Development
and Study of Wound Healing Activity of an Ayurvedic
Formulation
Swati Rawat1*
and Akhilesh Gupta2
1Shri Bhagwan
College of Pharmacy Aurangabad (M.S.)
2Kunwar Haribansh
Singh College of Pharmacy Jaunpur (U.P.)
ABSTRACT:
The effect of prepared
Ayurvedic formulation was evaluated on excision and incision wound
models in rats. The wound-healing activity was assessed by the rate of period
of epithelialization and skin-breaking strength.
Histological study of the granulation tissue was carried out to know the extent
of collagen formation in the wound tissue. The Ayurvedic formulation prepared was then promoted for
wound-healing activity in two wound models. The treated animals showed
significant reduction in the wound area up to (P<0.001) and faster rate of epithialisation (23.17±0.54). In an incision wound model, formulation
treated animals demonstrated significant skin-breaking strength up to 420.33±5.92. Histological studies of the tissue obtained
from the formulation treated group revealed that the activity was more significant in this
group. Our present study reveals that the Ayurvedic
formulation posses potent wound healing activity, which could be a good choice
of remedy for wound healing but less potent than standard Nitrofurazone.
KEYWORDS: Excision wound,
incision wound model, Ayurvedic medicinal plants
INTRODUCTION:
“Best practice requires the assessment of the whole
patient, not just the hole in the patient. All possible contributing factors
must be explored.” The therapeutic efficacies of many indigenous plants for
various diseases have been described by traditional herbal medicine
practitioners1. Natural products are a source of synthetic and
traditional herbal medicine2. The presence of various life
sustaining constituents in plants has urged scientists to examine these plants
with a view to determine potential wound healing properties. The Indian
traditional system of medicine described several drugs of plant,
mineral, and animal origin are in the Ayurveda for
their wound healing properties, Scientists who are trying to develop newer
drugs from natural resources are looking toward the Ayurveda
3.
Wound healing is the process
of repair that follows injury to the skin and other soft tissues. The objective
in wound management is to heal the wound in the shortest time possible, with
minimal pain, discomfort, and scarring to the patient.
At the site of wound closure
a flexible and fine scar with high tensile strength is desired. Understanding
the healing process and nutritional influences on wound outcome is critical to
successful management of wound patients. Researchers who have explored the
complex dynamics of tissue repair have identified several nutritional cofactors
involved in tissue regeneration, including vitamins A, C, and E, zinc, arginine, glutamine, and glucosamine 4.
Tissue injury initiates a
response that first clears the wound of devitalized tissue and foreign
material, setting the stage for subsequent tissue healing and regeneration. The
second phase of wound healing, the inflammatory phase, presents itself as erythema, swelling, and warmth, and is often associated
with pain5. In the late inflammatory phase, monocytes
converted in the tissue to macrophages, which digest and kill bacterial
pathogens, scavenge tissue debris and destroy remaining neutrophils.
The subsequent proliferative phase is dominated by the formation of granulation
tissue and epithelialization. Its duration is
dependent on the size of the wound. Collagen levels rise continually for
approximately three weeks. The amount of collagen secreted during this period
determines the tensile strength of the wound. The final phase of wound healing
is wound remodeling, including a reorganization of new collagen fibers, to form
more organized lattice structure that progressively continues to increase wound
tensile strength6.
MATERIALS
AND Methods:
This work
focus to evaluate the Ayurvedic ointment formulation,
which contains Jasat Bhasma
(20.00%), Gandhak (7.00%), Tankankhar
(2.00%), Ras kapoor
(0.10%), Base (q.s.)
Animals:
The Ethics Committee for
animal experimentation approved these study. Healthy
Albino rats weighing 150-200g were used for the study. They were individually
housed and maintained on normal food and water ad libitum.
Animals were periodically weighed before and after the experiment.
The wound site was prepared
following the excision wound model7. 3 groups of five animals each
were taken. The rats were anaesthetized prior to and during infliction of the
experimental wounds. The surgical interventions were carried out under sterile
conditions using diethyl ether. A wound was made on dorsal thoracic region (500
sq. mm). Animals were closely observed for any infection and those which showed
signs of infection were separated and excluded from the study and replaced. The
animal were observed for would closure and for period of epithelialisation
at the interval of 5, 10 and 15th day.
Albino rats (150-200 g) were selected for studies and
grouped to three, each containing five animals. The rats were anaesthetized
prior to and during creation of the wounds, with diethyl ether. The dorsal fur
of the animals was shaved with an electric clipper. A longitudinal paravertebral incision of 6 cm long was made through the
skin and cutaneous tissue on the back8. After the incision, the parted skin was sutured
1 cm apart using a surgical thread and curved needle. The wounds were left
undressed 9. Formulation was topically applied to the wound once a
day. The sutures were removed on 8th post wound day and continued
the application of the formulation. The skin-breaking strength was measured by
the method of Lee10 on the 10th day
evening after the last application.
Statistical analysis:
Results, expressed as mean ± SE were evaluated using
the t-test. Values of P<0.001 were considered statistically significant
Histopathological study:
The healing tissues obtained on the 10th day
from all three groups of animals of the incision wound model were processed for
histological study. The amount of collagen was quantified using Vangeison stain.
