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Development and Biological Evaluation of Herbal Anti-Acne Gel

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Abstract

Topical formulations (Gel) have been developed containing hydro-alcoholic extract of Ocimum sanctum, ethanolic extract of Tabernaemontana divaricata, Aloe vera concentrate gel powder and tea tree oil. In vitro antibacterial activity was performed against Propionibacterium acnes (P. acnes), a causative organism for Acne vulgar for the developed formulations using agar well diffusion method. The measured zones of inhibitions of the formulations were compared with standard antibiotic (tetracycline), standard marketed topical herbal preparation for acne and active ingredients of the formulations. Results of the investigation showed that formulation 5 has greater antibacterial activity (zones of inhibition >16 mm) than other formulations and which is comparable to that of standard marketed topical herbal preparation. When compared with hydro-alcoholic extract of Ocimum sanctum (Zone of inhibition < 9 mm), ethanolic extract of Tabernaemontana divaricata (Zone of inhibition < 8 mm) and tea tree oil (Zone of inhibition 9 mm-10 mm); all formulations had shown greater activity while the activity was found less on comparison with the standard antibiotic solution (zone of inhibition > 17 mm).
International Journal of PharmTech Research
CODEN (USA): IJPRIF ISSN : 0974-4304
Vol.2, No.3, pp 2028-2031, July-Sept 2010
Development and Biological Evaluation of Herbal
Anti-Acne Gel
H.A.Sawarkar*, S.S.Khadabadi, D.M.Mankar, I.A.Farooqui, N.S.Jagtap
Govt. College of Pharmacy,Pharmacognosy & Phytochemistry Department,
Kathora Naka, Amaravati- 444 604 (M.S.),India
*Corres. author: mrhemant1979@gmail.com
Contact No. 09890619536
Abstract: Topical formulations (Gel ) have been developed containing hydro-alcoholic extract of Ocimum sanctum,
ethanolic extract of Tabernaemontana divaricata,Aloe vera concentrate gel powder and tea tree oil. In vitro antibacterial
activity was performed against Propionibacterium acnes (P. acnes), a causative organism for Acne vulgar for the
developed formulations using agar well diffusion method. The measured zones of inhibitions of the formulations were
compared with standard antibiotic (tetracycline), standard marketed topical herbal preparation for acne and active
ingredients of the formulations. Results of the investigation showed that formulation 5 has greater antibacterial activity
(zones of inhibition >16 mm) tha n other formulations and which is comparable to that of standard marketed topical
herbal preparation. When compared with hydro-alcoholic extract of Ocimum sanctum (Zone of inhibition < 9 mm),
ethanolic extract of Tabernaemontana divaricata (Zone of inhibition < 8 mm) and tea tree oil (Zone of inhibition 9 mm-
10 mm); all formulations had shown greater activity while the activity was found less on comparison with the standard
antibiotic solution (zone of inhibition > 17 mm).
Keywords : Acne vulgaris, Propionibacterium acnes, Antibacterial assay.
Introduction
Acne vulgaris is an extremely common skin
disorder that affects areas containing the largest oil
glands, including the face, back, and trunk.
Propionibacterium acnes (P. acnes), an anaerobic
pathogen5, plays an important role in the pathogenesis
of acne. It is implicated in the development of
inflammatory acne by its capability to activate
complements and by its ability to metabolize
sebaceous triglycerides into fatty acids, which
chemotactically attract neutrophils1, 2
. Although not a
serious threat to general health, acne is one of the most
socially distressing skin conditions, especially for
adolescents, who must deal with a disfiguring disease
that erupts just when sexual maturity makes them most
sensitive about their appearance. Moreover, severe
acne can lead to permanent scarring of the skin that
carries the social distress throughout adulthood3. For
many years, antibiotics have been used to treat acne
vulgaris. However, antibiotic resistance has been
increasing in prevalence within the dermatologic
setting. The development of antibiotic resistance is
multifactorial, including the specific nature of the
relationship of bacteria to antibiotics, how the
antibacterial is used, host characteristics, and
environmental factors. To overcome the problem of
antibiotic resistance, medicinal plants have been
extensively studied as alternative treatments for
diseases1.
