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 Dedicated to my family

ϭ

 CONTENTS

CHAPTER I Formulation and evaluation of oral sustained Page 3

release drug delivery system using
Andrographolide
Jugnu Goyal, Pushpendra Garg, Anju Dhiman,
Munish Garg
CHAPTER II Phytochemical investigations and systematic Page 13
exploration of anticancer potential of leaves of
Terminalia bellerica
Parul Grover, Shashank Kumar Singh, Gulshan
Kumar Bansal
CHAPTER III Antimicrobial activity of Tecomella undulata Page 22
Ruby Rohilla, Hitender Sharma, Munish Garg
CHAPTER IV Antidiarrheal activity of Rajanyadi churna Page 32
alcohol extract in rats
Aswatha Ram H.N., Rupali Deshpande,
Shreedhara C.S., Saleemulla Khan
CHAPTER V Pharmacognostic study (morphology and Page 42
microscopy) of the leaves of Callicarpa arborea
Uma Chandur, Ganga Rao Battu, Ramadevi D,
Rambabu Dasari, Srinivas Duvvada, Praneeth
Dasari
CHAPTER VI Phytochemical analysis and estimation of Page 49
phenolic compounds of Mentha arvensis and
Mentha piperita
Madhu Gill, Balasubramanian Narasimhan,
Munish Garg
CHAPTER VII Plants as potential source of antimicrobials Page 63
Pooja Suneja Madan, Savita Jakhar and Pushpa
Dahiya
CHAPTER VIII Medicinal plants cultivation: An urge Page 76
Ashish Malik, Ravinder Kumar, Nidhi Verma,
Jyoti Ahlawat, Nidhi Rani, Anita R. Sehrawat

Ϯ

 CHAPTER I

FORMULATION AND EVALUATION OF ORAL SUSTAINED RELEASE

DRUG DELIVERY SYSTEM USING ANDROGRAPHOLIDE

Jugnu Goyal, Pushpendra Garg, Anju Dhiman, Munish Garg

Abstract. The ultimate of drug therapy has been to deliver drug in requisite quantity
exclusively to its therapeutic site or ensure a prolonged and controlled therapeutic
effect. Many herbal drugs such as andrographolide are known to have low water
solubility profile, unstable in gastric pH, low oral bioavailability that makes the
development of botanical pharmaceutical formulations difficult. To overcome these
demerits, particulate system like microparticles has been used as a physical approach
to alter and improve the pharmacokinetic and pharmacodynamic properties of this
herbal drug molecule. These have been used in vivo to protect the drug entity in the
systemic circulation, restrict access of the drug to the chosen sites and to deliver the
drug at a controlled and sustained rate to the site of action. Two polymers i.e.
cellulose acetate phthalate and ethyl cellulose have been used to formulate the
microparticles which were prepared by using double emulsion technique.
Keywords: Andrographolide, cellulose acetate phthalate, ethyl cellulose.
Microparticles.
1. Introduction
Microparticles are defined as particulate dispersions or solid particles with a size in
the range of 1-1000 ȝm. The drug may be dissolved, entrapped, encapsulated or
attached to a microparticle matrix (Kataria et al., 2011). Depending upon the method
of preparation, microparticles, microspheres or microcapsules can be obtained.
Microspheres are matrix systems in which the drug is physically and uniformly
dispersed. Microcapsules are the systems in which the drug is confined to a cavity
ϯ

surrounded by a unique polymer membrane. In recent years, biodegradable polymeric
microparticles, particularly those coated with hydrophilic polymer such as poly
ethylene glycol (PEG) known as long-circulating particles, have been used as
potential drug delivery devices because of their ability to circulate for a prolonged
period. Microparticles offer easy administration to deliver macromolecules by a
variety of routes and effectively control the release of drugs over the periods ranging
from few hours (h) to months, because of effective protection of encapsulated drug
against degradation (e.g. enzymatic) (Mehta et al., 2001).
Andrographolide is the main diterpenoid lactone contained in the leaves of
Andrographis paniculata (Acanthaceae). It is an herb originated from India and
widely distributed in southern China with annual growth of 0.30 - 0.70 m height
(Chao & Lin, 2010). It has been used in traditional systems of medicine to treat a
number of ailments including common cold, fever, diarrhoea, liver diseases, and
inflammation (Denial, 2009). Recent studies have revealed some cardiovascular
effects of this herb (Jarukamjorn & Nemoto, 2008). It is also found to be a promising
new way for the treatment of AIDS, and numerous symptoms associated with
immune disorders, works effectively as a immunostimulant (Panossian et al., 2000).
Three main diterpenoid lactones identified in the Andrographis paniculata leaves
were andrographolide, neo-andrographolide and deoxyandrographolide (Sheeja et al.,
2006). The molecular formula of andrographolide is C20H30O5 (Rawat & Vashistha,
2011). Andrographolide can be easily dissolved in methanol, ethanol, acetic acid and
acetone, but slightly dissolved in ether and water. The melting point of this
compound is 228o – 230oC (Avanigadda & Vangalapati, 2010).
2. Materials and methods
Andrographolide was obtained as a gift sample from the Yucca Enterprises, Mumbai.
Cellulose acetate phthalate, ethyl cellulose, methanol, liquid paraffin, chloroform,
cyclohexane were obtained commercially from Spectrochem Pvt. Ltd. All chemicals
used were of analytical grade.
2.1 Preparation of microparticles
ϰ

Microparticles were prepared through oil-in-oil (O1/O2 emulsion solvent evaporation
method) using different ratios of CAP and EC ratios (as shown in Tables 6.4 and 6.5
respectively). 500 mg ANDR was dispersed in 5 ml of mixed solvent system
(acetone: ethyl alcohol 4.5: 0.5). The drug suspension was then emulsified in liquid
paraffin under stirring at 600 rpm for 20 min. Then 25 ml of chloroform and
cyclohexane was added to harden the microparticles and stirring was continued for
further 20 min. The hardened microparticles were collected by filtration and washed
with three portions of 30 mL of non-solvent to remove any remained oily phase.
Microparticles, so formed, were dried for 12 h and stored in desiccator for further
evaluation (Jelvehgari et al., 2011).
2.2 Evaluation of microparticles
2.2.1 Determination of entrapment efficiency-
Accurately weighed 30 mg of ANDR loaded microparticles were dissolved in 100 ml
of phosphate buffer solution (pH 6.8) by shaking with magnetic stirrer for 24 h. 1ml
of this solution was taken and diluted up to 10 ml. The final solution was filtered and
aliquots were assayed spectrophotometrically for ANDR at 235 nm (Bhatt & Shah,
2011). The entrapment efficiency was calculated using the following formula:
ࢃࢋ࢏ࢍࢎ࢚࢕ࢌ࡭ࡺࡰࡾ࢏࢔࢓࢏ࢉ࢘࢕࢙࢖ࢎࢋ࢘ࢋ࢙
ࡱ࢔࢚࢘ࢇ࢖࢓ࢋ࢔࢚ࡱࢌࢌ࢏ࢉ࢏ࢋ࢔ࢉ࢟ሺΨሻ  ൌ  ൈ ૚૙૙
ࢃࢋ࢏ࢍࢎ࢚࢕ࢌ࡭ࡺࡰࡾࢌࢋࢊ࢏࢔࢏࢚࢏ࢇ࢒࢒࢟
2.2.2 Morphology
The shape and surface characteristics of microparticles were analyzed by scanning
electron microscopy (SEM). Samples were dusted on a double-sided adhesive tape
applied previously to an aluminium stub. Excess samples were removed and stub
sputter coated (Polaron Sputter 7040) with 30 nm layer of gold-palladium. Samples
were then observed with a scanning electron microscope (Leo 435 VP 501B).
Photographs of SEM analysis were shown in Figure No. 1 and 2.
2.2.3 Differential scanning calorimetry (DSC)
DSC measurements were carried out using DSC Q 10 9.0 Build 275, Water Ltd. Test
sample (approx. 2 mg) was placed in sealed aluminium pan and heated from 10ºC to

ϱ

300ºC at a rate of 20ºC/min under nitrogen atmosphere (100 ml/min), using empty
pan as reference. Photographs of DSC analysis were shown in Figure No. 3, 4 and 5.
2.2.4 Fourier transformed infrared (FTIR) spectroscopic analysis
Fourier transforms IR spectra of test samples was recorded on FTIR- Perkin Elmer,
Pyris Diamond, USA. 1 mg of the sample was placed on the platinum ATR and
scanned from 4000 cmع to 400 cmع. Photographs of FTIR analysis were shown in
Figure No. 6, 7 and 8.
2.2.5 In-vitro drug release study
In-vitro dissolution tests were performed for pure andrographolide and various
batches using USP dissolution test apparatus type II (paddle type) (Lab India DF
8000). An accurately weighed sample of microparticles was were suspended in the
dissolution media consisting of 500ml of 0.1 N (pH 1.2) hydrochloric acid without
enzymes and dissolution studies were carried out for 2 h. At the end of 2 h, 400 ml of
0.1M tribasic sodium phosphate was added to all dissolution vessels, the pH was
adjusted to 7.2 (±0.2) and the dissolution was continued until the microparticles were
depleted of drug or for 12h. 5ml aliquots of dissolution fluid were withdrawn after at
every 2h to assay the released ANDR spectrophometrically at 235 nm in both stages
of dissolution (Khan et al., 2010).
3. Results and discussion
The andrographolide microparticles were prepared by double emulsion technique.
Cellulose acetate phthalate was used as the main retardant polymer for pH dependant
release of andrographolide from microparticles. The drug was found to be
encapsulated above 80% in almost all the batches of microparticles, which shows that
there is no wastage of drug. Best two formulations having good entrapment
efficiencies were selected for the further studies like DSC, FT-IR, morphology and
Dissolution studies. Scanning electron micrographs of the prepared microparticles
reveals that they are discrete and spherical in shape. The microparticles showed the
same melting point as that of the pure sample of andrographolide. Compatibility
studies were carried out by preparing physical mixtures of andrographolide with CAP
and EC and the prepared microparticles were analysed using DSC, FT-IR. No extra
ϲ

or new peaks were observed in spectras of DSC, FTIR. The dissolution studies were
performed for B2 and B6 bathes of andrographolide microparticles in pH 1.2 buffers
for the first 2 hours and in phosphate buffer of pH 7.4 for the remaining 10 hours.
Out of all the batches, the microparticles having CAP (Batch-B2) is meeting both
enteric as well as sustained drug release pattern. The results showed that the drug
release in the acidic medium for the first 2 hours was found to be negligible. The
drug release in the remaining period in phosphate buffer started to release the drug.
The percentage of drug release was found to be 96.12% (B2). The kinetics of drug
release was found to be linear when log percentage of drug remaining was plotted
against time. This indicates that the drug release followed Higuchi’s square model
and the mechanism of drug release was diffusion.

Table 1: Composition of different batches of microparticles containing

andrographolide
Solvent
Batch Drug (Acetone: Stirring CAP EC
name (mgs) Ethanol) time (mgs) (mgs)
(mins)
B1 500 4.5: 0.5 40 250 -
B2 500 4.5: 0.5 80 250 -
B3 500 4.5: 0.5 40 500 -
B4 500 4.5: 0.5 80 500 -
B5 500 4.5: 0.5 40 - 250
B6 500 4.5: 0.5 80 - 250
B7 500 4.5: 0.5 40 - 500
B8 500 4.5: 0.5 80 - 500

ϳ

Figure 1: SEM image of B6 Figure 2: SEM image of B2

Figure 3: DSC curve of pure Figure 4: DSC curve of batch B2

andrographolide

Figure 5: DSC curve of batch B6 Figure 6: FTIR Spectra of pure

andrographolide

ϴ

Figure 7: FTIR Spectra of batch B2 Figure 8: FTIR Spectra of batch B6

Table 2: In-vitro release profile of pure ANDR and batches B2 and B6 (%

cumulative drug release)

Sample 0 0.5 h 1h 2h 4h 6h 8h 10 h
h
Pure 0 11.84 14.58 29.85 38.85 45.9 68.90 78.05
ANDR (1.45) (1.05) (1.18) (1.67) (1.24) (1.45) (1.23)
B2 0 12.37 30.67 44.66 64.34 82.4 95.20 96.28
(1.06) (1.34) (1.46) (1.76) (1.69) (1.86) (1.56)
B6 0 9.67 21.12 37.15 41.20 61.8 73.04 77.78
(1.44) (1.09) (1.1) (1.45) (1.26) (1.76) (1.24)

Figure 9: In-vitro drug release profile of pure andrographolide, B2 and B6

ϵ

 Higuchi plot
ϭϮϬ
%Age cumulative release
ϭϬϬ LJсϯϯ͘ϰϲdžͲ ϯ͘ϰϴϵ
ϴϬ ZϸсϬ͘ϵϴϱ
ϲϬ
ϰϬ
ϮϬ
Ϭ
ͲϮϬ Ϭ Ϭ͘ϱ ϭ ϭ͘ϱ Ϯ Ϯ͘ϱ ϯ ϯ͘ϱ
Square root of time

Figure 10: Fitting data into Higuchi’s Equation for B2

4. Conclusion
The results of this study, it is concluded that by the use of cellulose acetate phthalate
& ethlycellulose as excipients for the model drug, andrographolide, it is possible to
eliminate or minimize the gastrointestinal side effects and degradation of
andrographolide at gastric pH. Controlled release without initial peak levels achieved
with andrographolide microparticles formulation may reduce its dosing frequency,
decrease side effects and improve patient compliance.

ϭϬ

5. Bibliography
Avanigadda S, Vangalapati M. Experimental and modelling studies of
andrographolide extraction from A. paniculata. International Journal of
Chemical, Environmental and Pharmaceutical Research 2010; 1(1): 32-36.
Bhatt YA, Shah DA. Effect of processing variables in formulation and development
of biodegradable microparticles. International Journal of Pharmacy and
Pharmaceutical Sciences 2011; 3(4): 234-239.
Chao WW, Lin BF. Isolation and identification of bioactive compounds in
Andrographis paniculata (Acanthaceae). Chinese Medicine 2010; 5(17): 1-15.
Denial G. Medicinal uses of andrographis. International herb association 2009: 8-19.
Jarukamjorn K, Nemoto N. Pharmacological aspects of Andrographis paniculata on
health and its major diterpenoids constituent andrographolide. Journal of Health
Sciences 2008; 54(4): 370-378.
Jelvehgari M, Hassanzadeh D, Kiafar F , Loveymi BD, Amiri S. Preparation and
determination of drug-polymer interaction and in-vitro release of mefenamic
acid microspheres made of cellulose acetate phthalate and/or ethylcellulose
polymers. Iranian Journal of Pharmaceutical Research 2011; 10 (3): 457-467.
Kataria S, Middha A, Sandhu P, Bilandi A, Kapoor B. Microsphere: a review.
International journal of research in pharmacy and chemistry 2011; 1(4): 1184-
1198.
Khan SA, Ahmad M, Murtaza G, Aamir MN, Rehman NU, Kousar R et al.
Formulation of nimesulide floating microparticles using low-viscosity
hydroxypropyl methylcellulose. Tropical Journal of Pharmaceutical Research
2010; 9(3): 293-299.
Mehta KA, Kislalioglu MS, Phuapradit W, Malick AW, Shah NH. Release
performance of a poorly soluble drug from a novel Eudragit based multi-unit
erosion matrix. International Journal of Pharmaceutics 2001; 213(22): 7-1.
Panossian A, Hovhannisyan A, Mamikonyan G, Abrahamian H, Hambardzumyan
E, Gabrielian E et al . Pharmacokinetic and oral bioavailability of

ϭϭ

 andrographolide from Andrographis paniculata fixed combination Kan Jang in
rats and human. Phytomedicine 2000; 7(5): 351-364.
Rawat R, Vashistha DP. Common herbal plant in Uttarakhand. Used in the popular
medicinal preparation in ayurveda. International Journal of Pharmacognosy and
Phytochemical Research 2011; 3(3): 64-73.
Sheeja K, Shihab PK, Kuttan G. Antioxidant and anti-inflammatory activities of the
plant Andrographis paniculata Nees. Immunopharmacology and
Immunotoxicology 2006; 28(1): 129-140.

