Wound Healing
Activity of Poly Herbal formulation
Syed Safiullah Ghori1* , Mohammed Gouse2, Niranjan Panda3, Shaik
Khaled1, Syed Basheeruddin1,
Mirza Danish Baig1, Mohammed Yaqub1,
Mohammed Mufaqqum1, Arfa Nazneen1,
Amtul Zehra Butul1
1Pharmacology Research Lab, Anwarul Uloom College of
Pharmacy, New Mallepally, Hyderabad 50001, Telangana, India
2Almocare
Herbal and Ayurvedic Clinic, Chirag
Ali lane, Abids, Hyderabad, Telangana,
India
3Department
of Pharmaceutics, Anwarululoom College of Pharmacy,
New Mallepally, Hyderabad 50001, Telangana,
India
*Corresponding Author E-mail: safiullahghori@gmail.com
ABSTRACT:
Aim:
To evaluate the Wound healing potential of a Poly herbal formulation.
Material and
Methods: In
the present work a poly herbal Unani formulation;
beeswax (base), sesame oil (10%), olive oil (10%), honey (20%), psoraleac (5%), borax (10%), arnica (5%) and curcuma longa (1%) were used and the wound healing potential was
determined by both excision and incision wound healing models. Albino wistar rats were used for this purpose. The wound area in
excision wound model and tensile strength in incision wound model were
determined. Histopathological studies were also
carried out to determine the phases of wound healing.
Results: From the results obtained it
was evident that the PUF has shown wound healing activity in both excision and
incision wound models. In the EWM the beneficial effect of the PUF was evident
from the day of first measurement as the progressive decrease in wound area was
observed. On the 2nd day of wounding, the wound area shrinkage in
10% formulation treated group was more when compared to the other test groups.
The wound area was decreasing constantly and predominantly in all test treated
groups as compared to the negative control group. In the IWM there was decrease
in tensile strength of the animals treated with the 10% formulation and was
comparable with the standard group. In the histopathological
examination, the phases in the wound healing process were observed and it
showed that proliferation phase was the predominant stage of action of PUF. The
best re-modeling, particularly, re-epithelization
were detected with the 10% formulation group On the other hand; faster keratinization characterized with minor intraepithelial cornification was seen in 15% group.
KEYWORDS: Poly herbal Unani
formulation (PUF), Excision wound model (EWM), Incision wound model (IWM)
INTRODUCTION:
Several
natural products which are composed of active principles, like terpene alkaloids, flavonoids and
biomolecules have been reported to promote the
process of wound healing (Chitra et al., 1995). Herbs
increase the rate of tissue healing by providing different essential
substances, required at various steps of regeneration. These herbs being
cheaper and safer than allopathic drugs may be useful in veterinary practice,
especially in India where these are found in plenty. Moreover the current
methods used to treat chronic wounds include debridement, irrigation,
antibiotics, tissue grafts and proteolytic enzymes,
which possess major drawbacks and unwanted side effects (Nayak
et al, 2009).
The present study was designed the wound
healing potential of a poly herbal formulation; beeswax (base), sesame oil
(10%), olive oil (10%), honey (20%), psoraleac (5%),
borax (10%), arnica (5%), and Curcuma longa (1%).
MATERIALS AND PREPARATION:
The formulation for assessment of wound
healing potential was procured from a local Ayurvedic
practitioner Dr. Mohammed Gouse.
Preparation of Test Formulation:
Test
formulation was taken in three different proportions (5%, 10% and 15%). Cetostearyl alcohol (2.5 g) was melted on a water bath and
liquid paraffin (25 g) was added and heated to 60oC. Test cream was
dissolved in different proportions i.e. 5% (2.5 g), 10% (5 g), 15% (7.5 g) in
freshly boiled and cooled purified water and heated to 600C. This
solution was added to the oily mixture and stirred until cold. Cream was
transferred to a suitable container and was stored for further use.
