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Journal of Applied Sciences Research, 6(6): 674-682, 2010
© 2010, INSInet Publication
Comparative Efficacy of Ampucare and Silversulfadiazine against Burn Wound Rat
Vivek K.Dwivedi, Manu Chaudhary, Adnan Ahmad, Arvind Soni and Vijay Naithani
Pre clinical Division, Venus Medicine Research Centre, Hill Top Industrial Estate, Bhatoli Kalan,
Baddi, H.P. - 173205 India
Abstract : The present study was undertaken to investigate the comparative efficacy of ampucare and
silversulfadiazine drug on biochemical parameters in burn wound rat model. Burn injury was made on the
ventral shaven area of the rats under anesthesia and the animals were treated topically twice daily with
ampucare and silversulfadiazine drug for 14 days. Our results showed that in both treated groups, there
was statistically significant (p<0.001) decrease in xanthine oxidase, nitric oxide and malonaldialdehyde
levels along with significant (p< 0.001) increase in the ascorbic acid, protein and reduced glutathione level
after 14 days of treatment when compared with infected group. But these parameters showed better
response in ampucare treated group in comparison to silversulfadiazine treated group after 14 days
treatment. Therefore, these findings concluded that ampucare has antioxidant property that reduces
oxidative stress, improves enzyme activity and increases wound healing effect than silversulfadiazine drug
after burn injury.
Key words: wound healing, xanthine oxidase, nitric oxide, ampucare, silversulfadiazine
INTRODUCTION
discovered. A wounds is described as a' break in
continuity of tissue, from violence or trauma' and is
regarded as helped if there is restoration of the wound
and inflammed tissue to normal conditions. Wound
healing involves a complex interaction between
epidermal and dermal cells, the extra cellular matrix,
controlled angiogenesis and plasma- derived protein all
coordinated by an array of cytokines and growth
factors[10]. There are four distinct stages involved in
wound healing namely inflammatory stage, debridement
stage, proliferation stage and maturation/remodeling
stage[11]. Initial stages of wound healing involve an
acute inflammatory phase followed by synthesis of
collagen and other extracellular matrix which are later
remodeled to form scar[12]. Certain factors such as
bacterial infection, nutritional deficiency, drugs,
sterility, movement of wound, edges, site of wound and
wasting diseases distorts wound healing process during
burn injury[13].
Ampucare is a oil based drug formulation which is
used topically for the treatment of burn wounds. It is
a polyherbal drug with anti-inflammatory and
antimicrobial product, which improves blood flow. It
has an immunomodulatory action along with tissue
regeneration properties. Wound healing is an important
biological process involving tissue repair and
regeneration. The active components of ampucare are
Burn injury is accompanied by complex
pathophysiological alterations that exert deleterious
effect on various organ system[1]. Thermal injury
initiates systemic inflammatory response, generation
limitations of water vapour transport of oxygen radicals
leading to peroxidation of biomolecules. It produces
performed systematic changes such as anemia, renal
failure, and metabolic changes etc. Various biological
and metabolic alterations follow burn injury including
degradation of adenosine triphosphate and significant
reduction in polyunsaturated fatty acids in red cell
membrane[2,3]. It is related to alterations in proteins
structure and moist environment depends on the quality
of grossly visible change in the skin. The skin is one
of the largest organs in humans and accounts for 15%
of body weight and 10–25% of whole-body protein
turnover[4]. Tissue repair and wound closure may last
for weeks after burn injury, and closure usually
requires extensive surgery and skin grafting[5].Various
complications that are exacerbated or favored by
nutritional deficiencies can occur, such as infections,
delayed wound healing, and muscle wasting[6],
infections remain a leading cause of death after major
burns[7-9]. Healing of burn wound has still remained
challenge to modern medicine though many drug are
Corresponding Author: Vivek K.Dwivedi, Pre clinical Division, Venus Medicine Research Centre, Hill Top Industrial
Estate, Bhatoli Kalan, Baddi, H.P. - 173205 India,
Phone No: 91-1795-302127; Fax No: 91-1795 302133
E-mail: [email protected]
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J. Appl. Sci. Res., 6(6): 674-682, 2010
Azadirachta indica and Curcuma longa involved in
wound healing[14]. The present study was evaluate the
comparative efficacy of ampucare drug and its
comparison with SSD (Silversulfadiazine drug) on
oxidant enzyme as well as biochemical parameters in
burn wound rat model.
