N. A. Patel et al / International Research Journal of Pharmaceuticals (2011), Vol.

01, Issue 01

Research Paper

Formulation and Evaluation of Polyherbal Gel for

Wound Healing
Nikunjana A Patel*, Megha Patel, Rakesh P Patel

* Department of Pharmacognosy, S. K. Patel College of Pharmaceutical Education and Research, Ganpat University,
Ganpat vidyanagar, Kherva, Gujarat, India. E-mail: nikunjanapatel@rediffmail.com
Abstract
Terminalia arjuna, Centella asiatica and Curcuma longa are reported to possess wound healing, anti-inflammatory,
antioxidant and anti-bacterial activities. The carbopol 934 gel formulations containing different concentrations of extract of
the above mentioned herbs were formulated and their wound healing activity was studied on experimentally induced open
wounds in albino rats. The individual herbs were evaluated for their standard specification according to the Herbal
Pharmacopoeia of India. Extracts were obtained by established procedures. HPTLC analysis for the extracts was carried out
for identification of some known active constituents present in these herbs. Formulations containing 1% and 2% herbal
extracts were prepared and applied topically three times a day to open wounds for 24 days post-operatively and compared
with base control. The treated wounds showed a faster rate of wound contraction compared with controls. The wound
contraction studies revealed that the wound contractions increase with an increase in the herbal extract concentration.

Keywords: Terminalia arjuna; Centella asiatica; Curcuma longa; Wound healing; Polyherbal formulation

1. Introduction
In India, medicines based on herbal origin have been the
basis of treatment and cure for various diseases (Biswas et
al, 2004). Moreover, Indian folk medicine comprises
numerous prescriptions for therapeutic purposes such as
healing of wounds, inflammation, skin infections, leprosy,
diarrhea, scabies, venereal disease, ulcers, snake bite, etc
(Mukherjee et al, 2000). More than 80% of the world’s
population still depends upon traditional medicines for
various skin diseases (Babu et al, 2002). Herbal medicines
in wound management involve disinfection, debridement
and providing a moist environment to encourage the
establishment of the suitable environment for natural
healing process (Purna et al, 2000). A large number of
plants are used by folklore traditions in India for treatment
of cuts, wounds and burns.
The drugs selected for this work were Terminalia arjuna,
Centella asiatica and Curcuma longa. These three
important herbs are reported to have significant anti-
bacterial, immunomodulatory and anti- inflammatory
activities (Rahman et al, 2004; Phan et al, 2001; Liu et al,
2008) which are complementary to wound healing process.
The growing popularity of natural and herbal medications,
easy availability of raw materials, cost-effectiveness and
paucity of reported adverse reaction, prompted us
formulate a polyherbal topical preparation and assess its
wound healing ability. The combination is used in order to
enhance the wound healing activity.
2. Materials and methods
2.1. Materials
The plants were selected on the basis of their anti-
microbial activities and their medicinal uses reported in the
literatures. The herbs (Terminalia arjuna, Centella
asiatica and Curcuma longa) were purchased from plant
drug supplier Sanjivani Aushadhalay, Bhavnagar, Gujarat,
India. The marker compound curcumin, from Sigma
eldritch, Bombay, India while asaiaticoside and arjunolic
acid were purchased from Spic Pharma Ltd., Chennai,
India. All other chemicals were of analytical grade and
used without further purification.

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2.2. Monographic Analysis of Herbs Table 1: The Compositions of Gel Formulations

