. CHO GT Requiem
The parental CHO IFNγ and CHO GT Requiem cells were grown as a batch culture (Fig. 1) and the cell
viability and extent of apoptosis was confirmed by Annexin V and Propidium iodide for day 1, 3 and 5
samples. The cell numbers measured using the Haemocytometer count and viability was monitored using the
trypan blue exclusion assay. The cell count indicated lesser numbers for the GT cells than the parental. We
think it might be due to the aggregation of the GT cells that was observed in culture. This might have been
due to the fragility and delayed homeostasis of the GT cells to adapt to culture temperature (37°C) on thawing
from the frozen cell bank (-80°C). Parental cells seemed to adjust pretty easily on the temperature change
from freezer to culture.
389
Figure 1: Batch growth curve of CHO IFNγ and CHO GT Requiem cells. Culture period was 8 days. Cell numbers/
ml of culture is noted in the y axis and time in the x axis.
On the other hand, flow cytometer analysis using Annexin V-Propidium Iodide showed higher PI shift for
parental CHO IFNg compared to the CHO GT Requiem (Fig. 2D-F). The Annexin shift seems to be similar
for both parental and GT cell line (Fig 2A-C). These results confirm that CHO GT cells undergo lesser
extent of apoptosis than parental cells. The cell counts were not representative of the above as the clumped
cells tended to aggregate and were undercounted. Thus the data obtained from the bivariate analysis were
not significant enough to compare the extent of apoptosis between the parental CHO IFNγ and CHO GT
Requiem cell lines.
3.2. Annexin and Propidium Iodide Fluorescence Interference
As the aim of this study was to optimize the Annexin/PI assay for cell surface glycosylation analysis in
combination with lectin analysis, it was essential to evaluate various factors affecting the interference. Based
on the literature studies, it is known that lectins can induce apoptosis [3]. Hence it was necessary first to
validate the Annexin V/PI assay on CHO IFNγ parental and GT cell lines at different stages of the batch
curve. For our experiment, samples from Day 1, 3 and 5 were used. With increased age group of samples
we were able to infer significant shift in PI intensity data in both the cell lines. However the data obtained
from the bivariate analysis were not significant enough to compare the apoptotic level between the CHO
IFNγ Parental and CHO GT Requiem cell line. This might be due to fluctuations in the batch growth curve
as stated in the previously. The data obtained using this analysis can be used to compare with samples with
lectin treatment.
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Figure 2: A, B, C – Annexin V fluorescence for parental CHO IFNγ and CHO GT Requiem cell lines on Day 1, 3
and 5 respectively. D, E, F – Propidium Iodide fluorescence for parental CHO IFNγ and CHO GT Requiem cell lines
on Day 1, 3 and 5 respectively.
3.3. Optimization of Lectin concentration for the Lectin-Based Flow Cytometer Assay

The lectin concentration had to be optimized to get the lowest toxicity yet a good signal. This is because a direct
correlation of glycosylation with apoptosis cannot be established if the apoptosis is caused by the lectin itself.
Our optimization was done with lectin concentrations of 5, 10 and 25 μg/ml for the WGA, MAA and sWGA
lectins. We found that WGA had the highest cytotoxicity for all the three concentrations followed by MAA and
sWGA. MAA (10 μg/ml) showed Annexin and PI penetration almost equivalent to the non-lectin treated cells
(Figure 3B). sWGA did not show toxicity at all (Figure 3C). Thus for MAA and sWGA, 10 μg/ml was chosen
as the working lectin concentration. For WGA, 5 μg/ml showed slightly less albeit existent cytotoxicity (Figure
3A). During the course of our comparison experiments we further to the concentration to 1 μg/ml as during
the late stages of culture cytotoxicity due to lectin was more significant. Thus the working WGA concentration
was 1 μg/ml. Though the fluorescence signal did not approach saturation at the chosen concentration, they were
chosen on the basis of reduced cytotoxicity and since we were treating the each of the samples with the same
concentration of lectins the selection was justified.
Figure 3: A – Fluorescence intensity histograms from trivariate analysis on WGA treated CHO IFNγ Parental Cell
Line with concentrations – 25, 10, 5 μg/ml.
391
Figure 3: B – Fluorescence intensity histograms from trivariate analysis on MAA treated CHO IFNγ Parental Cell Line
with concentrations – 25, 10, 5 μg/ml.
Figure 3: C – Fluorescence intensity histograms from trivariate analysis on sWGA treated CHO IFNγ Parental Cell
Line with concentrations – 25, 10, 5 μg/ml. The fluorescence intensity is in the x axis and the count in the y axis. The
geometric means were taken as an indicator as it is a weighted average of fluorescence intensity with count.
3.4. Choice of Lectin-Detecting Streptavidin Conjugated Fluorophore
Having identified the optimal concentrations for the three lectins (WGA, sWGA and MAA), it is necessary
to optimize the best fluorophore which gave the most inferable and reproducible data. In this study, we
compared the fluorescence signal obtained using Streptavidin -Allophycocyanin conjugate and Streptavidin
-Alexa Fluor 647 conjugate. We used WGA and MAA lectin based trivariate flow cytometric analysis with
10 and 25μg/ml for comparison. Results with respect to Streptavidin -Allophycocyanin conjugate showed a
defined and more reproducible fluorescence population as seen in Figure 4A and B. Whereas in the case of
Streptavidin- Alexa Fluor 647 conjugate, the results showed a jagged signal for FL4-H (Lectin) and a higher
spectral overlap with propidium iodide.
392
Figure 4: A – Propidium iodide fluorescence intensity for parental CHO IFNγ cells treated with WGA- Streptavidin
–Allophycocyanin (APC) conjugate, B - WGA lectin fluorescence for Sample parental CHO IFNγ cells treated with
WGA- Streptavidin –APC conjugate, C – Propidium iodide V fluorescence for parental CHO IFNγ cells treated with
WGA- Streptavidin -Alexa Fluor 647 conjugate, D - WGA lectin fluorescence for parental CHO IFNγ cells treated with
WGA- Streptavidin -Alexa Fluor 647 conjugate.
3.5. Comparison of glycosylation of CHO Parent and

CHO GT Requiem cells
The optimized trivariate lectin-based flow cytometry assay was performed on CHO IFNγ parental cells
and CHO GT Requiem cells to compare the cell surface glycosylation patterns and extent of apoptosis.
The experiment was performed over 6 days. Samples were analyzed on Day 0 i.e. on the day of seeding,
day 2, day 4 and day 6. Samples were not taken for later time points as the culture crashed. Day 0 samples
for WGA was done using 10 μg/ml lectin concentrations but was later decreased to 1 μg/ml for subsequent
analyses. MAA and sWGA were probed with predetermined concentrations of 10μg/ml as they have less
or no cytotoxicity. The profiles are indicated in Fig 5.
Figure 5: Comparison of glycosylation profiles of CHO IFNγ parental cells and CHO GT Requiem cells using WGA
(binds sialic acid and N-acetylglucosamine), succinylated WGA (binds N-acetylglucosamine) and MAA (binds sialic
acid) lectins. A – WGA time course profiling for day 2, 4 and 6. B – sWGA time course profiling for day 0, 2, 4 and 6.
C – MAA time course profiling for day 0, 2, 4, 6. All plots are lectin fluorescence intensity histograms.
393
4. CONCLUSIONS AND FUTURE PERSPECTIVE

The lectin-based trivariate flow cytometry can be adapted to profile cell surface glycosylation at
distinct phases of culture and can be correlated to apoptosis using the Annexin-PI fluorescence data.
In our experiment we have optimized the assay, equipment settings and identified best working lectin
concentrations. We have further demonstrated the usage to probe sialylation and glycosylation patterns of
CHO IFNγ and CHO GT Requiem cells. We have also confirmed the earlier observations that decrease in
apoptosis are accompanied by concomitant increase in sialylation. We believe that the assay will prove a
valuable tool to correlate apoptosis and glycosylation in CHO cells. It might be interesting to study the
molecular pathways that link apoptosis and glycosylation. Our preliminary analyses indicates that the
sialyltransferase and sialic acid biosynthetic-enzyme coding genes are under the control of housekeeping
Generic Transcription Factors (GTF) one of which is the SP1 transcription factor [7,8]. SP1 is capable of
causing transcription of a multitude of genes on binding to other transcription factors. It is also interesting
to note that the SP1 factor is cleaved and rendered inactive by caspase 3, the predominant executioner
of apoptotic cascade [9]. Thus, we reckon that by knocking down Requiem gene in CHO GT Requiem
cells, we have decreased the expression levels of Caspase 3, which further makes more SP1 available
as it is not cleaved by Caspase 3. Thus the free SP1 can bind to the promoters of sialylation genes and
hasten their expression, leading to increased sialylation of cell surface proteins. On the contrary, the SP1
is also capable of binding to the promoter of sialidase, the enzyme that cleaves the sialic acid residues
from sialylated proteins [10]. Thus SP1 being a generic transcription factor might bind to its promoter and
interact with other factors whose levels might be influenced by the current cellular state. In the absence
of death signals or presence of growth signals, SP1 might cause transcription of sialylation genes. Thus
studying the molecular events and mediators might help us to understand the relationship between the two
pathways, whether the sialylation pattern influences the onset of apoptosis or is it otherwise.
ACKNOWLEDGEMENTS
We would like to thank Dr. Niki Wong for supervising our work and offering her comments and suggestions
from time to time. We would like to specially thank Prof. Miranda Yap for letting us work at the Bioprocessing
Technology Institute (BTI). Last but not least we greatly acknowledge the opportunity accorded to us by
the Singapore MIT Alliance and the A* STAR Agency for Science, Technology and Research through the
Singapore MIT Alliance graduate fellowship.
REFERENCES
1. Wong DCF, Wong KTK, Nissom PM, Heng CK and Yap MGS, 2006, Targeting early apoptotic genes
in batch and fed-batch CHO cell cultures, Biotechnol Bioeng (in press)
2. Hakomori S, 2002, Glycosylation defining cancer malignancy, New wine in an old bottle, PNAS 99:16
10231-10233
3. Batisse C, Marquet J, Greffard A, Fleury-Feith J, Jaurand MC and Pilatte Y, 2004, Lectin-based three-
color flow cytometric approach for studying cell surface glycosylation changes that occur during
apoptosis, Cytometry A. 62:2 81-88.
4. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL and Henson PM, 1992, Exposure of
phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal
by macrophages J. Immunol, 148: 2207-2216.
5. Scahill SJ, Devos J, Van der Heyden J and Fiers W, 1983, Expression and characterization of the
product of a human immune interferon cDNA gene in Chinese hamster ovary cells, PNAS 80:4654-
4658.
394
6. Vermes I, Haanen C, Steffens-Nakken H and Reutelingsperger C, 1995, A novel assay for apoptosis -
flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein
labelled Annexin V. J. Immunol, Meth. 184: 39 -51
7. Taniguchi A, Yoshikawa I and Matsumoto K, 2001, Genomic structure and transcriptional regulation of
human Galbeta1,3GalNAc alpha2,3-sialyltransferase (hST3Gal I) gene. Glycobiology. 11(3): 241-247
8. Taniguchi A, Saito K, Kubota T and Matsumoto K, 2003, Characterization of the promoter region of
the human Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase III (hST3Gal III) gene. Biochim Biophys
Acta. 1626(1-3): 92-96
9. Rickers A, Peters N, Badock V, Beyaert R, Vandenabeele P, Dorken B and Bommert K, 1999, Cleavage
of transcription factor SP1 by caspases during anti-IgM-induced B-cell apoptosis. Eur J Biochem.
261(1): 269-274
10. Champigny MJ, Johnson M and Igdoura SA, 2003, Characterization of the mouse lysosomal sialidase
promoter. Gene 319:177-187
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Packed Bed Column Adsorption of Cr(VI) onto Acid
Treated Codium tomentosum Biomass
P. Suresh Babu1, S. Eswaramoorthi2, T.P. Rajesh3 and B. Anandaraj4
1
Department of Biotechnology, Sree Sastha Institute of Engineering and Technology,
Chembarambakkam, Chennai–600123, TN
2
Department of Civil Engineering, BIT Campus, Anna University,
Tiruchirappalli–620024, TN
3, 4
Department of Biotechnology, BIT Campus, Anna University,
Tiruchirappalli–620024, TN
e-mail: dranandaraj74@gmail.com, Mob: 9790915036
Abstract:
Packed bed column study was conducted by using acid activated Codium tomentosum biomass for the
adsorption of chromium (VI) from aqueous solution. SEM analysis was carried out to study the surface
topology of the biomass and SEM micrographs are subjected to fractal analysis to examine the surface
roughness and porosity of the biomass. The influence of bed depth and flow rate was investigated. The
breakthrough curves obtained for the experimental data elucidate the importance of bed depth and flow rate.
To evaluate the adsorption column performance, Bohart-Adam’s, BDST, Thomas and Yoon–Nelson model
was used. BDST Model expressed the good linearity for the experimental data. Linear regression coefficients
(R2 values) of Thomas and Yoon–Nelson model showed a good linearity when compared with Bohart-Adam
model. The potential of the acid treated Codium tomentosum biomass in the removal of chromium (VI) was
found to be good. Hence, it was concluded that acid treated Codium tomentosum biomass can be used as an
effective adsorbent for the removal of chromium (VI) ions.
Keywords: Codium tomentosum, Cr(VI) removal, Column studies, Fractal analysis, Acid activation.
INTRODUCTION
Chromium is ubiquitous heavy metal posing high toxicity. Exclusive redox potential of chromium attracts
various industries like leather tanning, welding, painting, electroplating, dye, cement, iron and steel mills.
Metal laden effluents discharged from these industries are responsible for the reasonable increase of chromium
in the water bodies, which originates the environmental pollution. Thus, the continuous expulsion of chromium
starts to mobilise and accrues in the environment, which threatens the living biota [1-2]. As per IARC (2012)
[3] report, hexavalent chromium as severe skin irritant, teratogenic, mutagenic and carcinogenic. Prolonged
exposure leads to respiratory problems, malignant tumour and dermal allergies. The permissible limit of Cr(VI)
in waste water is only 0.05 mg/L as per BIS standards [4]. In order to reduce the chromium concentration
in the effluents, various conventional methods like chemical precipitation, ion exchange, electrochemical
deposition, solvent extraction, membrane filtration and adsorption were employed. Some of these methods
are not sustainable in terms of economic and viable for low concentrated effluents [5]. Among these methods,
adsorption was found to be cost-effective and feasible. Various materials were investigated and suggested as
effective adsorbents by the scientist community, still the growing demands insist on searching for low-cost
adsorbents in large quantities with higher efficiency. Some of the low cost and readily available adsorbents
are listed as follows: fly ash, activated sludge, sawdust, activated charcoal derived from agri-waste, peat moss,
plant biomass, microbial biomass (living/dead) and marine macroalgae [5-8].
Almost all the biomaterials are rich in cellulose, hemicellulose, lignin and lignin like polymers in trace
amounts [9-10]. Macroalgal species also contain various functional groups like hydroxyl, carboxyl, carbonyl,
sulfhydryl, primary and secondary amines, aldehydes and ketones in the surface morphology [6]. To enhance
the adsorption characteristics, surface morphology of the biomass can be subjected to physicochemical
treatments. Some of the adopted and available methodologies are thermal activation, steam activation, acid,
alkali and solvent based treatments, etc. [6]. Gonzalez et al., 2011 reported that adsorption mechanism does
not depend upon any metabolic activity, and it depends only on passive binding conditions [11]. Numerous
literatures pointed out that chemically treated biosorbent have shown enhanced adsorption capacities to
most of the metals under particular conditions. The enrichment of surface morphology helps in the removal
of existing ions and crusts present in surface and thereby inducing the stronger binding specificity of metal
ions and adsorbent.
In this present study, Codium tomentosum macroalgal biomass was selected, pretreated using 0.1N
sulphuric acid and investigated for its efficiency for the removal of chromium(VI) from aqueous solutions
in a fixed bed column.

Codium tomentosum, the marine green macroalgae, was collected from Mimisal coastal area (9˚ 54’ 58”
N; 79˚ 9’ 3”E) of Tamilnadu, south east coast of India during April 2012. The macroalgal specimen was
endorsed at the Centre for Advanced Studies in Marine Science, Annamalai University, Tamilnadu. The
extraneous materials present in the biomass was washed thoroughly with tap water and then rinsed with
distilled water. Further shade-dried biomass was powdered and sieved, and the portion ≤ 600 microns
was selected for the present study. Required quantity of Codium tomentosum biomass was treated using
0.1 M sulphuric acid and kept overnight at room temperature [10]. The residual acid present in the
pretreated biomass was washed by adding required amount of dilute and warm sodium bicarbonate
solution and by continuous rinsing until the pH of the decent reaches 7. The pretreated biomass was
dried in hot air oven at 105˚C till the complete removal of moisture and stored in an airtight container.
Stock solution (1000 mg/L) of chromium (VI) was prepared by dissolving 2.8287 g of K2Cr2O7 in
deionised water. The working solution was prepared before use by diluting the stock solution as required.
The initial pH of each Cr(VI) solution was adjusted as required using 0.1 M NaOH or 0.1 M HCl solution. All
the reagents used in the present study were of analytical grade procured from Merck Ltd., India. Lab India
UV-3092 double beam UV-Vis spectrophotometer was used for chromium (VI) estimation at a wavelength
of 540 nm [12]. Scanning electron microscope (Make: TESCAN VEGA 3, Czech Republic) was used to
study the surface morphology of the native and acid-activated Codium tomentosum biomass.
Column Experiments
Packed bed column studies were conducted at room temperature using a transparent cylindrical glass column
of internal diameter of 2.5 cm and 36 cm in length, 50 mg/L of Cr(VI) solutions at flow rates of 2 mL/min and
4 mL/min and column bed heights of 4 cm, 6 cm, 8 cm and 10 cm were used for the packed bed experiment.
Initial Cr(VI) pH of 2 was maintained for all experiments, which was predetermined from batch study. The
Cr(VI) aqueous solution was allowed to pass through the column in the downward direction by gravity flow.
The treated Cr(VI) solution was collected at different time intervals and analysed. Experimentation was
continued till the exhaustion of the adsorbent packed in the column. The influence of bed height and flow
rate on Cr(VI) adsorption were studied and evaluated in the form of breakthrough curves (Ct/C0 vs. t). The
following equations were used for column parameter calculations [13]:
397
Volume of Cr(VI) solution = Flow rate (mL/min) x Exhaustion time (min)

Cr(VI) uptake = Amount of Cr(VI) adsorbed (mg)/Adsorbent mass (g)
Adsorbed Cr(VI) (mg) = Concentration of Cr(VI) adsorbed (mg/L) x Volume of Cr(VI) (L)

Biosorbent Characterization using Scanning Electron Microscopy
SEM micrograph is taken to reveal the surface microstructure characteristics of the acid treated Codium
tomentosum (Fig.1a). From which the surface roughness and porosity were determined by Fractal
analysis using Image J software (Fig.1b and Fig.1c) following Giedraitis et al. (2010) and Impoco
et al. (2006) [14-16]. Since surface heterogeneity has a direct impact on adsorption characteristics,
the heterogeneity parameters of roughness and porosity were explored [16-17]. The roughness and
porosity introduce geometric heterogeneity on the adsorbent surface that largely promotes ionic
diffusion, capillary condensation and increases configurational entropy. The configurational entropy
is a component of energetic heterogeneity – since it depends on the way in which the adsorbed atoms
are arranged on the surface. This arrangement is again dependent on the electronic state of the surface
atoms of the adsorbent. Depending on the nature of the adsorbent molecules, the atoms or molecules
may exhibit periodic arrangement - thereby giving rise to fractal property to the adsorbing surface.
Since biomolecules of cellulose, hemicellulose and lignin are composed of repetitive units, their
periodic nature is expected to present fractal nature to the adsorbent. Thus, along with an estimation of
roughness and porosity, fractal dimension was also calculated using box-counting method [16, 17]. The
fractal dimension of the surface is 2.6410, indicating highly rough surface of the adsorbent that paves
the way for increased adsorption capacity [18]. The porosity was found to be 0.4108 with 2465 number
of detected pores in a 511 x 511 pixel image.
Effect of Bed Height
Removal efficiency of chromium(VI) from aqueous solution (initial concentration 50 mg/L; pH 2; Flow rate
2 mL/min ) was calculated for different bed depth of 4 cm, 6 cm, 8 cm, 10 cm respectively. Fig. 2 depicts
the breakthrough curve at varying bed depths. The results indicate that a slight variation in the breakthrough
curve with respect to the difference in column bed heights. The exhaustion time was lower for least bed depth
(4 cm) and higher for 10 cm bed depth, which indicates the earlier and extended saturation of the biosorbent.
For all the bed depth, maximum Cr(VI) uptake was observed at the initial stage. However, an exponential
rise in the chromium uptake was noticed after the breakthrough time (tb). Cr(VI) removal capacities vary for
each bed depth, and this may be due to the availability of more binding sites and surface area of the packed
biomass. Higher removal percentage was observed for the bed depth of 10 cm with the flow rate of 2 mL/
min, which indicates the increased surface area and the availability of binding sites of the biomass. Similar
results were observed by Rajesh Kannan et al. [13].
Table 1: Column data and parameters obtained at different flow rates and bed heights
Bed height Flow rate Uptake capacity tb (h) te (h) ∆t (h) Cr(VI) Removal
(cm) (mg/ml) (mg/g) percentage
4 2 24.40 7 33 26 80.54
6 2 21.64 9 40 31 83.47
8 2 17.67 9 41 32 80.70
10 2 15.44 14 46 32 83.54
10 4 14.20 8 23 15 81.43
398
4.3 Effect of Flow Rate
Flow rate is an essential criterion in selecting an adsorbent for continuous treatment of metal-rich industrial
effluents on the large scale. At a constant bed depth of 10 cm, the influence of flow rate was evaluated at two
different flow rates, 2 mL/min and 4 mL /min respectively. 50 mg/L of chromium(VI) concentration with an
initial pH of 2 was used in the present investigation. Higher removal efficiency was noticed at a flow rate of
2 mL/min when compared with 4 mL/min (Table 1). This increased removal efficiency may be due to the
increased residence time of adsorbate that tends to amplify adsorbent-adsorbate interactions. In the case of
higher flow rates, the column saturation time was shorter. Hence, the decrease chromium (VI) uptake was
observed for higher flow rate.
4.4 Column Data Modelling
The experimental data was evaluated using the Bohart-Adams model, BDST model, Thomas model and
Yoon-Nelson model, in order to predict the breakthrough curve and column parameters. The linear regression
coefficient (R2) was calculated using QTI plot software and the goodness of fit was compared.
a) Bed depth service time (BDST) model N0
BDST model is a simplified mathematical model which is widely accepted and used for evaluating the column
experimental data for adsorption studies. Hutchins (1973) modified the Bohart-Adams model and named as
BDST model, which is based on the different breakthrough values obtained for varying bed depth and flow rate
was used to predict the packed bed performance by simple physical calculations [19]. This model assumes that
adsorbate interacts only on the surface of the adsorbent and ignores both intraparticle mass transfer resistance
and external film resistance [20]. A graph was plotted against bed depth (Z) versus breakthrough time (t),
which gives a linear relationship. The process parameters like initial chromium(VI) concentration, flow rate
and adsorption capacity of the adsorbent was taken into account while correlating the service time and bed
depth. From the slope and intercept, the model constants N0 and Ka was calculated and presented in Table
2. While calculating the N0 value, it is to be assumed that the initial chromium(VI) ion concentration and the
linear velocity was constant during experimentation. However, Ka characterizes the solute transfer rate from
aqueous solution to adsorbent. By using the BDST model parameters, we can easily predict the possible values
for different flow rates without performing the experimentation, which helps in designing a large-scale column.
This model failed to predict the service time and bed depth for varying flow rates, due to the availability of
limited data.
b) Bohart-Adams model
Bohart-Adams model is used to evaluate the experimental data for the breakthrough curve of different flow
rate and bed depth [21]. Assuming that adsorption process is continuous, and saturation is not instant. The
linearised equation of Bohart-Adams model is
 Ct  kABN 0 Z
4
ln   = kABC 0(t ) −
 C0 L
where, C0, Ct is inlet and outlet Cr(VI) concentration (mg/L), kAB is rate constant (L/mg.min), L
is the linear velocity (cm/min), Z is the bed depth (cm) and N0 is the saturation concentration (mg/L).
From the graph plotted against ln(Ct/C0), and time (t), the N0 and kAB were calculated and tabulated
(Table 2) (Figures not shown). For the different bed depths, the values of N0 were found to be higher for
lower bed depth and gradually decreased for other bed depths. This result suggests that chromium (VI)
adsorption was not influenced by internal and external mass transfer, particularly at the initial stage of the
adsorption. The observed correlation coefficient (R2) values were compared for suitability. The results
failed to predict the overall Cr(VI) adsorption kinetics in the column and authenticated the unsuitability
of the model.
399
Figure 1: Surface morphological characteristics of acid activated Codium tomentosum biomass a) Scanning electron
micrographic image; b) three-dimensional (3D) interactive plots depicting the surface roughness and c) pore depth
c) Thomas model
Thomas (1944) proposed a model to predict the packed bed column behaviour from breakthrough curve
and the adsorption capacity of the adsorbent, which employs Langmuir’s law isotherm [22]. Assuming that
the packed bed column with a constant flow rate and no axial dispersion, And the adsorption follow second
order reversible reaction kinetics and Langmuir isotherm [23, 24]. This model is very much suitable for the
adsorption process where the intraparticle diffusion and external film resistance will not be rate limiting step.
The linearized equation of the model is expressed as follows
 C 0  kTh QeW
In  −1 = kTh C0 (t ) (5)
 Ct  F
Where kTh is the Thomas rate constant (mL/min.mg), Qe is the equilibrium of chromium(VI) uptake
(mg/g), C0 is the inlet chromium(VI) concentration (mg/L), Ct is the effluent chromium(VI) concentration
at time t (mg/L), W is the mass of adsorbent (g), F is the inlet flow rate (mL/min) and t is the flow time
(min). The value of C0/Ct is the ratio of inlet to outlet chromium(VI) concentrations. A graph was plotted
against ln [(C0/Ct) − 1] and time (t) to determine the values of Qe and kTh from the intercept and slope,
respectively (Figures not shown). The determined constants were tabulated in Table 2. From the results,
it is clear that when the bed height is increased subsequently from 4 cm to 10 cm, decreased kTh and Qe
values were observed.
400
Table 2: Constants of various column models for the Cr(VI) adsorption onto acid treated Codium tomentosum biomass
collected from Mimisal coast
Model Bohart-Adam Thomas Yoon-Nelson

C0 Z ν kAB x N0 R2 kTh x 10-5 Qe R2 kYN τ (min) R2
(mg/L) (cm) (mL / 10-5 (L / (mg) (mL/ (mg/g) (1/min)
min) mg. min) min.mg)
50 4 2 3.38 9.436 0.87 6.00 13.16 0.98 0.0031 1232.93 0.96
50 6 2 2.93 7.591 0.88 5.27 11.96 0.94 0.0026 1519.42 0.94
50 8 2 2.71 5.902 0.87 5.02 9.29 0.96 0.0025 1541.69 0.96
50 10 2 3.07 5.110 0.89 5.00 9.31 0.97 0.0025 1815.28 0.97
50 10 4 4.92 4.880 0.82 8.54 8.23 0.94 0.0043 802.91 0.94
Model Bed Depth Service Time (BDST)

Ka (L/mg/h) N0 (mg/L) χ2 RMSE RSS R2
1.20E-03 41.76 2.35 1.53 4.70 0.95
d) Yoon-Nelson model
For a single component system, the linearized form of Yoon Nelson model [25] is expressed as follows
Ct
ln = k t − τ k (6)
YN YN
C 0 − Ct
The values of kYN and τ for the given bed depth, flow rate and initial Cr(VI) concentration are calculated from
the slope and intercept respectively (Figures not shown). The calculated values are tabulated in Table XX.
While the results for varying bed depth resulted in the decreased values of rate constant (kYN) whereas τ is
found to be increased. With the increase in the flow rate, kYN showed a slight decrease and τ decreased. The
linear regression coefficient R2 for the bed depth and flow rate of 10 cm and 2 ml/min was found to be better
when compared with other bed depths and flow rate.
Figure 2: Breakthrough curve of acid treated Codium tomentosum at varying bed depth
401
CONCLUSION
The results of the present investigation insist that the acid treated Codium tomentosum biomass can be
utilized as an effective biosorbent to adsorb Cr(VI) ions in a packed bed column. Surface heterogeneity and
porous nature were revealed from the SEM image. The significance of the effect of flow rate and bed depth
was validated from the observed experimental results. Yoon-Nelson and Thomas model predicts well the
chromium(VI) adsorption characteristics onto the acid treated Codium tomentosum biomass, when compared
with Bohart-Adam’s model.
REFERENCES
1. Park D, Yun Y-S, Jo JH and Park JM, Biosorption Process for treatment of electroplating wastewater
containing Cr(VI): laboratory-scale feasibility test. Ind Eng Chem Res. 2006; 45(14):5059–65.
2. Suresh Babu P, Dinesh Kumar and Anandaraj B, Biosorption of Chromium from synthetic aqueous
solution using Codium tomentosum. Res. J Chem. Environ. 2013; 17(11): 82-87.
3. IARC Monographs – 100C: Chromium(VI) compounds. 2012; 147-167.
4. BIS, Bureau of Indian Standards, IS 10500, Manak Bhavan, New Delhi, India, 1991.
5. Das N, Vimala R and Karthika P, Biosorption of heavy metals- An overview, Indian Journal of
Biotechnology. 2008; 7: 159-169.
6. Fiset J-F, Blais J-F and Riveros PA, Review on the removal of metal ions from effluents using seaweeds,
alginate derivatives and other sorbents, Rev des Sci l’eau. 2008; 21(3):283-308.
7. Netaji S, Sivasamy A and Mandal AB, Preparation and characterization of corn cob activated carbon
coated with nano-sized magnetite particles for the removal of Cr(VI), Bioresour. Technol. 2013; 134:
94–100.
8. Erhan D, Mehmet K, Elif S and Tuncay O, Adsorption kinetics for the removal of chromium (VI)
from aqueous solutions on the activated carbons prepared from agricultural wastes, Water SA, 2004;
30:533-539.
9.
Siddhanta AK, Chhatbar MU, Mehta GK, Sanandiya ND, Kumar S, Oza MD, et al. The cellulose
contents of Indian seaweeds, J Appl Phycol, 2010; 23(5):919–923.
10. Ling WL, Tjoon TT, Anees A, Norha SM and Yee SW, A novel pretreatment method of lignocellulosic
material as adsorbent and kinetic study of dye waste adsorption, Water Air Soil Poll, 2011; 218(1-4),
293–306.
11. González F, Romera E, Ballester A, Blazquez ML, Munoz JA and Garcia-Balboa C, Algal Biosorption
and Biosorbents, in P. Kotrba, M. Mackova, T. Macek (Eds.), Microbial Biosorption of Metals, Springer,
New York, 2011, pp. 159-178.
12. APHA 2005. Standard methods for the examination of water and wastewater, 21st Edition, Washington,
D.C.
13. Rajesh Kannan R, Rajasimman M and Rajamohan N, Packed bed column studies for the removal of
dyes using novel sorbent, Chemical Industry & Chemical Engineering Quarterly. 2013; 19 (4) 461-
470.
14. Giedraitis A, Tamulevicius S, Gudaitis R and Andrulevicius M, Kinetics of growth and sorption
properties of evaporable barium getter films, Medziagotyra, 2010; 16: 12-23.
15. Impoco G, Carrato S, Caccamo M, Tuminello L and Licitra G, Quantitative analysis of a cheese
microstructure using SEM imagery. In Proceedings of SIMAI 2006 - Minisymposium on Image
Analysis Methods for Industrial Applications – Ragusa, Italy, May 22-26, 2006, pp.1-6.
402
16. Research Services Branch NIMH & NINDS. ImageJ - Image processing and analysis in Java. Web site:
http://rsb.info.nih.gov/ij/. (Accessed on 21 April 2014)
17. Millan H, Govea-Alcaide E and Garcia-Fornaris I, Truncated fractal modeling of H2O-vapor adsorption
isotherms, Geoderma. 2013; 206:14–23.
18. Wei FH, Hong CM and He CZ, Surface pore tension and adsorption characteristics of polluted sediment,
Sci. China Ser. G-Phys. Mech. Astron. 2008; 51: 1022-1028.
19. Hutchins RA, New method simplexes design of activated carbon system, Chem. Eng. J. 1973; 80:
133-138.
20. Vimala R and Das N, Mechanism of Cd(II) adsorption by macrofungus Pleurotus platypus, J Environ
Sci (China). 2011; 23(2):288–293.
21. Bohart GS and Adams EQ, Some aspects of the behaviour of charcoal with respect to chlorine, Journal
of the American Chemical Society, 1920; 42(3): 523–544.
22. Thomas HC, Heterogeneous ion exchange in a flowing system, Journal of the American Chemical
Society. 1944; 66(10): 1664–1666.
23. Trgo M, Vukojevi N and Peri J, Application of mathematical empirical models to dynamic removal of
lead on natural zeolite clinoptilolite in a fixed bed column, 2011; 18:123–131.
24. Ghasemi M, Keshtkar AR, Dabbagh R and Jaber Safdari S, Biosorption of uranium(VI) from aqueous
solutions by Ca-pretreated Cystoseira indica alga: breakthrough curves studies and modeling, J.
Hazard Mater. 2011; 189(1-2): 141–149.
25. Yoon YH and Nelson JH, Application of gas adsorption kinetics I, A theoretical model for respirator
cartridge service life, American Industrial Hygiene Association Journal, 1984; 45(8): 509–516.
403
Biotransformation of Clozapine to Norclozapine-an
Active Metabolite by Cunninghamella Elegans
S. Varalaxmi1 and M. Vidyavathi2
Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Viswa Vidyalayam,

1, 2
Tirupathi – 517 590, A.P., India

e-mail: vara6veda12@gmail.com, Mobile: 09849199376, 09949576350
Abstract:
The biotransformation of clozapine a CYP1A2 substrate was investigated using eight different fungal
cultures by fermentation technique. Among all the selected fungi Cunninghamella elegans has shown
the ability to biotransform the clozapine. The metabolite found from culture extracts of Cunninghamella
elegans was identified by HPLC analysis and further the metabolite was isolated, and structure of metabolite
was elucidated using Nuclear Magnetic Resonance (NMR) and mass spectroscopy. The metabolite
structure was confirmed as norclozapine by above methods. Hence Clozapine was transformed into its
active metabolite norclozapine by Cunninghamella elegans. The microbial transformation of clozapine
was similar to the metabolism of clozapine in mammals. Thus the Cunninghamella elegans can be used as
a complementary tool as an in vitro models of drug metabolism studies in mammals and can also be used
to produce an active metabolite for further pharmacological and toxicological studies.
Keywords: Clozapine, Norclozapine, Cunninghamella elegans, Demethylation, CYP1A2 substrate,
Microbial biotransformation.
INTRODUCTION
Clozapine is a tricyclic benzodiazepine neuroleptic drug [1] and is efficacious in the treatment of resistant
schizophrenia [2]. It is indicated for use in individuals with schizophrenia who are either resistant or
intolerant to other antipsychotic drugs [3, 4]. Clozapine has been also shown to be effective in individuals
with schizoaffective and psychotic mood disorders [5, 6]. Non-psychotic rapid cycling bipolar disorder [7],
and Parkinson’s disease with drug-induced and other [8, 9]. Furthermore, clozapine has been demonstrated
to be effective in individuals with brain injury [10, 11]and severe borderline personality disorder with
aggression and self-abusive behavior [12, 13].
Biotransformation is the process of conversion of substrates to desired products by using enzymes
(bacteria and fungi) [14, 15]. Biotransformation of compounds by the application of whole cell
microorganisms has more advantages when compared to isolated enzymes [16]. Now days, microbial
transformation is considered to be an inexpensive and economical technology for the biotechnological
specialists to produce pure useful metabolites [17]. The safety and efficacy of a drug are an important
factor in the evaluation of mammalian metabolism. The identification of metabolites from animal sources
and clinical samples may be hindered by insufficient quantities of metabolites formed. Microorganisms
can metabolize compounds similar to mammals that can be used to isolate mammalian drug metabolites
with low cost and easily. Some microorganisms especially the fungi belonging to Cunninghamella
species, possess Cytochrome P-450 monooxygenase systems analogous to those in mammals [18]. Hence
microbial biotransformation had been proposed as a complementary in vitro model for mammalian
drug metabolism studies. Microbial models are mainly used to produce a large quantity of metabolites
using preparative-scale fermentation technique for complete structural elucidation, pharmacological

and toxicological studies of metabolites which are difficult by chemical synthesis [19-21].

Fungi
The fungi used in the present study were Aspergillus flavus (MTCC 1783), Aspergillus ochraceus (NCIM
1140), Aspergillus terreus (NCIM 657), Cunninghamella blakesleeana (MTCC 3729), Cunninghamella
elegans (MTCC 4279), Cunninghamella elegans (NCIM 689), Gliocladium roseum (NCIM 1064). Rhizopus
stolonifer (NCIM 880). These cultures were maintained on potato dextrose agar slants at four °C and
subcultured for every 6 months for maintaining their viability.
Chemicals
Clozapine was obtained from Mylan laboratories, Hyderbad, India. Acetonitrile, Methanol and Water used
for analysis were HPLC grade. Media components were obtained from Qualigens, and SD fine chemicals,
Mumbai, India.
Media composition
Potato dextrose broth (Potato chips, 20 gm/100 ml (steamed for 30min), dextrose 2 gm, yeast extract 10 mg,
distilled water up to 100 ml) for Aspergillus flavus, Aspergillus ochraceus, Aspergillus terreus, Cunninghamella
elegans, Cunninghamella elegans, Gliocladium roseum, Rhizopus stolonifer, Cunninghamella elegans,
Cunninghamella elegans. Oatmeal flakes 3gm/100ml (steamed for 30min) for Cunninghamella blakesleeana.
Microbial fermentation
The fermentation was carried by 50ml potato dextrose broth medium in 250ml Erlenmeyer flasks incubated
for about 48 h, operated at 120 rpm at 37°C on an orbital shaker. Each study has three flasks labelled as
drug control, culture control and sample. The fermentation was performed by drug control which had only
drug; culture control inoculated with respective fungus and sample flask had drug along with fungus to study
clozapine biotransformation.
Extraction Procedure
Extraction was performed after 48hrs of incubation. The flasks were removed from shaker incubator and
kept on water bath at 45°C for 30 min for inactivation of microorganisms and was centrifuged at 3000 rpm
for 10 min (R8C: Remi instruments, Mumbai, India). The supernatant was collected and was extracted
with ethyl acetate for drug and its metabolite [22]. The organic layer was collected and was evaporated by
air drying. Further dried extract was reconstituted with mobile phase for HPLC analysis.
ANALYTICAL TECHNIQUES
High Performance Liquid Chromatography
The analysis of Clozapine and its metabolite was performed by High Performance Liquid Chromatography (HPLC)
method. The HPLC system (Waters, USA) consisted of Waters 515 solvent delivery module and Waters 2489
UV-visible spectrophotometric detector. The mobile phase comprised of Sodium acetate:Acetonitrile:Methanol
(45:20:35v/v) with a flow rate of 1ml/min. The column used was C-18 (stainless steel column of 25 cm length and
4.6 mm internal diameter packed with porous silica spheres of 5 µ diameter, 100 Å pore diameter – II 5C-18 rs –
100a, 5 µm, 4.6 x 250 mm). The wavelength was set as 286 nm and sensitivity at 0.001 a.u.f.s [23].
Mass spectroscopy
The sample of Cunninghamella elegans culture showed a metabolite peak at retention time of 3.5min in
HPLC compared to their controls. Hence, the metabolite was collected from HPLC elute and dried and
405
further analyzed using mass spectroscopy operating in the electron spray ionization (ESI) mode. Model was
Agilent 1100 Series mass spectrometer operating in the electron spray ionization (ESI) mode. Detector used
was ion trap detector, operated at positive mode, range: 50-700, spray voltage: 3.5 kV, capillary temperature:
325°C, nebulizer gas pressure: 210 psi.
PNMR Spectroscopy
The metabolite collected from HPLC elute metabolite was dried, and its structure was further confirmed by
PNMR spectroscopy by using BRUKER AVANCE 400 MHz (IICT, Hyderabad). Deuterated Methanol was
employed as a solvent to analyze PNMR spectra of clozapine and its metabolite.
Results
In the present study, the biotransformation of clozapine was performed using eight fungal organisms. The
HPLC analysis of clozapine and its metabolite in different culture extracts were conducted, and its results
are represented in Table.1. The peak at retention time of 2.2min represented solvent peak, peak at 4.2min
represented culture contents and peak at retention time of 8min represented clozapine based on the retention
time of pure clozapine. Interestingly the sample of Cunninghamella elegans shown an extra metabolite
peak at 3.5 min compared to its control as shown in Figure.1. The structure of metabolite was analysed and
confirmed by Mass spectroscopy and PNMR techniques.
Table 1: HPLC data of Clozapine and its metabolite from fungal culture extracts
Name of the Organism Retention time (min.)

(Blank I) Drug control (Blank II) Culture control (Control) Pure Clozapine Sample
Aspergillus flavus 2.0 2.0 - 2.0
[MTCC 1783] - 4.0 - 4.0
8 - 8 8
Aspergillus ochraceus 2.2 2.2 - 2.2
[NCIM 1140] - 4.2 - 4.2
8.2 - 8.2 8.2
Aspergillus terreus 2.1 2.1 - 2.1
[NCIM 657] 4.1 4.1 - 4.1
8.1 - 8.1 8.1
Cunninghamella 2.2 2.2 - 2.2
blakesleeana [MTCC - 4.2 - 4.2
3729] 8.2 - 8.2 8.2
Cunninghamella elegans 2.1 2.1 - 2.1
[NCIM 691] - 4.1 - 4.1
8.1 - 8.1 8.1
Cunninghamella elegans 2.2 2.2 - 2.2
[NCIM 689] - 4.2 - 4.2
8.2 - 8.2 8.2
- - - 3.5*
Gliocladium roseum 2.0 2.0 - 2.0
[NCIM 1064] - 4.0 - 4.0
8 - 8 8
Rhizopus stolonifer 2.1 2.1 - 2.1
[NCIM 880] - 4.1 - 4.1
8.1 - 8.1 8.1
406
Figure 1: HPLC chromatogram of Clozapine from culture extracts of Cunninghamella elegans.

The mass spectrum of metabolite and clozapine were obtained and compared. The mass spectrum of
Clozapine shown a molecular ion peak at m/z 327 (M+1) the fragment peaks are obtained at m/z 270, m/z
245. The mass spectrum of metabolite shown a molecular ion peak at m/z 313(M+1) which is equal to the
molecular weight of norclozapine, fragment peaks at m/z 227, m/z 192 as given in Figure.2.
Figure 2: Mass spectrum of Clozapine metabolite.
The PNMR spectrum of pure Clozapine shown a peak in the range of δ 4 is due to aromatic C-N proton,
δ 2.13 and δ2.79 are due to methylene group protons, δ2.26 is due to alkyl group protons, δ 6.57- δ 7.24 are
due to benzene protons. The PNMR spectrum of metabolite shown a singlet in the range of δ 1.91 which is
due to amine group protons i.e., demethylated clozapine which is not seen in clozapine. Thus, the metabolite
structure was proposed as demethylated form of Norclozapine.
407
Figure 3: PNMR spectrum of Clozapine metabolite
DISCUSSION
The potential of fungi for their ability to biotransform the drug clozapine to its metabolite was studied using
eight selected fungal cultures. The CYP450 enzymes required for biotransformation of clozapine may be
present in Cunnighamella elegans hence it has ability to metabolise clozapine to its active metabolite
norclozapine. The transformation of clozapine to its metabolite was screened using fermentation and
HPLC analysis. The sample of Cunnighamella elegans shown an extra metabolite peak at retention time
of 3.5min compared to its controls as given in Table 1 and Figure 1. The extra peak represented the
formation of Clozapine metabolite by Cunnighamella elegans. When samples of other organisms were
compared with their controls, shown identical peaks indicated no metabolite formation by those organisms.
The metabolite was analysed by mass spectroscopy and PNMR studies. The clozapine is extensively
metabolised to two stable main metabolites i.e., norclozapine(active metabolite) and clozapine N-oxide
in human and invitro human liver [24, 25]. The major metabolic pathway of clozapine by Cunnighamella
elegans was demethylation which was confirmed by PNMR and Mass spectra results. Mass spectra of
metabolite of clozapine showed 14Da lower than that of the clozapine which suggested the loss of a methyl
group from parent drug Figure 2. PNMR spectrum of metabolite when compared with the parent compound
had shown a singlet in the range of δ 1.91 Figure 3 which confirm the demethylation of clozapine. The
use of microorganisms and in particular the fungi belonging to the genus Cunninghamella as a model for
biotransformation studies has been well documented [18, 26, 27, 19]. Hence, the demethylated metabolite
formed on biotransformation by Cunnighamella elegans is similar to the phase I metabolite formed in
human beings.
CONCLUSION
It can be concluded that Cunninghamella elegans have the ability to metabolise clozapine to its active
metabolite norclozapine because of the presence of enzymes required for biotransformation. Hence, it can
be demonstrated that Cunninghamella elegans had potential for production of phase I metabolite similar to
mammals. The microbial models can be used in the early phase of drug development to predict potential routes
408
of mammalian drug metabolism. Required quantities of phase I metabolites could be isolated by microbial
metabolic models, and these can be used as standards for metabolite identification and determination in
mammalian biotransformation studies. As a result of this type of studies, animals can be substituted by
microbial models, and use of laboratory animals in preclinical studies might be decreased significantly.
Thus, the present study reveals that Cunninghamella elegans can be used for the preparation and isolation
of norclozapine for further pharmacological and toxicological studies and the development of new drug
entities.
REFERENCES
1. J. Claghorn, G. Honigfeld, F.S. Abuzzahab Sr., R. Wang, R. Steinbook, V. Tuason and G. Klerman,
(1987) The risks and benefits of clozapine versus chlorpromazine, J. Clin. Psychopharmacol 7, 377–384.
2.
J. Kane, G. Honigfeld, J. Singer, H. Meltzer (1988) and the Clozaril Collaborative Study Group,
Clozapine for the treatment-resistant schizophrenic, Arch. Gen. Psychiatry 45, 789–796.
3. Baldessarini, R.J. and Frankenburg, F.R. (1991), A novel antipsychotic agent, New England Journal of
Medicine, 324, 746–754.
4. Kane J., Honigfeld G., Singer J. and Meltzer H.Y., (1988), Clozapine for the treatment-resistant
schizophrenia, A double-blind comparison with chlorpromazine (Clozaril collaborative study), Archives
of General Psychiatry, 45, 789–796.
5. McElroy S.L., (1991), Clozapine in the treatment of psychotic mood disorders, schizoaffective disorder
and schizophrenia, Journal of Clinical Psychiatry, 52, 4111–4114.
6. Zarate, C.A., Jr. Tohen M. and Baldessarini R.J., (1995), Clozapine in severe mood disorder, Journal of
Clinical Psychiatry, 56, 411–417.
7. Suppes T., Phillips K.A. and Judd C.R., (1994), Clozapine treatment of non-psychotic rapid cycling
bipolar disorder, A report of three cases, Biological Psychiatry, 36, 338–340.
8. Friedman J.H. and Lannon M.C., (1989), Clozapine in the treatment of psychosis in Parkinson’s disease,
Neurology, 39, 1219–1221.
9. The Study Group, (1999), Low dose clozapine for the treatment of drug-induced psychosis in Parkinson’s
disease, New England Journal of Medicine, 340, 757–763.
10. Michals M.L., Crismon M.L., Roberts S. and Childs A., (1993), Clozapine response and adverse effects
in nine brain injured patients, Journal of Clinical Psychopharmacology, 13, 198–203.
11. Green A.L., Zimmet S.V., Strous R.D. and Schildkraut J.J, (1999), Clozapine for comorbid substance
use disorder and schizophrenia: Do patients with schizophrenia have a reward-deficiency syndrome
that can be ameliorated by clozapine? Harvard Review of Psychiatry, 6, 287–296.
12. Benedetti F., Sforzini L., Colombo C., Maffei C. and Smeraldi E., (1998), Low dose clozapine in acute
and continuation treatment of severe personality disorder, Journal of Clinical Psychiatry, 59, 103–107.
13. Chengappa K.N., Ebeling T., Kang J.S., Levine J. and Parepally H., (1999), Clozapine reduces self-
mutilation and aggression in psychotic patients with borderline personality disorder, Journal of Clinical
Psychiatry, 60, 477–484.
14. Muffler K., Leipold D., Scheller M.C., Haas C., Steingroewer J., Bley T., Neuhaus H.E., Mirata M.A.,
Schrader J. and Ulber R., (2011), “Biotransformation of triterpenes”, Process Biochemistry, 46, 1-15.
15. Baiping M., Bing F., Hongzhi H. and Yuwen C., (2010) “Biotransformation of Chinese Herbs and
Their Ingredients,” Mode Tradit Chin Med Mater Med 2010, 12, 150-154.
409
16. Demyttenaere J.C.R., (2001), “Biotransformation of terpenoids by microorganisms”, Studies in Natural

Products Chemistry, 25, 125–178.
17. Patel R.N., (2008), Synthesis of chiral pharmaceutical intermediates by biocatalysis, Coordination
Chemistry Reviews, 252, 659-701.
18. Abourashed EA, Clark AM and Hufford CD, Microbial 1999; models of mammalian metabolism of
xenobiotics: An updated review, Curr Med Chem 6: 359-74.
19. Rao GP and Davis PJ, Microbial 1997; models of mammalian metabolism, Biotransformations of HP
749 (besipirdine) using Cunninghamella elegans, Drug Metab Dispos 25: 709-15.
20. Moody JD, Freeman JP and Cerniglia CE, 1999; Biotransformation of doxepin by Cunninghamella
elegans, Drug Metab Dispos 27: 1157-64.
21. Duhart BT, Zhang D, Deck J, Freeman JP and Cerniglia CE, 1999; Biotransformation of protriptyline
by filamentous fungi and yeasts, Xenobiotica 29: 733-46.
22. Wohlfarth A, Toepfner N, Hermanns-Clausen M and Auwarter V., 2011 May; Sensitive quantification
of clozapine and its main metabolites norclozapine and clozapine-N-oxide in serum and urine using
LC-MS/MS after simple liquid-liquid extraction work-up., Anal Bioanal Chem. 400(3):737-46.
23. Guitton C, Kinowski JM, Aznar R and Bressolle F, 1997 Mar 7; Determination of clozapine and its
major metabolites in human plasma and red blood cells by high-performance liquid chromatography
with ultraviolet absorbance detection., J Chromatogr B Biomed Sci Appl. 690(1-2): 211-22.
24. Jeremy is g. Dain, Joseph nicoletti and Frances Ballard, (1997) Biotransformation of clozapine in
humans, Drug metabolism and disposition, vol. 25, no. 5, 603-609.
25. Pirmohamed M, Williams D, Madden S, Templeton E and Park BK, 1995, Mar; Metabolism and
bioactivation of clozapine by human liver in vitro., J Pharmacol Exp Ther. 272(3): 984-90
26. Hezari M. and P.J. Davis, (1993), Microbial models of mammalian metabolism, Furosemide glucoside
formation using the fungus Cunninghamella elegans, Drug Metab.Dispos. 21:259–267.
27. Moody J.D., J.P. Freeman and C.E. Cerniglia, (1999) Biotransformation of doxepin by Cunninghamella
elegans, Drug Metab.Dispos. 27:1157–1164.
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Production of Electricity from Municipal Waste Water
Using Microbial Fuel-Cells
J. Jayabarath1, K. Gobika, R. Yuvashri and K. Jeyaprakesh2
1
Head, Dept of Biotechnology, Pavendar Bharathidasan College of Engineering and
Technology, Mathur-Trichy-24
2
Head, Dept of Biochemistry, Sir Rajah’s Serafoji College of Arts & Science, Thanjavour
E-mail:barath_bio@yahoo.co.in
Abstract:
The world is facing an energy crisis as well as significant environmental problems. Good solutions
are needed to address these problems. It is a well known fact that fossil fuels (petroleum, natural
gas and coal) are the main resources for generating electricity. However, they have been major
contributors to environmental problems. One potential alternative to explore is the use of microbial
fuel cells (MFCs), which generate electricity using microorganisms. MFCs uses catalytic reactions
activated by microorganisms to convert energy preserved in the chemical bonds between organic
molecules into electrical energy. MFC has the ability to generate electricity during the waste
water treatment process which simultaneously treating the pollutants.
The present study employed both individual cultures and mixed culture consortia in an MFC
for electricity generation and pollutant removals. The MFC in this study was designed as dual-
chambered system, in which the chambers were separated by a salt bridge made of agar and sodium
chloride. Waste water from a nearby pond and 0.1 N potassium ferricyanide were taken as a medium
in anode and cathode chamber respectively. The maximum current generated was in the mixture of
E.coli and A. niger, 1.56 mA at 72 hrs with 66.42% COD removal. It is demonstrated that MFC
offers great potential to optimize power generation using mixed cultures in waste water.
Keywords: MFC, COD, Dual-chambered system.
INTRODUCTION
The increase in energy consumption particularly in the past several decades has raised fears of
exhausting the globe’s reserves of natural resources in the future. Due to industrializations and
population growth our economy and technologies today largely depend upon natural resources, which
are not replaceable. Approximately 90% of our energy consumption comes from fossil fuels. World
energy conservation predicted estimation about the rate of utilization of energy resources shows that
the coal deposits will deplete within the next 200 to 300 years, and petroleum deposits will deplete in
next few decades. So, encourage the research and development activities covering a broad spectrum of
possible renewable resources, as their contributions are substantial. In a microbial fuel cell operation,
the anode is the terminal electrode acceptor recognised by bacteria in the anodic chamber. Therefore,
the microbial activity is strongly depedent on the redox potential of the anode. In fact, it was recently
published that a michaelis menten curve was obtained between the anodic potential and power output
of an acetate driven microbial fuel cells. A critical anodic potential seemed to exists at which the maximum
power output of a microbial fuel cell is achieved.
Microbial Fuel Cells

A microbial fuel cells (MFC) or biological fuel cells are bio electrochemical system that drives a
current by mimicking bacterial interaction found in nature. Mediator-less MFCs are a much more recent
development and due to this the factors that affect optimum operation, such as the bacterial used in the
system, the type of ion membrane, and the system conditions such as temperature, are not particularly well
understood. Bacteria in mediator-less MFCs typically have electrochemically active redox enzymes such
as cytochromes on their outer membrane that can transfer electrons to external materials.
Working of MFCs
A microbial fuel cell is a device that converts chemical energy to electrical energy by the catalytic reactions
of microorganisms. A typical microbial fuel cells consists of anode and cathode compartments, and fuel
is oxidized by microorganisms, generating electrons and protons. Electrons are transferred to the cathode
compartments through the membrane. Electrons and protons are consumed in the cathode compartment,
combining with oxygen to from water. In general, there are two types of microbial fuel cells: mediator and
mediator-less microbial fuel cells.
Generating Electricity
When the microorganism consume a substrate such as sugar in aerobic conditions, they produce carbon-di-
oxide and water. However, when oxygen is not present they produce carbon-di-oxide, protons and electrons
as described below:
Anode: C6H12O6 + 6H2O 6CO2 + 24H+ + 24e-
Cathode: 24H + 24e- + 6O2 12H2O
Microbial fuel cells use inorganic mediators to tap into the electron transport chain of cells and steal the
electrons that are produced. The mediator process the outer cell liquid membrane and plasma wall; it then
begins to liberate electrons from electron transport chain that would normally be taken up oxygen or other
intermediates. The now-reduced mediator exists the cell laden with electrons that it shuttles to an electrode
where it deposits them; this electrode becomes the electro-generic anode (negatively charged electrode)
the release of the electrons means that the mediator returns to its original oxidised state ready to repeat the
process. It is important to note that this only happened under anaerobic conditions if oxygen is present them
it will collect all the electron as it has greater electro negativity than the mediators.
Methods and Materials

Every fuel cells has two electrodes, one positive and one negative, called, respectively, the cathode and
anode. They are connected by means of a salt bridge. The reactions that produce electricity take at the
electrodes. Every fuel cell also has an electrolyte, which carries electrically charged from one electrode to
the another and a catalyst, which speeds the reaction at the electrodes. Hydrogen is the basic fuel, but fuel
cells also requires oxygen. One great appeal of the fuel cell is that they generate electricity with very little
pollution much of the hydrogen and oxygen used in generating electricity ultimately combined to form a
harmless byproduct, namely water.
Experimental setup
The MFC reactor was constructed of two identical chambers separated by salt bridge. The two chamber of
MFC was designed and fabricated using Plexiglas material of 1000 ml volume, the electrodes are carbon
electrode (anode) and platinum electrode (cathode). The anode chamber is filled with waste water sample,
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inoculum, glucose and neutral red. The carbon electrode is immersed in the anode chamber. The chamber
is tightly sealed using rubber cork as anaerobic reactions take place within the anode chamber. The cathode
chamber is filled with a solution of 0.10 M potassium ferricyanide. The platinum electrode is immersed in
this solution to form a cathode chamber.
Salt bridge
The anode and cathode compartments separated by salt bridge (dia 2.2 cm). The salt bridge which acts as
proton exchange membrane was made of agar and NaCl. The agar gel was casted in the connecting bridge
by pretreating with 3 g of Nacl and 0.5 N H2SO4 at 50°C for 30 minutes to induce the salinity level in the salt
bridge and porosity level respectively.
Waste water samples
The waste water was collected from a local pond. The waste water was filtered before pre-treatment to
remove solid particulates. The waste water was pre-treated by heating at 121°C at 15 psi for 20 minutes. The
waste water samples were kept in a refrigerator at 4°C before use. The waste water was used as an innoculum
for MFC tests without any modifications such as pH adjustment or addition of nutrients, mediator or trace
metals. Experiments were conducted using full strength wastewater at 30°C.
Table 1: Design Criteria of Dual Chambered Microbial Fuel Cell
Reactor Configuration Dual Chamber
Anode Chamber Suspended growth
Cathode Chamber 0.1 M potassium ferricyanide
Mediator-Chamber Various with micro-organisms
Mediator-Cathode Air
Volume of Anode and Cathode (l) 0.7
Anode and Cathode Material Carbon rod, Platinum rod
Connecting Bridge Salt Bridge
Operating Temperature (ºC) 28±2
Operating pH 7 (Cathode)
Microbes in MFC
Usually mixed culture of microbes is used for anaerobic digestion of substrate as complex mixed culture
permits broad substrate utilisation. But there are some regular MFCs design which explore metabolic
tendency of single microbial species to generate electricity. Organic component rich sources of microbes
that can be used in MFCs catalytic unit. Bacteria used in MFCs with mediator or without mediator have
been extensively studied and reviewed. Several types of microbes are used for the generation of the
electricity such as Esherichia coli, Saccharomyces cerevisiae, Aspergillus niger.
Working principles
MFC explores metabolic potential of microbes for conversion of organic substance into electricity by
transferring electrons from cell to circuit. In anodic chamber, oxidation of substrate in the absence of oxygen
by respiratory bacteria produce electron and proton that are passed onto terminal e- acceptor. However, in
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the absence of e- acceptor in an MFC, some microorganism pass electron onto anode. An efficient electron
was shuttling mediators. Direct electron transfer to anode by bacteria requires some physical contact with an
electrode for the current generation.
In mediated electron transfer machinery, microbes produces acquire indigeneous soluable redox
compounds or synthetic exogenous mediators to shuttle electron between terminal respiratory enzymes
and anode surface. These mediators can diverts electrons from the respiratory chain by entering outer cell
membrane, becoming reduced, and state to shuttle electron to electrode.
Monitoring Electricity and COD

Voltage (V) and current (I) measurements were recorded using a digital multimeter by connecting with
100 external circuit.COD measurements were conducted using standard methods. All sample were filterd
through a 0.22 µm membrane filter prior to COD measurements. COD removal was calculated by standard
methods (FAS method).
Result and Discussion
Effect of Time on Individual culture in the Anode Chamber

Effect of time on the individual microbial culture in the anode chamber was found out. The media components
were same for all the three organisms. The pH was not altered in the anode chamber, and room temperature was
maintained. But in the cathode chamber pH7 was maintained 6g/l of glucose was added to the anode chamber
as nutritional substrate where as neutral red is used as the mediator here. The optical density was measured
using spectrophotometer at the 600nm for every 8 hours. The maximum OD for E.coli was found to be 0.94 at
72 hrs, for s.cerevisiae it was found to be 0.82 at 72 hrs and for A.niger it was found to be 0.88 at 72 hrs.
S.No. Time (hrs) Optical density 600nm

E.coli S.cere visiae A.niger
1 0 0 0 0
2 8 0.04 0.02 0.03
3 16 0.07 0.05 0.06
4 24 0.12 0.13 0.11
5 32 0.19 0.17 0.21
6 40 0.25 0.24 0.27
7 48 0.42 0.35 0.34
8 56 0.65 0.46 0.55
9 64 0.71 0.67 0.72

Figure 1: Effect of time on the individual
10 72 0.94 0.82 0.88 microbial cultures in the anode chamber
Effect of time on the microbial mixture consortia in the anode chamber

Effect of time on the microbial mixture consortia in the anode chamber was found out. The media component
was same for all the three mixtures. The media components were same for all the three mixtures. The pH was
not altered in the anode chamber, and room temperature was maintained. But in the cathode chamber pH 7
was maintained. 6g/l of glucose was added to the anode chamber as nutritional substrate where as Neutral
red is used as a mediator in the anode chamber. The mixtures used were mixture A (E.coli & S.cerevisiae),
mixture B (S.cerevisiae & A.niger), mixture C (E.coli & A.niger). The optical density was measured using
414
spectrophotometer at the 600nm for every 8 hours. The maximum OD for mixture A was found to be 0.97 at
72 hrs, for mixture B was found to be 0.76 at 72 hrs and for mixture C was found to be 0.82 at 72 hrs.
Table 2: Effect of time on the mixed culture consortia in the anode chamber
S.No. Time Optical density 600nm

(hrs) Mix A Mix B Mix C
1 0 0 0 0
2 8 0.03 0.01 0.05
3 16 0.09 0.07 0.12
4 24 0.15 0.13 0.18
5 32 0.23 0.22 0.27
6 40 0.36 0.34 0.39
7 48 0.52 0.49 0.57
8 56 0.59 0.54 0.69
9 64 0.71 0.67 0.76
Figure 2: Effect of time on the mixed culture consortia in the
10 72 0.82 0.76 0.97 anode chamber
Mixture A = Escherichia coli + Saccharomyces cerevisiae

Mixture B = Saccharomyces cerevisiae + Aspergillus niger
Mixture C = Escherichia coli + Aspergillus niger
Current generated by the individual microbial culture in MFC utilizing waste water
Current generated by the individual microbial culture in MFC utilizing waste water was found out. The
media components were saved for all three organisms. The pH was not altered in the anode chamber, and
room temperature was maintained. But in the cathode chamber pH7 was maintained. 6g/l of glucose was
added to the anode chamber as nutritional substrate whereas; Neutral red is used as a mediator here. The
current generated was measured using multimeter at 10Ω for every 8 hours. The maximum current generated
by E.coli was 1.24 mA at 72 hrs, similarily for S.cerevisiae it was 0.62 mA at 72 hrs, for A.niger it was 1.04
mA at 72 hrs.
Table 3: Current generated by the individual microbial culture in MFC utilizing waste water
S.No. Time Current generated (mA)

(hrs) E.coli S.cerevisiea A.niger
1 0 0 0 0
2 8 0.34 0.22 0.28
3 16 0.40 0.30 0.35
4 24 0.48 0.38 0.39
5 32 0.54 0.47 0.51
6 40 0.60 0.52 0.58
7 48 0.70 0.58 0.61
8 56 0.82 0.62 0.64
9 64 1.11 0.59 0.83

Figure 3: Current generated by individual culture
10 72 1.24 0.58 1.04
in the anode chamber
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Voltage produced by the mixed culture consortia in MFC using waste water
Voltage produced by the mixed culture consortia in MFCs utilising waste water was found out. The
media components were saved for all three mixtures. The pH was not altered in the anode chamber, and
room temperature was maintained. But in the cathode chamber pH 7 was maintained. 6g/l of glucose
was added to the anode chamber as nutritional substrate whereas; Neutral red is used as mediator here.
The three mixtures used were mixture A (E.coli & S.cerevisiae), mixture B (S.cerevisiae & A.niger),
mixture C (E.coli & A.niger). The voltage produced by the mixture A was 38 mV, 44 mV at 8 hrs
and 16 hrs respectively. Similarly, for mixtures B it was found 28 mV and 39 mV at 8 hrs and 16 hrs
respectively. The maximum voltage produced by mixture A it was 178 mV and for mixture C it was 248
mV at 72 hrs
Table 4: Voltage produced by the mixed culture consortia in MFC using waste water
S.No. Time Voltage generated(mV)

(hrs) Mix A Mix B Mix C
1 0 0 0 0
2 8 38 28 46
3 16 44 39 52
4 24 53 47 64
5 32 68 54 70
6 40 80 72 92
7 48 104 90 119
8 56 120 99 138
9 64 154 116 174

Figure 4: Voltage generated by individual culture in the
10 72 178 138 248
anode chamber
Varying COD levels in the waste water samples when individual microbial culture is
employed in MFC
Varying COD levels in the waste water samples when individual microbial culture is employed in
MFC was found out. COD measurements were conducted using standard methods. COD was found
1484(mg/l) and 1395(mg/l) at 8 hrs and 16 hrs respectively when E.coli was employed. COD was
found 1612(mg/l) and 1504(mg/l) at 8 hrs and 16 hrs respectively when Saccharomyces cerevisiae
was employed. COD was found 1547(mg/l) and 1413(mg/l) at 8 hrs and 16 hrs respectively when
Aspergillus niger was employed. At 72 hrs, COD was found 534(mg/l) when E.coli was employed.
Similarly it was found 852(mg/l) when Saccharomyces cerevisiae employed. It was found 788(mg/l)
when Aspergillus niger was employed.
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Table 5: Varying COD levels in the waste water samples when individual microbial culture is employed in MFC
S.No. Time COD(mg/ml)

(hrs) E.coli S.cerevisiea A.niger
1 0 1650 1650 1650
2 8 1484 1612 1547
3 16 1395 1504 1413
4 24 1301 1450 1349
5 32 1263 1317 1280
6 40 1165 1285 1181
7 48 1076 1197 1054
8 56 998 1054 998
9 64 856 982 863 Figure 5: Varying COD levels in the waste

water samples when individual microbial
10 72 534 852 788 culture is employed in MFC
CONCLUSION
This study documented the feasibility of bioelectricity generation from anaerobic waste water treatment
using an MFC fabricated by our own. Electricity was successfully generated with waste (as COD) removal
from the waste water using micro-organisms. The microbial electricity technology is still in an early stage
of development, but shows great promise as a new method to accomplish both waste water treatment and
electricity generation. The procedure was environmentally sound and sustainable due to utilization of waste
water as substrate. The dual activity could significantly reduce the cause associated with current waste water
treatment methods. Major issue to be solved for practical application are to overcome activity loss, cost
factors and incomplete utilization of waste water. MFCs offer a promising prospect in waste water treatment;
however improvements and optimization are needed to achieve better results for electicity and pollutant
removal.
REFERENCE
1. Abhilasha S Mathuriya and V.N. Sharma, (2009), Bioelectricity production from various waste water
through microbial fuel cell technology, J Biochem Tech (2009) 2(1):133-137.
2. Daie, Neda, Gholam Hosain Shahidi Bonjar, Abdol Ali Saberi and Sonia Aghighi, Structure and
components, Asian Journal of Information Technology 5(2):126-128.
3. Jin-Na Zhang, Qing-Liang Zhao, Peter Aeltereman, Shi-JieYou and Jun-Qui Yang, (2008), Electricity
generation in a microbial fuel cell with a microbially catalyzed cathode, (2008) 30:1771-1776.
4. Surajit Das and Neelam Mangwani, (2010), Recent development in a microbial fuel cells: A review
2010, PP 727-731.
5. A.K. Shukla, P. Suresh, S. Berchmans and A. Rajendran, (2004), Biological fuel cells and their application
vol.87, No.4, 25 august 2004.
6. Pranab K., Bairuva D. and Deka, (2010), Electricty generation from biological based microbial fuel
cells, Vol1.issue1.
417
Production of Antimicrobial Secondary Metabolites from
Marine Associated Fluorescent Pseudomonas
J. Jayabarath1, J. Jeny Joseline and S. Sivakavi
1
Head, Dept of Biotechnology, Pavendar Bharathidasan College of Engineering and
Technology, Mathur-Trichy-24
Email:jenyjose94@gmail.com
Abstract:
The production of antimicrobial secondary metabolites from marine associated fluorescent pseudomonas
was studied. Isolated of fluorescent pseudomonas strains from the marine source is rare. The isolates were
screened for antimicrobial activity against rhizoctoniasolani and staphylococcus sp. The culture filtrate and
ethyl acetate extract of the strains were also screened against microbial pathogens. An efficient strain was
selected, purified and mass cultured. Analysis of metabolite was carried out with plants growth chlorophyll,
carotenoid and protein amount estimation. The secondary metabolites were purified through thin layer
chromatography and silicon gel column chromatography, and the concentration was analysed through UV-
Vis spectroscopy.
INTRODUCTION
Microbiologist working in the field of secondary metabolism is confronted with the matter what might be
the role of secondary metabolites in the physiology and ecology of the producing organisms. The solution
to this problem would not only enlarge our understanding of an important biological phenomenon, but
it might also provide industrial microbiologists with strategies for increasing the yield of economically
important microbial products. In recent years, it has become obvious that there are as many answers as there
are secondary metabolites. It has also become apparent that an understanding of the controls affecting the
production of a particular secondary metabolite can only be reached by extensive studies on its biosynthetic
pathway, on regulatory mechanisms involved in its formation, and on the genetics and physiology of the
producing organisms.
It is obvious to choose secondary metabolites of industrial importance and strains with a high producing
capacity for studies on secondary metabolism. In the past, this approach has been followed by industrial
microbiologists, and, while leading to considerable practical success in increasing the yields of antibiotics,
it has failed to reveal the regulatory processes governing the production of secondary metabolites. There
are numerous reports offering phenomenological descriptions of the parameters affecting the formation of
secondary metabolites, but there is a definite lack of studies at the biochemical and genetics levels. In part,
this may be due to the complex biosynthetic pathways of many secondary metabolites, to the variety of
organisms studied, to the absence of suitable genetic systems, and to the association of the formation of
many secondary metabolites with processes of differentiation, adding a further dimension of complexity to
the regulatory mechanisms involved.
In the present contribution, we summarize information on the production of secondary metabolites by
pseudomonads, with special emphasis on the fluorescent pseudomonads. The more important groups of
secondary metabolites are discussed and, where available, information on their biosynthesis and their mode
of action is reviewed. Pseudomonads represent the major group of non-differentiating microorganisms
producing antibiotics. The lack of cytological and physiological differentiation makes it possible to study
the effect of various factors on secondary metabolism without interference from genetically programmed
development cycles. The pseudomonads may, therefore, offer experimental advantages over the filamentous
fungi, the actinomycetes, and the bacilli for studying certain facets of secondary metabolism.
Biocontrol properties
Some P. fluorescens strains present biocontrol properties, protecting the roots of some plant species against
parasitic fungi such as fusarium and pythium as well as some phytophagous nematodes. It is not clear
exactly how the plant growth – promoting properties of P.fluorescens are achieved ; theories include: the
bacteria might induce systemic resistance in the host plant , so it can better resist attack by a true pathogen ;
the bacteria might outcompete other (pathogenic) soil microbes, e.g., by siderophores, giving a competitive
advantage at scavenging for iron the bacteria might produce compounds antagonistic to other soil microbes ,
such as phenazine-type antibiotics or hydrogen cyanide. To be specific, certain P.fluorescens isolates produce
the secondary metabolites 2,4-diacrtylphloroglucinol (2,4-DAPG), a compound found to be responsible for
antiphytopathogenic and biochemical properties in these strains. The phl gene cluster encodes factors for 2,4-
DAPG biosynthesis, regulation, export, and degradation. Eight genes, phlHGFACBDE, are annotated in this
cluster and conserved organizationally in 2,4-DAPG producing strains of P.fluorescens. Of these genes, phlD
encodes a type III polyketide synthase, representing the key biosynthetic factor for 2,4-DAPG production.
phlD shows similarity to plant chalcone synthases and has been theorized to originate from horizontal gene
transfer. But phylogenetic and genomic analysis has revealed that the entire phl gene cluster is ancestral to
P.fluorescens, many strains have lost the capacity and exists on different genomic regions among strains.
There is experimental evidence to support all of these theories, in certain conditions; Hass and Defago write
a good review of the topic. The strain referred to as Pf-CL145A has proved itself a promising solution to
the invasive Driessena (zebra and quagga) mussels. A strain of the bacteria produces toxins that destroy the
digestive system of the mussels and produces a >90% kill rate. Several strains of “P.fluorescens’’ such as
PF-5 and JL3985, have developed a natural resistance to ampicillin and streptomycin. These antibiotics are
regularly used in biological research as a selective pressure tool to promote plasmid expression.
Materials and Method
Description of species
Pseudomonas fluorescence is gram negative rod shaped bacteria that inhibit soil plants and water surface.
The optimum growth temperature is between 25-30 degree Celsius. It resides in the plant rhizosphere and
produce a variety of secondary metabolites including antibodies against soil borne plant pathogens. The
significance of these organisms has increased because of their ability to degrade various pollutants and their
uses as biocontrol against pathogens.
Isolation
Fluorescent pseudomonas species were collected from rhizosphere of coastal sand dune plants such as
spinifex sp., and canavaliasp .and ipomoea sp. present along the coastal region of Kanchipuram district,
Tamilnadu. The strains were isolated using plant method is king’s B medium. The strains were incubated for
24 hours at 37. The strains were tested for fluorescence using UV transilluminator. 42 colonies that showed
fluorescence at 365 nm were selected and purified.
Screening of Isolates for Antimicrobial activity

Antifungal activity
Rhizoctoniasolani, a fungal pathogen was inoculated in potato dextrose agar. After a period of seven days
mycelial disc was taken and inoculated in potato containing wells of pseudomonas strains and the antifungal
419
activity was measured through well diffusion assay. The 20 strains which showed good inhibit on against
a fungal pathogen were isolated in king’s B broth, and culture filtrate was obtained through centrifugation.
The mycelial disc was taken and inoculated in plate containing the wells of culture filtrate of fluorescent
pseudomonas.
The 11 culture filtrate which showed good inhibition against a fungal pathogen were mixed with ethyl
acetate. The mixture was thoroughly mixed. The ethyl acetate extract was obtained by using a separation
column. The mycelial discs was inoculated in plates containing the ethyl acetate extracts of 11 strains.
Antibacterial activity
Three bacterial pathogens staphylococcus sp. and pseudomonas sp. was inoculated in nutrient broth. The
bacterial pathogens were swabbed in nutrient agar. The 42 fluorescent pseudomonas strains were inoculated
in wells for antibacterial activity. It was noted that strains showed inhibition against staphylococcus alone.
Staphylococcus sp. was swabed and in the plates of nutrient agar containing wells of pseudomonas strains
and the antibacterial activity was measured through well diffusion assay. The 20 strains which showed
good inhibition against bacterial pathogens were isolated in king’s B broth and culture filtrate was obtained
through the centrifugation. The mashed bacterial pathogens were taken and inoculated in plate containing the
wells of culture filtrate of fluorescent pseudomonas.
The 11 culture filtrate which showed good inhibition against a bacterial pathogen were mixed with ethyl
acetate. The mixture was thoroughly mixed. The ethyl acetate extract was obtained by using a separation column.
The bacterial pathogens were swabbed in plants containing the wells of ethyl acetate extracts of 11 strains.
Growth of plants and analysis of metabolites