ϭϮ

 CHAPTER II

PHYTOCHEMICAL INVESTIGATIONS AND SYSTEMATIC

EXPLORATION OF ANTICANCER POTENTIAL OF LEAVES OF
TERMINALIA BELLERICA

Parul Grover, Shashank Kumar Singh, Gulshan Kumar Bansal

Abstract. Fruits of Terminalia bellerica (common name Baheda) is traditionally used

as anticancer. There are some reports on evaluation of some extracts of this herbal
drug as anticancer. The present study reports the fractionation of different extracts of
this herb in order to isolate the anticancer compound(s). Powder of dried fruits of
Terminalia bellerica was extracted with solvent of varied polarity (petroleum ether,
chloroform, ethylacetate and aqueous methanol (HA)). The ethyl acetate extract was
fractionated through column chromatography while HA extract was fractioned
through partitioning with solvents of different polarity. Each extract was screened for
the phytoconstituents using standard methods. Each extract as well as fraction was
evaluated for its anticancer activity against HL60 cell lines. All extracts except
petroleum ether extract was found to exhibit potent anticancer activity. Ethyl acetate
and HA extracts showed maximum cytotoxicity against HL60 cell lines. The ethyl
acetate and n-butanol fractions of HA extract were found to be the most active while
the other fractions were found to be significantly less active (IC50 >20 µg/ml).
Fractionation of the ethyl acetate extract yielded 21 fractions. Amongst these, seven
fractions were found to be significantly active with cytotoxicity more than 90%. The
other fractions were found to be insignificantly active. Phytochemical screening of
the extract and active fractions suggests that the anticancer activity may be due to
glycosides, flavonoids and tannins in the extracts. Terminalia bellerica can serve as
an excellent source of potent and probably new anticancer lead(s), which can be
further modified chemically to produce a series of compounds for development of
potent and safe anticancer drugs.
ϭϯ

Keywords: Terminalia bellerica, anticancer, HL60 cell line, MTT assay.

1. Introduction
Cancer is one of the major diseases responsible for mortality and expenditure on
health care for human beings. It is the second leading cause of death worldwide.
About 12.7 millions of cancer cases and 7.6 million deaths were recorded worldwide
in 2008. The first line treatment of cancer involves extensive use of chemotherapeutic
agents such as antimetabolites, alkylating agents, antibiotics and various cytotoxic
agents. However, their use is associated with sufferings from various side effects
such as bone marrow depression, alopecia and depression (Kaefer & Milner, 2008).
Since times immemorial, plants have been used for treatment of cancer. National
Cancer Institute collected about 35,000 different plant samples from 20 countries and
screened 1,14,000 extracts for anticancer activity (Cragg & Boyd, 1996). Now-a-
days, about 60% of the anticancer drugs available in the market are of natural origin
(Boopathy & Kathiresan, 2010). Some of the plant derived anticancer drugs include
vincristine, vinblastine, taxol, paclitaxel, camptothecin, homoharringtonine,
flavopiridol. The search for more anticancer drugs is still on to discover molecules
exhibiting excellent potency and tumor specificity.
Terminalia bellerica Roxb (Combretaceae) is a perennial herb mainly distributed in
the tropical regions and commonly found in South-East Asia, including Thailand. It is
one of the ingredients of “triphala”, an Ayurvedic formulation that is believed to
promote health, immunity and longevity (Jagetia et al., 2002; Saraswati et al., 2012).
This formulation is frequently used in Ayurvedic medicine to treat many diseases
such as anemia, jaundice, constipation, asthma, fever, chronic ulcers (Sandhya and
Mishra, 2006) and cancer (Baliga, 2010). The fruit of Terminalia bellerica has been
used to treat various ailments in the folklore medicine (Bunyapraphatsara, 2000) and
is reported to have many activities such as antibacterial (Ahmad et al., 1998),
antidiabetic, antioxidant (Sabu and Kuttan, 2002), purgative (Chakravarti and Tayal,
1947), immunomodulatory (Choudhary, 2012), cardiac depressant and hypotensive
(Siddiqui, 1963). The major phytoconstituents in the fruit include ȕ-sitosterol, gallic

ϭϰ

acid, ellagic acid, ethyl gallate, chebulagic acid, mannitol, glucose, galactose,
rhamnose, and fructose (Row and Murty, 1970; Nandy et al., 1989). The present
study has been designed to evaluate anticancer activity of fruit extract of Terminalia
bellerica using solvents of varied polarity.
2. Materials and methods
2.1 Plant material
Fruits of Terminalia bellerica were purchased from local market in Sirsa, (Haryana)
and were authenticated by botanist Dr. H.B. Singh, NISCAIR, New Delhi.
(NISCAIR/RHMD/Consult/2011-12/1805/105 dated 26/08/2011).
2.2 Chemicals and equipments
The various solvents such as petroleum ether, ethyl acetate, chloroform, methanol (all
LR grade) used for extraction and fractionation as well as the other chemicals and
reagents for phytochemical screening were purchased from sd Fine Chemicals
(Mumbai, India). The various equipments used for the study include rotary vacuum
evaporator (Equitron, Mumbai, India), vacuum oven (Macro Scientific Works, New
Delhi), Hot air oven (Narang Scientific Works, New Delhi, India), UV chamber
(Perfit, Ambala, India), CO2 incubator (model 150i, Heracell, North Carolina, United
States) and ELISA reader (SunriseTM, Tecan Group Ltd., Mannedorf, Switzerland).
2.3 Contaminant analysis of plant material
The fruits were got analysed for contamination due to heavy metals, microbes,
pathogen and pesticides from Oscar Analytical Pvt. Ltd., Baddi, Solan.
2.4 Extraction and fractionation
The fruits were crushed and subjected to successive Soxhlet extraction (36-40 h for
each solvent) using solvents in order of increasing polarity starting with petroleum
ether followed by chloroform, ethyl acetate and finally 20% aqueous methanol
(hydroalcohol). The ethyl acetate extract was fractionated through gradient elution
(methanol and chloroform) using column chromatography. The elution was started
with chloroform (100%) and polarity was increased in steps using an increment of
0.5% upto 20% methanol followed by increment of 1% upto 25% methanol and
subsequently 100% methanol was run. Solvent of each polarity was run till no
ϭϱ

component was detected in the eluent as revealed by TLC. However, a minimum of
200 mL of solvent of each polarity was run on the column. The fractions were
collected and pooled on the basis of their similar TLC profiles. The gummy
condensed hydroalcoholic (HA) extract was dissolved in water and filtered. The
filtrate was partitioned successively with petroleum ether, chloroform, ethyl acetate
and n-butanol using separating funnel. Each organic layer was condensed on rotary
evaporator. The dried gummy four fractions of HA extract, and the pooled fractions
of ethyl acetate extract from column were stored at 0ƕC till use.
2.5 Phytochemical Screening
Each extract was screened for the phytoconstituents like alkaloids, tannins,
flavonoids, carbohydrates, glycosides, saponins, steroids, free amino acids, fats and
fixed oils and starch using standard methods (Farnswoth, 1966; Kokate, 2006).
2.6 Anticancer Activity
Each extract was evaluated on human leukemia cancer cell lines (HL-60) using MTT
assay (Mossman, 1983; Verma et al., 2008; Ghate et al., 2014). Briefly, the cells
(2000-3000 in 100 µl of foetal bovine serum) were placed in each well of a 96 well
plate and incubated for 24 h so that the cells adhered to the surface. 1 µl of each of
the four test solutions (of different concentrations i.e., 20, 2, 0.2 and 0.02 mg/ml) of
each extract as well as fraction prepared in DMSO (10%) was added separately into
the wells containing adhered cells/100 µl of the medium so that the final
concentration of the test solutions become 200, 20, 2 and 0.2 µg/ml, respectively. 1
µl of DMSO was added in the control well. Control and each test solution was treated
in triplicate (n=3). The plate was incubated at 37 °C for 48 h. Subsequently, in each
of the control and test treated well, 25 µl of (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) (2.5 mg/ml of phosphate buffer solution of pH
7.4) was added and incubated for 4 h. The medium as well as the excess dye was
removed by gentle pressing on soaking paper. The formazan produced by the viable
cells in each well was solubilized by addition of 100 µL of DMSO (1% v/v) and
shaken gently for 5 minutes. Absorbance of the solution in each well was recorded at
570 nm by ELISA reader. The percent cell viability was calculated and the IC50 value
ϭϲ

of each test solution was determined from the plot of % cell viability and
concentration.
3. Results
3.1 Contaminant Analysis and Phytochemical Screening
The fruits were found free from heavy metals, pathogens and pesticides. Total
microbial count as well as total yeast and mould count were also found to be well
within the limits. The phytochemical composition and yields of various extracts are
given in Table 1. The yield of ethyl acetate and hydroalcoholic extracts were
significantly higher than the other, which indicate that majority of the
phytoconstituents are polar in nature. Further, the phytochemical compositions of
ethyl acetate and hydroalcoholic extracts were exactly similar.
3.2 Anticancer Activity
Ethyl acetate, and hydroalcoholic extracts were found to exhibit good cytotoxicity
with IC50 of 1 and 4 µg/ml, respectively (Table 1). Petroleum ether extract was
inactive and chloroform extract was weakly active. Based on these results, HA and
ethyl acetate extracts were fractionated. Yield and anticancer activity of each fraction
of ethyl acetate extract and HA extract are given in Tables 2 and 3, respectively.
Fractions 2, 3, 10, 16, 17, 18 and 19 of ethyl acetate extract have shown 90-100 %
cytotoxicity against the cell lines. However, the yield of these maximally active
fractions was significantly less than the others (Table 2). The ethyl acetate and n-
butanol fractions of HA extract were found to be most active and equipotent to the
HA extract itself while the other fractions were found to be significantly less active
(IC50 >20 µg/ml) (Table 3).
4. Discussion
Phytochemical screening of the ethyl acetate and HA extracts suggested that the
cytotoxicity of the both the extracts may be due to glycosides, flavonoids and tannins
in the extracts. Because ethyl acetate and HA extracts were maximally active, these
was fractionated and each fraction was evaluated for the activity to isolate the active
fraction(s). The fractions showing cytotoxicity of >90% were considered maximally
active and selected for further studies. However, these were obtained in minute yields
ϭϳ

which suggested that though these fractions constitute only a meager part of the herb,
but are highly potent.
Table 1: Yield, anticancer activity and phytochemical screening of T. bellerica
extracts
Constituent Extract (Yield in %w/w), IC50 in µg/mL
Pet. Chloroform Ethyl Hydroalcoholic
Ether (10.0), 40 acetate (18.6), 4
(12.6), (15.5), 1
>100
Alkaloids - - - -
Carbohydrates - - + +
Glycosides - + + +
Flavonoids - + + +
Saponins - - - -
Steroids - - + +
Amino Acids - - - -
Starch - - - -
Diterpenoids - - - -
Condensed Tannins - - - -
Hydrolysable - - + +
Tannins
Phenols - + + +
Fats and Fixed oils + - - -

Table 2: Yield and anticancer activity of fractions of ethyl acetate extract

Fractions Yield % Fractions Yield (%) %
(%) cytotoxicity cytotoxicity
1 1.9 84 12 3.5 57
2 0.9 100 13 0.8 85
3 2.5 100 14 10.5 75
ϭϴ

 4 4.1 60 15 1.8 84
5 1.7 65 16 1.8 93
6 6.5 61 17 1.1 97
7 1.7 57 18 0.3 98
8 15.7 50 19 0.2 100
9 0.9 40 20 2.8 84
10 3.9 100 21 5.1 85
11 0.2 7

Table 3: Yield and anticancer activity of fractions of HA extract

Fraction of HA extract Yield (% w/w) IC50 (µg/mL)
Petroleum ether 1.2 50
Chloroform 1.9 20
Ethyl acetate 3.1 4
n-butanol 2.7 5
Aqueous 18.3 ND
Residue of HA extract 0.8 ND

5. Conclusion
The ethyl acetate and HA extracts of T. bellerica are found to be potent cytotoxics.
Seven of the 21 fractions of ethyl acetate extract were found to be significantly active
with cytotoxicity > 90%. Further, the ethyl acetate and n-butanol fractions of the HA
extract have potent anticancer activity. The active fractions of HA and ethyl acetate
extracts shall be further chromatographed to isolate the pure compound responsible
for the anticancer activity.
6. Acknowledgements: The authors are thankful to Council for Scientific and
Industrial Research (CSIR), New Delhi for the financial support vide Scheme No.
02(0132)/13/EMR-II, and to Punjabi University, Patiala and Lord Shiva College of
Pharmacy, Sirsa (Haryana) for providing the infrastructural facilities.

ϭϵ

7. Bibliography
Ahmad I, Mehmood Z, Mohammad F. Screening of some Indian medicinal plants for
their antimicrobial properties, Journal of Ethnopharmacology 1998; 62: 183-
193.
Baliga MS. Triphala, ayurvedic formulation for treating and preventing cancer: A
review, Journal of Alternative and Complementary Medicine 2010; 16(12):
1301-1308.
Boopathy NS, Kathiresan K. Anticancer drugs from marine flora: An overview.
Journal of Oncology 2010: 1-18.
Bunyapraphatsara N. Medicine plants. 4th edition Bangkok: Prachachon Publishing
Ltd; 2000. P. 477-482.
Chakravarti MD and Tayal JN. Preliminary examination of the roots of Terminalia
belerica Roxb. Science Culture 1947; 13: 122.
Choudhary GP. Immunomodulatory activity of alcoholic extract of Terminalia
belerica Linn in mice, Der Pharmacia Lettre 2012; 4: 414-417.
Cragg GM, Boyd MR. Drug discovery and development at the National Cancer
Institute: the role of natural products of plant origin. In: Balick, MJ, Elisabetsky
E, Laird SA. Medicinal Plant Resources of the Tropical Forest. New York:
Columbia University Press; 1996. P. 101-136.
Farnswoth NR. Biological and Phytochemical screening of plants. Journal of
pharmaceutical Sciences 1966; 55: 225-276.
Ghate NB, Hazra B, Sarkar R, Chaudhuri, Mandal N. Alteration of Bax/Bcl-2 ratio
contributes to Terminalia belerica-induced apoptosis in human lung and breast
carcinoma. In Vitro Cellular & Developmental Biology-Animal 2014; 1-11.
Jagetia GC, Baliga MS, Malagi KL, Kamath MS. The evaluation of radioprotective
effect of Tripala in the mice exposed to radiation. Phytomedicine 2002; 9: 99-
108.
Kaefer CM, Milner JA. The role of herbs and spices in cancer prevention. Journal of
Nutritional Biochemistry 2008; 19: 347-361.
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Kokate CK. Practical Pharmacognosy. 4th ed. Delhi: Vallabh Prakashan; 2006.
Mossman T. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. Journal of Immunological Methods 1983;
65: 55–63.
Nandy AK, Podder G, Sahu NP, Mahato SB. Triterpenoids and their glucosides from
Terminalia belerica. Phytochemistry 1989; 28: 2769-2772.
Row LR and Murty PS. Chemical examination of Terminalia belerica Roxb. Indian
Journal of Chemistry 1970; 8: 1047-1048.
Sabu MC and Kuttan R. Antidiabetic activities of medicinal plants and its
relationship with their antioxidant properties. Journal of Ethnopharmacology
2002; 81: 155-160.
Sandhya T and Mishra KP. Cytotoxicity of breast cancer line, MCF 7 and T47D to
tripala and its modifications by antioxidants. Cancer Letters 2006; 238: 304-
313.
Saraswati MN, Karthikeyan M, Kannan M and Rajasekar S. Terminalia belerica
Roxb-A phytopharmacological review. International Journal of Research in
Pharmaceutical & Biomedical Sciences 2012; 3: 96-99.
Siddiqui HN. Studies on Terminalia belerica Roxb. Effect on bile secretion and
pharmacodynamic properties. Indian Journal of Pharmacology 1963; 25: 297-
302.
Verma M, Singh SK, Bhushan S, Sharma VK, Dutt P, Kapahi BK, Saxena AK. In
vitro cytotoxic potential of Polyalthia longifolia on human cancer cell lines and
induction of apoptosis through mitochondrial-dependent pathway in HL-60
cells. Chemico-Biological Interactions 2008; 171: 45-56.

