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Pravin et al. European Journal of Pharmaceutical and Medical Research
248
EVALUATION OF WOUND HEALING POTENTIAL OF VARIOUS WOUND HEALING
CREAMS IN FRESH, DIABETIC AND BURN WOUND MODELS
Patil Pravin1*, Nagore Dheeraj2, Ambikar Digambar1, Patil Manohar1, Nipanikar Sanjay2, Kanjilal Sanjeevan2,
Kanjilal Anisha2
1Marathwada Mitra Mandal’s College of Pharmacy, S.No.4/17, Sector No.34, PCNTDA, Off Kalewadi Phata, Pimpri
Road, Thergaon (Kalewadi), Pune-411 033 (M.S.).
2Ari Healthcare Private Limited, R & D Centre, Unit No. 401, International Biotech Park, BTS 2 Building, Chrysalis
Enclave, 4th Floor, Plot No – 2A, MIDC Phase II, Hinjewadi, Pune – 411057.
Article Received on 05/11/2015 Article Revised on 26/11/2015 Article Accepted on 16/12/2015
INTRODUCTION
Wound is defined as a loss or breaking of cellular and
anatomic or functional continuity of living tissues[1] and
Wound healing involves platelet aggregation, blood
clotting, formation of fibrin, an inflammatory response to
injury, angiogenesis and re-epithelization.[2-4] This
complex cascade of event starts from the moment of
injury and continues for varying periods of time
depending on the severity of wounding. Normal wound
healing contains four highly integrated and overlapping
phases of cellular and biochemical activities including
hemostasis, inflammation, proliferation and maturation
or remodeling.[5] In spite of tremendous advances in
pharmaceutical drug industry, the availability of drugs
capable of stimulating the process of wound repair is still
limited.[6,7] Only 1–3% of the drugs listed in Western
pharmacopoeias are intended for use on wounds; on the
other hand, at least one-third of herbal remedies are
applied as wound healing agents.[7,8] Many traditional
practitioners across the world have valuable information
of many plants for treating wounds and burns. The
presence of bioactive constituents in plants has urged
researchers to screen medicinal plants with a view to
determine potential wound healing activities and isolate
chemical entities associated with wound healing.[9]
Wound healing process is promoted efficiently by the
use of traditional remedies which are mainly based on
plant sources. These remedies have been shown to affect
one or more stages of the wound healing process. In this
context, traditional medicines can provide a vast source
for the discovery of original drug leads.[10-12]
There is need for new cost-effective therapies with better
efficacy for wound healing. Medicinal plants are
important sources of new chemical substances that have
beneficial therapeutic effect.[13] Taking in to
consideration the need of today’s world, Ari Healthcare
Pvt. Ltd. has conceptualized and developed the wound
healing cream (WHC). Based on the traditional
documents on Indian medicine, various new wound
SJIF Impact Factor 2.026
Research Article
ISSN 3294-3211
EJPMR
EUROPEAN JOURNAL OF PHARMACEUTICAL
AND MEDICAL RESEARCH
www.ejpmr.com
ejpmr, 2016,3(1), 248-258
*Correspondence for Author: Patil Pravin
Marathwada Mitra Mandal’s College of Pharmacy, S.No.4/17, Sector No.34, PCNTDA, Off Kalewadi Phata, Pimpri Road, Thergaon
(Kalewadi), Pune-411 033 (M.S.).
ABSTRACT
Introduction: The present study was designed to investigate comparative efficacy of various wound healing
creams WHCs (F1 to F7) with placebo cream and marketed formulations (Mupirocin cream, Silver Sulfadiazine
Cream and Jatyadi oil) using excision, incision and burn wound models in normal and diabetic rats. Materials and
Methods: In-vivo excision, incision and burn wound models in normal and diabetic rats were used in order to
assess comparative efficacy of various wound healing creams WHCs (F1 to F7) with marketed formulations. In
case of the excision and burn wound models, wound contraction and period of epithelization were studied.