Plant growth
Four plants pearl millet, ragi, black-eyed pea and green gram were grown with four treatments each in
nutrient rich sand filled in plastic cups. The treatments includes plant grown in water with pathogen
(rhizoctoniasolani discs), plant grown water without pathogen, plant grown in a culture filtrate with pathogen
and plant grown in a culture filtrate without pathogen. Plants were grown in room temperature with the
action of direct sunlight.
The growth of plants was at keen observation. 25 seeds of each plant were grown in plastic plates with
the above treatments and the number plants grown calculated and tabulated. The weight of the leafs of each
sample was measured and tabulated. The shoot length and root length of each sample were measured and
tabulated. The chlorophyll, carotenoid and protein amounts were estimated with the following methods.
Chlorophyll estimation
The total chlorophyll amounts were estimated with the following method. 200 mg of the leaf samples of
the each plant was taken and grinded well. The extract was collected and centrifuged at 8000 rpm for three
minutes. 2 ml of the supernatant was taken, and absorbance was read at various OD values such as 662 nm,
645 nm and 470 nm. The values of total chlorophyll amounts were calculated as below
Chlorophyll a = (11.75 x OD at 662 nm) – (2.350 x OD at 645 nm)
Chlorophyll b = (18.61 x OD at 645 nm) – (3.960 x OD at 662 nm)
Total chlorophyll = chlorophyll a + chlorophyll b
Carotenoids estimation
For calculating the amount of total carotenoids, the chlorophyll a and chlorophyll b values were calculated
as above and the carotenoids amounts were calculated as follows Total carotenoids = {[1000 x OD at 470 nm
x chlorophyll a] – [81.4 x chlorophyll b/227]}
420
Protein estimation
The protein estimation amounts were calculated with Bradford method. This method relies on the binding
of the dye coomassie brilliant blue to protein. At low PH, the free dye has an absorption maxima at 470 and
650 nm, but when bound to protein has an absorption maximum at 595 nm. The practical advantage of the
method is that the reagent is simple to prepare and that the colour develops rapidly and stable. Although it is
sensitive down to 20 g protein cm^-3, it is only a relative method, as the amount of dye binding appears to
vary with the content of the basic amino acids arginine and lysine in the protein. This makes the choice of a
standard difficult. In addition, many proteins will not dissolve properly in the acidic reaction medium. 200
mg of the plant sample was mashed and dissolved in 10 ml of the distilled water. 1ml of this sample were
made up to 10 ml. From the made up solution 1 ml were taken and mixed with 10 ml of Bradford reagent.
The absorbance values for the 16 such samples were measured at 595 nm. The values were tabulated, and
the protein amounts were calculated.
Purification Techniques
Thin layer chromatography
A thin layer of the stationary phase is formed on a suitable flat surface. Since the layer is so thin, the movement
of mobile phase across the layer, by simple capillary action, is rapid there being little resistance to flow. As
the mobile phase moves across the layer from one edge to the opposite, it transfer any analytes placed on
the layer at a rate determines by their distribution coefficient kd, between the mobile and stationary phase.
In practice, the principle of the distribution process may be based on that of absorption, part ion, chiral, ion
exchange or molecular exclusion chromatography. Analyte movement cause either when the mobile phase
(solvent front) reaches the end of the layer and capillary action flows ceases or when the plate is removed
from the mobile phase reservoir. The movement of the analyte is expressed by it retardation factor, RF such
that RF = Distance moved by analyte from origin/Distance moved solvent front from origin A range of prepared
plate is available commercially called polyamide layer sheets which consists of poly-c-caprolactum coiled
onto tooth sides of a solvent-resistant polyester sheet are unusual in that they are semitants parent allowing
unknowns and standard run on opposite side of the plate to be compared they can also be rowed if change
immediately with ammonia –acetone. The sample is applied to the plate 2.0 to 2.5 cm from the edge by
mans of micropipette or micro syringe, for preparation TLC, the sample is applied as a plate rather transfer
single spot plate development. Separation is most commonly takes place in a stage tank that contains the
developing solvent (mobile phase) to a depth of about 1.5cm. The techniques were carried out with hexane
and ethyl acetate ratios 9:1, 6:4, 4:6 and 2:8.
Silica Gel Column Chromatography

The principle of column chromatography separation may be depreciated by considering a column packed
with a solid granular stationary phase to a height of 5 cm, surrounded by the mobile phase there is 1 cm
per cm of column as shown, if 32 g of a compound is added to the column in 1 cm of mobile phase, then,
at this 1 cm moves on to the column to occupy position A, 1 cm of mobile phase will have leave the
bottom of column. If the compound added has an effective distribution coefficient of 1, it will distribute
itself equally the solid and liquid phase. If a further 1 cm of mobile phase is introduced onto the column,
the mobile in section A will move down to B taking 16 g of the compound with it, leaving 16 g at A. At
both A and B a redistribution of the compound will occur so that there is 18 in the mobile phase and 8 in
the solid phase. The addition of a further 1 cm 1of mobile phase to column displaces the mobile phase
in A to B and in B to, giving the distribution of the compound as shown in stage. Addition of a further 1
cm of mobile phase leads to distribution shown at stage 4, and further 1 cm of mobile phase leads to the
distribution. It is apparent that after a relatively small number of equilibrations the compound distributes it
symmetrically bands. A silica gel column was made, and sample was included at the top and the different
421
ratios of solvents hexane and ethyl acetate ratios at 9:1, 6:4, 4:6, 2:8 and the extracted samples was then
analysed in TLC.
Determination of secondary metabolite concentration by UV-Vis spectroscopy

The functioning of this instrument is relatively straightforward. A beam of light from the visible and UV
light source (colored red) is separated into its component lengths by a prism or diffraction grating. Each
monochromatic (single wavelength) beam in turn is split into two equal intensity beams by a half mirrored
device. One beam, the sample beam (coloured magenta), passes through a small transparent container
(cuvette) containing a solution of the compound being studied in a transparent solvent. The other beam, the
reference (colored blue), passes then measured by electronic detectors and compared. Over a short period,
the spectrometer automatically scans all the component wavelengths in manner described. The UV region
scanned is normally from 200 to 400 nm, and the visible portion is from 400 to 800 nm. Different compounds
may have very different absorption maxima and absorbances. Intensely absorbing compounds must be
examined in dilute solution, so that significant light energy is received by the detector, and this requires
the use of completely transparent (non-absorbing) solvents. The most commonly used solvents are water,
ethanol, hexane, and cyclohexane. Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I)
are generally avoided. The absorbance were measured at different wavelength and the values are tabulated
RESULTS
Table 1 & Table 2: Antifungal activity of fluorescent pseudomonads strains Rhizoctoniasolani & Pseudomonads culture
filtrate against Rhizoctoniasolani zone of inhibition (cm) respectively.
Strain Zone of Strain Zone of Strain Zone of inhibition

inhibition inhibition S10 0.0
S04 0.1 S29 0.0
S13 0.8
S07 0.0 S30 0.4
S15 0.0
S08 0.0 S31 0.5
S16 0.0
S09 0.0 S32 0.0
S24 0.8
S10 0.2 S33 0.3
S11 0.0 S34 0.0 S25 0.6
S12 0.1 S35 0.2 S30 0.5

S13 0.5 S39 0.0 S31 0.6
S14 0.4 S40 0.4 S33 0.9
S15 0.0 S42 0.9 S35 0.5
S16 0.2 S43 0.2 S40 0.6
S17 0.0 S44 0.2 S42 1.0
S18 0.0 S46 0.9 S43 0.0
S19 0.0 S48 0.0
S44 0.0
S20 0.0 S62 0.0
S46 0.7
S23 0.0 S65 0.0
S66 0.0
S24 0.8 S66 0.2
S68 0.7
S25 0.8 S68 0.9
S26 0.0 S69 0.8 S69 0.6
S27 0.0 S70 0.2 S70 0.3
S28 0.0 S72 0.1 S72 0.3
422
Table 3: Antibacterial activity of fluorescent pseudomonads against staphylococcus, zone of inhibition (cm)
Strain Zone of inhibition Strain Zone of inhibition

Vertical Horizontal Vertical Horizontal
S 04 0.0 0.0 S 29 2.4 2.3
S 07 1.5 1.6 S 30 2.0 1.8
S 08 2.0 1.8 S 31 2.0 1.9
S 09 2.5 2.3 S 32 1.2 1.1
S 10 2.5 2.6 S 33 0.0 0.0
S 11 2.4 2.4 S 34 0.0 0.0
S 12 0.0 0.0 S 35 0.0 0.0
S 13 2.3 2.0 S 39 2.0 1.8
S 14 0.0 0.0 S 40 2.0 1.9
S 15 0.0 0.0 S 42 0.0 0.0
S 16 2.3 2.4 S 43 2.0 2.2
S 17 2.6 2.5 S 44 2.0 1.7
S 18 0.0 0.0 S 46 2.2 2.0
S 19 2.0 2.2 S 48 2.4 2.4
S 20 1.9 1.8 S 62 2.6 2.8
S 23 0.0 0.0 S 65 2.0 2.1
S 24 2.3 2.0 S 66 2.0 1.8
S 25 2.2 2.0 S 68 1.0 1.1
S 26 0.0 0.0 S 69 2.2 2.0
S 27 2.0 2.2 S 70 2.4 2.3
S 28 2.3 2.3 S 72 2.2 2.3
Table 4 & Table 5: Antibacterial activity of fluorescent pseudomonads culture filtrates against staphylococcus, ethyl
acetate extract of fluorescent pseudomonads against staphylococcus zone of inhibition (cm)
Strain Zone of inhibition Strain Zone of inhibition

Vertical Horizontal Vertical Horizontal
S 10 0.0 0.0 S 13 2.3 2.3
S 13 1.7 1.8 S 24 0.0 0.0
S 14 0.0 0.0 S 25 0.0 0.0
S 16 0.0 0.0
S 30 0.0 0.0
S 24 1.2 1.3
S 31 1.5 1.6
S 25 1.4 1.4
S 40 2.3 2.2
S 30 1.1 1.2
S 31 1.7 1.7 S 46 0.0 0.0
S 33 0.0 0.0 S 68 0.0 0.0

S 35 0.7 0.6 S 69 1.2 1.3
S 40 1.6 1.5 S 70 1.8 1.7
S 42 1.1 1.3 S 72 0.0 0.0
S 43 0.0 0.0
S 44 1.1 1.1
S 46 1.2 1.2
S 66 0.0 0.0
S 68 1.3 1.4
S 69 1.2 1.2
S 70 1.5 1.6
S 72 1.9 1.9
423
Table 6 & Table 7: Estimation of chlorophyll and proteins respectively
Plant Treatment Pathogen Chlorophyll Chlorophyll Total Chlorophyll

a b (mg/ml)
Pearl millet Water Absent 4.56 0.40 4.96
Present 0.35 14.55 14.90
Culture Absent 0.63 24.71 25.334
Present 1.63 22.05 23.68
Raggi Water Absent 0.88 15.23 16.11
Present 0.81 15.19 16.00
Present 0.82 16.02 16.84
Black eyed pea Water Absent 0.09 12.93 13.02
Present 0.31 2.74 3.05
Present 0.14 5.14 5.28
Green gram Water Absent 0.65 7.43 8.08
Present 0.67 8.56 9.23
Present 1.00 17.12 18.12
Plant Treatment Pathogen OD at 595 nm Total proteins (mg/ml)

Pearl millet Water Absent 0.278 1.011
Present 0.334 1.140
Culture Absent 0.281 1.093
Present 0.310 1.134
Raggi Water Absent 0.291 1.114
Present 0.328 1.139
Present 0.307 1.132
Black eyed pea Water Absent 0.757 1.304
Present 0.659 1.233
Present 0.581 1.214
Green gram Water Absent 0.457 1.192
Present 0.370 1.153
culture Absent 0.499 1.198
Present 0.532 1.209
DISCUSSION
72 strains were obtained and out of which 42 colonies that showed fluorescence at 365 nm were selected
and purified. Fluorescent pseudomonads resisted growth of Rhizoctoniasolani and staphylococcus sp.,
where assist could not inhibit the bacterial pathogens Enterococcus sp. and pseudomonads sp. 20 strains that
showed good inhibition against Rhizoctoniasolani and staphylococcus sp. were selected. 11 culture filtrates
which showed good inhibition against the two pathogens were selected. Ethyl acetate extract of strains 13
was found to have good inhibition.
Pearl millet, ragi, black-eyed pea, and green gram plants showed better growth with action of fluorescent
pseudomonas culture filtrate against the infection of the fungal pathogen, Rhizoctoniasolani. The chlorophyll,
carotenoid, and the protein amounts present in the plant samples were estimated and tabulated. The secondary
424
metabolite was isolated through thin layer chromatography and silica gel column chromatography with
solvent fraction of hexane: ethyl acetate in the ratios 9:1, 6:4, 4:6 and 2:8.The sample was analysed in UV-VIS
spectroscopy. The absorbance was measured at different wavelengths, and the values are tabulated. A graph
with wavelength along x-axis and absorbance along y-axis was obtained. Thus, the secondary metabolite was
analyzed in UV-Vis spectroscopy.
REFERENCES
1. Arimak and Imanaka H, (1964), pyrrolnitrin, new antibiotic substances, produced by pseudomonas,
journal of Agri. Biol. Chem.28:575-576 .
2. Calhoun and Garcia, (2001), The branch point for pyocyanine biosynthesis in pseudomonas aeruginosa,
Journal of Gen. microbial. 72:581-583.
3. Carson M. and Glick, (1998), Phenotypic recognition of pyocyanine mutants in pseudomonas aeruginosa,
Journal of bacterial. 117:312-314.
4. Castirc and Gamble, (1998), hydrogen cyanide, the secondary metabolites of pseudomonas aeruginosa,
Can. Journal of Microbiol.21:613-618
5. Cattelan, (1977), The secondary metabolism, Journal of Incorpo-VOL.43,1979 438.
6. Chain E.B, (1977), The structure of pseudomonic acid B, Journal of chem. soc. Perkin trans. 1, p. 318-
322.
7. Chain Mellows, (1977), the structure of pseudomonic acid A, a novel antibiotic produced by pseudomonas
fluorescens, Journal of chem.soc. Perkin Trans. 1:294-309.
425
HLA G 14bp Indel Polymorphism Implicated in Genetic
Predisposition to Asthma in Kodaikanal
Renuka S1, Thenmozhi M2 and V.J. Kavitha3
Research Scholar, 3Assistant Professor, Department of Biotechnology,

1, 2
Mother Teresa Women’s University, Kodaikanal – 624 101.

e-mail: kavithahla@gmail.com
Abstract:
Asthma is a common and complex condition, with considerable heterogeneity both in its phenotype and
the underlying pathophysiology. In India, an estimated 57,000 deaths were attributed to Asthma in 2004
(WHO 2004) and it was seen as one of the leading causes of morbidity and mortality in rural India. The
HLA complex is a 3.6 Mb high-density gene region located at the 6p21.3 chromosome region, encompassing
more than 200 genes. The HLA G gene is a nonclassical class I HLA locus is composed of eight exons
and seven introns. In the 3’UTR of HLA G is the 14bp is composed of eight exons and seven introns. In
the 3’UTR HLA-G polymorphism is the 14bp indel polymorphism that can influence HLA G expression
through mRNA stability. Recent studies in asthmatic families and a birth cohort at high risk for developing
asthma suggest a role for HLA G in asthma susceptibility. This study attempted to understand asthma (type I
hypersensitivity) in the context of HLA G in a hilly terrain because they more number of people affected by
Asthma in the hills when compared to the plains. We conclude that ‘ID’ genotype in HLA G 14 bp 3’ UTR
polymorphism predisposes to asthma. We suggest that there is heterozygote advantage through balancing
selection in asthmatic patients to other unresolved serious clinical conditions. Further studies are required as
to understand why the heterozygotes are in high frequency in asthma patients in Kodaikanal.
Keywords: HLA G, 14 bp indel, heterozygote advantage, balancing selection, polymorphism, asthma.
INTRODUCTION
Asthma is one of the most common chronic inflammatory lung diseases worldwide [1]. The Global
Strategy for Asthma Management and Prevention Guidelines define Asthma as a “chronic inflammatory
disorder of the airways associated with increased airway hyper-responsiveness, recurrent episodes of
wheezing, breathlessness, chest tightness, and coughing, particularly at night/early morning.” Asthma
is a complex condition, with considerable heterogeneity both in its phenotype and in the underlying
pathophysiology. According to World Health Organisation (WHO) estimates 300 million people suffer
from Asthma, 255, 000 people died of Asthma in 2005 and over 80% of Asthma deaths are reported
from low and lower-middle income countries. In India, an estimated that 57,000 deaths were attributed
to Asthma in 2004, and it was seen as one of the leading causes of morbidity and mortality in rural
India [2].
Several strategies have been applied to identify genes and genetic variants that predispose to asthma
and allergic diseases. These mainly include positional cloning, candidate gene approach, genome wide
association study. Asthma is the result of a complex interaction between environmental factors and genetic
variants that confer susceptibility. Studies of the genetics of asthma have previously been conducted using
linkage designs and candidate gene association studies. Using candidate gene association studies, more than
100 candidate genes have been studied because their biological function suggests that they could play a role
in the pathogenesis of asthma [3].
The HLA complex is a 3.6 Mb high-density gene region located at the 6p21.3 chromosome region,
encompassing more than 200 genes. The HLA-G gene is a nonclassical class I HLA locus, which, like
its classical counterparts, is composed of eight exons and seven introns. In contrast to classical class
I loci, HLA-G has a stop codon at exon 6, leading to a short cytoplasmic tail, exhibits a 5’ upstream
regulatory (or promoter) region (5’URR) extending at least 1.4 kb from the initial ATG [4] and presents an
extended 3’ untranslated region (3’ UTR). The HLA-G molecule is almost monomorphic and expressed in
both membrane-bound and soluble isoforms. Fifteen HLA G alleles, providing seven different transcript
isoforms, have been reported; these alleles result from alternative splicing of mRNA and are expressed as
membrane-bound proteins (HLA G1, HLA G2,HLA G3, HLA G4) and soluble molecules (soluble HLA
[sHLA G]–1/HLA G5, HLA G6, HLA G7) [5]. The 3’UTR region has a 14 bp indel polymorphism that
can influence HLA G expression through mRNA stability. The HLA G gene has a microRNA binding site
at its 3’ UTR region, less than 200 bp away from the 14 bp polymorphic site. This site is a potential target
for three microRNAs — miR-148a, miR-148b e miR-152 [6]. The 3142C/G influences miRNA targeting
of HLA G and is associated with risk of asthma.
Consistently, conflicting results have been obtained in different studies concerning the association of
the 14bp HLA G genotypes with autoimmune diseases, pathological pregnancy, recurrent spontaneous
abortions, and pre-eclampsia. Recent studies in asthmatic families and in a birth cohort at high risk for
developing asthma suggest a role for HLA G in asthma susceptibility [7]. HLA G may be an attractive
candidate molecule for modulating specific T cell profiles that are important in asthma.
Patients who have asthma vary in age of onset, course, sensitivity to specific environmental precipitants,
and response to therapy. Consequently, the relative contribution of genetic factors also may vary considerably
among patients. Genetic predisposition varies with race and ethnicity (i.e., genes associated with asthma
in one population may not be associated or may be less frequently associated with asthma in another
population). This study attempted to understand asthma (type I hypersensitivity) in the context of HLA G in
a hilly terrain because they more number of people affected by asthma in the hills. We compared the different
types of hypersensitivity patients and controls from published literature to determine predisposition to other
hypersensitivity types if any in the study population.

It was ensured the volunteers participating in this study were all informed of the purpose and outcome of
the study. For genotyping purpose random samples, one per household were selected. All the volunteers
were above 18 years of age. The diagnosed Asthma patients were selected from the Government hospitals
for the present study. Total of 36 samples was collected from Thandigudi, Pannaikadu, Kodaikanal. The
investigation was done in accordance with the ethical principles outlined by the Indian Council of Medical
Research (ICMR) guidelines for medical research involving human subjects. And informed consent was
obtained from volunteers.
DNA was extracted through a modified protocol adopted from Azubel et al., 2000 protocol [8].
Genotyping 14 bp indel polymorphism was done using specific primers to amplify the fragment from the
isolated DNA as per Wu et al., 2010 [9]. Data was collected from the published literature for the allele and
genotype frequencies of HLA G and compared with the study population. Frequency tables were created using
Excel (2007). Arlequin (v3.1) was used to calculate the genetic distances. Dendrograms were constructed
in Molecular and Evolutionary Genetics Analysis (MEGA v3.1). Fischer’s exact test was performed using
Graph pad.
427

To see the prevalence of the predisposing genotype or allele for the HLA G, the populations were grouped
into nine Groups on the basis of the geographical locations. The total number individuals complied for the
study was 5028 (Patients N = 2383, Controls N = 2645) and the 25 samples (15 Patients and 10 Control) of
the study population.
Figure 1: Gel Picture for HLA G 14 bp indel polymorphism in Asthma patients
Insertion/Insertion (II) Homozygotes had 224 bp band, Deletion/Deletion (DD) Homozygotes had
210 bp band, Insertion/Deletion (ID) Heterozygotes had both 224bp and 210bp bands (Fig 1). In the study
population, Asthma patients, the ‘D’ allele was found at a frequency of 57%. The ‘I’ allele was found at a
frequency of 43%. Both these frequencies fell in the mean frequency group. Asthma patients showed a high
frequency of 73 % for ‘ID’ genotype, while ‘DD’ genotype was low at 20% and ‘II’ genotype was even lower
at 7%. This was significantly different from the controls who showed a frequency of 40% for ‘ID’ genotype
while ‘DD’ genotype was 30%, and ‘II’ genotype was 30% (Fig 2).
Figure 2: Graphical Representation for study Asthma with disease population
It was observed that heterozygote frequency is higher in patients. Heterozygotes are selected by natural
selection through balancing selection as a defence against deleterious factors that both homozygotes might
encounter. This is termed as heterozygote advantage. The classical example for heterozygote advantage is
prevalence of sickle cell anaemia in an endemic malaria region. HLA locus is known for its balancing selection
428
in various environments as response to pathogens [7]. But it is evident that the heterozygote predisposes to
asthma in the study population. This heterozygote condition might be in linkage disequilibrium with other
alleles at the HLA G locus and/or upstream promoter polymorphisms [10]. Further case control studies are
required to validate if the linkage disequilibrium is the reason for heterozygotes being at a higher frequency
in asthma patients in the HLA G 14 bp locus and if there is heterozygote advantage through balancing
selection.
In cluster analysis, it was seen that there is a difference between patients and controls for asthma in this
study because they fell in separate clusters. In the Madison study, [10] asthma patients are distinct from
the controls but they do not form separate clusters. It was also observed that the asthma groups did not
cluster together (Figure 3). This was because of the ‘DD’ genotype being higher in Madison patients. Study
population Asthma and European Systemic Lupus Erythematosus (SLE) patient forms a separate cluster. The
study population can also be at risk for SLE.
Figure 3: Cluster analysis for various Disorders with corresponding control with study population Asthma
This again proves that asthma patients of Kodaikanal are distinct from Madison studied. This may be
due to heterozygote advantage of asthma patients in Kodaikanal, a hill station when compared to Madison’s
study where the patients are from plains. The Fischer’s test was statistically significant with a p value of 0.02.
The allele 14 bp insertions are low secretors, and 14bp deletions are high secretors. 14bp deletion is
known to be associated with a high plasma level of sHLA G. It has been suggested that the low secretory
allele (I) decreases the amount of sHLA G in heterozygous condition in because of the dominant effect of the
low secretors over the high secretors [11]. Though there might be heterozygote advantage in asthma patients
for an as yet unresolved serious clinical condition, the heterozygous condition itself decreases the amount of
sHLA G and causes the progression to the pathogenesis of asthma. Similar evidence for balancing selection
at HLA G 14 bp locus was found recently in south Amerindians [12].
CONCLUSION
This study has proved that ‘ID’ genotype in HLA G 14 bp 3’ UTR polymorphism predisposes to asthma.
We have observed a heterozygote advantage at a hill station. Further studies are required as to understand
why the heterozygotes are in high frequency and if there is linkage disequilibrium. Asthmatic patients
in hills and plains need to be studied separately, and their genotype frequencies compared for the better
understanding of the epigenetics in asthma. Different ethnic populations are required to be studied to get
429
a better view of HLA G 14 bp indel in Asthma in the Indian context. Measuring the secretory level of
HLA G (i.e., sHLA G levels) in Asthmatic patients and understanding their pathophysiology in disease
progression is the need of the hour.
REFERENCES
1. Li H, Xiaoyan D, Quanhua L, Jie L and Yixiao B, (2009), Single-Nucleotide Polymorphisms in Genes

Predisposing to Asthma in Children of Chinese Han Nationality J Investig Allergol Clin Immunol; Vol.
19(5): 391-395.
2. Smith KR, National Burden of disease in India from indoor air pollution, Proc Natl Acad Sci USA 2000,
Nov 21, 97(24), 13286-93.
3. Vercelli D, Discovering susceptibility genes for asthma and allergy, (2008) Nat Rev Immunol 8(3):169-
182.
4. Moreau P, Flajollet S and Carosella ED, (2009), Nonclassical transcriptional regulation of HLA- G an
update, J Cell Mol Med. 13(9B):2973–2989.
5. Hviid TV, Rizzo R, Melchiorri L, Stignani M and Baricordi OR, (2006) Polymorphism in the 5’ upstream
regulatory and 3’ untranslated regions of the HLA-G gene in relation to soluble HLA-G and IL-10
expression. Hum Immunol 67: 53-62.
6. Veit TD and Chies JA, (2009), Tolerance vs. immune response — microRNAs as important elements in
the regulation of the HLA-G gene expression, Transpl Immunol 20:229-231.
7. Tan Z, Shon AM and Ober C, (2005), Evidence of balancing selection at the HLA-G promoter region,
Hum Mol Genet 14:3619–3628.
8. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K, (2001), Current
Protocols in Molecular Biology (Wiley, New York).
9. Wu F, Wu L, Luo X, Tang Z, Yang M, Xia Y, Xie C, Liu N, Zeng X, Guan J and Yua G, (2010),
Association of HLA-G 14bp insertion/deletion polymorphism with autoantibody production in patients
with autoimmune rheumatic diseases, Journal of Clinical Immunology and Immunopathology Research
Vol. 2(2), pp. 15-21.
10. Tan Z, Randall G, Fan J, Camoretti-Mercado B, Brockman-Schneider R, Pan L, Solway J, Gern JE,
Lemanske RF, Nicolae D and Ober C, (2007), Allele-Specific Targeting of microRNAs to HLA-G and
Risk of Asthma, The American Journal of Human Genetics Volume 81.
11. Shankarkumar U, Pawar A, Gaonkar P, Parasannavar D, Salvi V, Ghosh K, (2012), HLA allele associations
in idiopathic recurrent spontaneous abortion patients from India, J Hum Reprod Sci, 1: 19-24.
12. Veit TJ, Cazarolli J, Salzano FM, Schiengold M and Chies JAB, (2012), New evidence for balancing
selection at the HLA-G locus in South Amerindians, Genetics and Molecular Biology, 35, 4 (suppl),
919-923.
430
Production and Characterization of Biosurfactant from
Hydrocarbon Contaminated Soil
S. Monisha, T. Supassri, M. Dhivya, D. Venkatesan
Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli

monsbio28@gmail.com
Abstract:
Biosurfactants are extracellular surface active compounds produced by bacteria, fungi and yeast. Most microbial
surfactants are complex molecules, comprising different structures it includes lipopeptides, glycolipids,
polysaccharide-protein complexes, fatty acids and phospholipids. In the past two decades, bio surfactants have
gained increasing attention due to their useful properties such as biodegradability, low toxicity, ecological
acceptability and ability to be produced from renewable and cheaper substrates. In this study we have taken,
and the total number of twelve soil samples were collected from the welding shop, petrol bunk and automobile
workshop from different parts of Tiruvannamalai. The bacterial colonies were obtained by serial dilution
technique. Thirteen isolated colonies were obtained by quadrant streaking method those isolated colonies were
subjected for surfactant production. The oil displacement technique was applied for identification of surfactant
production. Among them, one isolated was effectively displaced oil. The effective organism was identified
by various biochemical tests such as Gram staining, citrate utilization test, lactose fermentation test, Catalase
test, Haemolysis test, motility test. Based on the characteristics the organism was confirmed as Pseudomonas
aeruginosa.The produced biosurfactant was characterized by TLC and confirmed by GC-MS. The effective
validation of biosurfactant was performed by stain removal capacity and antimicrobial activity.
Keywords: Biosurfactant, Pseudomonas aeruginosa, Oil displacement test, TLC, GC-MS.
INTRODUCTION
Biosurfactants are surface-active substances synthesised by living cells. Interest in microbial surfactants has
been steadily increasing in recent years due to their diversity, environmentally-friendly nature, possibility
of large-scale production, selectivity, performance under extreme conditions, and potential applications in
environmental protection.
Biosurfactants enhance the emulsification of hydrocarbons, have the potential to solubilise hydrocarbon
contaminants and increase their availability for microbial degradation. The use of chemicals for the treatment
of a hydrocarbon polluted site may contaminate the environment with their by-products whereas biological
treatment may efficiently destroy pollutants while being biodegradable themselves. Hence, biosurfactant-
producing microorganisms may play an important role in the accelerated bioremediation of hydrocarbon-
contaminated sites. These compounds can also be used in enhanced oil recovery and may be considered for
other potential applications in environmental protection. Other applications include herbicides and pesticides
formulations, detergents, healthcare and cosmetics, pulp and paper, coal, textiles, ceramic processing and
food industries, uranium ore-processing, and mechanical dewatering of peat.
Several microorganisms are known to synthesise surface-active agents; most of them are bacteria and
yeast. When grown on hydrocarbon substrate as the carbon source, these microorganisms synthesise a wide
range of chemicals with surface activity, such as glycolipid, phospholipid, and others. These chemicals are
synthesised to emulsify the hydrocarbon substrate and facilitate its transport into the cells. In some bacterial
species such as, surfactants are also involved in a group motility behaviour called swarming motility [1,2,4].

Collection of sample
The Soil samples were collected from the oil spilled surface from the automobile workshop, welding shop
and petrol bunk in Tiruvannamalai Tamilnadu. The samples were collected in sterile polythene bag and were
taken to the laboratory for the isolation of bacteria.
Isolation and enumeration of bacteria from soil sample

1g of each collected soil samples were serially diluted in peptone water up to 10-8 dilution and 0.1 ml of
serially diluted peptone water was added to each nutrient agar plate pre-coated with motor lubricant oil and
it was spread plated. It was then incubated at 37°C for 24 hours. After incubation different colonies produced
from plate were selected, and individual colonies were isolated by quadrant streaking method for further
analysis.
Screening of biosurfactant producing organism

Each isolated individual colonies were inoculated in 50 ml of nutrient and incubated at 37°C for 72 hours
with shaking condition. After incubation, the broth was centrifuged at 10,000 rpm for 10 min at 4°C. The
supernatant was collected and used for screening and qualitative analysis.
Identification of biosurfactant producing organism by oil spreading technique

50 ml of distilled water was added to a large Petri dish (15 cm diameter) followed by the addition of 20 μl of
crude oil to the surface of water, ten μl of supernatant of the culture broth
Characterization of biosurfactant producing organism [5,9]

The screened biosurfactant producing organism was then characterized by using Different tests.
Gram staining
Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixing kills some
bacteria but is mostly used to affix the bacteria to the slide so that they don’t rinse out during the staining
procedure.
Citrate utilization test

Simmons Citrate agar slants contain sodium citrate (the only carbon source) and ammonium ions (the sole
nitrogen source). A pH indicator, Bromothymol Blue is also included. Bromothymol Blue is GREEN at pH
< 7.0 and BLUE at pH > 7.6. Organisms that utilize citrate for energy produce alkaline compounds as by-
products. Thus, a positive result for citrate utilization is the formation of a BLUE color
Lactose fermentation test

Various bacteria ferment lactose as you observed in the Methyl Red Test experiment. Besides utilizing methyl
red medium to determine the presence of Lactose fermenting bacteria, one may also utilize MacConkey
(MAC), the media indicate the growth of lactose fermenters on. The growth of lactose fermenters on MAC
is noted by the presence of creamy-pink colored colonies.
Heamolysis test
The fresh single colony from the isolated cultures were taken and streaked on Blood agar plates. The plates
were incubated for 48 hours at 37°C. The bacterial colonies were then observed for the presence of a clear
zone around the colonies.
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Catalase test
1. One drop of hydrogen peroxide solution is placed on a slide.
2. A small portion of the suspect colony is spotted onto the centre of a cover-slip.
3. Invert the cover-slip and place it on the drop of hydrogen peroxide solution.
4. Look for vigorous bubbling occurring within 10 seconds.
Motility test
Place a drop of bacterial suspension on the center of the cover slip; apply wax or soft paraffin over the
corners of the cover slip. Put a glass slide gently over the cover slip and hold it upside down. It should be in
such a manner that bacterial suspension should be hanging between the cover slip and glass slide. Examine
under a microscope, first under 10x, and then under 40x.
Extraction of biosurfactant
Ammonium sulphate precipitation method
The biosurfactant producing organism is inoculated in 100ml of nutrient broth shaken at 200 rpm at 37°C
for 96 hours. It was then centrifuged at 10000 rpm 4°C for 10 min. The supernatant containing biosurfactant
was precipitated with 40% (w/v) ammonium sulphate and incubated overnight at 4°C. The precipitate was
then collected by centrifugation at 10,000 rpm for 10 min at 4°C, and it was then extracted with acetone.
After the extraction process, the precipitate was dried inside fume hood until dry, white-colored powder was
obtained. [6,7,8]
Solvent extraction method

Cells were removed from the culture broth by centrifugation at 10 000 rpm 4°C for 10 min. The biosurfactant
was extracted from supernatants with a mixture of solvents in the following ratio (methanol/chloroform/1-
butanol, 1:1:1 by volume). The mixture was shaken continuously for 200 rpm at 30°C for 5 hours.
After 5 hours, two layers of precipitation were obtained. The upper layer was discarded, and the lower
layer was poured onto a clean glass Petri dish. The Petri dish was put inside the fume hood until dry, brown-
colored paste was obtained [10,11,12].
Characterization of biosurfactant by thin layer

Chromatography
Silica gel plates were prepared by mixing the silica powder with distilled water (1:2) and poured on the glass
plates. A spot of crude biosurfactant was placed on a silica plate. The biosurfactant was separated on the
plate using toluene, butanol, methanol (6:3:1). Iodine reagent was sprayed to detect glycolipid biosurfactant.
Qualitative analysis of biosurfactan by drop

Collapse test
The microtitre plate was coated with oil, and five µl of supernatant added and waited for 6 hours for detection
of biosurfactant. (Jain et al., 1991; Bo door and Miller-Maier, 1998) with [15,16].
Analysis of biosurfactant by GC-MS

The crude biosurfactant was used to identify the chemical constituents present in it by soxhlet apparatus.
Hexane as a prime solvent used for extraction.
The identification of chemical composition of hexane extracts of powdered biosurfactant were
performed using a GC–MS spectrograph (Agilent 6890/Hewlett–Packard, 5975) fitted with electron
impact (EI) mode. The hexane extract (3.0 µl) of powdered biosurfactant were injected with a Hamilton
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syringe to the GC–MS manually for total ion chromatin-125 graphic analysis in split mode. In quantitative
analysis, selected ion monitoring (SIM) mode was employed during the GC- MS analysis. SIM plot of the
ion current resulting from very small mass range with only compounds of the selected mass was detected
and plotted
Antimicrobial activity of biosurfactant

The chemical constituents present in crude biosurfactant were extracted with hexane by the soxhlet method.
The precipitate was collected in Petri plate after evaporation of solvent Sterile distilled water was added to
it at different concentrations (5, 10, 15, 20 µl).The biosurfactant disc was prepared and placed on Mueller –
Hinton agar plate swabbed with E.coli, B.subtilus, S.typi, S.aureus, The antimicrobial activity of biosurfactant
can be detected by the halo zone formed around the disc [17,10].
Detergent activity of biosurfactant

Two clean clothes were stained with ten µl of motor lubricant oil. To the first cloth the commercial detergent
only added and washed. For the second cloth, the biosurfactant along with detergent added and washed like
first cloth [18].

Isolation and enumeration of bacteria from hydrocarbon contaminated soil sample [6,7,8]
The serial dilution technique was adopted for enumeration of bacteria present in the soil. The bacteria present
in different samples were depicted in figure 5.1 to 1.1
Table 5.1 to 1.1: colonies formation in serial dilution
S.No Location of sample Bacterial Count (CFU/g)

1 Automobile Workshop 3.1×10
2 Welding shop 6.2×10
3 petrol bunk 4.6 ×10
Figure 5.1 to 1.1 : Colonies formed on nutrient agar (Automobile workshop)
Screening of biosurfactant producing organism

Oil displacenent technique
The culture S3 showed a zone of displacement in the motor lubricant oil. The biosurfactant producing
organism can only able to displace the oil. The results were
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5.2 to 1.2: Oil displacement test
S.No Culture Sample Zone Size(mm)

1 S1(welding shop soil) NO
2 S2(automobile workshop soil) NO
3 S3(petrol bunk soil) 4mm
From this, we came to conclude that sample S3 has the capability to produce biosurfactant.
Figure 5.6 to 1.3: oil displacement test
Characterization of biosurfactant producing organism

The screened biosurfactant producing organism was taken for the extraction of Biosurfactant. The organism
was identified by different biochemical tests the Results were tabulated in table 5.3 to 1.4
Table 5.3 to 1.4: biochemical characteristics of isolated organism
S.NO BIOCHEMICAL TESTS OBSERVATION INFERENCE

1 Gram staining Pink color rod appears Gram-negative
2 Citrate utilization test Blue color appears Positive
3 lactose fermentation test Creamy pink colored colonies Negative
4 Heamolysis test Clear zone formation Positive
5 Catalase test Bubble formation Positive
6 Motility test Brownian movement Positive
Extraction process
Solvent extraction method
In this extraction method, two layers were obtained (figure 5.11 to 1.1). The upper layer was discarded, and
the lower layer was poured onto a clean glass Petri dish. The Petri dish was put inside the fume hood until
dry, brown-colored paste (figure 5.12) was obtained.
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Ammonium sulphate precipitation method

Precipitate was extracted with acetone. After the extraction process, the precipitate was dried in a fume hood
until it reached a constant weight, white-colored powder (figure 5.13) was obtained.
Figure 5.13: powdered biosurfactant
The dry weight of the biosurfactant produced was 2.5g/L by ammonium sulphate precipitation method.
Characterization of biosurfactant
The biosurfactant produced yellow spots on a TLC plate while spraying iodine. It confirmed the presence of
biosurfactant (lipid) The TLC plate was shown
Appearance of a yellow spot in TLC
Qualitative analysis of biosurfactant
Drop collapase test

The drop of a sample collapses from the coated oil. It indicated the presence biosurfactant in the culture
broth.
Analysis of biosurfactant by GC- MS

In GC-MS analysis in addition to the biosurfactant (Dodecanoic acid is 99% the form of rhamnolipid)
various other
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S.No RT Name of the Compound Area (%) Quality

1 9.966 Decane, 2,3,5,8-tetramethyl Heneicosane 0.20 52
2 10.278 Nonane, 5-methyl-5-propyl Decane 0.37 72
3 11.140 Sulfurous acid, hexyl pentyl ester 0.19 58
4 12.67 Octacosyl trifluoroacetate 0.15 64
5 13.996 Sulfurous acid 0.18 58
6 14.823 Pentacosane 0.23 59
7 17.095 3-Ethyl-3-methylheptane 0.37 64
8 21.570 Eicosane 0.27 58
9 24.732 Trimethyl[4-(1-methyl-1-methoxyeth Phenoxysilane 0.19 35
10 28.532 Decanoic acid, 1,2,3-propanetriyester(biosurfactant) 3.55 46
11 28.817 Decanoic acid, 1,2,3 propanetriyester 8.67 55
12 29.116 Decanoic acid, 1,2,3 propanetriy ester 2.72 54
Antimicrobial activity of biosurfactant

The clear zone of inhibition observed against Staphylococcus aureus, E.coli, S. typi and Bacillus subtilus. It
proved the presence of antimicrobial activity in the biosurfactant.
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Figure 5.16: Antimicrobial activities of the bacterial organism
Detergent activity of biosurfactant
Figure 5.17: Stain removal with commercial Figure 5.18: stain removal with
detergent and biosurfactant commercial detergent only
In this two scenario, the detergent with biosurfactant showed fast stain removal efficacy when compared
with detergent al
CONCLUSION
The bacterium Pseudomonas aeruginosa was isolated from soil, and it produces biosurfactant. It is used
to clear oil stains in cloth; it rapidly removes the stains with detergent than commercial detergent alone.
Biosurfactant also has antimicrobial activity against various bacterial species such as Methicillin Resistant
Staphylococcus aureus (MRSA), E.coli and so on. So it can be used in the washing process thus a cloth
borne disease can be avoided by using the biosurfactant. According to the report of various researches
the biosurfactant are used for the other purposes such as in food industries, cosmetics, pesticides and
pharmaceutical industries. It is very easy for production, cost effective, eco-friendly when compared to
synthetic chemical used for this purpose. If the people concentrating on biosurfactant for various application
means definitely it preserve the environment.
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BIBILIOGRAPHY
1. Abalos A., Pinaso A., Infante M.R., Casals M., Garcia F. and Manresa A., (2001), ‘Physicochemical
and antimicrobial properties of new rhamnolipids by Pseudomonas aeruginosa AT10 from soybean oil
refinery wastes, Langmuir Vol.17, No.3, pp. 67–1371.
2. Aronstein, B.N.Y.M. Calvillo and M. Alexander, (1991), ‘Effect of surfactants at low concentration on
the desorption and biodegradation of sorbed aromatic compounds in soil, Environ. Sci. Technol. Vol.25,
pp. 1728–1731.
3. Aguilar M.I., (2004), ‘Methods in molecular biology: HPLC of peptides and proteins’ Methods and
Protocols Vol. 251.
4. Barkay T., Navon-Venezia S., Ron E. and Rosenberg E., (1999), ‘Enhancement of solubilization
and biodegradation of polycyclic aromatic hydrocarbons by the bioemulsifier Alasan, Appl Environ
Microbiol Vol. 65, pp. 2697–2702.
5. Besson F., Peypoux F., Michel G., and Delcambe L., (1976), ‘ Characterization of iturin A in antibiotics
from various strains of Bacillus subtilis’ Journal of Antibiotics, Vol.29, No.10, pp. 1043-1049.
6. Bharathi S. and Vasudevan N., (2001), ‘Utilisation of hydrocarbons by Pseudomonas fluorescens isolated
from a petroleum-contaminated soil.’ Environ. Int. Vol.26, pp. 413- 416
7. Chen S.Y., Wei Y.H. and Chang J.S., (2007), ‘Repeted pH- stat fed-batch fermentation for rhamnolipid
production with indigenous Pseudomonas aeruginosa S2, Applied Microbiol Biotechnol., Vol.76, No.1.
Pp 67-74.
8. Cameotra S.S. and Makkar R.S., (1998), ‘Synthesis of biosurfactants in extreme conditions.’ Applied
Microbiology and Biotechnology, Vol.50, pp.520-529.
9. Cerniglia C.E., (1992), ‘Biodegradation of polycyclic aromatic hydrocarbons’. Biodegradation No.3,
pp. 351–368.
10. C.G. Van Ginkel, Biodegradation, (1989), Vol. 151, No.7.
11. Desai J.D. and Banat I.M., (1997), ‘Microbial Production of Biosurfactants and their Commercial
Potential, Microbial Mol Biol Rev, vol.6 No1 pp. 47-64.
12. Deleu M. and Paquot M., (2004), ‘From renewable vegetabless resources to microorganisms: new
trends in surfactants. Comptes Rendus Chimie, Vol. 7, pp.641-646.
13. Dunlap L.E. and Beckmann D.D., (1988), ‘Soluble hydrocarbons analysis from kerosene / diesel. In
proceedings of the conference on Petroleum hydrocarbons and Organic chemicals in ground water’:
Prevention, Detection and Restoration, Dublin, Ohio, National Water Well Association.
14. Edwards D., Luthy R. and Liu Z., (1991), ‘Solubilization of polycyclic aromatic hydrocarbons in
micellar non-ionic surfactant solutions.’ Environ Sci Technol Vol.25, pp.127–133.
15. Gregory A., ( 2001), ‘Enhanced oil recovery—Ten years on.’ Petrol Rev Vol.55,No.655 pp. 40.
16. Georgiou G, S. Lin and Sharma M.M., (1992), ‘Surface-Active Compounds from Microorganisms.’
Bio/Technology, Vol.10, PP.60 – 65.
17. Gieg, L.M. R.V. Kolhatkar, M.J. McInerney, R.S. Tanner, S.H. Harris, J.r. K.L. Sublette, and J.M.
Suflita. (1999) ‘Intrinsic Bioremediation of Petroleum Hydrocarbons in a Gas Condensate Contaminated
Aquifer. Environmental Science and Technology, Vol.33, pp. 2550-2560.
18. Hewald, S. Josephs, K. and Bolker M., (2005), ‘Genetic Analysis of Biosurfactant Production in
Ustilago maydis’ Applied and Environmental Microbiology, Vol.71No.6, pp.3033-3040.
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Optimization of media for the production of keratinase
enzyme production from Proteus strain
B. Anandaraj1, T.P. Rajesh2, N. Ilavarasan3 and P. Saranya Devi4
Department of Biotechnology, 3, 4Department of Civil Engineering, Anna University, Trichy

1, 2
e-mail: Corresponding author: biotechraj@gmail.com
Abstract:
Keratin are insoluble fibrous proteins found in hair, wool, feather, nail, horns and other epithelial covering
which is rich in beta helical coil linked through cysteine bridges. Keratinase belongs to the class hydrolase
which are able to hydrolyse insoluble keratins more efficient than other proteases. The keratinous waste can
be biologically degraded by enzymes or microbes itself to form useful products. The ability of strain Proteus
mirabilis to utilize azokeratin as a substrate was tested. Among various carbon sources the maximum keratinase
activity was found in glucose and among various nitrogen sources the maximum keratinase activity can be found
in casein. Similarly optimum temperature and pH for the enzyme activity was found. Medium optimization was
carried out by using Plackett-Burmann method. Specific enzymatic assays demonstrate that keratinase can act
on a large variety of soluble and insoluble protein substrates. Extracellular keratinase may be a useful alternative
and eco-friendly route for handling the abundant amount of waste feathers or for applications in other industrial
processes. Keratinase also hydrolysed the outer epithelial sheath of hair roots provoking depilation. These data
suggest the potential of this enzyme for application in ecologically-friendly leather processing.
Keywords: Keratinase, Proteus mirabilis, Optimization, Plackett-Burmann method, Enzyme.
INTRODUCTION
An enormous amount of feather waste is generated by poultry processing industries and it is crucial industrial
waste over the world. According to a recent report in leading news paper India‘s contribution alone is 350
million tonnes. The poultry feathers are dumped, used for land filling, incinerated or buried, which involves
problems in storage, handling, emissions control and ash disposal. Discarded feather also causes various
human ailments including chlorosis, mycoplasmosis and fowl cholera. Considering its high protein content,
this waste could have a great potential as a source of protein and aminoacids for animal feed and for many
other applications. The million tons of feathers are currently converted to feather meal through steam
pressure and chemical treatment. This process of chemical treatment makes keratin waste more digestible,
but it is expensive and destroys certain aminoacids. Currently some industries produce feather meal by steam
pressure cooking, which require high-energy input.
Keratin is the most abundant structural protein in skin, hair, wool and feathers. Disulphide and hydrogen
bonds makes keratin a stable protein resistance to proteolytic hydrolysis. Feathers contain over 90% of crude
protein in the form of keratin. Feathers are formed from beta keratin, which is a fibrous protein that resists
chemical agents and enzymatic lysis. Keratinases are proteolytic enzymes in nature. It was classified as
proteinase of unknown mechanism as recommended by the Nomenculture Committee on the International
Union of Biochemistry (1978) with EC number 3.4.99 (Owen et al., 1983). Keratinase are capable of
degrading keratin, an insoluble structural protein cross linked with disulfide hydrogen bonds. Keratinases
are produced only in the presence of keratin containing substrate. It mainly attacks on the disulfide (-S-S-)
bond of the keratin substrate (Bockel et al.,1995).Keratinase production by microbes is influenced by several
facors such as temperature, pH carbon and nitrogen sources. These factors have varied effects in keratinase
production. (Singh et.al 1975)
Cultivation conditions are essential for successful production of enzyme and optimisation of parameters
such as pH, temperature, media composition. Keratinase production varies from organism to organism, so
studies on nutritional and environmental factors controlling the enzyme production are required. Optimisation
is one of the most important criteria for developing any new microbial process. Factor analysis and response
surface analysis are the most important tools to determine the optimal process conditions.
The use of these enzymes as an alternative to dehairing, catalyst in leather industry, in slow release
nitrogen fertilizers, cosmetics and biodegradable films. The applications of the keratin enzyme extended to
many field such as food, leather, beverages, medicinal, clinical and the list may extend to its own. (Adriano
Brandelli 2008).
MATERIAL AND METHODS

Sample collection
Chicken feathers and soil samples were collected from a local slaughter-house and local poultry processing
waste site at Namakkal District, India. The chicken feathers were washed thoroughly with tap water and
were air-dried. The dried feathers were defatted by soaking in diethyl ether for 24 hrs and were washed again
thoroughly with distilled water, air-dried and autoclaved. Finally, the feathers were cut into small pieces and
stored at room temperature.
Isolation of keratinolytic strains

The collected soil samples were serially diluted and plated in feather meal media containing (g/l): peptone-
5g, Yeast extract-5g, K2HPO4-0.1g, MgSO4.7H2O-0.2g, agar-18g and incubated for 2 days at 37°C.Distinct
colonies were identified and plated to obtain pure strains. The isolated strain were grown in Nutrient broth
and stored in glycerol stock for further use.
Screening of keratinolytic strains

Milk agar medium or casinolytic medium is used as the primary screening of keratinolytic organism. The
casinolytic medium was prepared and boiled to dissolve the contents nicely. Media containing the following
composition: (milk-15 ml, agar-0.8g, distilled water-85ml) was poured into the plates and allowed to solidify.
The inoculum was loaded into the wells and plates were incubated for 8 hr at 30°C and clear zone formation
was examined.
Degradation of feather
The strain Proteus mirabilis is inoculated in to 50 ml liquid medium containing (%, w/v): NH4Cl 0.05 g,
NaCl 0.05 g, K2HPO4- 0.03g, KH2PO4 0.04g, MgCl2.6H2O 0.024g ,Yeast extract 0.01g, Feather 1g ,pH 7.5
in a 250 ml-Erlenmeyer flask and is kept under incubation for 3 days under observation.
Inoculum Development:
The strain Proteus mirabilis is inoculated in to 50 ml liquid medium containing (%, w/v): NH4Cl 0.05g,
NaCl 0.05g, K2HPO4 0.03g, KH2PO4 0.04g, MgCl2.6H2O 0.024g, yeast extract 0.01g and feather meal 1g,
pH 7.5 in a 250ml Erlenmeyer flask. The culture was grown on a rotary shaker at 150 rpm and incubated at
37°C for 24 hours and used as an inoculum.
Production of enzyme
One ml of culture was inoculated into a 500 ml Erylen Meyer flasks containing the same 100ml liquid
medium. The flasks were shaken at 150 rpm for 3 days at 37°C. After 3 days of incubation, the culture
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medium was filtered through Whatman No.4 filter paper to remove the undergrated residues (Xiang et al;
1992). The filtrate was then subjected to centrifugation at 10,000 rpm for 10min to remove bacterial residue
and the crude enzyme used for enzyme assay.
Assay for keratinolytic activity

This procedure tested the keratinolytic activity of keratinase on azo keratin. 5mg of azokeratin was added to
a 1.5-ml centrifuge tube along with 0.8 mL of 50 mM potassium phosphate buffer, pH 7.5. This mixture was
agitated until the azo-keratin was completely suspended. 0.2-ml aliquot of supernatant of crude enzyme was
added to the azo keratin, mixed and incubated for 15 min at 50°C with shaking. The reaction was terminated by
adding 0.2 mL of 10% Tri Chloro Acetic acid (TCA). The reaction mixture was filtered and analyzed for activity
(Burtt et al., 1999). The absorbance of the filtrate was measured at 420 nm with a UV spectrophotometer. A
control sample was prepared by adding azokeratin with buffer solution without enzyme solution.
Effect of carbon source on keratinase production

To study the effect of different carbon sources on keratinase production the production medium was added
with different carbon source (1%) such as glucose, fructose, sucrose and starch for keratinase production.
Effect of nitrogen source on keratinase production

To study the effect of different nitrogen sources on keratinase production the production medium was added
with different nitrogen sources (1%) such as Ammonium sulphate, urea, casein and yeast for keratinase
production.
Effect of pH on keratinase production:

Effect of pH on the keratinalytic activity was determined using azokeratin as a substrate. Keratinolytic
activity was studied for various pH (4, 6, 8, 10) using the following buffers. For pH 4 and pH 6 Citrate buffer
is used. For pH 8 Phosphate buffer and for pH 10 Glycine - NaOH buffer was used.
Optimization of medium using Plackett–Burmann design

The important medium components with respect to their main effects were screened by Plackett–Burmann
design with a two-factorial design. It identifies the main physico-chemical parameters required for maximal
keratinases production by screening n variables in n + 1 experiments; each variable was examined at two
levels (Plackett et al.,). ‘Minitab 15’ was used to analyse the experimental plackett–Burmann design.

Isolation of keatinolytic organism
More than 30 organisms were obtained when the soil sample was spread on feather meal agar plate. From
that, the organism shown clear hydrolysis zone was selected.
Figure 1: Hydrolysis zone formation on feather meal agar plate
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Screening of proteolytic activity

Milk agar medium is used for primary screening of keratinolytic activity. The diameter of the hydrolysis
zone exhibited by the strain was measured. The maximum diameter of hydrolysis zone exhibited strain was
selected and is used for degradation process.
Figure 2: Screening on Milk agar plates
Degradation of feathers by Proteus mirabilis
The strain Proteus mirabilis exhibiting maximum hydrolysis zone was inoculated into a liquid me-
dium. It exhibits maximum growth and it results in complete degradation of feathers after 3 days when
incubated for 150 rpm at 37°C.
Figure 3: Degaradation of feather by strain proteus mirabilis
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Keratinase assay for Proteus mirabilis
Figure 4: Keratinase assay for strain tpr1
Fig 4 illustrates the maximum keratinase production by strain Proteus mirabilis was obtained in 69th hour
(106 U/ml) and the minimum keratinase production was obtained in 21st hour (43 U/ml).
Effect of carbon source on keratinase production
Figure 5: Effect of Various Carbon Sources on Keratinase Production
Fig 5 shows the effect of carbon sources on keratinase production for various incubation periods at 37ºC. The
maximum Keratinase production was recorded in dextrose supplemented medium and minimum Keratinase
production was recorded in fructose supplemented medium.
Effect of nitrogen source on keratinase production
Figure 6: Effect of Various Nitrogen Sources on Keratinase Production
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Fig 6 shows the effect of different kinds of nitrogen sources on keratinase production after various
incubation periods at 37ºC. The maximum amount of enzyme production was observed in casein
supplemented medium and minimum amount of keratinase production was observed in urea supplemented
medium.
Effect of pH on enzyme production
Figure 7: Effect of Various pH on Keratinase Production

Fig 7 shows the effect of various pH on keratinase production after 48 hours of incubation period at 37ºC.
The maximum keratinase production was observed at pH 8 and minimum amount of keratinase production
was observed at pH 4.
Effect of Temperature on keratinase production
Figure 8: Effect of various temperatures on keratinase production
Fig 8 shows the effect of various temperatures on keratinase production. The maximum keratinase production
was obtained at 45ºC and the minimum keratinase production was obtained at 4ºC.
Optimization of medium using Plackett-Burmann method

The keratinase activity of the cell free supernatant of strain culture at 48 h was maximum. The data
in Table 6 indicated that there was a wide variation of keratinase activity from 0.164U/mL to 160 U/
mL in twelve trials. This variation reflected the significance of factors on the enzyme activity. The
analysis of regression coefficients and t-value of seven ingredients are shown in Table. Generally, a large
t-value associated with a low P-value of a variable indicates a high significance of the corresponding
model term.
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Table 1: Plackett-Burmann design in coded units for seven variables along with the keratinase activity
S.No Feather Glucose casein CaCl2 MgSO4 NaCl KH2PO4 K2HPO4 Keratinase activity(U/ml)
1 1 1 -1 1 1 -1 1 -1 33.12
2 -1 1 1 -1 1 -1 -1 -1 119.2
3 -1 -1 -1 -1 -1 -1 -1 -1 01.99
4 -1 1 1 1 -1 1 1 -1 100.8
5 1 -1 1 1 -1 1 1 -1 27.63
6 1 -1 1 -1 -1 -1 1 1 38.62
7 1 1 1 -1 1 1 -1 1 160.05
8 -1 -1 1 1 1 -1 1 1 9.32
9 -1 1 -1 -1 -1 1 1 -1 117.3
10 1 -1 -1 -1 1 1 -1 1 1.66
11 1 -1 -1 1 -1 1 1 1 29.4
12 -1 1 -1 1 1 -1 -1 -1 0.164
Figure 9: Pareto Chart of the Effects for Keratinase Activity
The model equation for Keratinase activity

yield = 53.3 - 2.3 Feather + 44.3 Glucose + 18.9 Casein - 22.0 Cacl2 - 7.3 MgSO4 + 14.1 Nacl + 6.4
KH2PO4 + 11.2 K2HPO4
Neglecting the variables which were insignificant,the model equation for keratinase activity can be
written as yield = 53.3 + 44.3 Glucose.
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Table 2: Statistical analysis of Plackett Burmann design showing coefficient values, t- and P-value for each variable.
variable Coefficient t-stat value P-value Confidence limit

Constant 53.271 6.28 0.008 99.2
Feather -2.27 -1.71 0.778 22.2
Glucose 44.32 2.03 0.019 99.1
Casein 18.92 1.51 0.168 83.2
CaCl2 -21.996 -2.39 0.232 77.8
MgSO4 -7.29 -0.46 0.97 3
NaCl 14.12 0.98 0.241 76.9
KH2PO4 6.39 0.59 0.931 7.9
K2HPO4 11.22 0.41 0.607 40.9
Figure 10: Main effects of the medium constituents on keratinase production from
Plackett-Burman experimental results
The main effect of each constituent on the keratinase production was calculated as the difference
between the average measurement calculated at the higher (+) and lower (-) levels of the constituent. The
results showed that glucose, casein, Nacl, MgSO4.6H2O and KH2PO4 has the positive effect on keratinase
production. On the other hand Feather, CaCl2, K2HPO4 has the negative effect on the activity of keratinase.
Figure 11: Interaction plot for yield
Fig 11 illustrates the interaction between glucose and other factors in concentrations (g/l) on the keratinase
production. Finally, it can be concluded that the optimum formula of keratinase
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Production medium (g/l):

Feather - 2
Glucose - 7.5
Casein - 5
CaCl2 - 0.1
MgSO4 - 5
NaCl - 2
KH2PO4 - 10
K2HPO4 - 3
CONCLUSION
Utilization of these potential keratin degraders will definitely find biotechnological use in various industrial
processes involving keratin hydrolysis. It would also solve the waste disposal problem of poultry waste
and with limited resources recycling of keratinacious waste would be beneficial in both financially and
environmentally. Biodegradation of environmental wastes is preferred because it is safe, cheap and away from
pollution hazards. The ability of strain Proteus mirabilis to utilize as a substrate was tested. It was found that
maximum enzyme activity was 106 U/ml. Among various carbon sources the maximum keratinase activity
was found in glucose (89 U/ml) and among various nitrogen sources the maximum keratinase activity was
found in casein (57 U/ml). Similarly optimum temperature and pH for the enzyme activity was found to be
45°C and 8.0 respectively. The medium optimization was carried out using Placket Burmann method. In this
method the maximum keratinase activity was found in glucose.
ACKNOWLEDGEMENT
I thank Mr.N.Ilavarasan and Mr. T.P. Rajesh for their guidance and support for doing this project and I
wish to thank Ms. K. Malarvizhi for her support. Atlast I thank Department of Biotechnology and Civil
engineering for their excellent support.
REFERENCE
1. Anbu P., Gopinath S.C.B., Hilda A., Lakshmipriya T. and Annadurai G., ‘Optimization of extracellular
keratinase production by poultry farm isolate Scopulariopsis brevicaulis’, Bioresource Technology vol.
98 pp.1298-1303 (2007).
2. CHAO Ya-peng, XIE Fu-hong, YANG jing, LU Jing-hua and QIAN Shi-jun, ‘Screening for a new
streptomyces strain capable of efficient keratin degradation.’ Journal of Environmental Sciences 19
pp:1125-1128 (2007).
3. Deivasigamani B and Alagappan K.M., ‘Industrial application of keratinase and soluble proteins from
feather keratins’, Journal of Environmental Biology, 29(6), pp: 933-936 (2008).
4. Ekta Tiwary and Rani Gupta, ‘Medium optimization for a novel 58 kDa dimeric keratinase from Bacillus
licheniformis ER-15: Biochemical characterization and application in feather degradation and dehairing
of hides’, Bioresource Technology 101 pp: 6103–6110 (2010).
5. Emeka. A Okoroma, Hemda Garelick,Oduola O, Abiola, Diane Purchase “Identification and
Characterization of Bacillus licheniformis strain with profound keratinase activity for degradation of
melanised feather”, International Biodeterioration & Biodegradation 74 pp: 54-60 (2012).
6. Evgenia Vasileva-Tonkova and Adriana Gousterova Georgi Neshev ‘Ecologically safe method for
improved feather wastes biodegradation’ International Biodeterioration & Biodegradation 63 1008–
1012(2009).
448
7. Harde S.M., Balaji B. and Singhal R.S., ‘Optimization of fermentative production of keratinase from
Bacillus subtilis NCIM 2724’, Agriculture ,Food and Analytical Bacteriology 1(1) (2011).
8. Hong Gao, Mei Liu, Jintao Liu, Huanqin Dai, Xianlong Zhou, Xiangyang Liu, Ying Zhuo, Wenquan
Zhang and Lixin Zhang, ‘Medium optimization for the production of avermectin B1a by Streptomyces
avermitilis 14-12A using response surface methodology’, Bioresource Technology 100 4012–4016(2009).
9. Jin-Ha Jeonga, O-Mi Leeb, Young-Dong Jeona, Jeong-Do Kima, Na-Ri Leea, Chung-Yeol Leea
and Hong-Joo Sona, ‘Production of keratinolytic enzyme by a newly isolated feather-degrading
Stenotrophomonas maltophilia that produces plant growth-promoting activity’ Process Biochemistry 45
1738–1745(2010).
10. Lateef A, Oloke E.B. Gueguim kana,Sobowale B.O. and Ajao S.O., Keratinolytic activities of a
new feather-degrading isolate of Bacillus cereus LAU 08 isolated from Nigerian soil ‘International
Biodeterioration & Biodegradation 64 pp162-165 (2010).
11. Long-Xian Lv, Ming-Hao Sim, Yu-Dong Li, Jie Min, Wei-Hong Feng, Wen-Jun Guan and Yong-Quan
Li, ‘Production, characterization and application of a keratinase from Chryseobacterium L99 sp. nov.’,
Process Biochemistry 45 1236–1244(2010).
12. Sarita Agrahari and Neeraj Wadhwa ‘Degradation of Chicken Feather a Poultry Waste Product by
Keratinolytic Bacteria Isolated from Dumping Site at Ghazipur Poultry Processing Plant’, International
Journal of Poultry Science 9 (5): 482-489, (2010).
13. Subhasish Saha and Dharumadurai Dhanasekaran, ‘Isolation and Screening of Keratinolytic
Actinobacteria from Keratin Waste Dumped Soil in Tiruchirappalli and Nammakkal, Tamil Nadu, India’
Research Journal of Biological Sciences 2(2) pp: 124-131, (2010).
14. Sudhir K. Rai., Mukherjee and K. Ashis, (2011), ‘Optimization of production of an oxidant and detergent-
stable alkaline -keratinase from Brevibacillus sp. strain AS-S10-II: Application of enzyme in laundry
detergent formulations and in leather industry’Biochemical Engineering Journal 54 pp.47-56 (2011).
15. Tamil Kani P, Subha K, Madhanraj P, Senthilkumar G and Panneerselvam A, ‘Degradation of chicken
feathers by Leuconostoc sp. and Pseudomonas microphilus’, European Journal of Experimental Biology,
2 (2):358-362 (2012).
449
Enhancement of post-Harvest and Shelf Life of
Caricapapaya using Aloe Vera Gel based coatings
Nandakumar A1, Ebenezer Samuel King J2 and Arulvel R3
1, 2
M.tech-Biotechnology, 3Assistant Professor, Dept. of Biotechnology, K.S. Rangasamy College of Technology,
Tiruchengode-637215, Namakkal dist.
e-mail: nandhua91@gmail.com, Mobile No.: 7845659621
1
Abstract:
Papaya (Carica papaya) is one of the main tropical fruits of India. The desiccation of fruits and perishable
nature of papaya is a major drawback during transportation to distant markets and storage during glut in the
market. Aloe vera gel, mainly composed of polysaccharides, has been recently explored as an edible coating
owing to its antifungal activity. To improve the performance of edible coatings, various substances/chemical
additives have been incorporated. The present study was carried out to evaluate the ability of Aloe vera
gel based antimicrobial coatings to reduce/control the loss of post-harvest fruit quality in papaya. Freshly
harvested papaya fruits were coated with AGI (50% Aloe vera gel containing citric acid) and AGII (50%
Aloe vera gel containing tartaric acid). The coated and uncoated (control) fruits were stored at 30±3°C for 18
days. Physical (PLW, fruit size), chemical (pH, titrable acidity) and sensory characteristics (color, taste and
firmness), fruit disease index (FDI) and marketability were analyzed at regular intervals during the storage
period. The AGI coated fruits survived the storage period of 15 days, and the AGII coated fruits survived the
storage period of 18 days, whereas, all the uncoated controls decayed within ten days. The uncoated/control
fruits exhibited significantly greater changes in all the parameters tested. AGII coated fruits exhibited least
changes than AGI coated fruits. The coatings controlled the PLW, the ripening process (chemical changes,
color development and softening of fruit tissue) and decay to a greater extent and thereby extended the shelf
life quality of the fruits. Marketability was also found to be better for AGII coated fruits than AGI coated
fruits.
Keywords: Carica papaya, Aloe vera gel, Citric acid, Tartaric acid, PLW (Physiological Loss of Weight).
INTRODUCTION
One of the major growth segments in the food retail industry is fresh and minimally processed fruits and
vegetables. This new market trend has, thus, increased the demands for the food industry for seeking new
strategies to increase storability and shelf life and to enhance microbial safety of fresh produce. The most
important quality attributes contributing to the marketability of fresh and minimally processed produce
include appearance, color, texture, flavor, nutritional value, and microbiological safety. These quality
attributes are determined by plant variety, stage of maturity or ripening, and the pre- and postharvest
conditions, and all can change rapidly during post-harvest storage [1]. In general, desiccation and decay
are the two major causes of the termination of commercial life span of fruits and vegetables. The water
loss resulting from transpiration causes not only shrinkage and wilting, but also softening of produce. The
main tropical fruits produced in India include banana, mango, guava, pineapple and papaya. India is the
second amongst papaya producing countries and produces around 250lakh quintals of papaya annually [2].
The perishable nature of papaya is a major drawback for transport of the fruits to distant places and storage
during glut in the market. The estimated post-harvest losses of papaya fruits in India has been reported to be
between 40-100% [3]. The different storage methods used to preserve papaya fruits include low temperature
storage, controlled atmospheric storage, plastic film wraps and wax coatings. But there are many reports that
papaya in refrigerated storage is susceptible to fungal decay [4]. In controlled atmosphere storage, papaya
fruits were benefitted only slightly from storage in low oxygen (1-1.5%) conditions, at 13°C [5].
Gamma irradiations have also been used to increase the shelf life. However, higher doses caused
excessive softening of the flesh and low dose though increased shelf life reduced the level of ascorbic
acid. Polymeric films and wax coatings were found to be very effective in reducing water loss but were not
effective in preventing fruit decay.
There are few studies on the efficacy of edible coatings to reduce the perishability of papayas. Fruits
coated with these edible coatings were reported to extend the shelf life and delay the onset of fungal infection
[6]. Chitosan, a modified natural biopolymer with broad antimicrobial activity and film forming property [7]
has also been reported to be effective in extending the shelf life of papaya.
There is, hence, scope to study economically viable alternatives to improve the shelf life of papaya
fruits using other edible coatings that are biodegradable and eco-friendly. Recently, there has been increased
interest in using Aloe vera gel as an edible coating material for fruits and vegetables driven by its antifungal
activity [8-10]. Aloe vera gel based edible coatings have been shown to prevent loss of moisture and firmness,
control respiratory rate and maturation development, delay oxidative browning, and reduce microorganism
proliferation in fruits such as sweet cherry, table grapes and nectarines [11-13]. In addition to the traditional
role of edible coatings as a barrier to water loss and delaying fruit senescence, the new generation coatings
are being designed for incorporating and/or controlled release of antioxidants, nutraceutiacals, chemical
additives and natural antimicrobial agents [14-15].

