FINAL REPORT

Evaluation of Wound Healing Potential of

an Ayurvedic Formulation (Amrit TAIL
(Oil)) in Wistar Rats

A report submitted to Jain Pharmecy

By
Dr. A. Hannah Rachel Vasanthi
Associate Professor
Department of Biotechnology

Department of Biotechnology
School of Life Sciences
Pondicherry University
Puducherry - 605014

1
Evaluation of Wound Healing Potential of an Ayurvedic Formulation (Amrit Tail (Oil))
in Wistar Rats

Introduction
There are unavoidable events of life which arise due to physical, chemical or
biological agents occurring mostly due to injury such as a blow, a cut, chemicals, heat/ cold,
friction/shear force, pressure or as a result of disease, such as leg ulcers or carcinomas
(Hamdorf and Hall 2000). Further, injury refers to any kind of damage to the body caused by
accidents (Medline Plus 2016) which may be minor or fatal or may even lead to death. Minor
injuries can be cured with first aid, whereas severe injuries requires immediate medical
attention which can be life threatening, if left untreated. A wound, by definition is the
breakdown in the protective function of the skin or disruption of tissue integrity resulting in
the loss of continuity of epithelium, with or without loss of underlying connective tissue
(Leaper and Harding 1998).
Wound healing is a complex and dynamic biochemical process by which skin or body
tissues repair itself after trauma in an orchestrated sequence of events. The process of wound
healing is not only complex but also fragile as it is prone to failure which leads to the
formation of non-healing chronic wounds. Although the process of healing is continuous, it
can be arbitrarily divided into different phases in order to aid understanding of the
physiological processes that are taking place in the wound and healthy tissue(Richardson
2003). The process of healing consist of four integrated and overlapping phases which
interacts with each other in space and time: blood clotting (hemostasis), inflammation, tissue
growth (proliferation) and tissue remodelling (maturation). It also involves coordinated
interactions between immunological and biological systems and all these phases and their
biochemical functions must occur in the appropriate sequence, in a time frame, and continue
for a specific duration at an optimal intensity to repair the damage.
Wounds are almost unavoidable events in human life which arise due to physical,
chemical injury or microbial infection. Numerous modern medicines involved in curing
wounds are focused on drugs of mineral origin. This drug has many side effects and also has
less efficiency in curing wounds. Recent trend of wound cure is shifting towards herbal
extract i.e. drugs of plant origin. This is a completely new way of exploiting nature to heal
wounds. Although several plant based drugs used in wound healing have been studied
scientifically in different pharmacological models, the potency of many traditionally used
herbal agents still remains unexplored. Hence, in this study Amrit oil, a polyherbal

2
formulation of selected medicinal plants having a claim of to display wound healing activity
is taken to unravel the effect of the oil on experimentally induced wounds in rats.
Objectives
 To study the wound contraction parameters (wound area measurement,
percentage contraction and mean period of epithelization).
 To study the histopathological parameters of wound tissue sections
(Angiogenesis, Epithelization, Collagen Deposition and Neo- vascularization).
 To estimate the amount of DNA and Protein in the wound tissue.
 To study the expression of genes regulated during wound healing (MMP 2,
MMP 8, KGF and HSP 90)
Materials and Methods
Chemicals: Betadine, Xylazine-Ketamine, Trizol, Chloroform, Isopropyl alcohol, 75%
ethanol, RNase-free water, 90% ethanol, and 10% buffered formalin solution, Hematoxylin-
eosin dye.
Test Drug: The drug used in this study is a polyherbal formulation, Amrit Tail (Oil) supplied
by Jain Pharmecy. It is a combination of different phyto-extracts of various medicinal plants.
The composition of the polyherbal formulation is as follows:

Name Amount per 10ml

0.7g
Terminalia chebula
0.7g
Terminalia bellerica
0.7g
Emblica officianalis
0.7g
Pterocarpus santalinus
0.7g
Curcuma longa
0.4g
Adhatoda vasica
0.5g
Swertia chirata
0.6g
Azadirachta indica
10ml
Sesamum indicum

Animals: 3 – 4 weeks old female Wistar Rats weighing about 140 – 180 grams were obtained
from CPSCEA certified breeders and vendors and transported to Central Animal House of

3
Pondicherry University through a suitable car maintained at proper temperature in
polypropylene cages.
In-vivo Wound Healing Study – Excision Wound Model
Animal Information: 3 – 4 weeks old female Wistar Rats weighing about 140 – 180 grams
were kept in polypropylene cages at the Animal House, Pondicherry University.
Temperature of about 25±2oC and 12 hours of light and dark cycle were maintained. The rats
were fed with commercial pellet diet obtained from VRK Nutritional Solutions, Pune with
free access to water and were allowed to acclimatize for 1 week and examined by trained
veterinarian for any kind of abnormal behaviour. The feed and water intake are almost same
during acclimatization period. The animal care procedures and methods were approved by
Institutional Animal Ethics Committee (IAEC) with approval number (PU/SLS/16th IAEC
2016/AH/07).
Groupings: The rats were randomly grouped into 4 groups with 6 in each. One group was
kept as negative control, one as a positive control which was treated with quantum sufficient
amount of Betadine (Tork Zahrani, Amirali Akbari et al. 2002) and the remaining 2 groups
were subjected to test drug at different exposure of time according to the following design.

Groups Details Time of Drug

Negative Control (NC)

1 -
(i.e. excision wound created but not treated)

2 Positive Control (PC) Morning

(Q.S. Betadine)

3 Test drug applied once in a day (OID) Morning

4 Test drug applied twice in a day (BID) Morning Evening

Groupings and time of drug treatment.

Excision Wound Creation: Before excising the skin, the rat’s fur were fully clean shaved
manually with a clipper by marking a square (2 cm x 2 cm) and then subjected to 4 cm2
excision wound on the dorsal side of the animal by cutting with sharp surgical scissors.
Diethyl ether was used as pre-emptive analgesia. After that rats were randomized into 4
groups of 6 individuals in each groups using standard technique. (Saratha, Subramanian et al.
2010)

4
Drug Treatment: The drug was treated at two different interval of time in a day. The
morning time was maintained at 10:00 AM and the evening time at 4:00 PM with ± 10
minutes delay. Before drug treatment, animals were cleansed with 0.9 % saline so that no
residual husk is attached on the wound surface which may cause irritation to the rats. A
volume of 500 µl of test drug were applied topically each time and smoothly rubbed over the
wounded surface.

Body Weight Assessment: Body mass of each rats were recorded at every 0th, 7th, 14th and
21st of drug treatment over an electronic balance covered with a plastic container.

Wound Contraction Assay

In excision wound model, the animals were divided into 4 groups of 6 animals by complete
randomization. The area of wound was calculated using Adobe Photoshop CS5 software after
scanning (Prathibha, Tatiyana et al. 2015). Percentage of wound contraction was measured
using Wilson’s standard formula.

Period of Epithelization
Epithelization of the wounded tissue was analysed and measured from the first day till it
completely takes to repair (Nayak, Anderson et al. 2007).

Histopathology
The granulation tissue was excised from the wound site and after processing, the tissues was
stained with Hematoxylin-Eosin (H&E stain). The tissue sections were observed under light
microscope for its examination (Fischer, Jacobson et al. 2008).

Biochemical Analysis
RNA, DNA and Protein Estimation

RNA, DNA and Protein was isolated by Trizol reagent according to the (Hummon, Lim et al.
2007).