Histopathological study was conducted in order to assess fibroblast proliferation, collagen formation, angiogenesis
and epithelialization of wound. In incision wound model, tensile strength of wound was evaluated. Result: WHC
[Formulation No. 4 (F-4)] showed significantly better wound healing activity than placebo cream, other various
WHCs, Mupirocin Cream, Silver Sulfadiazine Cream and Jatyadi Oil. F-4 also showed complete epithelization and
good collagen deposition as compared to Placebo Cream, other various WHCs, Mupirocin Cream, Silver
Sulfadiazine Cream and Jatyadi Oil. Conclusion: F-4 showed statistically significant better wound healing activity
in excision, incision and burn wound models in normal and diabetic rats as compared to placebo cream, other
various WHCs, Mupirocin Cream, Silver Sulfadiazine Cream and Jatyadi Oil.
KEYWORDS: Burn wound model, Excision Model, Incision Model, Wound healing potential, diabetic wound
healing model, Silver Sulfadiazine Cream, Jatyadi Oil.
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Pravin et al. European Journal of Pharmaceutical and Medical Research
249
healing formulations were developed which contains
Jatyadi oil, Ficus religiosa, Ficus benghalensis, Centella
asiatica, Shorea robusta, Glycyrrhiza glabra,
Azadirachta indica, Pongamia glabra, and Yashad
Bhasma (Classical Ayurvedic Formulation). The study
was planned to evaluate wound healing activity of WHC
in comparison with various wound healing cream
(WHCs) (F1 to F7), Placebo Cream, marketed
formulations i.e. Mupirocin Cream, Silver Sulfadiazine
Cream and Jatyadi Oil by using excision, incision and
burn wound models in normal and diabetic rats.
MATERIALS AND METHODS
Preparation of formulations
WHCs were conceptualized and developed by Ari
Healthcare Pvt. Ltd. It contains Jatyadi Oil, Ficus
religiosa, Ficus benghalensis, Centella asiatica, Shorea
robusta, Glycyrrhiza glabra, Azadirachta indica,
Pongamia glabra and Yashad Bhasma (Classical
Ayurvedic Formulation). Seven different variants of
WHCs (F1 to F7) were prepared by using different
concentrations. Also placebo cream was prepared.
Jatyadi oil, Silver Sulfadiazine Cream and Mupirocin
cream were purchased from the market.
Experimental animals
Institutional Animal Ethics Committee approval was
taken (MMCOP/IAEC/001/2013) before initiation of this
study. Male Wistar Rats were procured and acclimatized
for 5 days. Animals were maintained at 25± 2oC and
relative humidity of 45 to 55% and under standard
environmental conditions (12 h light and 12 h dark
cycle). The food and water were provided ad libitum.
Excision wound model in normal rats
Rats were anaesthetized with ketamine 50-60 mg/kg. i.p
and xylazine 5-10 mg/kg. i.p. Animals were shaved on
right side. The area of the wound was outlined with
methylene blue using circular stencil. A full thickness
excision wound of around 300 mm2 and 2 mm in depth
was created to each animal. The entire wound was left
open. Treatments were given to respective group as per
study design (Table No. I). Treatments were given
topically twice a day. Wound area was measured and
recorded on day 1, 3, 7, 11 and 15. Wound area was
traced using transparent graph paper and permanent
marker. Assessment of the wound area was done.
Photograph of day 1, 9, 13, were taken. Day of complete
epithelization was recorded for each animal. Skin sample
of animals were collected and histopathological studies
were performed to observe Epithelization,
neoangiogenesis, neutrophil infiltration, collagen
deposition etc. as wound healing parameters.
Incision wound model in normal rats
Rats were anaesthetized with ketamine 50-60 mg/kg i.p.
and Xylazine 5-10 mg/kg i.p. Animals were shaved on
right side and placed in their respective group (Table No.
1). Paravertebral straight incision of 6 cm length was
made through the entire thickness of the skin. The wound
was closed by means of interrupted sutures placed at
approximately 1 cm apart. The treatment was done
topically twice daily. The wound breaking strength was
estimated on 10th day. The wound breaking strength was
evaluated by constant water flow technique. Each
anesthetized animal was secured to the operation table
and a line was drawn on either side of the wound 3 mm
away from the line. Two allice forceps were firmly
applied to the line facing each other. One of the forceps
was fixed while the other was connected to a freely
suspended light weight polypropylene graduated
container through a string run over to a pulley. Water
was allowed to flow from the reservoir slowly and
steadily into the container. A gradual increase in weight
was transmitted to the wound site for pulling apart the
wound edges. As and when the wound edges just opened
up, the water flow was arrested and the volume of water
collected in the container was noted. The average reading
of the group was taken as an individual value of breaking
strength.
