S. A.Thube et al.

/ Journal of Pharmacy Research 2012,5(6),3126-3130

Research Article Available online through
ISSN: 0974-6943 www.jpronline.info
Traditionally Used Indian Medicinal Plants: Antibacterial,
Antioxidant, Antiacne Activity And Formulation Development
S. A.Thube 1*, M. J.Patil2
1*
Department of Pharmacognosy, M.C.E.Society’s Allana College of Pharmacy,Azam Campus,2390 K.B.Hidyatullah Road,Camp, Pune 411 001.India
2
Department of Pharmacognosy,M.M’s College of Pharmacy, Tathawade, Pune, Maharashtra, India

Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012

ABSTRACT
Acne vulgaris is a chronic inflammatory disorder of the pilosebaceous follicle for which Propionibacterium acnes and Staphylococcus epidermidis have
been recognized as pus-forming bacteria triggering an inflammation in acne. The present work was undertaken to validate the folk use of five Indian
traditional plants in the treatment of acne vulgaris as a separate entity and as a combination by using scientific methods for the first time. The anti-acne
potential of ethanol extract of the plant materials were evaluated against P.acnes and S.epidermidis using cup plate diffusion method. Antioxidant activity
was screened by using DPPH and Hydrogen peroxide method. Topical gel formulations were developed and evaluated at different concentrations.
Amongst the plant materials screened Casuarina equisetifolia showed the strongest antibacterial activity (zone of inhibition =32.5 mm) followed by
Dalbergia sisso (zone of inhibition = 31.5 mm) against P.acnes. A good antioxidant activity was shown by Casuarina equisetifolia (IC50 = 0.055 mg/ml)
followed by Dalbergia sisso (IC50 = 0.13 mg/ml)by DPPH method and at IC50 0.09 and 0.15 by hydrogen peroxide method. Gel formulation F1 showed
greatest antibacterial activity against P.acnes and S.epidermidis (zones of inhibition >12 mm and 18 mm respectively) than other formulations. From the
stability studies, gels showed no change in pH, viscosity and spreadability after keeping at different temperatures for 90 days. The plant materials
Barleria prionitis, Butea monosperma, Casuarina equisetifolia, Dalbergia sympathetica and Lagenaria siceraria are effective in acne vulgaris as a
separate entity and in a combination. Since topical gels are very useful as palliative products and prove to be economical and safe, a simple gel formulation
was formulated with carbomer as a best gelling agent and evaluated.

Key words: Acne vulgaris, polyherbal, Propionibacterium acnes, Staphylococcus epidermidis, antibacterial, antioxidant

INTRODUCTION
Acne vulgaris is a chronic inflammatory disorder of the pilosebaceous follicle
characterized by comedones, papules, pustules, cysts, nodules and scars in
certain sites like face, neck, upper trunk and arms [1,2]. Propionibacterium
acnes and Staphylococcus epidermidis have been implicated as a causative
organism for acne vulgaris, which colonize in an environment rich in sebum
[3]
.The prevalence of the disease is that 85% adolescents experience it.
Prevalence of comedones (lesions) in adolescents approaches 100% and it
affects 8% of 25 - 34 year olds, and 3% of 35-44 year olds.
P.acnes produces substances that promote inflammation including
chemotactic factors along with lipolytic and proteolytic enzymes, which
convert triglycerides of the sebaceous glands into free fatty acids that
stimulate inflammation and edema that results into breakdown of the follicular
wal l[4,5].
lesion may be non-inflammatory called open or closed comedones or
inflammatory called papule, pustule or nodule. [6,7]
The treatment modalities for acne are usually aimed at decreasing the P.acnes
population, producing anti-inflammatory effect and decreasing the sebaceous
gland activity.[8]Since inflammatory conditions are associated with free radical
production, plant materials possessing antioxidant activity are beneficial.
For many years antibiotics and hormones were usually applied to treat
acne. [9,10] However, these agents are often accompanied by severe side
effects and drug resistance. [11,12] Therefore, phytotherapeutic approaches
with high antibacterial activity and without side effects have been extensively
studied as an alternative. In this context traditional herbs reported in the
traditional systems of medicine have been screened for the aforesaid antiacne
activity.