In the present study, 5 topical formulations (Gel)
have been developed containing hydro-alcoholic
extract of Ocimum sanctum, ethanolic extract of
Tabernaemontana divaricata,Aloe vera concentrate
gel powder and tea tree oil., which have been reported
for their antimicrobial6, 7, 8 , anti-inflammatory6, 7, 8, 9
and antioxidant activities6, 7, 8, 9. The developed
formulations were examined for antimicr obial
activities against microorganism frequently involved in
acne inflammation, P. acnes.
H.A.Sawarkar
et al
/Int.J. PharmTech Res.2010,2(3)
2029
Materials and Methods
Plants
The plants Ocimum sanctum and abernaemontana
divaricata were collected from local area of Amravati
district in the month of August, 2008. Dr. Prabha Y.
Bhogaonkar, Director, Governmevt Vidarbha Institute
of Science and Humanities had done authentication.
Aloe vera concentrate gel powder is obtained as a
free gift sample (10G) from Chaitanya Biologicals
Private limited, Malkapur, Buldhana (Maharashtra).
Tea tree oil (Dr.Urjita Jain) is purchased from retailer,
M/s, Sanjeevan medicals at Nagpur.
Preparation of Extracts
The fresh leaves of Ocimum sanctum and
Tabernaemontana divariacata were dried in shade &
powdered. The powdered plant material is defatted
with petroleum ether and then subjected to soxhlet
extraction till discolorisation for 4 hours to obtain
hydroalcoholic and ethanolic extracts resepectively.
The extracts thus obtained were filt ered, concentrated
on water bath to a thick paste & dried under vacuum.
Development of Formulation
Ingredients used for the development of
formulations are given in table no. 1.
Procedure
Aloe vera concentrate gel powder and Carbapol
934 were dissolved in sufficient quantity of water and
kept overnight. To this sodum hydroxide was added to
form a gel. Aloe vera gel and Carbapol 934 gel were
mixed together with vigorous stirring and kept in a
beaker. The beaker was kept on a water bath and the
temperature was allowed to reach above 50
0
c.To this
mixture the weighed quantities of extracts of Ocimum
sanctum and Tabernaemontana divaricata were added.
At the same time in another beaker weighed quantities
of methyl paraben and proyl paraben were added in
water and heated to dissolve. In another beaker
weighed quantities of propylene glycol and
polyethylene glycol were taken. To this weighed
quantity of Tea tree oil was added with continuous
shaking. Thus the mixtures obtained were finally
mixed to obtain a gel. Then remaining quantity of
purified water was added and PH was adjusted with
10% sodium hydroxide solution.
Microorganisms
P. acnes (MTCC 1951) was purchased from the
M.T.C.C., Institute of Microbial Technology,
Chandigarh (India). Blood agar base nutrient media
was used as a growth media.
TABLE NO. 1 DEVELOPMENT OF FORMULATION
Quantity taken per 100 g ( in grams)
Ingredients
1 2 3 4 5
Ocimum sanctum
extract
0.5 0.5 0.50 1.0 2.0
Aloe vera concentrate
gel powder
0.25 1.0 2.0 1.0 3.0
Tea tree oil 1.0 2.0 1.0 2.0 2.5
Tabernaemontana
divaricata extract
0.25 0.2 0.3 0.5 1.0
Methyl Praben 0.15 0.15 0.15 0.15 0.15
Propyl paraben 0.03 0.03 0.03 0.03 0.03
Carbapol 934 0.4 0.2 0.2 0.2 0.2
Propylene glycol 200 15.0 15.0 15.0 15.0 15.0
Polyethylene Glycol 5.0 5.0 5.0 5.0 5.0
Sodium Hydroxide
(10 %)
Quantity
sufficient
Quantity
sufficient
Quantity
sufficient
Quantity
sufficient
Quantity
sufficient
Pyrified water Quantity
sufficient
Quantity
sufficient
Quantity
sufficient
Quantity
sufficient
Quantity
sufficient
H.A.Sawarkar
et al
/Int.J. PharmTech Res.2010,2(3)
2030
Sample Preparations
Solutions of gels were prepared using 100 mg of
gel in 10ml of dimethyl sulfoxide (DMSO). Similarly,
Solution of marketed formulation was prepared.