Table No.1: Study design: Excision and incision wound models.
Gr. No.
Group Description
No. of Animals (N)
I
Normal Control (Base of formulation)
6
II
Formulation No.1 (F-1)
6
III
Formulation No.2 (F-2)
6
IV
Formulation No.3 (F-3)
6
V
Formulation No.4 (F-4)
6
VI
Formulation No.5 (F-5)
6
VII
Formulation No.6 (F-6)
6
VIII
Formulation No.7 (F-7)
6
IX
Positive control (Mupirocin cream)
6
X
Jatyadi Oil
6
Induction of diabetes
Animals were weighed and their fasting blood glucose
level was determined before induction of diabetes. The
animals were then injected with single dose of freshly
prepared streptozotocin 50 mg/kg in cold 0.1 M citrate
buffer pH 4.5 in tail vein to induce diabetes. Control
animals were injected with 0.1 M citrate buffer. Fasting
blood glucose was measured three days later to confirm
diabetes status of the animals. Blood was collected by
retro-orbital puncture method. After induction of
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Pravin et al. European Journal of Pharmaceutical and Medical Research
250
diabetes the same procedure that of normal rat for
conducting different models was followed.
Burn wound model in normal rats
Rats were anaesthetized with ketamine 50-60 mg/kg i.p.
and Xylazine 5-10 mg/kg i.p. Animals were shaved on
the neck region and placed in their respective groups
(Table No. 2). Partial thickness burn wounds were
inflicted by pouring hot molten wax (2 gm) at 80ºC. The
wax was poured with the spoon and spreading is
restricted using the stencil. The wax was allowed to
remain on the skin till it got solidified. The treatment was
given topically two times a day. Wound area was
measured on day 1, and every alternate day till the day of
epithelization. Measurement was done using
transparency sheet and permanent marker. Recordings of
the transparency were traced on the graph paper and
marked area on the graph paper were measured and
recorded. The day of Escher falling without any residual
raw wound was considered as the day of epithelization.
Table No. 2: Study design: Burn wound model in normal animals (rats).
Gr. No.
Group Description
No. of Animals (N)
I
Normal Control (Base of formulation)
6
II
Formulation No.1 (F-1)
6
III
Formulation No.2 (F-2)
6
IV
Formulation No.3 (F-3)
6
V
Formulation No.4 (F-4)
6
VI
Formulation No.5 (F-5)
6
VII
Formulation No.6 (F-6)
6
VIII
Formulation No.7 (F-7)
6
IX
Positive control (Silver sulfadiazine cream)
6
X
Jatyadi oil
6
Burn wound model in diabetic rats
Induction of diabetes mellitus in rats was done as
described earlier. Rats were anaesthetized with ketamine
50-60 mg/kg i.p. and Xylazine 5-10 mg/kg i.p. Animals
were shaved on the neck region and placed in their
respective group (Table No.3). Partial thickness burn
wounds were inflicted by pouring hot molten wax (2 gm)
at 80ºC. The wax was poured with the spoon and
spreading was restricted using the stencil. The wax was
allowed to remain on the skin till it got solidified. The
treatment was given topically two times as day. Wound
area was measured on day 1 and every alternate day till
the day of epithelization. Measurement was done using
transparency sheet and permanent marker. Recordings of
the transparency were traced on the graph paper and
marked area on the graph paper were measured and
recorded. The day of eschar falling without any residual
raw wound was considered as the day of epithelization.
Table No. 3: Study design: Burn wound model in diabetic rats.
Gr. No.