Pathogenesis and pathophysiology Traditionally the leaves of Barleria prionitis (Acanthaceae), leaves of Butea
Increase in the androgen mediated sebum production leads to the enlargement, monosperma (Papilionaceae), Casuarina equisetifolia bark (Casuarinaceae),
which leads to the keratinous obstruction of sebaceous follicle outlet. These Lagenaria siceraria fruit (Cucurbitaceae) and Dalbergia sisso bark
plugs containing anaerobic lipid-rich environment provides a growth medium (Papilionaceae) were used for the treatment of acne. [13] In present study an
for P. acnes. P.acnes population increases, which results in an inflammation. investigation had been made on these plants in terms of their ability to
Chemotactic factors attract neutrophils and depending on conditions, the reduce Propionibacterium acnes and Staphylococcus epidermidis population,
the causative organisms for acne and to act as antioxidants. The purpose of
*Corresponding author. the study was to prepare and evaluate an herbal formulation having a
S. A.Thube combined antibacterial, antioxidant, anti-inflammatory and astringent effect.
Department of Pharmacognosy,
M.C.E.Society’s Allana College of MATERIALS AND METHODS
Pharmacy,Azam Campus,2390 The leaves of Barleria prionitis (Acanthaceae), leaves of Butea monosperma
K.B.Hidyatullah Road,Camp, (Papilionaceae), Casuarina equisetifolia bark (Casuarinaceae), Lagenaria
Pune 411 001.India siceraria fruit (Cucurbitaceae) and Dalbergia sisso bark (Papilionaceae) were