Real Time PCR (qRT-PCR)

Quantitative real-time PCR (q-PCR) was performed on AbiPrism™ 7500 sequence
detection system (Applied Biosystems). Gene-specific primers such as MMP2, MMP8,
HSP90 and KGF were used. Specific genes were amplified in a reaction mixture containing
1μl of template (cDNA), 300 nM of forward and reverse primers and 1x SYBR green PCR
master mix and the reaction was performed as per the kit manufacturer‘s instruction
(Applied Biosystems). The change in fluorescence of SYBR green dye in every cycle

5
was monitored, and threshold cycle (ct) above background for each reaction was
calculated. Results are expressed as fold change in gene expression by comparing with the
controls.
Statistical Analysis
Statistical analysis was done by Graph pad Prism Software by one way ANOVA followed by
Turkey’s Multiple Comparison Test. The p value is < 0.05.

Results
In-vivo Wound Healing study
Wound Contraction
Wound healing is a response to any kind of tissue injury and is a complex multifactorial
process that results in the contraction of the wound. Wound contraction is an essential phase
in healing that leads to closure. Contraction commences approximately a week after
wounding, when fibroblasts have differentiated into myofibroblasts (Prathibha, Tatiyana et al.
2015). Thus, wound healing can be measured by assay in which the rate of contraction is
directly proportional to the amount of days expended after wounding. The rate of wound
contraction of the positive control (p.c.) and treated group (OID and BID) is outlined below.
The test drug produced a significant wound closure as compared to the p.c. which is revealed
in the results. Additionally, Group 3 (OID) treated rat’s yielded faster contraction of wound
than Group 4 (BID).
Wound area measurement, percentage wound contraction and period of epithelization
are some helpful data for analyzing wound closure.

Average Wound Area (in mm2)

Groups
Day 3 Day 7 Day 10 Day 14 Day 18 Day 21

I – NC 504.17 391.69 216.36 78.57 48.77 42.09

II - PC 519.35 483.55 352.22 133.2 100.98 30.14

III – OID 615.40 492.95 188.88 77.59 59.87 56.14

IV - BID 679.91 515.54 235.07 118.12 115.22 85.79

Average Wound Area Measurement Post-Wounding

6
Wound Area Measurement:
Average area of the wound tissue was recorded over 3rd, 7th, 10th , 14th, 18th and 21st days of
post-wounding using tracing paper followed by recording measurements of area using Adobe
Photoshop CS5 as follow (Lodhi, Pawar et al. 2006).
The following data mentions the significant reduction in area of wounded tissue in all
the groups. The result revealed that there is a significant reduction of wound area in Group 3
rats as compared to the positive (+ ve) control. While in Group 4, there is a reduction as
compared to control but not as much as Group 3. The graphical representation is outlined
below.

Wound Contraction Assay

800
Negative Control
Area of Wound (mm2)

Betadine
600
OID Treated
BID Treated
400

200

0
10

14

18

21
3

DAYS

Average Wound Area Measurement

DAY 18 DAY 21

Photographic Representation of Wound Contraction on Different Days of Control and Test

Drug Treated Wounds.

7
Wound Contraction Assay
Wound contraction is an essential process which leads to closure. Thus, visible appearances
and measurements of wound contraction become reliable parameters in macroscopic
evaluation of wounds. The percentage of wound contraction was measured on 3rd, 7th,
10th,14th,18th and 21st day of post-wounding using the Wilson’s standard formula mentioned
below and graph was plotted (Kant, Gopal et al. 2015).
0𝑡ℎ 𝑑𝑎𝑦 𝑤𝑜𝑢𝑛𝑑 𝑎𝑟𝑒𝑎 −𝑤𝑜𝑢𝑛𝑑 𝑎𝑟𝑒𝑎 𝑜𝑛 𝑎 𝑝𝑎𝑟𝑡𝑖𝑐𝑢𝑙𝑎𝑟 𝑑𝑎𝑦
% Wound Contraction = 𝑥 100
0𝑡ℎ 𝑑𝑎𝑦 𝑤𝑜𝑢𝑛𝑑 𝑎𝑟𝑒𝑎