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 CHAPTER III

ANTIMICROBIAL ACTIVITY OF TECOMELLA UNDULATA

Ruby Rohilla, Hitender Sharma, Munish Garg

Abstract. Tecomella undulata (Bignoniacae) is a deciduous or nearly evergreen tree

of dry regions of India. The plant is useful for planting in shelter belt plantations and
become an important agro-forestry tree in arid and semiarid areas for the production
of high quality timber, in addition to fuel, wood and fodder. Due to the presence of
various important secondary metabolites; Tecomella undulata has been used to treat
different ailments like spleen diseases, cancer, hepatoprotective, wound healer, anti-
inflammatory, anti-diabetic and antimicrobial. The present study has been carried out
to prove the antimicrobial effect of bark of the plant using different bacteria and
fungus. The hydro-alcoholic extract of bark of Tecomella undulata was fractionated
into petroleum ether, chloroform, acetone and remaining hydro-alcoholic fraction.
The petroleum ether fraction was obtained only in trace amount. Antimicrobial
activity of all other fractions of Tecomella undulata was performed on Gram +ve,
Gram –ve bacteria and fungal strain using Minimum inhibitory concentraction (MIC)
method. The bacteria used in the test were Staphylococcus aureus, Escherichia coli
and Bacillus subtilis and the fungus were Candida albicans and Aspergilus niger.
The antibacterial and antifungal activities of fractions were compared with standard
drug (Ciprofloxacin and Fluconazole). The chloroform fraction does not show any
activity against both bacterial and fungal strains. The acetone and hydro-alcoholic
fraction have shown activity against both bacterial and fungal strains. The hydro-
alcoholic fraction was the most potent against both bacterial and fungal strains. The
phytochemical screening indicated the presence of saponin, tannin, anthraquinones
and flavanoids in the hydro-alcoholic part. It is concluded that this fraction may
further be investigated for isolation of pure constituent responsible for activity.
ϮϮ

Keywords: Tecomella undulata, Minimum inhibitory concentration, hydro-alcoholic
fraction, Antimicrobial activity
1. Introduction
India has been identified as one of the top twelve largest biodiversity centers in the
world; occupies a vast area with great variations in climate, soil, altitude and latitude
and is the biggest repository of medicinal plants in the world (Meena et al., 2010).
Natural products obtained from plants are believed to be an important source of new
phytoconstituents with potential therapeutic efficacy (Ameh et al., 2009). Indian
treasure; Ayurveda mentions number of drugs which are most ancient and yet living
traditions practiced widely in different parts of world and has a sound philosophical
and experimental basis (Ullah et al., 2010). Tecomella undulata (Bignoniaceae), is
known as Rohida, ‘Nakacampanki’ in Tamil and ‘Rohita” in Sanskrit (Danya et al.,
2012).This is a medium sized tree (Khatri et al., 2009) found mostly in Thar Desert
regions of northwest and Western parts of India (Rana et al., 2008). Tecomella
undulata plays an important role in environmental conservation (Singh et al., 2011)
and has occupied a reputed position of having valuable medicinal properties in both
folklore and classical streams of indigenous medicinal systems (Wealth of India, Raw
material 1962) due to the presence of various secondary metabolites of
pharmacological importance. The plant has been extensively screened for its phyto-
constituents from its extracts. This has led to the isolation and identification of
pharmacologically relevant compounds from the heartwood such as (Rana et al.,
2008) and as an anti-cancer agent (Ravi et al., 2011). Ladies of tribal communities of
Samahni valley (Pakistan) take bark powder with hot milk for abortion (Muhammad
et al., 2008). Leaves have significant antimicrobial activity and are used in treatment
of AIDS (Nagpal et al., 2010; Azam, 1999). The paste of Tecomella undulata root is
given internally in leucorrhoea (Khare, 2004). Flower of the plant possess analgesic
activity (Ahmad et al., 1994). Different researchers have proved its biological
activities using suitable pharmacological models. Rat paw edema and tail immersion
method were used by Ahmad et al., 1994 for the analgesic and anti-inflammatory
activities using methanolic extract of the plant. The extract showed significant
Ϯϯ

analgesic potential compared to standard drug Aspirin. Goyal et al., 2012 has
investigated that Rohitaka ghrita which is an Ayurvedic formulation and is
recommended to be used in various clinical conditions including jaundice, cirrhosis
and cholestasis. It has a significant hepatoprotective potential against Paracetamol
induced hepatocellular damage in rats. Khatri et al., 2009 demonstrated the
hepatoprotective activity of stem bark of Tecomella undulata against Thioacetamide-
induced hepatotoxicity. Recently, Savjiyani et al., 2012 studied anticancer potential
of polyherbal formulation containing extract of stem bark of Tecomella undulata,
Bauhinia variegate, Oroxylum indicum and leaves of Indigofera tinctoria using in-
vitro and in-vivo experimental models. Since Tecomella undulata Linn. has wide
range of medicinal potential as documented in literature and studied clinically by
scientists still Tecomella undulata has not been systematically tested for their
biological activities in general and antimicrobial activity in particular. For these, We
examined the antimicrobial effects of various extracts of bark of Tecomella
undulata. After this; preliminary phytochemical screening of most potent fraction of
Tecomella undulata’s stem bark have been carried out.
2. Materials and methods
2.1 Preparation of extracts
The fresh aerial part of Tecomella undulata was collected from Bhiwani District
(Haryana). The collected plant material was authenticated by Dr. H.B. Singh Chief
Scientist & Head, Raw Materials Herbarium & Museum NISCAR, New Delhi. The
voucher specimen of the collected plant part was deposited in Pharmacognosy
Research lab, Department of Pharmaceutical Sciences, M. D. University, Rohtak. The
sample was shade dried and powdered in a Willey mill. Hydro-alcoholic extract was
prepared. The extract was filtered and concentrated using rotary evaporator and then
lyophilized. The percentage yield of dried hydro-alcoholic extract was calculated.
The above obtained hydro- alcoholic extract was further fractionated into petroleum-
ether, chloroform, acetone and remaining hydro-alcoholic fraction. All fractions of
hydro-alcoholic extract were used for antimicrobial study.
2.2 Test Microorganisms
Ϯϰ

The microorganisms were procured from the Department of Pharmaceutical Sciences,
Maharshi Dayanand University, Rohtak for the present study. These include bacterial
strains like Bacillus subtilis (Gram positive), Streptococcus aureus (Gram positive),
Escherichia coli (Gram negative), and fungal strains like Aspergillus niger and
Candida albicans respectively.
2.3 Antimicrobial Susceptibility Testing
Minimum Inhibitory Concentration (MIC) was determined by the standard method of
Van Den Berghe and Vlietinck, (1991) Nutrient broth and Subouraud dextrose broth
media were prepared and sterilized using autoclave. One ml of the prepared broth
was dispensed into the test tubes numbered 1-6 using micropipette. Petroleum ether
fraction collected was in trace amount. Stock solution of standard drug (100µg/ml of
Ciprofloxacin against antibacterial and Fluconazole against antifungal activity) and
of three remaining extracts (Chloroform, acetone and hydro-alcoholic fraction),
100mg/ml were prepared. Dropped 1ml of sterilized media serially (six in one row
with proper labeling). Added 1ml of stock solution of standard drug/fraction in 1st test
tube of each series respectively. Shake it properly so that it is diluted with 1ml media
already present in test tube. Transfer 1ml from this 2ml solution of 1st test tube to 2nd
test tube which is also having 1ml of sterilized liquid media. Again shake properly.
Then again 1ml from 2nd test tube is transferred to 3rd test tube having 1ml media.
And then to 4th and 5th test tubes in the same manner. In the 6th test tube 1ml of 2ml
is discarded outside. Now all six test tubes are having 1ml overall solution of
Nutrient/Subouraud broth and fractions of extract but with different concentration.
The final concentration of the plant extract in each of the test tubes numbered 1-6
after dilution 50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, 3.125mg/ml, 1.56mg/ml.
Now, added 0.1ml of normal saline solution having bacterial/ fungal sub-culture in
each test tube. Then plug these properly and incubate at specified time and
temperature (After 24hrs at 37˚C for bacteria, for fungi Candida albicans-37˚C for
2days, Aspergillus niger-25˚C for 7 days). Turbidity was noticed against white
background. The concentration, of the test tube which is previous to that specific test
tube showing turbidity is considered MIC (Minimum inhibitor concentration).
Ϯϱ

0.1 ml std. drug
1ml fraction+
1ml media+

1 2 3 4 5 6
1ml discard

3. Results and discussions

The antimicrobial studies of different fractions of this plant revealed that, the hydro-
alcoholic fraction had significant activity against most of the organism, while the
acetone fraction possessed moderate activity. The chloroform fraction did not show
any bacterial and fungal activity. As shown in Table 1 to 2 the most potent hydro-
alcoholic fraction was used for preliminary phytochemical screening. The
phytochemical screening indicated the presence of saponin, tannin, anthraquinones
and flavanoids as is given in Table 3.
Table 1: Antibacterial activity of fractions of hydro-alcoholic extract of
Tecomella undulata Linn.

Fraction Concentration Staphylococcus Bacillus Escherichia

aureus subtilis coli
Chloroform 50mg/ml ++++ ++++ ++++
25mg/ml ++++ ++++ ++++
12.5mg/ml ++++ ++++ ++++
6.25mg/ml ++++ ++++ ++++
3.125mg/ml ++++ ++++ ++++
1.56mg/ml ++++ ++++ ++++
Acetone 50mg/ml **** **** ****

Ϯϲ

 25mg/ml **** **** ****
12.5mg/ml ++++ ++++ ++++
6.25mg/ml ++++ ++++ ++++
3.125mg/ml ++++ ++++ ++++
1.56mg/ml ++++ ++++ ++++
Hydro- 20mg/ml **** **** ****
alcoholic 10mg/ml **** ++++ ****
5mg/ml ++++ ++++ ++++
2.5mg/ml ++++ ++++ ++++
1.25mg/ml ++++ ++++ ++++
0.625mg/ml ++++ ++++ ++++
Bacterial growth= ++++, MIC= ****

Table 2: Antifungal activity of fractions of hydro-alcoholic extract of

Tecomella undulata Linn.

Fraction Concentration Candida albicans Aspergilus niger

Chloroform 50mg/ml ++++ ++++
25mg/ml ++++ ++++
12.5mg/ml ++++ ++++
6.25mg/ml ++++ ++++
3.125mg/ml ++++ ++++
1.56mg/ml ++++ ++++
Acetone 50mg/ml **** ****
25mg/ml **** ++++
12.5mg/ml ++++ ++++
6.25mg/ml ++++ ++++
3.125mg/ml ++++ ++++
1.56mg/ml ++++ ++++
Hydro-alcoholic 20mg/ml **** ****

Ϯϳ

 10mg/ml ++++ ++++
5mg/ml ++++ ++++
2.5mg/ml ++++ ++++
1.25mg/ml ++++ ++++
0.625mg/ml ++++ ++++
Fungal growth= ++++; MIC= ****

Table 3: Phytochemical screening of hydro-alcoholic fraction of

Tecomella undulata Linn.
S.NO. Phytoconstitutents Qualitative test Hydro-
alcoholic
farction
Carbohydrates Molish ----
Fehling
Proteins and amino acids Nindydrin ----
Alkaloids Dragondroff ----
Hager
Mayer
Flavanoids Shinoda +++
Lead acetate
Tannins Exract+Ferric chloride +++
Phenolic compounds Ferric chlorides ++
Phytosterols and Libermann-Buchardt ----
triterpenoids test
Saponins Foam ++
Lead acetate
Anthraquinones Brontrager +++
Modified Brontrager
Bound anthraquinones Extract+ sulphuric ++
acid+benzene+ammonia

Ϯϴ

 Cardiac glycosides Legal test ----
Kedde test
(++) Presence of phytoconstitutents ; (+++) Presence of phytoconstitutents with
good indications; (----) Indicates absence of phytoconstituents.

4. Conclusion
The scientific investigation of traditional herbal remedies may provide valuable tool
for the development of alternative natural remedies. The results of the present study
support the folklore usage of the studied plants and suggest that the plant fraction
possess certain phytoconstituents with antimicrobial properties. This indicates that
the Tecomella undulata has broad inhibitory activities against pathogenic
microorganisms so it is a promising herbal remedy; hence it can be useful for treating
infectious diseases. It has higher potential against bacteria than that of fungus.
Tecomella undulata is rich in flavanoids, saponin, tannins and anthraquinones; out of
these any one or two could be responsible antimicrobial agent. Hence, the study has
provided some phytochemical basis for ethno-pharmacological claims of this plant in
the treatment and prevention of various diseases and disorders.

Ϯϵ

5. Bibliography
Ahmad F, Khan RA, Rashad S. Preliminary screening of methanolic extracts of
Celastrus peniculatus and Tecomella undulata for analgesic and anti-
inflammatory activities. Journal Of Ethanopharmacology 1994; 42: 193-198.
Ameh SJ, Obodozie OO, Afolabi EK, Oyedele EO, Ache TA, Onanuga CE et al.
Some basic requirements for preparing an antisickling herbal medicine-
NIPRISIAN. African Journal of Pharmacy and Pharmacology 2009; 3(5): 259-
264.
Azam MM. Anti-HIV agents and other compounds from Tecomella undulata.
Oriental Journal of Chemistry 1999; 15: 375-377.
Danya U, Udhayasankar MR, Arumugasamy K, BAluprakash T. Bioactivity of
Tecomella undulata (G.Don) Seem Bignoniaceae on Human pathogens. South
Asian Journal of Experimental Biology 2012; 2: 71-82.
Goyal R, Ravishanker B, Shukla VJ, Singh M. Hepatoprotective activity of Rohitaka
ghitra against Paracetamol induced liver injury in rats. Pharmacologia 2012; 3:
227-32.
Khare CP. Indian herbal remedies, rational western therapy, Ayurvedic and other
traditional usage. New Delhi (India): Society of new age herbals; 2004.
Khatri A, Garg A, Agarwal SS. Evaluation of hepatoprotective activity of aerial parts
of Tephrosia purpurea Linn. and stem bark of Tecomella undulata. Journal of
Ethanopharmacology 2009; 122: 1-5.
Meena KL, YAdav BL. Studies on ethnomedicinal plants conserved by Garasia tribes
of Sirohi district Rajesthan India. Indian Journal of Natural Product Research
2010; 1: 500-6.
Muhammad ICH, Khan MA. An ethanomeditional inventory of plants used for
family planning and sex disease in Samahni valley, Pakistan. Indian Journal of
Traditional Knowledge 2008; 7: 277-283.