In
both the models studied, significantly improved wound-healing activity has been
observed with the prepared ayurvedic ointment, compared to that of the
reference standard and control group of animals. In the excision wound model,
marketed formulation treated animals showed significant reduction in the wound
area (P< 0.001), faster rate of epithialisation (Table1), increased dry weight of the
tissue (P<0.001) and increased hydroxyproline
content (P<0.001) when compared with the control group of animals. Table 2 depicts the wound healing effect of marketed formulation in the incision wound model. The treated
animal’s demonstrated significant skin-breaking strength when compared to
control animals. Significant increase in the weight of the granulation tissue
(P<0.001) and hydroxyproline (P<0.001) content
were observed in animals treated with the Ayurvedic
formulation when compared to the control group of animals. Histological
studies of the tissue obtained from the treated
(Fig-II-c) group showed significant increase in collagen
deposition, few macrophages, tissue edema and more fibroblasts. It was more or
less equal to the animals treated with (0.2% w/w
nitro-furazone (Fig-II-b). The wound healing
was more significant in treated
group of animals. Fig-II-a: Granulation tissue of
group 1 animal (control) showing with less collagen and more macrophages (Vangeison stain). Fig-II-b: Granulation tissue of
group 2 (standard) animal showing moderate deposition collagen (Vangeison stain). Fig-II-c: Granulation tissue of
group 3 animal showing more collagen and less macrophages (Vangeison
stain).Wound healing is a process by which a damaged tissue is restored
as closely as possible to its normal state and wound contraction is the process
of shrinkage of area of the wound. It mainly depends on the repairing ability
of the tissue, type and extent of damage and general state of the health of the
tissue. The granulation tissue of the wound is primarily composed of
fibroblast, collagen, edema, and small new blood vessels. The undifferentiated mesenchymal cells of the wound margin modulate themselves
into fibroblast, which start migrating into the wound gap along with the fibrin
strands. The collagen composed of amino acid (hydroxyproline)
is the major component of extra cellular tissue, which gives strength and
support. Breakdown of collagen liberates free hydroxyproline
and its peptides; measurement of the hydroxyproline
could be used as an index for collagen turnover.
|
Group |
Wound area (mm2)Post wounding days |
Period of epithelialisation |
|||
|
0 |
5 |
10 |
15 |
||
|
Control |
502.6±5.37 (0.0) |
412.83±15.05 (17.86) |
304.66±10.82 (39.38) |
191.83±9.803 (61.83) |
26.26±0.40* |
|
Standard |
506.5±5.51 (0.0) |
294.5±17.30 (41.85) |
304.66±10.82 (39.38) |
1.003±0.82 (99.80) |
20.03±0.39* |
|
Ayurvedic formulation |
513.3±4.6* (0.0) |
399.66±6.86* (21.68) |
194.33±7.75* (61.92) |
49.66±4.41* (90.269) |
23.17±0.54* |
Values
are expressed as mean±SEM; n =animals in each group- 5; no in parenthesis indicates percentage
of wound contraction;* p£0.001 when compared to control group.
Fig-I – Comparison of wound site by excision wound model in
control, Standard and Test group
· Control (Simple
Ointment) Standard (0.2% w/w nitro furazone) Test (Jakhme-Ruz)
·
0th day 0th day 0th day
·
15th day 15th day 15th day
·
Fig II (a) Fig II (b) Fig II (c)
TABLE 2 -EFFECT OF
AYURVEDIC FORMULATION ON TENSILE STRENGTH OF RESUTURED INCISION WOUND ON 10th
POST WOUNDING DAY
|
Group |
Tensile strength (gm) |
|
Control |
277.86±03.19 |
|
Standard |
497.13±06.06 |
|
Ayurvedic formulation |
420.33±5.92*** |
Values
are expressed as mean±SEM; n =animals in each group-5;
***
p£0.001 when compared to control group
CONCLUSION:
The formulation promotes
wound-healing activity. It showed remarkable wound healing activity and it may
be suggested for treating various types of wounds in human beings. Further
studies with purified constituents are needed to understand the complete
mechanism of wound healing activity.
Reference:
1)
Natarajan V, Venugopal PV, Menon T. Effect of azadirachta (neem) on the growth pattern of dermatophytes.
Indian J Med Microbiol 2003; 21:98-101.
2)
Singh A, Singh DK.
Molluscicidal activity of Lawsonia
inermis and its binary and tertiary combinations
with other plant derived molluscicides. Indian J Exp Biol 2001; 39:263-268.
3)
Tuhin KB, Plant
Medicines of Indian Origin for Wound Healing Activity: A Review, The International Journal of Lower Extremity Wounds. 2003;
2(1): 25-39.
4)
Douglas M, Alan L,
Miller ND. Nutritional Support for Wound Healing. Alternative Medicine Review
2003; 8 (4): 359-377.
5)
Stadelmann WK, Digenis AG, Tobin GR.
Impediments to wound healing. Am J Surg 1998;
176:39S-47S.
6)
Stadelmann WK, Digenis AG, Tobin GR.
Physiology and healing dynamics of chronic cutaneous
wounds. Am J Surg 1998; 176: 26S-38S.
7)
Opara, IC. A preliminary investigation into the antiemetic
properties of the leaf extract of Ocimium gratissimum D. V. M. Project submitted to the Faculty
of Veterinary Medicine, University of Nigeria, Nsukka,
1999.
8)
EhrlichHP, HuntTK. Effect of cortisone
and vitamin A on wound healing.Ann Surg 1968; 167:324-328.
9)
Hukkeri VI, Nagathan CV, Karadi RV, Patil BS. Antipyretic
and wound healing activities of moringa oleifera lam. in rats. Indian J Pharm Sci 2006; 68:124-126.
10)
Lee KH. Studies on
mechanism of action of salicylates II. Retardation of
wound healing by aspirin. J. Pharm Sci., 1968;
57:1042-1043.
Received on 22.02.2011 Accepted
on 16.03.2011
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Asian J. Res. Pharm. Sci.
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