Group Description
No. of Animals (N)
I
Diabetic Negative Control (Formulation Base)
6
II
Diabetic Control (Formulation Base)
6
III
Formulation No.1 (F-1)
6
IV
Formulation No.2 (F-2)
6
V
Formulation No.3 (F-3)
6
VI
Formulation No.4 (F-4)
6
VII
Formulation No.5 (F-5)
6
VIII
Formulation No.6 (F-6)
6
IX
Formulation No.7 (F-7)
6
X
Positive control (Silver sulfadiazine cream)
6
XI
Jatyadi Oil
6
Excision wound model in diabetic rats
Induction of diabetes mellitus in rats was done as
described earlier. Rats were anaesthetized with ketamine
50-60 mg/kg. i.p and xylazine5-10 mg/kg. i.p. Animals
were shaved on a right side and placed in their respective
group (Table No. 4). The area of the wound was outlined
with methylene blue using circular stencil. A full
thickness excision wound of around 300mm2and 2 mm
in depth was created to each animal. The entire wound
was left open. Treatments were given to respective group
as per study design. Treatments were given topically
twice a day. Wound area was measured on day-1, 3, 7,
11 and 15 and recorded. Wound Area was traced using
transparency and permanent marker. Wound area was
measured using graph paper. Photograph of day 1, 9
and13 were taken. Day of complete epithelization was
recorded for each animal. Skin sample of animals were
collected and histopathological studies were performed
considering Epithelization, neoangiogenesis, neutrophil
infiltration, collagen deposition etc. as wound healing
parameters.
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Pravin et al. European Journal of Pharmaceutical and Medical Research
251
Table No. 4: Study design: Excision and incision wound models in diabetic rats.
Gr. No.
Group Description
No. of Animals (N)
I
Diabetic Negative Control (Formulation Base)
6
II
Diabetic Control (Formulation Base)
6
III
Formulation No.1 (F-1)
6
IV
Formulation No.2 (F-2)
6
V
Formulation No.3 (F-3)
6
VI
Formulation No.4 (F-4)
6
VII
Formulation No.5 (F-5)
6
VIII
Formulation No.6 (F-6)
6
IX
Formulation No.7 (F-7)
6
X
Positive control (Mupirocin Cream)
6
XI
Jatyadi Oil
6
Incision wound model in diabetic rats
Induction of diabetes mellitus in rats was done as
described earlier. Rats were anaesthetized with ketamine
50-60 mg/kg i.p. and Xylazine 5-10 mg/kg i.p. Animals
were shaved on right side placed in their respective
group (Table No 4). Paravertebral straight incision of 6
cm length was made through the entire thickness of the
skin. The wound was closed by means of interrupted
sutures placed at approximately 1 cm apart. The
treatment was done topically twice daily. The wound
breaking strength was estimated on the 10th day. The
wound breaking strength was evaluated by constant
water flow technique. Each anesthetized animal was
secured to the operation table and a line was drawn on
either side of the wound 3 mm away from the line. Two
allice forceps were firmly applied to the line facing each
other. One of the forceps was fixed while the other was
connected to a freely suspended light weight
polypropylene graduated container through a string run
over to a pulley. Water was allowed to flow from the
reservoir slowly and steadily into the container. A
gradual increase in weight transmitted to the wound site
pulling apart the wound edges. As and when the wound
was just opened up, the water flow was arrested and the
volume of water collected in the container was noted.
The average reading of the group was taken as an
individual value of breaking strength.
RESULTS
Burn model in normal rats
Among all groups, group V Formulation No.4 (F-4) took
less days (13) for complete wound healing in burn model
in normal rats. Formulation No. 4 (F-4) was statistically
significant effective wound healing agent as compared to
placebo cream. Also Silver sulfadiazine cream and
Jatyadi Oil was statistically significant wound healing
agents as compared to placebo group. When compared
between groups, Formulation No. 4 (F-4) was
statistically significant wound healing agent than Jatyadi
Oil and Silver sulfadiazine cream in burn model in
normal rats. The details are presented in Table No. 5 and
figure no 1.
Table No. 5: Effect of topical application of various wound healing formulations, placebo cream, Silver
Sulfadiazine cream (Positive control) and Jatyadi oil on days required for epithelization in burn model.
**P<0.01 significant as compared to placebo using one-way ANOVA followed by Dunnet test.
Gr. No.