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Nagpal N, Arora M, Rahar S, Swami G, Kapoor R. Pharmacological and
Phytochemical Review on Tecomella undulata. Research Journal of
Pharmacognosy and Phytochemistry 2010; 2(5): 354.
Rana MG, Katbamna RV, Duhrejiya AV, Sheth NR. Hepatoprotection of Tecomella
undulata against experimentally induced liver injury in rats.
Pharmacologyonline 2008; 3: 674-682.
Ravi A, Mallika A, Sama V, Begum AS, Khan RS, Reddy BM. Antiproliferative
activity and standardization of Tecomella undulata bark extract on K562 cells.
Journal of Ethnopharmacology 2011; 137: 1353-1359.
Savjiyani JV, Dave H, Trivedi S, Rachchh MA, Gokani RH. Evaluation of anticancer
activity of polyherbal formulation. International Journal of Cancer Research
2012; 8: 27-36.
Singh D, Gupta RS. Hepatoprotective activity of methanol extract of Tecomella
undulata against alcohol and paracetamol induced hepatotoxicity in rats. Life Sci
Med Res 2011; 26: 1-8.
The Wealth of India. A Dictionary of Indian Raw Material and Industrial Products-
Raw Material Series. New Delhi; NISCAIR (CSIR): 1962. P. 195(5).
Ullah MO, Uddin J, Hamid K, Kabir S, Rahman MA, Choudhuri MSK. Studies of
various biochemical parameters of rat plasma following chronic administration
of “Rohitakarista” An Ayurvedic formulation. Pakistan Journal of Biological
Sciences 2008; 11: 2036-39.
Van Den Berghe DA, Vlietinick AJ. Screening methods for antibacterial and antiviral
agents from higher plants. In: Dey PM, Harborne JB, Hostettman K, editors.
Methods in plant biochemistry. Assays for Biodiversity. London; Academic
press: 1991. P. 47-69 (Vol.6).

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 CHAPTER IV

ANTIDIARRHOEAL ACTIVITY OF RAJANYADI CHURNA ALCOHOL

EXTRACT IN RATS

Aswatha Ram H.N., Rupali Deshpande, Shreedhara C.S., Saleemulla Khan

Abstract. Rajanyadi churna (RC) is an Ayurvedic polyherbal classical formulation

prescribed for the treatment of digestive disorders, diarrhoea, fever, jaundice, anemia
and asthma. Objective: The present experiment describes the antidiarrhoeal activity
of Rajanyadi churna alcoholic extract (RCAL) in rats. Materials and Methods:
RCAL was investigated for acute toxicity in mice followed by antidiarrhoeal activity
in rats by castor oil-induced diarrhoea model. RCAL effects on movement of the
gastro-intestine, transit time of intestine and enteropooling were evaluated in rats.
Results: In castor oil induced diarrhoea, significant protection (P<0.001) was
observed with RCAL (100-400 mg/kg p.o.); transit time of the intestine was
inhibited, and gastric emptying rate was delayed. Loperamide (10 mg/kg p.o.)
showed (P<0.001), RCAL showed intestinal enteropooling and accumulation of fluid
significantly (P<0.001). RCAL reduced the volume of castor-oil induced fluid
secretion in the intestine, and decreased the fecal droppings, wet and fecal weight.
Conclusion: From this animal study, the experimental result indicates that RCAL
possesses anti-diarrhoeal activity in rats.Therefore, these findings suggest the
pharmacological reliance or and substantiate its traditional use.
Keywords: Antidiarrhoeal activity, Rajanyadi churna, RCAL
1. Introduction
As per WHO “Diarrhoea is a symptom of gastrointestinal infection which involves
three or more loose stools per day. Bacterial, viral and parasitic infections have been
the major causative factors in the onset of diarrhoea”. The condition can be
sometimes life-threatening especially in younger children due to the critical fluid
loss. Further the malnourished and (or) immunity compromised population is at a
ϯϮ

higher risk for fatal consequences due to the diarrhoeal diseases which are usually
associated with high morbidity and mortality rates. A concerted effort has been made
during the last four decades to combat the diarrhoeal diseases by a variety of
programs namely, sanitation improvement, awareness on rehydration and supply of
oral rehydration solution (ORS), vaccinations etc. A number of antidiarrhoeals have
been developed, for instance, anti-secretory, anti-motility agents, antibiotics, zinc
supplements and other controlling agents (Guerrant et al., 2003; Kelly, 2011;
UNICEF/WHO, 2009). The major drawback of present treatment method is the fact
that about 39% of the population of developing countries have no access to the
modern anti-diarrhoeals (UNICEF/WHO, 2009)
Rajanyadi churna (RC) is a polyherbal ayurvedic classical formulation used in
agnimandya (digestive impairment), atisara (diarrhoea), jvara (fever), kamala
(jaundice), pandu (anemia) and svasa (asthma) (Ayurvedic Formulary of India, 2003)
Literature survey so far have not revealed the biological activity of RC on a scientific
basis and hence the present study aims at the evaluation of anti-diarrhoeal activity of
RCAL.
2. Materials and Methods
2.1 Plant materials
The crude drugs used in the in-house RC formulation were purchased from the local
vendors in Udupi, Karnataka, India, and obtained an authentication of each drug from
the botanist Usha Rani S. Suvarna, Associate Professor and HOD of Botany, M.G.M.
College, Udupi, Karnataka. Voucher specimen (PP 532A, PP 577A, PP 588−PP 593)
of each crude drug was deposited in the Department of Pharmacognosy, Manipal
College of Pharmaceutical Sciences, Manipal.
2.2 Preparation of churna
The procedure given in The Ayurvedic Formulary of India, (2003) was followed for
the preparation of In-house RC churna in three different batches (Ayurvedic
Formulary of India, 2003). All the ingredients were pulverized and sieved (#80), and
the individual powders were thoroughly mixed in specified equal proportions (Table
1) to get uniformly mixed churna.
ϯϯ

 Table 1: Ingredients of Rajanyadi churna
Sanskrit name Botanical name Part used
Rajani (haridra) Curcuma longa Rhizome
Daru (devadaru) Cedrus deodara Heart wood
Sarala Pinus roxhburgii Heart wood
Sreyasi (gajapippali) Scindapsus officinalis Fruit
Brhati Solanum indicum Plant (whole)
Kantakari Solanum xanthocarpum Plant (whole)
Prsniparni Uraria picta Plant (whole)
Satahva Anethum sowa Fruit

2.3 Preparation of extract

Rajanyadi churna alcoholic extract (RCAL) was prepared by extracting 30 g of RC
with ethanol using soxhlet extraction process. The ethanolic extract was evaporated
to the dryness and the dried extract (yield 11.16%) was stored in the desiccators until
its use.
2.4 Animals
Healthy adult Wistar albino rats (weighing between 200-250 g) were used for the in
vivo animal experiment. The animals were placed in polypropylene cages (2 animals/
cage), and maintained standard conditions. Standard rat pellet were fed to the rats
and water ad libitum. This experimental study was approved with IAEC No.
IAEC/KMC/14/2013.
2.65Acute toxicity study
Safe dose was determined by carrying out acute toxicity studies in mice by staircase
method using graded dose up to 2000 mg/kg of RCAL as per OECD adoption 425.
After administration of the test substance, the animals were observed for till 72 h for
gross behavioral changes or mortality. The animals were kept under observation for
thirty days thereafter (Prabhakar et al., 2006).
2.6 Diarrhoea induced by castor oil in rats

ϯϰ

The animals used in the experiment were fasted for 16 h with free access to water.
The animals were randomly divided into five groups of six each. Group 1 was fed
with only propylene glycol vehicle, while, groups 2-4 were fed with RCAL extract
equivalent to 100, 200 and 400 mg/kg respectively. Group 5 was administered with a
standard antidiarrhoeal agent loperamide 10 mg/kg (p.o.). All the animals in each
group were individually placed in a separate cage having a transparent sheet at the
bottom after administration of toxicant. Thirty minutes after the pretreatment of
animals with vehicle the animals were administered with the doses of RCAL and
loperamide. Castor oil (3 ml/kg. p.o.) a toxicant to induce diarrhoea was fed orally to
all the experimental animals thereafter (Awouters et al., 1978). The animals were
then, observed continuously for 6 h for loose stools or diarrhoeal droppings. The
study evaluates the seriousness of the diarrhoea based on the recorded as numerical
scores which was marked depending on the consistency of stool (Dicarlo et al.,
1994). The scores were set as follows: 1 for normal stool (or lack of diarrhoea), 2 for
semi-solid stool and 3 for the watery stool. A corresponding purging index and
percentage was calculated using a formula described by Aye-Than et al. (1989) and
Mbagwu and Adeyemi (2008) in comparison with the control group rats. The time
taken (in minutes) between the administration of castor oil and the first visual of the
diarrheic stool was considered as occurrence of diarrhoea. The weight of fecal matter
collected after 6 h and the changes in body weight before and after 6 h in each group
were determined. A percentage inhibition of defecation and fecal weight were
calculated by using the following relation

ሾࢉ࢕࢔࢚࢘࢕࢒࢓ࢋࢇ࢔ െ ࢚࢘ࢋࢇ࢚ࢋࢊሺ࢚ࢋ࢙࢚ሻ࢓ࢋࢇ࢔ሿ
ൈ ૚૙૙
ࢉ࢕࢔࢚࢘࢕࢒࢓ࢋࢇ࢔
2.7 Intestinal transit, enteropooling and intestinal fluid in rats induced by castor
oil
The experiment was designed and carried out as described by Awe et al[11] briefly,
the experimental animals after 16 h fasting, were divided into five groups of six each
and individually placed in separate plastic cage lined with a transparent sheet at the
bottom. Group 1 rats were separately treated with 10 ml/kg (p.o.) of propylene
ϯϱ

glycol while the animals in the groups 2 to 4 received 10 ml/kg (p.o.) equivalent to
RCAL doses of 100, 200 and 400 mg/kg respectively. In the group 5 the animals
were fed with 10 mg/kg (p.o.) of loperamide. Animal in all the groups were then
received castor oil (3 ml/kg) and 30 minutes thereafter, 3 ml/kg (p.o.) of 10%
activated charcoal in physiological saline (0.9%, w/v sodium chloride solution). The
intestinal transit and volume of fluid in the intestine in rats then was measured as per
the methods of Dicarlo et al. 1994 with required modifications (Dicarlo et al., 1994).
All the animals in each group were sacrificed by cervical dislocation under ether
anaesthesia after forty minutes of charcoal meal. The rats were then individually
dissected in the lower abdominal region and the entire length of the small intestine
from pylorus to caecum was ligated and carefully removed. Peristaltic index (PI) i.e.,
the distance of charcoal meal movement of entire length of small intestine from
pylorus to caecum, was determined. This was expressed as percentage of full length
of the small intestine. The weight of the entire intestine along with its content was
recorded and then the intestine was evacuated by transferring its content in to a
measuring cylinder and the volume was measured, also the weight of the empty
intestine was noted. The difference in the weights of full and empty intestine was
calculated.
ࢀ࢚
Percentage transit inhibition = [To - ࢀ࢕ ] x 100
2.8 Data and statistical analysis
The Mean ± SEM for the number of rats used in each group (n = 6) has been
presented for the data. The results of the extract and the standard drug treated groups
were compared with the vehicle treated group (baseline group). The difference in the
results between the treated and control groups were then subjected to One-way
ANOVA and later by Dunnett’s post hoc test using software for statistics, SPSS
version 16.
3. Results
3.1 Acute toxicity study
Orally administered RCAL graded doses up to 2000 mg/kg neither produced any
mortality nor any symptoms of toxicity hence 2000 mg was considered safe and
ϯϲ

maximum tolerated dose is more than 2000 mg/kg. In the present study 1/5th, 1/10th
and 1/20th of the safe dose was selected as therapeutic doses.
3.2 Diarrhoea induced by castor oil in rats
Oral administration of castor oil produced extensive diarrhoea in the control group
after two-and-half hours as concluded from different tests. Pretreatment of rats with
RCAL (100, 200 and 400 mg/kg p.o.) delayed the onset of diarrhoea as well as
reduced the frequency of diarrhoea and defecation significantly. The wetness of the
fecal droppings was also reduced markedly. Loperamide (10 mg/kg, p.o.), the
standard antidiarrhoeal drug showed a better (p<0.001) inhibition of the effects
induced by castor oil in the group 5. The effect of loperamide on the studied
diarrhoeal parameters and that of RCAL are shown in Table 2.
Table 2: Effect of RCAL (100-400 mg/kg p.o.) on diarrhoea
induced by castor oil in rats
Wet stools
Dose in ml/kg Stools total
Treated group number % Protection
or mg/kg number

Vehicle control 10 7.50±2.09 7.80±1.98 0.00

100 5.33±0.33 1.33±0.56 82.91***
Extract 200 6.33±0.99 1.50±0.76 82.91***
400 5.17±0.98 0.83±0.31 89.32***
Loperamide 10 0.00 0.00 100.00***
Values are the Mean ± SEM, ***P < 0.001 vs Vehicle control

3.3 Intestinal transit in rats induced by castor oil

The intestinal transit study revealed a faster movement of charcoal meal in the
vehicle control group with 92.0% of the length of the small intestine in total when
compared to other groups. While in RCAL treated groups different doses, significant
reduction (p<0.001) in the intestinal motility and charcoal meal transit was evident.
Loperamide, on the other hand showed good anti-motility effect when compared to
RCAL treated groups. Results are shown in Table 3.

ϯϳ

 Table 3: Effect of RCAL (100-400 mg/kg p.o.) on Intestinal transit
in rats induced by castor oil
Treated Dose in ml/kg or Peristaltic Index Tranist Inhibition
group mg/kg (%) (%)
Vehicle control 10 86.48 0.00
100 57.40*** 33.6
Extract 200 55.02*** 36.4
400 21.21*** 75.5
Loperamide 10 6.94*** 92.0
Values are the Mean ± SEM, ***P < 0.001 vs Vehicle control

3.4 Enteropooling and fluid accumulation in rats induced by castor oil

Fluid volume in the intestine was increased significantly in castor oil alone treated
animals which was markedly prevented (p<0.001) by the test groups with RCAL
pretreated as shown in Table 4. The reference drug loperamide (10 mg/kg, p.o.), has
shown a significant inhibition of the accumulation of intestinal fluid (p <0.001).
Loperamide however showed a better inhibition when compared to RCAL treated
groups.
Table 4: Effect of RCAL (100-400 mg/kg p.o.) on enteropooling and fluid
accumulation in rats induced by castor oil
Dose in ml/kg Fluid in Inhibition of fluid volume in
Treated group
or mg/kg intestine (ml) intestine (% ml)
Vehicle control 10 3.55±0.72 0.00
100 1.7±0.21 52.11**
Extract 200 1.53±0.19 56.80**
400 1.43±0.15 59.62**
Loperamide 10 0.98±0.07 72.30***
Values are Mean ± SEM, **P<0.01, ***P<0.001 vs Vehicle control

4. Discussion
An active constituent of castor oil, ricinoleic acid (Gaginella et al., 1975) has been
known to stimulate the release of certain mediators namely, PGs, NO and PAF,
cAMP and also tachykinins (Izzo et al., 1999), which are thought to be the
underlying cause for onset of diarrhoea in castor oil treated animals. Diarrhoeal
ϯϴ

frequency score and wet stools were reduced significantly in RCAL treated groups.
Antidiarrhoeal effect of RCAL in diarrhoea induced by castor oil is similar to the
effects of loperamide and therefore it can be concluded that the action of RCAL
could be mediated by an antisecretory mechanism. This was also evidenced from the
inhibitiory activity (Meite et al., 2009), of castor oil-induced accumulation of fluids
by RCAL and the results were on par with loperamide.
The intestinal transit time test also testify the significant activity of RCAL which
slows down the transit of charcoal meal indicating the suppression of the driving
effect of charcoal meal by increasing the absorption of water and electrolytes (Meite
et al., 2009). A significant inhibition on enteropooling of RCAL induced by castor oil
proposes that the extract possibly act through the spasmolytic and antienteropooling
effect (Awe et al., 2011). Further, plant flavonoids (Dicarlo et al., 1994) and
phenolics (Yakubu et al., 2012), have been reported to possess antidiarrhoeal
properties. The flavonoid content (234.11mg/g) and total phenolic content (110.81
mg/g) were observed in the preliminary phytochemical screening of RCAL.
Therefore, it is easier to frame a theory that some of the active principles of RCAL
could have presented to the antidiarrhoeal activity.
5. Conclusions
To conclude, the current animal study clearly indicates the antidiarrhoeal potential of
RCAL in rats and provide evidence for the classically used Rajanyadi churna as
antidiarrhoeal agent. Further research is needed to find the actual constituents which
are responsible for the observed antidiarrhoeal activity.