Tetracycline (10mg/ml) was used as a positive control
and DMSO as a negative control. Solution of Tea tree
oil(10mg/ml), Aloe Vera gel(10mg/ml), hydroalcoholic
extract of Ocimum sanctum(10mg/ml and ) and
ethanolic extract of Tabernaemontana
divaricata(10mg/ml) was prepared in Dimethyl
Sufoxide (DMSO).
Antibacterial Assay
The antibacterial activity of different
formulations was determined by modified agar well
diffusion method6. In this method, nutrient agar plates
were seeded with 0.2 ml of 24 h broth culture of P
.acnes. The plates were allowed to dry for 1 h. A
sterile 8 mm borer was used to cut four wells of
equidistance in each of plates; 0.5 ml of solutions of
formulations, extracts, tea tree oil, Aloe vera gel,
marketed herbal formulation and tetracycline were
intoduced in to the wells at randomly. The plates were
incubated at 370c for 24 hours. The antibacterial
activity was evaluated by measuring the diamet er of
zones of inhibition (in mm).The experiments repeated
four times.
Results
The results of this investigation showed that all
developed formulations had inhibitory effect on the
P.acnes. Formulation 5 has higher activity than that of
other developed formulations. The activity of the
developed formulation 5 has been comparable to that
of marketed preparation. However, the activity of the
standard tetracycline was more than that of all
developed formulations, marketed herbal anti-acne
preparation, extracts, aloe vera gel and tea tree oil.
DMSO did not have significant activity. The diameter
of zones of inhibitions is given in table no.2.
Discussion
Acne vulgaris is an extremely common skin
disorder that affects virtually all individuals at least
once during life. The incidence of acne peaks at
teenage, but substantial numbers of men and women
between 20-40 years of age are also affected by the
disorder. Acne can have important negative
psychosocial consequences for the affected individual,
including diminished self-esteem, social withdrawal
due to embarrassment and depression3.
Herbal medication are considered safer than
allopathic medicines as allopathic medicines are
associated with side effects such as like contact
allergy, local irritation, scaling, photosensitivity,
itching, pruritus, redness, skin peeling, xerosis of the
skin etc3.
The present research work deals with formulation and
evaluation of herbal Anti- Acne gels. The Plant
material used for the formulations were hydro-
alcoholic extract of leaves of O.sanctum, ethanolic
extract of leaves of T. divaricata, tea tree oil and aloe
vera concentrate gel powder. Although various topical
herbal formulations for acne are available in the
market, we propose to make use of T. divaricata leaves
extract for the first time in the developed formulations.
The pla nt has been r eported in the literature as a good
antioxidant9 and anii-inflammatory agent9 . The
developed formulations were evaluated for their in
vitro antibacterial activity against P. acnes.
TABLE NO.2: RESULTS OF ANTI-BACTERIAL ACTIVITY
Zones of inhibitio ns in mm
S.No. Formulations
1234
Mean +S.D
(n = 4)
1Formulation 1 10.4 10.9 11.2 11 10.875 + 0.3403
2Formulation 2 12.3 11.7 12.0 12.6 12.15 + 0.3873
3Formulation 3 15 14.2 14.1 14.5 14.45 + 0.4041
4Formulation 4 15.3 15.4 16.0 15.7 15.6 + 0.3162
5Formulation 5 16.6 16.8 16.2 16.2 16.45 + 0.3
6Marketed herbal preparation 16.9 16.2 16.1 15.5 16.15 + 0.33
7Tetracycline(10mg/ml) 19 17.5 20.4 18.6 18.875 + 0.69
8Tea Tree Oil (10mg/ml) 9.2
9.5
9.8 9.5 9.5 + 0.24
10 O.sanctum extract (10mg/ml) 8.2 8.3 8.3 8.2 8.25 +0.057
11 T.divaricata extract (10mg/ml) < 8.0 < 8.0 < 8.0 < 8.0 < 8.0
12 A.vera gel(10mg/ml) 8.4 8.3 8.5 8.4 8.4 + 0.081
13 DMSO --- --- --- --- ---
H.A.Sawarkar
et al
/Int.J. PharmTech Res.2010,2(3)
2031
The Zones of inhibitions for the antibacterial activity
were compared with the standard tetracycline, herbal
marketed preparation for acne vulgaris, active
ingredients used in the formulation (A.vera gel, O.