Treatment
Days of Epithelization (Mean ± SEM)
I
Normal Control (Base of formulation
23 ± 0.70
II
Formulation No.1 (F-1)
21 ± 0.31
III
Formulation No.2 (F-2)
19 ± 0.61
IV
Formulation No.3 (F-3)
18 ± 0.80
V
Formulation No.4 (F-4)
13 ± 0.21**#
VI
Formulation No.5 (F-5)
21 ± 0.56
VII
Formulation No.6 (F-6)
16 ± 1.32*
VIII
Formulation No.7 (F-7)
17 ± 0.79
IX
Positive control (Silver sulfadiazine cream)
16 ± 0.33*
X
Jatyadi Oil Formulation
19 ± 0.81
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Pravin et al. European Journal of Pharmaceutical and Medical Research
252
Figure. 1: Effect of topical application of various wound healing formulations, placebo cream, silver sulfadiazine
cream (positive control) and Jatyadi oil on days required for epithelization in burn model.
Excision Wound Model in Normal Rats
Among all the formulation studied, Formulation No. 4
(F-4) required less days (15) for complete wound
healing. Formulation no. 4 (F-4) was statistically
significant wound healing agent as compared to placebo.
When compared between groups, formulation no 4(F-4)
was statistically significantly better wound healing agent
than Jatyadi Oil. Also Formulation 4 (F-4) was better
than Mupirocin cream in terms of wound healing
activity. The details are presented in Table No.6.
Placebo
Day 1
Placebo
Day 09
Placebo
Day 13
Formulation No. 4
Day 1
Formulation No. 4
Day 09
Formulation No. 4
Day 13
Silver Sulfadiazine
Day 1
Silver Sulfadiazine
Day 09
Silver Sulfadiazine
Day 13
Jatyadi Oil
Day 1
Jatyadi Oil
Day 09
Jatyadi Oil
Day 13
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Table No. 6: Effect of topical application of various wound healing formulations, placebo cream, Mupirocin
cream (positive control) and Jatyadi oil on days required for epithelization in excision model.
*P<0.05 **P<0.01 ***P<0.001significant as compared to placebo using one-way ANOVA followed by Bonferroni
post test. # P<0.05 Significant as compared to Formulation-4 using one-way ANOVA followed by Bonferroni post test.
Incision Wound Model in Normal Rats
The wound breaking strength of formulation no. 4 (F-4)
was higher (388.00 ± 46.38) as compared to any other
formulation used in this study. When compared between
groups, formulation no. 4.
(F-4) was having statistically more significant wound
breaking strength than Mupirocin cream. The details are
presented in Table No. 7.
Table No. 7: Effect of topical application of various wound healing formulations, placebo cream, Mupirocin
cream (positive control) and Jatyadi oil on wound breaking strength.
Gr. No.
Treatment
Wound breaking Strength (Mean ± SEM)
I
Normal Control (Base of formulation)
309.17 ± 19.85
II
Formulation No.1 (F-1)
256.67 ± 12.02
III
Formulation No.2 (F-2)
253.33 ± 8.43
IV
Formulation No.3 (F-3)
296.67 ± 18.56
V
Formulation No.4 (F-4)
388.00 ± 46.38**
VI
Formulation No.5 (F-5)
278.33 ± 46.76
VII
Formulation No.6 (F-6)
310.00 ± 30.22
VIII
Formulation No.7 (F-7)
250.00 ± 19.66
IX
Positive control (Mupirocin cream)
248.33 ± 19.56
X
Jatyadi Oil Formulation
313.33 ± 19.78
**P<0.01 significant as compared to Mupirocin using one-way ANOVA followed by Bonferroni post test.
Histopathology
In histological analysis of the wound, the wounds of
rats from control group had not been fully restored and
inflammatory cells in scar tissue were observed. Re-
epithelialization of the wounds showed incomplete.
When compared with that in control, the progress from
granulation formation to re-epithelialization was
accelerated in F-2, F-3, F-4, F-6, F-7, Mupirocin cream
and Jatyadi Oil groups.
Among all the formulations tested, Formulation No. 4
(F-4) was better in terms of wound healing parameters
viz. complete epithelization, more blood vessels and
more collagen fibers.
Microscopic observations of the wounds from animals
treated with Formulation No. 4 (F-4) showed complete
re-epithelialization and a normal epidermis covered the
wound area, collagen fibers were thick and were
denser as compared to that of Placebo Cream,
Mupirocin Cream and Jatyadi oil. The cutaneous
annexes, such as sebaceous glands and hair follicles in
the centre of the scar tissue were also well formed.