Materials
Freshly harvested papaya (Carica papaya) fruits were procured from the local market of Coimbatore. They
were selected on the basis of size, color and absence of external injuries. Fresh leaves of Aloe vera were
obtained from the GCT campus garden.
METHODS
Preparation of gel:
Aloe vera gel matrix was separated from the outer cortex of leaves, and this colorless hydro parenchyma was
ground in a blender. The resulting mixture was filtered to remove the fibers. The liquid obtained constituted
fresh Aloe vera gel. The gel matrix was pasteurized at 70°C for 45 min. For stabilizing, the gel was cooled
immediately to an ambient temperature and ascorbic acid (1.9-2.0 g L-1) was added. For AGI, citric acid
(4.5-4.6 g L-1) and for AGII, tartaric acid (4.8-4.9 g L-1) was then added to maintain the pH at four [16]. The
viscosity of the stabilized Aloe vera gel and its coating efficiency was improved by using 1% commercial
gelling agent and was used as Aloe gel coating.
Coating of the edible gel:

Before coating, papaya fruits were washed thoroughly and dried. The coating solutions used for papaya
fruits were 50% AGI and 50% AGII. The fresh fruits were dipped completely into the coating solutions at
room temperature for 15 min. They were allowed to drain and then dried at room temperature with forced air
drying to allow a thin film layer to be formed on the fruits. Fifteen fruits were used for each coating solution.
The fruits were then weighed and stored in bamboo baskets lined with newspapers at room temperature
(30±3°C). The experiments were replicated. Fruits without coating were used as controls and were stored
under same conditions as those for coated fruits. Various parameters were determined initially and at five
day’s interval. The parameters analyzed include Physiological Loss in Weight (PLW), fruit size, pH, titrable
451
acidity, sensory analysis for fruit quality (peel color, firmness, appearance and taste), marketability and
degree and rate of fruit spoilage.
Physiological Loss in Weight (PLW):

Water loss was calculated in terms of PLW by the following equation:
PLW = (A-B)/A * 100
where, A is the initial weight of fruits (0d) and B is the fruit weight after the storage period [16].
Size of Fruits:
The mean size of the fruits was measured by using Vernier calipers. Fruits were measured vertically and
across the fruit and the average value calculated [17].
Titrable acidity and pH:

Fruits were homogenized and the resultant pulp was filtered. The pH of the fruit juice was determined using
a pH meter. Ten mL of squeezed fruit juice was diluted with distilled water and titrated against 0.1 NaOH by
using phenolphthalein as indicator. The results were expressed as % total acids (titrable acidity) [18].
Sensory analysis for evaluating fruit quality:

Sensory analysis was carried out by 10 panelists. The fruits were randomly selected from each batch. The
sensory quality was evaluated visually in terms of peel color, fruit firmness and fruit taste as an indicator of
respiration rate and ripening [18]. Marketability was also determined. Table-1 shows the grades of various
sensory attributes.
Table 1: Grades of Various Sensory Attributes
Peel color Quality Marketability

Bright Green Very firm Excellent
Greenish Yellow Firm Good
Pale Yellow Moderate firm Fair
Yellow Soft Limited
Dark yellow Very soft Not marketable
Degree and rate of fruit spoilage:

The differently coated fruits were visually observed for fungal spoilage and fruit rots. The number of fruits
infected or spoiled was recorded periodically to assess the effect of the different coating on retarding fruit
spoilage [15]. It was reported as percentage disease index and calculated as follows:
% Disease index = {(0*a) + (1*b) + (2*c) + (3*d) + (4*e)} * 100
(a + b + c + d + e) * X
where 0, 1, 2, 3, 4, 5 are infection categories ( 0 – no lesions, 1– 5 to ≤ 15%, 2 – ≥ 15% to ≤ 25%,
3- >25% to 50%, 4- ≥ 50% to ≤ 75%, 5- ≥ 75%; a, b, c, d, e are number of fruits that fall into the infection
categories and X = maximum of infection categories.
RESULTS
The control fruits started deteriorating before 5 days and only few fruits survived up to 10 days. Shelf life
was extended to 15 days for AGI coated fruits, whereas 18 days for AGII coated fruits. Therefore, data for
uncoatedcontrol fruits were given only till 10 days storage period.
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Physical characteristics:
Abnormal loss of firmness due to over ripening and pitting of the skin causes changes in the weight and size
of fruits. The effect of selected edible coatings on PLW and fruit size was observed. PLW during storage was
found to be significantly different between the papaya fruits treated with the two different coatings and
from control fruits at the end of 10 day’s storage (Fig 1). PLW was observed to be 32%, 27% and 26%
for the control; AGI and AGII coated fruits, respectively. PLW of AGI and AGII were 38% and 33% after 15
days of storage. PLW of AGII was 35% at 18th day of the storage period.
Figure 1: Storage Days Vs PLW
The mean values of the fruit size are shown in Figure 2. The size of coated fruits was significantly different
from control fruits at the end of 10 day’s storage. The mean fruit size of coated and uncoated fruits after 10 days
was found to be 68.76±0.7, 104.64±2.6 and 108.17±2.1mm for control, AGI and AGII coated fruits, respectively.
The maximum reduction in size was seen in control and the least reduction in AGII coated fruits. The fruit size was
found to be 101.23±2.9 and 106.59±2.2mm for AGI and AGII coated fruits after 15 days of storage. The fruit size
was found to be 103.76±2.7mm for AGII coated fruits at 18th day of storage.
Chemical characteristics:
The values of the chemical parameters such as pH and titrable acidity determined are shown in Table 2.
Figure 2: Storage Days Vs Fruits Size
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Table 2: Effect of Edible Coatings on Chemical Parameters.
Storage Period (days) pH Titrable acidity

Control/ Coated Control/ Coated
Uncoated Uncoated
AGI AGII AGI AGII
0 5.65 5.65 5.65 0.2 0.2 0.2
5 5.78 5.74 5.67 0.18 0.19 0.2
10 7.1 5.85 5.71 0.12 0.15 0.18
15 * 5.92 5.74 * 0.08 0.15
16 * * 5.78 * * 0.13
17 * * 5.81 * * 0.10
18 * * 5.84 * *
0.08*Indicates complete decay of fruits.
Marketability:
At the end of 18th day storage period, AGII coated fruits possessed better marketability than AGI coated
fruits. Control fruits were completely decayed after 10th day of the storage period. Fruits coated with AGI
and AGII after 5 days of storage are shown in Fig 3 (a-c).
Fruit Disease Index (FDI):

FDI was used as a measure to indicate the effect of selected coatings on the microbial quality of fruits. Over
ripening and decay were greater in non-coated than in coated fruits (Fig 4). FDI was found to be 100, 40 and
30 for control, AGI and AGII coated fruits, respectively, after 10 days storage. FDI was found to be 50 and
35 for AGI and AGII coated fruits at 15th day of the storage period. At 18th day of the storage period FDI was
40 for AGII coated fruits.
a. Control / Uncoated fruit b. AGI coated fruit c. AGII coated fruit

Figure 3: a. Control, b. AGI and c. AGII Coated Fruit after 5 Days of Storage
Figure 4: Storage Days Vs FDI
454
DISCUSSION
The results have proved the ability of the various bio-based coatings used in the present study to the extent
the shelf life of papaya fruits. From the analysis, it can be observed that AGII coating had shown the
maximum effect in retarding the change in pH and titrable acidity and had the controlled color development
and softening of fruit tissue even at the end of 18th day to a greater extent than AGI. Maintenance of fruit
firmness could be related to lower PLW as reported earlier in sweet cherry treated with edible coating and
Aloe gel coated nectarine fruits [13]. The retardation of physiological changes in the fruits indicates the ability
of the selected coatings to modify the respiration rate and delay the ripening process, thereby extending the
shelf life and maintain the quality of the fruits. In climacteric fruits such as papaya, an increase in ethylene
production during ripening is a normal physiological process. Earlier studies have shown a reduction in
ethylene production in Aloe vera gel coated fruits. The retardation of the ripening process observed in the
present study may be attributed to reduced ethylene production caused by the modified atmosphere. Surface
coating has been reported to increase resistance of fruit skin to gas permeability, creating a modified internal
atmosphere and reducing the respiration rate. Reduced respiration rate has been reported in Aloe vera gel
coated sweet cherry [11] and table grapes [12]; in peach fruits coated with wax emulsions and in avocado
fruits coated with methyl cellulose. When compared to AGI, AGII had greatly delayed the physiological
changes, it can be used for fruits that need to be transported over longer distances and when longer shelf life
period is necessary.
In this study, Aloe vera based coatings showed the least disease index. Aloe vera gel based coatings
have been reported earlier to reduce microorganism proliferation in sweet cherries and table grapes. Earlier
in vivo studies had shown even unstabilised aqueous extracts of Aloe vera gel to have antifungal activity
against Colletotricum gloeosporioides, a major post-harvest pathogen of papaya fruit [15]. Antimicrobial
activity of Aloe vera inner gel against Gram-positive and Gram-negative bacteria has been also demonstrated
by Habeeb et al [22]. Coating of fruits with AGII resulted in better control of fruit decay compared to AGI.
CONCLUSIONS
Thus it is concluded that the combination of Aloe vera gel with tartaric acid has better quality maintenance
ability during post-harvest life of papaya than the combination of Aloe vera gel with citric acid. This AGII
combination could be used as a cost-effective simple method for extending the post-harvest shelf life and
quality of fruits and vegetables.
REFERENCES
1. Lin D and Zhao Y, Innovations in the development and applications of edible coatings for fresh and
minimally processed foods and vegetables, Comprehensive Rev Food Sci Food Safety, 2007, 60-75.
2. Chandra P, Issues and Solutions of fresh fruit export in India, Ministry of Commerce and Industries Data
Sheet, ICAR, GOI, 2005.
3. Singhal V, Papaya in Indian Agriculture, Indian Data Research Center, New Delhi, 1996, 169-170.
4. Mitra S, Papaya in Postharvest physiology and storage of Tropical and subtropical fruits CABI
publishing, India, 2005, 175-182.
5. Mattheis J and Fellman JK, Impact of modified atmospheric packaging and controlled atmospheres on
aroma, flavor and quality of Horticultural commodities, Hort Technol, 2007, 507-510.
6. Devlieghere F, Vermeulen A and Devebere J: Chitosan Antimicrobial activity, interaction with food
components and applicability as a coating on fruits and vegetables, Food Microbiol, 2004, 703-714.
455
7. Banos-Bautista S, Hernandez-Lauzardo M, Bosequez-Molina E and Wilson CE, Effect of chitosan and

plant extracts on growth of Colletotrichum gloeosporioides, anthracnose levels and quality of papaya
fruit, Crop Prot, 2003, 1087-1092.
8. Martinez-Romero D, Serrano M, Valero D and Castillo S, Application de Aloe vera como recubrimicento
stobre frutas Yhor taliza, Spain patent-200302937, 2003.
9. Saks Y and Barkai-Golan R, Aloe vera gel activity against plant pathogenic fungi, Postharvest Biol
Technol, 1995, 159-165.
10. Rodriguez de Jasso D, Hernandez-Castillo D, Rodriguez- Garcia R and Angulo-Sanchez JL, Antifungal
activity in vitro of Aloe vera pulp and liquid fraction against pathogenic fungi, Ind Crop Prod, 2005,
81-87.
11. Valverde JM, Valero A, Martinez-Romero D, Guillen F, Castillo S, et al, Novel edible coating based on
Aloe vera gel to maintain table grape quality and safety, J Agric Food Chem, 2005, 7807-7813.
12. Martinez-Romero D L, Alburquerque N, Valverde J M, Guillen F, Castillo S, et al, Postharvest cherry
quality and safety maintenance by Aloe vera treatment: A new edible coating, Postharvest Biol Technol,
2006, 93-100.
13. Ahmed M J, Singh Z and Khan A S, Postharvest Aloe vera gel-coating modulates fruit ripening and
quality of ‘Arctic Snow’ nectarine kept in ambient and cold storage, Int J Food Sci Technol, 2009, 1024-
1033.
14. Vargas M, Pastov C, Chirau A, Clements MC, Julian D and Gunzalez Martinez C, Recent advances in
edible coatings for fresh and minimally processed fruits, Crit Rev Food Sci Nutr, 2008, 496-511.
15. He Q, Changhong L, Kojo E and Tian Z, Quality and safety assurance in the processing of Aloe vera gel
juice, Food Control, 2005, 95-104.
16. El Ghaouth A, Arul J, Grenier J and Asselin A, Antifungal activity of chitosan on two post-harvest
pathogens of strawberry fruits, Phytopathology, 1992, 398-402.
17. Yaman O and Bayoindirli L, Effects of an edible coating and cold storage on shelf life and quality of
cherries,LWT-Food Sci Technol, 2002, 146-150.
18. Banks NH, Dadzie BK and Cleland DJ, Reducing gas exchange of fruits with surface coating, Postharvest
Biol Technol, 1993, 269-284.
19. Erbil HY and Muftugil N, Lengthening postharvest life of peaches by coating with hydrophobic
emulsions, J Food Proct Preserv, 1986, 269-279.
20. Maftoonazad N and Ramaswamy HS, Postharvest shelf life extension of avocados using methyl cellulose
based coatings LWT- Food Sci Technol, 2005, 617-624.
21. Habeeb F, Shakir E, Bradbury F, Cameron P, et al, Screening methods used to determine the antimicrobial
properties of Aloe vera inner gel, Methods, 42 (2007) 315-320.
22. Singh I, Biological control of Aspergillus flavus and aflatoxins: A comparative study of toxigenic and
non- toxigenic strains. Doctoral Thesis, University of Delhi, Delhi, 2001.
23. Abirami LSS, Efficacy of chitosan and natural plant extracts on the growth of selected fungal pathogens
and control of anthracnose disease of papaya, 2009.
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Biological Control of Damping off and Stem Rot of Tomato
(Lycopersicon Esculentum Mill.) using an Actinomycete,
Saccharopolyspora Erythraea
Prabha S.B and K. Murugesan
Centre for Advanced studies in Botany, University of Madras, Guindy Campus, Chennai – 600 025
Abstract:
Actinomycetes have been found to protect plants against their diseases. The present study aimed at the
isolation of actinomycetes from different rhizosphere soil of tomato plants and to study their antagonistic
effect against the phytopathogenic fungi, Rhizoctonia solani and Sclerotinia sclerotiorum. These species
of actinomycetes are found to cause stem rot and to damp off of tomato that is the major yield-limiting
factor. The selected isolates were further identified and characterized using physiochemical and biochemical
methods. Among the fifty strains isolated, the strain AMP14 showed significant activity against the two fungi,
R. solani and S. sclerotiorum on preliminary screening by dual culture plate method as well as crude was also
checked for its antagonistic effect. The presence of antimycotic compounds were analyzed using thin layer
chromatography (TLC). Based on the TLC analysis, further column was carried out using sephadex LH-20.
The purified compound was further confirmed by TLC and GC-MS, NMR, FTIR characterization. Further
for the species level identification of the actinomycete, AMP14, was identified using 16srDNA sequencing
was used and found to be Saccharopolyspora erythraea.
Keywords: Biological control, Saccharopolyspora erythraea, Phytopathogens.
INTRODUCTION
Actinomycetes are well known Gram-positive organisms know for their chemical and morphological
diversity (Goodfellow and Donnewell, 1989). Actinomycetes prefer to grow in soil constituents such as
humus, litter, dung and even on rock surfaces and are ubiquitous in nature (Lechevalier, 1981). Over 20
genera have been isolated from soil and viable counts of several million per gram soil are common (Williams
and Wellington, 1982). Actinomycetes, produce several secondary metabolites that have high DNA, G+C
content. Besides, they also produce antibiotics which affect fungal growth (Goodfellow and Williams, 1983).
Their metabolite formed potential applications in pharmacy, industry, agriculture and environment. The
ability of chitinase production enhances their synergistic effect by having the chitin as the main component
of cell wall (Goodfellow et al., 1988).
Tomato is one of the important and popular vegetable crops in India. Many diseases and disorders can
affect tomatoes during the growing season. In India, it is grown in 4.58 million hectares with a production of
74.62 million tonnes per season. Soil borne pathogens are complex not only in their biochemical constituents
but also in their behavioural pattern. R. solani is a plant pathogenic fungus with a wide host range and
worldwide distribution. It is the major fungus responsible for damping-off, black spot and root rot diseases
(Neha and Dawande, 2010) of several economically important crops throughout the world, causing severe
yield loss. R.solani is one of the dreadful phytopathogens that attack tomatoes cultivated worldwide causing
root and crown rot (Latorre, 2004).
The fungus S. sclerotiorum causes a stem rot disease condition in tomato plants called as timber rot. This
fungus attacks a wide host range of plants such as beans, cabbage, carrot, celery, cucumbers, lettuce, onions,
peas, pumpkins and squash. S. sclerotiorum is a non-host specific necrotrophic fungal pathogen, which is the
casual agent of stem rot and could attack 400 species worldwide and is considered as one of the serious threat
to many economically important crops (Boland and Hall, 1994; Hegedus and Rimmer., 2005).
Biological control of plant diseases, especially due to fungal infection have been achieved using
antagonistic micro-organisms such as Trichoderma sp, Pseudomonas sp, Bacillus sp and Streptomyces
sp. (Elad et al., 1980). In this study, the species of actinomycete, Saccharopolyspora erythraea against the
pathogens of tomato.

Sample collection
Soil samples from the rhizosphere of tomato plants in the fields from different districts i.e. Kancheepuram,
Villupuram and Dharmapuri of Tamil Nadu state in India were collected from the depth of 5-15cm in sterile
plastic bags and brought to the laboratory in aseptic condition.
Isolation of actinomycetes
Ten grams of the soil sample were taken in 95ml of water and placed in Shaker for 2 hours. The preparation
is known as master dilution. From the master dilution actinomycetes were isolated by serial dilution
technique and cultured on Starch casein Agar, International Streptomyces project Medium No. 2 (ISP2). All
supplemented with Fluconazole (20mg L-1) for the reduction of contamination caused by fungi (Labeda and
Shearer, 1990). The plates were incubated at room temperature for 14 – 21days. The selected colonies were
further streaked onto International Streptomyces project No. 2 Agar (ISP2). Thus, isolated colonies were
preserved in agar slants and glycerol stock and stored at -20°c.
Phytopathogenic fungi
R. solani that cause damping off disease and S. sclerotiorum which causes stem rot were isolated from
the soil near infected tomato plants. It is then identified in our lab and maintained in Potato Dextrose
Agar (PDA) slants.
Screening for biocontrol activity by dual culture method

The isolates were evaluated for its antagonistic activity against two phytopathogens namely R. solani and S.
sclerotiorum using dual culture technique (Huang and Hoes, 1976). The actinomycete culture was streaked
in Starch Casein Potato Dextrose Agar (SCPDA) and incubated for five days, after which the mycelial plug
of the fungi was streaked perpendicular to the actinomycete strain and incubated for 5 days. The results were
observed for 14 days.
Identification of the antagonistic actinomycete strain

The potent actinomycete strain which showed good antagonistic activity was characterized by physicochemical
and biochemical methods. Physiochemical methods consist of macroscopic and microscopic methods. Cover
slip culture method did the microscopic characterization. The mycelial structure, colour and arrangement of
the spore were observed through microscope. The observed structure was compared with Bergey’s manual of
Determinative Bacteriology, and the organism was identified. Gram staining, Acid fast staining and various
Biochemical tests, were performed.
Pigment production, starch hydrolysis, Casein hydrolysis, catalase test, Oxidase test and various
biochemical tests such as Indole, Methyl red, Voges proskauer, citrate utilization test and Urease production
458
Nitrate reduction tests were performed. 16s rDNA Sequencing was done for confirmation of the species.
Effect of different temperature, Nacl concentration and pH were also observed. The Chitinase activity of
AMP14 was also observed.
DNA isolation
Centrifugation harvested overnight cultured microbial cells, and cell pellets were suspended in TE buffer (10
mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 2 mg of lysozyme per ml in a total volume of 5 ml. Samples
were incubated at 30°C for 30 min, which was followed by addition of 1.2 ml of 0.5 M EDTA and protease
(final concentration, 0.2 mg/ml) and an additional 30 min of incubation. After addition of 0.7 ml of 10% (wt/
vol) sodium dodecyl sulfate (SDS), 1.8 ml of 5 M NaCl, and 1.5 ml of 10% CTAB-NaCl solution (10% [wt/
vol] hexadecyltrimethyl ammonium bromide in 0.7 M NaCl), the mixtures were incubated at 65°C for 20
min. Samples were extracted with chloroform-isoamyl alcohol (24:1, by volume), and the supernatants were
transferred to clean tubes. DNA was precipitated by addition of 0.6 volume of isopropanol and pelleted by
centrifugation (48,000 x g, 15 min). DNA pellets were rinsed with 70% (wt/vol) ethanol, dried, dissolved in
TE buffer, and quantified by absorption spectrophotometry at 260 nm.
Universal bacterial primers and PCR conditions

Each PCR mix contained ~25 ng DNA, 10 pm each forward and reverse primer, 20 mM dNTPs (Invitrogen), 2.5
mM MgCl2 and 1.0 U Platinum Taq DNA polymerase (Invitrogen) in the manufacturer’s buffer. All reactions
were done with a 3 min 95°C hot start followed by 35 cycles of 95°C for 1 min, the primer-specific annealing
temperature for 1 min, and 72°C for 2 min, followed by a 30 min 72°C extension. Primers designed to anneal
to conserved regions in actinomycetes were used to amplify bacterial 16S rDNA sequences that contained
intervening species-specific regions. The forward primer, 8F (5’-AGAGTTTGATCCTGGCTCAG- 3’), was
used with reverse primers 1492R (5’-GGGTTACCTTGTTACGACTT-3’) with an annealing temperature of
56°C and template DNA from the root-canal contents to amplify 1.3 and 1.4 kbp regions, respectively, of
bacterial 16S rDNA (Baker et al., 2003). The products of primers 8F and 1391R and the products of primers
8F and 1492R with pooled DNA from asymptomatic patients as template and the products of the same
primer pairs using DNA template pooled from symptomatic patients were cloned for sequence analysis. An
equivalent number of clones were analysed from both the symptomatic and asymptomatic patients, for a total
of ~1500 clones.
Optimization of the medium

Various Carbon sources such as glucose, starch, arabinose, fructose, galactose, mannitol, maltose, sucrose,
rhamnose and raffinose were given for growth and nitrogen sources such as peptone, yeast extract, L-tyrosine,
tryptone, L-asparagine, potassium nitrate, L-histidine and L-threnonine were given and the growth is
observed.
Different types of medium such as Starch Casein Agar, (International Streptomyces Agar medium ) Isp2,
Isp4, Glucose Asparagine Agar, Tryptic soy agar, Oatmeal Agar, Antibiotic Production Medium, Modified
Potato Dextrose Agar, Malt extract Agar and Modified Isp2 were used for the optimization of the medium.
Preparation and Screening of the antagonistic effect of Crude extract

The strain AMP14 was found to be very effective and was selected to carry out further studies. It was grown
in modified ISP2 medium and kept in a shaker under 290 g at room temperature (28 ± 2°C) for 14 days. After
14 days the culture filtrate was centrifuged at 10,000 g for 15 minutes and then extracted using ethyl acetate
with a separating funnel and it was evaporated using a rotary evaporator and dried. The dried crude extract
was checked for their antagonistic effect against the two phytopathogens R. solani and S. sclerotiorum using
well diffusion method.
459
The concentrated crude metabolites were mixed with silica gel (60-100 mesh) to prepare the metabolite-
silica gel slurry and air-dried. The slurry was fractionated by silica gel column chromatography using a
glass column. Silica gel of 60 – 100 mesh was packed in the column and washed with hexane. Then the
metabolites-silica gel slurry was loaded onto the column and were successively eluted initially with 100
% hexane followed by hexane: chloroform; chloroform: ethyl acetate; ethyl acetate: methanol and finally
with methanol (100 %). About 100 ml of each of the solvent system was used for elution. The presence of
the compounds was analyzed using thin layer chromatography (TLC). Based on the TLC analysis, Column
chromatography was carried out using silica gel, further column was carried out using sephadex LH-20. The
purified compound was further confirmed by T LC, NMR, FTIR and GC-MS characterization.
RESULTS
Sample collection
Soil samples were collected from 5-15cm depth into sterile plastic bags from different rhizosphere soil of
tomato fields from Kancheepuram, Villupuram and Dharmapuri districts and brought to the laboratory in
aseptic condition, and the pH of the soil samples was observed. (Table: 1)
Table 1: pH of soil samples collected from different districts
Collection of soil (Districts) Soil pH

Villupuram 7.2
Dharmapuri 7.8
Kancheepuram 5.8
Isolation of Actinomycetes
A total of fifty actinomycetes species were isolated from various rhizosphere (tomato) soils using different
selective media such as Starch casein Agar, International Streptomyces Project Medium No. 2 (ISP2) and
Glucose Asparagine Agar. They are designated as AMP-1 to AMP-50. (Table – 2)
Isolation of Phytopathogenic fungi

R. solani was isolated from infected plant, and its immediate soil samples, and S. sclerotiorum were isolated
from infected stem of tomato plants. It was identified in the lab and was maintained on Potato Dextrose Agar
(PDA) slants.
Biocontrol Screening for activity by dual culture method

The isolates were evaluated for its antagonistic activity against two phytopathogenic fungi namely R. solani and S.
sclerotiorum using dual culture technique (Huang and Hoes, 1976). Among all the fifty isolates of actinomycetes
tested, seven strains showed good antagonistic activity against R. solani and only one strain showed best
antagonistic activity against S. sclerotiorum. The strain AMP14 showed very good antagonistic effect against the
two phytopathogens, and it is chosen for further analysis.
460
Table 2: Colony morphology of 50 actinomycete species isolated from different rhizosphere soils (tomato)
Strain Size Shape Nature Appearance Aerial mycelium Sub mycelium
Amp 1 Moderate Concentric Powdery Pin drop Grey white Creamy
Amp 2 Moderate Concentric Powdery Pin drop White Brown
Amp 3 Moderate Concentric Powdery Pin drop Pinkish white White
Amp 4 Moderate Round Powdery Pin drop Pinkish white White
Amp 5 Moderate Round Powdery Flat Grey Red
Amp 6 Moderate Round Powdery Flat Pinkish white Brown
Amp 7 Moderate Round Powdery Flat Greyish white Creamy
Amp 8 Moderate Round Powdery Flat White Brown
Amp 9 Moderate Irregular Slimy with powder Pin drop Pinkish purplish powdery Creamy
Amp 10 Moderate Round Powdery Flat Brownish green Green
Amp 11 Smooth Round Powdery Flat Pin white White
Amp 12 Moderate Round Powdery Pin drop White Creamy
Amp 13 Smooth Round Powder slimy Pin drop White Creamy
Amp 14 Moderate Round Powdery Flat Brownish red Red
Amp 15 Moderate Concentric Powdery Pin drop Grey Red
Amp 16 Smooth Round Powdery Pin drop White Brown
Amp 18 Moderate Round Powdery Flat Pink white Creamy
Amp 19 Moderate Round Powdery Pin drop White Brown
Amp 20 Moderate Concentric Powdery Pin drop Grey white Red
Amp 21 Moderate Round Powdery Pin drop White Green
Amp 22 Smooth Round Powdery Pin drop White White
Amp 23 Smooth Round Powdery Pin drop White Creamy
Amp 24 Moderate Irregular Powdery Pin drop White Creamy
Amp 25 Smooth Round Slimy Pin drop Yellow cream Yellow cream
Amp 26 Moderate Round Powdery Flat Pinkish white Creamy
Amp 27 Moderate Round Powdery Pin drop White Cream
Amp 28 Moderate Round Powdery Pin drop Grey Red
Amp 29 Moderate Concentric Powdery Flat Dull white Creamy
Amp 31 Moderate Round Powdery Pin drop White Brown
Amp 32 Moderate Round Powdery Flat Pinkish white Creamy
Amp 33 Moderate Round Powdery Pin drop White Cream
Amp 34 Moderate Concentric Powdery Pin drop Pinkish white White
Amp 35 Smooth Concentric Powdery Pin drop Grey white Creamy
Amp 36 Moderate Round Powdery Flat Grey Grey
Amp 39 Smooth Concentric Slimy Pin drop Yellow cream Yellow cream
Amp 40 Moderate Round Powdery Flat Whitish pink Creamy
Amp 41 Moderate Concentric Rough Pin drop Violet Violet
Amp 42 Smooth Round Powdery Pin drop Orange White
Amp 43 Moderate Round Powdery Pin drop White White
Amp 44 Moderate Concentric Powdery Flat Dull white Creamy
Amp 46 Moderate Concentric Powdery Pin drop Grey Red
Amp 47 Smooth Round Slimy Pin drop White Brown
Amp 48 Smooth Round Powdery Flat White Grey
Amp 49 Moderate Concentric Powdery Pin drop White Grey
Amp 50 Smooth Round Powdery Pin drop Grey Brown
Identification of the antagonistic actinomycete strain
461
The potent strain AMP14 was characterized by physiochemical and biochemical methods. (Table – 3)
Table 3: Physiological and Biochemical characteristics of AMP14
Tests Characteristics
Gram staining Positive
Pigment production Positive
Starch hydrolysis Positive
Casein hydrolysis Positive
Catalase test Negative
Oxidase test Negative
Indole production Positive
Methyl red test Positive
Voges proskauer Positive
Citrate utilization test Positive
Urease test Positive
Nitrate reduction Positive
H2S production Positive
Chitin Positive
16s rDNA Sequencing

>Acti_16s_FWD_IX12
ACGCTGAAGCATCTTCGGGTGTGGATGAGTGGCGAACGGGTGAGTAACACGTGGGTAATC
TGCCCTGCACTCTGGGATAAGCCCGGGAAACTGGGTCTAATACCGGATAGGACACATGGC
CGCATGGTCTGTGTGTGGAAAGTTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTT
GTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGG
CCACACTGGGTCTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTGC
GCAATGGGCGAAAGCCTGACGCAGCAACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTA
AACCTCTTTCGACATCGACGAAGCCTTCGGGTGACGGTAGGTGTAGAAGAAGCACCGGCT
AACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGATTTATTGGG
CGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTTCGTGAAAACTGGAGGCTTAACCTTCA
GCTTGCGGTCGATACGGGCAGACTTGAGTTCGGTAGGGGAGACTGGAATTCCTGGTGTAG
CGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCCGAT
ACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAC
GCCGT
>Acti_16s_RvE_IX12
TCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCC
TGGTAGTCCACGCCGTAAACGTTGGGCGCTAGGTGTGGGGACTGTTTCCACGGTTCCTGT
GCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAA
AGGAATTGACGGGGGCCCGCTCAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGA
AGAACCTTACCTGGGTTTGACATGCACTAGACTGCCTCAGAGATGGGGTTTCCCTTGTGG
TTGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC
CCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCACGTAATGGTGGGGACTCGCGGGA
GACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCAACATGCCCCTTATG
TCCAGGGCTTCACANATGCTACAATGGCCGGTACAGAGGGTTGCGATGCCGTGAGGTGGA
462
GCGAATCCCTTAAAGCTGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGT
CGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTA
CACACCGCCNGTCACGTCATGAAAGTCGGTA
>Contig-0
ACGCTGAAGCATCTTCGGGTGTGGATGAGTGGCGAACGGGTGAGTAACACGTGGGTAATC
TGCCCTGCACTCTGGGATAAGCCCGGGAAACTGGGTCTAATACCGGATAGGACACATGGC
CGCATGGTCTGTGTGTGGAAAGTTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTT
GTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGG
CCACACTGGGTCTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTGC
GCAATGGGCGAAAGCCTGACGCAGCAACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTA
AACCTCTTTCGACATCGACGAAGCCTTCGGGTGACGGTAGGTGTAGAAGAAGCACCGGCT
AACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGATTTATTGGG
CGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTTCGTGAAAACTGGAGGCTTAACCTTCA
GCTTGCGGTCGATACGGGCAGACTTGAGTTCGGTAGGGGAGACTGGAATTCCTGGTGTAG
CGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCCGAT
ACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAC
GCCGTAAACGTTGGGCGCTAGGTGTGGGGACTGTTTCCACGGTTCCTGTGCCGTAGCTAA
CGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACG
GGGGCCCGCTCAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACC
TGGGTTTGACATGCACTAGACTGCCTCAGAGATGGGGTTTCCCTTGTGGTTGGTGTACAG
GTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC
GCAACCCTTGTCCTGTGTTGCCAGCACGTAATGGTGGGGACTCGCGGGAGACTGCCGGGG
TCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCAACATGCCCCTTATGTCCAGGGCTTC
ACANATGCTACAATGGCCGGTACAGAGGGTTGCGATGCCGTGAGGTGGAGCGAATCCCTT
AAAGCTGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTA
GTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCNG
TCACGTCATGAAAGTCGGTA
Figure 1: 16srDNA sequencing

Lane-1- 1 Kb ladder
Lane-2- PCR product
Plane3 PCR product
463
Different carbon and nitrogen sources

Among the various carbon sources tried, maximum growth was observed in1mM/2% starch, galactose and
glucose (Fig – 2) and in nitrogen source peptone (Fig – 3) at 2mM/2% supported maximum growth of
AMP14
Figure 2: Effect of different carbon sources on the growth of AMP14 strain
Figure 3: Effect of different nitrogen sources on the growth of AMP14 strain
Screening of the antagonistic effect of crude extract

The strain AMP14 was found to be very effective and was selected to carry out further studies. The dried
crude extract which was checked for their antagonistic effect against the two phytopathogens R. solani and
S. sclerotiorum using well diffusion method proved to be very effective against both the pathogens. It is
subjected to thin layer chromatographic technique.
Based on the TLC analysis, Column chromatography was performed using silica gel and a further
column was carried out using sephadex LH-20. The purified compound was further confirmed as Oleic acid
and L-Ascorbyl palmitate by TLC and NMR, FTIR, GC-MS characterization.
464
The main purpose of this study is to control two phytopathogenic fungi such as R. solani and S.
sclerotiorum which causes the dreadful disease such as damping off and Stem rot, which affects most of
the plants that lead to mortality of those plants. The present work states the invitro potential of the selected
actinomycete strain to control the above said dreadful fungal phytopathogens. The secondary metabolite
production is found abundant in the AMP14, and it supported in curbing the growth of the two dreadful
fungal pathogens R. solani and S. sclerotiorum. The compounds that are isolated from AMP14 such as Oleic
acid and L-Ascorbyl palmitate also found to be very effective against the two fungal pathogens.
ACKNOWLEDGEMENT
One of the authors SBP is thankful for the UGC- BSR for providing the Meritorious fellowship and to the
Director, CAS in Botany for the laboratory facilities.
REFERENCES
1. Baker G.C., Smith J.J. and Cowan D.A., (2003), Review and reanalysis of domain-specific 16S primers,
J Microbiol Methods 55, 541–555.
2. Boland GJ and Hall R, 1994, Index of plant hosts of Sclerotinia sclerotiorum, Canadian Journal of Plant
Pathology 16: 93–108.
3. Champness W.C. and Charter K.F., 1994, Regeneration and integration of antibiotic producing and
morphological differentiation in Streptomyces spp. In: Regulation of bacterial differentiation, (Eds.
Piggot, J. Moran, C. and Youagaman, P.), American Society for Microbiology, Washington, DC, USA.
4. Charter K.F. and Hopwood D.A., 1993, Streptomyces genetics, American Society for Microbiology,
Washington, DC, USA.
5. ELAD Y., Chet. I and Katan Y., (1980), Trichoderma harzianum a biocontrol agent effective against
Sclerotium rolfsii and Rhizoctonia solani, Phytopathology 70: 119-121.
6. Goodfellow M. and Williams S.T., (1983), Ecology of actinomycetes, Ann. Rev. microbiol., 37: 189-
216.
7. Goodfellow MS.T, Williams and M. Mordarski, 1988, Actinomycetes in Biotechnology, Academic press
Limited, London. 501p.
8. Goodfellow M. and A.G. O’Donnewell, 1989, Search and discovery of significant Actinomycetes, In
microbial products, New approach.44, The symposium of the society for General Microbiology ed.
Baumberg, S. Hunter, I and Rhodes M. Cambridge, U.K, Cambridge university press. pp:343-383.
9. Hegedus DD, Rimmer SR, 2005, Sclerotinia sclerotiorum: when “to be or not to be” a pathogen? FEMS
Microbiology Letters 251: 177–184.
10. Heydarian S.M., Ison A.P., Lilly M.D. and Ayazi Shamlou P.A., 2000, Turbulent breakage of filamentous
bacteria in mechanically agitated batch culture. Chem. Eng. Sci. 55: 1775-1784.
11. Huang M.C. and Hoes J.A., 1976, Penetration and infection of Sclerotinia sclerotiorum by Corinithurum
minitans. Can. J. Bot. 54: 406-410.
12. Jensen P.R., R. Dwight and W. Fenical, 1991, Distribution of actinomycetes in near-shore tropical marine
sediments, Appl. Environ. Microbiol, 57:1102-1108
13. Labeda D., 1993, DNA relatedness among strains of Streptomyces lavendulae phenotypic cluster group,
Int. J. System. Bacteriol., 43:822-825.
465
14. LATORRE and Bernardo, Enfermedades de las plantas cultivadas, Santiago; Ediciones Universidad
Catolica de Chile, 2004. 638 p. ISBN 956-14-0756-6. (Ref)
15. Lechevalier M.P., 1981, Ecological associations involving actinomycetes, Zentralblatt fur Bakteriologie,
Mikrobiologie Hygiene I Abteilung supplement 11, 159-166.
16. Neha Dev and A.Y. Dawande, 2010, Biocontrol of soil-borne plant pathogen Rhiozoctonia solani using
Trichoderma sp and Pseudomonas fluorescens, Asiatic J. Biotech. Res. 01: 39-44 (Ref)
17. Williams S.T. and Wellington E.M.H., 1982, Actinomycetes, In: page, A.L., Miller, R.H.) Keenchy, O.R.
(Eds.), Methods of soil Analysis, Part 2, Chemical and Microbiological properties, the second society of
America, Madison, pp.969-987.
466
Exploration for Antimicrobial Proteins
from Marine Bivalve
G. *Vinoth Kumar, M. Arputha Bibiana, C. Sherlina Daphny, P. Selvamani and S. Latha
Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli, Tamilnadu, India
e-mail: arputha_bibiana@yahoo.com
Abstract:
The microbial infections are abiding from ancient days to till date with different symptoms and severity.
Most of the modern medicines were failed to treat the infection without side effects. To overcome
all these effects the research was now focused upon bioactive natural products from various types of
natural sources especially on marine sources. The crude protein extracts of edible marine invertebrate
Perna perna collected from the coastal area of the Southern coast of Rameshwaram, Tamil Nadu, were
evaluated for its antimicrobial activity against a broad spectrum of bacterial pathogens. The crude
proteins were extracted from the flesh with 5% cold acetic acid. The crude was partially purified by
ammonium sulfate precipitation method and the precipitate was stored at -20°C. The antimicrobial
activity was done by well diffusion method at various concentrations. The Gram-negative bacteria
Shigella flexneri was inhibited with maximum effect of 13mm at concentration of 150μl extract and
11mm against Salmonella typhi. The other tested strains were inhibited with not less than 6mm.1.23mg/
ml protein was estimated in Perna perna acidic extract. The crude protein extracts with potent bactericidal
property may help to find a safe novel protein/peptide antibiotic, from edible bivalve.
Keywords: Edible bivalve, Marine invertebrate, Antimicrobial Assay, Antimicrobial Peptides.
INTRODUCTION
The marine resources are nowadays widely studied because of numerous reasons. One of the reason is as the
oceans cover more than 70% of the world surface and among 36 known living phyla, 34 of them are found
in marine environments with more than 300000 + known species of fauna and flora [3, 21].
The attention of finding drug from sea had started from 1970s. For instance, about 300 patents on bioactive
marine natural product have been issued between 1969 and 1999. So far, more than 10,000 compounds have
been isolated from marine organisms. Only 10% of over 25,000 plants have been investigated for biological
activity. The marine environment may contain over 80% of world’s plant and animal species. In recent years,
many bioactive compounds have been extracted from various marine animals like tunicates, sponges, soft
corals, bryozoans, sea slugs and marine organisms [1, 21].
The Marine ecosystem, which is considered as the ‘Mother of origin of life,’ is also a source of structurally
unique natural products which are mainly accumulated in marine organisms. Several of these compounds
show unique pharmacological activities and are helpful in the invention and discovery of bioactive
compounds that are mainly found abundantly in microorganisms, algae and invertebrates and vertebrates.
Modern technologies have opened vast areas of research for the extraction of biomedical compounds from
marine sources to treat deadly diseases [6]. A number of natural products isolated from marine organisms
increases rapidly and now exceeds 18,000, with hundreds of new compounds being discovered every year. A
large proportion of these natural compounds have been extracted from marine invertebrates, namely sponges,
ascidians, bryozoans and molluscs, and some of them are currently in clinical trials [2].
Among marine invertebrates, marine molluscs are the good source of bioactive metabolites. The
bioactive compounds extracted from many classes of molluscs exhibit antitumor, antileukemic, antibacterial
and antiviral properties. A marine invertebrate depends on innate immune mechanisms that include both
humoral and cellular responses. Humoral immunity in marine invertebrates is characterized by antimicrobial
agents present in the blood cells and plasma [5].
Protein is a major biochemical and bioactive constituent in all invertebrate, and it receives high attention
due to their potential bioactive and functional properties. However, interest in marine proteins might not
be only directly correlated to the intact protein, but also to the possibility of generating bioactive peptides.
In this sense, in the present study different peptides derived from marine proteins have been identified as
having antimicrobial activities. The isolated organisms were further evaluated for their ability to produce
bio-products having antimicrobial activity [14].

Collection of Marine Edible Bivalves
Edible variety of bivalve Perna perna was collected from the southern coast of Rameshwaram, Tamil Nadu,
India.
Preparation of Crude Protein Extract

Fresh samples of Perna perna collected were transferred to the laboratory immediately. The samples were
washed with distilled water, and the flesh was taken by breaking the shells of the bivalves. Microbicidal
proteins were prepared from the whole body tissue of bivalves using 5% acetic acid in water by simple
homogenization. The homogenized mixtures were centrifuged at 4°C in 5000 rpm for 15 min. The protein
in the supernatant is partially purified by ammonium sulfate precipitation method at 85% saturation and
again centrifuged at 4°C in 5000 rpm for 30 min. The precipitates were dialyzed extensively against double
distilled water and were stored at -20°C for further evaluation.
Antimicrobial susceptibility testing

The antimicrobial activity of the crude extracts was evaluated by well diffusion method. Nutrient agar plates
were swabbed with the log phase broth cultures of the organisms and kept for 15 minutes in the laminar
chamber for absorption of cultures. The wells were made in agar plates using a sterile cork borer of 5mm. The
crude extracts at different concentrations of 0, 10, 25, 50, 100 and 150 μl were evaluated for its antimicrobial
potency against the clinical isolates. The plates were incubated at 37°C for 24 hours, and the inhibition zones
were measured in millimeter.
Protein concentration Estimation

The concentration of protein in the crude sample was estimated by the Lowry’s method using BSA as
standard.
Analysis of crude protein using Paper Chromatography

A technique for the separation and identification of amino acids on partition chromatograms using circular
filter-paper discs as the stationary phase. N-butanol-acetic acid-water (4:1:5) ratio has been used as the
developing solvent. The solvent was allowed to run a distance of about 8-10 cm. When the solvent had run
the required distance, the paper was removed; the solvent boundary was marked with a pencil and dried.
After drying, the paper was sprayed with ninhydrin (0.1% in acetone or dipped into the reagent and dried at
65°C for 10-15 minutes. The presence of amino acids was shown by the formation of coloured concentric
circular zones, the radius of each vary with the type of amino acid. Based on the Rf values it predicts the type
of amino acid is concluded.
468
FTIR Analysis
The crude protein extract was subjected to FTIR characterization under suitable conditions. The Functional
groups of proteins were identified with the different wavelength and stretching obtained from the graph.

The crude extracts of the species showed promising antimicrobial susceptibility to all the tested strains at
the different concentration of 0, 10, 25, 50, 100, 150 μl well. All the tested pathogenic microbial cultures
were susceptible to the crude protein extracts of Perna perna. The maximum inhibitory effect of 13 mm was
observed with the Perna perna extract at150 μl concentration against Shigella flexneri (Tab: 1) and11mm
against Salmonella typhi. The deadly pathogenic Pseudomonasa eruginosa and Staphylococcus aureus were
found to be susceptible at 50 μl of the crude extracts. The other tested strains were inhibited with not less than
6mm of inhibitory effect by Perna perna extract. The highly pathogenic Gram positive and Gram negative
bacteria that were responded to the acetic acid extracts hopes for the development of new biopharmaceutical
product against the infectious pathogens.
Table 1: Antimicrobial activity of Bacterial pathogens with different concentrations.
S.No Bacterial pathogens Zone of Inhibition(mm) of crude protein extract

0 μl 10 μl 25 μl 50 μl 100 μl 150 μl
1 Salmonella typhi NA NA NA NA 7 11
2 Bacillus subtilis NA NA NA NA NA 6
3 E.coli NA NA NA NA NA NA
4 Pseudomonasa eruginosa NA NA NA 6 9 10
5 Staphylococcus aureus NA NA NA 6 10 10
6 Streptococcus pyogens NA NA NA 6 8 10
7 Proteus vulgaris NA NA NA NA 8 9
8 klebsiella pnemoniae NA NA NA 6 7 10
9 Shigella flexneri NA NA NA 7 10 13
NA – Not Active
The concentration of protein in the crude sample at 100 µl was estimated as 0.9 mg/ml. The values were
calculated from the standard graph plotted for BSA at different concentrations.
The analysis of paper chromatography concludes the presence of protein in the crude extract prepared
from the marine edible bivalve. The Rf value was measured as 0.65, and it shows the presence of possibilities
of aromatic aminoacids like phenylalanine, tryptophan, valine.
FTIR analysis of crude protein extract showed the presence of functional groups of protein like NH,
COOH, -CO-, CH, SH, C=O, C=C and other R groups at appropriate wavelength and stretching. The bend 3
in the graph depicts the presence of secondary amines which indicates the presence of proteins in the crude
extract. (Fig 1)
Figure 1: FTIR analysis of the crude protein extract
469
CONCLUSION
The protein extracted from the edible bivalve was found to be active against all the tested microbial pathogens.
The isolation and purification of the crude antimicrobial proteins may helps in synthesis of the potent peptide
antibiotics without any side effects.
REFERENCES
1. L.K. Mahalakshmi, M. Mythili, T. Vijayalakshmi and Radhakrishnareddy, Screening and Isolation of

Marine Bacteria with Biological Activity from Oil Contaminated Sea Water, International Journal of
Research and Reviews in Pharmacy, IJRRPAS, 2013, 1-44
2. Periyasamy N, Srinivasan M and Balakrishnan S, Antimicrobial activities of the tissue extracts of
Babylonia spirata Linnaeus, 1758 (Mollusca: Gastropods) from Thazhanguda, southeast coast of India,
Asian Pacific Journal of Tropical Biomedicine (2012)36-40
3. Jirge Supriya S and Chaudhari Yogesh S, Marine: The Ultimate source of Bio actives And Drug
metabolites, Review Article, 2010, 1(1) 55-62.
4. Kiran N, Siddiqui G, Khan AN, Ibrar K and Tushar P, Extraction and Screening of Bioactive Compounds
With Antimicrobial Properties From Selected Species Of Mollusk And Crustacean, Research Article,
2014, 5:1.
5. D. Martin and M.J. Uriz, Chemical Bioactivity of Mediterranean benthic organisms against embryos and
larvae of marine invertebrates, Research article, (1993) 11-27.
6. Samanta S. Khora, Marine fish-derived bioactive peptides and proteins for human therapeutics, Review
Article, 2013.
7. Madhu V.N, P. Sivaperumal, K. Kamala, Ajit A. Ambekar and B.G. Kulkarni, Antibacterial And
Antioxidant Activities of the Tissue Extract Of Perna virits Linnaeus, International journal of Pharmacy
And Pharmaceutical Sciences, 2014.
8. Chatterji A, Ansari.Z.A.et al.,. An extract obtained from marine animal having antiviral activities and
process for its extraction, Indian patent, 2000, 159.
9. S. Boobathy, T.T. Ajithkumar and Kathiresan, Isolation of Symbiotic Bacteria and Bioactive proteins
from the marine sponge Callyspongia diffuse. Indian Journal of Biotechnology 2009., 8:272-275
10. S. Anbuselvi, C. Chellaram, S. Jonesh, L. Jayanthi and Edward. Bioactive Potential of Coral Associated
Gastropod, Trochus tentorium of Gulf of Manner, Southeastern India, J.Med.Sci. 2009, 9(5) 240-244.
11. Rajeev Kumar Jha and Xu Zi-rong. Review Biomedical compounds from marine organisms, Marine
Drugs; 2004, 2: 123-146.
12. Jirge Supriya S and Chaudhari Yogesh S. Marine: the ultimate source of bio-actives and drug metabolites.
International Journal of Research in Ayurveda & Pharmacy, 2010, 1: (1), 55-62.
13. Marshall S.H and Arenas G, Antimicrobial peptides: A natural alternative to chemical antibiotics and a
potential for applied biotechnology. Electronic journal of Biotechnology, 2003, 6:1-14.
14. Carlos Lopez-Abarrategui et.al., Screening of antimicrobials from Caribbean sea animals and isolation
of bactericidal proteins from the littoral mollusk cenchritis muricatus, Curr Microbial; 2012, 64: 501-
505.
15. Hong Young Yan, Harvesting drugs from the seas and how Taiwan could contribute to this effort,
Changhua J Med 2004, 9:1-6.
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16. Charlet M, Chernysh S., et al., Innate immunity isolation of several cysteine-rich Antimicrobial peptides
from the blood of molluscs, Mytilus edulis, Journal of Biological Chemistry, 1996, 211:21808-21813.
17. C. Lopez-Abarrategui et al., Screening of Antimicrobials from Caribbean Sea Animals, Curr, Microbial,
2012, 64:501–505.
18. Bauer A.W, Kirley D, Sherris T.C and Truck M., Antibiotic susceptibility testing by Standardized single
disc diffusion method, American Journal of Clinical Pathology, 1996, 45, pp: 493-496.
19. Chatterji A, Ansari Z, et al., An extract obtained from marine animal having antiviral, Activities and
process for its extraction. Indian patent, 2000, 159.
20. Tincu AJ and Taylor SW, Antimicrobial peptides from marine invertebrates, Antimicrobial Agents and
Chemotherapy, 2004, 4
21. M. Arputha Bibiana, P. Selvamani and S. Latha, Identification and Appraisal of Crude Protein Extracts
From South Indian Marine Edible Bivalves For Their Potential Bactericidal Property, Vol 7, Suppl 1,
2014, 233-236.
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SECTION - IV
PHARMACEUTICAL
TECHNOLOGY
473
474
Phytochemical Investigation and In Vitro Anti Diabetic
Activity of Myxopyrum serratulum A.W. Hill
K. Kavitha1, K. Sujatha2, S. Manoharan3 and Sundara Ramaprabhu4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sri Ramachandra University,

1, 2
Porur, Chennai, Tamil Nadu, India

3
Department of Veterinary public health and Epidemiology, Veterinary College Research Institute,
Orathanadu – 614625, Thanjavur District
4
Alternative energy and nanotechnology laboratory, Department of Physics, IIT madras, Chennai
e-mail: kr.kavitha00@gmail.com, Mob: 9715342965
Abstract:
Myxopyrum serratulum A.W. Hill belongs to the family Oleaceae is a large scandent shrub, distributed
throughout Kerala. The whole plant is widely used as asthma, fever, cough, otitis, wounds and rheumatism.
The aim of the present study is to investigate the inhibitory activity of ethanolic extract of Myxopyrum
serratulum A.W. Hill on alpha amylase, alpha glucosidase and phytochemical studies of whole plant
extract. The phytochemical studies were carried out in terms of physicochemical standardization,
fluorescent analysis, preliminary phytochemical screening and quantification of important active
constituents present in the whole plant. The phytochemical study results showed that the Myxopyrum
serratulum A.W. Hill plant extract have rich amount of active constituents such as phenolic content
(78.09), flavanoid (51.2), flavonol (39.71) and tannin (86.25). Postprandial hyperglycemia is an early
defect in diabetes which increases the risk of diabetes related complication like cardiovascular diseases.
One important therapeutic strategy for decreasing the postprandial hyperglycemia is to inhibit the action
of hydrolyzing enzymes like alpha amylase and alpha glucosidase. The present study was to screen the
inhibitory activity of Myxopyrum serratulum A.W. Hill by means of in vitro studies. The in vitro assay
result suggests that the ethanol extract of the plant exhibit dose dependent inhibitory activity on alpha
amylase and alpha glucosidase enzymes.IC50 values were 435.02µg/ml and 688.61µg/ml for alpha
amylase and alpha glucosidase respectively. Acarbose used as a reference drug.
Keywords: Myxopyrum serratulum A.W. Hill, Oleaceae, Physicochemical studies, Quantification,
Antidiabetic activity.
Figure 1: Myxopyrum serratulum A.W. Hill flower

INTRODUCTION:
Diabetes mellitus is defined as a metabolic disease of numerous aetiology characterized by persistent
hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from deficiency in
insulin secretion, insulin action, or both [1]. The damage to the beta cells from islets of langerhans of pancreas
terminates in diabetes mellitus. The International Diabetes Federation (IDF) estimates that 6.4% of the world
population suffered from diabetes in 2010, and this will increase to 7.7% of the world population by 2030
[2]. About 90% percent of diabetic patients are diagnosed with type 2 diabetes (T2D) [3]. Early detection
and treatment is one approach proposed for reducing this trouble. The treatment of diabetes depends on the
severity and type of the diabetes. Diabetes mellitus is treated with insulin, weight reduction, diabetic diet
and exercise. When these procedures fail to control the high blood sugars, oral medications are used. If
oral medications are still insufficient, insulin medications are considered [4]. Anti-diabetic drugs can act in
different ways lower blood Glucose via multiple actions including insulin increasing, reducing glucagon,
and gastric emptying [5, 6]. But these antidiabetic agents lack efficacy and also produce some undesirable
side effects [7] like hypoglycemia, weight gain, kidney toxicity and gastrointestinal upset. Hence, there
is an urgent need to recognize and discover natural sources with fewer side-effects for the prevention and
treatment of diabetes. Medicinal plants continue to play a significant role in the treatment of a variety of
diseases due to several beneficiary compositions and having smaller or no side effect, plant-based medicines
can be used as different approaches to treat diabetes [8, 9].
Myxopyrum serratulum A.W. Hill (Oleaceae) is a large shrub growing in evergreen forests of Kerala (figure
1). It is commonly known as chaturamulla in Tamil [10]. In Ayurveda the plant leaves were used as acrid,
thermogenic, febrifuge, tonic and anodyne [11]. Chaturamulla is reported to have antioxidant, [12] wound
healing [13] and antimicrobial activity, [14] GC-MS analysis study also shows Myxopyrum serratulum A.W.
Hill have several important chemical constituents [15]. Some studies reported that polyphenols rich plant foods
produces some effects similar to insulin in the utilization of glucose and act as a good inhibitors in alpha
amylase and alpha glucosidase [16] and also polyphenols bioactivity in plants is related to their antioxidant
activity and many of these plant also have hypoglycemic activities [17]. There is no reports were available
regarding their antidiabetic activity and hence the present study aimed to investigate the antidiabetic activity of
Myxopyrum serratulum A.W. Hill by in vitro α-amylase and α-glucosidase inhibitory assay methods.
MATERIALS AND METHODS:

Plant materials:
The plant Myxopyrum serratulum A.W. Hill was collected from the tropical area in and around Tirunelveli
district, Tamil Nadu, Chennai and authenticated by Dr. P. Jayaraman, PARC, Chennai. The collected plant was
air dried, and it is powered. The plant powder was extracted with ethanol by cold maceration method [18, 19].
Physico chemical standardization:

The various physico chemical parameters [20] and extractive values for plant powder were performed
according to standard methods. The plant powder and extracts of Myxopyrum serratulum A.W. Hill were
treated with various reagents and the color produced was viewed under day, near and far UV [21].
Phytochemical screening of plant extracts:

For preliminary phytochemical screening the freshly prepared crude ethanolic extracts was tested for the
presence or absence of primary and secondary metabolites such as flavonoids, alkaloids, steroids, phenolic
compounds etc., using the standard procedure [22-23].
QUANTIFICATION OF ACTIVE CONSTITUENTS:

The important active constituents such as Alkaloids, [24] Phenolic content, [25] Flavonoids, [26] Flavonols
[27] and tannins [28] were quantitatively determined as per standard procedure.
476
IN VITRO ANTI DIABETIC ACTIVITY METHODS:

In vitro Alpha Amylase Inhibition Assay
The α-amylase inhibition assay was determined according to Miller (1959) using DNS method [29]. 500 μL
of plant ethanolic extracts of varied concentrations (1.95, 3.9, 7.81, 15.62, 31.25, 62.5, 125, 250, 500 and
1000 μg/ml) were added to 500 μL of sodium phosphate buffer 0.02 M (pH 6.9 containing 6 mM sodium
chloride) containing 0.04 units of α-amylase solution and incubated at 37°C for 10 min, After the incubation
period add 500 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) to all the test tubes.
The reaction was stopped by the addition of 1.0 mL of 3, 5-dinitro salicylic acid color reagent. All test tubes
were then incubated for 5 min in a boiling water bath and cooled to room temperature. The reaction mixture
was then diluted to 10 mL with distilled water, and absorbance was measured at 540 nm. The control samples
were also prepared without plant extracts and compared with the samples containing various concentrations
of the plant extracts. The results were expressed as a percentage of inhibition.
In vitro α-glucosidase inhibitor activity assay

The α-glucosidase inhibitory assay was determined according to Artanti et al [30]. 100µl of Sample at
different concentrations (1.95, 3.9, 7.81, 15.62, 31.25, 62.5, 125, 250, 500 and 1000μg/ml) were added to
100 µl of 20 mM pNPG (p-Nitrophenyl α-D-glucopyranoside) and 2.2 ml of phosphate buffer (100 mM) at
pH 7.0, and then incubated at 37°C for 5 min. The reaction was initiated by addition of 100 µl of enzyme
solution (1mg/0.1ml) followed by further 15 min incubation at 37°C. The reaction was stopped by addition
of 2.5 ml of Na2CO3 (200 mM). The absorbances were measured at 405 nm with a spectrophotometer.
Percentage of inhibition on the α-amylase and α-glucosidase activity was calculated by the equation
Abs (Control) − Abs ( Extract )

Percentage of inhibition = ×100
Abs (Control)

The crude plant powder contains inorganic radicals like carbonates, phosphates and silicates of potassium,
calcium, sodium. Determining these substances ash values were calculated to determining its purity and
quality. An accurately weighed quantity of drug was extracted with various solvents like ether, ethanol-water.
This is used to determine the quantity of active constituents extracted with solvents from a given quantity of
plant powders. The total moisture content of the crude drug was also determined, and the results were given
in table 1. In the fluorescent analysis, the plant powder and extract were treated with various solvents like
nitric acid, sulphuric acid, sodium hydroxide, ethanol and water; the color developed was visualized under
ordinary light, short UV (254nm) and long UV (366nm) in UV chamber. The results were recorded in table 2.
Table 1: Physiochemical parameters of Myxopyrum serratulum A.W. Hill
S.No Parameters Values

1. Ash values
Total Ash 8.6%
Acid insoluble ash 2.4%
Water soluble ash 3.9%
2. Loss on drying 3.70%
3. Extractive values
Ether soluble extract 0.24%
Ethanol soluble extract 3.92%
Water soluble extract 5.68%
477
Preliminary phytochemical screening of plant extract shows the presence of active constituents like
alkaloids, flavonoids, phenolic compounds, steroids and glycosides etc., (table 3.) Quantitative determination
shows Myxopyrum serratulum A.W. Hill plant have the high amount of active constituents, and the results
were given in table 4.
Table 2: Fluorescent analysis of Myxopyrum serratulum A.W. Hill powder and ethanol extract
Reagents Myxopyrum serratulum powder Myxopyrum serratulum extract

Day light UV light Day light UV light
At 254 At 366 At 254 At 366
Sample as such Green Brown Green Brown Dark brown Dark brown
Sample+methanol Greenish Yellow Dark Green Light Green Yellowish Orange Fluorescent Green Greenish brown
Sample+50% HNO3 Reddish orange Dark brown Yellowish green Yellowish green Brown Yellow
Sample+1N NaOH Fluorescent Green Dark Green Light Green Orange Dark brown Fluorescent Green
Sample+50% H2SO4 Light Green Dark Green Yellow Dark Green Brown Yellow
Sample+80% H2SO4 Brown Greenish Black Dark Green Brown Dark brown Dark Green
S a m p l e + C o n c . Brown Black Dark Green Reddish Brown Dark brown Brown
H2SO4
Sample+H2O Yellowish brown Dark Brown Yellowish green Brown Dark brown Dark brown
Table 3: Preliminary Phytochemical screening of Myxopyrum serratulum A.W. Hill
S.No Phytochemical constituents Myxopyrum serratulum

1. Alkaloids +
2. Carbohydrates _
3. Flavonoids +
4. Phenols +
5. Proteins And Amino acids _
6. Saponins test +
7. Tannins test +
8. Triterpenoids +
9. Steroids +
10. Glycosides +
(+) Presence (-) Absence
Table 4: Quantitative determination of active constituents
S. No Content Myxopyrum serratulum(mg/g)

1. Phenolic Content 78.09
2. Flavonoid 51.2
3. Tannin 86.25
4. Flavonols 39.71
5. Lipid 3.63%
6. Alkaloid 1.56%
Diabetes mellitus is a common endocrine disorder affects more than 100 millions of people worldwide.
Postprandial hyperglycemia is a major problem in diabetes mellitus, and it increases the risk of diabetes
associated secondary diseases and cause death [31]. Carbohydrate-rich diets cause the rapid absorption
in the intestine by means of hydrolyzing enzymes (α-amylase and α-glucosidase). These enzymes break
complex carbohydrates into absorbable monosaccharide [32]. Suppression of these enzyme activities would
delay the degradation of starch and oligosaccharides, which leads to decreases in the absorption of glucose
and, as a result, the reduction of postprandial blood glucose rise. This can be achieved by using α-amylase
and α-glucosidase inhibitors. Acarbose, voglibose and miglitol are the synthetic α-amylase α-glucosidase
inhibitors lowers the postprandial blood glucose by inhibiting the last step in carbohydrate digestion [33].
478
These inhibitors also produce some side effects like abdominal discomfort, diarrhea and flatulence. Hence,
there is a growing attention in discovering new and effectual α-amylase and α-glucosidase inhibitors from
plants with least or no side effects [34]. The results of in vitro studies (Table 5) showed that Myxopyrum
serratulum A. W. Hill inhibits alpha amylase and alpha glycosidase activity (Figure 2) compared to standard
drug acarbose (Table 6) The IC 50 values were 435.02µg and 688.61µg for alpha-amylase and alpha-
glucosidase respectively. The ethanolic extract of Myxopyrum serratulum A.W. Hill plant have the high
quantity of active constituents, these active constituents may be responsible for this inhibitory activity.
Further studies needed to isolate the respective carbohydrate hydrolyzing enzyme inhibitors.
Figure 2: In vitro antidiabetic activity of Myxopyrum serratulum A.W. Hill and Acarbose
Table 5: In vitro antidiabetic activity of Myxopyrum serratulum A.W. Hill
S.No Concentration (µg/ml) % Inhibition ±SEM

α-glucosidase inhibition IC50 α-amylase inhibition IC50
1 1.95 9.97±3.47 6.52±3.25
2 3.9 14.43±0.11 10.87±0.51
3 7.81 15.03±0.32 18.84±0.34
4 15.62 16.52±0.74 25.85±0.51
5 31.25 29.17±1.05 33.57±2056
6 62.5 33.76±0.42 38.78±0.34
7 125 36.95±0.21 42.58±0.34
688.61µg 435.02µg
8 250 40.74±0.42 48.44±0.34
9 500 45.93±0.00 56.14±1.20
10 1000 56.96±0.63 60.14±0.51
Table 6: In vitro antidiabetic activity of Acarbose
S.No Concentration (µg/ml) α- glucosidse α- amylase

1 20 38.45±0.00 25.30±0.56
2 40 46.75±0.48 31.82±0.98
3 60 59.78±0.56 58.30±0.98
4 80 72.48±0.68 65.42±2.38
5 100 89.32±0.56 73.32±0.70
Results expressed as Mean±SEM triplicates
479
CONCLUSION:
Our in vitro studies confirmed that Myxopyrum serratulum A.W. Hill plant have considerable α-glucosidase
and α-amylase inhibitory activity. The ethanolic extract of this plant have the potential to emerge as a new
therapy for the treatment of diabetes mellitus. Further studies need to be conducted to increases their activity
by converting into nanosized particles and also in vivo experiments can be performed on animal models to
confirm the hypoglycemic activity. It is a safe and effectual intervention for diabetes.
ACKNOWLEDGEMENT:
The author is thankful to Department of Science and Technology, for providing financial assistance in
the form of Inspire fellowship and Sri Ramachandra University for their kind support to carrying out this
research work.
REFERENCES:
1. World Health Organisation (WHO) & International Diabetes Federation (IDF) Definition and diagnosis
of diabetes mellitus and intermediate hyperglycaemia Geneva: WHO; 2006, [cited 01 Dec 2009].
2. J.E. Shaw, R.A. Sicree and P.Z. Zimmet, “Global estimates of the prevalence of diabetes for 2010 and
2030,” Diabetes Research and Clinical Practice, 2010; 87(1): 4–14
3. J.P. Boyle, M.M. Engelgau, T.J. Thompson, et al., “Estimating prevalence of type 1 and type 2 diabetes
in a population of African Americans with diabetes mellitus,” American Journal of Epidemiology, 1999;
149(1): 55–63.
4. Pallab Das Gupta and Amartya De, Diabetes Mellitus and its Herbal Treatment, International Journal of
Research in Pharmaceutical and Biomedical Sciences, 2012; 3(2):706-721.
5. D. Jones, “Diabetes field cautiously upbeat despite possible setback for leading SGLT2 inhibitor,”
Nature Reviews Drug Discovery, 2011; 10(9): 645–646.
6. J.M. Egan, A. Bulotta, H. Hui and R. Perfetti, “GLP-1 receptor agonists are growth and differentiation
factors for pancreatic islet beta cells,” Diabetes/Metabolism Research and Reviews, 2003; 19(2): 115–123.
7. H.C.S. Howlett and C.J. Bailey, “A risk-benefit assessment of metformin in type 2 diabetes mellitus,”
Drug Safety, 1999; 20(6): 489–503.
8. Grover JK, Yadav S and Vats V, (2002) Medicinal plants of India with anti-diabetic potential, J
Ethnopharmacol 81: 81-100.
9. Tamrakar AK, Yadav PP, Tiwari P, Maurya R and Srivastava AK, (2008), Identification of pongamol
and karanjin as lead compounds with antihyperglycemic activity from Pongamia pinnata fruits, J
Ethnopharmacol 118: 435-439.
10. C.P. Khare, Indian Medicinal plants an illustrated Dictionary, Springer, 2008; p. 431
11. Warrier PK, Nambiar VPK and Ramankutty C, Indian medicinal plants, Orient Longmann Ltd., Madras;
1994, Vol. IV. p.98
12. T. Sheela Rani, V. Gopal, S. Seetha Lakshmi and K. Chitra, Vitro anti-oxidant activity of Myxopyrum
serratulum A.W. Hill, Int J Pharm Sci, 2013; 5(4): 545-546.
13. S. Gopalakrishnan and R. Rajameena Rajangam, Wound Healing activity of the ethanol extract of the
leaves of Myxopyrum serratulum A.W. Hill in rats, Int. J. Pharm. Sci. Rev. Res., 2013; 22(1), 143-147.
14. S. Gopalakrishnan, R. Rajameena and E. Vadivel, Antimicrobial activity of the leaves of Myxopyrum
serratulum A.W. Hill, International Journal of Pharmaceutical Sciences and Drug Research, 2012; 4(1):
31-34.
480
15. S. Gopalakrishnan and R. Rajameena, GC-MS analysis of some bioactive constituents of the leaves of
Myxopyrum serratulum A.W. Hill, International journal of advances in pharmaceutical sciences, 2013;
4(5).
16. Reddy NVLS, Anarthe SJ and Raghavendra NM, J Res. Biomed. Sci, 2010; 1 (2):72-75
17. Ramkumar KM, Thayumanavan B, Palvannan T and Rajaguru P, Med. Chem. Res, 2010; 19:948-961
18. Kokate C.K., 1994, Kokate Practical Pharmacognosy Vallabh Prakashan, New Delhi. pp. 107–113,
19. Mukerjee P.M., 2005, Quality control of herbal drugs, India pg-273-274.
20. Wallis TE, A Practical Pharmacognosy, Pharmamed press, Hyderabad, 2011, 179-180.
21. Chase C.R. and Pratt R., Fluorescence analysis of powdered vegetable drug with particular reference
to the development of the system of identification, Journal of American Pharmaceutical sciences, 1949;
28: 324-341
22. Harborne J.B., 1998, Harborne Phytochemical Methods Chapman Hall, London,1998; pp 60–66.
23. Mukerje P.K., Quality control of herbal drugs, An Approach to evaluation of botanicals; business
horizons (ND), India, 2002, 132-144.
24. Mbaebie BO, Edeoga HO and Afolayan AJ, Phytochemical analysis and antioxidants activities of
aqueous stem bark extract of Schotia latifolia Jacq, Asian pacific journal of tropical biomedicine, 2012;
118-124.
25. Singleton V, Orthofer R and Lamuela-Raventos RM, Analysis of total phenols and other oxidation
substrates and antioxidants by means of folin-ciocalteu reagent, Methods Enzymol 1999; 99: 152-58
26. Chang C, Yang M, Wen H and Chern J, Estimation of total flavonoid content in propolis by two
complementary colorimetric methods, Journal of Food Drug Analysis, 2002; 10: 178-182.
27. Kumaran A and Karunakaran RJ, In vitro antioxidant activities of methanol extract of five phyllantus
species from India, LWT-Food Sci Technol 2007; 40: 344-352.
28. Jayaraman J, Laboratory manual in biochemistry, New Delhi: Wiley Eastern Limited, New Delhi (1981).
29. Miller G.L., 1959, Use of Dinitrosalicylic acid reagent for determination of reducing sugar, Analytical
Chemistry 31: 426-428.
30. Artanti N., Firmansyah T. and Darmawan A., Bioactivities Evaluation of Indonesian Mistletoes
(Dendrophthoe pentandra (L.) Miq.) Leaves Extracts. Journal of Applied Pharmaceutical Science, 2012;
2 (01): 24- 27
31. Slama G, Elgrably F, Sola A and Mbemba J, Larger E Postprandial glycaemia: a plea for the frequent
use of delta postprandial glycaemia in the treatment of diabetic patients. Diabetes Metab., 2006; 32:
187-192.
32. Kwon YI, Vattem DA and Shetty K, Evaluation of clonal herbs of Lamiaceae species for management
of diabetes and hypertension, Asia Pac J Clin Nutr 2007; 15:107-118.
33. Davis SN and Granner DK, Insulin, oral hypoglycemic agents and the pharmacology of endocrine
pancreas. In: Brunton LL, Lazo JS, Parker KL (Ed.), Goodman and Gilman’s: The pharmacological
basis of therapeutics, 11th ed. (McGraw-Hill Medical Publication Division: New York 2001; 1706-1707.
34. Shai LJ, Masoko P, Mokgptho MP, Magano SR, Mogale AM, Boaduo N and Ellof JN, Yeast alpha-
glucosidase inhibitory and antioxidant activities of six medicinal plants collected in Phalaborwa, South
Africa, S Afr J Bot 2010; 76: 465-470.
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In Vitro Antioxidant Activities, Total Flavonoids and Total
Phenolic Content of Ethanolic Extract from Whole Plant
of Lactuca runcinata (DC)
Jeyaraman Amutha Iswarya Devi and Arumugam Kottai Muthu
Department of Pharmacy, Annamalai University, Annamalai Nagar-608 002, Tamil Nadu, India
Abstract:
To evaluate the in vitro antioxidant activities and to estimate total phenol and flavonoids of the crude ethanolic
extract from the whole plant of Lactuca runcinata DC. The shade dried whole plant powder was extracted
with ethanol by using soxhlet extractor, and crude extract was used for in vitro antioxidant activity by DPPH,
nitric oxide, iron chelating, hydroxyl radical, superoxide radical scavenging, total antioxidant and FRAP
assay methods. Total phenols and flavonoids also estimated. The results revealed that L. runcinata ethanolic
extract have higher antioxidant property and contains many phenols and flavonoids. This study helps to
predict it is a better source of natural antioxidant and contains a large amount of phenols and flavonoids that
can be used as drugs, and further investigation may lead to the development of drug formulation.
Keywords: Ethanolic extract, In vitro antioxidant, Lactuca runcinata DC., Total flavonoids.
1. INTRODUCTION
Lactuca runcinata DC.; Synonyms, L. heyneana DC.. Family: Compositae; Asteraceae. This occurs many
parts of India, as a common weed. It is considered as valuable medicinal herb in traditional systems of
medicine in India. Action diuretic, slightly aperient. It is used as a diuretic in calculous affections, also
for chronic obstruction of liver and bowels [1]. A smaller var., found in western Uttar Pradesh, Rajasthan,
Saurashtra and the Deccan Penninsula, is equated with L. remotiflora DC.
However, no data are available in the literature on the antioxidant activity of whole plant of L.
runcinata. Therefore, we undertook the current investigation to examine antioxidant activities, total
flavonoids and total phenolic contents of ethanolic extract from the whole plant of L. runcinata through
various in vitro models.

2.1. Collection and preparation of plant material
The fresh whole plants of Lactuca runcinata DC were collected from the natural habitats of Kayathar,
Thoothukkudi district, Tamil Nadu, India. Taxonomic identification was made from Botanical Survey of
Medical Plants Unit Siddha, Government of India, Palayamkottai. The samples were washed thoroughly in
running tap water to remove soil particles and adhered a debris and finally washed with sterile distilled water.
The whole plants were shade dried and ground into a fine powder. The powdered materials were stored in
airtight polythene bags until use.
2.2. Plant sample extraction

The powder samples of Lactuca runcinata were extracted with ethanol at temperature between 60-65˚C by
hot continuous percolation method in Soxhlet apparatus [2] for 24 hrs. The extract was concentrated by using
a rotary evaporator and subjected to freeze drying in a lyophilizer till dry powder was obtained.
2.3. Evaluation of antioxidant activities by in vitro techniques

DPPH photometric assay
The effect of ethanolic extract on DPPH radical was assayed using the Mensor method [3]. A methanolic
solution of 0.5ml of DPPH (0.4mM) was added to 1 ml of the different concentrations of plant extract and
allowed to react at room temperature for 30 minutes. Methanol served as a blank and DPPH in methanol
without the extracts served as a positive control. After 30 min, the absorbance was measured at 518 nm and
converted into percentage radical scavenging activity as follows.
A 518 Control- A 518 Sample
Scavengingactivity (%) = × 100
A 518 Control
Where A518 control is the absorbance of DPPH radical+methanol; A518 sample is the absorbance of
DPPH radical+ sample extract/ standard.
Nitric oxide radical scavenging activity

Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with
oxygen to produce nitrite ions, which were measured by the Garrat method [4]. The reaction mixture (3ml)
containing 2 ml of sodium nitroprusside (10mM), 0.5 ml of phosphate buffer saline (1M) were incubated at
250C for 150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipetted and mixed
with 1 ml of sulphanilic acid reagent (0.33%) and allowed to stand for 5 min for completing diazotization.
Then 1 ml of naphthylethylene diamine dihydrochloride (1% NEDA) was added, mixed and allowed to stand
for 30 min. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric
oxide, which interacts with oxygen to produce nitrite ions that can be estimated by the use of Griess Ilosvay
reaction at 540 nm.
Iron chelating activity

The Benzie and strain method [5] was adopted for the assay. The principle is based on the formation of
O-Phenanthroline-Fe2+ complex and its disruption in the presence of chelating agents. The reaction mixture
containing 1 ml of 0.05% O-Phenanthroline in methanol, 2 ml ferric chloride (200µM) and 2 ml of various
concentrations of ethanolic extracts ranging from 125 to 1000µg was incubated at room temperature for 10
min, and the absorbance of the same was measured at 510 nm. EDTA was used as a classical metal chelator.
The experiment was performed in triplicates.
Hydroxyl radical scavenging activity

This was assayed as described by Elizabeth and Rao method [6]. The assay is based on quantification of
degradation product of 2-doxyribose by condensation with TBA. Hydroxyl radical was generated by the
Fe3+ -Ascorbate –EDTA –H2O2 system (Fenton reaction). The reaction mixture contained 0.1 ml deoxyribose
(2.8mM), 0.1 ml EDTA (0.1 mM), 0.1 ml H2O2 (1mM), 0.1 ml ascorbate (0.1mM), 0.1 ml KH2PO4-KOH
buffer, pH 7.4 (20mM) and various concentrations of ethanolic extract from L. runcinata in a final volume
of 1 ml. The reaction mixture was incubated for 1 hour at 370 C. Deoxyribose degradation was measured as
TBARS and the percentage inhibition was calculated.
483
Superoxide radical scavenging activity

Superoxide radical (O2-) was generated from the photoreduction of riboflavin and was deducted by nitro
blue tetrazolium dye (NBT) reduction method. Measurement of superoxide anion scavenging activity was
performed based on the method described by Winterbourne [7]. The assay mixture contained sample with
0.1ml of Nitro blue tetrazolium (1.5 mM NBT) solution, 0.2 ml of EDTA (0.1M EDTA), 0.05 ml riboflavin
(0.12 mM) and 2.55 ml of phosphate buffer (0.067 M phosphate buffer). The control tubes were also set
up where in DMSO was added instead of sample. The reaction mixture was illuminated for 30 min and
the absorbance at 560 nm was measured against the control samples. Ascorbate was used as a reference
compound. All the tests were performed in triplicate, and the results averaged. The percentage inhibition was
calculated by comparing the results of control and test samples.
Total antioxidant activity (Phosphomolybdic acid method)

The antioxidant activity of the ethanolic extract of L. runcinata was evaluated by the transformation of Mo
(VI) to Mo (V) to form phosphomolybdenum complex [8]. An aliquot of 0.4 ml of ethanolic extract was
mixed in a vial with 4 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate). The vials were capped and incubated in a water bath at 950C for 90 min. After the
samples had cooled to room temperature, the absorbance of the mixture was read at 695 nm against a blank.
The antioxidant activity was expressed relatively to that of ascorbic acid.
FRAP assay
A modified method of Benzie and Strain [5] was adopted for the FRAP assay. The stock solutions included
300 mM acetate buffer, pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution in 40 mM HCl and 20
mM FeCl3.6H2O. The fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ
and 2.5 ml FeCl3.6H2O. The temperature of the solution was raised to 370 C before using. Ethanolic extract
of L. runcinata (0.15 ml) was allowed to react with 2.85 ml of FRAP solution for 30 min in the dark
condition. Readings of the coloured product (Ferrous tripyridyltriazine complex) were measured at 593 nm.
The standard curve was linear between 200 and 1000 µM FeSO4. Results were expressed in µM (Fe (II) /g
dry mass and compared with that of ascorbic acid.
Total flavonoids
The measurement of total phenol is based on the method [9]. To 0.2g of the ethanolic extract of L. runcinata
was ground with ethanol-water in 2 different ratios namely 9:1 and 1:1 respectively. The homogenate was
filtered, and these two ratios were combined. This was evaporated to dryness until most of the ethanol has
removed. The resultant aqueous extract was extracted in a separating funnel with hexane or chloroform. The
solvent extracted aqueous layer was concentrated 0.5 ml of aliquot of the extract was pipette-out in a test
tube. 4 ml of the vanillin reagent (1% vanillin in 70% conc. H2SO4) was added and kept in a boiling water
bath for 15 mints. The absorbance was measured at 360 nm. A standard was run by using the catechol (110
µg/ml).
Total phenol
The measurement of total phenol is based on the method [10]. To 0.25g of ethanolic extract of L. runcinata,
added 2.5 ml of ethanol and centrifuged at 2°C for ten min. The supernatant was preserved. Then, the
ethanolic extract was re-extracted with 2.5 ml of 80% ethanol and centrifuged. The pooled supernatant was
evaporated to dryness. Then, added 3 ml of water to the dried supernatant. To which added 0.5 ml of Folins
phenol reagent and 2 ml of sodium carbonate (20%). The reaction mixture was kept in boiling water bath for
1 min. The absorbance was measured at 650 nm in a spectrophotometer.
484

DPPH is a stable free radical at room temperature often used to evaluate the antioxidant activity of several
natural compounds. The percentage of DPPH radical scavenging activity of ethanolic extract of L. runcinata
exhibited a maximum DPPH scavenging activity of 63.46% whereas for rutin (standard) was found to be
69.83% at 1000 µg/ml presented in Table 1. The IC50 of the ethanolic extract and rutin were found to be
510µg/ml and 480µg/ml respectively.
The percentage of nitric oxide free radical scavenging activity of ethanolic extract of L. runcinata shown
maximum activity was 57.85% and standard (ascorbate) was found to be 62% at 1000 µg/ml presented in
Table 1. The IC50 of the ethanolic extract and standard (ascorbate) were found to be 425µg/ml and 410µg/
ml better antioxidant is respectively.
Table 1: Effect of ethanolic extract of L. runcinata on DPPH assay & Nitric oxide free radical scavenging method
S.No Concentration % of activity (±SEM)* % of activity (±SEM)*

(µg/ml) Sample (Ethanolic Standard (Rutin) Sample (Ethanolic
Standard (Ascorbate)
extract) extract)
1 125 21.64±0.049 18.85 ± 0.076 36.71±0.09 27.63±0.076
2 250 30.36±0.087 22.08 ± 0.054 45.52±0.17 49.53 ±0.054
3 500 48.54±0.069 52.21 ± 0.022 52.67±0.11 55.12±0.022
4 1000 63.46±0.018 69.83 ± 0.014 57.85±0.09 62.00±0.014
IC50 = 510 µg/ml IC50 = 480 µg/ml IC50 = 425 µg/ml IC50=410 mg/ml
*All values are expressed as mean ± SEM for three determinations
Iron binding capacity of the ethanolic extract of L. runcinata and the metal chelator EDTA at various
concentrations were examined, and the values were presented in table 2. Maximum chelating of metal ions
at 1000µg/ml for ethanolic extract and EDTA was found to be 88.73% and 97.90% respectively. The IC50
value of ethanolic extract and EDTA were recorded as 425µg/ml and 65µg/ml respectively.
The highly reactive hydroxyl radicals can cause oxidative damage to DNA, lipids and proteins [11]. The
percentage of hydroxyl radical scavenging activity of ethanolic extract of Lactuca runcinata was presented
in Table 2. The ethanolic extract of L. runcinata was exhibited a maximum hydroxyl radical scavenging
activity of 62.24 % whereas for ascorbate (standard) were found to be 55.23 % at 1000 µg/ml. The IC50 of
the ethanolic extract and ascorbate were found to be 576µg/ml and 410µg/ml respectively.
Table 2: Effect of ethanolic extract of L. runcinata on Iron chelating method & Hydroxyl radical scavenging activity
S.No Concentration % of activity (±SEM)* % of activity (±SEM)*

(µg/ml) Sample (Ethanolic Standard (EDTA) Sample (Ethanolic Standard
extract) extract) (Ascorbate)
1 125 20.69 ± 0.081 58.68 ± 0.007 38.32 ± 0.072 26.87 ± 0.076
2 250 37.63 ± 0.021 65.87 ± 0.018 43.46 ± 0.048 30.30 ± 0.054
3 500 57.53 ± 0.014 83.83 ± 0.012 52.14 ± 0.045 60.64 ± 0.022
4 1000 88.73 ± 0.014 97.90 ± 0.019 62.24 ± 0.033 55.23 ± 0.014
IC50 = 425 µg/ml IC50 = 65 µg/ml IC50 = 576 µg/ml IC50 = 410 µg/ml
Percentage scavenging of superoxide anion examined at different concentrations of ethanolic extract of

L. runcinata was depicted in table 3. The maximum scavenging activity of ethanolic extract of L. runcinata
and quercetin at 1000 µg/ml was found to be 75.45% and 98.01% respectively. Superoxide scavenging
ability of plant extract might primarily be due to the presence of flavonoids [12]. The IC50 value of ethanolic
extract and quercetin were recorded as 220µg/ml and 60µg/ml respectively.
485
The percentage of total antioxidant activity of ethanolic extract of L. runcinata presented in Table 3. The
ethanolic extract of L. runcinata exhibited a maximum total antioxidant activity of 67.45 % at 1000 µg/ml
whereas for ascorbate (standard) was found to be 55.23 % at 1000 µg/ml. The IC50 of the ethanolic extract
and ascorbate were found to be 590µg/ml and 410µg/ml respectively.
Table 3: Effect of ethanolic extract of L. runcinata on superoxide anion scavenging activity & Total antioxidant activity
S.No Concentration % of activity(±SEM)* % of activity(±SEM)*

(µg/ml) Sample (Ethanolic Standard Sample (Ethanolic Standard
extract) (Quercetin) extract) (Ascorbate)
1 125 43.87 ± 0.054 73.81 ± 0.006 23.228 ± 0.032 26.87 ± 0.076
2 250 53.66 ± 0.034 91.31 ± 0.011 33.65 ± 0.046 30.30 ± 0.054
3 500 68.22± 0.046 92.99 ± 0.024 47.58 ± 0.034 60.64 ± 0.022
4 1000 75.45 ± 0.072 98.01 ± 0.012 67.45 ± 0.065 55.23 ± 0.014
IC50 = 220 µg/ml IC50 = 60 µg/ml IC50 = 590 µg/ml IC50 = 410 µg/ml
The antioxidant potential of Lactuca runcinata was ascertained from FRAP assay based on their ability
to reduce TPTZ-Fe (III) complex to TPTZ-Fe (II). The reducing ability of the ethanolic extract of L.
runcinata and ascorbate at various concentrations were examined, and the values are presented in Table 4.
The maximum reducing ability at 1000µg/ml for ethanolic extract and ascorbate were found to be 77.22%
and 98.07% respectively. The IC50 values of ethanolic extract and ascorbate were recorded as 175µg/ml and
50µg/ml respectively.
Table 4: Effect of ethanolic extract of L. runcinata on FRAP assay
S.No Concentration (µg/ml) % of activity(±SEM)*

Sample (Ethanolic extract) Standard (Ascorbate)
1 125 42.23 ± 0.022 72.04 ± 0.014
2 250 58.43 ± 0.032 82.05 ± 0.034
3 500 69.22 ± 0.034 86.04 ± 0.026
4 1000 77.22 ± 0.033 98.07 ± 0.041
IC50 = 175 µg/ml IC50 = 50 µg/ml
Flavonoids present in food of plant origin are also potential antioxidants [13, 14]. Most beneficial effects
of flavonoids are attributed to their antioxidant and chelating abilities [15]. The total amount of flavonoids
content of ethanolic extract of L. runcinata was presented in Table 5.
Phenols are very important plant constituents because of their scavenging ability due to their hydroxyl
groups [16]. Phenolic compounds are known as powerful chain breaking antioxidants [17]. The total amount
of phenolic content of ethanolic extract of L. runcinata was presented in Table 5. The ethanolic extract of
whole plant of was found higher content of phenolic components.
Table 5: Total flavonoids & total phenolic content of ethanolic extract of L. runcinata
S.No Extract Total flavonoids Total phenol content (mg/g

content (mg/g ±SEM)* of Catechol) ± SEM)*
1 Ethanolic extract of 2.978 ± 0.044 3.68 ± 0.023
Lactuca runcinata
486
4. CONCLUSION
From the results obtained in the present study, it is concluded that the whole plant of ethanolic extract of Lactuca
runcinata exhibits high antioxidant and free radical scavenging activities, which contains large amounts of
phenolic compounds. These in vitro assays indicate that this plant extract is a significant source of natural
antioxidant, which might be helpful in preventing the progress of various oxidative stresses. Therefore, further
investigations need to be carried out to isolate and identify the antioxidant compounds present in the plant
extract and vivo antioxidant activity of this extract needs to be assessed prior to clinical use.
REFERENCES
1. Khare CP, Indian Medicinal Plants, Springer; 2008. p. 357.