solidification and each animal was kept in other cages.
Experimental Protocol: Total eighteen wistar rats were
divided into three groups of six rats each as follow:
Group I : Burn wound without treatment
Group II: Burn wound treated with ampucare
Group III Burn wound treated with silver
sulfadiazine drug
Treatment with these drug was continued up to 14
consecutive days. Blood samples were collected on 14th
day. Blood sample was diluted 10 times in chilled
distilled water for the estimation of oxidant and
antioxidant enzyme levels in burn wound rat model.
All enzyme parameters were measured at 0-4 0C.
Xanthine oxidase enzyme activity was carried out
according to the method of Roussos[16]. One unit of
activity has been defined as change in absorbance at
290 nm within 1 minute by 1 ml of blood sample.
Protein was assayed by the method of Lowery et al
[17]
using bovine serum albumin as standard.
Malonaldialdehyde measurement has been utilized as
a maker of oxidative stress by the method of Ohkawa
et al.,[18] using thio barbituric acid reagent. 1,1,3,3
tetraethoxy propene (TEP) was used as standard.
Reduced glutathione was measured in the blood sample
by the method of Hissin and Hilf [19]. Ascorbic level
was estimated in the blood sample by the method of
Roe et al.[20] Nitric oxide concentrations was
determined in the blood sample by the method of
Griess reagent [21].
MATERIAL AND METHODS
Materials: 5,5 diathiobis (2 nitro benzoic acid )
(DTNB; 55495) Thiobarbaturic acid (TBA; 30231),
2,4 -Dinitrophynely hydrazine (52144), Xanthine
(03472), Bovine Serum albumin (BSA; 27423)reduced
glutathione (GSH; 13679) and other biochemicals were
procured from Himedia laboratories Ltd (Mumbai,
India) and Sigma, St. Louis, MO, U.S.A. Ltd.
Ketamine hydrochloride was purchased from Samarth
Life Science Pvt. Ltd. Mumbai All other chemicals
and reagents used were of analytical grade.
Drugs: Ampucare is polyherbal drug. It contains
Azadirachta indica, Berberis aristata, Curcuma longa,
Glycyrrhiza glabra, Jasminum officinale, Pongamia
pinnata, Rubia cordifolia, Terminalia chebula,
Trichosanthes dioica, Symplocos racemosa, Ichnocarpus
frutescens, Capsicum abbreviata, Nymphaea lotus etc.
ingredients. These pure ingredients were purchased
from authorized dealer and use as such. Ampucare drug
was obtained from Venus Remedies Ltd, Baddi, H.P.
and silversulfadiazine drug (SSD) was purchased from
a pharmacy (Chandigarh) for comparative efficacy of
respective drugs on burn rat wound.
Histopathological Analysis: After 14 days treatment,
animals were anesthetized and burn skin tissues were
collected in to 10% formalin solution for histological
examination. The samples were washed out with
distilled water and cut in 5 µm sections and stained
with hematoxylin and eosin (H&E), and examined by
light microscopy.
Experimental Animals: Total Eighteen male wistar
rats (250-270 gm) were selected for the present study.
They were housed in well-ventilated cages and fed
with normal mouse chow and water ad libitum. The
experimental room was air conditioned with
temperature 23 ± 2°C, humidity 65 ± 5%, and with
artificial fluorescent light (10 and 14 hours of light and
dark, respectively). Experiment was done after approval
from the institutional animal ethical committee.