Experimental
Animals:
Adult male wistar
rats weighing 150-200 gm were used to evaluate wound healing activity. The
animals were maintained under standard laboratory conditions in polypropylene
cages under 12 hours light/dark cycle, controlled temperature (24±2˚c),
fed with commercial pellet diet and water ad-libitum
in an animal house approved by committee for the purpose and supervision on
experiments on animals (1534/PO-/a/11/CPCSEA). All the animals were
acclimatized to the laboratory environment for 10 hours before commencement of
the experiment. The experiments were carried out in accordance with the
instructions of Institutional Animal Ethical Committee, Anwarul
Uloom College of Pharmacy, New Mallepally,
Hyderabad 500001, Telangana, India.
METHODOLOGY:
Draize test:
Male albino Wistar rats weighing (150-200
gm) with n= 6 per group were used in skin irritation test. Two patches each of
two square inch area were prepared by shaving the dorsal surface of one rat.
Patch made from two layers of light gauze was dipped in solutions containing
different concentrations -0% (Control), 5%, 10% and 15% of test extracts (PUF)
prepared in PG: EtOH (7:3). The animals were immobilized in the special holder during
the 24 hrs. patch exposure. Upon removal of the patches the animals were
observed for any sign of erythema or edema for a
period of 72 hrs. The observations were repeated after 72 hrs. (Kaushal et al., 2011).
Experimental
Design:
The animals were divided into 5 groups of
6 (n=6) rats each.
Group I- Negative Control: Ointment base
Group II- Standard: Soframycin
Group III- Test I: 5% w/w poly herbal
formulation
Group IV- Test II: 10% w/w poly herbal
formulation
Group V- Test III: 15% w/w poly herbal
formulation
Excision Wound
Model:
Animals
were anesthetized prior to and during creation of the wounds by diethyl ether
inhalation chamber. The rats were inflicted with excision wounds. The negative control group of animals
was treated with ointment base, whereas the 5%, 10%, 15% extracts ointments are
used for 3 test groups, the reference drug (5% poly herbal formulation) used
for standard group and the wound area was determined on days 0, 2, 4, 8, 12 and
16 using transparent paper and a marker. Change in wound area was calculated,
giving an indication of the rate of wound contraction. The day of scar falling
without any residual raw wound were considered as period of epithelialization
(Rawat et al., 2011).
Experimental
Design: The
animals were divided into 5 groups of 6 (n=6) rats each.
Group I- Negative Control: Ointment base
Group II- Standard: Soframycin
Group III- Test I: 5% w/w poly herbal formulation
Group IV- Test II: 10% w/w poly herbal
formulation
Group V- Test III: 15% w/w poly herbal
formulation
Incision Wound
Model
All
the animals were anaesthetized by chloroform inhalation chamber and the back
hair of the rats were shaved by using an electrical clipper. 5 cm long, two
linear-paravertebral incisions were made with a
sterile surgical blade through the full thickness of the skin at a distance of
1.5 cm from the midline of each side of the vertebral column (Ehrlich et al., 1968).
The
ointments are applied once daily as per schedule i.e. the negative control group of animals
was treated with ointment base, whereas
the 5%, 10%, 15% ointments were used for
3 test groups, the reference drug (Safromycin) used
for standard group, till 9 days. All the sutures were removed on the 9th post
wound day. On Day 10 all the animals were killed under anesthesia. The Tensile
Strength (in Newton) of one linear paravertebral
incised skin was measured using Tensiometer and
average value was taken as the tensile strength and the other paravertebral incised skin was taken carefully.
Experimental
Design:
The animals were divided into 5 groups of
6 (n=6) rats each.
Group I- Negative Control: Ointment base
Group II- Standard Safromycin
Group III- Test I: 5% w/w poly herbal
formulation
Group IV- Test II: 10% w/w poly herbal
formulation
Group V- Test III: 15% w/w poly herbal
formulation
Statistical analysis:
The
values were expressed as Mean ± SEM. P
< 0.05 was considered
significant, denoted by symbol
(*). The data was analyzed by
One-way Analysis of Variance followed
by Dunnett’s multiple comparison post-hoc tests using
Graph Pad Instat version 3.10 for Windows, Graph Pad
Software, San Diego California USA.
RESULTS:
Draize test:
No
signs of allergy (allergic spots or redness of skin) were observed on rat's
skin during the skin irritancy test. There were no cases of wound infection in
all the treated groups.