ϯϵ

6. Bibliography
Awe EO, Kolawole SO, Wakeel KO, Abiodun OO. Antidiarrhoeal activity of
Pyrenacantha staudtii Engl. (Icacinaceae) aqueous leaf extract in rodents.
Journal of Ethnopharmacology 2011; 137(1): 148-153.
Awouters F, Niemegeers CJE, Lenaerts FM, Janssen PAJ. Delay of castor oil
diarrhoea in rats: a new way to evaluate inhibitors of prostaglandin biosynthesis.
Journal of Pharmacy and Pharmacology 1978; 30: 41–45.
Aye-Than JH, Kulkarni W, Tha SJ. Antidiarrhoeal efficacy of some Burmese
indigenous drug formulations in experimental diarrhoea models. International
Journal of Crude Drug Research 1989; 27: 195–200.
Dicarlo GD, Mascolo N, Izzo AA, Capasso F, Autore G. Effects of quercetin on
gastrointestinal tract in rats and mice. Phytotherapy Research 1994; 8: 42–45.
Gaginella TS, Stewart JJ, Olsen WA, Bass P. Action of ricinoleic acid and
structurally related fatty acid on gastrointestinal tract. II. Effect on water and
electrolyte absorption in vitro. Journal of Pharmacology and Experimental
Therapeutics 1975; 195: 355–361.
Guerrant RL, Carneiro-Filho BA, Dillingham RA. Cholera, diarrhoea, and oral
rehydration therapy: triumph and indictment. Clinical Infectious Diseases 2003;
37: 398-405.
Izzo AA, Mascolo N, Capassco R, Germano MP, De pasuele R, Caspassco F.
Inhibitory effect of cannabinoid agonists on gastric emptying in rat. Naunyn-
Schmiedeberg's Archives of Pharmacology 1999; 360(2): 221–223.
Kelly P. Infection infectious diarrhoea. Medicine 2011; 39: 201-206.
Mbagwu HOC, Adeyemi OO. Anti-diarrhoeal activity of the aqueous extract of
Mezoneuron benthamianum Baill (Caesalpinaceae). Journal of
Ethnopharmacology 2008; 116: 16–20.
Meite S, N’guessan JD, Bahi C, Yapi HF, Djaman AJ, Guede Guina F.
Antidiarrhoeal activity of the ethyl acetate extract of Morinda morindoides in
Rats. Tropical Journal of Pharmaceutical Research 2009; 8(3): 201-207.

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Prabhakar KR, Veerapur VP, Bansal P, Vipan KP, Reddy KM, Barik A, et al.
Identification and evaluation of antioxidant, analgesic/anti-inflammatory activity
of the most active ninhydrin–phenol adducts synthesized. Bioorganic &
Medicinal Chemistry 2006; 14: 7113–120.
The Ayurvedic Formulary of India, 2nd ed. Part-I. Ministry of Health and Family
Welfare, Government of India: New Delhi 2003, 103, 115.
UNICEF/WHO. Diarrhoea: Why children are still dying and what can be done. WHO
Geneva, Switzerland: New report 2009, 1-68.
Yakubu Toyin M, Opakunle Khadijat F, Salimon Saoban S, Ajiboye Olakunle T,
Bamisaye Abraham F, Quadri Luqman A. Antidiarrhoeal activity of aqueous
leaf extract of Ceratotheca sesamoides in rats. Bangladesh Journal of
Pharmacology 2012, 7:14-20.

ϰϭ

 CHAPTER V

PHARMACOGNOSTIC STUDY (MORPHOLOGY AND MICROSCOPY) OF

THE LEAVES OF CALLICARPA ARBOREA

Uma Chandur, Ganga Rao Battu, Ramadevi D, Rambabu Dasari,

Srinivas Duvvada, Praneeth Dasari

Abstract. Callicarpa species have been Ethno medicinal significance in Asia. The
present study aims at studying the Pharmacognosy (morphology and microscopy) of
the leaves of Callicarpa arborea. Macroscopic characters were observed and noted
down. The leaf was found to be ovate, dorsiventral with reticulate venation, 35-42 cm
in length,15-16 cm in breadth; upper surface dark green ,smooth. Lower surface is
light green, velvety with prominent midrib. Base is asymmetric and apex is acute.
Microscopy studies were carried out by taking hand cut transverse sections as well as
powder using Phloroglucinol- hydrochloric acid as the staining agent. Nikon
photomicrograph was used to document the results. The transverse section exhibited
the typical arrangement of the dorsiventral leaf with the midrib and the lamina region
with abundant candelabra trichomes. Powder Microscopic: characters observed were
the candelabra trichomes, calcium oxalate crystals, wavy walled epidermis, stoma
very few surrounded by an indefinite number of epidermal cells, xylem and phloem
elements. Conclusions: The Pharmacognostic study (morphology and microscopy)
will help in the identification and standardization of the plant drug.
Keywords: Callicarpa arborea leaf, Pharmacognostic study, Morphology,
Microscopy.
1. Introduction
Callicarpa species (Family: Verbenaceae) have Ethno medicinal significance in china
and south Asia. They are used in the treatment of rheumatism, headache, fever,
indigestion, etc.Callicarpa arborea Roxb. Grows wild and was obtained from Araku
valley, Vishakapatnam, Andhra Pradesh.
ϰϮ

2. Materials and methods
Macroscopic characters weere observed and noted down. Microsscopy studies were
carried out by taking hannd cut transverse sections as well as powder using
Phloroglucinol- hydrochloricc acid as the staining agent. Nikon photomicrograph was
used to document the resultss.
3. Results
3.1 Morphology of Leaf
Type: dorsiventral, Venatioon: reticulate, Shape: ovate, Pediceel attached: 3-6cm
Length: 35-45cm mature leaf, Breadth: 15-16cm. Colour: 1.Uppper surface: dark
green, 2.Lower surface: lighht green. Margin: Sinuate, Base: asymm
metric, Apex: acute.
Texture: upper surface smoooth, Lower surface: velvety, glabrous, protruding mid rib
with prominent branches.

1.Dried leaff 2.Plant in haabitat

Figure 1: Morphology
M of leaf and plant in habitaat.
3.2 Microscopy
3.2.1 Transverse section
The transverse section exhibbited the typical arrangement of the doorsiventral leaf with
the midrib and the lamina region
r with abundant candelabra trichhomes on the lower
surface.The TS of the leaf caan be divided into mid rib and laminar portions.
a. Midrib

ϰϯ

Epidemis, the outer most layer consists of a single layer of compactly arranged cells
with abundant trichomes. Stomata are very few (almost absent) and surrounded by an
indefinite number of epidermal cells (anomocytic type). Trichomes are abundant in
the lower mid rib region and are of the candelabra type. Collenchyma were below the
epidermis is 3-4 layers of thick walled collenchymatous cells. Parenchyma below the
collenchyma several layers of thin walled loosely arranged parenchyma are found.
The central region of midrib consists of a continuous band of the vascular bundle
with xylem and phloem elements, surrounded by pericyclic fibres as shown in Fig
b. Lamina
The epidermis continuous over the laminar region. Spongy parenchyma below the
upper epidermis and above the lower epidermis are several layers' of thin walled
loosely arranged parenchymatous cells. Vascular strand consist the central region of
the lamina contains lignified vascular strands as shown in Fig 3.

Figure 2: Diagramatic representation showing T.S. of Callicarpa arborea leaf

(midrib and lamina).

ϰϰ

 T.S. of leaf laminna T.S. of uppeer midrib

T.S. showing central midrib T.S. of lower middrib trichomes

Figure 3: Photographs of the transverse section (T.S.) of Callicaarpa arborea leaf

3.3 Powder microscopy

The following are the impoortant powder drug characters of the leaf of Callicarpa
arborea. Epidermis consist several fragments of thin wavy walleed cells containing
very few stomata (almost absent). Stomata are very few and surrounded by an
indefinite number of epideermal cells. Trichomes are found inn abundant of the
candelabra type and are lignnified. Mesophyll several patches of meesophyll containing
chlorophyll. Vascular bundlles consisting of bundles of lignified phloem fibres and

ϰϱ

patches of xylem vessels, lignified with spiral walls were seen. In addition, calcium
oxalate was very few minute prisms.

Trichome (candelabra) Trichome

Trichome Xylem vessel (spiral Thickening)

Candelabra trichome Mesophyll tissue

ϰϲ

 Phloem Fibre Epidermis
Figure 4: Powder microscopy showing trichomes, mesophyll tissue, xylem,
phloem and epidermis Callicarpa arborea leaf

4. Conclusion
The present study will help in the authentication of the drug including detection of
adulterants and therefore the standardization of the plant drug.

ϰϳ

5. Bibliography
Jones W P & Kinghorn AD. Biologically active natural products of the genus
Callicarpa. Current bioactive compounds 2008; 4(1): 15.
Khandelwal KR. Practical pharmacognosy. Pune: Nirali Prakashan; 1998. P. 15-16.

ϰϴ

 CHAPTER VI

PHYTOCHEMICAL ANALYSIS AND ESTIMATION OF PHENOLIC

COMPOUNDS OF MENTHA ARVENSIS AND MENTHA PIPERITA

Madhu Gill, Balasubramanian Narasimhan, Munish Garg

Abstract. To study the phytochemical constituents along with qualitative and

quantitative determination of quercetin, rutin and gallic acid in the methanolic
extracts of leaf and stem of Mentha arvensis and Mentha piperita. Methods: A
preliminary phytochemical studies has been carried to identify the phytochemical
constituents present in the mentha species. Further, the quantitative determination of
quercetin, rutin and gallic acid was done with HPTLC. Results: Screening of
phytochemicals in mint samples showed the presence of flavonoids, tannins and
phenolic compounds. Furthermore Mentha arvensis leaf was found rich in total
phenolic content (4.40 mg/gm), quercetin (332.30 µg/gm), rutin (65.65 µg/gm) and
gallic acid (82.00 µg/gm) concentration as compare to other mint samples.
Conclusion: The present study reveals the presence of important phytochemicals in
mint samples which can be exploited for pharmaceutical application.
1. Introduction
The extensive use of natural plants as primary health remedies due to their
pharmacological properties is quiet common. Plants and plant-based medicaments are
the basis of many of the modern pharmaceuticals we use today for our various
ailments (Abraham, 1981). The discovery of medicinal plants has usually depended
on the experience of the populace based on long and dangerous self experiment.
Progress over the centuries towards a better understanding of a plant derived
medicine has depended on two factors that have gone hand in hand. One has been
the development of increasingly strict criteria of proof that a medicine really does
what it is claimed to do and the other has been the identification by chemical analysis
of the active compound in the plant (Holiman, 1989). Traditional medicine using
ϰϵ

plant extracts continues to provide health coverage for over 80% of the world’s
population, especially in the developing world (WHO, 2002). The medicinal value of
plants lies in some chemical substances that produce a definite physiologic action on
the human body. The most important of these bioactive compounds of plants are
alkaloids, flavonoids, tannins and phenolic compounds. The phytochemical research
based on ethno-pharmacological information is generally considered an effective
approach in the discovery of new anti-infective agents from higher plants
(Duraipandiyan et al., 2006). Knowledge of the chemical constituents of plants is
desirable, not only for the discovery of therapeutic agents, but also because such
information may be of value in disclosing new sources of such economic materials as
tannins, oils, gums, precursors for the synthesis of complex chemical substances. In
addition, the knowledge of the chemical constituents of plants would further be
valuable in discovering the actual value of folkloric remedies (Mojab et al., 2003).
Chemically constituents may be therapeutically active or inactive. The ones which
are active are called active constituents and the inactive ones are called inert chemical
constituents (Iyengar, 1995).
Mint species have been exploited by man for more than 2000 years. The genus
Mentha (Lamiaceae) is composed of 19 geographically wide spread species and 13
hybrids. Various species of mint have many constituents in common and all have
been held in high dietary medical repute (Gopalan et al., 2000). Three Mentha
species, M. x piperita L. (peppermint), M. arvensis L. (cornmint) and M. spicata
(spearmint) are commonly cultivated in the world for essential oil production that is
used extensively in the liquor and confectionary industries, flavoring, perfume
production and medicinal purposes (Rameshwar et al., 2012).Several workers have
separated and identified potential volatile components and also indicated the presence
of various phytochemical constituents from different extracts of mint. On the basis of
above, the need was though felt to authenticate the methanolic plant extracts for the
presence/absence of certain potent phytochemicals qualitatively and quantitatively.
The preliminary phytochemical studies were performed for the four mint samples
categorized as Mentha arvensis leaf, Mentha arvensis stem, Mentha piperita leaf and
ϱϬ

Mentha piperita stem followed by their total phenolic content estimation and
qualitative and quantitative studies of potential phytoconstituents responsible for
possible therapeutic activity.
2. Materials and methods
2.1 Plant material
Two different species of mint (Mentha arvensis and Mentha piperita) were collected
from Herbal garden of M. D. University, Rohtak. The plant was taxonomically
identified, authenticated and voucher specimen of the same has been retained in the
Department of Pharmaceutical Sciences M.D. University, Rohtak for the further
reference under number 101,102,103 and 104.
2.2 Preparation of plant extract
The leaves and stems of the plant were dried at room temperature under shade for 5
days and then pulverized into fine powder using mechanical grinder. Fifty grams of
these fine powders was first defatted with petroleum ether (40°-60°C) to remove
petroleum ether soluble components like fats, wax and chlorophyll and successively
extracted with 250 ml of methanol by using soxhlet extractor for 72 h at a
temperature not exceeding the boiling point of the solvent. The methanolic extracts
were again treated with petroleum ether to ensure complete removal of petroleum
ether soluble components. Then methanolic extracts were filtered using Whatman
filter paper (No: 1). Thereafter, methanolic extract was evaporated using a rotary
evaporator and freeze dried to give crude dried extract. Known amount of extracts
were taken and dissolved in HPLC grade methanol (final conc. 1mg/ml) and filtered
through 0.45 µm filter for HPTLC analysis
2.3 Phytochemical analysis
The mint extracts were subjected to phytochemical tests for plant secondary
metabolites, alkaloids, sterols, saponins, tannins, flavonoids, proteins, amino acids,
sugars and carbohydrates, glycosides, phenolic compounds and resins in accordance
with Trease and Evans, (1989) and Harborne, (1998).
2.4 Determination of total phenolic contents