2. Harborne J.B., Phytochemical methods 11 Edn., In Chapman & Hall, New York: 1984, 4-5.
3. Mensor L.L, Meneze F.S., Leitao G.G., Reis A.S., Dos Santor J.C., Coube C.S and Leitao S.G., Screening
of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method, Phytother.
Res.2001; 15, 127-130.
4. Garrat DC, The quantitative analysis of drugs, Champman and Hall, Japan, 1964; 3, 456- 458.
5. Benzie IEF and Strain JJ, The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant
power”: the FRAP assay, Anal Biochem.1996; 239, 70-76.
6. Elizabeth K and Rao MNA, Oxygen radical scavenging activity of curcumin, Int.J. Pharm., 1990; 58,
237-240.
7. Winterbourne C.C., Hawkins R.E., Brain M and Carrel R.W., The estimation of red cell superoxide
dismutase activity, J. Lab.chem.Med., 1975; 85, 337-341.
8. Prieto P., Pineda M. and Aguilar M., Spectrophotometric quantitation of antioxidant capacity through
the formation of a Phosphomolybdenum Complex: Specific application to the determination of vitamin
E. Anal. Biochem, 1999; 269, 337-341.
9. Cameron GR, Milton RF and Allen JW, Measurement of flavonoids in plant samples, Lancet., 1943, 179.
10. Mallick CP and Singh MB, Plant enzymology and Histoenzymology (eds), Kalyani publishers, New
Delhi, 1980, pp 286.
11. Spencer JPE, Jenner A, Aruoma OI, et al. Intense oxidative DNA damage promoted by L-DOPA and its
metabolites, implications for neurodegenerative disease, FEBS Lett 1994; 353:246–250.
12. Zheng W and Wang SY, (2001), Antioxidant activity and phenolic compounds in selected herbs, J. Agri.
Food. Chem., 2001; 49, 5165-5170.
13. Salah N, Miller NJ, Paganga G, Tijburg L, Bolwell GP and Rice Evans C, Polyphenolic flavonoids as
scavengers of aqueous phase radicals and as chain-breaking antioxidants, Arch Biochem Biophys, 1995;
322 (2), 339-346.
14. Van Acker SABE, Van den Vijgh WJF and Bast F, Stuctural aspects of antioxidant activity of flavonoids,
Free Rad Bio Med, 1996; 20 (3), 331-342.
15. Hassig A, Liang WX, Shwabl K and Stampfl K, Flavonoids and tannins: plant based antioxidants with
vitamin character, Med Hypotheses, 1999; 52, 471-481.
16. Hatano T, Edamatsu R, Mori A, et al. Effect of interaction of tannins with co-existing substances, VI.
Effects of tannins and related polyphenols on superoxide anion radical and DPPH radical, Chemical and
Pharmaceutical Bulletin, 1989; 37: 2016–2021.
17. Shahidi F and Wanasundara PKJPD, Phenolic antioxidants, Critical Reviews in Food 1Science and
Nutrition 1992; 32: 67–103.
487
GC-MS Characterization of Volatile Odorous Compounds
in Allium Cepa
N.C.J. Packia Lekshmi1, S. Viveka2, M.B. Viswanathan3, G. Manivannan4

and T. Mini Shobi5
Department of Biotechnology, Sathyabama University, Solinganallur, Chennai 600119, Tamil Nadu, India
1
2
Department of Biotechnology, Udaya School of Engineering, Vellamodi, Kanniyakumari 629204,
Tamil Nadu, India
3, 5
Department of Plant Science, Bharathidhasan University, Tiruchirappalli 620024, Tamil Nadu, India
4
Department of Microbiology, NMSSVN College, Nagamalai, Madurai 625019, Tamil Nadu, India
e-mail: packia_3779@yahoo.co.in
Abstract:
An analytical technique for the determination of volatile phytochemical compounds by GC-MS in onion,
Allium cepa L., was reported here. Three different cultivars of onion bulb (O1, O2 and O3) were extracted
with petroleum ether, and the volatile compounds were analyzed by GC-MS. Terpenes such as humulaneand
phytosterols and aldehydes are found common in all the three cultivars. Thiophenes and disulphides were
identified as major compounds in O1 and O3 respectively. Caryophyllene, a terpene was observed in both
O1 and O2. Squalene, allicin and sulphide compounds were identified specifically in O3. The study further
reported that the composition of volatile compounds in the cultivars is varied and these differences may be
due to the variations in geographic, climatic, soil fertility and cultivation practices. The intensity of flavour
is governed by hereditary factors and environmental conditions under which the onions grow.
Keywords: Phytochemicals, GC-MS, onion, Terpenes, Disulphides.
INTRODUCTION
Allium is the prime and most important genus in the family of Alliaceae with about 450 species. Technical
investigation on these plants was started in the middle of 19th century. Allium species are rich sources of
phytonutrients [1]. Onions occupy a major portion in food market. Cultivars and typologies of onion can
diverge from a chemical point of analysis and variations can be important in their classification [2].
Bulbs of Allium species are broadly used as food flavouring agents and as remedial agents because of its
valuable compounds such as allicin, alliin, flavonoid and their derivatives [3-7]. The oily substance which
has the characteristic onion odour from bulb of Allium cepa was di- and trisulphide [8] and thioaldehydes
[9]. These odoriferous volatile lipid compounds were analyzed by several research groups [10]. The nature
and composition of volatile components of onion be still contentious. Geographic, climatic and differences
in varieties may also play a vital part in the proportions of volatile compounds of onion [10].
The volatile lipid soluble organo sulphur compounds were analysed using GC-MS [11]. This method is
used to measure secondary metabolites formed during enzymatic/metabolic pathways such as disulphides
and trisulphides. The analytical strategy (GC-MS) used to analyse the volatile compounds, and selected
compounds were structurally analysed by MS transposing the method to GC-MS. In this paper, we evaluated
the organic volatile compounds which are responsible for biological activity, flavour and odour in this
particular medicinally valuable onion by GC-MS.

Extraction of organo-sulphur compounds (using non-polar solvent of petroleum ether)
The plant samples were collected from different cultivation localities such as Alankulam (O1) and Surandai
(O2) in Tirunelveli District and Vilathikulam (O3) in Tuticorin district of Tamil Nadu. The extraction of
organo-volatile compounds was accomplished using Soxhlet apparatus in which the polar barrier was broken
up and allowed the solvent to reach the non-polar compounds in order to achieve their extraction. Often, the
sample was dried, ground and prepared for the extraction.
GC-MS analysis
The filtered plant extracts were subjected to GC-MS analyses such as petroleum ether, chloroform, ethyl
acetate and methanol. GC-MS was carried out on an HP 5890 GC system coupled to a Quadrupole Mass
Detector. Helium was used as a carrier gas in a constant flow mode at 1ml/min. The initial temperature of
the column was 70°C, which was gradually increased by 10°C up to 280°C. The instrument was set to an
initial temperature of 70°C and maintained at this temperature for 2 min. At the end of this period, the Oven
temperature was raised up to 280°C, at the increased rate of 5°C/min, and maintained for 9 min. Injection
port temperature was ensured as 250°C and helium flow rate as 1 ml/min. The ionization voltage was 70eV.
Separation was achieved by RTS-volatile column about 30 m long. Quadrupole Mass Detector was employed
to detect compounds when they were vented from the column. Temperature of the detector was 300°C. Using
data library such as WILEY8.LIB and NIST 08 database, the mass spectra obtained through GC–MS, were
analysed, and the volatile compounds of the plant samples were identified.

The volatile organic compounds from petroleum ether extracts of onion cultivars were identified by GC-MS
analysis since this method was found to be a great value in the identification of organic volatile compounds
(Table 1).
Table 1: List of volatile compounds identified by GC-MS
S.No. R.Time Area Area% Compound name Present in

1 8.642 2697349 3.55 Undecane O1, O2 and O3
2 50.242 6081063 8.01 Gamma-Sitosterol O1, O2 and O3
3 19.605 829867 1.09 Phenol O1, O2 and O3
4 32.194 128551 0.17 Octadecanoic acid (Oleic acid) O1, O2 and O3
5 49.143 4996524 6.58 Ergost-8-en-3-ol O1, O2 and O3
6 49.606 9601417 12.65 9,19-Cycloergost-24(28(-en-3-ol O1, O2 and O3
7 48.008 172143 0.23 Cholesta-4,6-dien-3-ol O1, O2 and O3
8 48.609 2825006 3.72 17,(1,5-Dimethyl-hexyl)-10 O1, O2 and O3
9 48.457 341303 1.83 Cholesterol (Phytosterol) O1, O2 and O3
10 17.225 89109 1.13 Humulane (Sesquiterpenes) O1, O2 and O3
11 24.118 251871 0.33 Myristic acid O1, O2 and O3
12 50.247 106771 0.57 Fucosterol (Phytosterol) O1, O2 and O3
13 49.143 4996524 6.58 Ergost-8-en-3-ol, 14-methyl-, (3 beta,5 alpha.)- O1, O2 and O3
14 32.060 327236 1.76 Fumaric acid O1, O2 and O3
15 36.736 85464 0.46 Oxalic acid O1, O2 and O3
16 9.655 593153 0.78 2-Mercapto-3,4-dimethyl-2,3-dihydrothiophene O1
17 17.904 2533702 3.34 3(2H)-Furanone O1
18 19.007 224765 0.30 Thiophene O1
19 19.358 914661 1.20 Ethanone O1
20 22.177 543096 0.72 1,2-Dimethoxy-4-(2-methoxyethenyl)benzene O1
489
21 24.118 251871 0.33 Tetradecanal O1

22 29.625 110637 0.15 1,2-Benzenedicarboxylic acid O1
23 33.411 207295 0.27 Tricosane O1
24 35.110 384977 0.51 Octadecane O1
25 36.742 332279 0.44 Tetratriacontane O1
26 17.223 776709 4.17 Caryophyllene (Sesquiterpene) O1 and O2
27 35.432 1260074 1.66 Octanamide O1 and O2
28 28.400 1955725 2.58 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione O1 and O2
29 32.501 157762 0.21 Decanamide (Capramide) O1 and O2
30 29.388 187318 0.25 n-Hexadecanoic acid O1 and O2
31 50.851 14138860 18.63 9,19-Cyclolanost-24-en-3-ol O1 and O3
32 48.181 169854 0.22 Tetratetracontane O1 and O3
33 24.214 117164 0.63 Pentadecanal- O2
34 31.636 100535 0.54 1-Hexadecyne O2
35 29.249 1036092 5.56 Dibutyl phthalate O2 and O3
36 28.466 101132 0.54 Heptadecanoic acid O2 and O3
37 8.179 4447752 0.18 Diallyl disulphide (Sulfides) O3
38 8.17 311631 0.63 Allicin (sulphides) O3
39 44.108 100051 0.52 Squalene (Triterpene) O3
Most of the identified compounds were present in all the three cultivars. The important ones among
them were gamma-sitosterol, ergos-8-en-3-ol, cholesta-4,6-dien-3-ol, cholesterol, humulene, myristic acid,
fucosterol, fumaric and oxalic acids. Thiophene, an active compound specifically identified in O1. Similarly,
the volatile compounds, disulphides and thiophenes were identified in yellow onions by Jarvenpaa et al. [12]
in their solid phase micro-extraction combined GC-MS study and by Boelens et al. [13]. Caryophyllene, a
terpenoid was identified in both O1 and O2. Liu et al. [14] reported the presence of aromatic compounds
caryophyllene, thiophene and other terpenoids by GC-MS analysis (Figs.1-3).
Figure 1: GC-MS chromatogram of O1
490
491
The cultivar O3 showed the presence of volatile sulphide compounds –allicin, diallyl disulfide and a
terpeneof squalene. Keusgen [15] identified the volatile compounds of allicin and sulphides. Most of the
disulphides and thiol groups in onion were also determined by Marie Lokke et al. [16]. In this study, we
identified the precursor molecules for the synthesis of the volatile sulphide and terpenoids in all the three
cultivars. Besides, we also determined the presence of ethanol, isoamyl acetate, isobutyl alcohol, propyl
alcohol, palmitic acid, stearic acid, and lanosterol in the onion cultivars. Similar results were reported by
Ohta and Osajima [17]. They detected the typical sulphur containing aldehyde compounds, di- and trisulphide
in onion (Fig. 4).
Figure 4: Chemical structure of volatile compounds
The volatile compounds of onion were studied by many researchers [18-20]. The potential antibiotic
and flavour properties of these volatile compounds in onion received greater attention [21]. However, there
is a disparity in the identified compounds among the three cultivars. These variations may be due to the
variations in geographic, climatic, soil fertility and cultivation practices. The intensity of flavour is governed
by hereditary factors and environmental conditions under which the onions grow. The ability to intake sulfate
and the efficiency of producing flavour precursors differs among the onion cultivars [1]. Increased flavour
intensity in onion was contributed by improved sulphate fertility, dry growing conditions and increased
growing temperatures.
492
CONCLUSION
In the present study, an effective and easy method for the analysis of volatile compounds in red onion using
GC-MS is reported here. This method is very sensitive and reproducible to identify the phytocompounds
with accuracy and precision. Since this medicinal plant, onion, possesses a lot of phytochemicals, it can be
a superior and easily accessible material for nutraceuticals.
ACKNOWLEDGEMENT
The authors are thankful for the Jawaharlal Nehru University, New Delhi, for providing GC-MS analytical
data.
REFERENCES
1. Lanzotti V, The analysis of onion and garlic, J. Chromatogr. A., 2006; 1112: 3-22.
2. Bonaccorsi P, Caristi C, Gargiulli C and Leuzzi U, Flavanol glucosides in Allium species: A comparative
study by means of HPLC-DAD-ESI-MS-MS. Food Chem., 2008; 107: 1668-1673.
3. Block E, The organosulfur chemistry of the genus Allium— Implications for the organic Organic-
Chemistry of Sulfur. Angew. Chem. Int. Ed. Engl., 1992; 31:1135-1178.
4. Higuchi O, Tateshita K and Nishimura H, Antioxidative activity of sulphur-containing compounds in
Allium species for human low-density lipoprotein (LDL) oxidation in vitro, J.Agric. Food Chem., 2003;
51:7208-7214.
5. Fattorusso E, Iorizzi M, Lanzotti V and Tanglialatela-Scafati O, Chemical composition of shallot (Allium
ascalonicum Hort.), J Agric. Food Chem., 2002; 50:5686-5690.
6. Xiao H and Parkin KL, Antioxidant functions of selected Alliumthiosulfinates and S-alk(en)yl-L-
cysteine sulfoxides, J. Agric. Food Chem., 2002; 50:2488-2493.
7. Hertog MGL, Hollman PCH and Venema DP, Optimization of quantitative HPLC determination of
potentially anticarcinogenic flavonoids in vegetables and fruits. J. Agric. Food Chem., 1992; 40: 1591-
1598.
8. Semmler FW, The essential oil of onion (Allium cepa L.), Arch.Pharmacol., 1982; 230:434-443.
9. Kohman EF, The chemical components of onion vapors responsible for wound-healing qualities,
Science, 1947; 106:625-627.
10. Bandyopadhyay C, Srirangarajan AN and Sreenivasan A, Studies on flavour components of Onion
(Allium cepa) I, Thin Layer Chromatographic investigation of onion oil. J. Chromatogr. A, 1970;
47:400-407.
11. Deruaz D, Soussan-Marchal F, Joseph I, Desage M, Bannier A and Brazier JL, Analytical strategy by
coupling headspace gas chromatography, atomic emission spectrometric detection and mass spectrometry
application to sulfur compounds from garlic, J. Chromatogr. A, 1994; 677: 345-354.
12. Jarvenpaa EP, Zhang Z, Huopalahti R and King JW, Determination of fresh onion (Allium cepa L.)
volatiles by solid phase microextraction combined with gas chromatography-mass spectrometry. Z.
Lebens. Unters. Forsch. A, 1998; 207: 39-43.
13. Boelens M, De Valois PJ, Wobben HJ and Van Der Gen A, Volatile flavour compounds from onions, J.
Agric. Food Chem., 1971; 19: 984-991.
493
14. Liu C, Zhang J, Zhou Z, Hua Z, Wan H, Xie Y, Wang Z and Deng L, Analysis of volatile compounds and
identification of characteristic Aroma components of Toona Sinensis (A.Juss.) Roem. Using GC-MS and
GC-O, Food Nutr. Sci., 2013; 4:305-314.
15. Keusgen M, Volatile compounds of the genus Allium L. (Onions). Chapter 9, ACS Symposium Series.
ACS, 2011; 1068: 183-214.
16. Marie Lokke M, Edelenbos M, Larsen E and Feilberg A, Investigation of volatiles emitted from freshly
cut onions (Allium cepa L.) by real-time proton-transfer reaction-mass spectrometry (PTR-MS), Sensors,
2012; 12: 16060-16076.
17. Ohta H and Osajima Y, Volatile components recovered from fresh onion (Allium cepa L.) with a
combination of ethyl ether extraction and new cold trap apparatus, J. Fac.Agr. Kyushu Univ., 1992; 37:
125-131.
18. Brodnitz MH, Pollock CL and Vallon PP, Flavor components of onion oil, J.Agric.Food Chem., 1969;
17:760-763.
19. Mazza G, LeMaguer M and Hadziyev D, Headspace sampling procedure for onion (Allium cepa L.)
aroma assessment. Can.Inst.Food Sci.Technol.J., 1980; 13: 87-96.
20. Lukes TM, Thin-Layer chromatography of cysteine derivatives of onion flavour compounds and the
lachrymatory factor, J.Food Sci., 1971; 36:662-664.
21. Whitaker JR, Development of flavour, odour, and pungency in onion and garlic, In “Advances in Food
Research,” by CO Chichester, Academic Pres
494
Antimicrobial Activity Study of Flavonoids and Salicylic
Acid Extracted from Tagetes Erecta Linn
R. Devika1 and Y. Justin Koilpillail2
1
Research Scholar, Dept. of Biotechnology, Sathyabama University, Chennai
2
Dept. of Botany, St. Joseph’s College, Trichirappalli
e-mail: vineethdevika@gmail.com1, Justinkoilpillai@yahoo.co.in2
Abstract:
Plants are a great source of bioactive compounds that are rich sources of therapeutic agents. Out of 2, 50,
000 – 5, 00,000 plant species on Earth, roughly 10% have been studied chemically and pharmacologically.
Tageteserecta Linn., an ornamental plant used traditionally in all occasions has been selected to identify for
its medicinal value efficiency. A constant screening of the phytochemical constituents has been carried out
and Tageteserecta Linn. flower sample proved to have high therapeutic agents than stem, leaves and root
parts. The floral extracts by column chromatography were obtained, and TLC separated the phytochemical
constituents and then the individual constituents were screened, eluted and purified by standard methods
and were stored in airtight containers for future investigations. In the present investigation, the purified
flavonoids and salicylic extracts were subjected to antimicrobial activity with Acinetobacterbaumannii,
Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia and Candida
albicans by Kirby-Bauer disk diffusion method. Staphylococcus aureus recorded the maximum zone of
inhibition followed by Klebsiella pneumonia, Candida albicans, Pseudomonas aeroginosa and Bacillus
subtilis. Acinetobacterbaumannii showed resistant against flavonoids extract of Tageteserecta Linn. The
salicylic acid extract also registered the maximum zone of inhibition with Staphylococcus aureus, and other
organisms did not show any significant zone of inhibition.
Keywords: Therapeutics, Phytochemicals, Bioactive, Antimicrobial, Inhibition.
INTRODUCTION
Various parts of medicinal plants are highly potent source of antimicrobial and antipathogenic agents [1]
that may inhibit the growth or kill the pathogens or cause harm to the host [2, 3]. In India, different parts
of several medicinal plants are used for their therapeutic value from Ancient times to cure many diseases
[4], and they prove to be the blueprints for the modern medicine [5]. The phytochemical analysis of any
medicinal plants may help to determine its antimicrobial and antipathogenic activities [6]. These bioactive
compounds may inhibit the growth or kill the pathogens or cause harm to the host [7, 8] or sometimes they
become resistant to some drugs [9, 10]. Extracts of various medicinal plants containing flavonoids have been
reported to possess antimicrobial activity [11] wherein the activity has proved by inhibition of respiration
and reproduction of microbes [12]. Flavonoid has proved to be highly antiviral [13] bactericidal against
Gram- positive bacteria [14, 15].

In the present investigation, Tageteserecta Linn. an ornamental annual plant was selected to have high
therapeutic value. The various parts of the plant were segregated into leaves, stem, flower and root. Constant
screening (qualitative and quantitative) of bioactive constituents has been carried out earlier, and the floral
extract showed very high therapeutic value than leaf, stem and root [16, 17] of Tageteserecta Linn. The floral
extract was subjected to column chromatography, and the bioactive phytochemical were separated by Thin
Layer Chromatography (TLC). The individual constituents were screened, eluted and purified by Standard
methods and stored in air container for further investigations.
Material Required
• Nutrient broth
• Pure Test Cultures
• Muller Hinter Agar
• Extract dissolved in DMSO
Microbial Culture
• Acinetobacter baumannii
• Bacillus subtilis
• Candida albicans
• Klebsiella pneumonia
• Pseudomonas aeroginosa
• Staphylococcus aureus
Methods
A pure culture of the above target organisms were obtained from MTCC, IMTECH, Chandigarh and were
cultured in nutrient broth for antibacterial study in the present study. Antibacterial activity study of all the
organisms were conducted with Muller and Hinter Agar (Hi-Media Pvt. Ltd. Mumbai) by Kirby- Bauer Disk
Diffusion method [18]. The test organisms were swabbed onto the duplicate petri plates, and five wells were
made with the help of sterile cork borer. Control and the two target bioactive compounds (Flavonoids and
Salicylic acids) extracted from Tagetes erecta Linn. of 150 µg and 250 µg were pipetted out aseptically and
incubated for 24 hours at optimum temperature. The zone of inhibition (in mm diameter) were read after 24
hours and considered as the antimicrobial activity of the test organisms.

The pure culture of target test organisms like Acinetobacter baumannii, Bacillus subtilis, Candida
albicans, Klebsiella pneumonia, Pseudomonas aeroginosa, Staphylococcus aureus were collected from
Chandigarh and were subjected to antimicrobial activity against the purified flavonoids and salicylic acid
(Bioactive compounds) isolated from Tagetes erecta Linn. Standard procedures of Kirby-Bauer Disk
Diffusion Method [19] was followed aseptically throughout the present investigation. From the results
obtained, Staphylococcus aureus registered the maximum zone of inhibition of 14 mm and 18 mm in both
the concentrations (150 and 250µg) of flavonoids (Fig.1), respectively (Table. 1). Klebsiella pneumonia
registered the second maximum zone of inhibition (Fig. 2) with 14 mm and 16 mm of inhibition zones in
150 and 250 µg concentrations, respectively followed by Candida albicans (Fig 3) with 14 mm in both
the wells loaded with flavonoids. The next target test organism was Pseudomonas aeroginosa (Fig.4) that
registered 12 mm (150 µg) and 14 mm (250 µg). Bacillus subtilis (Fig.5) showed the maximum zone in
150 µg (16 mm) and lower zone in 250 µg (8 mm). Acinetobacter baumannii (Fig.6) did not show any
zone that indicates the resistance over flavonoids.
496
Table: 1. Antimicrobial activity of flavonoids and salicylic acid extracted from Tagetes erecta Linn.
S.No. Organisms Zone of Inhibition (mm)

Salicylic acid Flavonoids
150µg 250µg 150µg 250µg
1 Acinetobacter baumannii R 10 R R
2 Pseudomonas aeroginosa 8 8 12 14
3 Staphylococcus aureus 8 12 14 18
4 Bacillus subtilis 8 10 16 8
5 Candida albicans 8 10 14 14
6 Klebsiella pneumonia 8 10 14 16
Figure 1: Zone of Inhibition of S.aureus Figure 2: Zone of inhibition of K.pneumonia
Figure 3: Zone of Inhibition of C.albicans Figure 4: Zone of Inhibition of P. aeroginosa
Figure 5: Zone of inhibition of B. subtilis Figure 6: Zone of inhibition of A. baumannii
1 and 2: Zone of inhibition by Flavonoids

3 and 4: Zone of inhibition by Salicylic acid
C: Control
Salicylic acid extract from Tagetes erecta Linn. did not show many zones of inhibition when compared
to flavonoids extract. The highest zone of inhibition was recorded by Staphylococcus aureus (Fig.1) in 150
µg (8 mm) and 250 µg (12 mm), respectively followed by three other test organisms like Bacillus subtilis
497
(Fig.5), Candida albicans (Fig.3) and Klebsiella pneumonia (Fig.2) with 8 mm (150 µg) and 10 mm (250 µg),
respectively. Pseudomonas aeroginosa (Fig.4) recorded 8mm in both the concentrations, but Acinetobacter
baumannii (Fig. 6) showed resistant against 150 µg and registered 10 mm of zone in 250 µg of salicylic acid.
CONCLUSION
From the above investigation, the floral extract of Tageteserecta showed a significant response of flavonoids
and salicylic acid against the target organisms and proved to be more effective drug component in the
treatment of hypertrophic scars among the burnt patients. Further clinical and cell line study will a pave a
way for production of a new effective drug in the treatment of burn patients.
REFERENCES
1. Rathanavel, Sumitha J and Thillai Arasu P, Antimicrobial activity in selected Indian medicinal plant
extracts using disk diffusion method, Int J Pharm Bio Sci. 2014; 5 (!): 656-661
2. Sharkin AJ, Reich E, Frankin RM and Tatum EL, Effect of mitomycin C on Mammalian cells in culture,
Biochimica Biophysica Acta, 1962; 55(3): 277-289
3. Kalghatgi S, Spina CS, Costello JC, Liesa M, Ruben J, Ramirez M, Salmovie S, Molina A, Shirihai
OS and Collins JJ, Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in
mammalian cells, Sci Transl Med. 2013; 3(5): 192-194
4. Renisheya JeyJeba Malar T, Johnson M, Mary Uthith M and Arthy A, Antibacterial activity against
human pathogens, Asian Pacific J of Tropical Biomedicine, 2011; 576-578
5. Chitravadivu C, Manian S and Kalaichelvi K, Antimicrobial studies on selected medicinal plants Erode
region, Tamilnadu, India, Middle East J Sci Res. 2009; 4(3): 147-152
6. Banerjee RP, Banerjee S Sarkar P, Pradhan PK, Phytochemical analysis and antimicrobial activity of
natural resin (Laldhuna) from Shorearobusta (Sal), Int J of Pharm Sci and Hlth Care. 2014; 3(4): 52-60
7. Suginaka H, Ichikawa A and Kotani S, Penicillin resistant mechanisms in Pseudomonasaeruginosa:
Binding on Penicillin to Pseudomonas aeruginosa KM 338, Antimicrob Agents Chemotherapy, 1975;
7(5): 629-635
8. Peter C Appelbaum, Microbiology of Antibiotic resistance in Staphylococcus aureus, Clin Infect disease,
2007; 45:S165-S170
9. Shakya P, Barrett P, Diwan V, Marothi Y, Shah H, Chhari N, Tamhankar AJ, Pathak A and Cecilia S
Lundborg, Antibiotic resistance among E coli isolates from stool Samples of children aged 13- 14 years
from Ujjain, India, BMC Infectious Diseases, 2013; 13: 471-477
10. Khana P, Sharma OP, Sehgal M, Bhargawa C, Jain M, Goswami A, Singhvi S, Gupta V, Agarwal R,
Sharma P and Jain SC, Antimicrobials from tissue cultures of some plant species, 1980; 42: 113-114
11. Jasmine Mary S, John Merina A, 2012, Antibacterial activity of Bauhinia tomentosa Linn., In J of Appl
Res., 2012; 1(11): 1-2
12. Puszati R, Beladi I, Bakai M, Musci I and Eukan E, Study on the effect of flavonoids and related
substances, The effect of quercetin in different viruses, Ata Micro AcadSci Hung. 1966; 13(2): 133-135
13. Paul BD, Rao GS and Kapadia GJ, Isolation of myocardial, myricetin, taraxerol and taraxerone from
Myricacerifera L. root bark. J Pharm Sci. 1974; 63(6): 958-959
14. Aggag ME and Yousef RT, Study of antimicrobial activity of chamomile oil. Plant Med. 1992; 22(2):
140-144.
498
15. Eugene G and Weinberg, Antifungal agents, Principles of Medicinal chemistry Ed., William O. Foge,
Lea and Febiger. 1981; 809.
16. Devika R and Justin Koilpillai’ Phytochemical screening studies of bioactive compounds of Tageteserecta,
Int J Pharma Bio Sci. 2012; 3(4): (B), 596-602
17. Devika R and Justin Koilpillai, Screening and evaluation of bioactive components of Tageteserecta L.
by GC-MS analysis, 2014; 7(2): 58-60
18. Bauer AW, Kirby WM, Sherris JC and Turck M, Antibiotic susceptibility testing by a standard single
disk method, Am J Clin Pathol; 45: 493-49
499
Therapeutic Potential of Pleurotus Ostreatus: An Edible
Mushroom in Human Cancer Cell Line (MCF-7) and in
Rat Mammary Carcinoma
K. Deepalakshmi and S. Mirunalini*
*Correponding Author
Department of Biochemistry and Biotechnology, Annamali University, Chidambaram
e-mail: mirunasankar@gmail.com, Mob: 9442424438
Abstract:
Objective of this study was to explore the chemotherapeutic efficacy of Pleurotus ostreatus ethanolic extract
(POEet) against mammary cancer using in vitro and in vivo models. Preliminary phytochemical analysis of
mushroom was used in folklore has yielded a number of compounds with various pharmacological activities.
Thus in our study we first aimed to find out the active constituent (Ergosterol) in POEet by HPTLC fingerprint
and quantified in POEet with reasonable accuracy in a shorter time. In addition, POEet was investigated
in human mammary cancer cell line (MCF-7). As assessed from 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay, greater cytotoxicity was observed with an IC50 value of 1024.02
µg/ml. Further we extended our work in an animal model also. In order to confirm the chemotherapeutic
potential of POEet against 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary cancer on Sprague
dawley rats. After the end of the experimental period we estimate the lipid profile levels, of control and
experimental animals. The correlation of lipids and lipoproteins has been associated with the risk of breast
cancer. Thus the elevated levels of total cholesterol, phospholipids, triglycerides and free fatty acids in
plasma, liver and mammary tissues of DMBA induced cancer suffering animals. Moreover, the levels of
very low density lipoprotein (VLDL) and low-density lipoprotein (LDL) were increased and High density
lipoprotein (HDL) levels were decreased in cancer suffering animals. Whereas, oral administration of (600
mg/kg bwt) POEet were significantly reverted back to near normal level when compared to cancer animals
thereby reduced the cancerous risk. Taken together our finding revealed the chemotherapeutic potential of
POEet due to the presence of the active compound ergosterol which may responsible for the major impact
on human mammary cancer.
INTRODUCTION
Breast cancer is a much commonly diagnosed cancer that is a leading cause of death for women [1]. The
etiology of breast cancer is multifactorial. Significantly breast cancer risk factor include estrogenic food
additives, consumption of alcoholic beverages and the presence of estrogenic food contaminants, high fat
and reactive oxygen species (ROS) [2]. Lipids and hormones are another most important factors involved
in breast cancer development. Changes in lipid metabolism can affect cellular processes, including cell
growth, proliferation, differentiation and motility [3]. In recent years, natural dietary agents especially fruits,
vegetables, sprouts and mushrooms have drawn a great deal of attention both from researchers and from the
public because of their potential antioxidant and anticancer activities [4].
Pleurotus ostreatus (Jacq.ex.fr) P.kumm. is a cosmopolitan group of mushroom having highly nutrition
and also contains various bioactive compounds including terpenoids, steroids, phenols, alkaloids, lectins
and nucleotides, which have been isolated and identified from the fruit body, mycelium and culture broth of
mushrooms are shown to have promising biological effects [5]. Additionally, Gunde-Cimerman & Cimerman
postulated that the HMG-CoA-reductase inhibitor mevinolin (lovastatin) was detected in oyster mushroom
Pleurotus ostreatus (P. ostreatus), which could lead to a lipid-lowering effect [6].
The current investigation has been carried out to investigate the chemotherapeutic activity of POEet
in human mammary cancer cell line MCF-7 and the levels of lipid profile in DMBA induced rat mammary
carcinogenesis.

Chemicals
7,12 – dimethylbenz(a)anthracene (DMBA), 3-(4,5-dimethyl-2-thiozolyl)-2,5-diphenyl-tetrazolium bromide
(MTT), Tamoxifen were purchased from Sigma Chemical Pvt. Ltd., Bangalore, India. All other chemical
reagents were of analytical grade.
Material
Pleurotus ostreatus mushrooms were collected in and around areas of Udhagamandalam, Nilagiri district,
Tamil Nadu. The mushroom was taxonomically identified and voucher specimen (No: 233) was deposited in
the herbarium of Botany, Department of Botany, Annamalai University.
Preparation of mushroom Ethanolic extract

The fresh fruiting bodies of P. ostreatus were dried in shade conditions and the dried materials were pulverized
in a blender to get a coarse powder. For P. ostreatus fruiting bodies ethanolic extraction, five grams of the
powder were extracted with 100 mL of 95% ethanol using a Soxhlet apparatus. The solvent was evaporated
on a rotary evaporator (Buchi Rotarvapour, Switzerland) under reduced pressure and controlled temperature
(40-50°C) [7]. A dark semisolid material (6 % yield) thus obtained was stored at 4°C until use. A known
amount of the residual extracts was suspended in distilled water and was orally administrated to the animals
by gastric intubation.
HPTLC fingerprint analysis

Chromatography was performed on a 10×10cm preactivated HPTLC silica gel 60 F254 plates (Merk,
Darmstadt, Germany). Aliquots of the extract were separately applied (sample and standard) to the plate as
6mm wide band with an automatic TLC applicator Linomat-V with N2 flow (CA-MAG, Switzerland), 8mm
from the bottom. Densitometric scanning was performed on CAMAG scanner III at 490 nm. The plates
were prewashed by methanol and activated at 60ºC for 5min prior to chromatography. The slit dimension
was kept at 5×0.45 and 40mms-1 scanning speed was employed. The mobile phase consisted of ethylacetate:
chloroform (8:2) of mobile phase was used per chromatography. Liner ascending development was carried
out in 10×10cm twin glass chamber saturated with the mobile phase.
In vitro study
Cell lines and Culture conditions
Human mammary cancer cell lines (MCF-7) and normal vero cell lines were purchased from National
Centre for Cell Sciences (NCCS), Pune, India. The cells were maintained in Dulbecoo’s modified eagle’s
medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum (FCS), 3% L-Glutamine, 100
IU penicillin and 100g/mL of streptomycin. The cells were grown in 5% CO2 atmosphere at 37˚C and the
cells should have 80-90% confluence before they are subcultured for the experiments.
501
Cytotoxicity Assay
The Cytotoxicity of samples on MCF-7 & VERO was determined by the MTT assay (Mosman 1983) [8].
The effect of the samples on the proliferation of MCF-7 & VERO was expressed as the % cell viability, using
the following formula: % cell viability = A570 of treated cells / A570 of control cells × 100
In Vivo studies
Animals and diet
The experiment was carried out in six week old adult female Sprague dawley rats, weighing approximately
130-150g were obtained from National Institute of Nutrition, Hyderabad. All experimental procedures were
approved by the Institutional Animal Ethics Committee of Rajah Muthiah Medical College and Hospital
(Reg No. 160/1999/CPCSEA, Proposal number: 947), Annamalainagar. Sprague dawley rats were housed
in a room maintained at a controlled temperature (27 ± 2°C) and 12 h dark/light cycles. Food and water
provided ad libitum to all the animals.
Experimental design
Animals were assorted into six groups of six animals each according to the following experimental regimen.
Animals in Groups 1 were induced with DMBA 25 mg in 1mL of vehicle (0.5 mL of sunflower oil in 0.5 mL
of saline) [9]. In Group 2 and 3 along with DMBA will receive (600 mg/kg bwt) POEet extract as pre-initiation
and post-initiation. Group 4 will be treated with tamoxifen (TAM) (10 mg/kg bwt) along with DMBA. Group 5
will receive (600 mg/kg bwt) POEet alone and group 6 will be treated as control. The experiment will be first
terminated at the end of 15 weeks (pre-initiation phase) and subsequently terminated at the end of 27 weeks
(post-initiation phase). After the termination, all the rats were kept overnight fast and anesthetized using ketamine
chloride (24 mg/kg bwt) by intramuscular injection and sacrificed by cervical decapitation between 8.00 am to
10.00 am. Blood was collected in clean dry test tubes with few drops of heparin and plasma obtained was used for
various biochemical estimations. Tissues such as liver and mammary were homogenized in an appropriate buffer
and used for the estimation of various biochemical parameters.
Biochemical analyses
Lipid extraction was done by the method of Folch et al. (1957) [10]. Plasma and tissue total cholesterol,
triglycerides, free fatty acids and phospholipids were estimated by the methods of Allain et al. (1974), Foster
and Dunn (1973), Falholt et al. (1973), Zilversmit and Davis (1950), using reagent kit respectively [11-14].
Plasma high-density lipoprotein-C (HDL-C) was estimated by the method of Wilson and Spiger (1973) using
reagent kit [15]. Low-density lipoprotein-C (LDL-C) and very low density lipoprotein-C (VLDL-C) were
calculated by Friedwald’s (1972) formula [16].
Statistical analysis was performed using SPSS software package, version 16.0. The values were analysed by
One Way Analysis Of Variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). All these
results were expressed as mean ± SD for six rats in each group p-values<0.05 were considered as significant.
RESULTS
HPTLC fingerprint profile
HPTLC fingerprint patterns have been evolved for POEet. Ergosterol standard was quantitated accurately
using silica gel F254 HPTLC pre-coated plates with mobile phase ethyl acetate: chloroform (8:2 v/v), the
Rf value was about 0.88. The chromatographs of ergosterol and chloroform fraction of POEet are shown in
Fig.1. The standard ergosterol Rf value 0.90 were matched with the Rf value of POEet at 274 nm.
502
Cytotoxicity
The therapeutic activity of POEet (0-1040 µg/ml) were initially investigated for the cytotoxicity in normal
Vero cell line and human mammary cancer cell line (MCF-7) based on MTT assay (Fig. 2A, B). POEet
exposed to normal Vero cells did not show any significant cytotoxicity. In MCF-7 cells after 48 hrs of
treatment revealed that POEet at the higher concentration (1040µg/ml) significantly increased cytotoxicity
effect on MCF-7 cells when compared to low concentrations. Moreover, the exact IC50 value of POEet was
found to be 1020.02µg/ml could greater inhibit the cell growth. Whereas, untreated cancer cells did not
inhibit cell growth.
Effect of POEet on lipid profile on control and experimental animals

The levels of lipids (Total cholesterol (TC), phospholipids (PL), triglycerides (TG) and free fatty acids
(FFA)) in plasma, liver and mammary of control and experimental animals were depicted in Fig.3 and
Table 1, respectively. The levels of TC, PL, TG and FFA in plasma, liver and mammary were elevated
significantly in cancer suffering animals when compared to control animals. The plasma of cancer bearing
animals show a profound increment in the levels of very low-density lipoprotein cholesterol (VLDL) and
low-density lipoprotein cholesterol (LDL) and decreased high-density lipoprotein (HDL) (Table 2).Upon
oral administration of POEet (Pre and post-treatment) were significantly reverted back to the near normal
when compared to untreated cancer animals. Conversely, the effect of POEet at pre-treatment was found to
be more significant next to tamoxifen than post-treatment with POEet.
DISCUSSION
Phytoconstituents obtained from a natural source have been gaining importance in day to day because of the
vast chemical diversity. So, there is a need to ensure the quality, safety and efficacy of natural drugs. Planar
chromatography in the form of TLC/HPTLC has proved to be a valuable tool for the analysis of different
types of propolis and has been widely used for both qualitative and quantitative studies to determine the
chemical composition of the product [17]. Moreover, HPTLC fingerprinting is proved to be a linear, precise,
accurate method for herbal identification and can be used further in authentication and standardization of
the medicinally important plant [18]. Thus, the unique feature of the picture like image of HPTLC coupled
with digital scanning profile is gradually attractive to herbal analysis to construct the herbal chromatographic
fingerprint. This HPTLC could provide adequate information and parameters for comprehensive identification
and differentiation of two closely related herbal medicines [19]. The present study was intended, to identify
the active compound which is responsible for therapeutic activity of POEet in MCF-7 and rat mammary
carcinoma. By investigating the HPTLC analysis of POEet showed the presence of ergosterol. In this view,
Chiochhio et al. (2011) and Ana villares et al. (2014), have also reported that mushroom contain ergosterol
which is responsible for maximum antioxidant capacity [20, 21].
Accurate assessment of the anti-growth effects of chemotherapeutics is immensely importance in cancer
research about drug discovery and toxicology safety. In the present study, MTT cytotoxicity assay was
performed via the indirect measurement of the tumour cell number using some viability-related features of
the cells. On the basis of our result no cytotoxicity has been observed towards normal Vero cells. Whereas,
higher concentration of (1020.02µg/ml) of POEet treatment significantly showed increased cytotoxicity in
MCF-7 cells. Recent studies by Jedinak et al. (2008) demonstrated that the concentration of 1000 µg/ml
P.ostreatus methanolic extract inhibited the growth of MCF-7 cells and HT-29 cells with the proliferation
index percentage of 70% and 17% respectively [22]. Our results also inline with the above finding which
suggest that P. ostreatus suppresses proliferation of breast cancer cells without significant effect on
proliferation of normal human mammary cells.
503
Further we extended our work to investigate the chemotherapeutic potential of POEet on DMBA induced
mammary carcinogenesis in Sprague dawley rats. Previously numerous studies have indicated the correlation
of lipids and lipoproteins with the risk of breast cancer. In addition, Stepsenwol et al. (1966) also reported
that lipid might primarily affect the gonads and subsequently higher estrodiol secretion could influence the
development of malignancies in the mammary gland and lymphoid system [23]. Recent studies reported that
the alteration of lipid fluidity of the tumor cell membrane was due to the elevation of lipids (TC, PL, FFA and
TG), which can enhance the progression, and proliferation of tumor cell [24]. In our study DMBA induced
cancer-bearing rats also showed abnormalities in lipid metabolism as evidenced from increased TG, TC,
FFA, PL, VLDL-C and LDL-C levels and decreased HDL-C levels. Whereas oral administration of POEet
recouped back the deranged lipid metabolism found in cancer condition to normal. There is strong evidence
from the earlier report which proved that continuous treatment with oyster mushrooms improved the lipid
profile in rats [25]. Recently Schneider et al. (2011) also demonstrated that the presence of linoleic acid,
ergosterol and ergosta-derivatives of the oyster mushroom seem to have an additive effect in lowering serum
lipids [26]. Thus our results collaborate well with the above findings.
CONCLUSION
Our results clearly bring out that the ethanolic extract of P. ostreatus possesses anticancer activities,
attributed to its ergosterol content. Moreover, our study also suggests that P. ostreatus is a good source
of proteins and fibre and its inclusion in the diet may provide a unique effect against mammary cancer.
ACKNOWLEDGEMENT
The authors would like to acknowledge for the Finacial Support from University Grants Commission (UGC)
- Major Research project (F. NO: G7/17342/2012 (SR)), New Delhi, India to carry out the work successfully.
REFERENCE
1. Chen K, et al. Incidence, mortality and survival rates of female breast cancer in Tianjin China, Chin J
Oncol. 2002; 24(6):573-575.
2. Wolf MS and Weston A, Breast cancer risk and environment, Environ Health Perspect, 1997; 105(4):891–
896.
3. Santos CR and Schulze A, Lipid metabolism in cancer, FEBS J. 2012; 279(15):2610–23.
4. Amin AR, Kucuk O, Khuri FR and Shin DM, Perspectives for cancer prevention with natural
compounds, J Clin Oncol. 2009; 27(16):2712-25.
5. Deepalakshmi K and Mirunalini S, Pleurotus ostreatus: an oyster mushroom with nutritional and
medicinal properties, J Biochem Tech., 2014; 5(2):718-726.
6. Gunde-Cimerman N and Cimerman A, Pleurotus fruiting bodies contain the inhibitor of 3-hydroxy-3-
methylglutarylcoenzyme A reductase-lovastatin, Exp Mycol., 1995; 19(1):1–6.
7. Jayakumar T, Thomas PA and Geraldine P, In-vitro antioxidant activities of an ethanolic extract of the
oyster mushroom Pleurotus ostreatus, Innov Food Sci Emerg Technol, 2009; 10(2):228-234.
8. Mosmann T, Rapid colorimetric assay for cellular growth and survival: application to proliferation and
cytotoxicity assays, J Immunol Methods., 1983; 65(1-2): 55–63.
9. Samy RP, Gopalakrishnakone P and Ignacimuthu S, Anti- tumor promoting potential of luteolin against
7, 12-dimethybenz (a) anthracene-induced mammary tumours in rats, ChemBiol Interact, 2006; 164
(1-2):1-14.
504
10. Folch J, Lees M and Stanley GHS, A simple method for the isolation and purification of total lipids from
animal tissues, J Biol Chem., 1951; 226(1):497–509.
11. Allain EC, Poon LS, Chan CS, Richmond W and Fu FC, Enzymatic determination of total serum
cholesterol, Clin Chem., 1974; 20(4):470–5.
12. Foster LB and Dunn RT, Stable reagents for determination of serum triglycerides by a colorimetric
Hantazsch condensation method, Clin Chim Acta., 1973; 19(3):338–40.
13. Falholt K, Falholt W and Lund B, An easy colorimetric method for routine determination of free fatty
acids in plasma, Clin Chim Acta., 1973; 46(2):105–11.
14. Zilversmit DB and Davis AK, Micro-determination of plasma phospholipids bytrichloroacetic acid
precipitation, J Lab Clin Med., 1950; 35(1):155–9.
15. Wilson DE and Spiger MJ, A dual precipitation method for quantitatingplasma lipoprotein measurement
without ultracentrifugation, J Lab Clin Med., 1973; 82(3):473–82.
16. Friedwald WT, Levy RJ and Fredricken DS, Estimation of HDL-C in the plasma with-out the use of the
preparative ultracentrifuge, Clin Chem., 1972; 18(6):449.
17. Tang TX, Guo WY, Xu Y, Zhang SM, Xu XJ, Wang DM, Zhao ZM, Zhu LP and Yang DP, Thin-layer
chromatographic identification of Chinese propolis using chemometric fingerprinting, Phytochem Anal.,
2014; 25(3); 266–272.
18. Johnson M, Mariswamy Y and Gnaraj WE, Chromatographic fingerprint analysis of steroids in Aerva
lanata L by HPTLC technique, Asian Pac J Trop Biomed, 2011; 1(6): 428-433.
19. Kamboj A and Saluja AK, Development of validated HPTLC method for quantification of stigmasterol
from leaf and stem of Bryophyllum pinnatum, Arab J Chem., 2013; http://dx.doi.org/10.1016/j.
arabjc.2013.10.006
20. Chiochhio VM and Makkovic L, Determination of ergosterol in cellular fungi by HPLC a modified
technique, J Argent Chem Soc., 2011; 98:10-15.
21. Villares A, Mateo-vivaracho L, Garcia-Lafuente A and Gwillamon E, Stroage temperature and UV-
irradiation influence on the ergosterol content in edible mushrooms, Food Chem., 2014; 147: 252-256.
22. Jedinak A and Sliva D, Pleurotus ostreatus inhibits proliferation of human breast and colon cancer cells
through p53-dependent as well as p53-independent pathway, Int J Oncol., 2008; 33(6):1307–1313.
23. Stepsenwol J, Carcinogenic effect of cholesterol in mice, Proc Soc Exp Biol Med., 1966; 121(1):168–
171.
24. Damen J, Van Blitterswijk W, De Widt J and Hengeveld T, Accumulation of HDL like lipoproteins in
the plasma low-density fractions of tumor-bearing mice, Biochim Biophys Acta, Lipids Lipid Metab.,
1985; 833(3):495–8.
25. Hossain S, Hashimoto M, Choudhury EK, Alam N, Hussain S, Hasan M et al., Dietary mushroom
(Pleurotus ostreatus) ameliorates atherogenic lipid in hypercholesterolaemic rats, Clin Exp Pharmacol
Physiol., 2003; 30(7), 470–475.
26. Schneidera I, Kressela G, Meyerb A, Kringsb U, Bergerb RG and Hahna A, Lipid lowering effects of
oyster mushroom (Pleurotus ostreatus) in humans, J Functional Foods, 2011; 3(1):17-24.
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Figure 1: Three-dimensional HPTLC chromatogram of POEet at 274 nm, compared with Standard Ergosterol, Pink
colour: POEet; Green colour: Ergosterol
Figure 2: Cytotoxicity of POEet in A) Vero cells B) MCF-7 cells after 48 hrs. Data are expressed as mean±SD (n=3)
P<0.05 compared to control group
Table 1: Activity of POEet on tissues lipid profile of control and experimental animals
Groups Total Phospholipids (mg/g) Triglycerides (mg/g) Free fatty acids (mg/g)
cholesterol
(mg/g)
Liver Mammary Liver Mammary Liver Mammary Liver Mammary
DMBA 8.23±0.70 a
9.32±0.90a
48.32±3.94a
25.73±2.07a
9.83±0.83 a
9.78±0.94a
15.48±1.31 a
20.03±1.70a
DMBA+POE et 6.43±0.53b 7.62±0.72b 33.48±3.20b 20.63±1.57c 6.32±0.59b 7.85±0.77b 9.68±0.87b 16.23±1.51b
(pre-initiation)
DMBA+POE et 7.18±0.64c 8.43±0.79c 39.48±3.32c 22.84±1.94b 7.68±0.61c 8.85±0.84c 12.68±0.96c 18.23±1.52c
(post-initiation)
DMBA+TAM 5.43±0.43d 6.10±0.57d 28.86±2.74d 16.56±1.45d 5.55±0.53d 6.98±0.59d 8.43±0.75d 14.32±1.25d
(post-initiation) 4.86±0.41e 4.56±0.41e 24.68±1.97e 15.86±1.39d 4.80±0.45e 4.90±0.45e 7.18±0.63e 13.72±1.19d
POE et alone
Control 4.68±0.35e 4.08±0.35e 23.54±2.07e 14.98±1.28d 4.73±0.41e 4.21±0.36e 7.02±0.64e 13.03±1.16d
Values are expressed as mean ± SD for six animals in each group; values not sharing a common
superscript differ significantly at P < 0.05, (DMRT)
506
Figure 3: Effect of POEet on lipids in plasma of control and experimental animals. Values are expressed as mean ±
SD for six animals in each group. Values not sharing a common superscript differ significantly at P< 0.05, Duncan’s
multiple range test (DMRT)
Table 2: Influence of POEet on plasma lipoprotein level of control and experimental animals.
Groups HDL (mg/dL) VLDL (mg/dL) LDL (mg/dL)

DMBA 18.65±1.61a 32.84±2.83a 105.81±9.57a
DMBA+POEet 28.10±2.71b 19.50±1.51b 61.09±5.66b
(pre-initiation)
DMBA+POEet 23.68±2.12c 24.08±2.19c 77.69±7.27c
(Post-initiation)
DMBA+TAM (post- 32.68±3.14d 17.24±1.41d 46.38±3.07d
initiation)
POEet alone 36.58±3.35e 14.83±1.09e 27.73±2.02e
Control 38.72±3.53e 14.37±1.01e 28.77±2.37e
Values are expressed as mean ± SD for six animal in each group; values not sharing a common superscript
differ significantly at P< 0.05, (DMRT)
507
A Single Chemical Entity to Balance Anti-Amyloid and
Anti-Cholinesterase Capacity: In Silico Drug Design
Pavadai Parasuraman1, Ramalingam Suresh2 and Manathusamy Gopalakrishnan3
1, 2
Department of Pharmacy, 3Department of Chemistry, Annamalai University, Annamalai Nagar,
Chidambaram, Tamilnadu-608002, India
Abstract:
Alzheimer’s disease is currently the deliberations to be a syndrome, multiple complex factors; it is
unlikely to occur from a single causal factor; instead, there are a number of biological abnormalities
allies believe that puts them into its pathogenesis. This may explain why the drugs currently available,
developed according to the archetypal paradigm for drug discovery “one-molecule-one-target,” have
proven palliative. In the light of this, a combination of medicines that may work with different levels of
the neurotoxic cascade provides new horizons in the direction to the treatment of Alzheimer’s disease and
other neurological diseases. In such, a promising new strategy in the development of a chemical entity and
one can modify multiple targets simultaneously. This has led to a new paradigm in medicinal chemistry,
design and strategy “multi-target–directed ligand,” which has already been successfully exploited both
academic and industrial. 4-Piperidinone fused with acetylcholine mimic moiety was designed and docked
against AChE and BACE enzyme Using Schrodinger software. As a case study, we report here on Ethyl
2-(2,6-bis(4-bromophenyl)-3-butyl-4-oxopiperidine-1-carboxamido)acetate (KBr 5), a new molecule
developed following this strategy. The In-Silico profile of Ethyl 2-(2,6-bis(4-Bromophenyl)-3-butyl-
4-oxopiperidine-1-carboxamido) acetate demonstrates the suitability of the new strategy for obtaining
innovative drug candidates for the treatment of neurodegenerative diseases as it inhibits both AChE and
BACE enzyme through In Silico studies.
Keywords: In Silico, Alzheimer’s disease, 4-Piperidone, Multi-targeted Drug Design.
INTRODUCTION
Alzheimer’s disease (AD), the most widespread form of dementia, is a complex neurodegenerative disorder
[1, 2]. According to Alzheimer’s disease International, about 44.4 million peoples with dementia were
estimated worldwide in 2013. This statistic will increase to a level of 75.6 million in 2030 and 135.5 million
in 2050. By now, 62% of the populace with dementia survived in developing countries and expected to reach
71% by 2050. The fastest augmentation in the aged populace is taking place in China, India and their south
Asian and western Pacific neighbors.
The molecular etiology of Alzheimer’s is not fully understood, there exist several hypotheses surrounding
its causes. The oldest is the cholinergic hypothesis, which centers on the underproduction of acetylcholine
as the causative agent. It has also been postulated that it may cause what are known as plaques and tangles.
These are formations that are consistently found in the brains of Alzheimer’s patients upon autopsy. These
tangles, specifically intraneuronal fibrillary tangles, are the results of aggregations of misfolded tau protein
inside nerve cell bodies, and under the Tau hypothesis, this would be the earliest influence on the pathology of
Alzheimer’s. The plaques, however, consist of misfolded amyloidal beta peptide fragments, which aggregate
between neurons, and under the amyloid hypotheses, it is the deposition of these peptides which is the initial
influence. Although this is the dominant hypothesis, of course, there are others, as well as variations and
combinations of these.
Five FDA approved Alzheimer’s treatments fall into two categories, based on their mechanisms of
action. Considering that the cholinesterase hypothesis is the oldest etiological hypothesis; it makes sense
that the oldest of these and the majority are targeted to this characteristic of Alzheimer’s. Donepezil, Tacrine,
Galantamine and Rivastigamine acts as cholinesterase inhibitors, while the fifth, memantine, is the only
n-methyl-d-aspartate receptor antagonist.
Experimental evidence points to the involvement of several targets and pathways in the AD pathogenesis,
but all drugs developed to date are monofunctional, hitting only a single target among the many involved.
Therefore, these drugs are inherently insufficient for the treatment of complex diseases like AD, which have
multiple pathogenic factors. It is critical to recognize; however that combining two or more drugs always
raises the potential for greater side effects [3]. Another treatment option to emerge, based on the model
that a single molecule can be rationally designed to possess many of the characteristics associated with
biological properties. Clearly, treatment with a single drug, such multimodal have inherent advantages over
combined therapy [4]. Such therapy would avoid the challenge of managing multiple entities in a single
drug that could have distinctive bioavailability, pharmacokinetics, and metabolism. Furthermore, in terms
of pharmacokinetic and toxicological optimization, the clinical development of a drug able to hit multiple
targets should not, in principle, be different from the development of any other single lead molecule. This
therapy thus offers a far simpler approach than combination therapy. In addition, to reduce the potential
risk of drug interactions and therapeutic regimen greatly simplifying edition, with the potential to improve
patient compliance, which is a critical issue in the care of AD [5].
The fact that the complexity and make multiple etiologies of yield strategy AD difficult therapeutic
effect desirable objective, which makes choosing a Multi-Target-Directed Ligand (MTDL) is likely to be a
more effective strategy [6]. MTDL goal is to enhance remedial efficacy, is rationally designed to hit several
targets for a particular disease to improve pharmacological profiles [7-9].
Bioactive heterocyclic ring systems having the piperidine-4-one nucleus have aroused great interest in
the past and recent years due to their wide variety of biological properties such as antiviral, antitumour [10,
11], central nervous system [12], local anesthetic [13], anticancer [14], and antimicrobial activity [15], and
their derivative piperidine is also biologically important and act as neurokinin receptor antagonists [16],
analgesic and anti-hypertensive agents [17].
Based on the above facts, we focused on designing Multi-Target-Directed Ligands by incorporating
acetylcholine mimic to substituted piperidine-4-one to inhibit both Acetyl Cholinesterase and β-Secretase
and to assess its activity through in Silico molecular docking studies.

Protein structure preparation
The X-ray crystal structures of Acetyl Cholinesterase (PDB: 3LII) and β-Secretase BACE (PDB: 3U6A)
retrieved from the Research Collaboratory for Structural Bio informatics (RCSB) Protein Data Bank was
used for this study. The crystallization water molecules were removed from the compound, and the protein has
been optimized for connection to the utility of protein preparation and refinement provided by Schrödinger
LLC. Optimized protein was assigned partial atomic charges according to the force field OPLS-AA.
Ligand structure preparation

64 structures of piperidine-4-One analogues (Figure 1) were constructed using Maestro 9.3 (Schrodinger,
LLC) using the optimized simulation potential Atom-All liquids (OPLS-AA) force fields the steepest descent
509
followed by reduced Newton conjugate gradient protocol. Using the OPLS-AA force field partial atomic
charges were computed.
Molecular Modeling Studies

Molecular modeling studies were performed using GLIDE (Grid-based Ligand Docking with Energetics)
V5.5 software developed by Schrödinger runs on Red Hat Enterprise Linux 5 (RHEL5) workstation. Maestro
V9.3 has been used as a graphical user interface (GUI) workspace for all the steps involved in the preparation
of the ligand and the protein preparation and lead to additional coupling Extra precession docking (XP-
mode). Every single docking calculation have been performed using the ‘‘Extra Precision’’ (XP) mode of
GLIDE program. The finest docked structure was preferred using a GLIDE score function. Glide Score
(G-score) takes into account a number of criteria such as hydrogen bonds (H-bond), hydrophobic contacts
(Lipo), van-der-Waals (vdW), columbic (Coul), polar interactions in the binding site (Site), metal binding
term (Metal) and penalty for buried polar group (BuryP) and freezing rotatable bonds (RotB).
G-score = H bond + Lipo + Metal + Site + 0.130 Coul + 0.065 vdW – BuryP – RotB.
An additional scoring function utilized by GLIDE is E-model, which itself consequential from a
combination of the G score, Coulombic, van der Waals and the strain energy of the ligand [18].

All the designed ligands and standards were evaluated for docking studies against Acetyl Cholinesterase and
β-Secretase using GLIDE software. The docking results of the ligands with the top score when compared
with standard were selected and given in the Table I and Table II. The interaction energy comprises van
der Waals energy, electrostatic energy, as well as intermolecular hydrogen bonding, were calculated for
every minimized complex. Docking score using GLIDE varied between -0.04 Kcal/mol and -6.9 Kcal/mol
against Acetyl Cholinesterase and -0.7 Kcal/mol and -6.7 Kcal/mol for β-Secretase. The GLIDE Score for
the standards Donepezil, Galantamine, Rivastigmine and Tacrine docked with Acetyl Cholinesterase was
-4.9 Kcal/mol, -5.1 Kcal/mol, -2.6 Kcal/mol & -4.3 Kcal/mol relatively and with β-Secretase -5.8 Kcal/mol,
-5.4 Kcal/mol, -3.1 Kcal/mol & -6.1 Kcal/mol relatively. This proves that Piperidine-4-one analogues could
be potential drugs for Anti-Alzheimer drug development. The GLIDE score is capable of being used as a
semi-quantitative descriptor for the capability of ligands to bind to a specific conformation of the protein
receptor. Generally, the ligand having low GLIDE score, will have superior kinship towards the receptor
could be expected Ethyl 2-(2,6-bis(4-bromophenyl)-3-butyl-4-oxopiperidine-1-carboxamido)acetate (KBr
5) (Figure 2-6)showed the best inhibition for the Acetyl Cholinesterase with -6.8 Kcal/mol glide score and
β-Secretase with -6.3 Kcal/mol glide score protein receptor. We ascertain a very good concurrence between
the localization of the inhibitors upon docking and from the protein structures of Acetyl Cholinesterase and
β-secretase. Conformational analysis of docked complex shows that the residues THR 311, HIS 405(Stacking)
for KBr 5, PRO 368, ARG 296 and ARG 247 for Donepezil, GLU 313 and HIS 405 for Galantamine, ASN
233, GLU 313 and HIS 405 for Rivastigmine, GLN 413 and GLU 313 for Tacrine relatively against Acetyl
Cholinesterase and GLN 121, ASN 281, ARG 283 (Stacking) for KBr 5, GLU 278, ASP 80, TYR 119
for Donepezil, THR 280 for Galantamine, THR 120 and GLN 121 for rivastigmine, ASP 80 for Tacrine
relatively against β-Secretase plays a vital role in this receptor’s activity. From the fallout, we could monitor
that for thriving docking, intermolecular hydrogen bonding and lipophilic interactions linking the ligand
and the receptors are very important. Electro negativity of the substitution (Cl, Br, F and NO2) doesn’t have
much effect in the inhibition. Ligands with increased in chain length and with increased side chain have a
good impact in inhibition
510
Figure 1: Designed MTDL for inhibiting both AChE & BACE Figure 2: Structure of KBr 5 Molecule
Figure 3: Ligand interaction of KBr 5 with AChE enzyme Figure 4: Ligand interaction of KBr 5 with BACE
enzyme
Figure 5: Glide docking image of KBr 5 with AChE
Figure 6: Glide docking image of KBr with BACE

511
Table 1: In silico docking results against Acetyl Cholinesterase enzyme (PDB: 3LII)
S.No Compound code Glide score Hydrogen Bonding Interaction

1 KF 2 -6.7 GLN 121, THR 120, TYR 119(Stacking)
2 KBr 5 -6.3 GLN 121, ASN 281, ARG 283(Stacking)
3 K6 -6.0 GLN 121, THR 120
4 KF 6 -5.8 GLN 121, ASN 281, , THR 120, ARG 283( Stacking)
5 K5 -5.7 THR 280, GLN 121, ARG 283 (Stacking)
6 Donepezil -5.8 GLY 278, ASP 80 (Salt Bridge), TYR 119(Stacking)
7 Galantamine -5.4 THR 280
8 Tacrine -6.1 ASP 80
9 Rivastigmine -3.1 THR 120, GLU 121
Table 2: In silico docking results against β-Secretase enzyme (PDB: 3U6A)
S.No Compound Code Glide score Hydrogen Bonding Interaction

1 KBr 3 -6.9 THR 311, HIS 405(Stacking)
2 KBr 5 -6.8 THR 311, HIS 405(Stacking)
3 KF 3 -6.7 GLU 313
4 KBr 2 -6.7 THR 311, ARG 296, HIS 405(Stacking)
5 KOCH3 3 -6.5 ARG 296
6 Donepezil -4.9 ARG 247, ARG 296, PRO 368
7 Galantamine -5.1 HIS 405, GLU 313
8 Tacrine -4.3 GLU 313, GLN 413
9 Rivastigmine -2.6 ASN 223, GLU 313 (Salt Bridge), HIS 405 (Stacking)
CONCLUSION
In conclusion, we have identified a finest molecule of Ethyl 2-(2,6-bis(4-bromophenyl)-3-butyl-4-
oxopiperidine-1-carboxamido) acetate KBr 5 (Figure 2) as an innovative drug candidate who was docked
against Acetyl Cholinesterase and β-Secretase in a deliberate attempt to discover an MTDL, able to interfere
with diverse key target points of AD Neurodegeneration. This compound KBr 5 is well thought-out as an
optimal (lead) molecule, and we necessitate to synthesis and evaluate its potency against Alzheimer’s disease
through molecular level and in vivo studies.
REFERENCES
1. Ghosh AK, Kumaragurubaran N and Tang J, Recent developments of structure based beta-secretase
inhibitors for Alzheimer’s disease, Curr Top Med Chem., 2005; 5(16):1609-22.
2. Guo T and Hobbs DW, Development of BACE1 inhibitors for Alzheimer’s disease, Curr Med Chem.,
2006; 13(15):1811-29.
3. Toews ML and Bylund DB, Pharmacologic principles for combination therapy, Proc Am Thorac Soc.,
2005; 2(4):282-9.
4. Cavalli A, Bolognesi ML, Minarini A, Rosini M, Tumiatti V, Recanatini M and Melchiorre C, Multi-
target-directed ligands to combat neurodegenerative diseases, J Med Chem., 2008; 51(3):347-72.
5. Small G and Dubois B, A review of compliance to treatment in Alzheimer’s disease: potential benefits
of a transdermal patch., Curr Med Res Opin., 2007; 23(11):2705-13.
512
6. Morphy R and Rankovic Z, Designing multiple ligands - medicinal chemistry strategies and challenges,
Curr Pharm Des., 2009; 15(6):587-600.
7. Bolognesi ML, Cavalli A, Valgimigli L, Bartolini M, Rosini M, Andrisano V, Recanatini M and
Melchiorre C, Multi-target-directed drug design strategy: from a dual binding site acetylcholinesterase
inhibitor to a trifunctional compound against Alzheimer’s disease, J Med Chem., 2007; 50(26):6446-9.
8. Zhu Y, Xiao K, Ma L, Xiong B, Fu Y, Yu H, Wang W, Wang X, Hu D, Peng H, Li J, Gong Q, Chai Q,
Tang X, Zhang H, Li J and Shen J, Design, synthesis and biological evaluation of novel dual inhibitors
of acetylcholinesterase and beta-secretase. Bioorg Med Chem., 2009 Feb 15; 17(4):1600-13.
9. Marco-Contelles J, Leon R, de los Rios C, Samadi A, Bartolini M, Andrisano V, Huertas O, Barril X,
Luque FJ, Rodriguez-Franco MI, Lopez B, Lopez MG, Garcia AG, Carreiras Mdo C and Villarroya M,
Tacripyrines, the first tacrine-dihydropyridine hybrids, as multitarget-directed ligands for the treatment
of Alzheimer’s disease, J Med Chem., 2009; 52(9):2724-32.
10. El-Subbagh HI, Abu-Zaid SM, Mahran MA, Badria FA and Alofaid AM, Synthesis and biological
evaluation of certain α, β-unsaturated ketones and their corresponding fused pyridines as antiviral and
cytotoxic agents, J Med Chem, 2000; 43:2915-21.
11. Watson AA, Fleet GWJ, Asano N, Molyneux RJ and Nugh RJ, Polyhydroxylated alkaloids, Natural
occurrence and therapeutic applications, Phytochemistry 2001;56:265-95.
12. Ganellin CR and Spickett RG, Compounds are affecting the central nervous system, 1,4-piperidones and
related compounds. J Med Chem 1965; 8:619-25.
13. Hagenbach RE and Gysin H, Piperidones as potential antitumour agents, Experimentia 1952;8:184-87.
14. Ileana B, Dobre V and Nicluescu-Duvaz I, Spin labeled nitrosoureas, Potential anticancer agents, J Prakt
Chem 1985; 327:667-74.
15. Mokio IG, Soldatenkov AT, Federov VO Ageev EA, Sergeeva ND, Lin S, Stashenku EE and Prostakov
NS Andreeva EL, Antimicrobial activity of aliphatic-aromatic ketones, β-ketols and α-glycols, Khim
Farm Zh 1989; 23:421-27.
16. Dimmock JR and Kumar P, Anticancer and cytotoxic properties of mannich bases, Curr Med Chem
1997; 4:1-22.
17. Kubota H, Kakefuda A, Okamoto Y, Fujii M, Yamamoto O, Yamgiwa Y, Orita M, Ikeda K, Takenchi
M, Shibanuma T and Fsomura Y, Synthesis of (±)-N-[2-(3,4- dichlorophenyl)-4-(spiro-substituted
piperidin-1’- yl)butyl]-N-methylbenzamides and evaluation of NK[1]-NK[2] dual antagonistic activities,
Chem Pharm Bull 1998; 46:1538-44.
18. Parasuraman P, Suresh R and Premnath D, Balancing anti-amyloid and anti-cholinesterase capacity in a
single chemical entity: In-silico drug design, Int J Pharm Pharm
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A Calcium Channel Blocker - Amlodipine Attenuates
Acetic Acid Induced Ulcerative Colitis in Mice
Rajinikanth B* and Venkatachalam V V
Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram,

Tamilnadu - 608 002, India
e-mail: rajini_pharm@yahoo.co.in, Mobile: +919942885353
Abstract:
Objective - The objective of this study is to investigate the effect of Amlodipine, a calcium channel blocker
on acetic acid induced ulcerative colitis in Swiss Albino mice based on the hypothesis of calcium blocking
in the colon leads to decrease the neuropeptides that are responsible for ulcerative colitis. Methods - 0.1 ml
of (6% v/v) acetic acid (AA) was used to induce ulcerative colitis and assess clinical disease activity index
(DAI) every day like body weight loss, stool consistency and gross bleeding. After seven days treatment
with Amlodipine blood was collected from retro-orbital puncture, serum was separated and C - reactive
protein (CRP), Alkaline phosphatase (ALP) were measured. Colon was excised, washed and its length,
were also measured. Colon tissue homogenate was subjected to measure myeloperoxidase (MPO) assay,
lipid peroxidation and cytokines like IL-6 and TNF-α. Results - Intracolonic administration of Amlodipine
significantly reduced the severity of AA induced ulcerative colitis. It diminished the DAI from the fifth day
onwards, which shown that it suppresses body weight loss, stool consistency and gross bleeding. Serum
concentrations of CRP and ALP levels are reduced into normal levels. Amlodipine prevents tissue MPO
accumulation, colon shortening, lipid peroxidation effectively in inflamed colonic tissue and also reduced
inflammatory cytokines like IL-6 and TNF-α. Conclusion - The present study results confirmed that the
Amlodipine, a calcium channel blocker possess significant reduction in severity of acetic acid-induced
ulcerative colitis in mice.
Keywords: Amlodipine, Acetic acid, Myeloperoxidase, Neuropeptides, Ulcerative colitis and Cytokines
INTRODUCTION
Ulcerative colitis (UC) and crohn’s disease (CD) are chronic inflammatory disorders of gastrointestinal tract
known together as inflammatory bowel disease (IBD). The real causes of IBD are unknown, although abnormal
function of the immune system is clearly concerned. Suggested causes include chronic infection, genetic
defect, environmental factors, autoimmune and other abnormalities of the mechanisms of immunoregulatory
[1]. Recent statistics indicates that the risk of colon cancer for people with IBD increases from 0.5 to 1.0%
per year, and after 8-10 years of diagnosis [2]. Contemporary treatment for inflammatory bowel disease is
administration of non-steroidal drugs, immunosuppressive agents and antibiotics. However, these treatments
may results severe side effects [3].
In recent times, enteric nervous system has become a promising target in the treatment of many
gastrointestinal symptoms called as neuro- gastrointestinal pharmacology. Enteric nervous system (ENS)
is the major division of the peripheral and autonomic nervous system, and it consist of many different
types of nerve cells similar to that spinal cord and a group of neurotransmitters, neuromodulators similar to
those found in the central nervous system (CNS). Drugs act as in the enteric nervous system either directly
or indirectly act on a neuroeffector junctions by stimulating or inhibiting the synthesis, storage, discharge
or reuptake of neurotransmitters [4]. Both ENS and CNS can increase or modify aspects of intestinal
inflammation through the secretion of neuropeptides and it is released from the nervous system that serves
as a signaling molecule between cells and thought its play a potentially key role in IBD include substance
P, mu-opioid receptor agonists, corticotrophin releasing hormone, neurotensin and vasoactive intestinal
peptide. Among this group of neuropeptides substance P, neurotensin and gallanin increase the severity of
inflammatory bowel disease [5]. Particularly substance P and corticotrophin releasing hormone neuropeptide
receptors present on immune cells, suggesting that the paracrine action of neuropeptides has an important
immunomodulatory role.
Most studies on nerves and inflammatory bowel disease provide evidence for the role of neuropeptides
and receptors in the initiation and maintenance and exacerbation of the inflammatory response in the
gastrointestinal tract. Neuropeptides blockade is the new therapeutic approach in both CD & UC [6]. These
neuropeptides are released from large dense core vesicles (LDCVs) in the synaptic cleft in response to
calcium-dependent depolarization. Released neuropeptides from the nerve endings in the intestines diffuse
into surrounding tissues and bind to corresponding receptors affecting epithelium, endothelium, nearby
muscle and immune cells [7].
We hypothesized that calcium channels blockage in the intestines and colon will decrease the neuropeptides
levels responsible for the inflammatory bowel disease. L-, N-, P- and Q- type calcium channels have been
identified in enteric neurons [8] and particularly L type calcium channels are most abundantly present in
intestines and colon. So we are interested to investigate an L type calcium channel blocker amlodipine
for this study. L-type calcium antagonists previously reported as it inhibits the dynorphin neuropeptides
in dendrites [9], anti-inflammatory effect by suppressing plasminogen receptors on macrophages [10] and
suppress cytokine-induced activation of human neutrophils [11].