Journal of Pharmacy Research Vol.5 Issue 6.June 2012 3126-3130

 S. A.Thube et al. / Journal of Pharmacy Research 2012,5(6),3126-3130
collected from Ahmednagar district and authenticated at Botanical survey of Zone of Inhibition
India (Voucher specimen number SATBAP1, SATBUM 2, SATCAE3, Different concentrations of the ethanol extracts and the gel formulations
SATLAS4 AND SATDAS 5) vide letter No.BSI/WRC/Tech/2010. were screened for the antibacterial activity by cup plate method
[17]
.Clindamycin gel 1 % was used as a positive control. Ethanol was used as
Cultures of Propionibacterium acnes (MTCC No. 1951) and Staphylococcus a vehicle control. For P.acnes the plates were kept in the McIntosh Filde’s
epidermidis (MTCC No. 435) were procured from IMTECH, Chandigarh. anaerobic jar, filled twice with nitrogen gas after evacuating and incubated at
The culture media brain heart infusion broth for Propionibacterium acnes 37o C for 48 hrs. The zone of Inhibition was measured.
and Nutrient Broth for Staphylococcus epidermidis were obtained from
Himedia Laboratories Limited, Mumbai. DPPH ie.1,1Diphenyl-2-Picryl- Antioxidant activity
Hydrazyl radical was obtained from Sigma Aldrich, U.S.A. Hydrogen
Peroxide and Ascorbic acid AR grade were obtained from BDH, Mumbai. DPPH method
The in-vitro antioxidant activity of the ethanol extracts of the five plant
Table No.1 Plant materials taken for screening materials were established by their ability to scavenge a stable free radical
DPPH (1,1-Diphenyl-2-Picryl Hydrazyl). [18]The decrease in absorbance
Sr No. Plant Family Part used
of the DPPH solution brought about by the test solution was monitored
1. Barleria prionitis Acanthaceae Leaf spectrophotometrically using Jasco UV/VIS spectrophotometer at 516 nm
2. Butea monosperma Papilionaceae Leaves
3. Casuarina equisetifolia Casuarinaceae Bark 5 minutes after the addition of the test extracts. EC50 (ie. The concentration
4. Dalbergia sisso Papilionaceae Bark of the test solutions required to give a 50 % reduction in the absorbance of
5. Lagenaria siceraria Cucurbitaceae Fruit pulp the test solution s as compared to that of the blank solution) was calculated
Preparation of extracts by subjecting the reading for linear regression between 10-80 %.Ascorbic
The plant materials were dried, coarsely powdered and extracted by acid was used as a positive control.
continuous hot extraction (soxhlet) method using ethanol (95% v/v). The
extracts obtained were concentrated and subjected to phytochemical Hydrogen peroxide method
screening. [14] A solution of hydrogen peroxide (40 mM) was prepared in phosphate
buffer (50mM pH 7.4). The concentration of hydrogen peroxide was
Table No.2 Preliminary Phytochemical Screening
determined by absorption at 230 nm using a spectrophotometer. Extract
Phyto Test Barleria Butea Casuarina Dalbergia Lagenaria (20 - 60 g/ml) in distilled water were added to hydrogen peroxide and
chemical prionitis monosperma equisetifolia sisso siceraria absorbance at 230 nm was determined after 10 min against a blank solution
Carbohy- Molish test + + + + + containing phosphate buffer without hydrogen peroxide. The percentage of
drates hydrogen peroxide scavenging was calculated as follows:
Proteins Biuret test, - + - + +
Ninhydrin % Scavenged (H2 O2 ) = (A0 – A 1 / A 0 ) X 100
test Where A0 is the absorbance of control and A1 is the absorbance of test.
Alkaloids Dragen + + + - + Ascorbic acid was used as a positive control. [19]
droff’s test,
Mayer’s test
Tannins FeCl 3 test, + + + + + Development of the formulation
Lead acetate Literature survey showed that carbomers have many advantages as a gelling
test
Flavanoids Shinoda test + + + + + agent viz. high viscosity at low concentrations, stability to heat, unaffected
Steroids Liebermann - - - - - by aging, do not support microbial growth, nontoxic and nonirritant etc.
Burchard test [20,21]
Taking into consideration above facts carbomer 940 was taken for the
Saponins Foam test + + - - +
formulation of gel. Three different gel formulations were prepared containing
the extracts at different concentrations.
Antibacterial activity
Evaluation of the formulation
Minimum Inhibitory Concentration (MIC)
Standard cultures of the microorganisms were prepared by comparing against pH
McFarland Turbidity standard, such that it contained 1 X 105 cells/ml. [15] The pH of the various gel formulations was determined by using digital pH
The MIC(ie. The lowest concentration that can inhibit the growth of the meter.
microorganism) of the extracts were found out by Broth Dilution Technique Spreadability
[16]
. In the media Tween 80 (1%) and Thioglycollic acid (0.03%) were added It was determined by wooden block and glass slide apparatus. Weights
as reducing agents in Brain heart Infusion Broth. about 20 g were added to the pan and the time was noted for upper slide
(movable) to separate completely from the fixed slide. [22]
Table No. 3 Standard Protocols followed for collection and preservation
of microbial cultures
Consistency
Bacteria MTCC Growth Growth Incubation Incubation Subculture The measurement of consistency of the prepared gels was done by dropping
No condition media Temp time a cone attached to a holding rod from a fix distance of 10 cm in such way
that it should fall on the center of the glass cup filled with the gel. The
Propionibacterium 1951 Anaerobic Brain heart 37ºC 48 hrs 15 days
acnes infusion broth penetration by the cone was measured from the surface of the gel to the tip
Staphylococcus 435 Aerobic Nutrient broth 37ºC 24hrs 15 days of the cone inside the gel. The distance travelled by cone was noted down
epidermidis after 10 sec.

Tween 80 % and Thioglycollic acid were added as reducing agents in the Homogeneity
media for Propionibacterium acnes.
All developed gels were tested for homogeneity by visual inspection after

Journal of Pharmacy Research Vol.5 Issue 6.June 2012 3126-3130

 S. A.Thube et al. / Journal of Pharmacy Research 2012,5(6),3126-3130
the gels have been set in the container. They were tested for their appearance
and presence of any aggregates.

Skin irritation test

Test for irritation was performed on human volunteers. For each gel, five
volunteers were selected and 1.0 g of formulated gel was applied on an area
of 2 inch2 to the back of hand. The volunteers were observed for lesions or
irritation.