ϱϭ

Total phenolic content was estimated according to the method of Slinkard and
Singleton (Slinkard and Singleton, 1977) using Folin-Ciocalteu phenol reagent as a
standard phenolic compound. The phenolic content was expressed in terms of mg/g
of plant extract.
2.5 HPTLC procedure
2.5.1. Instrumentation and operating conditions
HPTLC system equipped with an automatic TLC sampler (ATS 4), TLC scanner 3
integrated with software (WinCATS version 1.4.2), UV cabinet and automatic
developing chamber ADC2 with humidity control facility was used for the analysis.
The samples were applied using automated TLC sampler in 10 mm bands at 10 mm
from the bottom, 15 mm from the sides and with 8 mm space between the two bands.
Plates were developed in software controlled automatic developing chamber ADC2
pre-saturated with the 10 mL of developing solvent phase for 30 min at room
temperature (25 ± 2 C) and relative humidity was maintained 45 ± 1%. The plates
were developed to a height of about 8 cm from the base in respective solvent systems.
After development, the plate was removed, dried and spots were visualized under UV
(254 and 366 nm) light. Quantitative evaluation of the plate was performed in the
reflectance/absorbance mode at 290 nm with following conditions: slit width 6 mm ×
0.3 mm, scanning speed 20 mm/s and data resolution 100 mm/step. To check the
identity of the bands, UV absorption spectrum of each standard was overlaid with the
corresponding band in the sample track.
2.5.2. Calibration and quantification
For the preparation of calibration curve, appropriate dilutions were made to get the
desired concentrations in the quantification range. The obtained working standard
solutions were then applied on the TLC plate for preparing five-point linear
calibration curves. Compounds were spotted as 2, 4, 8, 12, 24 ml. Sample solution (4
µl each) was applied on TLC plate (precoated silica gel 60 F 254 sheets (0.2 mm
thickness)) in triplicate with similar band pattern. The experimental parameters were
identical for all the above analysis.
2.6 Method validation
ϱϮ

2.6.1 Specificity
The specificity of the method was determined by analysing the bands of different
standards and samples. The bands for compounds in sample solution were confirmed
by comparing the Rf and UV spectra with the reference standards. The peak purity of
these compounds was assessed by comparing the spectra at three different levels, i.e.
peak start, peak apex and peak end positions.
2.6.2 Robustness
Robustness is a measure of the method to remain unaffected by small but deliberate
variations in the method conditions, and is an indication of the reliability of the
method. For the robustness, different parameters such as mobile phase composition,
developing TLC distance and different TLC plate lots were checked.
3. Results and Discussion
3.1 Phytochemical screening
Preliminary phytochemical screening of the methanolic leaf and stem extracts of mint
samples showed that the solvent extracts contain phytochemicals like flavonoids,
tannins, phenolic compounds and sterols. But phytochemicals alkaloids, reducing
sugars, glycosides, resins, amino acids, proteins and saponins are absent (Table 1).
Table 1: Results of preliminary phytochemical screening of mint samples
Chemical Constituents Mentha arvensis Mentha piperita
Leaf Stem Leaf Stem
Flavonoids + + + +
Alkaloids - - - -
Reducing Sugars - - - -
Glycosides - - - -
Tannins + + + +
Phenolic Compounds + + + +
Resins - - - -
Amino acids - - - -
Proteins - - - -
Saponins - - - -
Sterols + + + +

3.2 Total phenolic content

ϱϯ

To determine the total phhenolic content as gallic acid equivvalents (GAE), the
absorbance for standard ploot was measured as presented in Tablee 2 and calibration
curve of gallic acid was ploted as shown in Figure 1. Total content
c of phenolic
compounds mg/g of plant exxtract was found to be maximum in Mentha
M arvensis leaf
(4.4), followed by Mentha arvensis stem (2.63), Mentha piperiita leaf (2.46) and
minimum in Mentha piperitaa stem (2.3) (Table 3).
Table 2: Preparation of sttandard plot for determination of total phenolic
p contents
S. No. Concentration
n (µg/ml) Absorbance ± SEM
(n = 3)
1 0 0.000
2 5 0.063 ± 0.002
3 10 0.187 ± 0.003
4 15 0.347 ± 0.004
5 20 0.524 ± 0.002
6 25 0.605 ± 0.003

Figure 1:: Calibration curve of gallic acid to determine

the total phenolic content.

Table 3: Quantiitative determination of total phenolic content

c
Extracts Absorbance Content of Content of Total
T phenolic
± SEM GAE from cal. GAE from cal. c
contents mg/gm
(n=3) curve (ȝg/ml) curve (mg/ml) o plant extract
of
Mentha 0.306 ± 13.20 0.0132 4
4.40
arvensis 0.019
leaf
Mentha 0.168 ± 7.92 0.0079 2
2.63
arvensis 0.080
ϱϰ

 stem
Mentha 0.155 ± 7.42 0.0074 2.46
piperita 0.006
leaf
Mentha
piperita 0.142 ± 6.92 0.0069 2.30
stem 0.050

3.3 HPTLC analysis

In order to develop an effective solvent system for the separation of phenolic
compounds, various solvent systems (Table 4) were tried.
Table 4: Thin layer chromatographic solvent systems used in present study
S.No. Solvent System Ratio
1 Toluene: ethylacetate: acetic acid 30:40:5
2 Petroleum ether: ethylacetate: formic acid 30:10:10
3 Toluene: ethylacetate: formic acid 50:40:10
4 Toluene: ethylacetate: formic acid 35:10:10
5 Toluene: acetic acid 40:20
6 Cyclohexane: ethylacetate: formic acid 30:12:18
7 Cyclohexane: ethylacetate: acetic acid 31:14:5
8 Ethylacetate: glacial acetic acid: formic acid: water 100:11:11:26

9 Toluene: acetone: formic acid 30:10:10

10 n-Hexane: ethylacetate: acetic acid 31:14:5
11 n-Hexane: ethylacetate: formic acid 31:14:5
12 Carbontetrachloride: acetone: formic acid 30:10:10

The one containing Toluene: ethylacetate: formic acid (50:40:10) gave the best
resolution for quercetin (Rf = 0.79) while Ethylacetate: glacial acetic acid: formic
acid: water (100:11:11:26) gave the best resolution, with symmetrical and
reproducible peaks for rutin (Rf = 0.71) and gallic acid (Rf = 0.83) from the other

ϱϱ

components of the sample extracts and enabled their simultaneous quantification. The
standard chromatographic peak of quercetin, rutin and gallic acid can be seen in
Figure 2, 3 and 4 respectively.

Figure 2: A typical HPTLC chromatogram of standard quercetin (Rf 0.79)

Figure 3: A typical HPTLC chromatogram of standard Rutin (Rf 0.71)

ϱϲ

 Figure 4: A typical HPTLC chromatogram of standard gallic acid (Rf 0.83)

The plates were visualized at two different wavelengths 254 and 366 nm as the
compounds were found to absorb at variable spectrum range. In addition, this helped
in the generating a better fingerprint data whereby species could be well
differentiated on enhanced visual identification of individual compounds. The
method developed here was found to be quite selective with good baseline resolution
of each compound. The identity of the bands of compounds in the sample extracts
was confirmed by overlaying their UV absorption spectra with those of the standards
(Figure 5-7).

Figure 5: Superimposed UV spectra of samples and standard

of quercetin

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 Figure 6: Superimposed UV spectra of samples
and standard of rutin

Figure 7: Superimposed UV spectra of samples and standard

of gallic acid
The developed HPTLC method was validated for parameters like specificity and
robustness. The specificity of the method was ascertained by analyzing the standard
compounds and samples for the interference of other components. The bands for
compounds were confirmed by comparing the Rf and spectra of the bands with that of
standards. Absence of any interfering peak indicated that the method was specific.
The purity of bands was confirmed by overlaying the absorption spectra at the start,
middle, and end position of the bands. The content of quercetin, rutin and gallic acid
was estimated in the methanolic extract of mint samples by the proposed method and
the results obtained are summarized in Table 5.

Table 5: Concentration of quercetin, rutin and gallic acid

present in mint samples.
Samples Concentration(µg/g)

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 Quercetin Rutin Gallic acid
Mentha arvensis leaf 332.30 65.65 82.00
Mentha arvensis stem 79.62 16.00 17.75
Mentha piperita leaf 223.75 10.60 8.14
Mentha piperita stem 31.73 0.024 11.33

The results showed interesting differences in the amounts of these derivatives present
in mint samples. It is for the first time, a simple, accurate and rapid HPTLC method
has been developed for the simultaneous and comparative quantification of three
bioactive compounds in methanolic extracts of leaves and stems of Mentha arvensis
and Mentha piperita.
Several studies are on records which have established that phenolic components play
a vital role in the therapeutic efficacy of a drug. Flavonoids (group of polyphenolic
compounds) exhibit several biological effects such as anti-inflammatory, anti-
hepatotoxic, anti-ulcer, antioxidants, antiallergic, antimicrobial, antiviral actions and
some of them provide protection against cardiovascular mortality. In particular
quercetin, rutin and gallic acid have extensively been studied and proved to have
significant therapeutic potential. Quercetin and rutin are known to exhibit
antibacterial, antifungal and antiviral activity (Narayana et al., 2001). On the other
hand tannins have been reported to form irreversible complexes with proline rich
protein (Shimada, 2006) resulting in the inhibition of cell protein synthesis. Parekh
and Chanda (2007) reported that tannins are known to react with proteins to provide
the typical tanning effect which is important for the treatment of inflamed or
ulcerated tissues. Herbs that have tannins as their main components are astringent in
nature and are used for treating intestinal disorders such as diarrhoea and dysentery
(Dharmananda, 2003). The results of phytochemical screening of mint samples and
review of above facts inspire to further screen these samples for their therapeutic
activity potential.
4. Conclusion

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The chemical prospection of cleaned methanolic extracts of mint samples showed the
presence of flavonoids, tannins, phenolic compound and sterols. Total phenolic
contents of plant extract were found to be maximum in Mentha arvensis leaf and
minimum in Mentha piperita stem. The presented study clearly gave evidence of the
quantitative variation of quercetin, rutin and gallic acid in different species of the
same genus. The developed HPTLC method is an attractive technique in regard to its
simplicity, accuracy and selectivity. The method could be widely applied directly for
routine analysis and quality assurance of related extracts and drugs.

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5. Bibliography
Abraham Z. Glimpses of Indian Ethno botany, Oxford & Publishing Co: New Delhi;
1981, p. 308-320.
Dharmananda S. Gallnuts and the uses of Tannins in Chinese Medicine. In:
Proceedings of Institute for Traditional Medicine, Portland, Oregon. 2003.
Duraipandiyan V, Ayyanar M, Ignacimuthu S. Antimicrobial activity of some
ethnomedical plants used by paliyar tribe from Tamil Nadu, India. BMC
Complementary and Alternative Medicine 2006; 35(6): 1-7.
Gopalan C, Ramasastri BV, Balasubramanian SC, Narsinagarao BS, Deosthale YG,
Pant KC. Nutritive value of Indian foods. Hyderabad (India): National Institute
of Nutrition; 2000. P. 1-153.
Harborne JB. Phytochemical Methods - A Guide to Modern Techniques of Plant
Analysis. London: Chapman and Hall; 1998. p. 182-190.
Holiman A. Plants in Medicine. Chelsea Physic Garden: The Chelsea Physic Garden
Co Ltd; 1989.
Iyengar MA. Study of Crude Drugs. 8th ed. Manipal (India): Manipal Power Press;
1995. p. 2.
Mojab F, Kamalinejad M, Ghaderi N, Vahidipour H. Phytochemical screening of
some Iranian plants. Iranian Journal of Pharmaceutical Research 2003; 2: 77-
82.
Narayana K, Reddy MS, Chaluvadi MR, Krishna DR. Bioflavonoids Classification,
Pharmacological, Biochemical Effects and Therapeutic Potential. Indian Journal
of Pharmacology 2001; 33: 2.
Parekh J, Chanda S. In vitro antibacterial activity of crude methanol extract of
Woodfordia fruticosa Kurz flower (Lythacease). Brazilian Journal of
Microbiology 2007; 38: 2.

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Rameshwar Naidu J, Ismail RB, Chen Yeng, Sasidharan, Kumar. Chemical
composition and antioxidant activity of the crude methanolic extracts of Mentha
spicata. Journal of Phytology 2012; 4(1): 13-18.
Shimada T. Salivary proteins as a defense against dietary tannins. Journal of
Chemical Ecology 2006; 32(6): 1149-1163.
Slinkard K, Singleton VL. Total phenol analysis: automation and comparison with
manual methods. American Society for Enology and Viticulture 1977; 28: 49-55.
Trease GE, Evans WC. Textbook of Pharmacognosy. 12th edition London: Balliere
Tinadall Ltd; 1989.
WHO. Traditional Medicine: Growing Needs and Potential. WHO Policy
Perspectives on Medicines; Geneva: World Health Organization; 2002. P. 1-6.

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 CHAPTER VII

PLANTS AS POTENTIAL SOURCE OF ANTIMICROBIALS

Pooja Suneja Madan, Savita Jakhar and Pushpa Dahiya

Abstract. Microorganisms have the genetic ability to transmit and acquire resistance
to drugs, which are utilized as therapeutic agents. In recent years, due to
indiscriminate use of antimicrobial drugs, they have developed resistance to many of
these drugs as a result the drugs are losing their effectiveness. Strains of ȕ – lactam
resistant Satypholococcus aureus and methicillin resistant Satypholococcus aureus
(MRSA) are posing a serious problem not only in hospitalized patients, but also in
their caretakers. Therefore, efforts must be made to reduce this problem by
controlling the overuse of the drugs, developing research to understand the genetic
mechanisms of resistance and developing new drugs. Some of the strategies adopted
by pharmaceutical companies to supply the market with new antimicrobial drugs
include changing the molecular structure of the existing medicines in order to make
them more efficient or recover the activity lost due to bacterial resistance
mechanisms. Another approach is to identify new antimicrobials, especially those of
plant origin as they have a vast potential as curative medicine and are valuable source
of natural products for maintaining human health.
According to World health organization, more than 80% of the world population
relies on traditional medicine for their primary health care needs and also supports its
use provided they are proven to be efficacious and safe (WHO, 1985). The healing
properties of plants lie in their bioactive constituents that produce a definite
physiological action on the human body. Although some of the plants have been
screened for their antimicrobial potential, many still remain unexplored with regard
to this property. There is a continuous and urgent need to discover novel compounds
with diverse chemical structures for new and reemerging infectious diseases. The
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abundance of greenish plant life on the earth's surface has led to an increasing interest
in the investigation of different extracts obtained from traditional medicinal plants, as
a potential source of new antimicrobial agents. In the present scenario, looking for
new antimicrobial compounds in plants is therefore, essential and it needs to be
accompanied by awareness on the misuse of the drugs with the prospect to eliminate
the root cause of the problem.
Keywords: medicinal plants, antimicrobial activity, secondary metabolites, antibiotic
resistance, multi-drug resistant microorganisms
1. Introduction
Plant based medicine is undoubtedly the oldest form of medicine known to mankind
which are used in the treatment of various diseases and for revitalizing body systems.
The therapeutic properties of herbs were discovered probably due to their unique
action by trial and error. This knowledge has been preserved in a very systematic
manner in the written records. There is evidence that Neanderthals (present-day Iraq)
living 60,000 years ago used plants like hollyhock (Stockwell, 1988); and is still part
widely used around the world. The oldest written evidence of medicinal plants in the
preparation of drugs (approx. 5000 years old) has been found on a Sumerian clay slab
(Nagpur) and comprises 12 recipes for drug preparation referring to over 250 plants
(Kelly, 2009). The religious texts like Mahabharata, Ramayana, Charaka Samhita,
Ayurveda, Agnipurana and Bhavprakash also mentions abundant information on the
healing properties of the plants.
Hippocrates (5th century B.C.) listed nearly 300 to 400 medicinal plants. The
renowned medical writer Celsus (25 BC–50 AD) in his work “De re medica” quoted
approximately 250 medicinal plants such as aloe, henbane, flax, poppy, pepper,
cinnamon, the star gentian, cardamom, false hellebore, etc. (Tucakov, 1948).
Dioscorides wrote ‘De Materia Medica’, a medicinal plant catalogue in the first
century A.D and it became the precursor for all modern pharmacopoeias. Pliny the
Elder (23 AD-79) a contemporary of Dioscorides, wrote “Historia naturalis” in which
approximately one thousand medicinal plants are described. The work of Pliny’s and

