N. A. Patel et al / Intl. R. J. of Pharmaceuticals (2011), Vol. 01, Issue 01, pp.

15-20

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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 worlds 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 (Phan et al, 2001; Rahman et al, 2004 and
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 antimicrobial 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

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N. A. Patel et al / Intl. R. J. of Pharmaceuticals (2011), Vol. 01, Issue 01, pp. 15-20

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.
2.2. Monographic Analysis of Herbs
The individual herbs were evaluated with regard to their
standard specifications according to the Herbal
Pharmacopoeia of India (1996). The tests carried out were
loss on drying, extractive values and ash values.
2.3. Preparation of Herbal Extract
The dried powder of Centella asiatica leaves, Curcuma
longa rhizomes and Terminalia arjuna bark were
extracted with ethanol by maceration for 72 hours.
The extract obtained was concentrated in rotary evaporator
at a temperature not exceeding 60o C and stored in
desiccators.
2.4. Identification of Active Constituents in the Herbal
Extract
A HPTLC system equipped with linomate V sample
applicator and Camag TLC scanner III, using Camag Win
CATS software was used to identification of some known
active constituents in all the three extracts according to
procedures described in literature. (Singh et al, 2005;
Kalola & Rajani, 2006 and Paramasivam et al, 2008).
Mobile phases for curcuminoid, asiaticoside and arjunolic
acid were chloroform: ethanol: gl. acetic acid (95:5:1),
chloroform: Gl. Acetic acid:methanol:water (60:32:12:8),
toluene: ethyl acetate: formic acid : methanol (6:3:0.1:1.0)
respectively. TLC for curcuminoid was detected at 425 nm
without derivatization while for asiaticoside and arjunolic
acid TLC were detected at 550 nm and 598 nm
respectively after derivatization with anisaldehyde
sulphuric acid reagent.

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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
was shaved and prepared after washing with spirit. An area
of about 4 cm2 was defined with a marker on the shaven
back of the animals. The circular marked area was excised
with its full thickness using a surgical sterile blade and
scissors under ether anesthesia. The formulations G1 and
G2 were applied to the wounded rats of the respective
groups, three times a day. The wound contractions were
measured as the percentage of wound reduction in the
wound area for every four days. (Sunilkumar et al, 1998
and Charde et al, 2003). The reduction in the wound size
was calculated by the formula:

.(1)
A = Difference in the area of the wound in mm2 between the initial
and on a particular post-operative day
B = Area of the wound in mm2 immediately after the wound excision.
Table 1. The Compositions of Gel Formulations
Formulations

Ingredients

G1

G2#

C. asiatica extract (%w/w)

C. longa extract (%w/w)

T. arjuna extract (%w/w)

Carbopol 934 (%w/w)

2ml

2ml

Propylene glycol

5ml

5ml

q. s. to
neutralize the
gel base

q. s. to
neutralize the
gel base

q.s

q.s

Ethanol
Triethanolamine

2.5. Preparation of Topical Formulations

Water

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 and Srikanth et al, 2008).

G1#: Gel formulation with 1% extract

G2#: Gel formulation with 2% extract

2.6. Antimicrobial Activity

The anti-microbial activity of each formulation was
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 (Perumal et al,
1998 and Goel & Morris, 2007).

2.8. Statistical Analysis

The results were analyzed by one-way ANOVA and a P
value less than 0.01 was considered significant.

3. Results and Discussion

3.1. Monographic Analysis of Herbs
Table 2 shows the results of monographic analysis of the
herbs, performed according to the Indian Herbal

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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.
3.2. Preparation of Herbal Extract
The extract prepared had different color and odor
according to raw materials from which they are extracted.

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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-

Table 2. Monographic Analysis of Herbs

C. longa (%w/w)

Parameters

Obtained
Value

Pharmacopoeial
Limit

C. asiatica (%w/w)
Obtained
Value

Pharmacopoeial
Limit

T. arjuna (%w/w)
Obtained
Value

Pharmacopoeial
Limit

7.980.52

NMT 12

7.660.38

NMT 10

6.750.25

NMT 12

10.152.22

NLT 8

10.370.33

NLT 9.5

16.210.21

NLT 16

13.290.25

NLT 9

15.380.39

NLT 6

20.250.43

NLT 17

Total Ash

7.07 0.062

NMT 9

22.320.32

NMT 19

9.100.10

NMT 27

Acid Insoluble Ash

0.510.028

NMT 1

6.190.23

NLT 6

1.400.08

NMT 2

Loss on drying
Alcohol Soluble
Extractive
Water Soluble
Extractive

3.3. Identification of Active Constituents in the Herbal

Extracts
HPTLC analysis of the herbal extracts was performed
and the chromatograms of active constituents and extracts
are shown in Figure 1. It was found that the Rf values of
the active constituents in the chromatogram of
extract were match with chromatogram of active
constituents. It indicates the presence of active
constituent in extracts. The heights of peaks also indicate
the presence of active constituents in significant amount in
extract.
3.4. Preparation of Topical Formulations
The gel base without herbal extract was transparent and
had good viscosity. The color was changed to brown after
adding the extract and viscosity also slightly decreased due
to addition of extract.

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
Figure 2 and Figure 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 wound contraction is also increase.

Table 3. Antimicrobial Effect of Formulations

Test Organisms

Zone of inhibition (mm)

#

G1

G2#

Nitrofurazone Cream

Bacillus subtilis

130.2

190.7

220.9

Staphylococcus aureus

140.4

160.6

200.8

Escherichia coli

120.2

140.5

200.6

Pseudomonas aeruginosa

120.3

150.3

270.8

G1#: Gel formulation with 1% extract

G2#: Gel formulation with 2% extract

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

Wound Contraction (%)

Time (Days)
Simple Gel (Control)

Nitrofurazone Cream

G1#

G2#

11.28 0.095

12.96 0.066*

28.78 0.099*

32.57 0.163*

27.38 0.142

49.07 0.078*

51.16 0.160*

60.92 0.047*

12

46.17 0.213

63.730.064*

68.870.288*

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

Figure 1. HPTLC Chromatograms of A) C. longa Extract, B) Curcuminoids, C) C. asiatica Extract, D) Asiaticoside, E) T. arjuna Extract, & F)
Arjunolic Acid

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Figure 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

Figure 3. Effect of Different Gel Formulations and Standard on Wound Healing

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.

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N. A. Patel et al / Intl. R. J. of Pharmaceuticals (2011), Vol. 01, Issue 01, pp. 15-20

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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Paramasivam, M., Wasim, A., M., Poi, R., and Banerjee,

H. (2008) Bandyopadhyay A. Occurrence of curcuminoids
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