This indicated that Formulation No. 4 (F-4) stimulates
and accelerates the activities towards faster wound
healing and could play a role in the early stage of the
wound healing process as compared to Placebo Cream,
Mupirocin Cream and Jatyadi oil. Results are shown in
the figure no 2-5.
Gr. No.
Treatment
Days of Epithelization (Mean ± SEM)
I
Normal Control (Base of formulation)
27 ± 1
II
Formulation No.1 (F-1)
26 ± 1
III
Formulation No.2 (F-2)
18 ± 1**
IV
Formulation No.3 (F-3)
17 ± 1***
V
Formulation No.4 (F-4)
15 ± 0***
VI
Formulation No.5 (F-5)
25 ± 2
VII
Formulation No.6 (F-6)
19 ± 2 *
VIII
Formulation No.7 (F-7)
16 ± 1***
IX
Positive control (Mupirocin cream)
18 ± 2**
X
Jatyadi Oil
23 ± 2#
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254
Figure 2: Histopathological study of placebo cream
treated wound in rat (incomplete epithelialization
and presence of fibroblasts. H & E 20X).
Figure 3: Histopathological study of formulation-4
(F-4) treated wound in rat (skin –complete
epithelialization and presence of thick collagen
fibers. H&E 20X).
Figure 4: Histopathological study of Mupirocin
cream treated wound in rat (presence of
inflammatory cells, new blood vessels and
hemorrhages. H & E 20X).
Figure 5: Histopathological study of Jatyadi oil
treated wound in rat (skin –incomplete
epithelialization, presence of few inflammatory
cells. H & E 20X).
Burn model in diabetic rats
Among all groups, group VI (Formulation No. 4) took
less days (15) for complete wound healing in burn
model in diabetic rats. Formulation No. 4 was
statistically significant effective wound healing agent
as compared to placebo cream. Also, Silver
sulfadiazine was statistically significant wound healing
agent as compared to placebo group. When compared
between groups, Formulation No. 4 (Amarantha
Wound Healing Cream) was statistically significantly
better wound healing agent than Jatyadi Oil and Silver
sulfadiazine in burn model in diabetic rats. The details
are presented in Table No. 8 and figure no 6.
Table No. 8: Effect of topical application of various
Wound Healing formulations, Placebo Cream
Silver Sulfadiazine cream (Positive control) and
Jatyadi oil on days required for epithelization in
Burn Model.
**P<0.01 significant as compared to placebo using
one-way ANOVA followed by Dunnet post test.
#P<0.01 significant as compared to Silver Sulfadiazine
and Jatyadi oil using one-way ANOVA followed by
Bonferroni Test.
Gr. No.
Treatment
Days of Epithelization
(Mean ± SEM)
II
Diabetic Control
(Formulation Base)
27 ± 0.30
III
Formulation No.1 (F-1)
22 ± 0.15
IV
Formulation No.2 (F-2)
19 ± 0.25
V
Formulation No.3 (F-3)
17 ± 0.80
VI
Formulation No.4 (F-4)
15 ± 0.31**#
VII
Formulation No.5 (F-5)
22 ± 0.56
VIII
Formulation No.6 (F-6)
16 ± 1.43*
IX
Formulation No.7 (F-7)
17 ± 0.79
X
Positive control (Silver
sulfadiazine cream)
18 ± 0.33*
XI
Jatyadi Oil Formulation
21 ± 0.73
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255
Silver Sulfadiazine Day 1
Silver Sulfadiazine Day 09
Silver Sulfadiazine Day 13
Jatyadi Oil Day 1
Jatyadi Oil Day 09
Jatyadi Oil Day 13
Figure 6: Effect of topical application of various wound healing formulations, placebo cream silver sulfadiazine
cream (positive control) and Jatyadi oil on days required for epithelization in burn model.
Excision wound model in diabetic rats
Among all the formulation studied, formulation no. 4
required less days (15) for complete wound healing.
Formulation No. 4 was statistically significant wound
healing agent as compared to placebo cream. When
compared between groups, Formulation No 4 was
statistically significantly better wound healing agent than
Jatyadi oil. Also Formulation No 4 was better than
Mupirocin cream in terms of wound healing activity. The
details are presented in Table No. 9.