Average Wound Contraction (in %)

Groups
Day 3 Day 7 Day 10 Day 14 Day 18 Day 21
I – NC 0 22.31 57.09 84.42 90.32 91.65
II - PC 0 6.89 32.18 74.35 80.55 94.19
III – OID 0 19.89 69.30 87.39 90.27 90.87
IV - BID 0 24.17 65.42 82.62 83.05 87.38

Average Wound Contraction Post-Wounding

The results revealed that highest percentage wound contraction was observed in
Group-3 OID treated rats as compared to the positive control confirming that Amrit Oil
stimulates the wound contraction as seen from the percentage wounding wound contraction.
The graphical representation of the above data is given as follows.

Percentage Wound Contraction

150
Negative Control
Precentage Wound Contraction

Betadine
100 OID Treated
BID Treated

50

0
10

14

18

21
7

DAYS

Graphical Representation of Percentage Wound Contraction.

8
Period of Epithelization
Epithelization of the wounded tissue was analyzed by noting the number of days it takes for
the scar to fall away, leaving no raw wound behind. In simple words, it indicates the
formation of new epithelial tissue to completely cover the wounded surface (Sneha, Raju et
al. 2016). The epithelization was measured from the first day till the day of full healing. The
relative period of epithelization was pronounced to be 14 days for positive control, 10 days
for OID treated group and 14 days for BID which continues further until complete
epithelisation.
Biochemical Analysis
The DNA and Protein was quantified by Nano-drop by a ratio of a wavelength of
260nm/280nm and 260nm/230nm since. This is commonly used to assess DNA
contamination of protein solutions, since proteins containing aromatic amino acids absorbs
light at 280 nm(Sumitra, Manikandan et al. 2005)

Concentration
Groups
DNA(in µg/ml) Protein (in mg/ml)

I – NC 60.0 2.73

II – PC 228.13 9.96

III – OID 565.85 17.30

IV - BID 807.3 17.52

Biochemical estimation of DNA and Protein content in the wound tissue.

The results depict a gradual increase in the amount of DNA and protein content in a dose-
dependent manner which was in analogy to what was expected.
Histopathological Data
The Histopathological observations were made on 14th and 21st days according to different
groups as given below. Skin samples were collected on from normal area of experimental
animals showed intact normal stratified squamous epithelium and loose, thick, mature
collagen fibers in dermis as shown in Figure 1. The observations on 14th day are as follows:
Group-1 (Untreated): Wound area showed thick scab and necrotic debris on the surface.
The underneath granulation tissue had proliferating or new blood vessels, plumpy immature

9
fibroblasts and less collagen deposition as shown in Figure 2. Epithelization has started but
incomplete.
Group-2 (P.C.): Wound area showed granulation tissue with no scab on the surface. The
granulation tissue had numerous small blood vessels and had diffuse hemorrhage (may be
due to weak capillaries). Presence of immature fibroblasts and less collagen deposition was
observed as shown in Figure 3. Epithelization has started but incomplete.
Group-3 (OID): Wound area showed granulation tissue with minimal scab on the surface.
The underneath granulation tissue had new blood vessels mature fibroblasts and well-formed
collagen bundles as shown in Figure 4. Epithelization has started but incomplete.
Group-4 (BID): Wound area showed granulation tissue with thick scab on top. The
underneath granulation tissue had new blood vessels, immature fibroblasts and less collagen
as shown in Figure 5. Epithelization has started but incomplete.

Figure 1 – Histopathology of normal skin tissue A) 1x: Normal squamous epithelium, B) 10x:
Loose collagen bundles.

Figure 2- Histopathology of untreated control – A) 1x: Wound area B) 10x: Granulation

tissue showing thick scab C) 10x: Immature fibroblast and less collagen.

10
Figure 3- Histopathology of positive control (Betadine) – A) 1x: Wound area, B) 10x:
Hemorrhage in granulation tissue and C) 10x: Immature fibroblast.