Accelerated Stability studies

All the selected formulations were subjected to a stability testing for three Fig No. 4 Zone of inhibition of Lagenaria siceraria against P.acnes &
months as per ICH norms at a temperature of 40 ± 2ºC. Formulations were Staph.epidermidis
analyzed for the change in appearance, pH, homogeneity and consistency.

RESULTS AND DISCUSSION

Out of the five plant materials screened, Casuarina equisetifolia showed the
lowest MIC of 0.3 µg/ml and highest zone of inhibition against P.acnes.
Casuarina equisetifolia has been reported to contain a large amount of tannins
and flavonoid compounds which can be contributed to exhibit a strong
antibacterial activity.

Table No. 4 Antibacterial activity of the extracts

Fig No. 5 Zone of inhibition of Barleria prionitis against P.acnes
Sr.No Name of extract MIC (µg/ml) Broth Zone of Inhibition by
dilution technique Cup plate method in (mm)
P.acnes S.epidermidis P.acnes S.epidermidis

1. B.prionitis 1.2 1.4 26.2 24.2

2. B.monosperma 1.0 1.2 28.2 24.4
3. C.equisetifolia 0.3 0.4 32.5 29.7
4. D.sisso 0.7 0.8 31..5 29.7
5. L.siceraria 1.0 0.9 20.6 20.3
6. Standard (Clindamycin) 0.08 0.09 36.7 34.3

Fig No. 1 Zone of inhibition of Butea monosperma against P.acnes &

Staph.epidermidis

Fig No. 2 Zone of inhibition of Dalbergia sisso against P.acnes &

Staph.epidermidis

Fig No. 6 Zone of inhibition of the formulations against P.acnes &

Staph.epidermidis
Casuarina equisetifolia showed the best antioxidant activity with IC50 values
of 0.055 mg/ml by DPPH method and 0.09 mg/ml by hydrogen peroxide
method.The free radical scavenging activity and reported anti-inflammatory
activity of the extracts can be considered beneficial in reduction of the inflam-
Fig No. 3 Zone of inhibition of Casuarina equisetifolia against P.acnes & matory response in acne.
Staph.epidermidis
Journal of Pharmacy Research Vol.5 Issue 6.June 2012 3126-3130
 S. A.Thube et al. / Journal of Pharmacy Research 2012,5(6),3126-3130
Table No. 5 Antioxidant activity of the extracts by DPPH method & Table No.10 Stability studies
Hydrogen Peroxide method At ambient temperature
Sr.No Extract IC50 (mg/ml) ± SD Sr. Storage Evaluation parameters for polyherbal antiacne gel
DPPH method r* Hydrogen r* No period Appearance pH Viscosity (Cp)
Peroxide
method F1 F2 F3 F1 F2 F3 F1 F2 F3