Table No. 9: Effect of topical application of various wound healing formulations, Placebo Cream, Mupirocin
cream (Positive control) and Jatyadi oil on days required for epithelization in excision model.
Gr. No.
Treatment
Days of Epithelization
(Mean ± SEM)
II
Diabetic Control (Formulation Base)
26 ± 1
III
Formulation No.1 (F-1)
23 ± 1
Placebo Day 1
Placebo Day 09
Placebo Day 13
Formulation No. 4- Day 1
Formulation No. 4- Day 9
Formulation No. 4- Day 13
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Pravin et al. European Journal of Pharmaceutical and Medical Research
256
IV
Formulation No.2 (F-2)
17 ± 1**
V
Formulation No.3 (F-3)
18 ± 1**
VI
Formulation No.4 (F-4)
15 ± 1**#
VII
Formulation No.5 (F-5)
23 ± 2
VIII
Formulation No.6 (F-6)
18 ± 2 **
IX
Formulation No.7 (F-7)
17 ± 1**
X
Positive control (Silver sulfadiazine cream)
19 ± 2**
XI
Jatyadi Oil Formulation
24 ± 2
**P<0.01 significant as compared to placebo using one-way ANOVA followed by Dunnet test.
# P<0.01 significant as compared to Jatyadi oil using one-way ANOVA followed by Bonferroni Test.
Incision wound model in diabetic rats
The wound breaking strength of Formulation No. 4 was
higher (395.00 ± 16.18) as compared to any other
formulation used in this study. When compared between
groups, Formulation No. 4 was having statistically more
significant wound breaking strength than Placebo cream,
Mupirocin cream and Jatyadi Oil. The details are
presented in Table No. 10.
Table No. 10: Effect of topical application of various Wound Healing formulations, Placebo Cream, Mupirocin
cream (Positive control) and Jatyadi oil on wound breaking strength.
Gr. No.
Treatment
Wound breaking Strength
(Mean ± SEM)
II
Diabetic Control (Formulation Base)
289.17 ± 14.35
III
Formulation No.1 (F-1)
266.27 ± 11.32
IV
Formulation No.2 (F-2)
273.11 ± 5.93
V
Formulation No.3 (F-3)
256.76 ± 16.51
VI
Formulation No.4 (F-4)
395.00 ± 16.18**
VII
Formulation No.5 (F-5)
259.43 ± 32.76
VIII
Formulation No.6 (F-6)
287.00 ± 23.23
IX
Formulation No.7 (F-7)
263.00 ± 12.66
X
Positive control (Mupirocin Cream)
259.23 ± 18.26
XI
Jatyadi Oil
309.23 ± 17.87
**P<0.01 significant as compared to placebo using one-way ANOVA followed by Dunnet test.
Histopathology
In histological analysis of the wound of rats from control
group it was observed that wound was not fully restored
and inflammatory cells in scar tissue were observed. Re-
epithelialization of the wounds showed incomplete.
Among all the formulations tested, Formulation No. 4
(Amarantha Wound Healing Cream) was better in terms
of wound healing activity. As compared to positive
control (Mupirocin cream), Formulation No. 4 showed
complete epithelization, more blood vessels and more
collagen fibers. Results are shown in the figure. 7-10.
Figure No.7: Histopathological study of placebo
cream treated wound in rat (incomplete
epithelialization and presence of fibroblasts. H & E
20X).
Figure No. 8: Histopathological study of formulation-
4 treated wound in rat (skin –complete
epithelialization and presence of thick collagen fibres.
H & E 20X).
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Pravin et al. European Journal of Pharmaceutical and Medical Research
257
Figure No. 9: Histopathological study of Mupirocin
cream treated wound in rat (presence of
inflammatory cells new blood vessels and
hemorrahages and incomplete epithelization. H & E
20x).
Figure. 10: Histopathological study of Jatyadi oil
treated wound in rat (skin –incomplete
epithelialization, presence of thick collagen fibers. H
& E 20X).
Microscopic observations of the wounds from animals
treated with Formulation No. 4 showed complete re-
epithelialization and a normal epidermis covering the
wound area, collagen fibers were thick and were denser
as compared with that of placebo control and Jatyadi oil.