The individual herbs were evaluated with regard to their Formulations

Ingredients
standard specifications according to the Herbal G1# G2#
Pharmacopoeia of India. The tests carried out were loss on
C. asiatica extract (%w/w) 1 2
drying, extractive values and ash values.
C. longa extract (%w/w) 1 2
2.3. Preparation of Herbal Extract T. arjuna extract (%w/w) 1 2
Carbopol 934 (%w/w) 2 2
The dried powder of Centella asiatica leaves, Curcuma
longa rhizomes and Terminalia arjuna bark were extracted Propylene glycol 2ml 2ml
with ethanol by maceration for 72 h. The extract obtained Ethanol 5ml 5ml
was concentrated in rotary evaporator at a temperature not q. s. to
q. s. to neutralize
exceeding 60°C and stored in desiccators. Triethanolamine neutralize the
the gel base
gel base
2.4 Identification of Active Constituents in the Herbal Water q.s q.s
Extract G1#: Gel formulation with 1% extract; G2#: Gel formulation with 2%
extract
A HPTLC system equipped with linomate V sample
was shaved and prepared after washing with spirit. An area
applicator and Camag TLC scanner III, using Camag Win
of about 4 cm2 was defined with a marker on the shaven
CATS software was used to identification of some known
active constituents in all the three extracts according to back of the animals. The circular marked area was excised
with its full thickness using a surgical sterile blade and
procedures described in literature. (Singh et al, 2005;
scissors under ether anesthesia. The formulations G1 and
Paramasivam et al, 2008; Kalola et al, 2006). Mobile
G2 were applied to the wounded rats of the respective
phases for curcuminoid, asiaticoside and arjunolic acid
groups, three times a day. The wound contractions were
were chloroform: ethanol: gl. acetic acid (95:5:1),
measured as the percentage of wound reduction in the
chloroform: Gl. Acetic acid:methanol:water (60:32:12:8),
wound area for every four days. (Charde et al, 2003;
toluene: ethyl acetate: formic acid : methanol (6:3:0.1:1.0)
Sunilkumar et al, 1998). The reduction in the wound size
respectively. TLC for curcuminoid was detected at 425 nm
was calculated by the formula:
without derivatization while for asiaticoside and arjunolic
acid TLC were detected at 550 nm and 598 nm
respectively after derivatization with anisaldehyde ……..(1)
sulphuric acid reagent.
∆ A = Difference in the area of the wound in mm2 between
2.5. Preparation of Topical Formulations the initial and on a particular post-operative day

The topical gel was prepared by soaking the carbopol 934
in water for 24 h and their compositions are given in Table
1. The herbal extracts were incorporated into prepared gel
base in two different concentrations. (Sunilkumar et al,
1998; Srikanth et al, 2008).
2.6. Antimicrobial Activity
B = Area of the wound in mm2 immediately after the
wound excision.
2.8. Statistical Analysis
The results were analyzed by one-way ANOVA and a P
value less than 0.01 was considered significant.

The anti-microbial activity of each formulation was 3. Results and Discussion

assessed by cup plate method. The zone of inhibition was
measured in nutrient agar medium, employing Bacillus
subtilis, Staphylococcus aureus, Escherichia coli and
Pseudomonas aeruginosa as test organisms (Goel et al,
2009; Perumal et al, 1998).
2.7. Wound Healing Studies
An excision wound model was used for studying wound
healing activity. Wistar albino rats of either sexes
weighing 150-180 gm were randomly divided into 4
groups of six animals each. The back of the each animal
3.1. Monographic Analysis of Herbs
Table 2 shows the results of monographic analysis of the
herbs, performed according to the Indian Herbal
Pharmacopoeia and WHO guideline for quality control of
herbal raw materials. It was found that the moisture
content of all the extract were less than 10%. The
extractive values and ash vales of all the extracts were
within the pharmacopoeias limit. It indicates the good
quality of raw materials.

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 N. A. Patel et al / International Research Journal of Pharmaceuticals (2011), Vol. 01, Issue 01

Table 2: Monographic Analysis of Herbs

C. longa (%w/w) C. asiatica (%w/w) T. arjuna (%w/w)

Parameters Obtained Pharmacopoeial Obtained Pharmacopoeial Obtained Pharmacopoeial
Value Limit Value Limit Value Limit
Loss on drying 7.98±0.52 NMT 12 7.66±0.38 NMT 10 6.75±0.25 NMT 12
Alcohol Soluble Extractive 10.15±2.22 NLT 8 10.37±0.33 NLT 9.5 16.21±0.21 NLT 16
Water Soluble Extractive 13.29±0.25 NLT 9 15.38±0.39 NLT 6 20.25±0.43 NLT 17
Total Ash 7.07± 0.062 NMT 9 22.32±0.32 NMT 19 9.10±0.10 NMT 27
Acid Insoluble Ash 0.51±0.028 NMT 1 6.19±0.23 NLT 6 1.40±0.08 NMT 2

Table 3: Antimicrobial Effect Of Formulations

Zone of inhibition (mm)

Test Organisms #
G1 G2# Nitrofurazone Cream
Bacillus subtilis 13±0.2 19±0.7 22±0.9
Staphylococcus aureus 14±0.4 16±0.6 20±0.8
Escherichia coli 12±0.2 14±0.5 20±0.6
Pseudomonas aeruginosa 12±0.3 15±0.3 27±0.8

G1#: Gel formulation with 1% extract; G2#: Gel formulation with 2% extract

Table 4: The Effect of Formulations on Excision Wound Healing in Rats

Wound Contraction (%)