1. B.prionitis 0.045 ± 1.901 0.9356 0.09 ± 1.536 0.9584 1. Initial Tc Tc Tc 6.95 7.41 7.04 20.062 18.656 15.093
2. B.monosperma 0.26 ±2.6873 0.9458 0.1 ± 2.045 0.9646 2. 1 st month Tc Tc Tc 6.95 7.43 7.05 20.123 19.064 15.156
3. C.equisetifolia 0.055 ± 3.218 0.8997 0.09 ± 1.032 0.9980 3. 2 nd month Tc Tc Tc 6.96 7.44 7.05 20.215 19.087 15.189
4. D.sisso 0.13 ± 2.374 0.9587 0.15 ± 2.583 0.8667 4. 3 rd month Tc Tc Tc 6.98 7.42 7.08 20.221 19.120 15.231
5. L.siceraria 0.52 ± 1.647 0.8668 0.25 ± 1.943 0.9246
6. Standard 0.047 ± 2.698 0.8635 0.058 ± 3.546 0.8725 Tc – Translucent
(Ascorbic acid)
At elevated temperature (40 ± 10 C).
SD- Standard Deviation ; r* - correlation coefficient
Sr. Storage Evaluation parameters for polyherbal antiacne gel
Out of the three gel formulations, F1 showed maximum zone of inhibition No period Appearance pH Viscosity (Cp)
F1 F2 F3 F1 F2 F3 F1 F2 F3
and also a good antioxidant activity. The activity can be due to synergistic
effects of the extracts. The stability studies of the gels showed no change in
pH, viscosity and spread ability after keeping at different temperatures for 1. Initial Tc Tc Tc 6.95 7.41 7.04 20.062 18.656 15.093
2. 1 st month Tc Tc Tc 7.05 7.49 7.44 18.923 16.064 13.956
3 months. 3. 2 nd month Tc Tc Tc 7.12 7.52 7.56 18.115 15.087 13.109
4. 3 rd month Tc Tc Tc 7.26 7.67 7.78 17.561 14.790 11.631
Therefore, these plants can be used as an alternative treatment for acne,
having combined antibacterial, antioxidant and the reported anti-inflammatory At refrigerated temperature (5 ± 10 C)
activity. The ex-vivo antiacne activity is being carried out on rats. Sr. Storage Evaluation parameters for polyherbal antiacne gel
No period Appearance pH Viscosity (Cp)
F1 F2 F3 F1 F2 F3 F1 F2 F3
The further objective of this research work is to develop an antiacne gel
formulation with isolated chemical entities and also to compare their antiacne 1. Initial Tc Tc Tc 6.94 7.08 7.18 24.937 23.062 16.500
potential, so as to develop a well-accepted antiacne formulation. 2. 1 st month Tc Tc Tc 7.11 7.14 7.26 23.987 22.987 15.340
3. 2 nd month Tc Tc Tc 7.54 7.43 7.46 22.623 21.765 14.045
Table No. 6 Formulation development 4. 3 rd month Tc Tc Tc 7.62 7.61 7.51 21.089 20.165 13.941

Sr.no Ingredients F1 F2 F3
ACKNOWLEDGEMENT
1. Carbopol 940 2.0 % 2.0 % 2.0 % The authors kindly acknowledge the facilities provided by the Jawaharlal
2. Propylene glycol 10 % 10 % 10 %
3. Propyl paraben 0.08 % 0.08 % 0.08 % Nehru Technological University, Hyderabad.
4. Methyl Paraben 0.2 % 0.2 % 0.2 %
5. Triethanolamine q.s q.s q.s
6. Distilled water q.s q.s q.s REFERENCES
7. B.prionitis extract 1.2 % 1.2 % - 1. C.R..Karnick.(1994). Ancient Science of Life, ’Some clinical
8. B.monosperma extract 1.0 % -
9. C.equisetifolia extract 0.3 % 0.3 % 0.3 % observations on acne vulgaris’,13,286-292.
10. D.sisso extract 0.7 % - 0.7 % 2. Mamgain R.K.(2000).Acne vulgaris and its treatment by
11. L.siceraria extract 1.0 % - -
indigenous drugs SK-34 and SK-235’, The Antiseptic,97(3),76-
Table No. 7 Antibacterial activity of the formulation 78.
3. Ingham. E, Holland K. T, Gonland g.(1983). Studies of the
Formulation Zone of Inhibition by
Cup plate method in (mm) extracellular proteolytic activity produced by P.acnes. Journal of
applied bacteriology, 54,263-271.
P.acnes S.epidermidis
F1 33.5 30.7
F2 23.9 19.4 4. Kaur P, Kaur S, Kumar S, Singh P.(2010). Rubia cordifolia L. and
F3 20.4 17.7 Glycurrhiza glabra L. Medicinal plants as potential source of
Standard (Clindamycin) 35.8 32.5
COX-2 Inhibitors. Americal Journal of Biomedical Sciences,
Table No. 8 Antioxidant activity of the formulation 2(2),108-120.
Sr.No Formulation IC50 (mg/ml) ± SD 5. Yuangang Zu, Huimin Yu, Liang Lu, Yujie Fu, Efferth T, Liu X,
DPPH r* Hydrogen r* Nan Wu. (2010).Activities of Ten Essential Oils towards
method Peroxide
method Propionibacterium acnes and PC-3,A-549 and MCF-7 Cancer
Cells.Molecules,15,3200-3210.
1. F1 0.055 ±2.6873 0.9458 0.1 ± 2.045 0.9646 6. Cotterell J.A et al.(1972). British Medical Journal, Further
2. F2 0.045± 1.901 0.9356 0.9 ± 1.536 0.9584
3. F3 0.35 ± 3.218 0.8997 0.1 ± 1.032 0.9980 observations in the pathogenesis of acne,2,404-408.
4. Standard 0.047± 2.698 0.8635 0.058 ± 3.546 0.8725 7. Cunliffe W.J, Shusters.(1969). Pathogenesis of acne. Lancet,1,685-
(Ascorbic acid)
687.
SD- Standard Deviation; r* - correlation coefficient
8. Lucinda G.Miller.(1998).Herbal Medicals, A clinical guide.
Table No.9 Evaluation of gels Pharmaceutical products press, pp 253-267
Sr.No Formula- Colour Appear pH Viscosity Spread Homo Skin
tion ance (cp) ability geneity irrita 9. Poulin, Y.(2004).Practical approach to the hormonal treatment of
code tion acne. J. Cutan. Med. Surgery, 4,6–21.
Test
10. Tan A.W, Tan H.H.(2005). Acne vulgaris: a review of antibiotic
1 F1
Dark Translu 6.95 20.062 23.45 Good NIL therapy. Expert Opin. Pharmacotherapy, 6, 409–418.
brown cent
orange 11. Leyden, J.J.(2004).Antibiotic resistance in the topical treatment
2 F2 Orange Translu 7.41 18.656 25.45 Good NIL of acne vulgaris. Cutis, 73, 6–10.
cent
3 F3 Light Translu 7.04 15.093 22.87 Good NIL 12. Yemisci A, Gorgulu, A, Piskin, S.(2005) Effects and side-effects
orange cent