Statistical Analysis: The results are expressed in mean
± SD. Statistical evaluation of the data was performed
by one way- ANOVA followed by student NewmanKeuls using INSTAT 3.0 software package. The
statistical difference was analyzed between without
treated vs with treated drug groups. p<0.05 was
considered statistically significant.
Burn model: Skin burns were made as described by
Mohammed et al.[15]. All experimental animals were
anesthetized by kitamine hydrochloride (100mg/kg body
weight) and burn wounds were created on the shaved
ventral area of rats by pouring hot molten wax at 80
0
C. The wax was poured on the shaven area of the
animal through a cylinder of 300mm2 circulating
opening. The second degree of burn injury involving
approximately 30-35% of the total body surface area.
Wax was allowed to remain on the skin till it get
solidified. It was removed very carefully after
RESULTS AND DISCUSSION
Results: No mortality was seen in the animals during
study.
Histological evaluation, after 14 days treatment, it
was found that burn healing was found to be higher in
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J. Appl. Sci. Res., 6(6): 674-682, 2010
ampucare treated group than silversulfadiazine treated
group. The burned skin (control without treated group)
showed denuded epidermis, diffuse infiltration of
lymphocytes cells and polymorphs. Epidermis causes
the separation of epithelial cell in control without
treated group. Epidermis was found clear in ampucare
treated group than silversulfadiazine. The keratin layer
was observed thickened in silversulfadiazine treated
group. see Figure 1.
In the present study, wound diameter was found to
be statistically significant decreased in ampucare (p<
0.001) as well as silversulfadiazine treated group (p<
0.001) as compared to control without treated group
after 14 days treatment of respective drug. Wound
diameter was found lowered (p< 0.001) in ampucare
treated group in comparison to silversulfadiazine (SSD)
treated group after 14 days treatment. see Figure 2.
Xanthine oxidase enzyme activity was significantly
(p< 0.001) decreased in ampucare as well as in
silversulfadiazine treated group as compared to control
without treated group after 14 days treatment. This
enzyme activity was found to be lowered (p< 0.001) in
ampucare treated group in comparison to
silversulfadiazine treated group after treatment of 14
days. see Figure 3.
Protein level was significant evaluated (p< 0.001)
in ampucare treated group as well as silver sulfadiazine
treated group as compared with control without treated
group after 14 days treatment. The level of protein was
found to be statistically significant (p<0.001) increased
in ampucare treated group when compared to silver
sulfadiazine treated group after 14 days treatment. see
Figure 4.
Ascorbic acid level was significantly (p<0.001;
p<0.01) increased in ampucare treated group as well as
in silver sulfadiazine treated group when compared
with control without treated group after 14 days
treatment. When ampucare treated group was compared
with silversulfadiazine treated group, the level was
statistically significantly (p<0.05) increased in ampucare
treated group after 14 days treatment. (see Figure 5.)
Reduced glutathione level was significantly
(p<0.001) increased in ampucare treated group as well
as in silver sulfadiazine treated group when compared
to control without treated group after 14 days
treatment. When ampucare treated group was compared
with silversulfadiazine treated group, the level of
reduced glutathione was significantly (p<0.001)
increased in ampucare treated group. see Figure 6.
Nitric oxide and MDA levels were statistically
significantly (p< 0.001; p < 0.05; p< 0.001) decreased
in ampucare as well as in silversulfadiazine treated
group as compared with control without treated group
after 14 days treatment. When ampucare treated group
was compared with silversulfadiazine treated group, the
NO and MDA levels were statistically significantly
lowered in ampucare treated group after 14 days
treatment. see Figure 7 and 8.