Figure 4- Histopathology of OID treated – A) 1x: Wound area, B) 10x: Mature fibrocytes and
collagen and C) 10x: Scab.

Figure 5- Histopathology of BID treated – A) 1x: Wound area, B) 10x: Dense and thick scab
and C) 10x: Immature fibroblasts and collagen.

The histopathological observations on 21st day are as follows:

Group-1 (Untreated):
Granulation tissue surface was irregular, and had thick continuous scab and necrotic debris on
the surface. Epithelialization is partially complete. The area without epithelium is covered by
thick scab. The underneath granulation tissue was less stromal and more cellular with
numerous blood vessels, plumpy immature fibroblasts and inflammatory cells (+++)
suggesting active inflammatory and proliferative phase are shown in Figure 6A.
Group-2 (P.C.): Granulation tissue showed thick continuous scab and necrotic debris on the
surface. Epithelialization is partially complete. The area without epithelium is covered by
thick scab. The underneath granulation tissue was less stromal and more cellular with
numerous blood vessels and inflammatory cells (++++) suggesting active inflammatory phase
are shown in Figure 6B.
Group-3 (OID): Granulation tissue surface was regular with no scab on the surface.
Epithelialization was complete and keratinized. The underneath connective tissue few blood
vessels, mature fibroblasts, few inflammatory cells (+) and well-formed collagen bundles are
shown in Figure 6C.

11
Group-4 (BID): Granulation tissue surface was irregular, and had thick continuous scab and
necrotic debris on the surface. Epithelialization is partially complete. The area without
epithelium is covered by thick scab, necrotic debris and inflammatory cells. The underneath
granulation tissue was less stromal and more cellular with numerous blood vessels, plumpy
immature fibroblasts and inflammatory cells (++++) suggesting active inflammatory and
proliferative phase are shown in Figure 6D.
Based on the histopathological findings the wound healing process is graded as follows:
Group3 > Group 1>Group2>Group4

Figure 6 –A) Histopathology of Untreated, B) Histopathology of positive control (Betadine),

C) Histopathology of OID treated, D) Histopathology of BID treated all pictures were
observed under 10X magnification.
Gene Expression Studies
To explore the molecular mechanism underlying wound healing by the test drug in
excisional wound model of wistar rats, the fold change of mRNA levels of MMP-2, MMP-8,
HSP-90 and KGF were examined using quantitative RT-PCR method. The gene expression
of MMP-2, MMP-8, HSP 90 and KGF is shown in the following Figure 7A, 7B, 7C and 7D
respectively. As compared to normal skin tissue, the untreated control showed an obvious
higher amount of MMP-2 and MMP-8. It also revealed that the MMP levels decreases after
cell takes up tissue repair i.e. as the healing progresses as seen in the wound treated –
Betadine (positive control), Test Drug (OID and BID) (Armstrong, 2002).

12
 HSPs protect against tissue injury by maintaining synthesis and proper conformation
of proteins, repairing damaged proteins, and promoting wound-healing of injured tissue. In
figure 7C, the skin from normal tissue is treated as a reference control, and the expression of
HSP was found to increase in a trend to what was expected, during stress response of
wounding. BID shows the highest HSP activity due to increased HSP-protein expression for
restoration of wounded tissue due to application of drug-twice a day. But OID has relatively
lower expression when compared to other groups. Thus we can conclude that OID is more
effective than BID.
Keratinocyte growth factor-1 (KGF-1), a member of the fibroblast growth factor
(FGF) family and is expressed in normal and wounded skin. OID treated sample have the
highest expression of KGF relative to others, suggesting that it underwent massive wound-
healing validated by the role of KGF in modulating tissue proliferation and remodelling in
response to stress. This study demonstrated that OID-treated sample have augmented and
accelerated the wound healing process characterized by an increase in tensile strength and
breaking strength of wounds.