Time (Days)
Simple Gel (Control) Nitrofurazone Cream G1# G2#
4 11.28 ± 0.095 12.96 ± 0.066* 28.78± 0.099* 32.57 ± 0.163*
8 27.38± 0.142 49.07 ±0.078* 51.16± 0.160* 60.92± 0.047*
12 46.17 ± 0.213 63.73±0.064* 68.87±0.288* 82.69± 0.297*
16 61.4 ± .0172 86.22 ± 0.191* 87.2 ± 0.242* 96.31 ± 0.152*
20 74.82 ± 0.095 96.23 ± 0.161* 98.04 ± 0.176* 100 ± 0.080*
24 88.08 ± 0.196 100 ± 0.295* 100 ± 0.272* 100 ± 0.165*
*Significant difference between treatment groups and the control group (P ≤ 0.05)
G1#: Gel formulation with 1% extract; G2#: Gel formulation with 2% extract

3.2. Preparation of Herbal Extract matched with chromatogram of active constituents. It

indicates the presence of active constituent in extracts. The
The extract prepared had different color and odor height of peaks also indicates the presence of active
according to raw materials from which they are extracted. constituents in significant amount in extract.

3.3. Identification of Active Constituents in the Herbal

Extracts 3.4. Preparation of Topical Formulations

HPTLC analysis of the herbal extracts was performed and The gel base without herbal extract was transparent and
the chromatograms of active constituents and extracts are had good viscosity. The colour was changed to brown after
shown in Figure 1. It was found that the Rf values of the adding the extract and viscosity also slightly decreased due
active constituents in the chromatogram of extract were to addition of extract.

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Fig: 1 HPTLC Chromatograms of A) C. longa extract, B) Curcuminoids, C) C. asiatica extract, D) Asiaticoside, E) T. arjuna extract, & F)
Arjunolic acid

Fig: 2 Wound Healing Activity of Gel Formulation G2 On A) Zero Post Wounding Day, B) Fifth Post Wounding Day, C) Tenth Post
Wounding Day, D) On The Day Of Complete Healing

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 N. A. Patel et al / International Research Journal of Pharmaceuticals (2011), Vol. 01, Issue 01
Fig: 3 Effect of different gel formulations and standard on wound healing
3.5. Anti-Microbial Activity of the Formulations
The results of the antimicrobial activity of formulations are
illustrated in Table 3. The gel base without the herbal
extracts did not show any zone of inhibition while in case
of formulation G1 G2, the zone of inhibitions were found
to increase on increasing the concentration of extracts both
the formulations were shown the better activity against
Bacillus subtilis and Staphylococcus aureus than
Escherichia coli and Pseudomonas aeruginosa. Hence the
results of this study confirm that the herbs possess anti-
microbial activity and this will help to keep the wound
area sterile, thus promoting wound healing. This fact
supports a faster wound healing in the treated groups
compared with the control group.
3.6. Wound Contraction Studies
The results of wound contraction studies are shown in Fig.
2 and Fig. 3. From % wound contraction data, it is
concluded that formulation G2 produced greater wound
contraction compared with the other tested formulations
and wound contraction increases as the day passes. On the
basis of the results shown in Table 4, it is also conclude
that the gel formulations have significant (p < 0.001)
wound healing activity compared to control group.
The rate of wound contraction was found to reach a
maximum on the 16th day in the group treated with G2 (2%
extract) while in case of G1 (1% extract) it was 20th day. It
indicates as the concentration of extract increase the rate of
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wound contraction is also increase. On the basis of the
result obtained in the present investigation, it is possible to
conclude that the gel of the extracts of above plants has
significant wound healing activity. Several
phytoconstituents present in plants like asiaticoside,
curcuminoids and triterpenoids are known to promote
wound healing process due to their anti-oxidant and anti-
microbial properties. The study reveals that the better
activity of polyherbal formulation may be due to the
synergistic action of the plants constituents present in the
formulation.
4. Conclusion
From the above observations, it can be concluded that all
the monographic parameters of the selected herbs were
within in the Pharmacopoeial limit indicates the good
quality of raw materials. HPTLC studies of the extracts
showed the presence of selected active constituents in
significant amount. Their Rf values were similar to
standard Rf values. The microbiological studies indicated
that the formulations possess anti-microbial activity
against tested organisms. The wound contraction studies
revealed that the wound contraction increases on
increasing the concentration of herbal extract. The study
also reveals that the better activity of polyherbal
formulation may be due to the synergistic action of the
plants constituents present in the formulation. Thus, the
prepared topical gels possess a multifaceted approach in
healing the wound contraction.
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