Chemicals
Hexa decyl trimethyl ammonium bromides, O-dianisidine dihydrochloride, 2-Thiobarbituric acid were
purchased from Sigma-Aldrich chemicals Pvt Ltd, Bangalore, India. Serum estimations were carried out
by using commercially available kits (Agappe Diagnostics Ltd, Kerala, India). Cytokines like TNF-α, IL-6
enzyme immunoassay kits, were purchased from Peprotech (USA).
Animals
Swiss albino female mice (20-30g; n=6 per group) were obtained from for Laboratory Animal Sciences
at National Institute of Nutrition, Hyderabad. They were housed under standard laboratory conditions and
were given free access to regular laboratory chow diet and water. The study protocols were approved by
Institutional Animal Ethics Committee Rajah Muthaiah Medical College, Annamalai University, Proposal
number – 1009, Reg No.160/1999/CPCSEA.
Induction of experimental ulcerative colitis

Twenty-four hour fasted mice were lightly anesthetized with ketamine injection (24mg/kg IM). To induce
colitis 0.1 ml of acetic acid (6% v/v) solution is diluted with distilled water was slowly administered into the
lumen via the catheter fitted onto a 1-ml syringe. Animals were kept in a vertical position for thirty seconds
to prevent the leakage of intracolonic instill [13].
Experimental Design
Animals were divided into five groups, each consisting of minimum six in a group. Group-1 Control animals
received vehicle; Group-2 Colitis animal received vehicle, Group -3 Colitis animals received AML (0.65
515
mg/kg). Group -4 Colitis animals received AML (1.3 mg/kg).Group -5 Colitis animals received standard
drug Prednisolone (5mg/kg).
AML doses were fixed by calculating from human doses by using a standard formula [12] and made a
suspension by using water and polyethylene glycol. All treatments were administered daily for 7 days and
on the 8th day the animals were anaesthetized with ketamine (24mg/kg) and blood was collected by retro-
orbital puncture, serum was separated and stored under at -80° C Mice were sacrificed and their colons were
removed.
Evaluation of Disease
Disease activity index (DAI)
The clinical DAI which was took into the account of body weight loss (scored as: 0, none; 1, 1-5%;
2, 5-10%; 3, 10-20% 4, over 20%), stool consistency (scored as :0,1 well formed pellets; 2,3, loose
stools; 4,5 diarrhea) and presence or absence of fecal blood (scored as; 0, normal 1,2 hemoccult
positive; 3, 4, gross bleeding) [13].
Serum Estimation
The CRP, ALP were measured based on the procedure given in the kit using a Humalyzer 3000 (semi- auto
analyzer).
Biochemical Estimation
Assessment of colonic MPO activity
Colon tissue homogenate was centrifuged (800 × g) at 4°C for 30 min, and the supernatant was discarded.
In that pellet is mixed with 10 ml of ice-cold 50 mM potassium phosphate buffer (pH 6.0), containing
0.5% hexadecyltrimethylammonium bromide and 10 mM EDTA. It was subjected to one cycle of freezing,
thawing and a brief period (15seconds) of sonication. After sonication, again the solution was centrifuged at
13,100 ×g for 20 min. From this 0.1 ml of supernatant was combined with 2.9ml of 50mM phosphate buffer
containing 0.167 mg/ml of O-dianisidine hydrochloride and 0.0005% hydrogen peroxide and measured
change in absorbance at 460 nm spectrophotometrically. One unit of MPO activity is defined as the change
in absorbance per min by 1.0 at room temperature, in the final reaction [13]. It has been calculated by using
the following formula
MPO activity U/g = X/weight of the piece of tissue taken
Where X=10×change in absorbance per min/volume of supernatant taken in the final concentration.
Estimation of colonic Lipid Peroxidation (MDA)

Colon tissue homogenate (0.1 ml) is mixed with 2 ml of TBA–trichloroacetic acid–HCl reagent in 1:1:1 ratio
(0.37%TBA, 0.25MHCl and 15%TCA) and set aside for 15 min in a water bath, cooled and then centrifuged
at 3500 ×g for ten min at room temperature. Clear supernatant was subjected to measure the absorbance at
535 nm against a reference blank. Values were expressed as mM/100 g-tissue [13].
ELISA determination of TNF-α, IL-6 levels in colon tissues

Distal colon samples were weighed, homogenized and centrifuged at 12,000×g for 10 min, TNF- α, IL-6
levels were assayed by a quantitative TNF- α, IL-6 enzyme immunoassay kit (Peprotech USA) and the
values were expressed as pg/mg tissue. Absorbance was measured in an ELISA reader at 450 nm [13].
516
All data expressed as mean ± SEM statistical significance was measured by ANOVA followed by Dunnett’s
multiple comparison test using Graphpad prism version 5.0 and values of P<0.05 were considered as
statistically significant.
RESULTS AND DISCUSSIONS:
Figure 1: Changes in disease activity index were evaluated daily throughout the 7-day experimental period
Figure 2: Photographic representation of colon from each group
DAI, which took into account of body weight loss, stool consistency and gross bleeding, ; these are the
typical signs of human ulcerative colitis. AML treated groups significantly suppressed the DAI from the fifth
day onwards compared with AA induced colitis groups shown in Fig.1
Table 1: Effect of Amlodipine on CRP, ALP, TP and Colon length.
Groups CRP(mg/L) ALP(U/L) Colon length (cm)

Normal control 6.23 ± 0.44 296.7 ± 23.6 9.91±0.2
Colitis control 13.50 ± 0.34a 426.8 ± 23.02a 6.18±0.2a
AML(0.65mg/kg) 11.83±0.69b 321.4±29.53b 8.56±0.3e
AML(1.3mg/kg) 8.43±0.23 c
273±12.32 c
9.11±0.3c
Prednisolone (5mg/kg) 7.03 ± 0.28c 308 ± 21.4d 9.75±0.2c
Data are expressed as mean±SEM (n=6), aP<0.001 colitis control vs normal control, bP<0.05 colitis
control vs AML (0.65mg/kg), cP<0.001colitis control vs AML (1.3mg/kg), Prednisolone (5mg/kg), dP<0.01
colitis control vs Prednisolone (5mg/kg), ep<0.001 vs AML (0.65mg/kg), ns-no significant - colitis control
vs AML (0.65mg/kg)
517
Serum estimations results were shown in (Table 1). In acetic acid-induced colitis control (p<0.001) CRP
is increased significantly when compared with normal control. AML (0.65 mg/kg) significantly (p<0.05)
reduced the CRP levels compared with colitis control mice. AML (1.3mg/kg) and prednisolone (5mg/kg)
produce significantly (p<0.001) reduce CRP levels when compared with colitis control group. CRP is one
of the most important proteins that is rapidly produced by hepatocytes during an acute-phase response upon
stimulation by IL-6, TNF-α, and IL-1β originating at the site of inflammation or pathology. So it is an important
marker of inflammatory bowel disease [14]. ALP is an enzyme present in intestines, and it is increased in UC
patients. ALP levels were significantly increased in (p<0.01) colitis mice compared with control mice. AML
(0.65mg/kg) produces no significant changes in compared with control mice. AML (1.3mg/kg) significantly
reduced (p<0.05) ALP levels when compared with colitis control mice and prednisolone 5mg/kg (p<0.01)
significantly reduce ALP levels compared with the colitis control mice. Colon length (Table 1) is inversely
associated with severity of colitis. In acetic acid-induced, colitis groups colon shortening was observed when
compared with normal control group. AML at both dose levels (0.65mg/kg and 1.3mg/kg) and prednisolone
(5mg/kg) prevents colon shortening compared with colitis control group. Photographic representation of
colon from each group was shown in Fig.2.
Myeloperoxidase is an enzyme, an important marker of tissue inflammation present in neutrophils,
monocytes and macrophages. MPO activity level is directly proportional to the concentration of neutrophils
in inflamed tissue. So it is an important parameter for acute intestinal inflammation. [15]. MPO level (Fig. 3)
was raised significantly (p<0.001) in the colitis control group compared with normal mice, AML (0.65mg/kg)
produces significantly (p<0.05) reduced MPO levels compared with colitis control mice, AML (1.3mg/kg),
Prednisolone (5mg/kg) treated mice produce have significant (p<0.001) decrease in MPO when compared
with colitis control mice. MDA is the major end product of lipid peroxidation, which is usually measured
as a marker of oxidative stress. Glutathione normally protects the gastric mucosa and during experimental
colitis glutathione depletion takes place. MDA level was significantly increased in AA induced colitis mice
(p<0.001) when compared with the control mice. AML (0.65mg/kg) produced a significantly (p<0.05)
decrease MDA levels when compared with the AA induced colitis group. AML (1.3mg/kg) and Prednisolone
(5 mg/kg) showed significantly (p<0.001) reduce MDA levels when compared with the AA induced colitis
control mice (Fig 4).
Colonic IL-6 levels (Fig.5) are raised in AA induced colitis group compared with normal control group
(p<0.01). Treatment of AML (0.65mg/kg) produced (p<0.05) reduces colonic IL-6 levels when compared
with AA induced colitis group. Similarly AML (156 mg/kg) and Prednisolone (5 mg/kg) produced (p<0.01)
IL-6 levels when compared with AA induced colitis group. Colonic TNF-α levels are significantly increased
in AA induced colitis group compared with normal control group (p<0.001). AML (0.65mg/kg and 1.3mg/
kg) produced (p<0.01) reduces colonic TNF-α levels when compared with AA induced colitis group.
Prednisolone (5 mg/kg) produced (p<0.001) significantly reduced TNF-α levels to normal when compared
with AA induced colitis group shown in Fig. 6. TNF-α is a cytokine plays an important role in inflammatory
conditions and as a critical mediator of pathological inflammation in inflammatory
Figure 3: Colonic MPO level
518
Figure 4: Colonic MDA level
Figure 5: Colonic IL-6 levels
Figure 6: colonic TNF-α levels
Bowel disease. It’s major action is the induction of adhesion molecules, and chemokine expressions
lead to accumulation of inflammatory cells. TNF-α also stimulates the proliferation of fibroblasts, intestinal
smooth muscles to induce the synthesis of IL-6, IL-8, PGE2 and IL-1β. IL-6 production is increased in
inflammatory states such as rheumatoid arthritis, ulcerative colitis, mesangial glomerulonephritis, sepsis and
fever. Many reports had been stated that TNF-α, IL-6 blocking attenuates acute and chronic experimental
colitis [16].
CONCLUSION
From the results of this study suggest that AML dose-dependently decrease the severity of AA induced colitis
by inhibiting CRP, TNF-α, IL-6, neutrophil accumulation and MDA. A possible mechanism of action of
AML maybe it blocking the neuropeptides like substance P, vasoactive intestinal peptide, Calcitonin gene-
related peptide leads to decreased production and release of cytokines and chemokines and also suppress
cytokines mediated CRP production and inflammatory markers. However, further studies are required to
confirm the molecular mechanisms responsible for the effects of AML on neuropeptides blockade.
519
REFERENCES
1. Bickston SJ and Cominelli F, Inflammatory Bowel Disease: Short and Long Term Treatments, Disease
a month, 1998; 44(4):143-72.
2. Munkholm P, The incidence and prevalence of colorectal cancer in inflammatory bowel disease, Aliment
Pharmacol Ther., 2003; 18(2):1-5.
3. Yao J, Wang JY, Liu L, Zeng WS, Li YX, Xun AY, Zhao L, Jia CH, Feng JL, Wei XX and Wang
LS, Polydatin ameliorates DSS-induced colitis in mice through inhibition of nuclear factor-kappaB
activation, Planta Med., 2011; 77(5):421-7.
4. Sasselli V, Pachnis V and Burns AJ, The enteric nervous system, Dev Biol., 2012; 5:64-73.
5. Gross KJ and Pothoulakis C, Role of neuropeptides in inflammatory bowel disease, Inflamm Bowel
Dis., 2003; 13(7):918-32.
6. Kraneveld AD, Rijnierse A, Nijkamp FP and Garssen J, Neuro-immune interactions in inflammatory
bowel disease and irritable bowel syndrome: future therapeutic targets, Eur J Pharmacol., 2008; 585:361-
74.
7. Lundy FT and Linden GJ, Neuropeptides and neurogenic mechanisms in oral and periodontal
Inflammation, Crit Rev Oral Biol Med., 2004; 15(2):82-98.
8. Smith TK, Kang SH and Berghe PV, Calcium channels in enteric neurons, Curr Opin Pharmaco., 2003;
3:588-93.
9. Simmons ML, Terman GW, Gibbs SM and Chavkin C, L-type calcium channels mediate dynorphin
neuropeptide release from dendrites but not axons of hippocampal granule cells, Neuron; 1995:14(6):1265-
72.
10. Burke T, Wagoner DR and Plow EF, L-type calcium channel blockers exert an antiinflammatory effect
by suppressing expression of plasminogen receptors on macrophages, Circ Res., 2009; 105(2):167-75.
11. Medhi B and Prakash A, Practical manual of experimental and clinical pharmacology, 1st ed. Jaypee
brother’s medical publishers (P) ltd, New Delhi, (2010) 24-25.
12. Rajinikanth B, Venkatachalam VV and Manavalan R, Investigations on the potential of serratiopeptidase
– a proteolytic enzyme on acetic acid induced ulcerative colitis in mice, IJPPS.2014; 6(5):525-31.
13. Vermiere S, Assche GV and Rutgeerts P, C-Reactive Protein as a Marker for Inflammatory, Inflamm
Bowel Dis. 2004; 10(5):661-5.
14. Thippeswamy BS, Mahendran S, Biradar MI, Raj P, Srivastava K, Badami S, et al. Protective effect of
embelin against acid-induced ulcerative colitis in rats, Euro J Pharmacol., 2011; 654:100-5.
15. He J, Liang J, Zhu S and Zhao W, Protective effect of taurohyodeoxycholic acid from pulvis fellis
suis on trinitroben zene sulfonic acid-induced ulcerative colitis in mice. Pulmonary Gastrointestinal
Urogenital Pharmacol., 2011; 670:229-35.
520
Antioxidant Activity and Mosquitocidal Activity of
Allium sativum of Kodaikanal Hilly Areas
M. Razia*, K. Lavanya and B. Sri Shaila Devi
Department of Biotechnology, Mother Teresa Women’s University, Kodaikanal – 624 102, TamilNadu
*Corresponding authour
e-mail: razia581@gmail.com
Abstract:
Qualitative analysis of phytochemical compounds was studied in the plant Allium sativum using methanolic
extract. The following metabolites were presented tannins, saponin, flavonoids, phenols, glycosides,
alkaloids and reduced sugar namely. The nature of components present in the methanolic extract of A. sativum
was analysed by GCMS. FTIR spectroscopy can be used the presence of characteristic functional groups
Carboxylic acids, amines, amides, sulphur derivatives, polysaccharides, organic hydrocarbons; halogens
were identified in FT-IR analysis.
Reducing power assay and hydrogen peroxide scavenging assay was performed. A. sativum showed
lower reducing power activity when compared with synthetic standard ascorbic acid. Methanolic extract
showed 82% activity. The IC 50 value of standard was (0.14 mg/ml), aqueous extract (0.03 mg/ml) and
methanolic extract (0.15mg/ml) respectively. For hydrogen peroxide, radical scavenging activity methanolic
extract 90% activity was obtained. IC 50 values are 0.12, 0.03 and 0.18 for ascorbic acid, aqueous and
methanol extract respectively. The present study exhibits the mosquitocidal properties in the plant extract
suggesting their use in mosquito population control. The mortality rate of larval instars of A. aegypti, C.
quinquefasciatus and C. tritaeniorhynchus at 0.5% concentration was significantly higher than the mortality
rates at 0.1%, 0.2%, 0.3% and 0.4% concentrations of crude plant extract at 24, 48 and 72 h of exposure.
Higher mortality rate was also recorded at 72 h bioassays than those at 24 h and 48 h. Therefore, these
extracts could be used for further isolation and purification of active compound
INTRODUCTION
Garlic (Allium sativum) is the edible bulb from a plant of the Allium genus, commonly used for flavouring in
cooking and for its beneficial effects for human health. Although garlic cloves are usually eaten raw or cooked
different garlic dietary supplements including dried or powdered formulations, oils and liquid extracts have been
recently incorporated into the market to satisfy the demand of consumer for garlic bioactive compounds [1].
Antioxidants are compounds that are having the ability to trap free radicals. Plant secondary metabolites
are major sources of the natural antioxidants. It can be phenolic compounds (tocopherols, Flavonoids,
and phenolic acids) nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids and amines) or
carotenoid as well as ascorbic acids [2].
Garlic has the potential to kill mosquito larvae in an excellent way. It is a major vector for several deadly
diseases such as malaria, dengue fever, yellow fever, filariasis, schistosomiasis and Japanese encephalitis. To
prevent proliferation of mosquito-borne diseases and to improve the quality of the environment and public
health, mosquito control is essential. The major tool in mosquito control operation is the application of
synthetic insecticides such as organochlorine and organophosphate compounds. But the drawback of these
synthetic insecticides are non-biodegradable, cause environmental pollution, affects non-target organisms
and also costly. Plants play pivotal roles in ecological systems [3]. They may provide potential alternatives to
currently used insect-control agents because they constitute a rich source of bioactive chemicals [4]. Studies
were undertaken for the mosquito larvicidal activity of some common spices (Cuminum cyminum, Allium
sativum, Zingiber offinale, Curcuma longa)and vegetable waste (Solanum tuberosum germinated tuber) on
Culex quinquefasciatus and Anopheles stephensi [5]. In view of the increasing interest in developing plant
origin insecticides as an alternative to chemical insecticide, this study was undertaken to assess the larvicidal
potential of the extracts from A. sativum against mosquito larvae.

Collection and Preparation of extract
The bulbs of Allium sativum was collected from fields of Kodaikanal. The dry skins of the bulb were
removed before use, and then the cloves were peeled and dried at 40°C in hot air oven. 30g of dried sample
was extracted with 300 ml methanol in soxhlet apparatus. The extract was evaporated and concentrated in a
rotary evaporator. Aqueous extract also prepared.
The methanolic extracts of A. sativum were studied for their phytoconstituents using standard phytochemical
tests [6].
GC-MS and FT – IR analysis

1µl of methanolic extract of A. sativum was injected into GC-MS (PerkinElmer Clarus 500).The compounds
were detected in the range by matching with NIST library [7]. FTIR analysis was performed using Perkin
Elmer Spectrophotometer system, which was used to detect the characteristic peaks and their functional
groups.
Antioxidant activity
Determination of the free radical scavenging activity of the aqueous and methanolic extracts was carried out
using Reducing power assay. [8] An aliquot of each sample (1mL), with different concentrations (0.1 – 0.5
mg/ ml), was mixed with 2.5 mL of phosphate buffer (0.2 M,pH 6.6) followed by 2.5 mL of 1% potassium
ferricyanide. The mixture was incubated for 20 min in a water bath at 50°C. After incubation, 1mL of 10%
trichloroacetic acid was added, followed by centrifugation at 3000gfor 10 min. The supernatant (2.5 mL)
was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride, and then the absorbance was
read at 700 nm and Hydrogen peroxide-scavenging activity [9]. A solution of H2O2 (40 mM) was prepared
in phosphate buffer (pH 7.4). Examined extracts in different concentrations (0.1 – 0.5 mg/ ml) were added
to3.4 ml of phosphate buffer, together with 0.6 ml of H2O2 solution. The absorbance value of the reaction
mixture was recorded at 230 nm.
Larvicidal activity
Test Mosquitoes
Larvae of Aedes aegypti, Culex quinquefasciatus and Culex tritaeniorhynchus were collected from Indian
Council for Medical Research (ICMR), Madurai. Larvae of mosquito species were kept separately in different
plastic trays and fed with the mixture of dog biscuits and dried yeast powder at the ratio of 3:1.
Larvicidal Bioassay
Larvicidal bioassay was performed according to the Standard WHO protocol [10]. Different concentrations
(0.1 % - 0.5%) of crude extract of garlic was transferred into a sterile 250 ml glass beakers. Twenty-five
522
3rd instar larvae of each mosquitos were separately introduced into different flasks containing appropriate
concentrations and control was made up of methanol in distilled water. Mortalities were recorded at different
(24, 48 and 72 hours) exposure periods. Dead larvae were identified when they failed to move after probing
with a needle in the siphon or cervical region.

Qualitative analysis of phytochemical compounds present in the A. sativum showed in the table 1.
Tannin +
Saponin +
Flavonoids +
Carotenoids -
Phenols +
Glycosides +
Phlobatannins -
Alkaloids +
Terpenoids -
Reducing sugar +
Table 1: Phytochemical analysis of Allium sativum
GC- MS analysis
The nature of components present in the methanolic extract of A. sativum was analysed by GCMS (Fig.1). The
major compounds of GC-MS analysis and their retention time were listed in Table 3. These compounds have
several activities that are involved in health care. N,N’- Diacetylethylenediamine (0.3%), Diallyl disulphide
(0.8%) have Antimicrobial, Anticancer, Cancer preventive, Diuretic Antiinflammatory, insecticide activity.
1,3-Dioxepin, 4,7-dihydro- (0.1%), Palmitic acid (2.02 %), 4-methoxy-1-(2-oxobut-3-enyl) azetidin-2-one
(1.1 %), Ricinoleic acid (2.26 %) showed antimicrobial and anti-inflammatory activity, Cyclohexanone,
2-ethyl-4-ethoxy- (0.1 %), 8-(Trimethylsilyl)-6-octenteneitrile (2.9 %), Proline, 3,4- didehydro (19.3 %),
4, Amino-3- methoxypyrazolo (25.8 %). All these compounds are of pharmacological importance as they
possess the properties such as analgesic, anti-diabetic, antibacterial, and antifungal activity (Table2).
Table 2: Identification of bioactive compounds in A. sativum using GC-MS
S.No R.T Name of the compound Peak Area Molecular Molecular

% Formula Weight
1 12.38 N,N’-Diacetylethylenediamine 0.3474 C6H12N2O2 144
2. 16.98 Diallyl disulphide 0.8102 C6H10S2 146
3. 17.38 1,3-Dioxepin, 4,7-dihydro- 0.1179 C5H8O2 100
4. 17.53 Palmitic acid 2.02 C19H38O4 330
5. 19.19 4-methoxy-1-(2-oxobut-3-enyl) azetidin 1.12 C8H11NO3 169
6. 21. 93 Ricinoleic acid 2.26 C18H34O3 298
7. 22.27 Cyclohexanone, 2-ethyl-4-ethoxy- 0.1455 C9H16O2 156
8. 23.82 8-(Trimethylsilyl)-6-ethanenitrile 2.92 C5H11NOSi 129
9. 24.88 Proline, 3,4- didehydro 19.35 C5H7NO2 113
10. 29.26 4, Amino-3- methoxy pyrazolo 25.81 C6H7N5O 165
523
Figure 1: GC-MS analysis of methanolic extract of A. sativum
FT-IR spectral features of A. sativum

The spectral feature of garlic was shown in figure 2. Totally 13 peaks were obtained in FT – IR analysis. The
broad peak showed the presence hydroxyl group, N-H stretching, C=O stretching mode, carboxylic acids,
P=O stretching, C-O-C stretching of polysaccharide, CH bending respectively. FTIR spectroscopy can be
used as an additional tool to screen vegetables for their content of phenolic compounds. The functional
groups were presented carboxylic acids, amines, amides, sulphur derivatives, polysaccharides, organic
hydrocarbons namely, and halogens were identified in FT-IR analysis. These compounds are responsible for
various medicinal properties of A. sativum (Table3).
Table 3: Functional Group Analysis of A. sativum by FT-IR
PEAK FUNCTIONAL GROUP

3392.90 O-H stretching
2922.25 N-H stretching
2852.81 C-H symmetric stretching
1637.62 C=O stretching
1581.68 C=C
1406.16 C-Cl stretching
1404.16 COO-
1080.17 P=O symmetric stretching
1045.45 C-O-C stretching
931.65 C-H bending
879.57 Si-H stretching
721.40 C-S
667.39 Halogen compounds
Figure 2: FT-IR Spectra of methanolic extract of Allium sativum

524
Antioxidant activity
Results of reducing power assay and hydrogen peroxide scavenging assay were shown in Figure 3. Tested
samples showed lower reducing power activity when compared with synthetic standard ascorbic acid.
Ascorbic acid showed 88% antioxidant activity at the concentration of 0.5 mg/ml.
Methanolic extract showed 82% activity. The IC 50 value of standard (0.14 mg/ml), aqueous extract (0.03
mg/ml) and methanolic extract (0.15 mg/ml) and this activity was dose dependent. For hydrogen peroxide,
radical scavenging activity methanolic extract showed more activity than aqueous and standard ascorbic
acid. In methanolic extract, 90% activity was obtained. IC 50 values are 0.12, 0.03 and 0.18 for ascorbic acid,
aqueous and methanol extract respectively. Whole garlic and aged garlic extract exhibit direct antioxidant
effects and enhance the serum levels of two antioxidant enzymes, catalase and glutathione peroxidase [11].
A comparative study was conducted for antioxidant activity of onion and garlic. In this, garlic showed (IC50
= 0.95 mg/mL) highest activity than onion [12].
Figure 3: a) Reducing Power assay b) Hydrogen Peroxide scavenging activity
Larvicidal bioassay
Larvicidal activity of crude extract of A. sativum against A. aegypti and C. quinquefasciatus and C.
tritaeniorhynchus were presented in table 3 and 4. The present study exhibits the mosquitocidal properties in
the plant extract suggesting their use in mosquito population control. The mortality rate of larval instars of A.
aegypti, C. quinquefasciatus and C. tritaeniorhynchus at 0.5% concentration was significantly higher than
the mortality rates at 0.1%, 0.2%, 0.3% and 0.4% concentrations of crude plant extract at 24, 48 and 72 h of
exposure. Higher mortality rate was also recorded at 72 h bioassays than those at 24 h and 48 h.
The toxicity of garlic bulb extracts to the second, third and fourth instar larvae of C. quinquefasciatus
was studied [13]. The results showed that ethanol garlic bulb extract was very effective against C.
quinquefasciatus larvae. Similarly, studied the use of garlic and lemon peel extracts as C. pipens larvicides
in which the interaction and persistence of the extracts with the organophosphate resistance mechanism were
observed [14].
Table 4: Mortality of A. aegypti, C. quinquefaciatus and C. tritaeniorhynchus exposed to crude extract of A. sativum.
Mosquitoes Time (Hours) Concentrations (mg/l)

100 200 300 400 500
A. aegypti 24 4±1 4 ± 1.15 32 ± 1.52 40 ± 1.52 48 ± 2
48 12 ± 1.15 36 ± 1.15 64 ± 1.15 60 ± 2.64 84 ± 3.21
72 20 ± 1.15 56 ± 3.05 80 ± 1.15 84 ± 3 96 ± 0.57
C. quinquefasciatus 24 8 ± 1.52 24 ± 1 36 ± 1.52 40 ± 1.52 44 ± 1
48 20 ± 2.64 44 ± 1.52 64 ± 2.64 68 ± 2.30 76 ± 0
72 32 ± 4.16 64 ± 1.52 80 ± 1 80 ± 1 92 ± 1
C. tritaeniorhynchus 24 4±1 20 ± 1.52 32 ± 1 40 ± 1 48 ± 1.15
48 16 ± 1.15 44 ± 0.52 68 ± 1.52 80 ± 2.30 88 ± 3.05
72 28 ± 3.05 76 ± 1 80 ± 2.51 92 ± 2.51 96 ± 0.57
525
CONCLUSION
A.sativum extracts exhibit presence of phytochemical constituents, antioxidant and mosquitocidal activity.
Plant secondary metabolites are major sources of the natural antioxidants. It can be phenolic compounds
for antioxidant property. Further studies on A. sativum could be used for isolation and purification of active
compounds for antinsecticidal property.
REFERENCES
1. Cobas AC, Martinez M and Villamiel M, (2010), A comprehensive survey of garlic functionality, Nova
science publishers, Inc.
2. Abubakar EM, (2009), Efficacy of crude extracts of garlic (Allium sativum Linn.) against nosocomial
Escherichia coli, Staphylococcus aureus, Streptococcus pneumonieaand Pseudomonas aeruginosa,
Journal of Medicinal Plants Research, 3(4):179-185.
3. Garcıa M, Gonzalez-Coloma A, Donadel OJ, Ardanaz CE, Tonn CE and Sosa ME, (2007), Insecticidal
effects of Flourensia oolepis Blake (Asteraceae) essential oil, Biochemical Systematics and Ecology,
35: 181–187.
4. Qin W, Huang S, Li C, Chen S and Peng Z, (2010), Biological activity of the essential oil from the
leaves of Piper sarmentosum Roxb, (Piperaceae) And its chemical constituents on Brontispa longissima
(Gestro) (Coleoptera: Hispidae), Pesticide Biochemistry and Physiology, 96:132–139.
5. Singha S and Chandra G, (2011), Mosquito larvicidal activity of some common spices and vegetable
waste on Culex quinquefasciatus and Anopheles stephensi, Asian Pacific Journal of Tropical Medicine,
288-293.
6. Ayoola GA, Coker HAB, Adesegun SA, AdepojuBello AA, Obaweya K, Ezennia EC and Atangbayila
TO, (2008), Phytochemical screening and antioxidant activities of some selected medicinal plants used
for malaria therapy in southwestern Nigeria, Tropical Jouranl Pharmaceutical Research 7(3):1019‐1024.
7. Massada Y, (1976), Analysis of Essential Oils by Gas Chromatography and Mass spectrometry, John
Wiley and Sons, New York, U.S.A.
8. Yen GC and Chen HY, (1995), Antioxidant activity of various tea extract in relation to their antimutagenic,
Journal of Agricultural and Food Chemistry, 43: 27–32.
9. Ruch RJ, Cheng SJ and Klaunig JE, (1989), Prevention of cytotoxicity and inhibition of intracellular
communication by antioxidant catechins isolated from Chinese green tea, Carcinogenesis, 10: 1003–1008.
10. WHO, (1996), Report of the WHO informal consultation on the evaluation and testing of insecticides,
CTD/WHOPES/IC/96, 1. Geneva: Control of Tropical Diseases Division.
11. Yamasaki T and Lau BH, (1997), Garlic compounds protect vascular endothelial cells from oxidant
injury, Nippon Yakurigaku Zasshi 110 Suppl 1:138-141.
12. Othman, Idid, Suleiman Koya, Rehan and Kamarudin, (2011), Antioxidant Study of Garlic and Red
Onion: A Comparative Study, Pertanika Journal Tropical Agriculture Science 34 (2): 253 – 261.
13. Kalu G, Ofoegbu U, Eroegbusi J, Nwachukwu CU and Ibeh B, (2010), Larvicidal activities of ethanol
extract of Allium sativum (garlic bulb) against the filarial vector, Culex quinquefasciatus, Journal of
Medicinal Plants Research 4(6): 496-498.
14. Claire JT and Amanda C, (1999), The Use of Garlic (Allium sativa) and Lemon Peel (Citrus limon)
Extracts as Culex pipiens Larvacides: Persistence and Interaction with an Organophosphate Resistance
Mechanism. Chemosphere 39(14): 2489-2496.
526
Phytochemical and Antimicrobial Investigations in
Pseudarthria viscida (Fabaceae)
S. Tamil Selvi1, M. B. Viswanathan2, * and M. Venkatesan3
1, 2
Centre for Research and Development of Siddha-Ayurveda Medicines (CRDSAM), Department of Plant Sci-
ence, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India
3
Department of Botany, Sourashtra College, Madurai 625004, Tamil Nadu, India
e-mail: vinaabdu@gmail.com, Tel: +91 431 2407061, fax: +91 431 2407045
Abstract:
Priliminary phytochemical analysis of the various solvent extracts of Pseudarthria viscida indicated the
presence of steroids, triterpenoids, phenols, flavonoids, tannins, quinones and catechins. Agar well-diffusion
method was employed to test solvent extracts against 12 human pathogenic bacteria such as Bacillus cereus
(MTCC 430), Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 96), Staphylococcus epidermis
(MTCC 435), Aeromonas hydrophila (MTCC 646), Escherichia coli (MTCC 724), Klebsiella pneumoniae
(MTCC 432), Proteus mirabilis (MTCC 425), Proteus vulgaris (MTCC 426), Pseudomonas aeruginosa
(MTCC 741), Salmonella paratyphi (MTCC 735) and Salmonella typhi (MTCC 733). Petroleum ether,
chloroform and ethanol extracts of the root, stem and leaves were tested wherein root extract showed the
highest activity against Gram-negative than Gram-positive bacteria.
Keywords: Pseudarthria viscida, Fabaceae, Root, Stem, Leaves, Phytochemistry, Human pathogenic
bacteria, Antibacterial activity.
1. INTRODUCTION
Pseudarthria viscida (L.) Wight and Arn. (Syn. Hedysarum viscidum L.) belonging to the family of Fabaceae
is a subshrub which is the preferred source of the raw drug, Saliparni in Ayurvedic System of Medicine. The
roots constitute an important ingredient of the well-known Ayurvedic formulation in Dasamoola aristam.
It is administered to relieve biliousness, rheumatism, breathing difficulties, excessive heat, fever, diarrhea,
asthma, heart diseases, worms and piles. The roots are stringent, thermogenic, digestive, anthelmintic,
antiflammatory, diuretic, aphrodisiac, nervine, cardio and rejuvenating tonic. Due to its scarcity, other
trifoliate leguminous plants, particularly the species of Desmodium and Uraria are used as substitutes. As
there have been no phytochemical or biological studies available they are reported here for this Ayurvedic
Medicine.

2.1. Plant material
The plant material was collected from the Perumal hills in Tiruchirappalli District and Kolli hills in Namakkal
District between December 2008 and September 2009. A voucher specimen (MBV & TS 540) collected from
the Perumal Hills in Tiruchirappalli District was identified and authenticated by Prof. M.B. Viswanathan
and has been deposited in the Herbarium of the Centre for Research and Development of Siddha-Ayurveda
Medicines (CRDSAM), Department of Plant Science, Bharathidasan University, Tiruchirappalli. Shade-
dried and coarsely powdered root, stem, and leaves were successively extracted with petroleum ether,
chloroform and ethanol using Soxhlet apparatus. The solvents were removed by vacuum distillation in a
rotary evaporator at 60°C and the dried substance was dissolved in suitable solvents and stored in cold room
for future use.
2.2. Phytochemical studies
The solvent extracts were tested for preliminary phytochemical screening (1). Phytochemical constituents
were isolated and purified by chromatographic techniques (2).
2.3. Antibacterial studies
Different concentrations such as 50, 25, 12.5 and 6.25 mg/ml of the various solvent extracts of root, stem
and leaves were prepared and tested.
2.3.1. Test microorganisms
Bacterial strains were procured from Microbial Type Culture Collection (MTCC) at the Institute of
Microbial Technology (IMTECH), Chandigarh. The test organisms were: Gram-positive bacteria – Bacillus
cereus (MTCC 430), Bacillus subtilis (MTCC 441), Staphylococcus ssaureus (MTCC 96), Staphylococcus
epidermis (MTCC 435) and Gram-negative bacteria – Aeromonas hydrophila (MTCC 646), Escherichia
coli (MTCC 724), Klebsiella pneumoniae (MTCC 432), Proteus mirabilis (MTCC 425), Proteus vulgaris
(MTCC 426), Pseudomonas aeruginosa (MTCC 741), Salmonella paratyphi (MTCC 735), Salmonella typhi
(MTCC 733). Muller-Hinton Broth and Muller-Hinton Agar (MHA) were used to test antibacterial activity.
2.3.2. Determination of antimicrobial activity

The antibacterial activity was determined by Agar well-diffusion method (3). Muller - Hinton Agar No. 2
(MHA) plates were swabbed (sterile cotton swabs) with overnight broth culture of respective bacteria. Five
wells (8 mm diameter) were made in each of these plates using sterile cork borer. Different concentrations
of petroleum ether, chloroform and ethanol extracts of root, stem, and leaves were added to the wells using
sterilized dropping pipettes and allowed to diffuse at room temperature for 2 h. The plates were incubated
at 37°C for 18-24 h. Respective proper controls of ethanol were also maintained and average values were
calculated for recording antibacterial activity.
3. RESULTS
Preliminary phytochemical analysis of the different solvent extracts such as petroleum ether, chloroform and
ethanol of the root, stem and leaves in Pseudarthria viscida indicated the presence of secondary metabolites in
a different manner. Steroids are present in all the solvent extracts of root and petroleum ether and chloroform
extracts of the stem and leaves. Triterpenoids, phenols, flavonoids, tannins and catechins are present in the
ethanol extracts of root, stem and leaves. Petroleum ether and chloroform extracts of the root, stem and
leaves indicated the presence of quinones. Sugars, alkaloids, saponins, anthroquinones, amino acids, lignins
and coumarins are absent in all the extracts. Petroleum ether: ethyl acetate 98:2 yielded a triterpene for
which structural elucidation is in progress. The root extracts were expressed antibacterial activity (Table 1).
However, more inhibitory activity recorded for Gram-negative than Gram-positive bacteria. Escherichia
coli showed significant inhibitory activity to all the solvent extracts ranging from 18 - 21 mm. More activity
recorded for petroleum ether extract (20 and 21 mm) than chloroform (19 and 20 mm) and ethanol (18 and 19
mm) extracts. Proteus vulgaris showed more activity ranging from 15 - 21 mm to the ethanol extract. More
or less similar activity recorded such as 12 – 17 mm in ethanol extract, 12 – 15 mm in chloroform extract and
10 – 16 mm in petroleum ether extract to Salmonella typhi. Aeromonas hydrophila showed more activity to
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petroleum ether extract such as 10 – 16 mm. This is followed by chloroform extract such as 11 – 15 mm and
ethanol extract such as 11 – 14 mm. Inhibition of K. pneumoniae was 12 – 15 mm to petroleum ether extract,
13 – 14 mm to ethanol extract, and 12 – 14 mm to chloroform extract. Stem (Table 2) also showed more
activity to Escherichia coli such as 17 – 19 mm in ethanol extract, 16 – 18 mm in chloroform extract and 16 –
17 mm in petroleum ether extract. Only Aeromonas hydrophila exhibited 14 – 16 mm in chloroform extract,
10.5 – 13 mm in ethanol extract and 10 – 12 mm in petroleum ether extract. Maximum inhibition for the
remaining Gram-negative bacteria was 14 mm to Aeromonas hydrophila and minimal was 8.5 mm each to
Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhi. Gram-positive bacteria expressed
minimal inhibition of 8.5 mm and maximum inhibition 12 mm. Leaves (Table 3) showed more activity to
Proteus mirabilis such as 12 – 15 mm in chloroform extract, 11 – 15 mm in petroleum ether extract and
12 – 14 mm in ethanol extract. Pseudomonas aeruginosa showed more activity to ethanol extract such as
9 – 15 mm. In the case of Salmonella paratyphi, Klebsiella pneumoniae and Proteus vulgaris, minimum
inhibition was 9 mm and a maximum of 14 mm. Aeromonas hydrophila exhibited 10 – 12 mm in ethanol
extract, 9 – 12 mm in petroleum ether and chloroform extracts. Escherichia coli showed more activity to
ethanol extract such as 9 – 13 mm, petroleum ether extract such as 11 – 12 mm and 9 mm in chloroform
extract. Staphylococcus aureus and Salmonella paratyphi exhibited 9 – 13 mm in ethanol extract, 9 – 11 mm
in chloroform extract and 9 – 10 mm in petroleum ether extract.
4. DISCUSSION
Preliminary phytochemical analysis showed the presence of steroids, triterpenoids, phenols, flavonoids,
tannins, quinones and catechins. The presence of alkaloids and the absence of steroids were reported in this
plant (4). In the present study also, similar results were recorded. Ethanol leaf extract of Khaya senegalensis
against Staphylococcus aureus showed 10 mm of inhibitory zone (5). But, in the present study, more activity
of 13 mm inhibition zone was recorded against it to the ethanol leaf extract. Antibacterial activity for the
ethanol extracts of stem and leaves were reported in Nerium indicum and Hibiscus rosa-sinensis (6). Ethanol
extracts of stem in Nerium indicum and Hibiscus rosa-sinensis inhibited variedly such as 10 mm and 8 mm
against Bacillus subtilis, 10 mm and 14 mm against Escherichia coli, 8 mm each against Staphylococcus
aureus, and 12 mm and 8 mm against Pseudomonas aeruginosa, respectively. In the case of their leaf extract
against similar bacteria, inhibitions were recorded such as 8 mm and 10 mm, 12 mm and 8 mm, 10 mm each,
and no activity and 8 mm, respectively. In the present study, ethanol extracts of stem and leaves inhibited
such as 9 mm and 13 mm against Bacillus subtilis, 19 mm and 13 mm against Escherichia coli, 12 mm
and 13 mm against Staphylococcus aureus, and 10 mm and 15 mm against Pseudomonas aeruginosa. In
addition, there was no activity reported for Salmonella typhi by them but in the present study maximum
activity of 12 mm to the stem and 17 mm to the leaves was recorded (Tables 2, 3). Antibacterial activity of
petroleum ether extracts and chloroform extracts of leaves in Eupatorium triplinerve was reported such as 12
mm and 20 mm against Bacillus cereus, 13 mm and 20 mm each against Bacillus subtilis and Staphylococcus
aureus, 11 mm and 19 mm against Escherichia coli, 11 mm and 20 mm against Salmonella paratyphi, and
11 mm and 19 mm against Salmonella typhi and 10 mm and 19 mm against Pseudomonas aeruginosa (7).
In the present study, antibacterial activity of petroleum ether extract and chloroform extract of leaves was
recorded such as 10 mm and no activity against Bacillus cereus, 11 mm and 10 mm against Bacillus subtilis,
10 mm and 11 mm against Staphylococcus aureus, 12 mm and 9 mm against Escherichia coli, 11 mm each
against Salmonella paratyphi, and 10 mm and 11 mm against Salmonella typhi and 11 mm and 12 mm
against Pseudomonas aeruginosa (Table 3). Antibacterial activity of the chloroform root extract in Boscia
angustifolia against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa was reported
such as 24.7 mm, 22.8 mm and 25 mm respectively (8). In the present study, the chloroform root extract
exhibited 15 mm, 20 mm and 13 mm activity against these bacteria (Table 1).
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Table 1: Antibacterial activity of the root extracts in Pseudarthria viscida
Bacterial Petroleum ether (mg/ml) Chloroform (mg/ml) Ethanol (mg/ml) Standard

cultures
50 25 12.5 6.25 C 50 25 12.5 6.25 C 50 25 12.5 6.25 C
Gram-positive
B. cereus 14 12 11 10 - 15 12 11 10 - 16 13 11 11 - 33 (T)
B. subtilis 12 11 11 10 - 13 12 11 10 - 13 12 12 10 - 30 (A)
S. aureus 14 13 12 11 - 15 14 13 12 - 14 13 13 12 - 45 (M)
S. epidermis 14 13 12 11 - 15 13 13 12 - 14 13 13 13 - 40 (T)
Gram-negative
A. hydrophila 16 12 11 10 - 15 13 10 11 - 14 13 12 11 - 20 (Tr)
E. coli 20 20 21 21 17 20 20 20 19 17 19 19 18 18 17 30 (K)
K. pneumonia 15 14 13 12 - 14 13 13 12 - 14 13 13 13 - 30 (K)
P. mirabilis 19 15 12 11 - 15 12 11 11 - 16 15 13 12 - 25 (E)
P. vulgaris 16 15 12 11 - 12 15 11 11 - 20 17 16 15 - 30 (T)
P. aeruginosa 12 11 10 9 - 13 11 10 9 - 15 13 12 11 - 20 (K)
S. paratyphi 13 12 11 11 - 14 12 11 11 - 13 12 11 9 - 35 (G)
S. typhi 16 13 11 10 - 15 14 13 12 - 17 14 13 12 - 20 (Na)
- No activity; C - DMSO (Dimethyl sulphoxide); measurements are given in mm; Ampicillin (A);
Erythromycin (E); Gentamicin (G); Kanamycin (K); Methicillin (M); Nalidixic acid (Na); Tetracycline (T);
Trimethoprin (Tr).
Table 2: Antibacterial activity of the stem extracts in Pseudarthria viscida

cultures 50 25 12.5 6.25 C 50 25 12.5 6.25 C 50 25 12.5 6.25 C
Gram-positive
B. cereus 10 9 9 8.5 - 9 8.5 9 9 - 11 11 9 9 - 33 (T)
B. subtilis 11 11 10 10 - 12 10 10 9 - 9 9 8.5 8.5 - 30 (A)
S. aureus - - - - - - - - - - 12 11 10 10 - 45 (M)
S. epidermis 12 12 11 10 - 12 11 11 10 - 11 10 9 9 - 40 (T)
Gram-negative
A. hydrophila 12 11 10 10 - 16 15 14 14 - 13 12 11 10.5 - 20 (Tr)
E. coli 17 16.5 16 16 - 18 17 17 16 - 19 18 18 17 - 30 (K)
K. pneumoniae 10 9 9 8.5 - 11 10 11 10 - 13 12 11 11 - 30 (K)
P. mirabilis 12 9 9 9 - 11 9 9 9 - 14 13 13 10 - 25 (E)
P. vulgaris - - - - - - - - - - 12 11 10 10 - 30 (T)
P. aeruginosa 10 9 9 8.5 - 12 10 9 9 - 10 11 10 10 - 20 (K)
S. paratyphi 12 12 11 10 - 10 10 9.5 9 - 13 12.5 12 11 - 35 (G)
S. typhi 10 10 10 10 - 11 10 10 10 - 12 11 9 8.5 - 20 (Na)
Trimethoprin (Tr).
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Table 3: Antibacterial activity of the leaf extracts in Pseudarthria viscida

cultures 50 25 12.5 6.25 C 50 25 12.5 6.25 C 50 25 12.5 6.25 C
Gram-positive
B. cereus 10 9 9 9 - - - - - - 13 11 10 9 - 33 (T)
B. subtilis 11 11 11 11 - 10 11 11 10 - 13 12 12 10 10 30 (A)
S. aureus 10 9 9 9 - 11 10 9 9 - 13 12 10 9 - 45 (M)
S. epidermis 12 11 11 12 - 12 11 10 9 - 13 12 11 10 - 40 (T)
Gram-negative
A. hydrophila 12 11 10 9 - 12 12 11 9 - 12 12 11 10 - 20 (Tr)
E. coli 12 11 11 11 - 9 9 9 9 - 13 12 11 9 - 30 (K)
K. pneumonie 12 11 11 10 - 12 11 11 9 - 14 13 12 11 - 30 (K)
P. mirabilis 15 13 12 11 - 15 12 12 12 - 14 12 12 12 - 25 (E)
P. vulgaris 13 12 11 10 - 12 10 10 9 - 14 12 11 10 - 30 (T)
P. aeruginosa 11 10 10 9 - 12 11 10 9 - 15 13 11 9 - 20 (K)
S. paratyphi 11 10 10 9 - 11 11 10 10 - 14 13 12 11 - 35 (G)
S. typhi 10 10 9 9 - 11 11 9 9 - 13 12 9 9 - 20 (Na)
Trimethoprin (Tr).
5. CONCLUSION
Preliminary phytochemical studies and antibacterial activity of Pseudarthria viscida are reported here for the
first time to this Ayurvedic medicine.
REFERENES
1. Brindha P, Sasikala B and Purushothaman KK, Pharmacognostic studies on Merugan kizhangu, Bull
Medico-Ethnobotanical Res, 1982; 3:84-96.
2. Harborne JB, Phytochemical methods: A Guide to Modern Techniques of Plant Analysis, 3rd ed. New
York: Chapman and Hall; 1998.
3. Perez C, Pauli M and Bazerque P, An antibacterial assay by agar well diffusion method, Acta Bio Et Med
Exp. 1990; 15:113-115.
4. Mathew GM and Sasikumar JM, Antioxidant activity of Pseudarthria viscid, Indian J Pharma Sci 2007;
69:581-582.
5. Makut MD, Gyar SD, Pennap GRI and Antony P, Phytochemical screening and antimicrobial activity
of the ethanolic and methanolic extracts of the leaf and bark of Khaya senegalensis, Afr J Biotechnol
2008; 7:1216-1219.
6. Naqvi BS, Rafi Shaikh M, Maleka FA and Shaikh D, Studies of antibacterial activity of ethanolic extracts
from Nerium indicum and Hibiscus rosa-sinensis, J Islamic Acad Sci 1994; 7:167-168.
7. Rahman MDS and Junaid M, Antimicrobial activity of leaf extracts of Eupatorium triplinerve Vahl
against some human pathogenic bacteria and phytopathogenic fungi, Bangladesh J Bot 2008; 37:89-92.
8. Hassan SW, Umar RA, Lawal M, Bilbis LS, Muhammad BY and Dabai YU, Evaluation of antibacterial
activity and phytochemical analysis of root extracts of Boscia angustifolia, Afr J Biotechnol, 2006;
5:1602-1607.
531
Extraction and Antimicrobial Evaluation of Buffer
Extracts of Marine Invertebrate Donax cuneatus
M. Arputha Bibiana, C. Sherlina Daphny, M. Umamageshwari, P. Selvamani

and S. Latha
Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli-620024

e-mail: arputha_bibiana@yahoo.com
Abstract:
Five different phenotypes of the marine edible bivalve Donax cuneatus were collected from the coastal area
of Cuddalore, 11°46’N latitude, 79°45’E longitude, Tamil Nadu, India. The five species were named as S1,
S2, S3, S4 and S5 and were homogenized for crude protein with buffers Tris Hcl, Tris base and Phosphate
Buffer in three range of pH (4.5, 7.4 and 9.0). The crude protein was further partially purified by ammonium
sulfate precipitation method the precipitate was diluted and stored in -20°C until antimicrobial evaluation.
All the crude buffer extracts showed promising activity against highly pathogenic organisms considerably.
The acidic and alkaline extract of S4 sample showed maximum zone of inhibition against Gram positive
Staphylococcus sp and Gram negative Bacillus sp. The phenotypes S3 and S5 also inhibit the growth of
pathogens in considerable ranges. The protein concentration was 1.0 mg/ml in S4. The SDS PAGE analysis
confirms the presence of proteins by colored bands in the gel and FTIR analysis showed the presence of the
protein in the crude bivalve extract by their identified functional groups.
INTRODUCTION
Ocean has plenty of organisms which are being used as medicine. Among the marine sources the marine
invertebrates belonging to category bivalves and crustaceans are large sized and are commonly consumed
as edible food sources by the coastal area people of India [1, 2]. The rich proteins, glycogens and minerals
present in them are naturally built as defenders of microbial infections. The marine ecosystem was enriched
with tremendous of invertebrate species that are still unexplored for their property as biopharmaceuticals.
The amazing property of invertebrates is the innate immunity developed within them to act against many
infectious agents rather than active immunity [3, 4]. This property of marine invertebrates was the main
attention for most of the researchers to explore the marine invertebrates for antimicrobial peptides against
the human microbial pathogens [5].
Antimicrobial peptides are defined as the compounds of molecular weight below 10 kDa with
antimicrobial properties of rapid response against microbial infections. The specific physicochemical nature
of antimicrobial peptides lies within them is the hydrophilic and hydrophobic regions all over the surfaces
irrespective of their structure and size [6, 7].
The switching of pathogenic strains from sensitive to resistant forms was being a major challenge for
the scientists in search of new antibiotics. In this regard the research is now greatly focused upon exploring
the marine sources for developing it into antimicrobial pharmaceuticals. As marine sources are highly
proteinaceous in nature the proteins inbuilt within them plays a lead role in immunity. Identifying the
antimicrobial effect of such proteins may helps to develop new biopharmaceuticals for the serious problem of
antimicrobial resistance [11-13]. The main aim of the present study is to identify the effective antimicrobial
properties of the proteins extracted from the edible marine bivalve.

Collection of bivalve
Five different phenotypes of marine edible bivalves were collected from the coast of Cuddalore, Tamil
Nadu and they were authenticated by Dr. A. Shanmugam, Professor, CAS in Marine Biology, Annamalai
University, Parangipettai. The samples were identified as different phenotypes of Donax cuneatus.
Figure 1: Five different phenotypes of Donax cuneatus
Preparation of crude protein extract

The collected bivalves are brought to the laboratory and shells were broken and soft body was removed. The
collected sample was extracted using various types of buffer solution. The extracts were homogenized at
1500rpm for 15mins and the supernatant was collected. Tissue seperated from bivalve and 10ml of 3 types
of buffers (tris-hcl,tris buffer,phosphate buffer) were added with 5g of tissue.Homogenize the tissues in
manual homogeniser and centrifuge the homogenised tissue at 7500 rpm for 15minutes at 4°C.Supernatent
was removed and store at -20°C in another tube.
Partial purification of protein

The volume of the supernatant was measured and calculate the required amount of ammonium sulphate salt
needed to Saturate the solution at 100%. 60gm ammonium sulphate salt is needed for 100ml of saturated
solution. Add the required salt to the solution slowly in small quantities and mix well continuously in ice
bath supported magnetic stirrer after each addition. After the addition is completed and the salt is completely
dissolved, centrifuge the sample at 7500 rpm for 30 min. Discard the supernatant and dissolve the precipitate
in few ml of distilled water.
Estimation of protein by lowry’s method

Lowry’s method is commonly used to estimate the concentration of protein. Different dilutions of BSA solutions
are prepared by mixing stock BSA solution (1 mg/ ml) and water in the test tube as given in the table. The final
volume in each of the test tubes is 5 ml. The BSA range is 0.05 to 1 mg/ml. From these different dilutions,
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pipette out 0.2 ml protein solution to different test tubes and add 2 ml of alkaline copper sulphate reagent
(analytical reagent). Mix the solutions well. This solution is incubated at room temperature for 10 mins. Then
add 0.2 ml of reagent Folin Ciocalteau solution (reagent solutions) to each tube and incubate for 30 min. Zero
the colorimeter with blank and take the optical density (measure the absorbance) at 660 nm. Plot the absorbance
against protein concentration to get a standard calibration curve. Check the absorbance of unknown sample and
determine the concentration of the unknown sample using the standard curve.
Antimicrobial activity was determined by agar well diffusion method. Nutrient agar plates were swabbed
with the respective broth culture of the organisms and kept for 15 minutes in laminar chamber for absorption
of cultures. Wells were made in agar plates using a sterile cork bore of 5mm and 200µl of crude protein
extract was added to each well.The plates were incubated at 37°C for 24 hours and the diameters of the
inhibition zone were measured in millimeter.
SDS-PAGE analysis
The gels typically consist of acrylamide, bisacrylamide, SDS, and a buffer with an adjusted pH. The
solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization.
Alternatively, butanol may be added to the resolving gel after it is poured, as butanol removes bubbles and
makes the surface smooth. An ammonium persulfate and TEMED are added to initiate polymerization. The
polymerization reaction results in a gel because of the added bisacrylamide, generally about 1 part in 35
relative to acrylamide, which can form cross-links between two polyacrylamide molecules. The acrylamide
concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels
are better for resolving very high molecular weight proteins, while much higher percentages are needed to
resolve smaller proteins. Determining how much of the various solutions to mix together to make gels of
particular acrylamide concentration is possible.
FTIR-analysis
The crude protein extract was subjected to FTIR characterisation under suitable conditions. The Functional
groups of proteins were identified in a different wavelength.

Antimicrobial activity test
All the five crude buffer (Tris HCl, Tris Buffer, Phosphate Buffer) extracts at different pH ranges were
evaluated for its antimicrobial activity against pathogenic microorganisms.
Activity of Tris Hcl Buffer extract

The results of Tris HCl buffer extracts of all the five samples can be interpreted from Table:1
Table 1: Zone of inhibition of TRIS HCl buffer extract
S.No Bacterial Zone of inhibition (mm) of crude protein extract

pathogens P1 P2 P3 P4 P5
pH4 pH7 pH9 pH4 pH7 pH9 pH4 pH7 pH9 pH4 pH7 pH9 pH4 pH7 pH9
1 Salmonella sp 10 6 11 10 7 9 10 11 12 8 9 10 12 12 13
2 Bacillus sp 8 6 8 11 10 9 15 11 13 14 12 15 11 16 14
3 E.coli 4 4 5 6 5 6 6 5 5 7 6 9 10 9 8
4 Pseudomonas sp 9 4 4 8 4 3 7 3 4 6 4 5 8 7 6
5 Staphylococcus sp 11 13 15 12 11 15 14 13 12 16 13 14 12 11 13
6 Streptococcus sp 4 4 3 3 4 4 4 4 4 4 4 5 3 4 3
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The Tris HCl buffer extract of Donax cuneatus showed promising antimicrobial effect against the tested
bacterial and fungal cultures. All the pathogens were responded to the extracts to a minimum range of 3mm.
A maximum of 9mm was observed with the acidic pH extract of phenotype P4 and a minimum of 6mm was
observed with the neutral pH of extracts P2 and P5. Comparingly the Gram negative organism Bacillus sp
also showed better antimicrobial response to the alkaline and acidic extracts of P3 and P4. All the extracts
showed a minimal inhibitory zone against the tested pathogens but the acidic and alkaline extracts of P4 was
specifically good in susceptibility comparing the other extracts. The highly pathogenic Gram positive and
Gram negative bacteria that was responded to the Tris Hcl buffer extracts hopes for the development of new
biopharmaceuticals against the infectious pathogens.
Activity of Tris Buffer extract

The results of Tris buffer extracts of all the five samples can be interpreted from Table 2.
Table 2: Zone of Inhibition of Tris Buffer Extract
S.No Bacterial pathogens Zone of inhibition(mm)

P1 P2 P3 P4 P5
1 Salmonella sp 5 4 6 4 3 7 4 3 5 4 3 6 4 3 6
2 Bacillus sp 3 4 4 4 3 4 3 4 4 4 3 4 3 3 4
3 E.coli 4 3 5 4 3 5 4 3 5 4 3 5 4 5 6
4 Pseudomonas sp 3 4 4 3 4 3 3 4 4 3 4 4 3 4 4
The Tris buffer crude extract at three different pH evaluated for its antimicrobial activity also showed
antimicrobial response against the tested microbial strains. The alkaline extract of P4 showed the maximum
zone of 8mm against the Staphylococcus sp. All the other extracts of P4 also showed better inhibitory zone
against Staphylococcus sp. 7mm zone was the next maximum inhibitory level against the Gram positive
pathogen Staphylococcus sp. The Gram negative bacteria Salmonella sp responded upto a maximum of 7mm
zone of inhibition by the alkaline extract P2. In overall the extracts of all the phenotypes showed minimum
of 1mm zone and maximum of 8mm zone against the tested strains.
Activity of Phosphate Buffer extract

The results of Phosphate buffer extracts of all the five samples can be interpreted from Table 3.
Table 3: Zone of Inhibition of Phosphate Buffer Extract
S.No Bacterial Zone of inhibition(mm) crude protein extract

pathogens P1 P2 P3 P4 P5
1 Salmonella sp 8 4 9 8 5 7 8 9 10 6 7 8 10 10 11
2 Bacillus sp 6 4 6 9 8 7 13 9 11 12 10 13 9 14 12
3 E.coli 4 4 3 4 3 4 4 3 3 5 4 7 8 7 6
4 Pseudomonas sp 7 3 3 6 3 4 5 4 4 5 4 3 6 5 4
The phosphate buffer extract of all the phenotypes showed potent anti-infectious effects against
Staphylococcus sp like the other two extracts. Here the maximum of 10mm and 9mm zone was observed with
the acidic extract of P4 and alkaline extract of P1, P2 respectively. The minimum of 5mm zone was observed
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with the acidic, alkaline and neutral extracts of P1, P2 and P5. All the extracts also showed minimum zone
of inhibition against the tested strains upto 1mm level
Protein estimation
The Tris buffer extracts of marine bivalve were estimated for their protein concentration by Lowry’s method.
The concentration of crude protein was calculated from the standard graph of BSA. The optical density of
the extracts at various concentrations revealed the amount of protein in the extract. The optical density of P1,
P2, P3, P4 and P5 Tris buffer extracts of different pH were observed at 640nm. The maximum concentration
of 1.0mg/ml of protein was observed with P4 crude extract. All the other extracts have protein concentration
of not less than 0.6 mg/ml.
SDS-PAGE analysis
The P5 crude extract (Tris HCl, Tris base and phosphate buffer extract) of edible bivalve was subjected
to molecular weight determination by SDS - PAGE analysis. Six bands were observed as a result of the
analysis. The bands were separated at range of molecular weight 14, 17, 28, 38, 62 and 188 kda (Fig:1).
These were compared with the protein marker and the separated proteins were identified to resemble the
molecular weight of Lysozyme, Myoglobin red, Carbonic anhydrase, Alcohol dehydrogenase, BSA and
Myosin respectively.
Figure 1: SDS-PAGE (MM – Molecular Marker, Lane 1 - Acidic Tris Hcl extract, Lane 2 - Acidic Tris base extract,
Lane 3 – Acidic Phosphate buffer extract
FTIR Characterization
FTIR characterization of crude protein showed the presence of functional groups of protein like NH, COOH,
-CO-, CH, SH, C=O, C=C and other R groups at appropriate wave length and stretching. The results can be
inferred from the Table: 4 and Fig:2
Table 4: IR analysis of crude protein
S.No Functional Group Absorption Spectrum

1 Amino (NH) 3417
2 Carboxylic acid (COOH) 1778
3 Amide 1635
4 Aromatic C=C 1529
5 Alkyl Subtituted ether 1197
6 Disulfide 617
7 Polysulfide 428
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Figure 2: FTIR Analysis report
CONCLUSION
Marine sources are rich of many biopharmaceuticals and used as a drug for most of the microbial infections.
The marine bivalve Donax cuneatus collected for our study was extracted with various buffers and partially
purified by ammonium sulfate precipitation and the diluted precipitate was evaluated for its antimicrobial
property against few of the microbial pathogens like Gram positive and Gram negative bacterial cultures.
All the crude buffer extracts showed promising activity against highly pathogenic organisms even mildly.
The acidic and alkaline extract of P4 sample showed maximum zone of inhibition against Gram positive
Staphylococcus sp and Gram negative Bacillus sp. The phenotypes P3 and P5 also inhibit the growth of
pathogens in considerable ranges. The protein concentration was 1.0 mg/ml in P4. The SDS PAGE analysis
revealed the presence of proteins by colored bands in the gel and FTIR analysis showed the presence of the
protein in the crude bivalve extract by their identified functional groups.
The present study identifies concentrated protein content in the crude marine bivalve extract which
was potential against the growth of many pathogenic microbial strains. The protein was characterized
and confirmed by many analyses. In future the antimicrobial proteins identified now could be positively
developed into antibiotic or biopharmaceutical against the microbial pathogens.
REFERENCES
1. Rajeev Kumar Jha and XuZi-rong, Review Biomedical compounds from marine organisms, Marine
Drugs 2004; 2: 123-146.
2. Jirge Supriya S and Chaudhari Yogesh S: Marine: the ultimate source of bioactives and drug metabolites,
International Journal of Research in Ayurveda & Pharmacy, 2010; 1: (1), 55-62.
3. Marine Invertebrate Zoology, Phylum Mollusca: Bivalves. 2011 Oct 21; Available from: URL:http://
www.cfcc.edu_rogers_msc174_Bivalves.org/
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4. Prof. A. Shanmugam and Prof. S. Rajagopal, Molluscs, Centre of Advanced Study in Marine Biology,
Annamalai University. Available from: URL:http://www.ocw.unu.edu_international-network-on-water-
environment-and--heath_unu-inweh-course-1-mangroves_Molluscs/
5. M.J. Abad, L.M. Bedoya and P. Bermejo, Marine Compounds and their Antimicrobial Activities, Science
against microbial pathogens: communicating current research and technological advances. A. Mendez-
Vilas (Ed.)@FORMATEX 2011: 1293-1306
6. K.H. Wong and Peter C.K. Cheung, Nutritional evaluation of some subtropical red and green seaweeds
Part II. In vitro protein digestibility and amino acid profiles of protein concentrates. Food Chemistry
2001; 72: 11-17.
7. T. Unci, Dilek Nal and Atakan Sukatar, Antimicrobial Activities of the Extracts of Marine sources from
the Coast of Urla (Uzmir, Turkey).Antimicrobial Agent and Chemotherapy, Mar.1975,P.320-321.
8. James T. Richards, Earl R. Kern, Lowell A. Glasgow, James C. Overall, JR., E. Frank Deing and
Melvin T. Hatch, Antiviral Activity of Exracts from Marine invertebrates, Antimicrobial Agents and
Chemotherapy, July 1978, p.24-30.
9. Arakawa Y, Shibata N, Shibayama K, Kurokawa H, Yagi T, Fujiwara H and Goto M, 2000, Convenient
test for screening metallo-beta-lactamase-producing Gram-negative bacteria by using thiol compounds.
J ClinMicrobiol 38: 40-43.
10. Faulkner DJ, Harper MK, Haygood MG, Salomon CE and Schmidt EW, 2000, Symbiotic bacteria in
sponges: sources of bioactive substances. In N Fusetani, Drugs from the sea, Karger, Basel, p. 107-119.
11. Gerwick WH, Coates RC, Engene N, Gerwick L, Grindberg RV, Jones AC and Sorrels CM, 2008, Giant
marine cyanobacteria produce exciting potential pharmaceuticals. Microbe 3: 277-284.
12. Netz DJA, Bastos MCF and Sahl HG, 2002, Mode of action of the antimicrobial peptide aureocin A53
from Staphylococcus aureus. Appl Environ Microbiol 68: 5274-5280.
13. Mahenthiralingam E, Campbell ME, Foster J, Lam JS and Speert DP, 1996, Random amplified
polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic
fibrosis, J ClinMicrobiol 34: 1129-1135.
14. Petrucci, et al., General Chemistry (buffers): Principles & Modern Applications, 9th ed. Upper Saddle
River, New Jersey 2007.
15. Aceret TL, Sammarco PW, Coll JC and Uchio Y, 2001, Discrimination between several diterpenoid
compounds in feeding by Gambusia affinis. Comp Biochem Physiol 128C: 55-63.
16. Almeida AMP, Berlinck RGS and Hajdu E, 1997, Alcaloidesalquilpiridinicos de esponjasmarinhas,
Quim Nova 20: 170-185.
17. Amsler CD, Iken KB, McClintock JB and Baker BJ, 2001, Secondary metabolites from Antarctic marine
organisms and their ecological implications, In: McClintock JB and Baker JB (Eds.) Marine Chemical
Ecology, Boca Raton, CRC Press, p. 267-300.
18. Andersen RJ and Stonard R, 1979, Clionamide, a major metabolite of the sponge Clionacelata Grant.
Can J Chem 57: 2325-2328.
19. Andersen RJ, van Soest RWM and Kong F, 1996, 3-Alkylpiperidine alkaloids isolated from marine
sponges in the order Haplosclerida, In: Pelletier SW (Ed.) The Alkaloids: Chemical and Biological
Perspectives. New York, Pergamon Press, Vol. 10, p. 301-355.
538
20. Bandaranayake WM, Bennis JE and Bourne DJ, 1996, Ultraviolet absorbing pigments from the marine
sponge Dysideaherbacea: isolation and structure of a new mycosporine. CompBiochemPhysiol 115C:
281-286.
21. Becerro MA, Uriz MJ and Turon X, 1994, Trends in space occupation by the encrusting sponge
Crambecrambe: variation in shape, as a function of size and environment. Mar Biol 121: 301-307.
22. Becerro MA, Turon X and Uriz MJ, 1995, Natural variation of toxicity in encrusting sponge
Crambecrambe (Schmidt) in relation to size and environment, J ChemEcol 21: 1931-1946.
23. Becerro MA, Uriz MJ and Turon X, 1997, Chemically-mediated interactions in benthic organisms: the
chemical ecology of Crambecrambe (Porifera, Poecilosclerida). Hydrobiol 355: 77-89.
24. Becerro MA, Paul VJ and Starmer J, 1998, Intracolonial variation in chemical defenses of the sponge
Cacospongia sp. and its consequences on generalist fish predators and the specialist nudibranch predator
Glossodorispallida. Mar EcolProgSer 168: 187-196.
25. http://www.wpi.edu/Academics/Depts/Chemistry/Courses/General/tlc.html
539
Evaluation of Anticholinesterase Activity of Cadaba indica
by TLC Bioautography Method and Isolation of Active
Phytoconstituents by Bioassay Guided Fractionation
P.S. Dhivya, P. Selvamani, S. Latha, M. Omrohini and M. Sobiya
Department of Pharmaceutical Technology & Centre for Excellence in Nanobio Translational Research,
Anna University, Bharathidasan Institute of Technology Campus, Tiruchirappalli - 620 024, Tamil Nadu
corresponding author
email id : dhivyapsundaram@gmail.com
Abstract:
Alzheimer disease is a slowly progressive neurodegenerative disorder , characterized by the loss of memory
and progressive decline of cognitive abilities. Alzheimer’s disease is neuropathologically characterised by
the occurrence of β - amyloid plaques and neurofibrillary tangles. Epidemiological data indicate a potentially
considerable increase in the prevalence of the disease in the next two decades. Alzheimer’s disease can affect
different people in different ways, but the most common symptom pattern begins with gradual worsening
difficulty in remembering new information. This is because disruption of brain cells function usually begins
in the regions involved in forming new memories. The patients with Alzheimer’s disease suffer from marked
reduction of cholinergic neuronal function resulting in a deficiency in acetylcholine concentration in the
brain. The aim of the current work is to investigate the Anticholinesterase activity of Cadaba indica for
the treatment of Alzheimer`s disease by TLC autobiographic method and isolate the active components
responsible for the cholinesterase inhibitory activity by GC-MS Analysis. In the current study, the GC-MS
spectrum of the active compound was matched with NIST database spectrum was found to be 8-Amino-6-
methoxy-4-trifluromethylquinoline, we conclude that the Cadaba indica belonging to Capparaceae family
showed significant inhibitory against Acetylcholinesterase enzyme in a dose dependent manner. Finally the
results of the work suggest that the Cadaba indica would be a valuable source of anticholinesterase agents
with a potential use in cognitive disorders.
Exrtraction, TLC Bioautographic assay, Isolation & GC-MS Spectrum Matched with NIST Database
INTRODUCTION
Alzheimer’s disease is a neurodegenerative disorder of the brain which mainly affects the elderly population
with an unidentified etiology. Initially patients exhibit some degree of forgetfulness which evolves into short
term memory and eventually leads to long term memory deficits. Alzheimer’s disease is characterized by
accumulation of intracellular or extra cellular protein aggregates [1]. Neuropathology of the Alzheimer’s
disease contributes to β-amyloid (Aβ) and tau protein aggregation which reduces the acetylcholine level
[2]. Progressive neurodegeneration leads to death in various brain regions particularly in hippocampus,
which is the site specific for memory and cognition [3]. As a result of cholinergic deficits leads to decreased
number of cholinergic neurons in the basal fore Brain nuclei, increased level of acetylcholinesterase and
reduced level of choline acetyl transferase in the frontal and temporal cortices [4]. In the current clinical
strategy there is no cure available to seize the disease but there are drugs for symptomatic treatment for
mild to moderate cases [5]. This is mainly based on replacement therapy for deficits in central cholinergic
transmission, which improve the Alzheimer’s disease patients to recall the old memories and involved in
formation of new memory and learning by using Acetylcholine inhibitors [6]. In India plant products are the
main ingredient in most of the day to day diets often used as energy supplement, nutraceuticals, preventive
aids for many human ailments including degenerative disease. Current drug strategies which are approved
by FDA are of plant origin, for example Galanthamine, and it is one of the few drugs originated from plants
with its potential acetylcholinesterase inhibitory activity. Hence there is still a need of exploration to identify
the most potent natural sources with minimum side effects and better cure in the treatment of Alzheimer’s
disease.
Cadaba indica is mostly distributed throughout the world mostly tropical and sub-tropical
regions. It is a common shrub of the arid plains of Sind world and Baluchistan provinces of Pakistan.
They are common not only in large depressions but also found on sandy silts of valleys, around
temporary ponds and on stabilized dunes, where there is subsoil rich in fine particles containing
termite mounds. Cadaba indica belong to the family capparaceae is an unarmed shrubs or trees with
older, smooth, purplish, younger, pubescent, yellowish brown Stems. The crude extracts of Cadaba
indica were previously reported to possess significant Hepatoprotective activity, Anti-oxidant activity,
Anti-protozoal activity, Schistosomicidal activity, Antifungal activity, Anticancer activity, Cytotoxic
activity, Anti bacterial activity, Wound healing activity [7]. Leaf juice of Cadaba indica is used as a
remedy for dysentery, stimulant, purgative, fever, cough and lungs problem. It was reported to possess
stachydrine, 3-hydroxystachydrine from the stem, roots and cadabine from leaves [8].
MATERIALS AND METHOD

Plant Materials
Leaves of Cadaba indica were collected at the end of flowering stage in the month of June from various
districts of Tamilnadu. The collected plant are identified and authenticated by Botanical Survey of India,
Coimbatore, Tamil Nadu, India. Voucher Specimen of the plants was submitted in the Department of
Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli, Tamil Nadu.
Chemicals
Acetylthiocholine iodide (Sigma A5751), 5,5’-Dithiobis (2-nitrobenzoic acid) (Himedia RM1677),
Cholinesterase, acetyl (E.C No.3.1.1.7) Type VI-S From electric Eel (sigma), and TLC Silica gel 60 F254
(Merck 1.05554.007). Ethanol and all other organic solvents of analytical grade were purchased from Merck.
541
Preparation of ethanolic extracts and Isolation of active phytoconstituents