MMP-8
MMP-2 B
A 20
15 ***
*** 15
***
Fold Change
Fold Change

10
10 ***
***
5 5
***
0 ***
0
ID
ed

ne

ID
n
ki

B
ie
at
ID
ed

ne

ID
n

ad
re
ki

B
ie
at

al
S

nt

et
ad
re

m
al

B
nt

et

or
m

N
or
N

Groups
Groups

KGF
HSP 90 D 2.0
C 1.5 ***
***
1.5
Fold Change
Fold Change

1.0 ***
1.0
** ***
0.5
0.5
*** ***
***
0.0 0.0
ID
ed

ne

ID
n
ID
ed

ne

ID
n

ki

B
ie
at
ki

B
ie
at

ad
S

re
ad
re

al
al

nt

et
nt

et

m
m

B
U

or
or

N
N

Figure 7: Comparative gene expression studies of MMP-2, MMP-8, HSP-90 and KGF in
normal skin tissue and wounded tissue.

13
References

1. Hamdorf, J. and J. C. Hall (2000). "Acquiring surgical skills." British Journal of

Surgery 87(1): 28-37.
2. Leaper, D. J. and K. G. Harding (1998). Wounds: biology and management, Oxford
University Press, USA.
3. Richardson, M. (2003). "Acute wounds: an overview of the physiological healing
process." Nursing times100(4): 50-53.
4. Tork Zahrani, S., S. Amirali Akbari and N. Valaie (2002). "Comparison of the effect
of Betadine and water in episiotomy wound healing." KAUMS Journal (FEYZ)5(4):
80-85.
5. Saratha, V., S. Subramanian and S. Sivakumar (2010). "Evaluation of wound healing
potential of Calotropis gigantea latex studied on excision wounds in experimental
rats." Medicinal chemistry research19(8): 936-947.
6. Prathibha, M., M. Tatiyana, K. Bairy and S. Adiga (2015). "Wound healing activity
of oil extract of tectona grandis leaves in wistar rats." Research Journal of
Pharmaceutical, Biological and Chemical Sciences6(1): 423-428.
7. Nayak, B., M. Anderson and L. P. Pereira (2007). "Evaluation of wound-healing
potential of Catharanthus roseus leaf extract in rats." Fitoterapia78(7): 540-544.
8. Fischer, A. H., K. A. Jacobson, J. Rose and R. Zeller (2008). "Hematoxylin and eosin
staining of tissue and cell sections." Cold Spring Harbor Protocols2008(5): pdb.
prot4986.
9. Hummon, A. B., S. R. Lim, M. J. Difilippantonio and T. Ried (2007). "Isolation and
solubilization of proteins after TRIzol® extraction of RNA and DNA from patient
material following prolonged storage." Biotechniques42(4): 467.
10. Lodhi, S., R. S. Pawar, A. P. Jain and A. Singhai (2006). "Wound healing potential of
Tephrosia purpurea (Linn.) Pers. in rats." Journal of Ethnopharmacology108(2): 204-
210.
11. Kant, V., A. Gopal, D. Kumar, N. N. Pathak, M. Ram, B. L. Jangir, S. K. Tandan and
D. Kumar (2015). "Curcumin-induced angiogenesis hastens wound healing in
diabetic rats." Journal of Surgical Research193(2): 978-988.
12. Sneha, B., M. G. Raju and V. Babu (2016). "WOUND HEALING ACTIVITY OF
AQUEOUS EXTRACT OF LEPTADENIA RETICULATA IN WISTAR ALBINO
RATS." International Journal of Pharmaceutical Sciences and Research7(3): 1240.

14
13. Su`mitra, M., P. Manikandan and L. Suguna (2005). "Efficacy of Butea monosperma
on dermal wound healing in rats." The International Journal of Biochemistry & Cell
Biology37(3): 566-573.

15