Journal of Pharmacy Research Vol.5 Issue 6.June 2012 3126-3130

 S. A.Thube et al. / Journal of Pharmacy Research 2012,5(6),3126-3130
of spironolactone therapy in women with acne. J. Eur. Acad. 18. D’Mello P.M, Jadhav M.A, Jolly C.I.(2000). Free radical
Dermatol. Venereol, 19, 163–166. scavenging activity of Syzigium cumini and Ficus bengalensis-
13. K.R.Kirtikar and B.D.Basu.(2005) Indian Medicinal Plants. plants used in ayurveda for Diabetis mellitus. Indian Drugs .37,
International Book Distributors, Dehradun, Second Edition, 518-520
pp.785,817,819-820,1116,1877,2351 19. Ruch RJ, Cheng SJ, Klaunig JE. (1989). Prevention of cytotoxicity
14. C.K. Kokate, A.P. Purohit, S.B.Gokhale.(1998) Pharmacognosy, and inhibition of intracellular communication by antioxidant
Nirali Prakashan, twenty fourth edition,pp.212,214,215,398- catechins isolated from Chinese green tea. Carcinogenesis, 10,
400,395-397,437,424-425,181,184,186,216-219,518- 1003-1008.
520,372,373,400-402. 20. Hazra, B., Basu, S., Ghosh, A.(2005). Evaluation of the
15. A.W. Baueg, M.D.K. Kirby, J.C. Sherries, M. Truck. (1996). Antibacterial Activity of Ventilago madraspatana Gaertn. Rubia
Antibiotic susceptibilities testing by standard single disc diffusion cordifolia Linn. and Lantana camara Linn, Isolation of Emodin
method. American Journal of Clinical Pathology, 45(4),493-496 and Physician as Active Antibacterial Agents. Phytotherapy
16. B.J.Wadher,G.L.Bhoosreddy.(1995) Manuals of Diagnostic research. 19, 888-894
Microbiology, Himalaya Publishing House, first edition,Pg.62- 21. Singh A.P.(2005).Current status and future direction of herbal
67. medicine. Trends in Pharmacological sciences, 23(8), 347-348.
17. Barry, B.W. (1983), Dermatological Formulations, Marcel 22. G.D.Gupta, R.S. Gound.(1999). Release rate of nimesulide from
Dekker., Inc., New York, Basel, 18,96-115. different gellants. Indian J Pharm Sci. 61, 229-234.
Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 6.June 2012 3126-3130