The plant materials were collected, shade dried at room temperature, crushed to coarse powder by a
mechanical grinder and stored at ambient temperature prior to extraction. Accurately weighed coarse powder
of the desired plant parts were charged in a soxhlet extractor and continuously extracted with hot ethanol.
The obtained extracts were then filtered, concentrated and evaporated to dryness under vacuum to give
crude ethanolic extracts. The ethanolic extract of Cadaba indica was suspended on water and extracted
successively with n-hexane, chloroform, to yield n-hexane and chloroform fractions. Chloroform soluble
fraction was subjected to thin layer chromatography for acetylcholinesterase inhibitory assay [9].
Thin layer Chromatography

TLC was performed on a pre-coated silica gel TLC plates grade F254 to determine the number of compounds
present in the chloroform extract. A total of 5 µl (10 mg/ml) of sample was spotted at 1 cm from the bottom
of silica gel plates using capillary tubes. Different solvents at various combinations and concentrations
were used for metabolites profiling. Development of the chromatogram was done in closed tanks, in which
the atmosphere has been saturated with eluent vapour by wetting a filter paper lining. The chromatogram
was visualized under UV light (365 nm and 254 nm), white light and iodine vapour. The Rf values of the
compounds were calculated using the following formula.
distance travelled by the compound
Rf =
distance travelled by the solvent front
Bioautography method for detection of AChE inhibition

The TLC with bioassay detection for AChE inhibition was modified from the study of Rhee et al., 2001
[10] and Kornakanok Ingkaninan, et al., 2003 [11]. A 2.5mm silicasilica gel plate F254 no. 5554 was used
as a stationary phase. In the mobile phase was hexane and ethyl acetate 7:3 (v/v) was used. Five microliter
of chloroform extract was spotted on to the plate. After the plate had been developed, it was dried at room
temperature and then sprayed with 30mM ATCI followed by 20mM DTNB. The plate was dried at room
temperature for 15 min, and then sprayed with 10.17 U/ml AChE. After 5 min, the plate was observed under
visible light. A positive spot indicating AChE inhibitor was a colorless spot on the yellow background.
Different spots were detected and calculated their Rf values.
Autobiography derived compound isolation

Fresh TLC plate were spotted with chloroform extract and developed using the same solvent system of
bioassay detection of AChE inhibition. Using the positive spot Rf value, most active spot was scrapped. The
TLC scrapped powder was dissolved in chloroform and centrifuged at 10,000 rpm for 5 min. The supernatant
was used for the compound identification using GC-MS analysis.
Gas Chromatography mass spectrometry analysis

An Agilent 6890 gas chromatograph was equipped with a straight deactivated 2 mm direct injector liner
and a 15m Alltech EC-5 column (250μ I.D., 0.25μ film thickness). A split injection was used for sample
introduction and the split ratio was set to 10:1. The oven temperature program was programmed to start at
35°C, hold for 2 minutes, then ramp at 20°C per minute to 260°C and hold for 5 minutes. The helium carrier
gas was set to 2 ml/minute flow rate (constant flow mode). A JEOL GC mate II benchtop double-focusing
magnetic sector mass spectrometer operating in electron ionization (EI) mode with TSS-20001 software
was used for all analyses. Low-resolution mass spectra were acquired at a resolving power of 1000 (20%
height definition) and scanning from m/z 25 to m/z 700 at 0.3 seconds per scan with a 0.2 second inter-scan
delay. High resolution mass spectra were acquired at a resolving power of 5000 (20% height definition) and
scanning the magnet from m/z 65 to m/z 750 at 1 second per scan.
542
Mass spectrometry library search

Identification of the components of the purified compound shall be matched with their recorded spectra and
with data bank mass spectra of NIST library V 11 provided by the instruments software.
RESULTS AND DICUSSIONS

Preparation of ethanolic extracts and Isolation of active phytoconstituents
A total of 1.75 gram of ethanolic extract was obtained and fractionated compounds from hexane (0.19 g) and
chloroform (0.4 g) was also obtained from the crude extract of Cadaba indica.
Thin layer Chromatography standardization

Among the solvent system tested, the solvent system, hexane: ethyl acetate (7:3) has shown 10 compounds
with different Rf values from chloroform extract. The developed chromatogram was visualized under
different light source and shown in Figure 1.
Figure 1: Developed chromatogram of chloroform extract using hexane: ethyl acetate (7:3)
TLC Autobiographic assay for anticholinesterase inhibition

The TLC autobiography of antiacetylcholinesterase assay on chloroform extract showed active spots at
different Rf values (Table 1 & Figure 2).
Figure 2: Bioautogram of Cadaba indica
543
Table 1: TLC autobiography of anti-acetylcholinesterase activity of chloroform extract
Compounds Rf values AChE inhibition

01 0.06 -
02 0.21 ++
03 0.34 -
04 0.38 -
05 0.46 -
06 0.55 -
07 0.72 +++
08 0.74 -
09 0.80 +
10 0.91 -
+ inhibition; - no inhibition
GC-MS analysis of the active compound 7

Based on the in-vitro antiacetylcholinesterase activity, the compound seven was taken for further
studies. The GC-MS spectrum and was found to be the active compound 7 was matched with NIST
database spectrum of 8-Amino-6-methoxy-4-trifluromethylquinoline (Figure 3& 4).
Figure 3: GC-MS total ion chromatogram of active compound

GC-MS Spectrum of the Active Compound Matched With NIST Database
Figure 4: GC-MS spectrum of the Active compound matched with NIST Database.
544
CONCLUSION
Thus it can be concluded that the Cadaba indica belonging to Capparaceae family showed positive white
spots by TLC autobiographic assay. Further analysis of the isolated compound confirms that the GC-MS
spectrum of the active compound which was matched with NIST database spectrum was found to be
8-Amino-6-methoxy-4-trifluromethylquinoline. With this, we conclude that the further research related
to the isolation of the bioactive constituents through bioassay-directed fractionation studies could lead to
discovery of bioactive compounds which may helpful in treatment of diseases related to cognitive disorder.
REFERENCES
1. Mark S Forman, John Q Trojanowski and Virginia M-Y Hee, Neurodegenerative disease: A decade of
discoveries paves the way for therapeutic break through. Natural Medicine 2004; 10(10):1038-11130.
2. Kashani A, hepicard E, Poirel O, Videau C, David, Fallet JP et al. Loss of VGLUT1, and VGLUT2 in the
prefrontal context is correlated with cognitive decline in Alzheimer’s disease, Neurobiol Aging 2008;
29: 1619-1630.
3. West MJ, Coleman PD, Flood DG et al., Differences in the pattern of hippocampal neuronal loss in
normal aging and Alzheimer’s disease, Lancet 1994; 344:7692-772.
4. Francis PT, Palmer AM, Snape M and Wilcock GK, The cholinergic hypothesis of Alzheimer’s disease:
review or progress. Journal of Neurology, Neurosurgery and Psychiatry 1999; 66, 137-147.
5. Orhan I, Kartal M, Tosun F and Sener B, Screening of various phenolic acids and flavonoid derivatives
for their anticholinesterase potential, Z Naturforsch C, 2007; 62(11-12):829-32.
6. Alan M. Palmer Pharmacotherapy for Alzheimer’s disease: Progress and Prospects. Trends in
Pharmacological Sciences. 2002; 23(9): 426-433.
7. Umesh B. Telrandhe and Vaibhav Uplanchiwar, Phyto-Pharmacological Perspective of Cadaba farinosa
forsk, American Journal of Phytomedicine and Clinical Therapeutics, ISSN 2321 – 2748.
8. Watt JM and Breyer-Brandwijk MG, Medicinal and Poisonous Plants of Southern and Eastern Africa,
2nd ed. E&S Livingston, Ltd., London.1962; pp-160.
9. Esra Kupeli Akkol, Ilkay Erdogan Orhan and Erdem Yesilada, Anticholinesterase and antioxidant effects
of the ethanol extract, ethanol fractions and isolated flavonoids from Cistus laurifolius L. leaves. Food
Chemistry 131 (2012) 626–631
10. Rhee I.K., Van der Meent M., Ingkaninan K. and Verpoorte R., Screening for acetylcholinesterase
inhibitors from Amaryllidaceae using silica gel thin-layer chromatography in combination with bioactive
staining. Journal of Chromatography 2001: 915:217-223.
11. Kornakanok Ingkaninan, Prapapan Temkitthawon, Kanchanaporrn Chuenchom, Thitaree Yuyaem
and Warawit Thongnoi, Screening for acetylcholinesterase inhibitory activity in plants used in Thai
traditional rejuvenating and neurotonic remedies, Journal of Ethanopharmacology.2003:89: 261-264.
545
Energy Conservation Opportunities in Paper
Plant using Combined Heat and Power for
Pharmaceutical Application
Dr. N. Stalin
Assistant Professor, Department of Petrochemical Technology, Anna University, BIT Campus,

Tiruchirappalli - 620024, TN
e-mail: mnstalin@gmail.com, Mob: 9976577636
Abstract:
Energy crisis and the exponential hikes in the cost of energy, the energy audit is of prime importance in all
industrial sectors. Energy audit paves the way to understand the areas where energy and fuel be consumed
in the industry, identify the areas where wastage of energy can occur and where the scope of improvements
exists. Energy audit in a nutshell quantifies how and where energy is being spent in industries. The paper
industry is one of the most energy-intensive industries in the manufacturing sector. The energy consumed
by the utilities alone would be over 50% of the total energy requirement of the site. Energy audit of a paper
plant in which the raw material used is old corrugated paper, used newsprint, Kraft waste and used paper
boards. Overall energy consumption and the specific energy (both electrical and thermal) consumption are
also calculated and compared with the Standard SEC. The performance analysis of major energy utilizing
equipments is also done comprehensively, and their operating equipment efficiency is determined so as to
undertake some improvements or replacing the whole equipment itself.
The present turbine is analyzed for its optimum use and proposed a new turbine to increase the power
output. In addition to that, some of the Energy conservation proposals are proposed to reduce Energy
consumption and increase cost savings. Each and every Energy conservation proposals is discussed in depth
along with detailed backup calculations and simple payback Analysis.
Keywords: Energy Conservation, Paper Plant, CHP, Operating Efficiency.
INTRODUCTION
Energy is an essential input to all the productive economic activity and the process of economic development
in the country, which inevitably demands increasingly higher level of energy consumption. The Indian
industrial sector contributes significantly to the national exchequer through a variety of direct/indirect
taxation and by exports. With a highly competitive global market and pressure towards lowering margins,
the industry will be compelled to cut costs. Therefore, in energy intensive industries, ways and means will
have to be found to significantly reduce energy costs.
Potential for energy conservation

The potential for energy conservation is around 25% in Indian Industries and 23% for whole Indian economy.
India’s energy intensity per unit of GDP is higher compared to Japan, US and Asia by 3.7, 1.55 and 1.47
times respectively. This indicates an inefficient use of energy but also substantial scope for energy saving.
The conservative estimate of potential of energy saving in India is creating nearly 25,000 MW of new
capacity. Sector wise potential for energy conservation are Industrial (25%), Transport (20%), Agricultural
(30%), Domestic and Commercial (20%), Economy as a whole (23%).
Energy is a manageable expense, and it can be easily controlled through dedicated efforts. Energy
Conservation is the quickest, cheapest and most practical method of increasing the productivity. Hence,
Energy Conservation will be one of the most important factors to increase the productivity in the future.
Manufacturing process
Plant produces newsprint, Kraft paper, cream wove, copier paper, SS Maplitho and other grades of paper.
There are three paper machines. Newsprint is made in paper machine three mainly and others varieties at
paper machine 1, 2 & 3[1-3] The manufacturing process is given below. Plant manufactures pulp from four
different raw materials. They are:
• Rice Straw
• OCC/Kraft waste
• White cuttings and
• Old newspapers & magazines by de-inking process
Salient Details of CHP Cogeneration plant
MW Co-generation plant
Boiler 4 is rated at 36 Tph, 64 Bar, and 480+10°C. Boiler 4 is supplies steam to the turbine to generate
power. Turbine is an extraction cum condensing type. Design specification of turbine is given below.
Table 5.1: Turbine Design Details
Description Unit Turbine input Extraction Condensing

Steam quantity Tph 36 20 16
Temperature °C 485 305 45.8
Pressure Bar 64 10 0.1
Rated power output from Turbo Generator (TG) for the above specification is 5.75 MW Boiler
4 is designed to operate with either coal or combination of both coal and rice husk. As per turbine
performance curve, the power generated from condensing the steam is 206 kWh/ton. Presently 5.75
MW TG set operates independently with EB power. 5.75 MW TG set is exclusively feeding power to
paper machine 3. During starting of 5.75 MW TG set, the auxiliary power requirement is met from EB.
After TG stabilization, TG is synchronized with EB power and then EB power is disconnected. Since
TG set is running on islanding mode, any change in the demand pattern of the connected equipment
will directly affect the loading of TG set. Also, the nature of the power demand for the paper machine
is variable, and, therefore, TG set operates at varying load. Average loading of TG set for 2009-2010
is 4.86 MW.
Separate cooling tower is in place for condenser cooling. Connected load for condenser cooling
tower is 285 kW (2*110 kW pump and 3*15 kW cooling tower fan, 22 kW condenser extraction
pump). The steam generated from no.4 boilers is used to generate power through a steam turbine
(self-generation, single extraction cum condensing turbine 1 unit which can produce 5.74 MW (actual
production varies from 4.9 to 5.1 MW)).
547
MASS AND HEAT BALANCE
Bar °C
Tph kJ/kg
548

ENERGY CONSERVATION PROPOSAL
Insulate the hot primary air ducts near the boiler 4
Present System
The Forced Draft (FD) fan in the boiler supplies a part of the fresh air to the Primary Air (PA) fan. The air
supplied to the PA fan gets heated up by the hot flue gas so the temperature of the primary air rises above
100 degree Celsius. A common header is provided, which gets the hot air from the two PA fans. This header
then divides into three separate lines and each of them further divides into four ducts. So totally twelve ducts
are provided to supply primary air to the combustion chamber of the boiler. The surface temperatures of the
ducts, (which are not insulated), were measured to be around 80 deg C. The ambient temperature is taken as
30 deg C for calculations.
Proposal
It is proposed to insulate the twelve hot primary air ducts, near the boiler. The length of the ducts was
measured to be 6.5 m, the circumference of the duct was 0.53 m and the surface area requiring insulation
calculated to be around 3.49 m2 for each duct. Since there are twelve ducts, the total surface area requiring
insulation was calculated to be 41.3 m2. The heat loss before insulation is calculated to be 650 W/m2.
Expecting the surface temperature to be 10 deg C above the ambient temperature, the heat loss after
insulation will be only around 100 W/m2. Hence, the annual energy saving after insulation will be around
6706.466 lac. kJ. The details are enclosed in base data and the backup calculation. The cost of insulating
material is estimated to be Rs. 650/m2.
Cost Benefits
• Annual Energy Savings: 1.863 lac. kWh
• Expected Annual Cost Savings : Rs.0.85 lac
• Investment : Rs.0.27 lac
• Payback: 4 months
Base Data
S.No. Description Unit Value

1 Average ambient temperature °C 30
2 Surface temperature of the duct °C 80
3 Surface temperature of the duct after insulation °C 40
4 Coal cost Rs./ton 1700
5 Calorific value of coal kJ/kg 13395.2
6 Circumference of the duct m 0.53
7 Length of the duct m 6.50
8 Total number of ducts to be insulated Nos 12
9 Surface area requiring insulation m2 41.5
10 Annual operating hours Hrs 8160
CALCULATION:
FORMULA:
Heat loss = {(0.548*E)*(((Ts+273.16)/55.55) ^4 -((Ta+273.16)/55.55)^4) +

(1.957*(Ts- Ta) ^ (5/4)) + ((196.85*Vw+68.9)/68.9)^0.5 }
549
Where,
Heat loss in W/m2
E = emissivity (assumed as 0.95)
Ts = Surface temperature, in °C
Ta = Ambient temperature, in °C
Vw = Wind velocity (assumed as 0 m/sec)
Heat loss before insulation= 650 W/m2
Heat loss after insulation = 100 W/m2
Total energy savings = (650-100)*41.5*8160 = 1.863 lacs. kWh
Total Energy Saving in Joules = 1.863*10^5 *860*4.186 = 6706.466 lac kJ
Fuel Saving = (670.646*10^6)/13395.2 = 50.1 Tons
Cost Saving = (50.1*1700)/10^5=Rs 0.85 lac.
Payback = (0.27/0.85)*12 = 4 months
Description Unit Value

Surface area requiring insulation m 2
41.5
Avg. ambient temperature °C 30
Avg. surface temp. before insulation °C 80
Heat loss before insulation* W/m2 650
Expected surface temp. after insulation °C 40
Heat loss after insulation* W/m 2
100
Energy savings W/m2 550
Total energy savings kW 22.83
Annual operating hours hrs 8160
Annual heat savings Mkcal 160.19
Coal cost Rs./ton 1700
Calorific value of the coal kcal/kg 3200
Heat cost Rs./Mkcal 531
Annual cost savings lac Rs. 0.9
Investment lac Rs. 0.3
Payback Months 3.8
ENERGY CONSERVATION PROPOSAL : [2]

Control continuous blow down by incorporating TDS detection system in Boiler [4]
Present system:
At present Continuous Blowdown (CBD) is adopted which blows down certain quantity of steam at regular
intervals thereby keeping the total dissolved solids (TDS) in feed water to the required level. The blowdown
steam has been used to preheat the feed water to some extent. The quantity of blowdown steam, the quantity
of feed water and the temperature rise achieved by the feed water are not being measured.
Proposed system:
The existing system can be made fully automatic by installing a TDS level monitoring facility. The control
system modulates the blow down value based on the inputs from the TDS level detector located in the cooled
550
blow down side steam. Also consider installing a silica monitoring system. By incorporating this system,
the quantity of blowdown can be better controlled than the existing system. This is likely to reduce the blow
down quantity considerably and achieve the steam savings up to 25%.
Cost Benefits:
• Annual water savings: 1799 m3
• Annual steam Savings: 1799 tons
• Annual Cost Savings: Rs. 8.4 lakhs
• Investment (estimated): 6 lakhs
• Payback: 8.6 months
BASE DATA
Sl. No. Description Unit Value

1 Temperature of blow down condensate °C 280
2 Pressure of blow down condensate Bar 64
3 Sensible heat of blow down condensate @ 280°C & 64 bar kJ/kg 1236
4 Sensible heat of steam @ 100°C & 1 bar kJ/kg 419
5 Latent heat of flash steam @ 1 bar kJ/kg 2257
6 Measured quantity of condensed water @100°C m3/day 13.5
7 Specific enthalpy of condensate @280°C kJ/kg 2780
8 Annual operating days Days 340
9 Coal cost Rs./ton 1700
10 Net Calorific Value (NCV) kcal/kg 3200
ENERGY SAVING CALCULATION:

Percentage of flash steam = (1236-419)*100/2257 = 36%
Blow down quantity of flash steam &condensate@280°C = 13.5/0.64 = 21.1m3/hr
Total heat content in the condensate = (21.1*2780)/*4.1861000 = 14.05 MJ/day
Expected Annual saving in heat content=14.05*0.25*340 = 1194 MJ/day
Fuel (coal) saving at 80% boiler efficiency = (1194*1000)/(0.8*3200) = 466 Tons
Annual coal cost saving = 466*1700 = 8 lacs
Expected steam saving per Annum =21.1*0.25*340 = 799 Tons
Expected annual DM water saving =1799
Annual DM water cost saving = 27.5*1799 = 0.49 lac
Net annual cost saving = 8.5 lacs
Investment = 6 lacs
Payback = (6*12)/8.5 = 8.5 months
Description Unit Value

Sensible heat of blow down condensate @ 280°C & 64bar kJ/kg 1236
Sensible heat of steam @ 100°C & 1 bar kJ/kg 419
Latent heat of flash steam @ 1 bar kJ/kg 2257
Percentage of flash steam from the condensate % 36%
Percentage of drained water @100°C % 64%
Measured quantity of condensed water @100°C m3/day 13.5
Blow down quantity of flash steam &condensate@280°C m /day
3
21.16
Specific enthalpy of flash steam & condensate @280°C kJ/kg 2780
551
Total heat content in the condensate MJ/day 58.8

Equivalent heat content Mkcal/day 14.05
Expected percentage saving in blow down % 25%
Expected steam saving per day t/day 5.29
Expected annual steam saving tpa 1799
Expected DM water saving per day m3/day 5.29
Expected annual DM water saving m 3
1799
DM Water cost Rs./m3 27.5
Annual operating days days 340
Annual water cost saving lac Rs. 0.49
Expected saving in heat content Mkcal/day 3.51
Expected annual saving in heat content Mkcal 1194
Coal cost Rs./t 1700
Net Calorific Value (NCV) kcal/kg 3200
Fuel (coal) saving at 80% boiler efficiency T 466
Heat cost Rs./ Mkcal 531
Annual coal cost saving lac Rs. 8
Net annual cost saving lac Rs. 8.4
Investment (estimated) lac Rs. 6
Payback Months 8.55
ENERGY CONSERVATION PROPOSAL [3]

Install Variable Frequency Drive (VFD) for FD fan of Boiler [4]
Present System:
The nominal capacity of Boiler # 4 at Coastal plant is 36 tph of steam
@65 Bar and 460°C. The maximum capacity achieved so far is 38 tph. A Forced Draft (FD) fan meets
the combustion air needs of the boiler, which consists of primary air required for combustion of coal and air
required to keep the bed in the fluidized condition. The air volume drawn is controlled through an inlet guide
vane. The design conditions of the FD fan are 15.8 m3/sec, 700 mmwc static head and 150 kW motor rating.
The required air volume (based on the average monthly coal used and with 4.4% of O2 content in flue gas)
is 14.78 m3/sec with 590-600 mmwc static head and 154.1 kW power consumption. This indicates that there
is scope for speed reduction of the fan based on the volume of air supplied.
Proposal:
It is recommended to install a Variable Frequency Drive (VFD) for the FD fan motor to regulate the speed
and thereby regulate the volume of air supplied. This would practically eliminate the usage of the inlet
guide vane. 93.5% speed reduction is for the required flow of 14.78 m3/s. With this 93.5% speed reduction,
the expected static head is 612 mm which is adequate, and the power consumption reduces to 132.8 kW;
producing a saving of 21.3 kW (nearly 14%). Necessary by-pass arrangements are to be incorporated in the
drive, so as to continue the operation with the inlet guide vane if and when the VFD fails.
Cost Benefits:
• Expected annual energy savings: 1.7 lac kWh
• Expected Annual Cost Savings : Rs. 6.8 lacs
• Investment : Rs. 5.1 lacs
• Payback : 9 months
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BASE DATA
S.No. Description Unit Value
1 Design volume of air m3/s 15.8
2 Required volume of air m3/s 14.78
3 Rated static pressure mmwc 700
4 Type of control - Inlet Guide vane
5 Pressure drop across inlet guide vane mmwc 75
6 Motor rating kW 150
7 Inlet vane opening % 40
8 Average air box pressure mmwc 590-600
9 Energy consumption kW 154.1
ENERGY SAVING CALCULATION:

Possible speed reduction = (14.78*100)/15.8)
= 93.5%
Expected power consumption with VFD = (154.1*0.935^3)/0.95
= 132.6 kW
Power Saving = 154.1-132.78
= 21.32 kW
Annual Cost savings = (154.1-132.78)*8160*3.9/10^5
= 6.8 lacs
Investment = 5.1 lacs
Payback = (5.1*12)/6.8
= 9 months

The paper plant is analysed with complete process description, and the Energy consumption of the plant
is studied and compared with the recommended Specific energy consumption. The Mass and heat balance
of the cogeneration plant are studied, and the required calculation for the balance is calculated. The energy
saving proposals is developed after undertaking a complete energy audit of the plant. The various energy
saving proposals developed will increase the total profitable ratio of the plant after its implementation. It
also helps the plant to be in track for meeting the International standards also. New turbine is proposed with
modification of process demand in place of present one. The cost savings will be 430 lacs and payback will
be 24 months. Various inefficient pumps have replaced with new energy efficient pumps. Lighting energy
saver and new energy efficient lights with electronic choke is proposed. Various uninsulated areas in the
boiler are identified, and insulation is proposed.VFD for pumps and FD fan in the boiler is proposed with
the necessary feedback.
REFERENCE
1. Chemical Recovery in the Alkaline Pulping Process (R.P. Green and G. Hough, Eds.), TAPPI PRESS,
Atlanta, 1992.
2. Lavigne J.R., Instrumentation Applications for the Pulp and Paper Industry, Miller Freeman, San
Francisco, 1979.
3. http://www.census.gov/epcd/ec97/def/322.htm
553
4. http://www.industrialcenter.org/GasIRPaper/Learn%20About/Paper_Manufacture.html
5. www.kpatents.com
6. Pulp and paper plant materials issues addressed by x-ray and neutron diffraction methods C.R. Hubbard,
R.A. Peascoe and J.R. Keiser
7. Research and development sponsored by US Department of Energy through: Industrial Materials for the
Future (IMF) Program and Chemical Industry of the Future, Office of Industrial Technologies, Energy
Efficiency and Renewable Energy.
8. Stelling O. and Vegeby A.P.
554
Energy Management and Integration of PEM Fuel Cell
with UAV for Pharmaceutical Application
Dr. N. Stalin
Assistant Professor, Department of Petrochemical Technology, Anna University, BIT Campus,

Tiruchirappalli - 620024, TN
e-mail: mnstalin@gmail.com, Mob: 9976577636
Abstract:
The main objective of this study is to replace PEM Fuel Cell as an alternative energy source to Li batteries
to power up Unmanned Aerial Vehicle and other possible applications. The author has developed a new
software tool using Visual Basic to identify the performance of PEM Fuel cell using graph obtained
from tested data. The Compatibility analysis made in this paper and other research and development
related with PEM fuel Cell were analyzed. Hydrogen Generator using the sodium boro hydride which
is compatible with 100 Watts PEM fuel cell design and development were analyzed in detail. 200 Watts
PEM fuel Cell, which is Compatible with UAV design, structure and its performance, were studied. Mass
and energy balance of PEM fuel cell are calculated. The possibility of static power generation using fuel
cell was explained, and another recent technological development in PEM Fuel Cell, and its benefits were
explained.
Keywords: PEM Fuel Cell, Li batteries, Hydrogen Generator, UAV design.
INTRODUCTION
INTRODUCTION TO UAV FOR INDIAN ARMED FORCES:
The capability provided by an unmanned system is game changing. It is possible to have your adversaries
under surveillance 24/7. No armed force can do without unmanned systems, and they have proved their
worth in Iraq, Afghanistan and the Balkan conflict. Unmanned systems not only provide a platform for
precision attacks; they go a big way in saving human lives and preventing collateral damage.
The Indian Army and Paramilitary forces encounter terrorists, insurgents and other asymmetric
and urban warfare threats in cities as well as in the challenging terrains of North and the North East
parts of India. An unmanned system goes a long way in assisting the forces to keep control. Unmanned
systems are a strong focus in the prospective plans for Indian defense forces as well as homeland security
agencies. India is building significant unmanned capability for battlefield management for conventional,
asymmetric and urban warfare. Current Indian plans include unmanned systems for ground, underwater
and air superiority.
FUEL CELL BASED UAV IN OTHER COUNTRIES:

The long endurance unmanned aerial vehicle (UAV) has significant value as a low-cost, autonomous
reconnaissance and remote sensing platform for research, commercial and military missions. Fuel cell
power plants are of interest in this application because of the potential to construct power plants of high
specific energy, low noise, low thermal signature, and improved environmental compatibility. Because of
these performance advantages, fuel cells have found their first aviation applications as power plants for
small-scale long-endurance and long-range UAVs. Table 1 lists the demonstrated fuel cell powered UAVs
known to the authors. In 2003, Aero Vironment Inc., a vehicle design and manufacturing company in
Monrovia, California, built and flew the first fuel cell powered aircraft. Its monopoles polymer electrolyte
membrane (PEM) fuel cell system consumes hydrogen from a sodium borohydride reaction vessel. Between
those first flights and the present, a number of researchers and commercial entities have developed fuel
cell powered UAVs of increasing scale and capability. A majority of the demonstration aircraft have
used a PEM fuel cell. A variety of hydrogen storage systems has been used including gaseous pressure
vessels, chemical hydrates and low-pressure cryogenic liquid hydrogen tanks. A notable technological
outlier is the propane-fueled, solid-oxide fuel cell (SOFC) UAV that was constructed in 2006 by Advanced
Materials Inc.

Compatibility Analysis of PEM Fuel Cell for Unmanned Aircraft instead of Li Batteries
UAV:
The Unmanned Aircraft System (UAS) consists of an Unmanned Aircraft (UA) which is essentially a robot
plane together with a Ground Control System (GCS). The Unmanned Aircraft contains a flight control
computer, precision navigation (GPS and an Inertial Measurement Unit) and flight control electronics, a
low vibration engine (such as a Wankel engine) and a payload, such as a high resolution camera. The UA
represents a new, cost effective and more environmentally responsible approach to aerial reconnaissance and
geophysical survey work.
Any aerial application, in which the payload weighs less than the average adult male (say 85 Kg, although
the US military allows a “worst case “soldier weight of 136 Kg) could be performed less expensively and in
a more environmentally friendly way, through the use of an Unmanned Air Vehicle.
Unmanned Aircraft have a historical military presence, in the form of the German V1 flying bomb
of Second World War vintage, followed by the modern turbine-powered cruise missile, such as the US
Tomahawk cruise missile shown below, made by Raytheon. There are also some differences between the
V1, the cruise missile and the UAV: the Unmanned Aircraft returns for reuse. The early civilian Unmanned
Aircraft was in essence a radio controlled aeroplane.
FUEL CELL FOR UAV:

HYDROGEN GENERATION FOR 100 WATTS PEMFC:
A hydrogen generator that generates hydrogen gas from the aqueous solution of sodium borohydride is
developed. The performance testing of the hydrogen generator using a cobalt-phosphorous (Co-P) catalyst
supported on a porous nickel foam is discussed. The microstructures and catalytic activities of the electroless-
deposited Co- P catalysts are analyzed as a function of the electroless deposition conditions such as the pH
and temperatures of heat treatment. The catalytic activity of the supported Co- P catalyst depends largely
on the bath pH and calcinations conditions. For the optimization of the hydrogen generator, hydrogen
generation rate was measured as a function of NaBH4 concentration, NaOH concentration and flow rate of
NaBH4 solution. When the NaBH4 concentration in aqueous solution (< 25 wt.%) is increased, the hydrogen
generation rate linearly increases, but too high NaBH4 concentration (> 25 wt.%) causes clogging of the
reactor because of increased solution viscosity. The hydrogen generation rate is also enhanced when the
flow rate of the solution is increased. The response of hydrogen generation is improved by pumping liquid
catalyst into the reactor before starting feeding the NaBH4 solution.
556
The hydrogen generator successfully provided a stable hydrogen generation rate of 0.5 to 2 L/min. The
hydrogen generator is used to supply hydrogen to a PEMFCs stack, and about 100 W power is produced at
a constant loading of 6.5 A.
HYDROGEN GENERATION:
Figure 2.1: Schematic of the 100 W-scale NaBH4 hydrogen generator
CONNECTING TO PEMFC STACK:

The hydrogen generation system was connected to a commercially-available PEMFC stack (Horizon Fuel
Cell Technologies). The full power output of PEMFC stack is 16 V and 6.5 A. The open-circuit voltage is 24
V. The PEMFC stack was made by joining 22 single cells, each single cell had area of 20 cm2. Hydrogen was
fed to the anode at a flow rate of 1.4 L/min while air was fed to the cathode at 6 L/min. Gases were fed to the
stack without passing through the operating humidifier. Tests were made of power generation at a constant
current of 6.5 A operated by an electronic load (6051A System DC Electronic Load, Agilent).
RESPONSE OF HYDROGEN GENERATOR:

The hydrogen generation rate as a function of time for 20 wt.% NaBH4 solutions and constant feed of 1.5 ml/
min are shown in Fig. 11. The hydrogen starts to generate when the NaBH4 solution is fed into the reactor
by switching on the pump. The hydrogen rates reach a constant value of 0.8 L/min after 30 s without liquid
catalyst and ten switch liquid catalyst, respectively. The hydrogen generation cease in 10 s by switching off
the pump. After the reactor rests for 1 hour, the NaBH4 solution is fed into the reactor by re-switching on
the pump. The hydrogen rates reach a constant value of 0.8 L/min after 120 s without liquid catalyst, and
10 s with liquid catalyst, respectively. The results indicate the response of hydrogen generation with liquid
catalyst being pumping into the reactor is faster than that without liquid catalyst being pumping into the
reactor.
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Figure 2.2: Effects of NaBH4 concentration on hydrogen generation rate of Co-P/Ni catalysts. NaBH4 concentration:
(a) 5, (b) 10, (c) 15, (d) 20, (e) 25 and (f) 30 wt.%. NaOH = 3wt.%, flow rate = 1.5 ml/min
Figure 2.3: Effects of NaOH concentration on hydrogen generation rate of Co-P/Ni catalysts. NaOH concentration: (a)
3, (b) 5, (c) 10, (d) 15 and (e) 20 wt.%. NaBH4 = 20 wt.%, flow rate = 1.5 mL/min
Figure 2.4: Variation of hydrogen generation rate with the flow rate of NaBH4 solution. Flow rates: (a) 0.5, (b) 1.0, (c)
1.5, (d) 2.0, (e) 2.5 and (f) 3.0 ml/min. NaBH4 = 20 wt.%, NaOH = 3 wt.%, flow rate = 1.5 ml/min.
Figure 2.5: Power performance of the PEMFC stack.
558
200 WATTS PEMFC DESIGN:

Membrane Electrode Assembly:
• Electrodes
Anode
Cathode
• Electrolytes
The electrolyte is a thin membrane of a plastic-like film that ranges in thickness from 50 to 175 (microns).
These membranes are composed of perfluoro sulfonic acids, which are Teflon-like fluorocarbon polymers
that have side chains ending in sulfonic acid groups
Figure 2.6: PEMFC Stack Exploded View

Flow Field Plates:
Figure 2.7: Flow Field Plates
559
• The flow fields plates channel fuel and oxidant to opposite sides of the MEA.
• Coolant plates placed between each fuel cell
• Seals between the graphite plates ensure that these flow streams do not mix.
Figure 2.8: Unipolar Plate, Bipolar Plate, Cooling Plate
PEMFC:
A schematic presentation of the principle is illustrated in Figure 1 for an acid electrolyte fuel cell. Hydrogen
is catalytically oxidized at the anode to give protons and electrons. Since the electrolyte is a non-electronic
conductor, the electrons flow away from the anode via the external circuit to the cathode. The protons pass
through the electrolyte to the cathode, where the oxygen gas reacts with the incoming electrons and protons
to produce water. The overall reaction of the fuel cell is the sum of the anode and cathode reactions, i.e. a
combination of hydrogen and oxygen to produce water and energy:
Anodic reaction: H2 → 2 H+ + 2e- E0anode = 0
Cathodic reaction: ½O2 + 2e- + 2H+ → H2O E0cathode = 1.229 V
verall reaction: H2 + O2 → 2H2O E0cell = 1.229 V
Fuel cells are generally classified according to the electrolyte and its ionic conductivity, which can
be hydrogen ions or protons (H+) in Polymer Electrolyte Membrane Fuel Cells (PEMFC) and Phosphoric
Acid Fuel Cells (PAFC) or hydroxyl ions (OH-) in Alkaline Fuel Cells (AFC), or carbonate ions (CO32-) in
Molten Carbonate Fuel Cells (MCFC) or oxide (O2-) in Solid Oxide Fuel Cells (SOFC). The PEMFC is
attractive for portable electronics and transportation applications, and is a major competitor for stationary
power applications less than 100kW.
An exploded view of PEMFC components is shown in Figure 2.9 below. The components include:
(1) the ion exchange membrane; (2) an electrically conductive porous backing layer; (3) a catalyst layer
(the electrodes) sandwiched between the backing layer and the membrane; (4) gaskets for gas tight seal
and electrical insulation; (5) cell plate hardwares with gas channels on one side (end plates or plates for
560
single cells) or on both sides (bipolar plates) that delivers the fuel and oxidant to the reactive sites; (6) other
materials for e.g. interconnecting, cooling, manifold and others.
Figure 2.9: PEMFC Exploded view
Some instruments for fabricating and testing the PEMFC components such as tape-casting machine,
hot-press and test station are shown in Figure 2.9.
The key component in a PEM fuel cell is the proton exchange membrane. The most widely used is
perfluoro sulfonic acid (PFSA) membranes with perfluorinated backbones and sulfonic acid as the terminal
group, and The membranes conductivity is dependent on a certain amount of absorbed water on the sulfonic
sites. This strong dependence of conductivity on water content makes it necessary for the PEMFC system to
manage the water balance carefully through evaporation and condensation of water at a temperature close
to its boiling point. This links up the air humidity, the airflow rate, system pressure and stack temperature
during the PEMFC construction and operation. The presence of water in the system also limits the operational
temperature, typically set at 80°C. At this temperature, the PEMFC has poor tolerance to fuel impurities
(e.g. CO), which results in bulky and complex fuel processing units for CO cleanup when hydrocarbons or
alcohols are used as fuel via reforming.
Figure 2.10: PEM Fuel Cell
561
Figure 2.13: Cooling Airflow Requirement
Hybrid operating option to deliver 600 Watts with Li-Battery:

It can deliver a peak power output of 600Watts in combination with Li battery to meet the high power
requirements during UAS take-off or climbs. This is realized by connecting a lithium-polymer battery in
parallel hybrid configuration. A power management card combines the total power output from fuel cell and
hybrid battery before delivering to the load. It also recharges the battery when excess power is available from
the fuel cell during cruise.
Figure 2.14: Performance of PEM Fuel Cell at Various Altitudes
Plotting PEM Fuel Cell Performance Graph for Energy Management using Software Program
REQUIRED GRAPHS IN ANALYSIS OF PEM FUEL CELL DESIGN:
• Graph for Performance of PEM Fuel Cell
• Graph for Activation Losses as a function of Current Density
• Graph for Activation Losses as a function of Temperature
• Chart Director is an additional software required to plot the Graph
562
GRAPH FOR PERFORMANCE OF PEM FUEL CELL:
Figure 4.1: Time, Voltage, Current Data Form Window:
VB Coding Used for Input Form Window:
GRAPH FOR ACTIVATION LOSSES AS A FUNCTION OF CURRENT DENSITY:
Formula used:
The Butler-Volmer equation is valid for both anode and cathode reaction in a fuel cell. It states that the
current produced by an electrochemical reaction increases exponentially with activation overpotential. This
equation also says that if more current is required from a fuel cell, voltage will be lost. The Butler-Volmer
equation applies to all single-step reactions, and some modifications to the equation must be made in order
to use it for multistep approximations
563
As shown above similarly, we can write VB Coding for this one but the formula only varies we can change the
program according to the requirement.
Typical voltage losses seen in a fuel cell are illustrated. The single fuel cell provides a voltage dependent
on operating conditions such as temperature, applied load and fuel/oxidant flow rates. The standard measure
of performance for fuel cell systems is the polarization curve, which represents the cell voltage behavior
against operating current density. When electrical energy is drawn from the fuel cell, the actual cell voltage
drops from the theoretical voltage due to several irreversible loss mechanisms. The loss is defined as the
deviation of the cell potential
B = R × T /(2 × Alpha × F)
Voltage loss = B × log10 (I / io)
Where n is the number of exchange protons per mole of reactant, F is Faraday’s constant, and a is the
charge transfer coefficient used to describe the amount of electrical energy applied to change the rate of
the electrochemical reaction. The exchange current density, io, is the electrode activity for a particular
reaction at equilibrium. In PEM fuel cells, the anode io for hydrogen oxidation is very high compared
to the cathode io for oxygen reduction; therefore, the cathode contribution to this polarization is often
neglected. Intuitively, it seems like the activation polarization should increase linearly with temperature
based upon Equation
Figure 4.2: Activation Losses as a function of Current density
564
GRAPH FOR ACTIVATION LOSSES AS A FUNCTION OF TEMPERATURE:
Formula Used:
Where n is the number of exchange protons per mole of reactant, F is Faraday’s constant, and a is the
charge transfer coefficient used to describe the amount of electrical energy applied to change the rate of the
electrochemical reaction26. The exchange current density, io, is the electrode activity for a particular reaction
at equilibrium. In PEM fuel cells, the anode io for hydrogen oxidation is very high compared to the cathode io
for oxygen reduction; therefore, the cathode contribution to this polarization is often neglected. Intuitively, it
seems like the activation polarization should increase linearly with temperature based upon Equation
Figure 4.3: Activation Losses as a function of Temperature
From these graphs, we can simply determine the performance analysis of PEM Fuel Cell.
565
CONCLUSION
Data presented in the paper shows that refinements can be made to the power plant and propulsion system to
improve the performance of the UAV. The primary components in which refinement of the design can lead
to climbing rate and acceleration rate increases are the electric motor and propeller. Improving the hydrogen
utilization of the fuel cell power plant can increase the endurance of the aircraft. These types of trade-offs
identified in this study illustrate the need to integrate detail subsystem models with system-level optimization
in the design viable fuel cell aircraft.The development and refinement of the fuel cell demonstrator aircraft
will continue with the goal of developing long endurance fuel cell UAVs based on the technologies and
lessons learned of this project.Future work can be done in determining a suitable set of design requirements
for an optimum vehicle by conducting a conceptual design study of a UAV using SOFC fuel cell technology.
REFERENCES
1. Velev O., “Summary of fuel cell projects: AeroVironment 1997-2007.” National Hydrogen Association
Fall 2007 Topical Forum, Charlotte, SC, Oct. 2007.
2. AeroVironment Press Release, “AeroVironment flies world’s first liquid hydrogen-powered UAV,”
Jun. 2005.
3. Scheppat B., “Betriebsanleitung fuer das brennstoff-zellenbetriebene Modellflugzeug,“ Fachhochschule
Wiesbaden, 2004.
4. Kellogg J., “Fuel cells for micro air vehicles,” Joint Service Power Exposition, Tampa, Florida,
May 2005.
5. Crumm A., “Solid Oxide Fuel Cell Systems,” Proceedings of the Fuel Cell Seminar, Honolulu, HI,
Nov. 2006
6. Bradley T.H., Moffitt B., Mavris D. and Parekh D.E., “Development and Experimental Characterization
of a Fuel Cell Powered Aircraft,” Journal of Power Sources, Vol. 171, 2007, pp. 793-801.
7. California State University Press Release, “Cal State L.A.’s fuel-cell plane passes key flight test,”
Sep. 2006.
8. Deutsches Zentrum fuer Luft- und Raumfahrt Press Release, “Erfolgreicher erstflug des Hyfish,”
April 3, 2007.
9. Herwerth C., Chiang C., Ko A., Matsuyama S., Choi SB., Mirmirani M., Gamble D., Arena A., Koschany
A., Gu G. and Wankewycz T., “Development of a Small Long Endurance Hybrid PEM Fuel Cell Powered
UAV,” Society of Automotive Engineers Paper 2007-01-3930, Sep. 2007.
10. Kwon S., “PEM fuel cell system for UAV,” Available online at http://rocket.kaist.ac.kr/03_sub_08.htm
11. “AeroVironment’s unmanned aircraft achieves record flight,” Fuel Cells Bulletin, Vol. 2007, No. 8,
2007, p. 8.
12. Friend M.G. and Daggett D.L., “Fuel cell demonstrator airplane,” AIAA Paper 2003-2868, Jul. 2003.
13. Kohout L.L. and Schmitz P.C., “Fuel cell proplulsion systems for an all-electric personal air vehicle.”
AIAA Paper 2003-2867, Jul. 2003.
14. Baldock N., and Mokhtarzadeh-Dehghan M.R., “A study of solar-powered, high-altitude unmanned
aerial vehicles,” Aircraft Engineering and Aerospace Technology: An International Journal, Vol. 78,
No. 3, 2006, pp. 187–193.
15. Nickol C.L., Guynn M.D., Kohout L.L. and Ozoroski T.A., “High altitude long endurance air vehicle
analysis of alternatives and technology requirements development,” AIAA Paper 2007-1050, Jan. 2007.
16. Burke K.
566
Pharmacognostical Evaluation of Duranta repens Leaves
Emdad Hossain, Ranadheer Rai and Rishikant Tripathi
Pharmacy College, Azamgarh 276128, Uttar Pradesh, India

e-mail: emdad@rediffmail.com
Abstract:
Under the verbena family (Verbenaceae), Duranta repens Linn is commonly known as Golden dewdrop, is
found in India, Pakistan, Egypt, West Indies, as well as in Central and South America. It is shrubs to small tree
and cultivated mostly as an ornamental plant bearing blue purple flowers. The infusion of leaves and juices
of the fruits are used as diuretic. Stem and fruit extracts also possess mosquito repellent property. Several
compounds like durantanin, campenoside I, cistanoside E, acteoside, iridoid glycosides as durantosides I, II,
III, IV, and lamiide were isolated from different parts of the plant. Though the plant have so much potentiality,
but proper pharmacognostical standardization of the plant parts was still required.
Different pharmacognostical parameters were evaluated including macroscopical and microscopical
characters along with powder microscopy of the powdered leaves. Other pharmacognostical parameters like
physical constant values, extractive values and behavioral aspects of powdered leaves with different chemical
reagents were also determined by standard methods. In the microscopical features of Duranta repens leaves,
6-13 longitudinal arrangement of xylem was present and it was highly compacted with phloem cells at the
centre of the midrib. Epidermal hairs were found to be present. In the powder microscopy, starch grain,
unicellular covering trichome and glandular trichome etc. were seen. Thus the present study will provide
significant information in respect to the proper identification of the plant for suitable utilization in different
herbal formulation.
Keywords: Duranta repens, Glandular trichome, Pharmacognostical character, Verbenaceae.
INTRODUCTION
Since the time immemorial plants and different plant products have been used extensively for treating various
illnesses for ruminants and human. Authentication of the starting crude material is essential to ensure the
reproducible quality of herbal products. According to World Health Organization (WHO), the macroscopic
and microscopic description of a medicinal plant is the most necessary step towards establishing the identity
as well as the degree of purity of such materials and should be carried out before any tests are undertaken [1].
Under the family (Verbena family) Verbenaceae, Duranta is an important genus of containing flowering
plants. It contains for about 17 species of shrubs and small trees that are native from southern Florida to
Mexico and South America. They are commonly cultivated as hedges and ornamental plants in tropical
and subtropical countries [2]. Duranta repens Linn is an important ornamental plant commonly known as
Golden dewdrop and well cultivated throughout India. It is evergreen shrub to small tree, usually matures
with a height 1-3 m. Crowns of golden dewdrop plant are replete with fine branches and twigs that are often
form thorny shape. The bark is light gray, becoming rough when old. Light-blue, blue purple or white,
tubular flowers are borne on terminal or axillary racemes. The yellow or yellow-orange fleshy fruits are
grown in hanging clusters [3].
As per literature, it is extensively used to treat malaria and intestinal worms [4]. Ethyl acetate and
aqueous extracts of the leaves showed significant antimalarial activity [5]. a-Amyrin, β-sitosterol,
scutellarein, 4′-methoxy scutellarein, pectolinarigenin were isolated from leaves [6]. Two new cytotoxic
triterpene saponins, named durantanin IV and V were isolated from powdered leaves of the plant [7].
The present research work is associated with characterization of different pharmacognostical parameters
of Duranta repens leaves, included botanical identification, macroscopical study, photomicroscopic study,
powder characteristics, leaf constants, analytical standardization, florescence study etc. Hence, all the
experimentations were done in this regard have been described below.
Botanical identification
Duranta repens leaves were collected from Chandeshwar, Azamgarh, Uttar Pradesh, India during December
2012 and the botanical identification was done by Dr N.K. Dubey, a taxonomist and Professor, Department
of Botany, Banaras Hindu University, Varanasi, India. The voucher specimen (no. Verb. 2013-34) was
deposited in the herbarium of Natural Products Laboratory, Pharmacy College, Azamgarh, India for future
reference.
Macroscopical studies
The leaves of the plant were studied for their macroscopic characters such as size, shape, margin, apex,
surface, color, odor, taste, nature and texture as per given procedure in WHO guidelines on quality control
methods for medicinal plants materials [8].
Photomicroscopic study
Collection of Specimens
For the photomicroscopic study, the leaves of Duranta repens were freshly collected from selected healthy
plants. Followed by collection the leaves were fixed in alcoholic formalin acetic acid solution prepared by
mixing formalin, acetic acid and 70% ethyl alcohol at the ratio of 1:1:18 [9].
Preparation of Sections
After 18 h of fixation, the specimens were sectioned with the help of sharp razor and about 10-20 µm
thickness of the sections was made. The section was treated with chloral hydrate followed by that; the sections
were stained with safranin. However, whenever necessary, sections were also stained with phloroglucinol-
hydrochloric acid and iodine-potassium iodide. For studying the stomatal morphology, venation pattern,
paradermal sections (parallel to the surface of leaf) were made and then cleared with 5% sodium hydroxide
or epidermal peeling was prepared through partial maceration by employing Jeffrey’s maceration fluid [10].
Cleared sections were then mounted with glycerin for microscopical observation. The different cell types
were later observed at lower and then higher magnification for the report.
Powder Microscopy
The powdered materials of leaves of the plant were also cleared with chloral hydrate, further treated either
with acetic acid or with phloroglucinol and hydrochloric acid or with iodine-potassium iodide solution as per
requirement and then mounted in glycerin medium.
Photomicrographs
For microscopic description of tissues, photomicrographs of different magnification were taken with
Samsung S750 digital microscopic unit. Descriptive terms of the anatomical features were given as per
standard anatomy books [9, 11].
568
Leaf constants
Determination of quantitative microscopical constants like vein-terminal number, vein-islet, palisade ratio
and stomatal index were done according to the established procedures [11].
Analytical parameters
Various analytical parameters like foreign matter, ash value, foaming index, swelling index and water content
in the powdered raw materials were determined by following the WHO guidelines [8]. In earlier study it was
found that different plant secondary metabolites showed specific color with different chemicals [11]. In this
regard, powdered material of and Duranta repens leaves, was mixed with different chemical agents like
dilute hydrochloric acid, concentrated hydrochloric acid, dilute sulphuric acid, concentrated sulphuric acid,
5 & 10% potassium hydroxide, ferric chloride, distill water, ammonia, iodine and observed under normal
light and ultra violet light (254 nm and 365 nm) for the effect of different chemical agents on powdered crude
drug [8]. This preliminary study helps for standardization of the crude drug as well as further processing of
the sample with some indication regarding the nature of chemical compounds present in it.
Fluorescence Study
Coumarins, flavonoids and other different compounds produce specific florescence characteristics which
are helpful for preliminary chemical study as well as for standardization of specific plant materials. For
florescence study, the powdered leaves were extracted with different solvents like petroleum ether (40-60°C),
cyclohexane, ethyl acetate, acetone, methanol, water in cold maceration technique for 24 h; the sample were
filtered and filtrate was seen under ultra-violet rays at 365 nm for observing any specific florescence.
RESULT
Macroscopical Features
The shrub has the simple and opposite leaves which are pinnate in venation and light green in appearance. It
is elliptic to ovate in shape, acute in apex, petiolet, entire to partially serrate in margin and the length of leaf
is 1.5-10.1 cm long and 0.9-3.6 cm in width. Dried leaves are grayish green in color, rough texture, slight
bitter in taste and brittle in nature.
Microscopical Description
Leaflets
The leaflets of the plant were dorsiventral in symmetry with circular midrib and thin lamina forming wings
on either side of the midrib (Figure 1). The midrib has consisted of outer ground tissue and complex vascular
structure. Epidermal layer was thin with small cell and prominent cuticle. The upper epidermis was found
to be formed of two layers of epidermal cells whereas lower epidermis has consisted of single layer of
epidermal cells. The inner portion of the abaxial part consisted of fairly wide zone of collenchyma cells
which have thick walls without intercellular space and somewhat starch-grains found to be present. The
vascular tissues formed a complex structure. These types of cells are very big in size with compare to
cells present in between upper and lower epidermis. The midrib consisted of a circular vascular bundle
consisting of 6-13 longitudinal arrangement of xylem strand present in the centre of midrib along with highly
compacted phloem cells. Diameter of xylem cells was about 3-6 mm (Figure 3a). Unicellular sharp pointed
trichomes were also found above the upper epidermis at midrib (Figure 1).
Lamina
The lamina was 250-340 µm thick. The adaxial epidermis was thin with prominent cuticle. The epidermal
cells were about 12-14 µm thick. Inner to the epidermis, there was one layer of sub-epidermal cells found
569
somewhere. 2-3 layers of palisade parenchyma were found in adaxial portion of lamina. Thin epidermis cells
were found at abaxial side. Epidermal hairs were found present on the lamina surface. Spongy parenchyma
was found in abaxial part with less intercellular space (Figure 2).
Epidermal Cells and Stomata

In surface view, the adaxial epidermis appeared small, polygonal in shape, thin walled and straight; no
stomata occurred on the adaxial epidermis (Figure 3d). Abaxial epidermis was found wavy walled in shape.
Stomata occurred in the abaxial part. The abaxial epidermal cells were narrow, with straight, thick anticlinal
walls. The stomata were dense; they were anomocytic or cyclocytic type i.e. the stoma was surrounded by
circle of subsidiary cells (Figure 3e). Glandular trichomes were also frequently seen in the abaxial epidermis.
They were short with 2-4 cellar glandular head.
Powder Microscopy
In powder microscopy of Duranta repens leaves different characteristics were found are as follows:
Starch granules: They were made up of 6.5-10 µm mostly circular in shape.
Trichome: Covering trichome was multi-cellular, unilayered non-lignified. Grandular trichome was also
found attached with epidermis.
Stomata: Anomocytic type of stomata was found in powder microscopy.
Physical Parameters and Extractive Values

Different physical parameters and extractive values for leaves of Duranta repens were represented in Table
1 and 2.
Other Phytochemical Parameters

The results of florescence study were depicted in Table 3. The result of effects of different chemical agents
of powdered leaves of Duranta repens, was displayed in Table 4.
DISCUSSION
An interest in herbal drugs have increased tremendously because of various reasons and scientists working
with medicinal plants are sincerely trying to rejuvenate several plant drugs of traditional system of medicine
through experimentations using modern tools for the last two decades. It is well known that for identification of
plant drugs, the pharmacognostical study is one of the most important criteria [12]. Hence, it is evident that the
present study would provide various resourceful information in relation to pharmacognostical identification
of Duranta repens leaves. Furthermore, information regarding physicochemical characteristics of such plant
leaves and nature of chemical constituents present in them would also be useful for standardization of such
herbal drugs of folk medicinal practice of present era and enrichment of Ayurvedic Pharmacopoeia. It would
also help scientists to utilize such needful information regarding the plants identity and characteristics in
building new poly herbal formulations.
ACKNOWLEDGMENT
We are thankful to Mr. Bajrang Tripathi, Manager, Pharmacy College, Azamgarh for his kind support.
REFERENCES
1. World Health Organization. Quality control methods for medicinal plant materials, Geneva: WHO
Library, 1998, pp.1-115.
570
2. Duranta [Cited 12 March 2013]. Available from: http://en.wikipedia.org/wiki/Duranta

3. Francis JK. Golden dewdrop [Cited 8 March 2013]. Available from: http://www.fs.fed.us/global/iitf/pdf/
shrubs/Duranta%20erecta.pdf
4. Whistler WA, Tropical ornamentals, a guide. Portland: Timber Press, Inc. 2000, p. 542.
5. Castro O, Barrios M, Chinchilla M and Guerrero O, Chemical and biological evaluation of the effects of
plant extracts against Plasmodium berghei. Revista de Biologia Tropical 1996; 44(2A): 361-367.
6. Makboul AM and Abdul BAM, Flavonoids from the leaves of Duranta repens, Phytoterapia 1981; 52:
219-220.
7. Ahmed WS, Mohamed MA, El-Dib RA and Hamed MM, New triterpene saponins from Duranta repens
Linn and their cytotoxic activity. Molecules 2009; 14: 1952-1965.
8. World Health Organization, Geneva, Quality Control Method for Medicinal Plant Materials, New Delhi;
A.I.T.B.S. Publisher and Distributors, 2002, pp.8-24.
9. Asoka J, Botanical microtechnique: Principles and practice, First edition, Chennai; Plant Anatomy
Research Centre, 2006, pp. 7-86.
10. Sass JE, Elements of Botanical Microtechnique, New York; Mc Graw Hill Book Co, 1940, p. 222.
11. Khandelwal KR, Practical Pharmacognosy, Fifteenth edition, Pune; Nirali Prakashan, 2006.
12. Kulkarni YA, Yele SU, Gokhale SB and Surana SJ, Pharmacognostical studies of leaves of Machilus
macrantha Nees. Phcog Mag 2008; 4(13): 83-88.
Table 1: Physical parameters for leaves of Duranta repens
S.No. Parameters Results

1. Foreign matter None
2. Total ash (mg/g) 7.8 ± 0.03
3. Loss on drying 11.9%
4. Foaming index <100
5. Swelling index 1.0 cm
Table 2: Extractive values for various solvents for leaves of Duranta repens
S.No. Solvent Extractive value (% w/w)

1. Water 32.75
2. Methanol 25.50
3. Chloroform 4.50
4. Petroleum ether (40-60°C) 3.01
5. Acetone 6.75
Table 3: Florescence Study for various solvents having extract of leaves of Duranta repens
S.No. Solvent Florescence

1. Petroleum ether (60-80°C) Light orange
2. Cyclohexane Light yellow
3. Ethyl acetate Light yellow
4. Acetone Greenish yellow
5. Methanol Greenish yellow
6. Aqueous Deep grey
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Table 4: Effect of Different Chemical Agents on Powdered Duranta repens leaves
Treatment Normal light Ultra violet light

254 nm 366 nm
Blank Grayish green Dark grey Black
Powder + Dilute hydrochloric acid Yellow Green Black
Powder + Conc. hydrochloric acid Green Green Black
Powder + Dilute sulphuric acid Yellow Green Dark green
Powder + Conc. sulphuric acid Brownish black Black Black
Powder + 5% potassium hydroxide Light brownish yellow Greenish black Brownish black
Powder + 10% potassium hydroxide Brownish yellow Greenish yellow Black
Powder + 5% Ferric chloride Light black Greenish black Black
Powder + Distill water Greenish yellow Black Black
Powder + Ammonia Brownish yellow Greenish brown Yellowish black
Powder + Iodine Light black Light yellow Black
Table 5: Leaf constants for Duranta repen

Sl. No. Parameters Value
1. Stomatal number Lower surface 112-138
Upper surface Nil
2. Palisade ratio 2.67-2.83
3. Vein – islet number 31-42
4. Vein-termination number 12-17
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Figure 1: Transverse Section of midrib
Ep: Epidermis; Ph: Phloem; X: Xylem; Co: Collenchyma
Figure 2: Transverse Section of lamina

573
UE: Upper Epidermis; Pp: Palisade Parenchyma; Sp: Spongy Parenchyma; LE: Lower Epidermis
Figure 3: Different anatomical features
Figure 4: Different features in powder microscopy
574
Screening of Aegle marmelos for Antidiabetic Activity in
Streptozotocin Induced Diabetic Rats
S. Jebaseelan1, Dr. P. Ramasubramanian2 and Dr. Venkatesh3
1
Professor, Dept. of Pharmaceutical Chemistry, Ultra College of Pharmacy, Madurai, Tamil Nadu, India
2
Professor, Dept. of Pharmaceutics, 3Professor, Dept. of Pharmaceutical Chemistry, Sankaralingam
Bhuvaneshwari College of Pharmacy, Sivakasi, Tamil Nadu, India
Corresponding Author e-mail: jebaseelanmpharm2000@rediffmail.com
Abstract:
Background: Diabetes is a chronic metabolic disorder that has emerged as one of the main alarms to human
health in the 21st century. Many allopathic medicines are available to treat, but treatment is associated
with many side-effects that necessitate the replacement of allopathic medicine with natural drug. As an
alternative medicine to treat diabetes mellitus, many herbal drugs are being studied throughout the world.
Aim: In the present study an attempt was made to investigate the anti-diabetic activity of Aegle marmelos in
streptozotocin-induced diabetic rats. Materials and Methods: The study was conducted among five groups
with three albino rats in each group. The ethanolic and aqueous extracts were administered for seven days.
Results: The plasma glucose concentration significantly decreased by both the extracts when compared with
the control. Conclusion: The study shows that the ethanolic and aqueous extracts of Aegle marmelos has
antihyperglycemic activity, can ameliorate the diabetic state and is probably the source of hypoglycemic
compound.
Keywords: Aegle marmelos, Diabetes, Streptozotocin, Extracts.
INTRODUCTION
Diabetes mellitus is a complex chronic condition that is a major source of ill health worldwide. This
metabolic disorder is characterized by hyperglycemia and disturbances in carbohydrate, protein and fat
metabolisms, secondary to an absolute or relative lack of the hormone insulin. Besides hyperglycemia,
several other factors including hyperlipidemia or dyslipidemia are involved in the development of micro
vascular and macro vascular complications of diabetes, which are the major causes of morbidity and
death [1]. The incidence of diabetes mellitus is increasing all over the world and is becoming a problem of
significant importance. The World Health Organization (WHO) has declared India as the diabetic capital
of the world. Globally diabetes affects 246 million people, which is about 6% of the total adult population.
It is fourth leading cause of death by disease and every 10 seconds, a person dies from a diabetes-related
cause in the world [2].
The growing number of diabetic patients and the current probable future health care constraint will mean
that high-quality diabetic care will become increasingly difficult to be delivered in the future. This requires
identification and implementation of cost-effective treatment measures that will improve diabetic care in the
long term, decrease the associated morbidity and reduce the high direct and indirect costs or its management.
This can be possible by incorporation of cost effective and efficient medicinal plants in the management of
diabetes [2]. Long term complications arising due to diabetes mellitus and serious side effects shown by
synthetic hypoglycemic agents has continued the search for more effective and safer anti diabetic agents
[3]. Oral hypoglycemic agents are useful in the treatment of diabetes, but their use is restricted by their
pharmacokinetic properties, secondary failure rates and accompanying side effects [4].This has highlighted
the importance and relevance of traditional medicinal plants [2]. World Health Organization expert committee
on diabetes has listed as one of its recommendations that traditional methods of treatment for diabetes should
be further investigated [4]. Herbal medicinal products are defined as any medicinal product, exclusively
containing one or more active substances. WHO report 80% of the world population relies on the drug from
natural origin. A large number of plants are used in the treatment of diabetes. Less toxicity, better therapeutic
effect, good patient compliance, and cost-effectiveness are the reasons for choosing a drug from natural
origin [5]. India has about 45,000 plant species and several thousand have been claimed to possess medicinal
properties. Among Indian traditional medicinal plants, several potential anti-diabetic plants and herbs are
being used as part of our diet since prehistoric times [2]. In the Ayurveda system of medicine, as ents and
various Asthanga sangraha, there are about 600 plants that are stated to have antidiabetic property. In the
traditional system of Indian medicine plant formulation and several cases, combined extracts if plants are
used as a choice rather than individual plant [4].
REVIEW OF LITERATURE
Aegle marmelos (Linn.) Correa
Aegle marmelos (linn) Correa commonly known as Bael (or Bel) belonging to the family rutaceae, is a
moderate-sized, slender and aromatic tree. A number if chemical constituents and therapeutic effects of
A.marmelos have been reported by different workers. Extensive investigations have been carried out on
different parts of A.marmelos have been carried out on different parts of the plant and as tannins, carotenoids
and seed oils and other miscellaneous compound properties like antidiabetic, antiulcer, antioxidant,
antimalarial, anti-antifungal, antiviral and antibacterial activities [6].
Sharma et al. reported hypoglycemic and hypolipidemic effect of A.marmelos (L.) leaf extract on
streptozotocin-induced diabetic mice [7].
The antidiabetic activity of the leaves of A.marmelos was reported in alloxan induced diabetic rats. The
methanolic extract of the leaves reduced the blood sugar level. Reduction in blood sugar could be seen from
6th day after continuous administration of the extract, and 12th-day sugar levels were found to be reduced
by 54% [8].
The hepatoprotective effect of the leaves of A.marmelos were reported in alcohol-induced liver injury
in albino rats [9].
The leaves of A.marmelos were reported to possess analgesic activity. The methanol extract of the leaves
significantly reduced the writhing induced by acetic acid in swiss mice [10].

