INTRODUCTION:
Wound healing is a complex process of repairing
cellular structures and tissue layers. The
process of wound healing occurs in three different phases: inflammatory phase, proliferative phase, and the
remodeling phase1. Within these the remodeling phase is a complex and
coordinated series of events that include chemotaxis, phagocytosis,
neocollagenesis, collagen degradation, and collagen remodeling2. Many
investigators evaluated the wound healing properties of medicinal plants using
animal models. Medicinal plants seem to
possess moderate efficacy with no or less toxicity as compared to synthetic
drugs which make them remarkable applications for drug development research
programs3, 4. The scientific assessment of medicinal plants may
result in the discovery of novel, reasonably applicable medicinal plants to
treat wounds5.In India; there has been interest in the potential of
natural products obtained from plants and animals for development of drugs with
wound healing properties6.
Cleome
viscosa Linn (Family; Capparidaceae) has long been used in Ayurveda
for therapeutic purposes. All parts of the plants are reported to be
medicinally important for the treatment of various diseases7. This
herb is found throughout the greater part of India, often in waste places. The
whole plant is sticky in nature and has a strong odour resembling asafoetida. As
a medicinal plant, it has been reported to possess anti-malarial, bronchitis, anti-diarrheal, anti-pyretic9, analgesic10, anti-microbial, and Hepato protective11, 12 properties. The leaves are
used as an external application to wounds and ulcers. The juice of the leaves
has been used to relieve ear ache. The seeds are anthelmintic, carminative,
rubefacient and vesicant13. On the basis of traditional use, we have
made an attempt to screen the wound healing property of C.viscosa on
albino wistar rats.
MATERIALS
AND METHODS:
Plant
material:
An
aerial parts and roots of C.viscosa were collected from the premises of
Loyola Academy, Hyderabad and the plant was
authenticated, a voucher of specimen Herbarium was deposited in the department.
Preparation
of plant extracts:
Shade
dried aerial parts and roots of C.viscosa were powdered
separately and extracted with methanol (90%). The solvent was completed removed
by vacuum and a semisolid mass was obtained (9.8%, w/w). The phytochemical
screening gave positive results for steroids, flavonoids and tannins. A weighed
quantity of methanol extract of aerial parts and roots were suspended in 1%
Carboxy methyl cellulose
for the experiment.
Animals:
Male
albino Wistar rats weighing 180–200 g were purchased from National Institute of
Nutrition. The rats acclimatized to animal house conditions, and fed with
pellet chew (Hindustan Lever Ltd., Bangalore,
India). All the animal experiments were conducted according to the ethical
norms approved by ministry of social justices and empowerment, Government of
India and institutional animal ethics committee guidelines. The animals were
anesthetized prior to and during the experiment.
Wound
Healing Activity:
Excision
and incision wound models were used to evaluate the wound-healing activity of C.viscosa
Excision
study:
Excision
wounds were made as described by Morton and Malone14. Male Albino
Wistar rats weighing 180-200g were divided into four groups of 6 rats each and
received the following treatment schedule.
Group I
|
Control animals treated with
topical application of 1% CMC
|
|
Group
II
|
Animals
were treated with topical application of soframycin ointment.
|
|
Group
III
|
Animals
were treated with topical application CVAM Ext (500 mg/kg b.wt).
|
|
Group
IV
|
Animals
were treated with topical application of CVRMExt (500 mg/kg b.wt).
|
Animals
were anaesthetized with anaesthetic ether and placed in operation table in its
natural position. The dorsal fur of the animal was shaved and a full thickness
of the excision wound of 6 cm in width and 0.3 cm depth was created (Fig.1).
The wounds were treated with topical application of the 500 mg/kg b.wt of
extracts, wound contraction was studied by tracing the raw wound area
subsequently on day 0, 4, 8, 12and 16 on graph paper. Scar residue, area and
time for complete epithelialisation were also measured. The percentage of wound
closure and period of epithelialisation were recorded.
The wounds were monitored and the area of
wound was measured on 0, 4, 8, 12, 16 post-wounding days and the mean % wound
closure is reported in Table 1. The period of epithelization was calculated as the number
of days required for falling of the dead tissue remnants without any residual
raw wound.
Wound healing rate
% of wound closure =
Wound area on day 0 − wound area on
day n
Wound area on day 0 × 100
Where n =number of
day 4, 8, 12, and 16.
Incision
wound study:
The
method of Ehrlich and Hunt15, was adopted for incision wound study.
Male Albino Wistar rats weighing 180-200g were divided into four groups each
group contains six animals and received the following treatment schedule.
|
Group I
|
Control animals treated with
vehicle for 9 days.
|
|
Group
II
|
Animals
were treated with topical application of soframycin ointment for 9 days.
|
|
Group
III
|
Animals
were treated with topical application CVAMExt (500 mg/kg b.wt) for 9
days.
|
|
Group
IV
|
Animals
were treated with topical application CVRMExt (500 mg/kg b.wt) for 9
days.
|
The
animals were anesthetized under light ether and the back of the animal was shaved
and washed with spirit. 6 cm long paravertebral parallel incision was made
through the entire thickness of the skin on right side of vertebral column with
the help of a sharp blade. The wounds were closed with three surgical
interrupted sutures of 1 cm apart (Fig.2). All the sutures used in the
experiments were non-absorbable braided non-capillary and siliconized. Then,
500 mg/kg b.wt of extracts were topically applied on the incision wounds and
Sutures were removed on 9th day and breaking strength was measured on 11th day
post wounding by continuous water flow technique16. Three readings
were taken on each wound and the mean of six such readings in each animal was
used for statistical analysis. Subsequently animals were sacrificed by light
ether anesthesia.