Discussion: Wound healing is a major issue in burns,
and delayed healing with graft failure is a serious
problem. Burns involving >20% of the body surface
result in extensive inflammatory, endocrine, metabolic,
and immune changes[22]. Infection is also another major
complication during burn injury[23]. Healing of burn
tissue is a complex process, that involves in reepithelisation, granulation, tissue formation and
remodeling of extracellular matrix. Burn injury initiates
inflammatory response, generation of oxygen free
radicals leading to peroxidation of biomolecules.
Various biological and metabolic changes occur during
burn injury including degradation of adenosine
triphosphate (ATP) and significant reduction in
polyunsaturated fatty acids in red cell membrane[2,3].
Oxygen- derived free radicals play a significant role in
the pathophysiology of thermal injury. There are
several report suggested that the involvement of
superoxide radical in the pathogenesis of burn injury[24].
Free radicals as well as histamine and prostaglandins
released from the burn wound cause lipid peroxidation
in the skin tissue. These radicals appear to be involved
in the appearance of lipid peroxidation products
extractable from burned skin and distant organs[25-27].
Lipid peroxidation is a most spectacular manifestation
of cell damage which causes the transformation of
membrane polyunsaturated fatty acids to hydroperoxides
and degradation to low molecular species, respectively.
In burn injury condition, the adenosine tri phosphate
(ATP) levels gradually fall, and increased adenosine
mono phosphate (AMP) is converted to hypoxanthine
and xanthine. Simultaneously xanthine dehydrogenase
is converted by selective proteolysis into xanthine
oxidase. Xanthine oxidase (XO) is important source of
free radical generating enzyme which acts on xanthine
and hypoxanthine to produce uric acid and oxygen free
radicals. During burn injury, the ratio of free radical
generating enzyme and antioxidant defense mechanism
may altered which may lead to several
pathophysiological state. Because xanthine oxidase has
been reported to be an important source of oxidants
involved in cell and tissue damage in situation of
ischemia followed by reperfusion. Till et al.,[28] has
been reported that xanthine oxidase may also play a
role in thermal injury of skin. Burn injury also leads
to increase oxidative stress in the cells as seen by
decreased endogenous non enzymatic and enzymatic
antioxidant activity. Adequate antioxidant defense is
also required for the normal wound healing process.
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J. Appl. Sci. Res., 6(6): 674-682, 2010
Fig. 1: Histopathological section of skin after 14 days treatment. Slide A Shows control thermally burned animal
without treatment and slide B and C shows thermally burned animal treated with ampucare and
silversulfadiazine ( H E) 10X E; epidermis, I; inflammation
Fig. 2: Values are presented in mean ± SD. C= Control without treated group, A = ampucare treated group, SSD=
silversulfadiazine treated group. P***< 0.001 highly significant; P** < 0.01 significant; P* < 0.05
significant; P > 0.05 insignificant (ns).
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J. Appl. Sci. Res., 6(6): 674-682, 2010
Fig. 3: Values are presented in mean 1 SD. C= Control without treated group, A = ampucare treated group, SSD=
silversulfadiazine treated group. P***< 0.001 highly significant; P** < 0.01 significant; P* < 0.05
significant; P > 0.05 insignificant (ns)
Fig. 4: Values are presented in mean ± SD. C= Control without treated group, A = ampucare treated group, SSD=
silversulfadiazine treated group. P***< 0.001 highly significant; P** < 0.01 significant; P* < 0.05
significant; P > 0.05 insignificant (ns)
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J. Appl. Sci. Res., 6(6): 674-682, 2010
Fig. 5: Values are presented in mean ± SD. C= Control without treated group, A = ampucare treated group, SSD=
silversulfadiazine treated group. P***< 0.001 highly significant ; P** < 0.01 significant; P* < 0.05
significant; P > 0.05 insignificant (ns)
Fig. 6: Values are presented in mean ± SD. C= Control without treated group, A = ampucare treated group, SSD=
silversulfadiazine treated group. P***< 0.001 highly significant; P** < 0.01 significant; P* < 0.05
significant; P > 0.05 insignificant (ns)
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J. Appl. Sci. Res., 6(6): 674-682, 2010
Fig. 7: Values are presented in mean ±SD. C= Control without treated group, A = ampucare treated group, SSD=
silversulfadiazine treated group. P***< 0.001 highly significant; P** < 0.01 significant; P* < 0.05
significant; P > 0.05 insignificant (ns)
Fig. 8: Values are presented in mean ± SD. C= Control without treated group, A = ampucare treated group, SSD=
silversulfadiazine treated group. P***< 0.001 highly significant ; P** < 0.01 significant; P* < 0.05
significant; P > 0.05 insignificant (ns)
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J. Appl. Sci. Res., 6(6): 674-682, 2010
Ascorbic acid is an essential cofactor for the synthesis
of collagen and other organic components of the
intracellular matrix of tissues such as bones, skin,
capillary walls, and other connective tissues.