Plant Material:
Aegle marmelos leaves were collected from Madurai district of Tamil Nadu, India. The species were identified
and authenticated at the Department of Botany, American College, Madurai, Tamil Nadu
Extraction:
The fresh leaves were washed under running water followed by rinsing with distilled water; shade dried and
pulverized in a mechanical grinder to obtain coarse powder. The dried powdered leaves were extracted with
different solvents such as petroleum ether, chloroform, ethanol and aqueous water by soxhelation process at
a temperature not exceeding the boiling point of solvent (table 1)
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Table 1: Percentage Yield of Different Extracts
Sl.No Solvent Colour of the Extract Percentage of Yield

1 Petroleum ether Dark Green 4%
2. Ethyl acetate Dark Green 3%
3 Ethanol Brown 4%
4 Aqueous Dark Brown 3%
Preliminary Phytochemical Investigations

Preliminary phytochemical screening of Aegle marmelos Linn leaf powder was done following standard
methods and the results are presented in table 2.
Table 2: Result of Preliminary Phytochemical Investigations of Aegle marmelos Linn
Name of the Pet.Ether Ethyl acetate Ethanol Aqueous

constituent Extract Extract Extract Extract
Sterols - - + -
Terpenoids - + + +
Carbohydrate - + - +
Flavonoids - + + +
Proteins - + - +
Alkaloids - + + -
Glycosides - + + -
Tannins - - + +
Saponins - + + +
Phenolic - + + +
Compounds
Fixed oil & Fats - - + +
Cumarin - - - +
(+) Presence of constituents (-) Absence of constituents
LCMS STUDY
UHPLC – ESI MS/MS Study on Ethanolic Extract of Aegle marmelos
The LCMS study of the ethanolic extracts of Aegle marmelos study identified the following compounds:
1) Quercetin: Molecular Formula : C15H10O7

Structural Formula:
577
2) Rottlerin: Molecular Formula: C27 H26 O7

Structural Formula
3) Rutin: Molecular Formula: C27 H30 O16

Structural Formula:
Table 3: UHPLC-ESI MS/MS Analysis of Ethanolic extract of Aegle marmelos
[M-H] M/Z (g/mol) Retention Time (min) Fragmentation In MS HPLC –ESI Identity Molecular Formula
–MS
505.3 16.6-17.0 155.2,191.2,285.2 Rottlerin C27H26O
301.2 16.0-16.5 125.2,139.2,189.3 Quercetin C15H10O7
509.5 24.0-24.2 169.1,271.3,300.3 Rutin C27H30 C16
LCMS PEAK OF ETHANOLIC EXTRACT OF AEGLE MARMELOS
578
Experimental Animals:
Male albino rats weighing between 150-250 gm were chosen for the study. They were allowed free access
to food (standard pellet diet) and water ad.linbitum. The animals were housed in cages under ambient
temperature and lighting. The experimental protocol was approved by the Institutional Animal Ethical
Committee. The animals were divided into five groups of three rats each. All the chemicals used for the
study were of analytical grade.
Induction of Diabetes:
Diabetes was induced in rats by the intraperitoneal injection of streptozotocin at a dose of 150mg/kg b.w.
Dissolved in citrate buffer. Each animal with a blood glucose level greater than 200mg/dl was considered to
be diabetic.
Experimental Design:
The fasting blood glucose levels of all the rats were determined before the start of the experiment. The rats
were divided into five groups consisting of three rats each.
Group I: Normoglycemic control group, administered vehicle served as control
Group II: Diabetic control, administered streptozotocin served as control
Group III: Diabetic rats treated with Glibenclamide 10mg/kg
Group IV: Diabetic rats treated with ethanolic extract 400mg/kg once daily
Group V: Diabetic rats treated with aqueous extract 400mg/kg
The five groups were given the agents for seven days. The blood glucose levels of animals were recorded
on day 0, 3, and 7. The blood was collected by puncturing the tail vein
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Table 4: Hypoglycemic Activity of Ethanolic and Aqueous Extracts of Aegle marmelos
S.No Group Serum glucose mg/dl

0 day 3rd day 7th day
1 Untreated control 89.17 ± 5.1* 89.50 ± 5.2* 89.75 ± 4.5*
2 Diabetic control 275.60 ± 5.3 343.45 ± 5.5 354.25 ± 5.6
3 Diabetic + glibendamide (10 mg/kg) 260.17 ± 4.5* 145.25 ± 2.9 105 ± 4.4*
4 Diabetic+ Ethanolic Extracts (400 mg /kg ) 255.70 ± 3.5 160.35 ± 4.4 120.27 ± 3.5*
5 Diabetic +Aqueous Extract (400 mg/kg) 260.74 ± 4.4 164.75 ± 5.2 125.35 ± 3.5*
All value are expressed as mean ± SEM (n=3). The statistical analysis was carried out by one way ANOVA
and the *P value < 0.1 considered as significant
RESULTS
Preliminary phytochemical analysis of Aegle marmelos showed the presence of steroids, flavonoids, fatty
acids, tannins, saponins, phenolic compounds in an aqueous and ethanolic extract.The following three
compounds Quercetin, Rottlerin, Rutin were identified by the LCMS study. Aqueous extract and ethanolic
extract of Aegle marmelos exhibited significant hypoglycemic activity against streptozotocin-induced
hyperglycemia. The activity was found to be dose-dependent that is the activity increased with dose and
was comparable to the standard drug Glibenclamide. Hence, the ethanolic extract of Aegle marmelos has
more effect than the aqueous extract of Aegle marmelos. The ethanolic extract of Aegle marmelos at the
doses 500mg/kg exhibited significant anti-diabetic activity. The activity was comparable to the reference
drug of Glibenclamide.
DISCUSSION
Diabetes mellitus of long duration is associated with many complications and have long been assumed to be
related to chemically elevated blood glucose level. Diabetes mellitus causes disturbances in the uptake of
glucose as well as glucose metabolism. The current study findings that the ethanolic and aqueous extracts
of the leaves of Aegle marmelos significantly decreased the serum glucose levels in streptozotocin-induced
rats is supported by previous works done by Sabu & Kuttan [8] and Sharma et al [7]. Certain plants like
Momordica charantia, Glymen sylvestre, Madhu indica, Tinospora cardifolia, Ocimum sanctum and Costus
speciosus have found to be effective in reducing blood sugar level in diabetic rats [11].
CONCLUSION
We conclude that the ethanolic and aqueous extract of Aegle marmelos leaves was effective in decreasing the
serum level in diabetic rats. This evidence suggests that the leaf of Aegle marmelos could be beneficial for
the protection and alleviation of diabetic complications. Further studies need to be carried out to strengthen
the current evidence.
BIBLIOGRAPHY
1. Sikarwar MS and Patil MB, (2010), Antidiabetic activity of Pogamia pinnata leaf extracts in alloxan-
induced diabetic rats. Int J of Ayurveda Res. 1(4): 199-204 [doi: 10.4103/0974-7788.76780]
2. Apparna K, Spandana S and Poshadri A, (2010), Medicinal plants for Diabetes mellitus, Health Action.
32-34.
3. Juvekar AR and Bandawane DD, (2009), Antihypergycemic and Antihyperlipidemic activity of Aele
marmelose (L.) leaf extract I streptozotocin induced diabetic rats, Indian Drugs. 46(7): 43-49.
580
4. Sikarwar MS and Patil MB, (2010), Preparation and evaluation of antidiabetic polyherbal formulation,
Indian Drugs.47(12):27-34.
5. Chandira M and Jayakar BB, (2010), Formulation and evaluation of herbal tablets containing Ipomoea
digitata Linn. Extract. Int J of pharm Sci Rev and Res. 3(1):101-110.
6. Dhankar S, Ruhil S, Balhara M, Dhankar S and Chhillar AK, (2011), Aegle marmelos(Linn.) correa:
A potential source of phytomedicine. Journal of Medicinal Plants Research.5(9):1497-1507 [Available
online at http://www.academicjournals.org/JMPR].
7. Sharma B, Satapathi SK and Roy P, (2007), Hypoglycemic and hypolipidemic effect of Aegle marmelos
(L.) leaf extract on streptoszotocin induced diabetic mice, Int J of Pharmacology, 3:444-452 [doi:
10.3923/ijp.2007.444.452].
8. Sabu MC and Kuttan R, (2003), Indian J Physiol. Pharmacol., 48:81-88.
9. Singanan V, Singanan M and Begum H, (2007), International Journal of Science & Technology.2:83-92.
10. Shankarananth V, Balakrishnan N, Suresh D, Sureshpandian G, Edwin E and Sheeja E, (2007),
Fitoterapia., 78:258-259
11. Pavan Kumar K, Vidyasagar G, Ramakrishna D, Madhusudhana Reddy I, Gupta Atyam VSSS and
Sarva Raidu Ch, (2011), Screening of Madhuca indica fr antidiabetic activity in streptozotocin and
streptozotocin-nicotinamide induced diabetic rats. International Journal of Pharm Tech Reseearch,
3(2):1073-1077.
581
Anthelminthic Activity of Acalypha indica Belongs to the
Family Euphoriaceae
S. Kalimuthu, V. Balasubramanian and R. Xavier Arulappa
S.A Raja Pharmacy College-Vadakangulam, Tiruenlveli Dist-627116

e-mail: kalimuthupharma017@gmail.com
Abstract:
Plants have formed the basis for traditional medicinal systems for thousands of years, with the first records
dating from about 2600 BC in Mesopotamia. Peoples used oils from cedar and cypress, licorice, myrrh, and
poppy juice, among other things, substances that are still in use today for the treatment of a variety of illnesses
and infections. Ancient Egyptian, Chinese, and Indian documents show that medicine in these societies
included numerous plant-based remedied preventives. The Greeks and Arabs both contributed substantially to
the assimilation, codification, and development of plant-based medicines. The isolation of the active principles
from the plants and herbs such as strychnine, morphine, and colchicin began in the early 1800s.
Acalypha indica occurs widely throughout the tropics of the Old World. In Africa it occurs in Nigeria
in West Africa and further widely throughout tropical Africa and the Indian Ocean islands. It also occurs in
India, South East Asia, and Oceania. It has been introduced to areas of the new world with favorable climates
1. INTRODUCTION
Helminthiasis or infection with parasitic worm is pathogenic for human beings. Immature forms of the parasites
invade human beings via the skin or gastrointestinal tract (GIT) and evolve into well differentiated adult worms
that have characteristics tissue distribution. Anthelmintics are drugs that may act locally to expel worms from
the GIT or systematically to eradicate adult helminths or development forms that invade organs and tissues.
Most of the existing anthelmintic produces side effects such as abdominal pain, loss of appetite, nausea,
vomiting, headache and diarrhoea1.Chemotherapy is the only treatment and effective tool to cure and control
helminths infection, as effective vaccines against helminths have not been developed so far. Indiscriminate use
of synthetic anthelmintic can lead to resistance of parasites2. Most diseases caused by helminths are of a chronic
and debilitating in nature3 and could be of value in preventing the development of resistance4.
Helminthitic infections are among the most common infections in human beings, affecting a large
proportion of the world’s population. In developing countries they pose a large threat to public health and
contribute to the prevalence of anaemia, malnutrition, eosinophilia and pneumonia. Although the majority
of infections due to worms are generally limited to tropical countries, they can occur to travelers, who
have visited those areas and some of them can be developed in temperate climates1. The helminthes which
infect the intestine are cestodes e.g. Tape worms (Taenia solium) nematodes e.g hookworm (Ancylostoma
duodenale), roundworm (Ascaris lumbricoids) and trematodes or flukes (Schistosoma mansoni and
Schistomosa hematobolium). The diseases originated from parasitic infections causing severe morbidity
include lymphatic filariasis, onchocercasis and schistosomiasis. These infections can affect most populations
in endemic areas with major economic and social consequences. Helminthes also affect millions of livestock
resulting in considerable economic losses in domestic and farm yard animals. Because of limited availability
and affordability of modern medicines most of the world’s population depends to a greater extent on
traditional medical remedies. The traditional medicines hold a great promise as source of easily available
effective anthelminitic agents to the people, particularly in tropical developing countries, including India.
It is in this context that the people consume several plants or plant-derived preparations to cure helminthic
infections2. Ideally an anthelminitic agent should have broad spectrum of action, high percentage of cure
with a single therupuetic dose, free from toxicity to the host and should be cost effective. None of the
synthetic drug available meets this requirement. Even most common drugs like piperazine salts have been
shown to have a side effects like nausea,intestinal disturbances and giddiness3. Resistance of the parasites
to existing drugs4 and their high cost warrants the search for newer antheminitic molecules. The orgin of
many effective drugs is found in the traditional medicine practices and in view of this several researchers
have undertaken studies to evaluate folklore medicinal plants for their proclamined anthelminitic efficacy5.
In the treatment of parasitic diseases, the anthelmintics drugs are used indiscriminately. Recently the use
of anthelmintics produces toxicity in human beings. Hence the development and discovery of new substances
acting as anthelmintics are being derived through plants which are considered to be the best source of
bioactive substances. Various plants were used in veneral diseases, to promote healing of wounds, swellings,
abscesses, rheumatism and treating pain in lower extremities, skin diseases, leucorrhoea, dysentery, dysuria
and fever10, 11. Anthelmintics are those drugs that are used in expelling out the worms that are parasitic in
nature by either stunning them or by killing them. They are also known as vermifuges or vermicides.
1.2 Herbal drugs and its importance

In the recent years, the importance of Herbal drugs in Medicine has tremendously increased because of
their fewer side effects. Consequently, the demand for the herbal formulation is increasing day by day. The
phytochemical constituents and their standardization are accelerated with the development of instrumental
analysis and this field becomes important and new for investigation.
As the half of world suffering from bacterial and Helminthes infection, the source of infection being
very common due to poor sanitation, poor family hygiene, malnutrition, and crowded living conditions7. The
sources of infections are:
1) Human being: The commonest source of infection is human being themselves.
2) Animals: Many pathogens are able to infect both human being and animals. Animals act as source of
human infection.
3) Insects: Blood sucking insect may transmit pathogen to human beings. Besides acting as vector, some
insect may also act as a reservoir for hosts.
4) Soil and water: Some pathogens can survive in the soil for very long periods. Water may act as the source
of infection either due to contamination with pathogenic microorganism or due to presence of aquatic
vector.
5) Food: Contaminated food act as a source of infection8.
So there is a need to develop antibacterial and anthelmintics drug from herbal source.
1.3 Natural Anthelminitics
Natural anthelmintic includes the following list of components:
• Tobacco
• Walnut
• Wormwood
• Clove
• Kalonji seeds
• Garlic
• Malefern
• Pineapple
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• Diatomaceous earth
• Soya and other legumes
• Honey, water and vinegar are mixed with warm water act as vermifuges.
1.4 Different types of worms

This includes the intestinal nematodes (roundworms), trematodes (flukes) and cestodes (tapeworms).
(i) Roundworms
The common infections occurring with intestinal worms include Ascaris lumbricoides, Trichuris trichiura,
Necator americanus and Ancylostoma duodenal with the household aggregation of infection.
(ii) Flukes
Flukes are the parasitic trematodes of Schistosoma species which are transmitted through direct contact with
fresh water. They penetrate into the intact human skin and enter the capillaries and then migrate to the central
and portal system where they mature.
(iii) Tapeworms
Humans are the intermediate host for the Taenia solium with the development of the tissue cysts. After the
ingestion of the uncooked beef (T. saginata) or pork it develops the cysts and it causes the mild abdominal
symptoms.
1.5 Classification of anthelmintic drugs

Anthelmintics are the broad and wide range of drugs and are separated into classes on the basis of similar
chemical structure and mode of action.
(i) Piperazine
Piperazine was first used as an anthelmintic in 1950s and it is still the active constituent as over the counter
drug and is used in the remedies for thread worm infection in children.
(ii) Benzimidazole
Benzimidazole compounds which showed a number of different biochemical effects. The anthelmintic efficacy
of benzimidazoles is due to the ability of compromising the cytoskeleton through a selective interaction with
β‐tubulin factor15. This showed the effects of benzimidazoles on the species of C. elegans, which includes
the locomotion impairment, reproduction and detrimental effects on oocytes with the disruption of processes
thus requires the integral microtubules.
(iii) Levamisole, Pyrantel and Morantel

These anthelmintics are the nicotinic receptor agonist 19 which causes spastic muscle paralysis due to which
the prolonged activation of the excitatory nicotinic acetylcholine (nAch) receptors on muscle occurs.
(iv) Paraherquamide
Paraherquamide is a drug of oxindole alkaloid family and marcfortine A are isolated from Penicillium
paraherquei and Penicillium roqueforti, respectively23.
(v) Ivermectin
Ivermectin is a semi‐synthetic derivative of avermectin which is introduced as anthelmintic .It is a potent drug
and its discovery led to the development of ivermectin analogues which include moxidectin, milbemycin
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oxime, doramectin, selamectin, abamectin and eprinomectin28. Ivermectin causes the paralysis of pharyngeal
and body wall musculature29.
(vi) Emodepside
Emodepside is cyclodepsipeptide molecule and semi‐synthetic derivative obtained through fermentation
from the fungus, Mycelia sterilia. It is effective against isolates of parasites that are resistant to the molecule
having pore‐forming properties.
(vii) Nitazoxanide
Nitazoxanide is a pyruvate ferredoxin oxidoreductase inhibitor which acts against broad spectrum of protozoa
and helminths that occur in the intestinal tract.
1.5.1 Mechanism of Anthelminitic Drugs

They are used to treat infections with parasitic worms. It may act by causing:
1. Paralysis of the worm.
2. Damaging the worm leading to partial digestion or rejection by immune mechanisms.
3. Interfere with the metabolism of the worm.
1.6 Future Scope of the Anthelmintic Drugs

The challenges for the companies that are engaged in anthelmintic behavior of the drugs are not shared by
wealthy phase in the pharmaceutical industry because anthelmintic drugs market is cost effective and it is a
prime concern.
The widest concern for the future is the emergence of resistance to all the anthelmintics that are used
currently including ivermectin a pivotal in defining the mode of action for the majority of these drugs and can
also contribute in understanding mechanisms of resistance especially through the ‘model‐hopping’ approach.
In future it is to be hoped that new anthelmintics that have novel modes of action will solve the problem
of anthelmintic resistance. More targeted approaches to anthelmintic drug discovery are also ongoing and
thus providing the fundamental information with the aim not to target peptidergic signaling pathways which
translated a marketable drug49.
1.7 Material and Methods

The chloroform and methanol extracts were suspended in 1% gum acacia and used for anthelmintic activity.
Earth Worms
Adult Indian earth worms (Pheretima posthuma) collected from the local earth worm breeder in the outskrits
of Madurai were used for the study.
1.8 Anthelmintic Activity
The anthelmintic activity was evaluated on earth worms (8 ± 1cm in length) washed with normal saline
to remove all the extrageneous matter as previously described. The assay was performed on adult Indian
earth worm due to its anatomical and physiological resemblance with the intestinal round worm parasite of
human beings. The worms were divided into eight groups with six worms in each group and released into
appropriately labeled petri dishes with the solvent composition shown in Table 1 that were made up to 50 ml
with normal saline and then evaluated for anthelmintic activity.
Observations were made for the time taken for paralysis and death of individual worms to occur. For
each worm, paralysis was said to have occurred when it was not able to move even in normal saline and
death was concluded when it lost its motility followed with fading away of its body color. Death was also
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confirmed by dipping the worm in slightly warm water and the mortality of the parasite was assumed to
have occurred when all signs of movement had ceased. The mean paralysis time and mean lethal time of the
worms for the standard and each extract were recorded.
Table 1.1: Treatment groups of the worms and the solvents into which they were kept
Group Worm released into

1* 50 ml of 1% gum acacia in normal saline
2** 50 ml albendazole 10 mg/ml
3 50 ml chloroform extract 10 mg in 1% gum acacia in normal saline
6 50 ml methanol extract, 10 mg in 1% gum acacia in normal saline
*control; **standard
Table 1.2: Anthelmintic activity of various extracts of Acalypha indica. (L.f.) Pres
Treatment Time for paralysis (min) Time taken for death (min)
I Control - -
II Albendazole 36.19 ± 0.14 63.12 ± 0.31
III Chloroform extract 48.75 ± 0.16 98.33 ± 0.32
IV Chloroform extract 27.10 ± 0.21 44.29 ± 0.41
V Chloroform extract 14.34 ± 0.04 26.43 ± 0.32
VI Methanol extract 65.80 ± 0.17 113.38 ± 0.29
VII Methanol extract 41.54 ± 0.12 65.57 ± 0.37
IX Methanol extract 21.98 ± 0.15 42.95 ± 0.49
Values are expressed as mean ± S.E.M (n=6) Control worms were alive up to 24 hours of the experiment.
Results are expressed as mean ± SEM were evaluated by one way ANOVA followed by Newman Kew’s
multiple range tests. Values of P < 0.001 were considered statistically significant.
586
Anthelmintic activity in Acalypha indica (L.f) Pers
Figure 1: Figure 2:
Figure 3:
587

The times for paralysis and when death of the worms occurred are provided in Table No 5.2. Both chloroform
and methanol extracts of A.indica aerial parts exhibited dose dependent and significant anthelmintic activity
as compared with standard drug, Albendazole. Chloroform extract required least time to causes paralysis and
death of the earth worm followed when compared to the extracts from methanol suggesting either higher
concentrations of the compounds producing anthelmintic activity or more compounds with the activity.
The results of this work is limited to inability to report the actual compounds responsible for the activity
reported because they were not isolated or investigated. However, certain intermediate polar constituents
may be responsible for anthelmintic activity than polar constituents.
CONCLUSION
The present study therefore justifies it use in foklore remedies as an anthelmintic drug of natural origin. The
traditional use of plant Acalypha indica as a powerful anthelmintic have been confirmed as the chloroform
extract displayed profound anthelminitic activity in the study. Further it would be interesting to isolate the
possible Phytoconstituents which may be responsible for the anthelmintic activity and to possible mechanism
of action.
REFERENCES
1. James WT and Lesile TW, Drugs used in the chemotherapyof helmenthiasis, In: Goodman, L.S., Gilman,
A., editors., Goodman and Gilman’s the pharmacological basis of therapeutics 10th Ed. New York: Mc
graw Hill medical publishing division; 2001.p.1121-1140.
2. Singh D, Swarnkar CP and Khan FA, Anthelmintic resistance in gastro intestinal nematodes in live stock
in India. J Vet Parasite 2002; 16: 115-130.
3. Dewanjee S, Maiti A and kundu M, Evaluation of anthelmintic activity of crude extracts of Diospyros
peregrine. Dhaka Univ J Pharm Sci 2007; 6: 121-123.
4. Hammond DA, Feilding D and Bishop SC, Prospectus for plant anthelmintics in tropical veterinary
medicine. Vet Res Com 1997; 21: 213 – 228.
5. Bundy DAP, immunoepidemiology of intestinal helminthic infection, Trans Royal soc Trop Med Hygine,
8 (1994) 259-261.
6. Satyavati GV, Use of plant drugs in Indian Traditional System of medicine and their relevance to
primary health care, In:Economic and medicinal plant research by fransworth NR and waggner H(Eds),
Academic press Ltd, London,(1990) 190-210.
7. Litu LX and Weller PF, An update on antiparasitic drugs, N England Journal Medicine,334 (1996),1178.
8. Walter PJ and Prichard KK, Chemotheraphy of parasitic infections, In; W.C. Camphell and L.S.
Rew(Eds), Plenum, New York, (1985),278-539
9. Temjenmongla and Yadav A, Anticestodal efficacy of folklore medicinal plants of Nagatribes in North-
East India, African Journal Traditional cam,2(2)(2005)129-133.
10. Anisuzzaman M, Rahman AHMM, Harun‐or‐Rashid M, Naderuzzaman ATM and Islam AKMR, An
Ethnobotanical study of mdahupur, Tangail. Jounal of Applied Science Research (3)(2007): 519‐530.
11. Vijayan A, Liju VB, Reena John JV, Parthipan B and Renekac, Traditional remedies of kani tribes of
kotor reserve forest, Agasthyavanam, Thiruvanathapuram kerala. Indian Journal of Traditional remedies
.(6) (2007) 589‐594.
588
12. Basu and Sharma, Tropical gardening plant in India. (2005). 17‐18
13. Ananthnarayan V and Thomson C, Structure function studies on Hsp47: pH dependent inhibition of
collagen. Journal of Biochemistry (349), (2000 ) 877‐83.
14. Borgers M and De Nollin S, Ultra structural changes in Ascaris suum intestine after mebendazole
treatment in vivo. Journal Parasitol , (61)(1975) 110–122.
15. Aceves J, Erlij D and Martinez‐Maranon R, The mechanism of the paralyzing action of tetramisole on
Ascaris somatic muscle. British Journal of Pharmacology. (38)(1970)602-607
16. Zinser EW, Wolfe ML, Alexander BSJ, Thomas EM, Davis JP, Groppi VE and Lee BH, Anthelmintic
paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals, Journal of Vet
Pharmco Therapy (25) (2002) 241–250.
17. Haber CL, Heckaman C L, Li GP, Thompson DP, Whaley HA and Wiley VH, Development of a
mechanism of action‐based screen for anthelmintic microbial metabolites with avermectin‐like activity
and isolation of milbemycin‐producing Streptomyces strains. Antimicrob Agents. Chemother (35)
(1991) 1811–1817.
18. Brownlee DJ, Holden‐DL and Walker RJ, Action of the anthelmintic ivermectin on the pharyngeal
muscle of the parasitic nematode, Ascaris suum. Parasitology(135) (1997) 553–561. Pemberton DJ,
Franks CJ, Walker RJ, Holden DL. Characterization of glutamate‐gated chloride channels in thepharynx
of wild‐type and mutant Caenorhabditis elegans delineates the role of the subunit GluCl‐alpha2 in the
function of the native receptor. Mol Pharmacol (59) (2001) 1037–1043.
19. Greenwood K, Williams T and Geary T, (2005), Nematode neuropeptide receptors and their development
as anthelmintic screens, Parasitology (131) (2005) S169–S177.
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Spectroscopic Studies of Plant Extract of Ixora coccinea
(L.F) belongs to the Family Rubiaceae
V. Balasubramanian, S. Kalimuthu and N. Balakrishnan
S.A Raja Pharmacy College-Vadakangulam, Tiruenlveli Dist-627116

e-mail: kalimuthupharma017@gmail.com
ABSTRACT
1. MEDICINAL PLANTS:
Plants have formed the basis for traditional medicinal systems for thousands of years, with the first records
dating from about 2600 BC in Mesopotamia. Peoples used oils from cedar and cypress, licorice, myrrh,
and poppy juice, among other things, substances that are still in use today for the treatment of a variety of
illnesses and infections. Ancient Egyptian, Chinese, and Indian documents show that medicine in these
societies included numerous plant-based remedied preventives. The Greeks and Arabs both contributed
substantially to the assimilation, codification, and development of plant-based medicines. The isolation of
the active principles from the plants and herbs such as strychnine, morphine, and colchicin began in the early
1800s [Newman et al., 2000; Dev, 1999; Fallarino, 1994.
Today approximately 80% of the world’s population relies on traditional plant based medicines for
primary health care. The remaining 20% of the world’s population also depends on plant products for health
care [Arvigo&Balick, 1993; Farnsworth et al., 1985.
About 25% of prescription drugs dispensed in the United States contains plant extracts or active ingredients
derived from plants. Out of a total of 520 new drugs approved for commercial use between 1983 and 1994,
30 were new natural products and 127 were chemically modified natural products. Despite the great successes
already achieved in natural products chemistry and drug development, we have barely begun to tap the potential
of our molecular diversity. Only an estimated 5% to 15% of the 250,000 species of higher terrestrial plants in
existence have been chemically and pharmacologically investigated in systematic fashion. The percentage of
insects, marine organisms, and microbes investigated is far lower still. In the case of microbes, it is estimated
that 95% to 99% of existing species are currently not even known, never mind analyzed. There is currently great
interest in exploring extreme habitats for useful enzymes from microbes, including acid ophiles (from acidic
sulfurous hot springs), alkalophiles (from alkaline lakes), halophiles (from salt lakes), thermophiles (from deep
seavents), and psychrophiles (from extremely cold waters). For example, albuterol is based on the hormone
adrenaline and binds to the same receptor. Today, more systematic approaches are used. High-throughput
screening is used to test thousands of potential targets with thousands of diverse chemical compounds in order
to identify promising lead compounds (chemical entities that interact with targets and therefore have potential
as drugs). The alternative method of rational drug design involves the design and synthesis of compounds based
on the known structure of either a specific target or one of its natural ligands. The results of the Human Genome
Project and Human Pathogen Genome projects provide many new potential drug targets. For this reason, target
identification must be followed by target validation, which confirms the likelihood that interfering with the
target protein will impact on the disease. The development of a new therapeutic drug is a complex, lengthy and
expensive process. It can take from 10-15 years and over 500 000 000 $ to bring a drug from concept to market.
This includes 2-4 years of pre-clinical development, 3-6 years of clinical development and additional time for
dealing with the regulatory authorities.
In southern Africa, a large proportion of the population still uses traditional remedies. More than 700
plant species are being traded for medicinal purposes throughout South Africa, in the informal medicinal
plant market (Dold and Cocks, 2002).
This vast usage of and great dependence on traditional plants as the preferred form of health care is
aided by the fact that most of these plants are widely available and affordable, and additionally encompasses
practices based on the social-cultural norms and religious beliefs. It is evident that, even though scientific
advances have been made in our quest to understand the physiology of the body, biotechnology and the
treatment of disease, natural products are main a crucial component of the comprehensive health care
strategy for the future.
1.1. WHO Definition:
The World Health Organization (WHO) defines traditional medicine as the “diverse health practices,
approaches, knowledge and beliefs incorporating plant-, animal- and/or mineral based medicines, spiritual
therapies, manual techniques and exercises applied singularly orin combination to maintain well-being,
as well as to treat, diagnose, or prevent illness”. It’s clear, however, that there is a need to validate the
information through an organized infrastructure for it to be used as an effective therapeutic means, either
in conjunction with existing therapies, or as a tool in novel drug discovery. Traditional medicine utilizes
biological resources and the indigenous knowledge of traditional plant groups, the latter being conveyed
verbally from generation to generation. This is closely linked to the conservation of biodiversity and the
related intellectual property rights of indigenous people (Timmermans, 2003).
Although it is these traditional medicines that provided the link between medicine and natural products,
it was not until the 19th century that active compounds were isolated and principles of medicinal plants
identified (Phillipson, 2001). The Greek physician Dioscorides compiled an extensive listing of medicinal
herbs and their Materia Medica, and remained the authority in medicinal plants for over 1500 years
(Mendonca-Filho, 2006). Another Greek physician, Galen (AD 129-200), devised the pharmacopoeia
describing the appearance, properties and use of many plants of his time. It was the discovery of medicines
such as those listed, that sparked an interest in the study of plants as medicinal agents; with the isolation of
morphine from opium by Serturner (1805) being the start of natural product chemistry (Patwardhan et al.,
2004).
Despite the discovery of natural products from higher plants, the interest of chemists, pharmaceutical
scientists and pharmacologists turned to the production of synthetic compounds. In the late 19th century,
research was focused mainly on the modification of natural products, in an attempt to enhance biological
activity, to increase selectivity and to decrease toxicity and side effects. Aspirin is one such example and
was the earliest of the semodified natural products. In more recent years, however, industry has once again
turned its interests to natural product research (Phillipson, 2001). This is as a result of the development of
drug-resistant micro-organisms, side effects of modern drugs and emerging diseases for which no medicine
is available. Phytoconstituents are the main effective compounds of medicinal plants. They have various
pharmacological activity used for the treatment of various diseases. At present thousands of Phytoconstituents
is being successfully used in the treatment of variety of diseases. According to an estimate 80% of the
world’s population relies upon plant for their medication (Akerele 1993). Many plant components are now
synthesized in large laboratories for use in pharmaceutical preparation, for example vincristine (an anti-tumor
drug), digitalis (a heart regulator) and ephedrine (a bronchodilator used to decrease respiratory congestion)
were all originally discovered through research on plants.
There is abundant evidence from epidemiological studies that the Phytoconstituents in fruits, plants,
and vegetables can significantly reduce the risk of cancer, probably due to polyphone antioxidant and anti-
inflammatory effect.
Phytoconstituents have been used as drugs for millennia. For example; Hippocrates in 400BC used to
prescribe willow tree and later synthetically produced to become the staple over the counter drug called
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aspirin.
Number of chemical tests was available for the identification and quantification of Phytoconstituents.
The plant material is subjected to preliminary phytochemical screening for the detection of various plant
constituents. Instrumental analyses such as fluorescence analysis, colorimetriy, or ultra-violet absorption
were also used for the detection of plants. Among the several of separating, the chromatography procedure
originated by Tswett is one of the most commonly used techniques of general application.
S.No Active constituent Pharmacological activity Botanical name
1. Atropine Anti cholinergic Atropa belladonna
2. Colchicine Anti tumor, anti gout agent Colchicum autumnale
3. Digitoxin Cardio tonic Digitalis purpurea
4. Morphine Analgesic Pap paver somniferum
5. Pilocarpine Parasymphathomimetic Pilocarpus Joborardi
Ayurveda, Siddha, Unani and Folk (tribal) medicines are the major systems of indigenous medicines.
Among these systems, Ayurveda is most developed and widely practiced in India. Ayurveda dating back to
1500-800 BC has been an integral part of Indian culture. The term comes from the Sanskrit root Au (life)
and Veda (knowledge). As the name implies it is not only the science of treatment of the ill but covers the
whole gamut of happy human life involving the physical, metaphysical and the spiritual aspects. Ayurveda
recognizes that besides a balance of body elements one has to have an enlightened state of consciousness, sense
organs and mind if one has to be perfectly healthy. Ayurveda by and large is an experience with nature and
unlike in Western medicine, many of the concepts elude scientific explanation. Ayurveda is gaining prominence
as the natural system of health care allover the world. Today this system of medicine is being practiced in
countries like Nepal, Bhutan, Sri Lanka, Bangladesh and Pakistan, while the traditional system of medicine
in the other countries like Tibet, Mongolia and Thailand appear to be derived from Ayurveda. Phytomedicines
are also being used increasingly in Western Europe. Recently the US Government has established the “Office
of Alternative Medicine” at the National Institute of Health at Bethesda and its support to alternative medicine
includes basic and applied research in traditional systems of medicines such as Chinese, Ayurvedic, etc. with
a view to assess the possible integration of effective treatments with modern medicines. The development of
systematic pharmacopoeias dates back to 3000 BC, when the Chinese were already using over 350 herbal
remedies. Ayurveda, a system of herbal medicine in India, Sri Lanka and South East Asia has more than
8000 plant remedies and using around 35,000-70,000 plant species. China has demonstrated the best use
of traditional medicine in providing the health care. China has pharmacologically validated and improved
many traditional herbal medicines and eventually integrated them in formal health care system. Green plants
synthesize and preserve a variety of biochemical products, many of which are extractable and used as chemical
feed stocks or as raw material for various scientific investigations. Many secondary metabolites of plant are
commercially important and find use in a number of pharmaceutical compounds. However, a sustained supply
of the source material often becomes difficult due to the factors like environmental changes, cultural practices,
diverse geographical distribution, labour cost, and selection of the superior plant stock and over exploitation
by pharmaceutical industry. Plants, especially used in Ayurveda can provide biologically active molecules and
lead structures for the development of modified derivatives with enhanced activity and /or reduced toxicity.
The small fraction of flowering plants that have so far been investigated have yielded about 120 therapeutic
agents of known structure from about 90 species of plants. Some of the useful plant drugs include vinblastine,
artemesinin, vincristine, taxol, podophyllotoxin, camptothecin, digitoxigenin, gitoxigenin, digoxigenin,
tubocurarine, morphine, codeine, aspirin, atropine, pilocarpine, capscicine, allicin, curcumin and ephedrine
among others. In some cases, the crude extract of medicinal plants may be used as medicaments. On the other
hand, the isolation and identification of the active principles and elucidation of the mechanism of action of
a drug is of paramount importance. Hence, works in both mixture of traditional medicine and single active
compounds are very important. Where the active molecule cannot be synthesized economically, the product
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must be obtained from the cultivation of plant material. About 121 (45 tropical and 76 subtropical) major drugs
have been identified for which no synthetic one is currently available
1.1.1. Ethno pharmacological research:

The study of plants used in traditional medicine requires the effective integration of information on chemical
composition of extracts, pharmacological activities of isolated compounds, as well as indigenous knowledge
of traditional healers. The acquisition of ethno botanical information remains an empirical aspect in any such
study (Soejarto, 2005)
The process of isolating and identifying lead compounds from a complex mixture requires a number of
specific resources, including comprehensive knowledge, specialized equipment and skill. The urgency of
the discovery of new agents is a result of impenetrable factors that come into play, including the emergence
of new killer diseases, known antimicrobial drug-resistance, the inefficiency of synthetic drug discovery
and the high cost of bringing to market a single drug. A shift towards natural product research, which is
further driven by remarkable advances in plant extract technology, biotechnology and analytical chemistry,
is therefore inevitable. There is a great need and ethical obligation to accurately document investigative
findings on plants used for health purposes. This will additionally aid in the efficient preservation and
conservation of traditional knowledge, thereby preventing the further disappearance of indigenous systems
of medicine, which may potentially benefit society in general. According to the Southern African Trade
Directory of Indigenous Natural Products, more than 1000 species of plants are used traditionally in southern
Africa (Izimpande, 2005) of which the genus Ixoracoocinea.
1.1.2. An introduction to the Family Euphorbiaceae of the Genus Ixora:

Although there are around 500 species in the genus Ixora, only a handful are commonly cultivated, and the
common name, Ixora, is usually used for I. coccinea. I. coccinea is used in warm climates for hedges and
screens, foundation plantings, massed in flowering beds, or grown as a specimen shrub or small tree. In
cooler climes, it is grown in a greenhouse or as a potted house plant requiring bright light. I. coccinea is also
grown in containers, looking very distinguished as a patio or poolside plant. This tight, compact shrub is
much branched and tolerates hard pruning, making it ideal for formal hedges, although it is at its best when
not sheared. There are numerous named cultivars differing in flower colour (yellow, pink, orange) and plant
size. Several popular cultivars are dwarfs, usually staying under 3 ft (1 m) in height. Ixora ‘Nora Grant’
is a popular dwarf and ‘Super King’ is a popular hybrid with much larger flower clusters than the species.
Many new cultivars and hybrids of I. coccinea have come to market in the last couple of decades, leading to
resurgence in popularity for the beautiful flame-of-the-woods.
2. PLANT PROFILE
2.1. Ixora coccinea(L.f)
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Nomenclature :
Kingdom: Plantae
Sub Kingdom:Angiosperms
Tribe: Ixoreae
Genus: Ixora
Order: Gentianales
Subfamily: Ixoroideae
Family: Rubiaceae
Botanical name: Ixora coccinea
Description:
I. coccinea is a dense, multi-branched evergreen shrub, commonly 4–6 ft (1.2–2 m) in height, but capable of
reaching up to 12 ft (3.6 m) high. It has a rounded form, with a spread that may exceed its height. The glossy,
leathery, oblong leaves are about 4 in (10 cm) long, with entire margins, and are carried in opposite pairs or
whorled on the stems. Small tubular, scarlet flowers in dense rounded clusters 2-5 in (5–13 cm) across are
produced almost all year long.
Uses:
The flowers, leaves, roots, and the stem are used to treat various ailments in the Indian traditional system
of medicine, the Ayurveda, and in various folk medicines. The fruits, when fully ripe, are used as a dietary
source.
3.1. Experimental procedure:
3.1.1. Selection, collection, identification, authentication, and extraction of plant material:

Based on the traditional uses reported on the literature review, the following plant of the different genus were
selected for the study
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Ixora coccinea(L.f) belongs to the family Rubiaceae
Ixora coccinea(L.f):
The leaves and aerial portions of Ixoracoccinea which has been predominantly grown in water which were
procured from Kavalkinaru, Tirunelveli district of Tamil Nadu.
3.1.2. Garbling process:
Garbling refers to the separation of particular portion of the plant to be used from other parts of the plants,
like dirt and other extraneous matter. This step is often done during the collection process. Usually it is
performed by hand. After removing all such unwanted adhered materials; the required plant parts were then
spread over trays and dried under shade, with regular sifting of collected plant materials everyday in order to
avoid fungal growth. Such shade dried bark/leaves/aerial portions of plant were ground to powder and then
it was subjected for extraction.
3.1.3. Plant material:

Ixora coccinea (Rubiaceae) was collected from Kavalkinaru ,Tirunelveli District in Tamil nadu in the month
of September-November. A small unarmed middling-sized tree with young parts puberulous: leaflets are
acute, terminal one sub sessile. This is a middling-sized tree. The whole plant was then dried under air, shade
dried and mechanically powdered separately to obtained a coarse powder which then subjected to extraction.
3.1.4. Soxhlet Extraction:

The powdered leaves of known quantity was taken in a Soxhlet apparatus and extracted with absolute ethanol
.The material was extracted continuously for 72 hours. The excess solvent present in the crude extracts were
removed by distillation and concentrated under vacuum and then dried.(Trease&Evans 2006)
Preliminary phytochemical tests procedure for the presence of various Phytoconstituents present in
the plant extract of Ixoracoccinea.
S.No. Procedure Observation Inference
Tests for alkaloids
1. Mayer’s test:
To 2 ml solution of the extract add 0.2 ml of dil. Yellowish-buff colored Presence of alkaloids
HCl and 0.1 ml of Mayer’s reagent precipitate
2. Dragendroff’s test:
To 2 ml solution of the extract add 0.2 ml of dil. Orange brown precipitate Presence of alkaloids
HCl and 0.1 ml of Dragendroff’s reagent
3. Wagner’s test:
To 2 ml solution of the extract add 0.2 ml of dil. Reddish brown precipitate Presence of alkaloids
HCl and 0.1 ml of Wagner’s reagent
4. Hager’s test:
To 2 ml solution of the extract add 0.2 ml of dil. Yellowish precipitate Presence of alkaloids
HCl and 0.1 ml of Hager’s reagent
5. To 2 ml solution of the extract add 0.2 ml of dil. Buff colored precipitate Presence of alkaloids
HCl and 0.1 ml of phospho-molybdic acid reagent
Tests for amino acids
595
10. Shinoda test:
To 1 ml solution of the extract add a small piece Magenta color Presence of Flavanoids
of Mg metal and Con. HCl was added drop wise
and heated
11. Fluorescence test:
Alcoholic solution of the extract was observed Green fluorescence Presence of Flavanoids
under UV light
12. Zn–HCL reduction test:
To 1 ml solution of the extract add a pinch of Zn Magenta color Presence of Flavanoids

dust and few drops of Con. HCl
13. Boric acid test:
To 1 ml solution of the extract add equal volume Yellow color Presence of Flavanoids
of HBO3 solution
14. To 1 ml solution of extract add Con. H2SO4 Color change from yellow to Presence of Flavanoids
orange
15. To 1 ml solution of the aqueous extract add 1% Yellow color Presence of Flavanoids
aluminum solution
16. Wolfram test:
To 2 ml solution of the extract add sodium Pink color Presence of isoflavones

amalgam and HCl
17. To 1 ml solution of the extract add equal volume Yellow color Presence of isoflavones
of Con. HNO3
Tests for glycosides
18. Legal’s test: Pink color was formed Presence of glycosides
To 1 ml solution of the extract add 1 ml of
pyridine and a few drops of sodium nitroprusside
solution then it was made alkaline with sodium
hydroxide solution
19. Baljet test: Yellowish orange color was Presence of glycosides
formed
To 1 ml solution of the extract add 1 ml of
sodium picrate solution
20. To 1 ml solution of the chloroform extract add Pink color was formed Presence of glycosides
equal volume of dil. NH3 solution
21. 0.1 gm of the powder was boiled for 2 min Pink color or cherry red color Presence of anthraquinone
with dil.HCl& few drops of ferric chloride and was formed glycosides
filter. After cooling, extract it with benzene and
separated. Equal volume of NH3 solution was
added to the benzene extract and shaken well.
Tests for gum and mucilage
22. 1 ml solution of extract was extracted with Swelling observed indicates the Presence of mucilage
absolute alcohol, stirred and filtered. The presence of gums and mucilage
filtrate was dried and examined for its swelling in the extracts.
properties.
23. Few ml solution of the aqueous extract prepared Reddish pink color was formed Presence of mucilage
was treated with rheuthenium red solution.
Test for proteins
24. Biuret test:
To 1 ml solution of the extract add equal volume Violet color was obtained Presence of proteins
of 5% w/v sodium hydroxide and 4 drops of 1%
copper sulphate solution
596
25. Millons test:
To 1 ml solution of the extract add equal volume Red precipitate was obtained Presence of proteins
of Millon’s reagent
26. Xanthoprotein test:
To 1 ml solution of the extract add Con.nitric An orange color was obtained Presence of proteins
acid, boil for 1 min, cooled and add Con. NH3 till
alkaline.
27. To 1 ml solution of the extract add 10% tannic acid White precipitate was Presence of proteins
solution obtained
Tests for reducing sugars
28. Fehling’s test:
To 5 ml solution of the extract add 5 ml Fehling’s Brick red colored precipitate Presence of reducing
A and B solution and boiled for 5 min on boiling sugars
water bath
29. Benedicts test:
To 5 ml solution of the extract add 5 ml of Brick red colored precipitate Presence of reducing
benedicts reagent and boiled for 5 min on boiling sugars
water bath
30. Molisch test:
To 5 ml solution of the extract add 2 drops of Violet colored ring was Presence of reducing
5% alpha-naphthol solution (freshly prepared) and formed of at the junction of sugars
added 1 ml of H2SO4 on the sides of the test tube two liquids
Test for Saponins
31. Foam test:
To 1 ml solution of the extract was diluted to 20 Stable foam was formed Presence of Saponins
ml with distilled water, shake vigorously in a
graduated cylinder for 15 min
Emulsion test:
32. Dil. aqueous extract of the sample was shaken Formation of emulsion. Presence of Saponins
vigorously to form a stable persistent froth. The
frothing was mixed with 3 drops of olive oil and
shaken vigorously.
Test for starch
33. Iodine test:
To1 ml of solution extract adds 1 ml of dil. iodine Bluish black color Presence of starch
solution.
Tests for steroids
34. Liebermann Burchard test:
10 mg of the extract was dissolved in 1 ml of Reddish purple color Presence of Steroids

chloroform add 2 ml of Liebermann Burchard
reagent
35. Salkowski’s test: Chloroform layer acquired
reddish brown color and
10 mg of the extract was dissolved in 1 ml acid layer showed green Presence of steroids
chloroform then add 1 ml of sulphuric acid fluorescence
Tests for tannins
36. To 1 ml solution of the extract add 1 ml of 5% Greenish black precipitate Presence of tannins
ferric chloride solution
597
6. Ninhydrin test:
To 1 ml solution of the extract add ninhydrin Purple color Presence of amino acids
solution and adjust the pH between 4 and 8
Test for Anthraquinones
7. 5 ml solution of the extract was hydrolyzed with Presence of
dil.H2SO4 and extracted with benzene. Add 1 ml of anthraquinones
dil.NH3 solution to the benzene layer. rose pink coloration
Tests for Flavanoids
8. 10 ml solution of extract was hydrolyzed with 10% Greenish yellow Presence of Flavanoids
H2SO4. Then it was extracted with ether and the
ether extract was divided into three portions Pale yellow color
Add 1 ml of dil. NH3 solution Yellow color

Add 1 ml of dil.Na2CO3 solution
Add 1 ml of dil.NaOH solution

9. To 1 ml solution of the extract add 25% basic Yellow color Presence of Flavanoids
PbOAc solution
37. To 1 ml solution of the extract add 1 ml of 10% Yellow precipitate Presence of tannins
lead acetate solution
38. To 1 ml solution of the extract add 1 ml of 15% Yellowish brown precipitate Presence of tannins
potassium dichromate solution
39. To 1 ml solution of the extract add 1 ml of aqueous Brown precipitate Presence of tannins
bromine solution
40. To 1 ml solution of the extract add potassium ferric Characteristic red color Presence of tannins
cyanide followed by NH3
Tests for terpenoids
41. Noller test:
To the extract add few ml of chloroform and filter. Pink color appears Presence of terpenoids
To the filtrate add tin and thionyl chloride warmed
gently.
UV VISIBLE SPECTROSCOPY:
Ultra violet visible spectroscopy is routinely used in the qualitative determinations of solutions of transition
metal ions and highly conjugated organic compounds .organic compounds with a high degree of conjugation
absorb light in the UV or visible region of the electromagnetic spectrum. Ethanol was used as a solvent.
Sample preparation:
Sample preparation was carried out by taking the known weight of dry sample (1mg) in a clean 100ml
standard flask. Add small amount of solvent like ethanol and stirred well until to get the clear solution.
Then volumes make up to 100ml with ethanol. The absorbance was measured at 250-400nm .Scanning sped
is fast .Sampling interval is 0.5mins, and auto sampling interval is enabled. The instrument type was UV
1800 series. Measuring mode was absorbance. Slit width 1.0mm. Light source change wavelength 340.8nm.
(Wikipedia.org)
598
INFRA RED SPECTROSCOPY:

Infra red spectroscopy is a technique which a can be used to identify compounds or investigate sample
composition. Infrared spectroscopy exploits the fact that molecules have specific frequencies at which they
rotate or vibrate corresponding to discrete energy levels (vibrational modes). These resonant frequencies
are determined by the shape of the molecular potential energy surfaces, the masses of the atoms and, by
the associated vibronic coupling. In order for a vibrational mode in a molecule to be IR active, it must be
associated with changes in the permanent dipole.
Sample preparation:
Sample preparation was carried out by taking known weight of the dry sample(1mg) in a smooth agate
mortar and mixed thoroughly with 2.5 mg of dry potassium bromide (KBr) using a pestle. The powder
was filled in the micro-cup of 2mm internal diameter to obtain the diffuse samples. The IR spectra was
recorded at room temperature (26°C ± 1°C) in the mid infra red range (4000-400 cm-1) using FTIR -8201
1PC.Fourier transform infra red spectrometer (SHIMADZU).Typically, 20 scans were signal averaged
for a single spectrum .Each spectrum is displayed in terms of absorbance .spectrum using the hyper IR
Software .To minimize the difficulties arising from unavoidableshifts, baseline correction was applied.
Each spectrum was normalized as normalization procedures spectrum in which maximum value of
absorbance becomes 2 and minimum value 0. To improve the resolution of complex bands the digitalized
original spectrum was smoothed on noisy spectrum using Kubelka Munk algorithm and converted into
its second derivative using the Savitzky- Golay algorithm using 21-point smoothing. The instrument type
was infrared 2010 series (Shimadzu).
NMR SPECTROSCOPY
Proton NMR (alsoHydrogen-1NMR or H1 NMR) and the Carbon-13 NMR or C 13 NMR are the application
of Nuclear Magnetic Resonance spectroscopy with respect to hydrogen-1, Carbon-13 Nuclei within the
molecules of a substance, in order to determine the structure of its molecules.
Sample preparation:
When preparing the solution for NMR spectroscopy care should be taken to ensure cleanliness of
apparatus and absence of contamination by solvent impurities. The Solute of Interest (5-10mg) can be
dissolved in a separate glass vial using less than the final volume of deuterated solvent (Ex: CDCl3),
required to make the NMR sample. After the solute has been dissolved it can be transferred to a NMR
tube by passing the solution through the cotton wool. The sample can be adjusted by adding the remaining
solvent to the NMR tube that the final sample volume of 700µl, or a sample height of 55m is reached
followed by vigorous shaking of the sample be effectively mixed its content .The suggested sample
volume is 700µl.It is important to keep the NMR tubes free from dust, grease etc. but they should never
be cleaned with chromic acid, as this may leave behind paramagnetic impurities in the glass which
makes them unusable. The commercial cleaning agent “decon 90” is a very effective for contamination
especially grease. After soaking in a suitable solution, tube can be rinsed with distilled water and then
acetone. An effective method is to blowing on or air through the tube, with a pipette while, warming
it gently for a few minutes or to leave under vacuum for some hours. The NMR tubes is not subjected
to high temperature for a long periods of time this will cause them to distort and become unusable
.For obtaining the high resolution it is necessary that the NMR sample is free of suspended particles.
Suspended material can be easily removed from the NMR sample by constructing a filter using cotton
599
wool as a filtering agent. The used NMR tube is Wilmad 507-PD or New – Era ML5 or MP5 must be
used. To obtain the beautiful spectra distill the solvent; avoid vacuum grease and crystalline solids. The
instrument type was Astra 2010 series (Shimadzu).
Insertion of sample:
The NMR tube is inserted into a spinner. The spinner is then inserted into a sample gauge, and is then
checked that the sample is in the region, of the lines. The solvent height is at least 4 cm from the bottom of
the tube. The bottom of the tube is 2cm below the coil centre. The sample depth is less than 4cm then the
centre of the solution should be placed at the coil centre. Before inserting the sample ensures that there is not
already a sample inside. Remove the previous sample and put the new sample in place. Press on lift on again
and the sample sinks into the magnet. The temperature maintained at 298.0k or 24.85°C (room temperature).
Then on the control panel for correcting the magnetic field homogeneity.
Initial acquisition:
The spectral region and the acquisition are appropriate. Sometimes, the fid may be truncated and ringing
will appear in the spectrum that the signals may be broad washing time on acquiring noise or there may be
signals outside the range. After the acquisition a Fourier transform is carried out converts the acquired signal
into the spectrum.
Liquid chromatography-Mass spectroscopy: (LC-MS)

Liquid chromatography-Mass spectroscopy is a technique which combines high performance liquid
chromatography (HPLC), a powerful analytical separation technique with mass spectroscopy, a powerful
analysis and detection technique.
The following conditions are maintained in running the sample on LCMS 2010, SHIMADZU, JAPAN
HPLC Conditions:
The C18 column can be used. The mobile phase is Methanol: water in the ratio of 90:10 .The flow rate is
0.2ml/min. Sample is dissolved in methanol and the volume of sample is injected is 5µlts. The UV-VIS
detector detects the response in the wavelength of 254 nm.
Mass spectroscopy conditions:

There are two common atmospheric pressure ionization were employed. The atmospheric chemical ionization
is mainly used for analyze the non polar compounds and the electron spray ionization is mainly used for
analyze the polar compounds. Positive chemical ionization which gives protonated M+1 values, and the
negative chemical ionization which gives the deprotonated M-1 values and the positive or negative ionization
and the type of probe can be identified in the data file name. In presence of halogens the values will show M
and M+2 in positive, M and M-2 in negative.
Adductions:
In presence sodium salts M+23 sodium adduct ions, in presence methanol mobile phase M+32 adduct ions,
in some cases both M+55 will form. These adduct ions mainly shows in electron spray ionization (ESI).
Dimerisation shows 2M+1 or 2M+23 adductions
RESULTS
600
7.1. Results of phytochemical tests for the presence of various Phytoconstituents Present in Ixoracoccinea:
S.No. Phytoconstituents LP
1. Alkaloids +
2. Amino Acids -
3. Anthraquinones -
4. Flavonoids +
5. Glycosides +
6. Gums and Mucilage +
7. Proteins -
8. Reducing sugars +
9. Saponins -
10. Starch +
11. Steroids +
12. Tannins +
13 Terpenoids +
(+) - Indicates Present

(-) - Indicates Absent
Characterization of Extracts of Ixoracoccinea belongs to the family Rubiaceae:
Figure 1: UV Spectrum of Extracts of Ixoracoccinea
601
Figure 2: IR Spectrum of Extracts of Ixoracoccinea
Regions of Proton NMR spectra
Table 6:
S.No Region Frequency Intensity

HZ PPM
1. 12150.3 1464.959 7.3200 0.20
2. 12254.9 1453.427 7.26214 10.58
3. 15309.2 1116.678 5.5798 0.15
4. 17850.6 836.487 4.1797 0.13
5. 17916.8 829.193 4.1433 0.13
6. 17982.2 821.976 4.1072 0.18
7. 18094.3 809.620 4.0455 0.16
8. 18139.5 804.634 4.0206 0.14
9. 18498.8 765.020 3.8226 0.17
10. 21106.7 477.495 2.3859 0.19
11. 21172.8 470.215 2.3495 0.30
12. 21239.0 462.911 2.3131 0.30
13. 21314.8 454.552 2.2713 0.74
14. 21339.6 451.817 2.2576 0.70
15. 21787.2 402.472 2.0111 0.29
16. 22321.7 343.544 1.7166 1.04
17. 22843.8 285.986 1.4290 1.31
18. 23078.3 260.127 1.2998 2.62
19. 23162.2 250.880 1.2536 21.00
20. 23552.3 207.865 1.0387 0.51
21. 23611.2 201.375 1.0062 0.52
Figure 5:
Liquid chromatography-Mass spectroscopy positive mode spectrum of isolated compound
602
Figure 13:
Discussion
The plant Ixora coccinea belongs to the family of Rubiaceae was found in pond, lakes and water stagning
areas. It is a herbs, sometimes shrubs , rarely trees ; mostly autotrophic, less often hemiparasitic or parasitic.
Stipules absent. Leaves alternate, opposite, whorled, or basally opposite and apically alternate, simple
or sometimes pinnately dissected. Inflore scencesracemes, spikes, or thyrsoid panicles, determinate or
indeterminate, or flowers solitary. Flowers perfect, usually zygomorphic, rarely actinomorphic. (Zipcodezoo.
com). Then the collected plant were cleared, grabled, and dried under shade with regular shifting. Accurately
weighed quantity (100 gm) of the powdered shade dried bark/ leaves were extracted in a soxhlet apparatus
with absolute ethanol exhaustively for a period of 72 hours. The excess solvent present in the crude extract
thus obtained were removed by distillation under vacuum and dried.
Various standard phytochemical tests were performed for the ethanolic extract of Ixora coccinea and the
results were reported in Table. The phytoconstituents were detected by using various reagents.
The phytoconstituents present in the plant Ixora coccinea were alkaloids, glycosides, flavanoids, gums
and mucilage, reducing sugars, starch, steroids, tannins, terpenoids. The phytoconstituents such as flavanoids,
terpenoids, glycosides, steroids present in the Ixora genus which was reported to possess various therapeutic
properties
603
Characterization of isolated compound from the plant extract of Ixoracoccinea

8.4.1. Ultra violet visible spectroscopy:
The UV Spectrum showed λ max at 430 nm and 670 nm for a п → п* and n→п* transition indicates the
presence of a chromophoric group probably (c=o).The π→π* and the n→π* transitions occurs in the near uv
and visible region (180-700nm). Molecule exhibits n→π* transition include molecules that contain C=O,
NO, N=N.
8.4.2. Infra red spectroscopy:

The IR spectrum showed C-H stretching bands at 3370,2924,2856 cm-1 and C-H bending bands at
1735,1651,1543,1453 cm-1 that are characteristic of aliphatic hydrocarbon .O-H carboxylic stretching
bands at 3373 cm-1 that are characteristic of acid group.
8.4.3. NMR spectroscopy:
The H1 NMR Exhibits peaks at ∂ 0.851 showing the presence of methyl groups. The strong peak at ∂ 1.597
indicates the presence of a long chain of methylene groups .The peaks at δ 2.039 is due to methylene groups
adjacent to carbonyl group.
The C13 NMR Exhibits peaks at ∂ 26.9 for a methyl group and the peaks at ∂ 20.12, 23.07, 29.74, 32.31
are due to long chain methylene groups.
Liquid chromatography mass spectroscopy:
The LC-MS showed that the molecular weight of the compound is 450 obtained from the APCI-LCMS
negative mode spectrum m/z 480, for [M-H] – ion.
CONCLUSION
Ixorais a genus of flowering plants. It includes 800 species which are herbs, sometimes shrubs, rarely
trees; mostly autotrophic, less often hemi parasitic or parasitic. Stipules absent. Leaves alternate, opposite,
whorled, or basally opposite and apically alternate, simple or sometimes pinnately dissected. In several of
these species flavanoids, terpenoids, essential oils, hydrocarbon containing fatty acids have been studied and
identified by various chromatographic and spectroscopic techniques.
The UV technique reported by Maitreyi Zaveri, et al.,(2008) is precise, specific, accurate technique used
for the study of phytoconstituents and the determination of absorbance of compound present in the extract
(λ max ) in the UV and visible region. The IR, NMR, CHN (Elemental analysis) technique reported by
Rajesh Gupta, et al., (2009) add the knowledge on qualitative and quantitative estimation of the various atom
present in the extract . The results obtained from LC-MS add Knowledge on the molecular weight and nature
of the ion present in theextract .
REFERENCES
1. T.E Wallis, Text book of pharmacognosy, 5th edition, Page no. 497-501.
2. Trease and Evans Pharmacognosy14th edition 2000 Page no. 42; 289; 474; 493.
3. Raw materials The Wealth of India, A dictionary of Indian raw materials and industrial products, Volume
II 1950 Page no. 313-314.
4. M Nadkarni Indian Materia Medica Page no. 167-172
604
5. C. Katal; bm. Kapur, Cultivation utilization of Medicinal plants. Regional Research laboratory. Jammu
–Tawi 1982 Page no. 33-34.
6. Publication and information directorate CSI the useful Plants of India Page no. 138 R New Delhi -1986.
7. A. Solkar, Kakkar and Chakre, 2nd supplement to glossary of Indian medicinal plants with active
principles part I A-K Page no. 224-227. Publication and information directorate 1992 New Delhi
8. Editor. R.P Rastogi, CSIR Luck now Compendium of Indian medicinal plants Publication and
information directorate Vol .2 Page no. 204 -207, 1970-1979, 1993 New Delhi
9. Kirtikar and Basu, Indian medicinal plants Vol 2, 2nd Edition Page no. 525-528;
10. Elien Jobarran, Lance R. Patterson and Syndey Finegold Methods for testing anti microbial effectiveness,
(Chapter 14) Bailey and Scotts diagnostic microbiology; Page no. 168-188.
11. Elmer Koneman, William M. Janda, Hebert M. Sommers, Stephen D. Allen and V.R. Dowell, Colour
Atlas of text book of diagnostic microbiology 3rd edition Page no. 487; 492 -497.
12. Antibiotic Susceptibility testing by a standardized single disc method. The American Journal of clinical
pathology Vol 45 Page no. 493-496
13. M.L. Dhar, M.M. Dhar, B.N Dhawan, B.N. Mehrotra and C. Ray, Screening of Indian plants for
biological activity part I. Indian .J. of experimental biology Vol 6. 1968, 232-247.
14. Chemical abstracts American chemical society Vol., 77; 1972, Page no. 217, and 210-211.ss
15. F. Genetet, N. Meyer and R. Wolf, The estimation of cholesterol distribution in the paper electropherogram
of serum. The biochemical journal Vol 89, 1963 Page no. 21
16. Hack seang Kim, Myeon –woo-chaung and Young choong Kim, Antihepatotoxic Activity of Bergenin,
the major constituent of Mallotus Japonicus on carbon tetra chloride intoxicated hepatocytes. Journal of
ethno pharmacology 69 (2000) Page no. 79-83
17. Narayan das prajapati: A Hand book of medicinal plants; A complete source book (2003) page no: 316
18. Deil U, A review on habitats plant traits and vegetation of ephemeral wetlands, A Global perspective
phytocoenologia (2005) 35 (2-3), page no: 533-705.
19. Jang D.S.a, Su B.-N.a.e, Pawlus A.D.a and Jones W.P.ae, Ixoracoccinea ketone, a highly oxygenated
phenolic derivative from limnophilageoffrayi; Journal of natural products ,(2005), 68(7), page no:1134-
1136.
20. Lin Y.a Wang, G.-X.b c, Li, W. c Ito, M.b Secondary structure prediction of Acetolactate synthesis
protein in sulfonyl urea herbicidal resistantIxoracoccinea; Journal of pesticide science,(2004) 29 (1)
page no:1-5.
21. Petersen M.J.a, Gelhaus J.K.b and Bernard E.C.c, New species and records of crane flies from great
smoky mountains National park Tennessee and north carolna, U.S.A; Transactions of the American
entomological society,(2004), 130 (4) page no: 439-445
22. Bui M.–L.ab, Grayer R.J.b, Veitch N.C.b and Kite G.C.b, Uncommon n8- oxygenated Flavonoids from
Ixoracoccinea (Rubiaceae). Biochemical systematics and ecology, (2004) 32 (10), page no: 943-947.
23. Bramachari G., Gorai D., Chatterjee D., Mondal S. and Mistri, A novel flavonoids constituent of Ixora
coccinea; Indian journal of chemistry – section –b organic and medicinal chemistry (2004), 43(1), page
no: 219-222.
24. Bramachari G., sohel S.M.A., Gorai D., Mondal S. and Mistri B., An ethylenedioxy flavonoids carboxylic
acid from Ixora coccinea; Journal of Chinese chemical society (2003), 50 (2), page no: 325-328.
605
Diuretic Activity of Ethanolic Extract of Aristolochia
indica Linn. in Rats
R. Mohan Kumar1, M. Balakumaran2, P. Selvamani3, N. Subramanian4

and K. Ruckmani5
5
Author for correspondence, Prof & Head, Director – CENTRE,
Department of Pharmaceutical Technology, BIT Campus, Anna University, Tiruchirappalli-620024,
1, 2, 3, 4, 5
Tamilnadu, India
e-mail: 5hodpharma@gmail.com, Mobile: +919842482568
Abstract:
Aristolochia indica Linn is used in the traditional medicine as a diuretic. In the present study, the ethanolic
extract of the plant of Aristolochia indica linn was studied, and the activity was compared with furosemide as
standard. The doses 250 and 500 mg/kg bodyweight exhibit significant diuretic activity as evidence increased
by total urine volume and the urine concentration of Na+, K+ and Cl-. .These results that support the use of
Aristolochia indica Linn. as a diuretic agent.
Keywords: Aristolochia indica Linn, Diuretic, Furosemide, Aristolochiceae.
INTRODUCTION:
Several specious of the genus Aristolochia are employed in the folk medicine of many countries to treat
a variety of diseases such as anthelmintic, stomachic, cardiotonic, purgative, anti-inflammatory, diuretic,
sudorific, febrifuge, anti-periodic, emmenagogue and tonic [1].
The genus Aristolochia consisting of approximately 300 species is distributed throughout India at low
elevations, on hedges and bushes and throughout the subcontinent, mainly in the plains and lower hilly
regions from Nepal to Bangladesh. Leaves simple, alternate, short petioled, the blade ovate or somewhat
wedge-shaped, very variable in shape and size. The young leaves are light purplish. Leaves are used to treat
cholera, bowel complaints and intermittent fevers in childrens. The fresh juice of the leaves is a popular
antidote to snake poison. A paste made of leaves is reported to be useful as a remedy for itches and insect;
bites and can also be mixed with castor oil and applied to control eczema. The flowers greenish, white
or light purplish in axillary cymes or fascicles with swollen or inflated basal part. The fruit is a capsule,
roundish or oblong and hexagonal, 2.5 - 4 cm long and slightly less broad, with shallow grooves and six
valves, containing many triangular seeds. The young roots are light brown and fairly smooth, whereas the
older ones are comparatively rough due to the development of cork, lenticles and the presence of scars of
rootlets. The cork layer somewhat friable. In a freshly coloured strip, surrounding! A wide woody core. The
wood has a light yellow colour and appears highly porous, with the pores being sufficiently large to be easily
visible with the naked eye; the medullary rays are soft and creamy white in colour; there is no pith in the
centre. The root is pungent, bitter, alexiteric, emmenagogue, useful in “tridosha,” bowel troubles of children
[2]. Tuberous roots are crushed and applied on the body for treating itching [3]. Here the presence study has
been undertaken to investigate the diuretic properties of ethanolic extract of Aristolochia indica (EEAI) in
standard animal models.

Plant material
The whole plant of the Aristolochia indica Linn was collected from Krishnagiri district of Tamil Nadu, India,
in the month of august 2009 and authenticated by Dr. G.V.S. Murthy, Join director, Botanical survey of India,
Southern circle, TNAU campus, Coimbatore. A voucher specimen (BSI/SC/5/23/09-10/Tech.331).
Preparation of extract
The Plant materials were dried under shade and powdered to moderately coarse powder and was
extracted successively with petroleum ether (60-80ºC) and ethanol, using soxhlet apparatus. The extracts
were dried under vacuum (yield 10.78%, 9.47 % respectively) then suspended in 20% v/v propylene glycol
water to give concentration of 500mg / ml [4, 5].
Diuretic activity
Albino rats of both sexes (150-250g) were collected and housed under standard laboratory conditions.
They were fed with standard rat feed and water adlibitum. The experimental protocols were approved by
institutional animal ethics committee (JKKMMRFCP/IAEC/MP/PCOL/01/2009-2010). The method of
Lipchitz. et. al, [6] was employed for the evaluation of diuretic activity. The animals were divided into four
groups (six in each) deprived of food and water for 18h prior to the experiment. On the day of the experiment,
the group I animals received normal saline (20ml/kg.p.o), a group II animals received furosemide (20mg/kg.
i.p.); a group III and IV animals received Ethanol extract 250mg/kg and 500mg/kg respectively. Immediately
after the administration, the animals were kept in metallic cages (two per cage) specially designed to separate
urine and feacal matter and kept at room temperature (20±0.5ºC). The total volume of urine was collected
at the end of 5h. During this period, no water and food was made available to the animals. The parameters
accounted for ascertaining the diuretic activity are total volume of urine and the urine concentration of Na+,
K+ and Cl-. The Na+ and K+ were measured by flame photometry [7], and Cl- concentration was estimated by
titration [8] with silver nitrate solution (N/50) using three drops of potassium chromate as an indicator. The
student “t” value was employed mean ±S.E.M.P<0.05 (compared to control was considered significant), [9].

The preliminary phytochemical analysis showed the presence of glycosides, flavonoids, terpenoids and fixed
oils. The extract at the dose of 500 mg/kg showed increasing the urine volume and also the sodium, K+
& Cl- ions in urine (Table: 1). It was previously reported the flavonoids; glycoside are endowed diuretic
activity [10]. Therefore, we concluded that the diuretic activity of Aristolochia indica Linn may be due to the
presence of flavonoids in the extract. It was also concluded the whole plant of Aristolochia indica possess
hyper chlorimea, hypernatremia, hyper kaelemie diuretics. The present study justified that the traditional
use of Aristolochia indica Linn as a diuretic agent and warrants future detailed investigation as a premising
diuretic agent.
Treatment Dose Total Urine Total Na+ Total K+ Total Cl- Na+/K+
Volume (Ml/24h) (moles/kg) (moles/kg) (moles/kg) Ratio
Normal saline 25ml/kg.p.o. 16.5±0.67 76.27±0.26 72.52±0.56 650.92±0.59 1.05
Furosemide 20mg/kg.i.p. 41.5±0.27* 160.45±0.92* 135.58±0.76* 2913.45±0.82* 1.18
Ethanol extract 250mg/kg.p.o 25.6±0.36 * 125.76±0.86* 117.6±0.52 * 2106.57±0.92 * 1.06
Ethanol extract 500mg/kg.p.o 32.7±0.36* 142.91±0.86 * 126.69±0.52* 2506.54±0.93* 1.11
Mean ± SEM, n = 6, Student’s “t” test, *P< 0.001 (Compared to control) was considered significant
607
REFERENCES
1. Anonymous, The Indian medicinal plant, a compendium of 500 species, Publications and Information
Directorate, New Delhi (2005) vol: III, 199.
2. Kritikar KR and Basu BD, Indian Medicinal Plants, Vol III. 2nd ed, Bishen Singh Mahendra Pal Singh,
Dehradun (1975) 2122 – 2124.
3. Anonymous, The Wealth of India Raw materials, Publications and Information Directorate, New Delhi
(2006) vol: I, 83.
4. Vogel GH and Vogel WH, Drug Discovery and Evaluation, Pharmacological Assays, IIIrd Ed, Springer
– Verlag, Berlin, Heiderberg, New York, 2002, 323-324.
5. Singh GK and Dixit VK, Diuretic and Anti-inflammatory activity of Trianthema portulacastrum Linn.
Indian Drugs, 1992, 30(4), 170–172.
6. Lipschitz WT, Haddian Z and Kepscar A, Bioassay of diuretics. J Pharmacol Exp Ther 1943; 79: 110.
7. Vogel, Textbook of Quantitative Analysis of Chemical Analysis, Vth Ed, Addition Wesley Longman
Ltd., England.
8. Becket BH and Stenlake JB, Practical Pharmaceutical Chemistry, Part I, Ist Ed, CBS Publishers and
Distributors, New Delhi, 1997, 197.
9. Ghosh MN, Fundamentals of experimental pharmacology, IInd Ed., Scientific Book Agency, Calcutta,
1984, 156-157.
10. Kavimani S, Ilango R, Gurubatham J, Jaykar B, Majumder UK and Gupta M, Acetylcholine antagonistic
action of aqueous extract of orthosiphon thymiflorus, Indian J Pharm Sci 1997, 59, 271-272.
608
In Vitro Antioxidant and Hepatoprotective Activity of
Ethanolic Extract of Sesbania Grandifolia Linn Leaves
K.S. Sridevi Sangeetha and S. Umamaheswari
Department of Pharmacology, Faculty of Pharmacy, Sri Ramachandra University, Porur, Chennai

e-mail: sangeethsb@gmail.com, Mobile:+91 9884023926
Abstract:
Sesbania grandiflora Linn. is an Indian medicinal plant commonly called as agathi that belongs to family
Fabaceae. It is mostly cultivated in south and west India in the Ganga valley and Bengal. Traditionally Sesbania
grandiflora was used for colic disorder, jaundice, small-pox, antipyretic, leprosy, night blindness and gout.
Various part of the plant showed hepatoprotective activity but in vitro hepatoprotective activity in ethanolic
extract of the leaves have not been studied so far, the present study was designed to investigate the qualitative
analysis of phytochemical constituents, in vitro antioxidant and in vitro hepatoprotective activity. Sesbania
grandiflora leaves are collected in Tambaram and authenticated. Acetaminophen was used as hepatotoxicity
inducing agent, and it was carried out by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay in Chang liver cell line. The phytochemical analysis of the plant showed the presence of flavonoids,
saponins, tannins and steroids. The invitro antioxidant was confirmed by DPPH (1-diphenyl-2-picrylhydrazyl)
free radical scavenging activity. The leaves were found to have dose dependent hepatoprotective activity in
Chang liver cell line (Figure 1). The result of this study indicates the leaf extract of Sesbania grandiflora has
good potentials against hepatic disease.
Keywords: Agathi, Sesbania, Acetaminophen, Hepatoprotective.
INTRODUCTION
Hepatic system of an organism is involved in various metabolic activities.. In this process, it may expose to various
challenges and hence, hepatic system is not only evolved to perform its function but also to protect itself to various
challenges like exposure to antibiotics, chemicals, etc. Liver is such an organ that its physiological role and its
self-protective mechanism are well developed and orchestrated. In spite of such balanced internal milieu, hepatic
aberration, damage and necrosis commonly occurring due to over exposure to hepatotoxic causes to such an extent
that it over powers the mechanism. However, there are several herbs and herbal formulation which are found to be
claimed for treating hepatic disorders.
Nature has provided an excellent storehouse of remedies to cure all the ailments of mankind. Plants have been
used for healing and treatment of disease from time immemorial. Even when the modes of medicine have changed
from time to time, plants continue to be the main stay of medicine. India is considered to be an emporium of herbal
drug for the prevention and treatment of disease [1].The plant Sesbania grandiflora (L.) Poir (Fabaceae) is a short
lived and fast growing tree, native of India and Malaysia. In India it is present in Punjab, Dehli, Bengal, Assam and
Tamil Nadu. Sesbania grandiflora, commonly known as “Agathi,” is used in Indian traditional medicine for the
treatment of several diseases. Different parts of Sesbania grandifolia Linn was used as folk medicine for various
purposes. The roots were used in rheumatism, inflammation and painful swelling, bark as astringent, in snake bites,
small pox and also as antipyretic and anthelmintic agent [2]. Leaves were used as tonic, antipyretic, leprosy, night
blindness and gout [3]. It is useful in ophthalmia and poultice for bruise. Flowers were used in eyes to relive dimness
of vision and to improve appetite. Fruits were used as laxative, cures fever, pain, anemia, and also to improve taste
and thirst [4].
Various research have been done in the different parts of the Sesbania grandiflora and has shown anti
inflammatory, anti-arthritic [5], antiulcer [6], [7],analgesic, antidiarrheal, antibacterial, antifungal activity
[8], antihelmintic [9], hypolipidemic [10], [11], diuretic, CNS depressant and laxative [12] actions. But there
are very few reports on the hepatoprotective activity on Sesbania grandiflora. Despite the large number
of compounds available in the market, there is still a need to identify potent leads with chemotherapeutic
significance. From the literature review, it was concluded that Ethanolic extract of Sesbania grandifolia
(EESG) leaves against acetaminophen induced hepatoxicity in Chang cell line was not yet studied.

Plant collection and authentication
Sesbania grandiflora leaves were collected around Tambaram, India during April. The plant material was
identified and authenticated by Prof. P Jayaraman, Plant Anatomy Research Centre, Chennai, Tamil Nadu.
(Reg.No: PARC/2010/577). The leaves were washed with double distilled water to remove dirt and then
shade dried. The dried leaves were powdered.
Extraction
The powdered materials of Sesbania grandiflora was extracted with 70% ethanol by Soxhlation. The extracts
were filtered and concentrated using rotary flask evaporator and dried over hot water bath.
Cell lines and growth media

Chang liver cells (normal human liver cells) were purchased and cultured in Dulbecco’s modified eagles
medium (DMEM) supplemented with fetal bovine serum (10%), penicillin G (100 IU/ml) and streptomycin
(100 μg/ml). Cells were maintained at a temperature of 37°C in a 5% CO2 atmosphere with 95% humidity.
Qualitative Phytochemical Analysis

The individual extract was subjected to the qualitative phytochemical screening for the presence of some
chemical constituents. Phytochemical test were carried out adopting standards procedure [13].
Antioxidant assay by DPPH method [14]

The DPPH radical scavenging assay was first described by Blois (1958), and this was modified later by
many researchers. The free radical scavenging activities of ethanolic extracts and the standard L-ascorbic
acid (vitamin C) were measured in terms of hydrogen donating or radical scavenging ability, using the stable
radical (DPPH) 1, 1-diphenyl-2-picrylhydrazyl. DPPH, being a stable free radical it is frequently used to
determine radical scavenging activity of natural compounds. DPPH (radical form) absorbs at 517 nm, but
upon reduction with an antioxidant, its absorption decreases. It is due to the formation of its non-radical form
DPPH–H.
The reaction mixture contained 2 mL of 0.1 mol/L DPPH in ethanol and 2 mL of the serial dilution of
the extract at the concentration of 50, 75, 100, 125, 150 and 200. The mixture was incubated at 25 °C for 15
min, and the absorbance was determined at 517 nm. Distilled water was used as a control, and L-Ascorbic
acid was used as standard antioxidant. The scavenging activity of DPPH radicals in samples was calculated
according to the following equation:
(absorbance at blank) − (absorbance at test)
Percentage antioxidant activity = × 100
(absorbance at blank)
The experiment was repeated in triplicates and the results were expressed in mean. The IC50 values were
determined as the concentration of the test mixture that gave 50% reduction in the absorbance from a control
blank. The results were computed for analysis.
610
In vitro cytotoxicity assay [15], [16]:

The Cytotoxicity of ethanolic extract of Sesbania grandifolia leaves on Chang Liver cells was determined by
the MTT assay. It is based on the concept that dead cells and their products do not reduce the tetrazolium.
The number of viable cells is proportion to the extent of formazan formation. The monolayer cell culture was
trypsinized and cell count was adjusted to 1x105 cells/ml. To each well of the 96-well tissue plate, 0.1 ml
diluted suspension was added and incubated for 24 hr. The supernatant was removed and the monolayer was
washed once and to this 100µl of different concentration of the extract was added and incubated at 37°C for
72 hours in 5% CO2. The plant extract in the wells was discarded. Add 50 µL MTT solution (5mg/ml ) were
added to each well and Incubated at 37°C for 3 hours. The supernatant was removed. Add 50µL propanol
and the plates were gently shaken ( to dissolve the formazan salt) and the absorbance was measured at 540
nm. The effect of the samples on the proliferation of Chang Liver cells was expressed as the % cell viability,
using the following formula:
% cell viability = Mean OD of Test group / Mean OD of control group × 100
In vitro hepatoprotective activity against Acetaminophen induced toxicity in Chang liver

cells by MTT assay[17], [18].
Chang liver cells (1x105cells/well) were maintained in culture media containing 125 μg/ml of acetaminophen,
in the presence of Ethanol extract of Sesbania grandifolia leaves at the concentrations of 100, 50, 25, ten μg/
ml for 24hrs. Later, supernatants from each well were removed and the % of cell viability was calculated.
The monolayer cell culture was trypsinized and cell count was adjusted to 1x105 cells/ml. To each well of the
96-well plate, 0.1 ml diluted suspension was added and incubated for 24 hr. The supernatant was removed
and the monolayer was washed once. 100µl of different concentration of the extract was added to the well
plate and incubated 24 hours. After 24 hours pretreatment with the extract, 100 µl of Acetaminophen ( )
was added to well and incubated at 37°C for 72 hours in 5% CO2. The supernatant was discarded. Add 50
µL MTT solution (5mg/ml ) were added to each well and Incubated at 37°C for 3 hours. The supernatant
was removed. Add 50µL propanol and the plates were gently shaken( to dissolve the formazan salt). The
absorbance was measured at 540 nm.
100 − Mean OD of individual Test group
% Growth inhibiton = × 100
Mean OD of the control group
STATISTICAL ANALYSIS
Data are expressed as mean ± SEM.

The preliminary phytochemical screening of Sesbania grandifolia leaf has shown the presence of saponins,
flavonoids, tannins, alkaloids and steroids shown in table1.The % inhibition of DPPH free radical by different
concentration of the standard ascorbic acid and ethanol extract of the plants were depicted in table 1and the
IC50 values were calculated. The reducing capacity of the plant extract was found to be increasing with dose
dependent manner when compared with ascorbic acid. The plant extract showed dose dependent scavenging
effect and it increase with an increase in concentration, but the activity was less when compared to standard
ascorbic acid (Figure 1 and Table 1) and the IC50 value of ethanolic leaf extract was found to be 135.91 μg/ml.
The cytotoxicity study result showed that the plant extract has good cytoprotective activity in Chang liver
cells. The in vitro hepatoprotective assay, the toxicant acetaminophen showed the percentage viability of
40% while the cells that are pretreated with extract showed an increase in percentage viability of 55% at the
concentration of 10µg/ml of EESG (Table 2 and Figure 2 )The plant showed the maximum Hepatoprotective
action of 71% at the concentration of 100µg/ml.
611
Table 1: In vitro antioxidant activity of ethanolic extract of Sesbania grandifolia (EESG ) by DPPH assay
Concentration Percentage of inhibition

in (µg/ml)
Ethanolic extract of Sesbania grandifolia Ascorbic acid
Leaves (EESG)
50 26.62±0.21 86±0.34
75 34.43±0.36 88±0.12
100 42.62±0.29 90±0.21
125 48.08±0.12 92±0.42
150 55.37±0.22 94±0.29
200 64.32±0.16 96±0.33
IC50 135.91
Average of three independent determinations, values are mean ± S.E.M
Figure 1: Histogram showing the antioxidant activity of ethanolic extract of Sesbania grandifolia (EESG) by DPPH
method
Table 2: Protective effect of given extracts on acetaminophen induced toxicity in Chang liver cells
S.No Treatment Concentration (µg/ml) % Cell Viability

1 Control - 100
2 Acetaminophen 125 39.92±31.22
3 Acetaminophen 100 71.32±15
+ 50 65.86±23
Sesbania ethanolic leaf extract 25 60.40±36
10 55.90±29
Average of six independent determinations, values, are mean ± S.E.M.
612
Figure 2: In vitro hepatoprotective activity of ethanolic extract of Sesbania grandifolia (EESG) against acetaminophen
in Chang liver cell line
In the present study the extract was subjected phytochemical screening, it was observed that the
extract has shown a marked presence of flavonoids, tannins and steroids. Hepatoprotective activity was
associated with antioxidant activity since it is free radical mediated damage. The probable mechanism by
which Sesbania grandiflora (Linn) exerted its protective action might be due to the stimulation of hepatic
regeneration through an improved synthesis of proteins, or with interference with the liberation of microsomal
activation to toxicants. Flavonoids and tannins were reported to possesses various of pharmacological
activity including hepatoprotective activity. In the present investigation, preliminary phytochemical study
on Sesbania grandiflora (Linn) gave positive tests for flavonoids and tannins. This could be the reason for
the dose dependent hepatoprotective property of the leaf extract.
CONCLUSION
The present investigation revealed that the ethanolic extract of leaves of Sesbania grandiflora Linn exerted
dose dependent protection against hepatotoxicity. The extract has also shown good in vitro cytoprotective
activity. Our results indicated that the potent hepatoprotective activity of the extract might be due to its free
radical scavenging properties which may be due to presence of flavonoids in the plant. Further investigation
has to be done to identify the active constituent responsible for the activity and to evaluate the in vivo
hepatoprotective property using animal models. Also, the underlying mechanism of action contributing to
the hepatoprotective activity has to be enumerated and validated.
REFERENCES
1. Mala Agarwal, Medicinal plants, RBSA publishers, 2002, 1-2

2. Vaidyaratnam PSV, Indian Medicinal Plants, A Compendium of 500 Species, Vol. V, Orient Longman,
Madras, India, 1996; 117-118
3. Kirtikar KR and Basu BD, Indian Medicinal Plants. 2nd ed., Vol-I, 1999; 735-73
4. V.K. Singh, JN Govil and Shamima Hashmi, Recent progress in medicinal plants volume 7, Ethnomedicine
and pharmacognosy II, 2003, studium press LIC USA, 247
5. Patil R., Nanjwade K., et al.,. Effect of Sesbania grandiflora and sesbania sesban bark on Carrageenan
induce acute inflammation and adjuvantinduced arthritis in rats. IJPS 2010; Vol 1: pp 75-89.
6. Bhalke R., Giri A., et al.,. Antiulcer activity of ethanol extract of leaves of Sesbania grandiflora. IJPPS
2010:Vol 2: pp 206-208.
613
7. Sertie A.A., Wiezel G,. Et al,. Antiulcer activity of etrhanol extract of Sesbania grandiflora. Brazilian
journal of pharmaceutical sciences 2001; Vol 37: pp 1-10
8. Subramanian H., Varghese S,. etal, Pharmacological screening of Sesbania grandiflora poiret extracts.
Natural products sciences 2003; Vol 9: pp 154-157.
9. Jalapure.S., Alagwadi.K,. et al,. In vitro antihelminitic property of various seed oils. Iranian journal of
pharmaceutical research 2006; Vol 4: pp 281-284
10. Saravanakumar A, Vanitha S., et al,. Hypolipidemic activity of Sesbania grandiflora in triton wr-1339
induced hyperlipidemic rats. IJOP 2010; Vol2: pp 52-58.
11. Ramesh T., Mohaideen V., et al,. Hypolipidemic effect of Sesbania grandiflora on cigratte smoke
exposed rats. Pharmacologyonline 2006; Vol 3: pp 309-323
12. Rajasakaran A and Murugusen S Diuretic, CNS depressant and laxative evaluation of the leaf extract of
Sesbania grandiflora. Int J chem.Sci 2003; Vol 4: pp 436-439.
13. Hedge IC. Labiatae, In Flora of Pakistan.Ali SL, Nasir YJ(Ed) University of Karachi, Department of
Botany, Karachi, 953:3-25
14. Gu¨lçın, _I., Oktay, M., Kıreçcı, E., & Ku¨frevıo˘glu, O¨ . _I (2003). Screening of antioxidant and
antimicrobial activities of anise (Pimpinella anisum L.)seed extracts. .Food Chemistry, 83, 371e382
15. Mosmann T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation
and Cytotoxicity Assays. Journal of Immunology Methods 1983; 65: 55-63.
16. Masters RW. Animal cell culture, Cytotoxicity and viability assays. 3rd ed., 2000:202-203.
17. Vijayan P, Kumar VS, Dhanraj SA, Badmani S and Suresh B, In Vitro Cytotoxicity and Anti-tumor
Properties of the Total Alkaloid Fraction of Unripe Fruits of Solanum pseudocapsicum. Pharmaceutical
biology 2002:406-456.
18. Beena P, Purnima S and Kokilavani R, In vitro hepatoprotective activity of ethanolic extract of Coldenia
procumbens Linn. J Chem Pharm Res 2011; 3(2):144-149.
614
Synthesis and Characterization of Novel Amino Acid
Prodrug of Rosiglitazone
S. Vijayaraj1, G. Kalyana Chakravarthi2 and R. Shanmugam3
1, 2, 3
Department of Pharmaceutical Analysis, Sree Vidyanikethan College of Pharmacy, Sree Sainath Nagar,
Tirupati, Andhra Pradesh, India
1
Department of Pharmaceutical Sciences, NIMS University, Jaipur, Rajasthan, India
e-mail: vijaysurender@yahoo.co.in, Mob +919032774923
Abstract:
Diabetes mellitus is the most common endocrine disorder. Rosiglitazone, a thiazolidinedione class of
antidiabetic drug widely used to treat type-II diabetes. Rosiglitazone being classified under Biopharmaceutical
Classification System class II drug do have poor solubility. As solubility is an important parameter in
enhancing bioavailability, majority of R&D focused pharma companies are targeting class II category of
drugs to overcome the solubility barrier. Prodrug approach is a promising effort to overcome this barrier.
Hence the present work aims to prepare novel amino acid prodrug of rosiglitazone. The synthesized prodrugs
were characterized by spectral and physicochemical techniques. Chemical hydrolysis study was performed
to ensure the stability of the prodrugs. The prepared prodrug was found to stable in simulated gastric fluid
(SGF) and simulated intestinal fluid (SIF). Reduction in Log P value 0.39 of prepared prodrug compared to
2.4 of drug indicates increase inhydrophilicity. Aqueous solubility study confirms 4.66 fold enhancement in
solubility of amino acid prodrug of rosiglitazone compared to pure drug. Thus the synthesized amino acid
prodrug of rosiglitazone has good potential for further optimization and development.
Keywords: Rosiglitazone, Aminoacid prodrug, Solubility, Chemical hydrolysis.
INTRODUCTION
Rosiglitazone (RG) chemically, 5-[(4-{2-[methyl (pyridin-2-yl) amino]ethoxy} phenyl)methyl]-1,3-
thiazolidine-2,4-dione acts as hypoglycemic agent Fig.1.RG falls under BCS class II drugs which do have
poor solubility and good permeability [1-3].Solubility is one of pharmacokinetic barrier to be encountered in
screening studies of new chemical entities as well as in formulations design and development. Though various
physical and chemical methods like hydrotropic solubilization, solid dispersions, melt sono crystallization
were available in solubility enhancement of RG [4-6], prodrug approach is one of the most attractive method
in which active drug is masked to alter its undesirable properties. Prodrug is a chemically modified inert
drug originator, which upon biotransformation liberate the pharmacologically active parent compound [7].
Amino acids are widely used ionizable group introduced to the parent drug molecule to compensate for poor
aqueous solubility. As amino acids are biocompatible moieties, they are metabolized and excreted without
much safety concerns [8]. To the best of our knowledge there were no reported methods for synthesis of
amino acid prodrug of RG. Hence the aim of the present research is to synthesize and characterize a novel
water soluble amino acid prodrug of RG.