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Dioscorides’ incorporated complete knowledge of medicinal plants of his time
(Toplak, 2005).
The cultivation of plants possessing medicinal properties, preparation of plant based
drugs and skill of healing moved to monasteries in the Middle Ages. Medicinal plants
such as Greek seed, tansy, sage, anise, mint, savory etc. were grown within the
monasteries and used to prevent or cure infectious conditions. The 19th century
witnessed substantial expansion in the use and sharing of knowledge of medicinal
plants with the discovery, substantiation and isolation of bioactive compounds. In
19th century, phytoconstituents isolated in pure form increasingly substituted or
displaced the crude drugs. In the early 20th century, focus was laid on standardizing
methods for stabilizing labile components obtained from medicinal plants. Gradually,
more interest developed in identifying proper conditions suitable for the cultivation
of medicinal plants and manufacturing drugs from them. The continuous
development this field has made modern and sophisticated means available for the
processing and usage of plant based drugs.
2. Secondary metabolites
Plants are storehouses of different secondary metabolites such as phenols, flavonoids,
terpenoids, alkaloids and tannins. These are organic compounds that are not directly
involved in the normal growth and development of the plant but have important
defensive role in the plant system. Secondary metabolites serve as chemical signals
enabling the plant to respond to environmental cues or functioning in the defense of
the producer against herbivores, pathogens, and other competitors.
They also provide protection from radiations from the sun and assist in
pollen and seed dispersal. They are produced at various sites within the cell and
stored primarily within the vacuole. They have been shown to be promising as plant
and human disease-controlling agents. Man uses them as medicines, flavorings and
recreational drugs. These active compounds are important for the maintenance of
human health and may potentially control the growth of microorganisms in diverse
situations. These phytochemicals have demonstrated their potential as antimicrobial
when used alone and as synergists or potentiators of other therapeutic agents beyond
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doubt. These natural products also provide clues to synthesize new structural types
of antimicrobial and antifungal chemicals that are relatively safe to man (Kalimuthu,
et al., 2010).
Phenolics are the organic compounds found across the plant kingdom. They are an
essential part of the human diet and are of considerable interest due to their multiple
biological effects. Structurally, they consist of a hydroxyl group (-OH) bonded
directly to an aromatic hydrocarbon group. They vary from simple, low molecular
weight, single ringed structure (coumarins and benzoic acid) to large complex
polyphenols (flavanoids and tannins). They have multiple roles assigned to them like
they provide odour, colour and flavour to attract symbiotic insects; are
phagostimulants; act as allelopathic agents to the vegetation growing in their vicinity.
They are also effective against a number of pathogens for example caffeic acid is
effective against bacteria (Brantner et al., 1996), fungi (Duke, 1985) and viruses
(Wild, 1994). They are also believed to play an important role in preventing the
development of chronic diseases such as cancer, heart disease, stroke, Alzheimer's
disease, rheumatoid arthritis, and cataracts. (Devare et al., 2012).
Flavonoids are polyphenolic secondary metabolites having C6–C3–C6 ring structure.
They are also known as Vitamin P or citrin. The major classes of flavonoids are
flavonols (colourless to pale yellow pigments), anthocyanins (red to purple
pigments), proanthocyanidins (PAs) or condensed tannins and flavanols (colourless
pigments that turn brown after oxidation). They protect plants from attacks by
microbes, fungi and insects. The mechanisms could be inhibition of the oxidation,
possibly through reaction with sulfhydryl groups or through more nonspecific
interactions with the proteins (Mason and Wasserman, 1987). Flavonoids also
possess anti-oxidant, anti-proliferative, anti- tumor, anti-inflammatory and pro-
apoptotic activities. Besides, they also have important role in defense, allelopathy and
modulating the levels of reactive oxygen species (Taylor and Grotewold, 2005;
Treutter, 2005; Bais et al., 2006). Flavonoids inhibit the pro-inflammatory activity of
enzymes involved in free radical production such as cyclooxygenase, lipooxygenase
or nitric oxide synthase (lzzi, 2012; Gomes, 2012). The health-promoting effects of
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flavonoids may relate to interactions with key enzymes, signaling cascades involving
cytokines and transcription factors or antioxidant systems (Polya 2003).
3. Plants vs. microbes
The struggle between man and microbes is evident since time immemorial and
probably the first weapon used by ancient human for treatment of various diseases
was plants (Cowan 1999). Looking at the importance of the medicinal properties of
plants, the employment and development of antimicrobial drugs continued
throughout different civilizations and is also evident today. As long as no synthetic
medicines were available, the plant kingdom contributed immensely to human health.
However, with the advent of new facets of technology, man gradually became more
and more dependent on synthetic and semi-synthetic antibiotics with a belief of fast
recovery.
Plants have the ability to produce a large array of secondary metabolites and this
provides them with the potential to overcome the continuous attacks of
microorganisms. Microorganisms on the other hand continue to invade the defense
strategies of plants by breaking down the secondary metabolites. As a result of this
everlasting battle, plant kingdom develops a vast number of biochemical defense
compounds. Similarly, the conflict between humans and pathogenic microorganisms
also continues. As new drugs are developed to fight the disease, microorganisms
develop new ways to strengthen themselves and overcome the resistance. So in
nature, a competition between microbes and plants is an ongoing process, one trying
to overcome the defense strategies and the other trying to protect itself.
4. Resistance in microorganisms
The discovery of antibiotics came as a boon to mankind and they have been hailed as
miracle drug due to their ability to eliminate bacteria without causing much harm to
the patient. They retard bacterial proliferation by entering the microbes and
interfering with the production of many components required for the growth of the
bacteria. For instance, the antibiotic tetracycline binds to ribosome and thus impairs
the protein synthesis; penicillin and vancomycin on the other hand impede the
synthesis of bacterial cell wall. People however, failed to realize that although
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antibiotics help to control the bacterial infections but they have broad, undesirable
effects on the microbial ecology. They can produce a long-lasting change in the
types and proportions of bacteria not only in the treated individual but also in the
environment. The antibiotics should thus be used only when they are truly needed
and prescribed by a medical practitioner. Although, antibiotics are available only by
prescription, but this does not ensure their proper use. As the patient gets some relief,
he often fails to finish the complete course of treatment. Patients then stockpile the
leftover doses and medicate themselves, or their family members and friends, in
unregulated doses. In both circumstances, the improper usage fails to eliminate the
disease agent completely. Moreover, they are also administered for viral infections,
over which they have no control. The antibiotics are therefore used incorrectly many
a times; as a result, the targeted bacteria directly adapt and develop resistance.
Due to its inherent property of rapid multiplication, bacteria passes resistant genes
through plasmid exchange, leading to an increasing prevalence of multi-drug resistant
infections. This encourages the growth of resistant strains, which may produce hard-
to treat disorders thus increasing bacterial resistance to many antibiotics that once
cured diseases readily. This looming threat of incurable multi drug resistant strains is
the latest twist in health sector as it is increasingly limiting the effectiveness of
current antibiotics leading to the failure of treatment of various infections (Hancock,
2005). Many strains of S. aureus have developed resistant to all antibiotics except
vancomycin worldwide. In addition, strains of at least three bacterial species capable
of causing life threatening illnesses (Enterococcus faecalis, Mycobacterium
tuberculosis and Pseudomonas aeruginosa) have also evaded every antibiotic
available with the clinician’s. Due to rise in resistance to antibiotics, the death rates
for some communicable diseases (tuberculosis) have started to rise again, after
having declined in the industrialized nations. Incidents of epidemics due to such drug
resistant microorganisms are now a common global problem causing enormous
public health concerns (Iwu et al., 1999). The misuse of antibiotics by human, the
employment of antibiotics in veterinary practices and the growing presence of

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antibiotics in water, soil and food contribute to the problem of antibiotic resistance
(Moshirfar et al., 2006).
Bacteria develop resistance to antimicrobials either by modification of target sites or
enzymatic inactivation of the drug or extrusion by efflux (Davies, 1994; Spratt, 1994;
Nakaido, 1994). Resistance of Streptococcus species to macrolides, lincosamide and
streptogramin B antibiotics (MLSB resistance) is a result of methylation of the N6
amino group of an adenine residue in 23S rRNA. This mechanism of resistance is
also responsible for ȕ -lactam resistance in non ȕ -lactamase producing Haemophillus
influenza (Matic et al., 2003). The resistance to ȕ -lactam antibiotic by both gram
negative and gram positive bacteria have long been attributed to the activity of ȕ –
lactamases (Frere, 1995). The other strategy employed by a number of pathogens in
evading the effect of antibiotics is by production of hydrolytic enzymes and group of
transferases (Wright, 2005).
Apart from that house-keeping genes that are widespread in bacterial genomes is
responsible for constitutive expression of efflux pump proteins which is responsible
for the phenomenon of intrinsic antibiotic resistance (Lomovskaya and Bostian,
2006). This mechanism of antibiotic resistance could be applicable for all antibiotics
(Li et al., 1994; Gill et al., 1999; Lin et al., 2002), since the majority of the efflux
systems in bacteria are non-drug-specific proteins that pump out a broad range of
chemically and structurally unrelated compounds from bacteria in an energy-
dependent manner, without drug alteration or degradation (Kumar and Schweizer,
2005).
In order to overcome the problem of drug resistance, improved management of
antibiotic use and restoration of the environmental bacteria susceptible to these drugs
are required. The presence of susceptible bacteria is important because if they are
not available then resistant forms would not face any competition for survival and
would persist indefinitely. Now, we should accept the fact that bacteria are normal,
generally beneficial organism of the world, and not to eliminate them except when
they are causing harm or a disease. Concerted efforts to educate the world’s

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populations about drug resistance and the impact of improper antibiotic use is the
best way that will help in controlling drug resistance.
5. Plants as potential source of new antimicrobials
The healing properties of medicinal plants were identified by mankind; noted through
advanced civilizations and passed on to the successive generations. The knowledge of
one society was conveyed to another which upgraded the old properties, discovered
new ones, and this process exists even today. Plants thus have been a source of
inspiration for novel drug compounds and plant derived medicines (phytomedicines)
has contributed significantly to human health and well-being. Many commercially
proven drugs used in modern medicine were initially used in crude form in traditional
or folk healing practices or for other purposes that suggested potentially useful
biological activity. As plants are the largest biochemical and pharmaceutical stores
known to mankind, it is estimated that they provide models for 50% of drugs
manufactured by western countries. Phytomedicines usually have multiple effects on
the body as their actions often act beyond the symptomatic treatment of disease. For
example, Hydrastis canadensis extract has significant antimicrobial activity and it
also increases blood supply to the spleen and promote optimal activity of the spleen
to release mediating compounds. Similarly, isoquinoline alkaloid emetine obtained
from the underground part of Cephaelis ipecacuanha has been used as amoebicidal
drug as well as for the treatment of abscesses due to the spread of Escherichia
histolytica infections. Another important drug of plant origin is quinine, a alkaloid
which occurs naturally in the bark of Cinchona tree. Apart from its continued
usefulness in the treatment of malaria, it can be also used to relieve nocturnal leg
cramps. Aspirin from Salix purpurea (willow) is used for treating inflammation, pain
and thrombosis. In addition, plants have also made important contributions in the
areas beyond antiinfectives such as cancer therapies; antileukaemic alkaloids
(vinblatine and vincristine) are obtained from Catharanthus roseus - the Madagascan
periwinkle (Nelson, 1982).
Plants based drugs have gained and retained their importance as they are not only
effective in the treatment of infectious diseases but also extenuates many of the side
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effects that are usually associated with synthetic compounds. Medicinal plants
therefore work in an integrated or pro-biotic approach with little or no adverse effects
on the body. The combination between the plant extracts of antimicrobial potency
with some antibiotics can be of great value, as it may alter the mode of action and
enhance the effectiveness of the drug. In this techno-era, with the help of advanced
methods of analytical, biotechnological, genomics, proteomics and metabolomics,
medicinal plant research can contribute significantly to the advancement of
alternative natural antimicrobials.
6. Conclusion
Plants are the tremendous source for the discovery of new products of medicinal
value. There are two reasons to investigate plants: first, it is possible that these
phytochemicals may provide with antimicrobial drugs and second, because of
problems with the over prescription and misuse of traditional antibiotics. Although,
the pharmacological activity of many medicinal plants has been studied, majority still
remains to be explored with regard to this property. Among the estimated 250,000-
500,000 plant species, only a small percentage of plants have been investigated
phytochemically and the fraction submitted to biological or pharmacological
screening is even smaller. India, one of the twelfth mega biodiversity centres is a
potential source of therapeutically useful agents due to its rich flora. India has about
4.5 million plant species and among them, several thousands have been claimed to
possess medicinal properties against human diseases. In addition, the rich traditional
knowledge will also be of great help in search for new antimicrobials and evaluating
plants from the traditional Indian system of medicine can provides useful clues for
their use of in the treatment of disease. This perhaps will also instigate serious
research into the old manuscripts on medicinal plants as potential sources of
contemporary pharmacotherapy. In the present scenario where 80% of the world
population has no access to the benefits of western medicines due to financial
constraints, it is necessary to emphasize the importance of plant products as remedies
which constitute a major part of the health care system in the developing countries
and are also entering the therapeutics in the developed countries. Considering the vast
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potentiality of plants as sources for antimicrobial drugs, a systematic investigation
should be undertaken to screen the local flora for their antibacterial and antifungal
properties. A sense of urgency however in this regards is required as the pace of
species extinction is occurring at a very fast rate and we should unwrap the vast
potential of nature before losing it.