Ascorbic acid deficiency causes abnormal collagen
fibers and alterations in the intracellular matrix that
manifests as cutaneous lesions, poor adhesion of
endothelium cells, and decreased tensile strength of
fibrous tissue. Glutathione play a significant role in the
detoxification of foreign compounds, hydrogen peroxide
and free radical[29]. Reduced glutathione (GSH) enzyme
is free radical scavenging enzyme which quench the
superoxide radical and thus prevent the damage of the
cell caused by free radical. In the present study, the
MDA, NO levels and free radical generating enzyme
xanthine oxidase were significantly lowered in
ampucare as well as in silversulfadiazine treated group
when compared to control (infected) without treated
group after 14 days treatments. Due to enhancement of
MDA level and xanthine oxidase enzyme activity, it
may be confirmed that free radical mediated damge
occur in control without treated group during burn
injury. The ascorbic acid, reduced glutathione and
protein level was found higher in ampucare and SSD
treated group as compared to control without treated
group after 14 day treatment. It means that both drugs
had increased cellular proliferation and collagen
synthesis at the burn wound site which has been
evidenced by increasing protein levels in both treated
group. It was also found that the levels of MDA, NO
and xanthine oxidase enzyme activity were decreased
along with increased in the ascorbic acid, protein and
reduced glutathione level in ampucare treated group in
comparison to silversulfadiazine treated group after 14
days treatment.
Curcuma longa and Azadirachta indica are the
active constituents of the ampucare and these
constituents play a significant role in wound healing
process. Curcuma longa is considered to have natural
medicinal properties, including antibacterial, antiinflammatory, antineoplastic, and analgesic activities
because it contains a number of moniterpenoids,
sesquiterpenoids, and curcuminoids[30-32]. Azadirachta
indica possesses significant anti-inflammatory and
hepatoprotective activity[33,34]. The anti-inflammatory
and antioxidant properties of curcumin, an active
component of turmeric suggest that curcumin may exert
anti-ulcer activity and wound healing property through
scavenging reactive oxygen species (ROS) and also
regulating MMP activity[32]. Several studies have been
reported that curcumin treated wound heal much faster
as indicated improved rates of epithelialisation, wound
contraction and increased tensile strength[35]. Curcumin
treatment caused significant increase in free radical
scavenging enzymes along with decrease in the MDA
level thus accelerating wound healing process.
Ampucare treatment caused significant increase in
antioxidant enzyme activities (ascorbic acid and GSH)
along with significant decrease in MDA, NO levels and
xanthine oxidase enzyme activity in blood when
compared with silversulfadiazine treated group. With
these findings, it is concluded that ampucare possesses
free radical scavenging property and plays a significant
role in the treatment of burn wounds. It has a better
therapeutic and faster burn wound healing effect than
silversulfadiazine drug due to the presence of curcumin
and gallotannins.
ACKNOWLEDGMENTS
Authors are thankful to financial department of
Venus Medicine Research Centre for financial support.
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