Instruments and Chemicals
Melting points were determined on a digital capillary melting point apparatus. Aluminum sheets coated
with silica gel 60 F254 of Merck were used for TLC. Photo microscopic images were taken using Olympus
research microscope. Elemental analysis was performed using Carlo-Erba model 1108. The IR spectra were
recorded in KBr discs on FT-IR Bruker IFS 55 spectrophotometer and wavenumbers are reportedin cm-1.
The 1H1NMR spectra were obtained on a Bruker DRX-300 spectrometer (75 MHz) in DMSO. Chemical
shifts were recorded in ppm (d) relativeto TMS as an internal standard. High resolution mass spectra were
recorded on an Agilent 5975 MSD series Direct Inlet Probe system using electro spray ionization technique.
All chemicals used were of analytical grade procured from SD fine, Himedia and E. Merck while standard
drug of RG was purchased from Yarrow Chem Products, Mumbai.
Synthesis of Amino acid prodrug

Boric acid catalyzed amidation was adopted for synthesis of amino acid prodrug of RG. 5mM of RG, 5mM
of tyrosine, 5mM boric acid and 126ml of xylene were taken in round bottomed flask and reflux condenser
was assembled. The resulting mixture was condensed about 1 hour followed by re-crystallization. Ethanol
was added for complete solubilization of product when it sticks to the walls of round bottom flask. Solvent
was evaporated, and filtered by vacuum filtration. Residue was dried, product was collected and percentage
practical yield was calculated. [9-11] Fig. 2
Spectral and thermal characterization

Pressed pellet technique was adapted for FT-IR analysis, drug admixed with KBr were made in to disc
and was analysed in spectral range of 4000 to 400 cm-1and IR spectrum was recorded. 1H1, Mass and DSC
studies were carried out for the synthesized prodrug.
Partition Coefficient
The partition coefficient of product was determined in n-octanol/water system (10:10) by standard technique.
Product (drug or prodrug) was accurately weighed (10 mg) and added to 10 ml each of n-octanol and aqueous
phase. The mixture was shaken using mechanical shaker for 24 hrs until equilibrium was reached. Phases
were separated by separating funnel and aqueous phase was analyzed for amount of product after appropriate
dilution. Procedure was performed in triplicate [12].
Where kd is partition Coefficient

Co = Concentration of solute distributed organic phase
Caq= Concentration of solute distributed in aqueous phase
Aqueous solubility
Equilibrium solubility was determined by a “shake-flask” method [13].The aqueous solubility of compound
was determined by adding excess amount of drug beyond its saturation limit in sealed conical flask containing
10 ml of water. This conical flask is placed in a mechanical shaker for 48hrs (This duration was previously
tested to be sufficient to reach equilibrium). The solvent was filtered through Whatmann filter paper
No.42 and the portion of the filtrate was suitably diluted with water. Solutions were analyzed by using UV
spectrophotometer at 242 nm, which was the absorption maxima and drug concentrations were calculated.
616
Chemical Hydrolysis Study

The rate of chemical hydrolysis of the prodrug was determined in Simulated Gastric Fluid (SGF, pH 1.2)
and Simulated Intestinal Fluid (SIF, pH 7.4) at 37°C. Solution of 10 mg of synthesized prodrug was placed
in dissolution basket containing 90 ml of SGF/ SIF individually.An aliquot of 15 ml of this solution was
withdrawn repeatedly and kept in test tubes maintained at 37 ± 0.5ºC. At a definite interval of time (0.5, 1,
2 up to 8 h), an aliquot was withdrawn to different test tubes and was transferred to micro centrifuge tubes
followed by addition of methanol to make up the volume. The tubes were placed in freezing mixture in
order to arrest further hydrolysis, followed by vortexing at high speed for 5 min. After vortexing, the tubes
were centrifuged at high speed 3000 rpm for 5 min. A 5 ml of clear supernatant obtained from each tube was
measured on UV spectrophotometer for the amount of free drug released after the hydrolysis of prodrug at
273 nm and 306nm in SGF and SIF respectively [14].

Aminoacid prodrug of RG was synthesized by using boric acid catalyzed amidation method and its percentage
yield was found to be 85.28%.TLC studies have shown the Rf values of RG and its aminoacid prodrug to be
0.78cm and 0.53cm respectively, which ensures the formation of prodrug.
Photo microscopic images of RG and its aminoacid prodrug were observed at 45X. Fig. 3. Morphology
of the synthesized aminoacid prodrug was different from that of RG. Elemental analysis of synthesized
prodrug shows C: 60.24, H: 5.32, N: 10.44, O: 18.01, S: 5.47. Fig. 3.
IR spectrum of aminoacid prodrug exhibits broad peak in the range of 3200-2200 cm-1 because of the
presence of O-H stretch, whereas in the IR spectrum of rosiglitazone, it has been short peak in the range of
3200-2200 cm-1because of the absence of O-H stretch.RG shows NH- stretch at 3447 cm-1compared with
aminoacid prodrug at 3199cm-1. Aminoacid prodrug shows C=N stretch at 1684cm-1, which absent in drug.
This interpretation indicates NH group is involved in bond formation Fig. 4, 5.
1H1 NMR of Aminoacid prodrug of RG (CDCl3 and DMSO) δ in ppm: 1.11 (s,1H, R- NH2), 3.3 (s,5H,
CH3 protons), 3.8 (s,3H, CH3 protons), 4.0 (t, 2H, CH2 proton), 4.4 (m,1H, R-NH2), 6.5 (d,2H, Ar-OH
protons), 7.4 (t, 1H, Ar-OH protons), 8.0 (d, 1H, CH protons of pyridine). 1H1 NMR of RG (CDCl3 and
DMSO) δ in ppm:2.9 (q, 1H, CH3), 3.1 (s,3H, R-NH2 protons), 3.2 (d, 1H, CH3 protons), 3.9 (t, 2H, CH2
proton), 4.1 (t,2H, R-NH2), 4.36 (q,1H, Ar- OH protons), 7.0 (d, 2H, Ar-OH protons), 8.0 (d, 1H, CH protons
of pyridine) Fig. 6, 7.Chemical shift of N-H proton in drug at 4.1 and in aminoacid prodrug at 4.4. Aminoacid
prodrug havetotal of 28 protons (i.e., aromatic and amine) compared to RG having 19 protons. Fig. 6.
The mass spectrum of RG represents characteristic parent ion peak at m/z = 357 (M+ peak, C18H19N3O3S)
and base peak at m/z value = 121(C4H9N) Fig. 9. The mass spectrum of synthesized amino acid prodrug of RG
exhibits parent ion peak at m/z = 535 (M-2 peak, C27H26N4O6S), from parent peak, 5-(4-(2-(methyl(pyridine-
2-yl)amino)ethoxy)thiazolidine-2,4-dione (C18H18N3O3S, m/z = 355) has been cleaved. Further N, N-dimethyl
pyridine-2-amine (C7H10N2)and p- cresol were fragmented (m/z = 107, C7H8O, base peak) Fig. 7, 8.
From the IR, NMR & Mass studies, the molecular structure for synthesized amino acidprodrug was
predicted and the proposed structure was confirmed to be amino acid derivative of RG & the molecular formula
was found to be C27H28N4O6S.
Digital capillary melting point apparatus was used to study the melting point of the synthesized prodrug
in relation to the individual drug. Melting point of RG was found to be 1220C, while amino acid prodrug
shows melting point at 1780C. The Melting point of synthesized prodrug was distinct, with a different melting
transition from that of individual drug. This indicates the formation of novel prodrug.
Log P value was calculated from Partition coefficient studies and was found to be 2.26 and 0.39 for drug and
prodrug respectively. Decrease in log P values ensures increase in hydrophilic character of synthesized prodrug.
617
Aqueous solubility of RG and amino acid prodrug was calculated in mg/ml and was found to be 3.023
and 13.961 for RG and prodrug of RG respectively Fig. 9.
Chemical hydrolysis study of amino acid prodrug in buffer solutions (Simulated Gastric Fluid, pH 1.2
and Simulated Intestinal fluid, pH 7.4) at 37°C was performed. The Rate of hydrolysis, Hydrolysis constant
and t½ of synthesized amino acid prodrug was found to be 68.75% and 93.29% at 180 min and 210 min, 3.96
× 10-3and 3.01 × 10-3, 64.30 and 67.30 min in SGF and SIF respectively. Table 1.
CONCLUSION
Novel amino acid prodrug of RG was synthesized by using boric acid catalyzed amidation. The prepared
prodrug exhibits good solubility, reasonable in vitro chemical stability in acidic and alkaline medium. Partition
coefficient studies ensure the increase in hydrophilicity of the synthesized prodrug. Aqueous solubility of
synthesized prodrug was enhanced by 4.66 folds to that of RG. These properties make the novel amino acid
prodrug of rosiglitazone effective in treating diabetes with enhanced bioavailability.
REFERENCES
1. Pubchem Compound, http://pubchem.ncbi.nlm.nih.gov/summary/cid=77999 (accessed 4th August 2013)

2. Ajjan RA and Grant PJ, The cardiovascular safety of Rosiglitazone. Expert opin. Drug saf.2008; 7 (4):367-76
3. Radhakrishna T, Satyanarayana J and Satyanarayana A, LC determination of rosiglitazone in bulk and
pharmaceutical formulation. J Pharm Biomed Anal 2002; 29:873-80.
4. Jagtap V.A, Vidyasagar G and Dvivedi S.C., Solubility enhancement of rosiglitazone by melt sono
crystallization technique, J Ultrasound. 2014; 17:27-32.
5. Sherje A P and Desai K J, Spectrophotometric determination of poorly water soluble drug rosiglitazone
using hydrotropic solubilization technique. Indian J Pharm Sci 2011;73:579-82
6. Punitha K, Daisy C.K, Venkatesh Kumar V and Suresh Kumar S, Enhancement of solubility of
Rosiglitazone through solid dispersion technique: in vitro and in vivo permeation study analysis. Der
Pharma Chemica, 2010; 2(5): 190-200.
7. Bundgaard H., Design of Prodrug. Amsterdam: Elsevier; 1985.
8. Vig B.S, Httuunen KM, Laine K and Rautio J, Amino acids as promoieties in prodrug design and
development. Adv Drug Deliv Rev. 2013; 65(10):1370-85.
9. Tang P, Boric Acid Catalyzed Amide Formation From Carboxylic Acids And Amines: N-Benzyl-4-
Phenylbutyramide. Org. Synth. 2005; 81, 262
10. Bhattacharya A and Bandichhor R, Green Technologies in the Generic Pharmaceutical Industry. In
Green Chemistry in the Pharmaceutical Industry, Dunn, P.; Wells, A.; Williams, M.T., Eds.; Wiley-VCH,
Weinheim, 2011; Chapter 14, 289-309.
11. Tang P.W., Preparation of Novel Delivery Agents for Delivery of Macromolecular Drugs Using Boric Acid
Mediated Amidation of Carboxylic Acids and Amines. In Advances in Controlled Drug Delivery; Dinh, S.
M.; Liu, P., Eds.; ACS Symposium, American Chemical Society: Washington, DC, 2003; Vol. 846, Chapter 8,
103–112.
12. OECD, Guidelines for the Testing of Chemicals, Partition Coefficients. 1981.
13. Baka E, Comer JEA and Takacs-Novak K. Study of equilibrium solubility measurement by saturation shake-
flask method using hydrochlorothiazide as a model compound.J. Pharm. and Biomed. Anal.2008; 46, 335-341.
14. Mahdi MF and Alsaad HN, Design, Synthesis and Hydrolytic Behavior of Mutual Prodrugs of NSAIDs
with Gabapentin Using Glycol Spacers. Pharmaceuticals.2012; 5, 1080-1091.
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Table 1: % Hydrolysis of amino acid prodrug of rosiglitazone
% Hydrolysis Kobs
Media pH t1/2 (min)
30 60 90 120 150 180 210 (min-1)
SGF 1.2 1.2 55.68 57.38 57.95 60.79 65.90 68.75 3.96 ×10-3 64.30
SIF 7.4 34.75 40.54 44.41 76.93 83.94 86.58 93.29 3×10-3 67.30
Figure 1: Chemical structure of RG
Figure 2: Chemical scheme for preparation of aminoacid prodrug of RG
Figure 3: Microscopical images of RG (I) and Amino acid prodrug (II)
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Figure 4: FT-IR spectra of RG
Figure 5: FT-IR spectra of Aminoacid prodrug
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Figure 6: 1H NMR spectra of RG (I) and Amino acid prodrug (II)
Figure 7: Mass spectra of RG
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Figure 8: Mass spectra of Aminoacid prodrug
Figure 8: Mass spectra of Aminoacid prodrug
Figure 9: Aqueous solubility study
622
Assessment of Carcinogenicity Potential by In Vitro
Methods: Towards Better Strategy
Darshan T. Valani, Dr. S. Rajesh Sundar, Dr. Mukul R. Jain and Dr. Sonal S. Bakshi
Zydus Research Centre, Ahmedabad, India

Institute of Science, Nirma University, Ahmedabad, Gujarat, India
e-mail: sonal.bakshi@nirmauni.ac.in
Abstract:
Genotoxicity assays are considered as surrogate to carcinogenicity studies. Most genotoxic agents are human
carcinogens but not all carcinogens are genotoxic in terms of mode of action. Although the current laboratory
assays are based on the association of genotoxicity with carcinogenicity, the importance of monitoring
human genotoxicity has been recognized not only for cancer as a consequence, but also as a risk factor
causing inheritable genetic change if germ cells are affected. The available data raises concern regarding the
sensitivity of the current genotoxicity test battery in detecting carcinogens owing to the frequent incidences
of false positives and false negatives. This battery of tests is also not adequate to identify the potentially non-
genotoxic carcinogens. In accordance with the 3 ‘R’ principle i.e. reduce, refine, and replace, it is desirable to
develop new in vitro assays with high throughput, better predictability for carcinogenicity in order to facilitate
the timely conduct of clinical trials, reduce the use of animals, and save drug development resources. This
review takes a stock of the current knowledge regarding novel approaches for the genotoxicity assessment
that are proposed alternative or complementary laboratory endpoints. These are of particular interest for
researchers and regulatory communities involved in forming guidelines on planning, performing, and
interpreting the studies on monitoring the groups of individuals exposed to genotoxic agents.
Keywords: Genotoxicity, Non-genotoxic carcinogens, Carcinogenicity, ICH S2 (R1)
INTRODUCTION
The study of the chemical carcinogenesis mechanisms and efficient prevention strategies and measures are of
crucial importance to protect human health [1]. The current chemical and pharmaceutical industries and units
involved in manufacturing engineered nanomaterials/particles (ENMs/ENPs) are facing critical challenges
on genotoxicity and carcinogenicity assessment for a candidate drug compound or new nanomaterials.
Owing to the mechanistic associations of DNA damage and cancer in addition to practical limitations of
carcinogenicity assays, results of in vitro genotoxicity have been used as a surrogate for carcinogenicity,
which are necessary for initiation of clinical trials. The standard genotoxicity testing battery of assays as per
the revised ICH S2 (R1) guideline is summarized in Table 1[2].
Table 1: Standard genotoxicity assays used for testing of pharmaceuticals (as per revised ICH S2 (R1) guideline).
TEST CATEGORY OPTION 1 OPTION 2

In vitro gene mutation in Bacterial Reverse Mutation (Ames) Test Bacterial Reverse Mutation (Ames) Test
bacteria
in vitro metaphase chromosome
aberration test
or
In vitro cytogenetic test for
in vitro micronucleus test NOT REQUIRED
chromosomal damage
or
in vitro mouse lymphoma Tk gene
mutation assay.
In vivo test for genotoxicity In vivo cytogenetic assay using rodent With two different tissues, usually:
hematopoietic cells. an assay for micronuclei using rodent
hematopoietic cells and
DNA strand breakage assay in liver
Although the link between DNA damage and cancer is well documented, the potential to predict the
outcome of trials in terms of carcinogenicity using genotoxicity test data may not be precise enough for
direct extrapolation [3]. The reliability of in vitro genotoxicity testing represents many issues. Particularly,
Kirkland et al. [4, 5] have shown that the current in vitro tests (Bacterial reverse mutation (Ames) test,
Mouse Lymphoma Assay (MLA), Micronucleus (MN) test and Chromosome aberration (CA) assay) have
high false-positive rates, thus low specificity and high sensitivity in prescreening for the prediction of
carcinogenicity.Table-2 depicts some representative examples for comparison of current genetic toxicology
and carcinogenicity test results.
Table 2: Representative examples for comparison of current genetic toxicology and carcinogenicity test results.
TEST DATA
DRUG THERAPEUTIC
Genotoxicity Carcinogenicity
EXAMPLE/S AREA
In vitro In vivo Rodent Human
+ve -ve +ve -ve Chloramphanicol Antibiotic
+ve -ve -ve -ve Ciprofloxacin Antibiotic
Pioglitazone Diabetes
Atorvastatin Dyslipidemia
Pravastatin Cholesterol Lowering
-ve -ve +ve +ve
Simvastatin Hypolipidemic
Rosuvastatin Hypolipidemic
Eplerenone Chronic Heart Failure
-ve +ve -ve -ve Diflunisal Anti-inflammatory
Note: +ve, positive; -ve, negative

Table 2: data are from Reference No. : 16, 47, 48
Snyder and Green (2001) reported that about 50% of non-carcinogenic marketed pharmaceuticals
had positive results in at least one of the regulatory in vitro genotoxicity tests [6]. Genotoxic potential as
identified by current in vitro tests, has been reported in as many as one third of the compounds reaching
preclinical regulatory evaluation, and about 12% of drug candidates are abandoned due to safety concerns
[6]. The false positive findings leading to many promising drug molecules being discarded at an early stage
of development involves huge monitory waste as well as research efforts. The equivocal conclusions also
trigger additional testing of apparently genotoxic candidates, both in vitro and in animals, to confirm if the
suggested hazard is genuine [7] before starting the clinical trials [8]. The discrepancy is due to limitations of
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genotoxicity endpoints and assays viz., insufficiency of certain in vitro assays to simulate the in vivo target
organ situation and the complexity of carcinogenic mechanisms. Moreover newer formulation techniques
like, nanotechnology prompt to revisit the current genotoxicity assessment methodology. Additionally as on
today, no definitive conclusion can be drawn concerning the genotoxic potential of engineered nanomaterials/
particles (ENMs/ENPs), essentially because of inadequate characterization and pitfalls in experimental
approaches. Newer methodologies are warranted other than focusing on reactive oxygen species estimation
including validation trials with known positive nanomaterials. Unless the ENP has been functionalized
leaving a reactive ‘organic’ surface, or contains impurities, no information has been recently published
that provides compelling evidence that poorly soluble ENPs behave differently to other well characterized
insoluble fine particulates with respect to causing DNA damage.
Therefore, an increase in the specificity of the current in vitro genotoxicity test battery by replacing,
optimizing, or adding new short term tests is urgently needed [9]. This may help achieve considerable
reduction in animal studies, avoid unnecessary in vivo follow-up studies and also replacement of in vivo
studies, thereby contributing to the promotion of 3Rs (reduction, replacement and refinement) in the
regulatory safety testing [10].
The fact that only one-third of marketed drugs found to be positive or equivocal for carcinogenicity were
also positive for genotoxicity indicates the existence of a considerable number of non-genotoxic carcinogens
[8]. Non-genotoxic carcinogens have been shown to act as tumor promoters, endocrine-modifiers, receptor-
mediators, immuno-suppressants or inducers of tissue-specific toxicity and inflammatory responses [11].
The diversity of modes of action of non-genotoxic carcinogens, the tissue and species specificity, and the
absence of genotoxicity makes predicting their carcinogenic potential extremely challenging [12].
Various non-conventional methods of in vitro genotoxicity have been reported that improve the
predictability for carcinogenic potential. The current review outlines some of the methods that need to be
explored to produce larger data.
GreenScreen HC Assay:
GADD45a (growth arrest and DNA-damage-inducible, alpha) gene is one of many genes that show increased
transcript levels following stressful growth conditions and treatment with DNA-damaging agents. It plays a
central role in DNA stress, damage and repair cascades within the mammalian cells. Mutagens, clastogens
and aneugens all cause increased expression of the mammalian GADD45a gene [8]. This feature has been
exploited in the development of a green fluorescent protein (GFP) reporter based assay (GreenScreen) for the
gene using human lymphoblastoid cell line TK6 by Hastwell et al [3]. TK6 cells have a wild type p53 tumor
suppressor gene [12]. This attribute make this cell line very useful for assessing the genotoxic response
properly.
The assay protocols described in detail by Hastwell et al [3] are summarized here. Assay uses 96 well
plate formats, in which four compounds are each tested in two series of nine two-fold dilutions. One series
is tested using the TK6 cell line expressing the GADD45a-GFP reporter (test cells), and another is tested
using TK6 cells in which the reporter has a non-expressed GFP gene (control cells). Assay can be done in
presence and absence of metabolic activation system. Data from the control cells identify compounds that
are themselves fluorescent, or induce cellular auto-fluorescence, and thus allow for normalization with the
test-cell data. Data from S9-treated samples are collected using flow cytometry [14], and data from samples
not treated with S9 can also be collected using fluorescence/absorbance spectrometry [3] in addition to flow
cytometry as both give comparable results [14]. The GreenScreen assay is reported to exhibit sensitivity and
high specificity for various classes of genotoxins comparable with other in vitro mammalian cell assays [3,
15, 16]. International multi-laboratory ‘ring trials’ have demonstrated transferability of assay versions both
with and without S9 metabolic activation [17].
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Pig – a Gene Mutation Assay

A cytoplasmic membrane bound glycolipid structure, termed glycosyl phosphatidylinositol (GPI) anchors
that link various protein markers to the surface of several types of mammalian cells, including hematopoietic
cells [18]. An enzyme required at very initial step for the synthesis of GPI is governed by the endogenous
phosphatidylinositol glycan complementation group A gene (Pig-A) [19, 20]. In addition to Pig –A many
genes are required for GPI synthesis (e.g., Pig-B and Pig-C). The Pig-A gene is located on the X chromosome
[21, 22] hence, single mutation is sufficient to cause alteration in cell phenotype. Numerous protein markers
bind with GPI on cell surface of various tissues [23, 24]; thus, mutations in the Pig-A gene could result
in the lack of GPI synthesis thus resulting in deficiency in GPI-anchored proteins. Rapid flow-cytometric
methods for measuring the frequency of rodent and nonhuman primate peripheral blood cells deficient in
GPI-anchored proteins (presumed Pig-A mutant cells) have been developed. One common approach is to
evaluate the expression of proteins (in rats, usually CD59) anchored by glycosyl phosphatidyl inositol (GPI)
to the surface of peripheral red blood cells (RBCs).
The detail procedure is mentioned by Miura et al [18]. Briefly, the animals are dosed for single or multiple
treatment regimens with test item. At the end of treatment peripheral blood is collected in anticoagulant and
diluted in PBS and the cells are labeled with anti-rat CD45 antibody. After incubation for 1 hr in the dark
at room temperature, the cells are washed, re-suspended in PBS, and assayed for CD59-negative RBCs or
reticulocytes using flow cytometer. The change in CD59-negative RBCs or reticulocytes in treatment group
as compared to those of control group is analyzed as end point.
The Pig-A assay is reported to be a promising tool for evaluating in vivo mutagenicity, hence the recent
draft guideline for impurity testing, ICH M7 (Step 2 version) includes Pig-A assay to assess the in vivo
relevance of in vitro mutagens detected with positive bacterial mutagenicity [25].
γH2AX by flow assay

DNA double-strand breaks (DSBs) are the toxic lesions that can drive genetic instability [26]. Histone variant
H2AX is a key component of DNA damage response. It becomes rapidly phosphorylated at the carboxyl
terminus to form the so-called γH2AX at DSB sites [27]. The measurement of serine139-phosphorylated
histone H2AX provides a biomarker of DNA double-strand breaks (DSBs) and thus identify potential
genotoxic activity [28]. The γH2AX facilitates the repair of DNA double-strand breaks (DSBs) as an integral
component in the DNA damage response machinery of mammalian cells [27]. Flow cytometric measurement
of γH2AX is a novel approach for detection of genotoxic potential of a compound. The assessment of
γH2AX over conventional assays like mouse lymphoma assay (MLA) and chromosome aberration (CA)
assay is reported to be more advantageous [29, 30]. The genotoxic signatures in the form of DNA damage
response and cell cycle information are readily elucidated, low amount of compound is required and there is
rapid and high throughput data acquisition [28].
The detailed assay protocol reported by D.J. Smart et. al [28] includes Mouse lymphoma L5178Y cells
(tk+/−). This assay can be done in presence and absence of S9. Relative cell counts (RCC; % control) using
coulter counter provide an index of cytotoxicity [31].
To evaluate γH2AX, cells are lysed to separate the nuclei which are resuspended in PBS containing anti-
human γH2AX-FITC antibody and 7-AAD (7-Aminoactinomycin D) to form a suspension of single nuclei.
Nuclear i.e. γH2AX and genomic DNA are labeled with green and red fluorescence, and forward/side light
scatter from 104 nuclei are measured using a flow cytometer with standard emission filters. The difference in
fluorescence values are expressed as relative fold-change in γH2AX fluorescence over control in correlation
with relative cell count as a measure of cytotoxicity in terms of % control. The data are used to determine
whether a compound induced a positive, negative or equivocal response in the assay.
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Many genotoxins and non-genotoxins are reported to be identified by γH2AX flow assay, including a
wide range of chemicals viz., alkylating agents, aromatic amines, amides, nitroso, epoxides, hydroperoxides,
N-nitrosos, with both non- and structurally-alerting chemicals (based on SAR using DEREK for Windows).
Thus the flow-cytometric assay of γH2AX offers broad range genotoxicity assay [28]. It has shown high
sensitivity to detect DNA-reactive genotoxic compounds though the utility for metabolic activation
dependent compounds is limited and needs to be improved. Newer approaches for in vitro improvement in
sensitivity (i.e. toxicogenomics) may be combined with this automated assay to obtain improved efficiency
in genotoxicity testing [9].
Cell Transformation Assays (CTAs)

Transformation as one of the key events in the carcinogenesis process can be assessed in vitro by Cell
Transformation Assay30. In vitro cell transformation is, to date, the only well-established methodology that
has the potential to detect both genotoxic and non-genotoxic carcinogenic compounds 31(11th reference). As a part
of the strategy to predict carcinogenicity, the CTA may be helpful as a follow-up assay to reduce the number
of irrelevant false in vitro genotoxicity positives [32]. These assays are done in vitro, generally using SHE
cells or BALB/c 3T3 cell line
The primary cells derived from SHE cells are used, and the colony formation of transformed cells is
detected as the endpoint [33]. The cells at clonal density were treated with the test chemicals for 7 days in
Dulbecco’s modified Eagle’s medium at pH 6.7 or 7.0 containing fetal bovine serum (FBS). Subsequently
the colonies are fixed, stained, and scored for morphological transformation [33]. Another cell line employed
is Balb/c 3T3 (fibroblast cell line derived from Balb/c mouse embryo), clone A31-1-1 that retain the contact
inhibition. When cell proliferation lead to cell crowding and saturation in culture dish, growth comes to
halt in a monolayer, which is a feature of normal cells. However, when the cells lose contact inhibition
after treatment with tumor promotors, disordered cell proliferation is induced and the cells tend to pile up
in a random criss-cross fashion. Such transformed cells also exhibit a morphological change which shows a
distinctive shape (spindle-shape). Tumor promoting potential of chemical is determined by enumerating the
foci of transformed cells, the detail procedure has been reported by Sasaki et al [34].
Several CTA protocols have been developed which are extensively reviewed in the OECD [35, 36].
The Syrian hamster embryonic (SHE) CTA and the BALB/c 3T3 CTA, were selected for the performance
assessment in the ECVAM (European Centre for the Validation of Alternative Methods) pre-validation study
[37]. On the basis of conclusion on performance the OECD Washington expert group recommended that the
SHE and the Balb/c 3T3 CTAs should be developed in to OECD test guidelines [36].
The performance of the Syrian Hamster Embryo (SHE) cell transformation assay as a predictor of
carcinogenic potential has been established on hundreds of chemical carcinogens and has been reviewed in
a database summarized in the OECD Detailed Review Paper (DRP) 31 [35,36]. An ECVAM study [38] that
addressed the availability of standardized protocols, their transferability within- and between-laboratories
reproducibly, in combination with the results of the DRP31, supports the use of the in vitro CTA for the
assessment of carcinogenic potential [37]. However, subjectivity involved in identifying morphologically
transformed colonies could impair the performance of the CTAs, the assay performed based on the OECD
guideline [39] or relevant standard literature is able to provide reliable result for carcinogenicity prediction.
Toxicogenomics is the integration of genomics to toxicology that involves the study of gene expression
patterns after exposure to a test item with the objective of gaining a deeper mechanistic understanding
of toxic actions, and developing predictive tools to rank, select, and evaluate new drugs [40]. The use of
toxicogenomics also offers the most promising results in terms of reliability and reducing the number of
animals, time, and cost [12]. With recent progress in molecular technologies it has become possible to study
complex network of cellular pathways at genomic level in response to treatment with chemicals assuming
that gene expression changes precede histopathological or clinical pathological variations. [8].
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The studies can be done in vitro using various cell lines like TK6, L5178Y, HepG2 etc., and also
isolated tissues of target organ from treated animals. The toxicogenomic data enables to differentiate
between DNA reactive vs. DNA non-reactive mechanism, which is essential for critical risk assessment
following positive findings of carcinogenicity and genotoxicity study [41, 42]. Toxicogenomics can also be
helpful in detection of genes related to cell receptor-ligand modulation, transporter modulation, metabolic
transformation, inhibition of apoptosis, increased cell proliferation, hormonal perturbations, regenerative
proliferation, receptor activation, peroxisome proliferation, endocrine disruption, DNA repair, inflammatory
responses, p53 regulation, oxidative DNA damage response, mitochondrial function, stress response etc.
In addition to this, recent molecular techniques offer various tools to reliably detect epigenetic markers
like DNA methylation, histone modifications etc. The main issue with toxicogenomic and microarray data
is the interpretation because treatment with a compound lead to up and down regulation of hundreds of
genes. With prototype chemicals of specific mode of actions, efforts are constantly being made to simplify
toxicogenomic data analysis.
A significant challenge in applying predictive toxicogenomics is to identify specific gene expression
changes that are highly correlated and predictive of a toxicological reaction. These gene expression changes
are often termed ‘signatures’ [43]. In the vast majority of cases where predictive toxicogenomics has been
successful, this has been accomplished by first establishing toxicogenomic databases of gene expression
profiles as reported by Yang et al [44]. Ideally, these databases consist of many known pharmaceutical
agents, toxicants, and control compounds, at multiple doses and time points, with biological replicates for
each condition [43]. The reference compounds profiled in the database usually consist of different structure–
activity relationships and represent a variety of toxic mechanisms. Often, pharmaceutical companies or
laboratories will establish their own toxicogenomic databases, which allows for the flexibility of building
the database using proprietary compounds within their libraries [43] In other cases, a number of open or
commercial databases are available. The advanced technologies are promising for future applications and
have impacted the manner in which discovery toxicology is used.
CONCLUSION
The field of in vitro genotoxicity testing is progressing towards mechanistic approach. Growing concern over
reducing animal testing also prompted identifying new assays that can replace or complement conventional
methodologies. Many newer and better in vitro techniques to predict carcinogenicity in precise way are
under development or validation. Structure activity relationship (SAR) tools in the field of computational
biology (in silico methods) are also being updated and adapted for better prediction of carcinogenicity.
Conflict of Interest:
ACKNOWLEDGEMENT:
Authors are thankful to Mr. Chetan K. Kajavadara, Mr. Kaushal N. Joshi and Mr. Dolatsinh B. Zala for
critical reading and valuable suggestions for the manuscript. The communication number from zydus
research centre for this publication is 454.
REFERENCES:
1. R. Benigni, C. Bossa and O. Tcheremenskaia, In vitro cell transformation assays for an integrated,
alternative assessment of carcinogenicity: a data-based analysis, Mutagenesis 28 (2013) 107-116.
2. L. Muller, Y. Kikuchi, G. Probst, L. Schechtman, Y. Shimada and T. Sofuni, ICH harmonized guidances
on genotoxicity testing of pharmaceuticals evolution, reasoning and impact. Mutat. Res. 436 (1999)
195-225.
628
3. P.W. Hastwell, L. Chai, K.J. Roberts, T.W. Webster, J.S. Harvey, R.W. Rees and R.M. Walmsley,
High-specificity and high-sensitivity genotoxicity assessment in a human cell line: validation of the
GreenScreen HC GADD45a-GFP genotoxicity screening assay, Mutat. Res. 607 (2006) 160–175.
4. D. Kirkland, M. Aardema, L. Henderson and L. Muller, Evaluation of the ability of a battery of three in
vitro genotoxicity tests to discriminate rodent carcinogens and noncarcinogens. I. Sensitivity, specificity
and relative predictivity, Mutat. Res. 584 (1–2) (2005) 1–256.
5. D. Kirkland, S. Pfuhler, D. Tweats, M. Aardema, R. Corvi, F. Darroudi, A. Elhajouji, H. Glatt, P. Hastwell,
M. Hayashi, P. Kasper, S. Kirchner, A. Lynch, D. Marzin, D. Maurici, J.-R. Meunier, L. Müller, G.
Nohynek, J. Parry, A. Parry, V. Thybaud, R. Tice, J. van Benthem, Ph. Vanparys and P. White, How to
reduce false positive results with in vitro genotoxicity testing and avoid unnecessary follow-up animal
tests: report of an ECVAM workshop, Mutat. Res. 628 (2007) 31–55.
6. J. Aubrecht J, JJ. Osowski, P. Persaud, JR. Cheung, J. Ackerman, SH. Lopes and WW Ku, Bioluminescent
Salmonella reverse mutation assay: a screen for detecting mutagenicity with high throughput attributes,
Mutagenesis 22 (2007) 335-342.
7. R .M. Walmsley and N. Billinton, How accurate is in vitro prediction of carcinogenicity? Br J Pharmacol.
162(2011): 1250–1258.
8. H. Ellinger-Ziegelbauer, J. Aubrecht, JC. Kleinjans and HJ. Ahr, Application of toxicogenomics to study
mechanisms of genotoxicity and carcinogenicity, Toxicol Lett. 186 (2009) 36–44.
9. M. Tsamou, D. G. J. Jennen, S. M. H. Claessen, C. Magkoufopoulou, J. C. S. Kleinjans and J. H. M. van
Delft, Performance of in vitro γH2AX assay in HepG2 cells to predict in vivo genotoxicity, Mutagenesis
27 (2012) 645-652.
10. J. Scheel and C. Brekelmans, Implementation of the 3Rs in European regulation - activities of Working
Group 4 of the European Partnership for Alternative Approaches to Animal Testing I. Impact of liability
issues and the precautionary principle, II. Evaluation of statistical reporting for measuring the uptake of
3Rs in regulatory testing, AATEX 14 (2007) 775–778.
11. GM. Williams, Mechanisms of chemical carcinogenesis and application to human cancer risk assessment,
Toxicology 161 (2001) 3-10.
12. L.G. Hernández, H. van Steeg, M. Luijten, G. Lya and J. Benthem, Mechanisms of non-genotoxic
carcinogens and importance of a weight of evidence approach, Mutat. Res. 682 (2009) 94–109.
13. A.W. Knight, L. Birrell and R.M.Walmsley, Development and validation of a higher throughput
screening approach to genotoxicity testing using the GADD45a-GFP GreenScreen HC assay, J. Biomol.
Screen. 14 (2009) 16–30.
14. C. Jagger, M. Tate, P.A. Cahill, C. Hughes, A.W. Knight, N. Billinton and R.M. Walmsley, Assessment
of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay,
Mutagenesis 24 (2009) 35–50.
15. L. Birrell, P. Cahill, C. Hughes, M. Tate and R. M. Walmsley, GADD45a-FP GreenScreen HC assay
results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of
new genotoxicity tests, Mutat. Res. 695 (2010) 87–95.
16. P.W. Hastwell, T.W. Webster, M. Tate, N. Billinton, A.M. Lynch, J.S. Harvey,R.W. Rees and R.M.
Walmsley, Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP ‘GreenScreen HC’
genotoxicity assay, Mutagenesis 24 (2009) 455-463.
17. N. Billinton, P.W. Hastwell, D. Beerens, L. Birrell, P. Ellis, S. Maskell, T.W.Webster, S. Windebank, F.
Woestenborghs, A.M. Lynch, A.D. Scott, D.J. Tweats, J. van Gompel, R.W. Rees and R.M. Walmsley,
629
Interlaboratory assessment of the Green-Screen HC GADD45a-GFP genotoxicity screening assay: an

enabling study for independent validation as an alternative method, Mutat. Res. 653 (2008) 22–33.
18. D. Miura, VN. Dobrovolsky, Y. Kasahara, Y. Katsuura and RH. Heflich, Development of an in vivo
gene mutation assay using the endogenous Pig-A gene: I. Flow cytometric detection of CD59-negative
peripheral red blood cells and CD48-negative spleen T-cells from the rat, Environ. Mol. Mutagen. 49(8)
(2008) 614-21.
19. J. Nishimura, Y. Murakami and T. Kinoshita, Paroxysmal nocturnal hemoglobinuria: an acquired genetic
disease, Am. J. Hematol. 62 (1999) 175–182.
20. R.A. Brodsky and R. Hu, PIG-A mutations in paroxysmal nocturnal hemoglobinuria and in normal
hematopoiesis, Leuk. Lymphoma. 47 (2006) 1215–1221.
21. J. Takeda, T. Miyata, K. Kawagoe, Y. Iida, Y. Endo, T. Fujita, M. Takahashi, T. Kitani and T. Kinoshita,
Deficiency of the GPI anchor caused by somatic mutation of the PIG-A gene in paroxysmal nocturnal
hemoglobinuria, Cell 73 (1993) 703–711.
22. K. Kawagoe, J. Takeda, Y. Endo and T. Kinoshita, Molecular cloning of murine Pig-a, a gene for GPI-
anchor biosynthesis, and demonstration of interspecies conservation of its structure, function, and
genetic locus, Genomics 23 (1994) 566–574.
23. M.G. Low, The glycosyl-phosphatidylinositol anchor of membrane proteins, Biochim. Biophys. Acta.
988 (1989) 427–454.
24. G.A.M. Cross, Glycolipid anchoring of plasma membrane proteins, Annu. Rev.Cell. Biol, 6 (1990)
1–39.
25. ICH (2013) Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit
potential carcinogenic risk, M7 Step 3 version dated 23 April.
26. J. R. Chapman, M.R.G. Taylor and S. J. Boulton, Playing the End Game: DNA Double-Strand Break
Repair Pathway Choice, Mole. Cell 47 (2012) 497-510.
27. W.M. Bonner, C.E. Redon, J.S. Dickey, A.J. Nakamura, O.A. Sedelnikova, S. Solier and Y. Pommier,
GammaH2AX and cancer, Nat. Rev. Cancer 8 (2008) 957–967.
28. D.J. Smart, K.P. Ahmedi, J.S. Harvey and A.M. Lynch, Genotoxicity screening via the γH2AX by flow
assay, Mutat. Res. 715 (2011) 25– 31.
29. X. Huang, H.D. Halicka, F. Traganos, T. Tanaka, A. Kurose and Z. Darzynkiewicz, Cytometric assessment
of DNA damage in relation to cell cycle phase and apoptosis, Cell. Prolif., 38 (2005) 223–243.
30. D.J. Smart, Genotoxicity of topoisomerase II inhibitors: an anti-infective perspective, Toxicology 254
(2008) 192–198.
31. S. Galloway, E. Lorge, M.J. Aardema, D. Eastmond, M. Fellows, R. Heflich, D. Kirkland, D.D. Levy,
A.M. Lynch, D. Marzin, T. Morita, M. Schuler and G. Speit, Workshop summary: Top concentration for
in vitro mammalian cell genotoxicity assays; and report from working group on toxicity measures and
top concentration for in vitro cytogenetics assays (chromosome aberrations and micronucleus), Mutat.
Res. 723 (2011) 77-83.
32. Ph. Vanparys, R. Corvi, M.J. Aardema, L. Gribaldo, M. Hayashi, S. Hoffmann and L. Schechtman,
Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals,
chemicals, food products and cosmetics, Mutat. Res. 744 (2012) 111-116.
33. K. Ohmori, In vitro assay for prediction of tumorigenic potential of non-genotoxic carcinogens, J.
Health. Sci. 55(1) (2009) 20-30.
630
34. K. Sasaki, S. Bohnenberger, K. Hayashia, T. Kunkelmann, D. Muramatsu, P. Phrakonkham, A. Poth, A.

Sakai, S. Salovaara, N. Tanaka, B. C. Thomas and M. Umeda, Recommended protocol for the BALB/c
3T3 cell transformation assay, Mutat Res. 744 (2012) 30-35.
35. OECD, Organisation for Economic Cooperation and Development, Detailed review on cell transformation
assays for detection of chemical carcinogens, OECD Environment, Health and Safety Publications,
Series on Testing and Assessment, No. 31 (2007).
36. P. Vasseur and C. Lasne, OECD Detailed Review Paper (DRP31) on “cell transformation assays for
detection of chemical carcinogens”: main results and conclusions, Mutat. Res. 744 (2012) 8–11.
37. R. Corvi, M.J. Aardema, L. Gribaldo, M. Hayashi, S. Hoffmann, L. Schechtman and Ph. Vanparys,
ECVAM prevalidation study on in vitro cell transformation assays: general outline and conclusions,
Mutat. Res. 744 (2012) 12–19.
38. ECVAM (2011) recommendation concerning the cell transformation assays using Syrian hamster embryo
cells (SHE) and the BALB/c 3T3 mouse fibroblast cell line for in vitro carcinogenicity testing. Annex
I: ESAC opinion on the ESAC peer review of an ECVAM-coordinated prevalidation study concerning
three protocols of the cell transformation assay (CTA) for in vitro carcinogenicity testing. http://ihcp.jrc.
ec.europa.eu/our activities/alt-animal-testing.
39. OECD (2012) In Vitro Carcinogenicity: Syrian Hamster Embryo (SHE) Cell Transformation Assay,
Draft Test Guideline dated 5 June.
40. P. Ancian, S. Leuillet, S. Arthaud, J.J. Legrand and R. Forster, Toxicogenomics for Regulatory Use The
View from the Bench, in : S. C. Sahu (Eds.), Toxicogenomics: A Powerful Tool for Toxicity Assessment,
John Wiley & Sons Ltd, 2008, pp. 365-375.
41. A. Jacobs and D. Jacobson-Kram, Human carcinogenic risk evaluation, Part III: Assessing cancer hazard
and risk in human drug development, Toxicol. Sci. 81 (2004) 260-262.
42. J. S. MacDonald, Human carcinogenic risk evaluation, Part IV: Assessment of human risk of cancer from
chemical exposure using a global weight-of-evidence approach, Toxicol. Sci. 82 (2004) 3–8.
43. M. J. Liguori, A. C. Ditewig and J. F. Waring, Application of Toxicogenomics in Drug Discovery, in: S. C.
Sahu (Eds.), Toxicogenomics: A Powerful Tool for Toxicity Assessment, John Wiley & Sons Ltd, 2008, pp.
269-285.
44. Yang Y, Xia M, Jin Q, Bendahhou S, Shi J, Chen Y, Liang B, Lin J, Liu Y, Liu B, Zhou Q, Zhang D, Wang R,
Ma N, Su X, Niu K, Pei Y, Xu W, Chen Z, Wan H, Cui J, Barhanin J and Chen Y., Identification of a KCNE2
gain-of-function mutation in patients with familial atrial fibrillation, Am. J. Hum. Genet. 75(2004) 899-905.
45. G. Brambilla, F. Mattioli, L. Robbiano and A. Martelli, Update of carcinogenicity studies in animals and
humans of 535 marketed pharmaceuticals, Mutat. Res. 750 (2012) 1–51.
46. G. Brambilla and A. Martelli, Update on genotoxicity and carcinogenicity testing of 472 marketed
pharmaceuticals, Mutat. Res. 681 (2009) 209–229.
631
Antimicrobial Activity of Asystasia Gangetica
(L.) T. Anderson Flower Extracts
R. Jayalakshmi, P. Selvamani, K. Ruckmani and N. Subramanian
National Facility for Drug Development for Academia, Pharmaceutical & Allied Industries,
Centre for Excellence in Nanobio Translational Research, Department of Pharmaceutical Technology,
Anna University, BIT Campus, Tiruchirappalli – 620 024, Tamil Nadu, India
Corresponding author
e-mail: rjayalakshmi.au@gmail.com
Abstract:
In present n-hexane, ethyl acetate, ethanolic and water extracts of Asystasiagangetica (L.) T. Anderson flowers
antimicrobial activities against Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Escherichia
coli, Proteus vulgaris, Shigelladysenteriae, Salmonella typhi, Klebsiella pneumonia and Pseudomonas
aeruginosa was investigated. The ethyl acetate and ethanolic flower extract of Asystasiagangetica (L.)
T. Anderson exhibited the strong antimicrobial activity against both Gram positive and Gram negative
organisms when compared with Amoxicillin as standard whereas n-hexane and water extract has not shown
any significant activity. The analysis reveals that Asystasia gangetica (L.) T. Anderson flowers possess
effective antimicrobial activity against a broad spectrum of microbes.
Keywords: Asystasiagangetica, Flower, Antibacterial activity and Agar well diffusion assay.
INTRODUCTION
Herbal medicine represents one the most significant fields of traditional medicine in the world. The study of
medicinal plants is very much needed to improve the proper usage and to establish their potential as sources
for new drug leads. Recently there has been an increased interest in exploration of plant materials as sources
of new antibacterial agents. Antimicrobials of plant origin have vast therapeutic potential when compared with
synthetic antimicrobials. Therefore, traditionally practiced natural medicine system is a never ending source for
identification and development of new antimicrobial compounds against highly pathogenic organisms [1-6].
Asystasia gangetica (L.) T. Anderson (Chinese violet or Asystasia coromandeliana Wight ex Nees) belongs
to the family of Acanthaceae. It is a fast growing shrubby herb with generally ascending, branched, quadrangular
stem up to 2 m long, often rooting at the lower nodes [7 and 8]. It is an herbaceous groundcover that grows from
300 – 600 mm in height and has oval-shaped green leaves with lime white or pale purple blue to violet colour
flower, capsules are 2.5-3.5 cm long with the wide base and the seeds are 5 mm in diameter [9-11]. The plant is
mainly distributed in tropical areas like Africa and Asia and also countries like Arabia [12].
The plant contains biologically active substances such as CHO, proteins, alkaloids, tannins, steroidal
aglycones, saponins, flavonoids and triterpenoids and some minerals like Ca, P, Na, Mn, Cu, Zn, Mg, Fe [13
and 14]. A. gangetica (L.) T. Anderson is used in the treatment of heart pains, stomach pains, rheumatism,
mild hypoglycaemia, asthma and anthelmintic [15-17]. It also has an antibacterial, antifungal, antidiabetic,
antihypertensive and anticancer (specifically against epidermoid carcinoma of nasopharynx & Gastric
cancer) activity [18 - 20]. Different types of compounds have been isolated from the plant out of which
only a few biological activities are well studied [21]. A. gangetica(L.) T. Anderson flowers contain the
7-O-glucoside and isosalispuroside.
The flowers were used as an intestinal astringent and also have in-vitro anti-inflammatory activity [22-25].
The present study is aimed to investigate the antimicrobial activity of A. gangetica (L.) T. Anderson flower extract
against the Gram positive and Gram negative organism for the identification of new antimicrobial drug molecule.
Fig. 1: Asystasiagangetica (L.) T. Anderson
MATERIAL AND METHODS

Collection and authentication of plant material
The plant of Asystasia gangetica(L.) T. Anderson were collected from Sri Adhivaraganallur village,
Cuddalore District, Tamil Nadu, India, on January 2014. The plant herbarium was prepared and deposited
at Botanical Survey of India, Southern Regional Centre, Coimbatore and was identified and authenticated by
Dr. M. Palanisamy, voucher specimen No. BSI/SRC/5/23/2013-14/Tech/1922.
Freshly collected flowers were shade dried for one week and pulverized to a fine powder. The powdered
material was stored at 4°C for further use.
Extraction of plant material

About 200 g of the dried flower powder was extracted with different solvent systems in the order of increasing
polarity. The solvents used were n-hexane (Hex), ethyl acetate (EtOAc), ethanol (EtOH) and water (H2O).
The extraction was done successively (hot extraction) for five days using a sohxlet apparatus. The extracts
were concentrated in a rotary evaporator under reduced pressure and completely dried over boiling water to
afford dryness of the extract. The dried extracts were stored at 4°C for further use.
Bacterial strains, culture medium and inoculum preparation

Nine clinically isolated test microorganisms were selected for this study. The Gram-positive organisms involved
in this study were Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis and Gram-negative
organisms were Escherichia coli, Proteus vulgaris, Shigellady senteriae, Salmonella typhi, Klebsiella pneumonia
and Pseudomonas aeruginosa. All the bacterial species were sub-cultured on a suitable fresh agar plate 24 h prior
to antimicrobial test. The bacterial inoculum was prepared by transferring single colonies of microbes to 10 ml of
sterile broth and the suspension was mixed properly to give a final density of 5 x 105 CFU/ml.
Preparation of crude extracts and antibiotics

From each n-hexane, ethyl acetate, ethanol and water extract of the flower 100μg/ml was weighed and
dissolved in dimethylsulfoxide (DMSO) and the standard reference drug Amoxicillin (0.025 μg/ ml) was
also prepared and used for further proceedings.
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The antimicrobial assay was performed for all four extracts of Asystasia gangetica (L.) T. Anderson flowers
by agar well diffusion method [26]. 200 ml molten sterilized Mueller Hinton Agar (MHA – Himedia,
Mumbai) was prepared and poured in 10 sterile petri plates 15-20 ml each. All the plates were allowed to
solidify for 5-10 min. For the identification the negative (DMSO) control n-hexane, ethyl acetate, ethanol,
water extracts and positive (amoxicillin) control were marked on the plate as (1, 2, 3, 4, 5and 6). 0.1 % of
24h freshly prepared inoculum was swabbed uniformly and was allowed to dry for 5 min. In each plate 6
wells punctured with the help of a cork-borer. Four extracts along with positive (amoxicillin) and negative
(DMSO) control compounds (100 μl/ well) were introduced into the well. The plates were incubated for 24
h at 37 °C. The experiment was repeated three times to confirm the result.

In the present study n-hexane, ethyl acetate, ethanol and water extracts of A. gangetica(L.) T. Anderson
flowers were assayed for its in vitro antibacterial activity by employing agar well diffusion method. The
inhibitory effects of our flowers extracts against nine microbes were calculated by measuring zone of
inhibition on the plate. The results obtained by measuring the zone diameters are summarized in Table 1. In
all the four extracts the ethyl acetate and ethanolic extracts of A. gangetica(L.) T. Anderson flowers showed
significant antimicrobial activity when compared with n-hexane and water extracts. When antimicrobial
activity was compared between ethyl acetate and ethanolic extracts, ethyl acetate extract showed more
considerable antimicrobial activity. Sudhakar et al. had already reported about the antimicrobial activity of
the ethanolic extract of A. gangetica (L.) T. Anderson flowers against Staphylococcus aureus, Pseudomonas
aeruginosa, Proteus vulgaris and Escherichia coli [27]. In addition to that the zone of inhibition value
exhibited by the extracts against the test organisms was somewhat equal to the standard drug Amoxicillin.
Fig. 2: The figure shows the antimicrobial activity of n-hexane, ethyl acetate, ethanol and water extracts of Asystasia
gangetica (L.) T. Anderson flowers at 100μg/ml, concentrations against both Gram positive and Gram negative bacteria
which was significantly higher than that of standard Amoxicillin.
634
Note: (A).Pseudomonas aeruginosa, (B). Proteus vulgaris, (C). Escherichia coli, (D). Klebsiella
pneumonia , (E). Shigellady senteriae,(F). Salmonella typhi,(G). Streptococcus pyogenes, (H).
Staphylococcus aureus and (I). Bacillus subtilis
Table 1: In vitro Antimicrobial activity of n-hexane, ethyl acetate, ethanol and water extracts of
Asystasiagangetica(L.) T. Anderson flowers
Microorganisms Zone of Inhibition in Diameter (mm)

DMSO n-Hexane Ethyl acetate Ethanol Water Amoxicillin
Staphylococcus aureus - - 8 1 - 5
Streptococcus pyogenes - 5 2 5 - 5
Bacillus subtilis - - 3 8 - 7
Escherichia coli - - 6 15 - 5
Proteus vulgaris - - - - - -
Shigelladysenteriae - - 8 4 - -
Salmonella typhi - - 8 - - 10
Klebsiella pneumonia - - - - - 14
Pseudomonas aeruginosa - - 6 4 - 8
CONCLUSION
In summary, results presented in this study confirmed that ethyl acetate and ethanolic extracts of A. gangetica
(L.) T. Anderson flowers have potential antimicrobial activity against both gram-positive and negative
organisms. In future in-depth investigation on ethyl acetate and ethanolic extracts of A. gangetica(L.) T.
Anderson flowers may open up an opportunity to identify strong antimicrobial agents active against a wide
range of infectious microorganisms.
ACKNOWLEDGEMENT
The authors are grateful for DST sponsored National Facility for Drug Development for Academia,
Pharmaceutical & Allied Industries, Department of Pharmaceutical Technology, Anna University, BIT
Campus, Tiruchirappalli, TN, India to have provided enormous backend support.
REFERENCES
1. El-Faky FK, Attif O, Aboul Ela M and Gaanem N, Antimicrobial evaluation of extracts from some
Yemeni plants. Alexanderian J. Pharm. Sci. 1995,9: 35-37.
2. Recio MC, A review of some antimicrobial compounds isolated from medicinal plants reported in the
literature 1978-1988. Phytotherap. Res. 1989, 3: 1445-1453.
3. Cragg GM, Newman DJ and Snader KM, Natural products in drug discovery and development. J. Nat.
Prod. 1997, 60: 52-60
4. Awadh Ali, NA, Juelich WD, Kusnick C and Lindequist U, Screening of Yemeni medicinal plants for
antibacterial and cytotoxic activities. J. Ethnopharmacol.2001, 74: 173-179.
5. Tomoko N, Takashi A, Hiromo T and Yuka I, Antibacterial activity of extracts prepared from tropical
and subtropical plants on methicillin-resistant Staphylococcus aureus. J. Health Sci. 2002, 48: 273-276.
6. Barbour EK, Al-Sharif M, Sagherian VK, Habre AN, Talhouk RS and Talhouk SN, Screening of selected
indigenous plants of Lebanon for antimicrobial acitivity. J. Ethnopharmacol. 2004, 93: 1-7.
7. Saunder HN, A Handbook of West African Flowers, Oxford University Press, 1958.
635
8. Ensermu K, A revision of Asystasia gangetica (L.) T. Anders. (Acanthaceae). In: Seyani, J.H. and
Chikuni, A.C. (Editors). Proceedings of the 13th plenary meeting of AETFAT, Zomba, Malawi, 2-11
April, 1991. Plants of the people, National Herbarium and Botanic Gardens of Malawi, Zomba, Malawi,
1994, 1, 333-346.
9. Kamemoto H and Storey WB, Genetics of Flower Color in Asystasia gangetica, Linn. Published with
the approval ofthe Director of the University of Hawaii Agricultural Experiment Starion as Technical
Paper No. 301. Manuscript received December 16, 1953.
10. Smith CW, Impact of alien plants on Hawaii’s native biota in Hawaii is terrestrial ecostims: preservation
and management, cooperative national park resources studies unit, University of Hawaii, Manoa, 1985;
180.
11. Sykes WR, Contributions of the flora of nine New Zealand Department of scientific and industrial.Res
Bull 1970, 37:200.
12. Daziel JM, The useful plants of West Africa, Crown Agents, London, 1937.
13. Odhav B, Beekrum S, Akula U and Baijnath, H, Preliminary assessment of nutritional value of traditional
leafy vegetables in KwaZulu-Natal, South Africa. J Food Compos Anal. 2007, 20, 430-435.
14. Hamid AO. Aiyelaagbe O, Ahmed RN, Usman LA and Adebayo SA, Preliminary Phytochemistry,
Antibacterial and Antifungal Properties of extracts of Asystasia gangetica Linn T. Anderson grown in
Nigeria.IJAAS, 2011, 2 (3): 219-226.
15. Chopra B, Further notes on CrustaceaeDecapoda in the Indian Museum. V, On
EutrichochelesModestus(Herbst): Family Axiidae. Records of the Indian Museum, 1933, 35, 2277-2281.
16. Kokwaro JO, Medicinal plants of East Africa, General Printers Ltd, Kenya, 1976, Pp. 12.
17. Akaha PA, Ezike AC, Nwafor SV, Okoli CO and Enwerem NM, Evaluation of the anti-asthmatic
property of Asystasia gangetica leaf extracts. J Ethnopharmacol. 2003, 89, 25-36
18. Kirtikar KR and Basu BD, Medicinal plants in India, vol.-I, Pullaiah Regency publication, New Delhi,
1998 and 1892.
19. Reddy SNVL, Anarthe SJ and Raghavendra NM, In Vitro Antioxidant and Antidiabetic activity of
Asystasia gangetica (Chinese Violet) Linn. (Acanthaceae). J Pharm Biomed Sci. 2010, 1 (2).
20. Praphaporn Stewart, Patcharee Boonsiri, Songchan Puthong and Panadda Rojpibulstit, Antioxidant
activity and ultrastructural changes in gastric cancer cell lines induced by Northeastern Thai edible folk
plant extracts Praphaporn Stewart, CAM. 2013, 13:60
21. Mugabo P and Raji IA, Effects of aqueous leaf extract of Asystasia gangetica on the blood pressure and
heart rate in male spontaneously hypertensive Wistar rats. CAM. 2013, 13:283
22. Anonymous. The Wealth of India, I: Raw Material, CSIR, New Delhi. 1964, p.134.
23. Harborne JB, Phytochemstry.1996, 5(11).
24. Nair AGR and Subramaniam SS, Phytochemstry, . 1969, 8 (319).
25. Sethuraman MG and Vigneshwari K, Studies on the flowers of Asystasia gangetica. Asian. J. chem.
1998, 10(4); 1029-1031.
26. Perez C, Paul M and Bazerque P, An Antibiotic assay by the agar well diffusion method. Acta. Bio. Med.
1990, 15: 113-115. 6.
27. Sudhakar M, RaoCh V, Rao PM, Raju DB and Venkateswarlu Y, Antimicrobial activity of
Caesalpinia pulcherrima, Euphorbia hirta and Asystasia gangeticum. J Fitoterapia 2006, 77: 378-80.
636
Computational Graphical User Interface Tool
Development for Personalized Drug Selectivity and
Designed Pharmacology
M. Poornima, P. Selvamani and S. Latha
Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli

e-mail: pselvamani@hotmail.com
Abstract:
For a given drug product, it is of interest to study how the drug moves through the body and the processes
of movement such as absorption (A), distribution (D), metabolism (M), and excretion (E) after drug
administration. The key concept of a pharmacokinetic (PK) study is to study what the body does to the drug
(which is usually characterized by ADME of a drug product after administration), while the key concept
of a pharmacodynamic (PD) study is to study what the drug does to the body. This leads to the study of
PK or population PK. The goal of a PK/PD study is to study the relationship between dose and response,
which provides insightful information regarding: (i) how best to choose doses at which to evaluate a drug,
(ii) how best to use a drug in a population, and (iii) how best to use a drug to treat individual patients or
subpopulations of patients.
Clinically, it may be necessary to apply pharmacokinetic principles to determine dosing regimens for
individual patients when one or more of the following criteria are met: (i)A drug has a narrow therapeutic
range, and serious clinical consequences result when the plasma concentrations are outside the range. (ii)
A drug displays wide interpatient variability in its pharmacokinetic parameters. (iii) A patient possesses a
characteristic that is frequently associated with altered pharmacokinetics. This may include renal disease,
hepatic disease, and low activity of a drug-metabolizing enzyme due to genetic factors or concomitant
medications, and low transporter activity.Drugs that are commonly subject to pharmacokinetic-based dosage
individualization include aminoglycosides, phenytoin, lithium, immunosuppressants, and digoxin.
Keywords: Pharmacokinetics, Pharmacodynamics , Individualization, Drug
INTRODUCTION
Pharmacokinetic-based dosage individualization covers empirical PK, numerical methods for PK parameter
estimation, physiological aspects of PK, modeling the distribution process, and PK/PD modelling. Mastering
the decision and selection process of a drug of choice in an appropriate dose requires continuous assessment
and adjustment based on the corresponding PK data review and data integration processes and calculations.
This article implements part of the proposed graphical user interface tool programmed using
html and javascript integrating the reported mathematical models for PK parameters for personalized
drug selectivity and to tailor real time dosage regimen.The object oriented language allows to create
modular programs with reusable code
It is our strong conviction that the proposed tool would be beneficial to clinicians, pharmaceutical
scientists/researchers and biostatisticians who are engaged in the areas of pharmaceutical research
and development and could be applied to optimizedspecifically for an individual patient through
therapeutic drug monitoring, Bioavailability / Bioequivalency, compartmental modeling etc., will
ensure the highest patient safety and best clinical care.New and innovative medication.
Generalized therapy often will not include customized disease prevention strategies and thus compromise
safety of the critical care patients and may require more time, cost, failure of therapy or sometime loss of
life which is very huge globally. Individualized therapy could predict drug susceptibility to disease, improve
disease detection and will offer better disease-combating strategies and might avoid unwanted side effects.
Also it may eliminate trial-and-error inefficiencies that inflate health care costs.
Personalized drugselection and dosage regimen fixation involves integration of empirical PK,
numerical methods for PK parameter estimation, physiological aspects of PK, modelling the distribution
process, and PK/PD modelling.
Mastering the decision and selection process of a drug of choice in an appropriate dose requires
continuous assessment and adjustment based on the corresponding PK data review and data integration
processes and calculations. Performing these activities manually is much more cumbersome.
DESIGN AND IMPLEMENTATION

Design
Table 1: Formula used in calculation
Characteristic Description Formula Terms description

Dosing Rate Amount of drug administered =Target Cp*CL/F Target Cp steady-state
plasma concentration
CL clearance
F bioavailability
T1/2 The time required for the concentration of =0.693* Vss/CL Vssvolume of
the drug to reach half of its original value. distribution at steady state
CL clearance
Css,max Maximum plasma concentration =(F.dose/Vss)/1-exp(-kT) Fbioavailability
k0.693
Tdosing interval
Css,min minimum plasma concentration ==Css,max*exp(-kT)
Fig 1: snapshot of the PK details screen
638
Implementation
Html and javascript is Programming language used to implement part of this tool.
Html
HTML or HyperText Markup Language is the standard markup language used to create web pages. A web
browser can read HTML files and compose them into visible web pages. The browser does not display the
HTML tags, but uses them to interpret the content of the page. HTML describes the structure of a website
semantically along with cues for presentation, making it a markup language rather than a programming
language. HTML elements form the building blocks of all websites. HTML allows images and objects to be
embedded and can be used to create interactive forms. It provides a means to create structured documents by
denoting structural semantics for text such as headings, paragraphs, lists, links, quotes and other items. It can
embed scripts written in languages such as JavaScript which affect the behavior of HTML web pages.Web
browsers can also refer to Cascading Style Sheets (CSS) to define the look and layout of text .
Javascript
JavaScript is a lightweight, interpreted programming language. Designed for creating network-centric
applications,Complementary to and integrated with Java,Complementary to and integrated with HTML,Open
and cross-platform.
A JavaScript consists of JavaScript statements that are placed within the <script>... </script> HTML
tags in a web page.
You can place the <script> tag containing your JavaScript anywhere within you web page but it is preferred
way to keep it within the <head> tags.
The <script> tag alert the browser program to begin interpreting all the text between these tags as a script.
So simple syntax of your JavaScript will be as follows
<script ...> JavaScript code </script>
Program Code
<!DOCTYPE html PUBLIC “-//W3C//DTD XHTML 1.0 Strict//EN”
“http://www.w3.org/TR/xhtml1/DTD/xhtml1-strict.dtd”>
<html xmlns=”http://www.w3.org/1999/xhtml” lang=”en-US” xml:lang=”en-
US”>

<script type=”text/javascript”>
function getText3(){
var in1=document.getElementById(‘in1’).value;
var in3=(0.88*100)/in1+0.33;
var val=in3.toFixed(2)+”ml/min”;
document.getElementById(‘in3’).value=val;
639
}
var in3val=in3.replace(/[A-Za-z$-///]/g, “”);
var age=document.getElementById(‘in1’).value;
var in6=in4*in3val*age*60/in5;
val1=in6.toFixed(2)+”mg/min.kg”;
document.getElementById(‘in6’).value=val1;
}
var clcr=document.getElementById(‘in2’).value;
var age=document.getElementById(‘in1’).value;
var in7val=clcr.replace(/[A-Za-z$-///]/g, “”);
var vss=(3.12*100/age+3.84)*age;
var val1=vss.toFixed(2)+”liters”;
document.getElementById(‘in7’).value=val1;
}
var cl=document.getElementById(‘in3’).value;
var vss=document.getElementById(‘in7’).value;
var in8val=vss.replace(/[A-Za-z$-///]/g, “”);
var halftime=0.693*in8val/cl;
}
</script>
<body>
<table>
<tr>
<td><b>
Patient details</b>
</td>
</tr>
</table>
<table>
<tr>
<td>Name</td>
<td><input type=”text” id=”name”/></td>
</tr>
<tr>
<td>Age</td>
<td><input type=”text” id=”in1”/></td>
</tr>
<tr>
<td>Weight</td>
<td><input type=”text” id=”weight”/></td>
</tr>
640
<tr>
<td>Gender</td>
<td><select>
<option value=”male”>male</option>
<option value=”female”>female</option>
</select></td>
</tr>
</table>
CONCLUSION
Every person has their own unique variation of the human genome. Individualized dosage proposes the
customization of healthcare with medical decisions, practices, and/or products being tailored to the individual
patient.The highly personal, nature of exchange between the therapist and the client allows for specific
focus on the issues present. Quantity of drug for each client differs in their age,weight,gender, same health
problems. Some of the uses of individualized dosage are
• Predict susceptibility to disease, improve disease detection
• Customize disease-prevention strategies
• Prescribe more effective drugs and avoid prescribing drugs with predictable side effects
• Reduce the time, cost, and failure rate of pharmaceutical clinical trials, and
• Eliminate trial-and-error inefficiencies that inflate health care costs.
A fully fledged tool can be developed in future in continuation of this tool.
REFERENCES
1. Goodman and Gilman’s, The Pharmacological Basis of Therapeutics

2. Ariëns, E.J. Affinity and intrinsic activity in the theory of competitive inhibition: I. Problems and
theory. Arch. Intern. Goodman & Gilman’s The Pharmacological Basis of Therapeutics Pharmacodyn.
Ther., 1954, 99:32–49. [PMID: 16026107]
3. Pharmacokinetics and Pharmacodynamics of Abused Drugs Edited by Steven B. Karch, MD, FFFLM
Consultant Pathologist and Toxicologist Berkeley, California
4. Text book of personalized medicine by Kewal .K.Jain
5. Computational pharmacokinetics Editor-in-Chief Shein-Chung Chow, Ph.D. professor Department of
Biostatistics and Bioinformatics Duke University School of MedicineDurham, North Carolina, U.S.A.
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Nanobio Pharmaceutical Technology

Applications and Perspectives

© 2014 International Conference on Innovations and Research Dimensions in

All rights reserved.

Published by Elsevier, a division of Reed Elsevier India Private Limited

Printed and bound in India.

PROCEEDINGS EDITORIAL BOARD CHAIRPERSON

PROCEEDINGS EDITORIAL BOARD MEMBERS

INTERNATIONAL SILVIO DUTZ TANAJI T. TALELE

1 In vitro Antimicrobial Efficacy of Zinc Doped Nano Hydroxyapatite

SECTION-II NANO DRUG DELIVERY SYSTEMS

1 M odified Release Multiparticulate Delivery System for Tramadol HCL

5 Formulation and In Vitro - In Vivo Evaluation of Wedelolactone Loaded Nanosuspension

20 Formulation and Evaluation of Stavudine Loaded Niosomes and Quantitative

1 Comparison of Cell Surface Glycosylation on Apoptotic Cells 387

SECTION-IV PHARMACEUTICAL TECHNOLOGY

1 Phytochemical Investigation and Invitro Anti Diabetic Activity of Myxopyrum

19 Invitro Antioxidant and Hepatoprotective Activity of Ethanolic Extract of

R. Baskar1, G. Devanand Venkatasubbu2 and K. Umamaheswari3

MATERIALS AND METHODS

Characterization of Zinc doped Hydroxyapatite Nanocrystals

Antimicrobial Testing by Microbroth dilution Technique

RESULTS AND DISCUSSION

Figure 1: XRD pattern of (a) 2% Zn-HA nanocrystals (b) 5% Zn-HA nanocrystals

Figure 2: HRTEM image of (a) 2% Zn-HA nanocrystals (b) 5% Zn-HA nanocrystals.

Table 1: ICP-OES values of (Weight %) of Zn in Zn-HA nanocrystals.

S. No Sample Zn Concentration (Weight %)

Table 2: Minimum Inhibitory Concentration (MIC) (µg/mL).

S. No Organism Tested MIC80 (µg/mL)

Chitra N,1 B.K. Nayak* and Anima Nanda

MATERIALS AND METHODS

Isolation of Alternaria sp.

Synthesis of Silver Nanoparticles

Characterization of silver nanoparticles

Antibacterial study of AgNPs

RESULTS AND DISCUSSION

Figure 1: Synthesis of silver nanoparticles from Alternaria sp.

Figure 2: UV–vis spectrum of silver nanoparticles synthesized from Alternaria sp.

Figure 3: FESEM analysis of silver nanoparticles synthesized from Alternaria sp.

Figure 4: XRD analysis of silver nanoparticles synthesized from Alternaria sp.

Pathogens AgNPs AMC AMC+AgNPs OX OX+AgNPs

1. Chauhan A, Zubair S, Tufail S, Sherwani A, Sajid M, Raman SC and Azam A, Fungus-mediated

8. Majeed S, Nanda A and Thirunavukarasu K, Evaluation of antimicrobial activity of biologically

E. Gomathi1, P. Rajesh Prasanna2 and P. Selvamani3

MATERIALS AND METHODS

Preparation of Ester and Amino Terminated PAMAM Dendrimers

Characterization of Ester and Amino Terminated PAMAM Dendrimer

Scanning Electron Microscope (SEM)

RESULT AND DISCUSSION

Scanning Electron Microscope (SEM)

Fourier transforms infrared spectroscopy

Figure 7: FTIR Spectrum of G2 PAMAM Dendrimer

Characterization of Synthetic Solutions

Adsorption properties of SiO2-G0–SiO2-G2

Table 1: Shows decrease in concentration of Mercury

Generation Concentration of Mercury (mg/l)

Figure 8: Generation of Dendrimers Vs Concentration of Mercury

Generation Concentration of Chromium (mg/l)

Figure 9: Generation of Dendrimers Vs Concentration of Chromium

Muthukrishnan Lakshmipathy and Anima Nandaa*

MATERIALS AND METHODS

Synthesis & Characterization

Single Cell Gel Electrophoresis (Comet Assay)

1 In vitro Antimicrobial Efficacy of Zinc Doped Nano Hydroxyapatite

1 M odified Release Multiparticulate Delivery System for Tramadol HCL

5 Formulation and In Vitro - In Vivo Evaluation of Wedelolactone Loaded Nanosuspension

20 Formulation and Evaluation of Stavudine Loaded Niosomes and Quantitative

1 Comparison of Cell Surface Glycosylation on Apoptotic Cells 387

1 Phytochemical Investigation and Invitro Anti Diabetic Activity of Myxopyrum

19 Invitro Antioxidant and Hepatoprotective Activity of Ethanolic Extract of

1. Chauhan A, Zubair S, Tufail S, Sherwani A, Sajid M, Raman SC and Azam A, Fungus-mediated

8. Majeed S, Nanda A and Thirunavukarasu K, Evaluation of antimicrobial activity of biologically

1. T. Hyeon, Chem.Commun, 8 (2003) 927.