sanctum hydro alcoholic extract, T.divaricata ethanolic
extract and tea tree oil). Formulation 5 has shown
comparable zones of inhibitions to that of the marketed
preparation. All the formulations have shown greater
zones of inhibitions than that of the tea tree oil. Zones
of Inhibitions for tetracyclline were found to be greater
than that of all the for mulations, marketed preparation
and tea tree oil. Tea tree oil with active constituent 1,
4-terpeniol 11, 12 may be responsible for the significant
antibacterial activity. The zones of inhibition for tea
tree oil are less than 9.8 mm, while the zones of
inhibitions for all the formulations are greater than
10.4 mm. This suggests that the other active
ingredients of the formulations containing secondary
metabolites like triterpenoids, flavonoid, tannins and
sapononis may have contributory antibacterial
activity. Their activity is probably due to their ability
to complex with extracellular and soluble proteins and
to complex with bacterial cell walls. The mechanism of
action of terp enes is not fully understood but is
speculated to involve membrane disruption by the
lipophilic compounds13.P. acnes, an anaerobic
pathogen, is implicated in the development of
infla mmatory acne. The formulations having
antibacterial agents inhibiting the P.acnes, may also
reduce the development of inflammatory acne.
References
1. Chomnawong Mullika Traidej, Surassmo Suvimol,
Nakoolkarn Veena , Gritsanapan Wandee,
Antimicrobial effects of Thai medicinal plants
against acne inducing bacteria, Journal of
Ethnopharmacology,2005, 101,330-333.
2. Chomnawong Mullika Traidej, Surassmo Suvimol,
Nakoolkarn Veena , Gritsanapan Wandee , Effect
of Garcinia mongostana on inflammation caused
by Propionibacterium acnes, Fitoterapia, 2003,
78,401-8.
3. Gopal M.G, Farahana B, Dr. Kala Suhas Kulkarni,
Effectiveness of Herbal Medication in treatment of
Acne vulgaris- A pilot Study,The Indian
Practioner; 2001, 54, 10, 723.
4. Jappe Utta, Pathological Mechanism of Acne with
special emphasis on P.Acnes and related Therapy,
Acta Derm Venerol, 2003,83,241-8.
5. Propionibacterium, Microbe Wiki, the student
edited Microbiology Resource; A microbial
Biorealm page on the genus Propionibacterium.
www.google.co.in
6. WHO Monograph on selected Medicinal Plants,
Department of essential Drugs and medicines
Policy, World Health Organization.
www.google.co.in., 172-178
7. Josias A Hamman. Composition and Application
of Aloe vera leaf gel. Molecules, 2008, 13,1599-
1616.
8. WHO Monograph on selected Medicinal Plants.
Department of essential Drugs and medicines
Policy, World Health Organization.
www.google.co.in. ,206-216.
9. Wasana Pratchayasakul, Anchalee Pongchaidecha,
Nipon Chattipakorn and Siriporn Chattipakorn,
Ethnobotany and Ethnopharmacology of
Tabernaemontana divaricata, Ind J Med research,
April 2008, 127,317-335.
10. Perez C, A. Pauli, P. Bazerque, An antibiotic assay
by agar well diffusion method, Acta Biol Med
Exp., 1990,15,113-115.
11. Sean D Cox et al., Determining the Antimicrobial
actions of tea tree oil. Molecules, 2001, 6, 87-91.
12. Carson CF, Riley TV., Susceptibility of
Propionibacterium acnes to the essential oil of
Melaleuca alternifolia. Letters in Applied
Microbiology, 1994, 19:24-25.
13. Marjorie Murphy Cowan,Plant Products as
Antimicrobial Agents, Clinical Microbiology
Reviews, 1999, 12(4), 564-582.
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