Statistical
analysis:
All
data were expressed as Mean ± S.D. Tukey-Kramer multiple comparisons test, one
way analysis of variance (ANOVA). A value of P<0.05 was considered
significant.
RESULTS:
Excision wound study:
A significant increase in the wound-healing activity
was observed in the animals treated with the CVAMExt compared with CVRMExt.
The mean percentage of closer of excision wound area was calculated on day 1,
4, 8 and 16 post wound healing days as shown in Table1.
Rats treated with CVAMExt 500 mg/kg bw showed
faster wound contraction 43.88 % on day 4, 56.11% on day 8, 78.84% % on day 12
and 91.53% on day 16, with a short period of epithelialization (17.86±2.01).
The results were similar (p<0.01) to the healing potential of Soframycin
treated group, which showed 33.10 % on day 4, 51.10% on day 8, 95.91% on day 12
and 100 of wound contraction on day 16. While CVAMExt 500 mg/kg bw
treated groups also showed better wound contraction (p>0.05) i.e. 30.72 % of
wound contraction on day 4, 49.21% on day 8, 71.47% on day 12 and 90.75% of
wound contraction on day 16 (Fig.3).
In this model, the CVAMExt and CVRMExt
treated animals showed a more rapid decrease in wound size and a decreased time
of epithelialization compared with the control rats which treate with vehicle.
Histopathological
section of wounds treated with CVAMExt showed increased and well
organized regenerated tissues, more fibroblasts and new blood vessels. While
the section of rats treated with CVRMExt promoted epithelialization,
fibrosis with underlying inflammatory cells. The section of rat tissue treated with soframycin
showed normal architecture. While, the section of control rats showed more
inflammatory cells and less fibroblasts (Fig.4).
Incision
wound study:
The incision wound study was also carried out to
measure the breaking strength of the regenerated tissue. C.vescosa
methanolic extracts significantly (P<0.01) increased wound breaking
strength compared to that of control (Table 2). The best re-modeling,
particularly, breaking strength were noticed in CVAMExt treated group
strength (414.37±3.0) which was more or less similar to Soframycin treated
group (p<0.01). On the other hand, animals treated with CVRMExt also
showed better wound healing activity (385.23±2.28). On the other hand, the
granulation tissue of rats treated with CVAMExt showed plenty of
fibroblasts and thick bundles of collagen tissue, whereas the section of rats
treated with CVRMExt also showed macrophages and plenty of fibroblasts.
While Soframycin treated group of rats showed normal histopathological
architecture. And the section of control group rats showed a mild retard
re-epithelialization with chronic inflammatory cells (Figure 5).
Table:
2: Effect of topical application C.vescosa on incision wound model
|
Groups
|
Treatment
|
Breaking strength (g)
|
|
Group I
|
CMC
|
292.58±4.51
|
|
Group II
|
Soframycin
|
425.74±3.10
|
|
Group III
|
CVAMExt 500 mg
|
385.23±2.28
|
|
Group IV
|
CVRMExt 500 mg
|
414.37±3.0
|
Values
are expressed as mean ± S.D (n=6). Statistically significant alterations are
expressed as *p<0.05. Significant; Group III and IV are compared with group
I
C. vescosa promoted the wound healing activity significantly in excision
and incision wound models. High rate of wound contraction, decrease in period
of epithelialization, increased dry weight of granulation tissue and high
breaking strength were observed in rats treated with CVA MExt when
compared to rats treated with CVR MExt.
DISCUSSION:
Wound
healing process begins with the renovation of a damaged tissue as closely as
possible to its normal state17. The healing mainly depends on the type and degree of
damage. The granulation tissue of the wound is chiefly composed of fibroblast,
collagen and new blood vessels18, 19. Moreover, phytochemical analysis of aerial parts of C.viscosa
revealed the presence of steroids,
flavonoids and tannins. A number of
studies indicate that plant products are potential agents for wound healing and
largely preferred because of their widespread availability, absence of unwanted
side effects and their effectiveness20. In present study, CVAMExt and CVRMExt promoted significant wound healing activity in
excision and incisional models. In excision
wound model topical application of CVAMExt markedly improved the wound contraction, and the
effects were distinctly visible from day 4. In
incision wound model the same extract treated group showed significantly
increased breaking strength, which was more or less similar to the Soframycin
treated group.
Flavonoids
are known to endorse the wound-healing process due to their antimicrobial and
astringent properties, which appears to be responsible for wound contraction
and epithelization21.22. Flavonoids and their derivatives are also known to
decrease lipid peroxidation by preventing or slowing down the progress of cell
necrosis23. Therefore, wound healing potential of C.viscosa
may be attributed to the phytoconstituents present in the aerial parts and
roots. In conclusion, the present
investigation demonstrated that the aerial parts and roots of C.viscosa
promote wound healing activity in albino wistar rats and it may be suggested
for treating various types of wounds. But further investigation is needed for
the absorption capacity of the formulation and the mode of its action in tissue
repair mechanism.
ACKNOWLEDGEMENT:
We
gratefully acknowledge Dr. V Kalyan Chakravarthy, M.D Pathology, Siddhardha
Medial College, Vijayawada, for his involvement in histopathological
examination and interpretation
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