The cutaneous annexes, such as sebaceous glands and
hair follicles in the center of the scar tissue were also
well formed.
DISCUSSION
In the present study it was observed that formulation no.
4 was statistically significant wound healing agent as
compared to Placebo Cream, Mupirocin Cream and
Jatyadi oil in Excision Wound Model. The
histopathological observations of wounds confirmed that
formulation no. 4 is having better wound healing activity
than Placebo Cream, Mupirocin Cream and Jatyadi Oil
as complete epithelization, thick and denser collagen
fibers, well-formed cutaneous annexes, such as
sebaceous glands and hair follicles were observed in
samples of skins rats. The wound breaking strength of
formulation no 4 was also statistically significantly better
than Mupirocin cream. The wound healing action of
Formulation No.4 was superior to various wound healing
creams, Placebo cream, Mupirocin Cream and Jatyadi
oil.
The results of the study showed that except formulation
no.1, 5 and Jatyadi Oil, all other formulations showed
statistically significant decrease in days required for
complete re-epithelization as compared to placebo in
excision model in diabetic rat. This activity in
formulation no. 2, 3, 4 and 7 is even better than the
Jatyadi oil formulation. Moreover, formulation No. 4
showed statistically significant increase tensile strength
in incision wound model as compared to Mupirocin
Cream and Jatyadi Oil. Formulation no. 4 also showed
complete epithelization and good collagen deposition in
the histopathological studies as compared to placebo
cream, Mupirocin Cream and Jatyadi Oil.
It was also observed that Formulation No. 4 (Amarantha
Wound Healing Cream) is statistically significant wound
healing agent than Jatyadi Oil and Silver sulfadiazine in
burn model in normal and diabetic rats.
The better efficacy of Formulation No. 4 over other
formulations may probably be due to the
phytoconstituents present in the plant or could be a
function of either the individual or the additive effects of
the phytoconstituents present in formulation no. 4.
It has been observed in scientific studies that most of the
ingredients of the Formulation No. 4 (Amarantha Wound
Healing Cream) possess anti-bacterial and anti-fungal
properties, which help in keeping the wound sterile
during wound healing process. Also it has been
established in the scientific studies conducted on
individual ingredient of the Formulation No. 4
(Amarantha Wound Healing Cream) that almost all the
ingredients have wound healing property, which help to
accelerate the healing process. Jatyadi oil is one of the
important ingredients of formulation no. 4. Jatyadi Oil
has been used to treat Dagdha (burns & scalds). Also
Mandukaparni (Centella asiatica) is useful in partial
thickness burns. Mandukaparni has shown to have burn
wound healing activity in mice. It has ability to heal
wounds by increasing synthesis of collagen and
intracellular fibronectin contents. It also possesses
wound healing and anti-microbial properties.
Yashtimadhuka (Glycyrrhiza glabra) is useful in burn
wound infection caused due to variety of organism such
as Pseudomonas aeruginosa, Staph. aureus, etc.
Ashvattha (Ficus religiosa) and Nimba (Azadirachta
indica) are used to treat burns. Yashad Bhasma
(Ayurvedic classical formulation) one of the important
ingredients of formulation no. 4, improves the binding
power of the cells of skin, soft tissues, improves cell
migration and cell regeneration and hastens wound
healing process.
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Pravin et al. European Journal of Pharmaceutical and Medical Research
258
Thus synergistic activity of ingredients of formulation no
4 would have exerted the superior results in healing of
excision, incision and burn wounds in normal and
diabetic rats.
CONCLUSION
These finding concludes that Formulation No.4 is
statistically significant better wound healing agent as
compared to various wound healing creams, Placebo
Cream, Silver sulfadiazine and Jatyadi oil in burn wound
models in normal rat and diabetic rat. Also Formulation
No.4 is statistically significant better wound healing
agent as compared to various wound healing creams (F-1
to F-3, F5, F6, F7) Placebo Cream, Mupirocin Cream
and Jatyadi Oil in excision and incision would models in
normal rat and diabetic rat. Thus formulation no. 4
(Amarantha Wound Healing Cream) is best alternative
solution in treating cut, wounds and burns.
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