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7. Bibliography

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spina-christi mill. Journal of Ethnopharmacology 1996; 52: 119–122.
Cowan MM. Plant products as antimicrobial agents, Clinical Microbiology Review
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Davies J. Inactivation of antibiotics and the dissemination of resistance genes.
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Fessenden RJ and Fessenden JS. Organic chemistry. 2nd ed. Boston: Willard Grant
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Frere JM. Beta-lactamases and bacterial resistance to antibiotics. Molecular
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Gill MJ, Brenwald NP, Wise R. Identification of an efflux pump gene pmrA,
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Hancock EW. Mechanisms of action of newer antibiotics for Gram-positive
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Izzi V, Masuelli L, Tresoldi I, Sacchetti P, Modesti A, Galvano F, Bei R. The effects
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Kelly K. History of medicine. New York: Facts on file; 2009. P. 29–50.
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Nakaido H. Prevention of drug access to bacterial targets: Permeability barriers and
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 CHAPTER VIII

MEDICINAL PLANTS CULTIVATION: AN URGE

Ashish Malik, Ravinder Kumar, Nidhi Verma, Jyoti Ahlawat,

Nidhi Rani, Anita R. Sehrawat

Abstract. Herbalism is the study and use of plants for medicinal purposes. Plants
have been basis for medical treatments through much of human history and such
traditional medicines is still widely practiced today. Several plant species of
medicinal importance have been heavily toiled upon and had lead to their depletion in
several regional habitats. Therefore, to meet the continuous demand of medicinal
herb could be solved by encouraging their cultivation. However, cultivation of
medicinal plants is not easy but rather a challenging task. Cultivation can be lucrative
only if it has necessary support of technology as well as finance. It is worthwhile to
examine the cultivation cost, yield of botanical raw materials/products and gain in
financial terms with respect to important medicinal and aromatic plants that has
become endangered as well as those that can be grown in India. Thus, it is imperative
to promote cultivation of medicinal plant species through appropriate financial
incentives, policy, infrastructural and marketing support in a synergistic manner.
Keywords: Cultivation, conservation, commercialisation, exploitation medicinal
herbs.
1. Introduction
Medicinal plants are the local heritage having a global importance. India has a vast
array of biodiversity of 15 agro climatic zones in which 17000 – 18000 flowering
plant species grow and out of them 6000 – 7000 plant species have the medicinal
utility in folk and documented systems of medicine, like Ayurveda, Unani, Sidha and
Homoeopathy (AYUSH) (Sharma, 2004). Thus, medicinal plants form the major
resource base of our indigenous health care traditions. As per World Health
Organization estimation the demand for medicinal plants is continuously increasing,
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which at present is approximately US $14 billion per year and is more likely to reach
to US $5 trillion by 2050 (Prahalathan, 2004). In India, about 960 species of
medicinal plants are in trade which estimated to be approximately US $1 billion per
year.Presently this share of India at world trade is quite low and the demand for
medicinal plant-based raw materials is growing 15 to 25% annually (Kaushik P,
Dhiman, 1999)
More than 90% of the medicinal plant species in trade continue to be sourced from
the wild. About 2/3rd are over – harvested from wild by destructive means for
preparing medicine by various industries which results into loss of their existing
population. Therefore, cultivation of medicinal plants is the key to meeting the raw
material needs of the AYUSH industry along with opportunities for higher levels of
income, crop diversification and growth of exports (Kala, 2005). Cultivation of
medicinal plants proves to be an efficient and specific tool to overcome all the
problems of wild harvesting and conservation of their genetic resources. A good and
managed cultivation is a very tough task as various challenges and constrains are
being present there such as improper identification and selection of planting material,
slow rate of production of many medicinal plants, long gestation period, shortage of
suitable cultivation technology, production of small quantity, unscientific harvesting,
inefficient processing techniques, fluctuation in demand and supply, poor marketing
infrastructure, lack of cultivation oriented research and above all inappropriate
government policies (Uniyal et al., 2000)
Farming is imperative as it conserves the wild genetic diversity of medicinal plants
and produces uniform material from which standardized products can be consistently
obtained. Thus, the development in medicinal plant cultivation led to affordable
health care, conservation of precious biodiversity and sustainable economic
development.
2. Wild harvesting vs cultivation
Wild harvesting is an inefficient tool on the large scale due to problems of
misidentification of plant species, genetic and phenotypic variability, occurrence of

ϳϳ

contaminants, high energy consumption, loss of genetic biodiversity and cost
effectiveness.
2.1 Misidentification
Plant species cannot be identified properly due to lack of knowledge and limited skill
of the collector which led to eradicating out of the morphologically similar species
from the wild. Wrong identification may cause accidental poisoning. The overlapping
names of many medicinal herbs depending on their location make further the
identification of correct plant species very confusing (Kala, 2000). Thus, a
knowledgeable, skilled, experienced and trained person is required for proper
identification of the medicinal plant species so that required plant material can be
obtained with minimum loss to the biodiversity. Cultivation provides the same and
out limits its use.
2.2 Variability
Variability at the genotypic and phenotypic level due to different climatic and
edaphic factors is another delimiting factor for wild harvesting. Plant extract obtained
from these highly variable plant species cannot meet the required demand. Each plant
responds and accumulates secondary metabolites and other active ingredients
according to its surrounding conditions. Thus, the same plant species obtained from
two different habitats cannot possess the same required active ingredients and results
into undesirable variability (Oostermeijer et al., 2003). Cultivation leads to
uniformity and specificity in achieving a particular plant and plant product without
causing the variations in plant species. The desired variability can be achieved at
genotypic level by using genetic engineering before cultivating a plant material
obtained from wild. Phenotypic variance in the cultivated plants can easily be seen by
varying the growth conditions during cultivation (Kala, 2004).
2.3 High energy consumption
High energy is consumed in collection and selection of a specific plant from the wild
due to their different habitat and random presence. Presence of unwanted or
adulterated material along with the required plant further makes the selection process
an unreliable task. A rarely occurring medicinally important plant species cannot be
ϳϴ

easily accessible from the wild. Lot of energy, manpower and time is consumed in
the collection of raw material of such species so farming of this particular species
eliminates such kinds of problems (Rao et al., 2004). Hence cultivation provides an
efficient and less energy consuming system along with a reliable botanical
identification.
2.4 Contamination
In wild, occurrence of a plant in isolation is not possible and co – existence of many
plant species causes contamination which is a common problem during harvesting.
Contamination not only limits the efficiency of wild harvesting but proves to be
harmful as it can be poisonous or affect human health on consumption (McChesney,
1999). Cultivation generates pure elite plant in a selected region.
2.5 Cost effective
Sometimes wild harvesting of the plant species cannot provide a regular supply of the
required plant material in desired amount due to dependence of growth on many
uncontrolled factors. Irregular supply directly affects the cost of the raw material.
When wild resources decline with over-harvesting, raw material prices increase and
cultivation becomes economically feasible. Economical feasibility is the main
rationale before bringing a plant species under cultivation. Cultivation guarantees a
steady source of raw material and this continuous supply of the required plant
material proves to be cost effective (Mashelkar, 2005).
2.6 Biodiversity loss: A major concern
A threat to the biodiversity in terms of loss of existing population of medicinal plant
species by overexploitation and continuous demand of plants is the major problem
associate with the wild harvesting. The demand of the medicinal plants has led to the
over-harvesting of many plants from their terrestrial habitat. Some of notable
example are Aconitum heterophyllum, Himalayan yew (Taxus baccata), Dactylorhiza
hatagirea, Nardostachys grandiflora, Polygonatum verticillatum, Gloriosa superba,
Megacarpoea polyandra and Arnebia benthamii have been gathered from the wild
and and today have been enlisted as rare and endangered (Kala, 2003, Hamilton,
2003). Over-exploitation and continuous depletion of an estimated 4,000 to 10,000
ϳϵ

medicinal plants species have been facing a local, regional, national and global
extinction with subsequent serious consequences for loss of genetic diversity,
livelihoods, economies and health care systems. Cultivation conserves the genetic
biodiversity at their best and maintains the ecological balance (Kala, 2006).
3. Challenges and opportunities
A lot of factors need to be considered and focused before we cultivate medicinal
plant species. We need to be completely knowledgeable in terms of their
identification, seed availability, seed maturity, favourable environmental conditions
for growth, processing, post harvest management and their well managed marketing
for a proper cultivation.
3.1 Identification
First of all, a plant should be well identified as a medicinal plant and its importance
for human welfare. Proper identification and knowledge is the key step for further
cultivation of plant. Selection of planting material for large-scale farming can be
done by identifying its medicinal properties. Therefore the planting material should
be of good quality, rich in active ingredients, pest- and disease-resistant and
environmental tolerant. Cultivation permits better species identification, improved
quality control, and increased prospects for genetic improvements (Schippmann et
al., 2002).
3.2 Agrotechniques
Agrotechniques should be one of the thrust areas for research, since it has been
found that knowledge and information on how the propagation of medicinal plants
should be done is available for less than 10% and agro-technology is available only
for 1% total global medicinal plants. Strategies for the effective cultivation as well as
conservation of medicinal plants should be followed such as integration of shade
tolerant medicinal plants as lower strata species in multistrata system. Similarly,
cultivation of short cycle medicinal plants as intercrops in existing stands of tree
crops, inter-planting medicinal plants with food crops and growing medicinal tree as
shade providers and boundary markers etc (Nautiyal, 2004).
3.3 Post harvest management
ϴϬ

Storage facilities for the plant and its products after harvesting play an important role
in cultivation for getting proper benefits. The importance can be felt from the fact
that as high as 30% of the raw material that reach the manufacturers is of poor quality
and therefore rejected. Therefore, it is of utmost importance that cultivation of
medicinal plants is further supported with infrastructure for ware housing grading,
drying, storage and transportation for adding value to the product and increasing of
the medicinal plants which may further lead to increasing profitability and reducing
losses (Peter et al., 2005). A good processing of the plant product is another factor
that needs attention. The processing unit should preferably be set up within the
existing industrial estates, which have the necessary infrastructure of power, road
network and linkages with rail head/sea ports such that minimum loss occurs during
transport of plant material to the market sector and farmers get more benefits. With
the help of an efficient processing technique good quality products with high yield
can be obtained (Swaminathan, 1995).
3.4 Marketing
The marketing system in medicinal plants sector is largely unregulated and
inequitable. There are numbers of stakeholders ranging from herb gatherers,
manufacturers, exporters, local middlemen, urban traders, wholesalers and herbal
healers in the medicinal plants trade sector. Generally, in medicinal plants sector,
there is a top down approach and even the many stakeholders at the bottom are not
aware of the rising demand of their product and the availability of its market. The
most effected people are medicinal plant collector. Generally, the medicinal plant
collectors are the marginal farmers and labourers who are often unaware about the
real market prices of many medicinal plant species. Their income is less than
sufficient to meet their basic requirements for food, health and children education by
selling medicinal plants. (Cunningham, 1998, Gupta, 1998). A strengthen
infrastructure of wholesale markets, agriculture mandies and herbal mandies should
be built. Farmers should be properly informed about market, prices, market trends
such that they are enable in selection of appropriate medicinal crops. Therefore,

ϴϭ

generation of the well managed market for the cultivated plants is equally important
for improved cultivation.
3.5 Government policies
There is a need of supportive and informative government policies that focuses on the
conservation of medicinal plants by prompting enhancement of more and more plants
to be cultivated should be adopted. This is tough challenge for both policy makers
and for economists to formulate policy that reduce the pressure on various forest
products, especially on the medicinal plants and is equally acceptable (Vines, 2004;
Hamilton, 2004). In order to meet these desired policy and their implementation
National Medicinal Plants Board (NMPB) was set up through a Government
Resolution on 24th November 2000 under the Chairmanship of Union Health &
Family Welfare Minister that have responsibility for coordination of all matters
relating to medicinal plants, such as drawing up of policies and strategies for
conservation of medicinal plants, cost-effective cultivation, proper harvesting
methods, processing, marketing of raw material and encouraging research and
development in the field of medicinal plants in order to promote and develop this
sector.
4. Use of biotechnology
In the present era of biotechnology, different biotechnological tools are helpful in
selection, multiplication, improvement and analysis of the medicinal plants. Wild
harvesting of the medicinal plant cannot harness the benefits of biotechnology and
eradicates the plant species in a non specific and random way. Cultivation of plants
under regulated growth conditions can solve the problem to some extent. But with the
use of biotechnological tools, its specificity and stability can be improved at a higher
rate (Khan & Khanum, 2000). Biotechnology involves creation of genetic
manipulation among the wild plants and their cultivation under desired conditions.
Production of transgenic plant with agronomic characters under cultivation, in vitro
culturing of rarely occurring wild plant either by micro propagation or by somatic
embryogenesis, DNA profiling for simultaneously analysis of multiple genes and
their expression and marker assisted selection of a plant with desirable character with
ϴϮ

the help of molecular techniques are gaining importance (Dubey & Guerra, 2002;
Bernath, 2002; Sharma, 1996].
Genetic transformation of a plant for the content of most active compound has been
done by direct manipulation of its DNA sequences to alter the gene expression. It not
only increase potency, uniformity, stability and predictability of extracts but reduces
the toxin level (Yaseen et al., 2009). A considerable interest in manipulating plant
biosynthetic pathways for the production of drug precursors, food components and
secondary metabolites is developing at fast. For example; Mentha spp (mints),
biosynthetic pathways have been engineered to modify essential oil production in the
trichomes and to enhance the resistance of the plant to fungal infection and abiotic
stresses (Veronese et al., 2001).
Plant tissue culture is a viable alternative for rapid propagation of the plant species as
it provides the solution to the problems related to the propagation of the plant like;
late flowering, seed dormancy, seed dispersal and other biotic and abiotic stress. It
also promotes genetic disturbances, which result into generation of somaclonal
variation and availability of the range of useful variation to the breeder (Bajaj, 1998).
Plant regeneration through somatic embryogenesis from the stem, petiole and leaf
explants of Indian chicory, Cichorium intybus L. establishes its breeding material
from wild populations and a mass-producing material for selection or engineering
(Ferreira & Duke, 1997).
The micropropagated plants are independent of climatic changes or soil conditions
and thus can be cultivated elsewhere regardless of the environmental conditions.
Different valuable medicinal herbs are under in vitro mass multiplication like; Aegle
marmelos, Acorus calamus, Celastrus paniculatus, Commiphora mukul, Peganum
harmala, Prosopis cineraria, Simmondsia chinensis, Spilanthes acmella, Stevia
rebaudiana, Sapindus mukorossi etc (Rout et al., 2000; Chand & Sahrawat, 2002;
Yadav & Singh, 2001b; Yadav et al., 2011).
Marker assisted selection of a trait recognize desirable genotypes at an early stage
and speed up the selection process. A specific DNA sequences that are either gene
directly concerned with the trait or that are closely linked to such genes are detected.
ϴϯ

A high degree of similarity in the DNA sequences of functional genes between
different plant species results into similar DNA probes of one species to be used to
identify homologous sequences in another closely related species (Joshi et al., 2004).
However, there have been relatively few reports of molecular marker-based
approaches to medicinal plant improvement as whole genome sequencing of
Arabidopsis and Oryza sativa and partial genome model of only few plant species
Medicago, Lycopersicon (tomato) and Populus (poplar) is being available (Bell et al.,
2001; Gilmore & Peakall, 2003).
4.1 Cultivation and economics:
Herbal medicine has become a topic of increasing global importance impacting both
world health and international trade. Medical and economic benefits of plant based
medicines are recognized by both the developing and industrialized countries. As per
World Bank reports trade in medicinal plants, botanical drug products and raw
material is growing at an annual growth rate between 5 to 15%. The Global
pharmaceutical market has risen from US $550 billion in 2004 worth to a close to
US$900 billion in the year 2008. In India the value of botanicals related trade is about
US$10 billion per annum with annual export of US$1.1 billion (Dhyani & Kala,
2005).
India has a great potential to keep pace with this trade as it has varied agro climatic
condition in which plenty of plants can be grown. Cultivation of medicinal plants is
one of the measures to be taken under consideration both for human welfare and
economic feasibility. Cultivation is a labour intensive process and generates
employment opportunities from the local farmers to the scientists at its each stage
(Raven, 1998). In selecting a plant for cultivation to the delivering of the plant
product to the users, employment opportunities are sufficient enough to raise the
national economy. Farmers which are growing traditional crops in their lands are less
benefitted than those cultivating the medicinal plant even on the comparatively small
land area. It not only enhances the income of the farmers but also improves their
socio – economic prospective in the society. After farming a particular plant,
maintenance and optimization of a good quality product with high yield is essential
ϴϰ

as various industries are to be established based on these cultivated plant material
which requires a large employment among local peoples. It is helpful in poverty
alleviation and up gradation of local farmers, labourers and other middle man of the
country. Up gradation of a farmer means the upliftment of the country. Thus,
cultivation enhances employment opportunities, maintains the sustainable growth and
provides best health care system which can raise the national economy to the highest.
5. Conclusion
In spite of the loss of medicinal plant species, there is a huge demand rate attributed
to their valuable medicinal properties. Although adequate protection of some species
can be achieved through increased regulation and the introduction of sustainable wild
harvesting methods but a more viable long-term alternative is to increase domestic
cultivation of medicinal plants. Cultivation also opens up the possibility of using
biotechnology to solve problems that are inherent in the production of herbal
medicines including species misidentification, genetic and phenotypic variability,
variability and instability of extracts, toxic components and contaminants. Cultivation
offers the opportunity to optimize yield and achieve a uniform, high quality product.
The prospective cultivator of medicinal plants must be aware of particular species to
grow in a rapidly shifting, and fashion-prone, market.

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