Excision Wound Model
In the excision wound model the
pharmacological activity was observed and the wound healing potential of the
poly herbal formulation has shown promising effects as shown in the Table 1.
The beneficial effect of poly herbal formulation was evident from the day of
first measurement as the progressive decrease in wound area was observed. On
the 2nd day of wounding, the wound area was decreasing constantly
and predominantly in all test treated group as compared to the negative control
group. The P value was < 0.01, considered highly significant. Variation
among column means is significantly greater than expected by chance.
Table 1: Effect Of PUF on wound healing
activity using Excision wound model
|
Groups |
Day 2 |
Day 4 |
Day 6 |
Day 8 |
Period of Epithelization |
|
-ve Control |
468.4
± 7.30 |
200.7
± 14.43 |
159 ±
2.93 |
192.1
± 20.78 |
18.3
± 1.14 |
|
Standard |
496.9
± 3.15 |
268.0
± 7.50 |
221.7
± 2.52 |
136.4
± 3.94 |
12.8
± 0.94 |
|
Test
I |
297.2
± 0.48 |
267.6
± 0.50 |
191.1
± 0.32 |
213.3
± 0.25 |
18.1
± 0.94 |
|
Test
II |
296.3
± 0.96 |
254.1
± 0.34 |
174.9
± 0.48 |
165.48
± 0.36 |
20.5
± 0.76** |
|
Test
III |
253.8
± 0.57 |
294.9
± 1.69 |
151.2
± 0.36 |
132.1
± 0.17** |
18.3
± 0.88** |
**P
< 0.01
Figure 1: Effect of PUF on wound healing activity using Excision wound model
Incision Wound
Model
Poly herbal formulation has shown wound
healing activity in the incision wound model as shown in Table 2. The results
of the measurements of tensile strength of the animals treated with the 15%
test were the next to the highest value of the standard group. The P value of
the test treated groups was also P < 0.01, which is considered as
pharmacologically significant range.
Table 2:
Effect of PUF on wound healing activity using Incision wound model
|
Groups |
Tensile
Strength |
|
Negative Control |
13.88±0.2587 |
|
Standard |
28.76±0.2507** |
|
Test I |
21.05±0.3026** |
|
Test II |
27.44±0.3295** |
|
Test III |
27.50±0.2610** |
**P<0.01
DISCUSSION:
Wound healing process begins with the
restoration of a damaged tissue as closely as possible to its natural state and
wound contraction is the course of shrinkage in wounded area (Clark R.A
2001). The healing primarily depends on
the repairing ability of the tissue in addition to type and degree of damage
and general health status of the tissue.
The collagen is the main constituent of extra cellular tissue, which is
responsible for support and strength. The poly herbal cream has shown
considerably good wound healing activity in Albino Wistar rats. The poly herbal
cream has been compared to that of standard ointment and negative control
groups. The ingredients used in this poly herbal cream have been reported to
have wound healing activity and used traditionally.
The measurements of the progression of
wound healing induced by the test cream, reference drug, negative control
groups were observed in this studies. In the excision wound model, the 10% test
cream to treated groups of animals showed 56.5% contraction on the wounds at
Day 6. The same test cream demonstrated 80.6% contraction on the day 12, and
healed completely on day 14 which was close to contraction value of the
reference drug soframycin. However, the 5% concentration
of test cream presented no significant results. Further, upon increasing the
concentration to 15% of extract did not show any increase in contraction value.
In the incision wound model measurements of tensile strength (in Newton’s) showed
significant results. Tensile strength of the animals treated with the 10% test
cream demonstrated the highest value (38.9%) at day 10. Topical application of
the 10% test on the incision wound model demonstrated a remarkable improvement
in wound tensile strength compared to other groups.
ACKNOWLEDGMENT:
The authors are thankful to the management
of Anwarul Uloom College of
Pharmacy (affiliated to Jawaharlal Nehru Technological University, Hyderabad)
for providing facilities to perform animal experimentation.
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Received on 04.04.2015 Modified on 15.04.2015
Accepted on 28.04.2015 ©A&V Publications All right reserved
Res. J. Pharm.
Dosage Form. & Tech. 7(2): April-June, 2015; Page 125-128
DOI: 10.5958/0975-4377.2015.00018.X