Asian Journal of Biological Sciences

Year: 2010 | Volume: 3 | Issue: 1 | Page No.: 18-27
DOI: 10.17311/ajbs.2010.18.27

Efficacy of Ampucare: A Novel Herbal Formulation for Burn Wound Healing Versus Other Burn Medicines

A. Soni, V.K. Dwivedi, M. Chaudhary, S.M. Shrivastava and V. Naithani

Abstract: This study was designed to determine the efficacy of ampucare against oxidative organ damage distant from the original burn wound. Under brief ketamine anesthesia, the shaved dorsum of the rats was exposed to 80°C (burn group) by hot wax for 5 min. The rats were fed standard pelleted diet and water ad libitum. The test room was air conditioned with temperature 23±2°C, humidity 65±5% and with artificial fluorescent light (10-14 h of light and dark), respectively. Rats were left for 24 h after burn injury and blood were taken for the determination of superoxide dismutase (SOD), catalase (CAT) activity, malonaldialdehyde (MDA), myeloperoxidase (MPO), xanthine oxidase (XO) and total protein level. Present findings showed that activities of antioxidant enzymes were significantly decreased along with increased level of MDA, MPO and XO in untreated group at 7th and 14th day of study. Similarly, the level of total protein was also found to be significantly lowered in untreated group at 7th and 14th day. These antioxidant enzymes (SOD, Catalase) along with MDA, MPO and XO were improved and come back to normal level. In conclusion, ampucare scavenges free oxygen radicals, decreases MDA or MPO level in blood and increase the antioxidant enzyme activity by preventing its inhibition. Considering our results, ampucare would be a beneficial for humans who suffer from thermal injury.

Keywords: Ampucare, wound healing, antioxidant enzymes and malonaldialdehyde

INTRODUCTION

A burn is a type of injury that can be caused by heat, cold, electricity, chemicals, light, radiation, or friction. Burns can be highly variable in terms of the tissue affected, the severity and resultant complications. Muscle, bone, blood vessel and epidermal tissue can all be damaged with subsequent pain due to profound injury to nerves. Depending on the location affected and the degree of severity, a burn victim may experience a wide number of potentially fatal complications including shock, infection, electrolyte imbalance and respiratory distress (Sevitt, 1979). The type and severity of the burn depend on the number of layers of skin affected. Most burns are mild, but some may be severe. Most importantly, 75% of burns are estimated to be preventable. Burns have tremendous medicolegal importance as they may be considered to be the commonest cause of unnatural death in India. Often, the circumstances of burns are enveloped in mystery, obscurity and unreliable statements. The reason behind this action may be personal, domestic, occupational or social tragedy and more recently dowry death (Mohanty et al., 2005). The overall mortality of 105 victims was 26.0% with males comprising 32.4% and females 67.4% reported by the various authors (Usama et al., 2008).

Development of hydrops in burn patients are mainly due to the inflammatory response which activates cytokines, with subsequent stimulation of phagocytic cells. This results in the formation of Reactive Oxygen Species (ROS) leading to lipid peroxidation; another source of ROS in burns is the enzyme Xanthine Oxidase (XO), produced from xanthine dehydrogenase under ischemic conditions producing ROS, which causes lipid peroxidation (Sahib et al., 2009) and is responsible for local and systemic complications in burns, including increased vascular permeability (Saes et al., 1984). The changes in the superoxide dismutase (SOD) concentrations after burn injury vary according to the type of SOD and also the type of tissues. As a result, the SOD concentrations may play some role in the early response to thermal trauma (Saitoh et al., 2001; Santos et al., 1995). On the contrary, the antioxidant system of tissues is damaged by injury and cannot cope against ROS in the following period (Demling and LaLonde, 1990; Dubick et al., 2002). The combination of increased oxidant with decreased endogenous non-enzymatical and enzymatical antioxidant activity corresponds to a decrease in cellular energetics and cell membrane lipid peroxidation. Membrane lipid peroxidation can lead to changes in membrane fluidity and permeability with increased rates of protein and nucleic acid degradation which finally leads to cell lysis. Therefore, detrimental effects of burns are not only limited locally to the skin, but they also affect distant organs.

Ampucare is a oil based formulation product which applied externally for the treatment of wounds. It is an antimicrobial and anti-inflammatory herbal formulation with proven activity against E. coli, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus, Enterococcus aerogenes and Candida albicans (Saurab et al., 2008). It is herbal combination with Azadirachta indica and Curcuma longa as active ingredients. These two components have anti-microbial, antioxidant, fungicidal and free radical scavenging properties (Dwivedi et al., 2008). These components are also reported to have an anti-inflammatory, immuno-modulatory and tissue regeneration properties. Ampucare is also reported for the treatment of dermal infections (Chaudhary et al., 2008a, b) and enhance the healing of different wound (Chaudhary et al., 2008c). In this study, we determined the efficacy and burn wound healing effect of ampucare as compared to other commercially available burn medicine at 7th and 14th day of burn.

MATERIALS AND METHODS

Study Conduct
The study has been carried out from 10th March 2009 to 20th June 2009 in pre-clinical unit of Venus Medicine Research Centre, Venus Remedies Ltd. Baddi (India).

Plant Materials
Azadirachta indica, Curcuma longa and other herbs were used in the formulation of ampucare. These raw material were purchased from local dealer which was identified and approved by the botanist.

Chemicals
All the biochemicals used in the experiment were procured from Sigma, St. Louis, MO, USA. Other chemicals purchased locally were of analytical grade.

Study Drug
The commercially available burn medicine such as OBM1, OBM2, OBM3, OBM4 and OBM5, were purchased from dealer and ampucare was obtained from Venus Remedies Limited India.

Experimental Animals
The 100-120 days old male albinos rats of weighing between 200-250 g, were housed in separate cages under standard conditions, with a 14/12 h light-dark regimen. The rats were given standard rat chow and water ad libitum. The protocol was approved by institutional animal ethics committee.

Study Design
The rats were divided into seven groups of six rats each as given below.

All these burn medicines were applied topically on burned wound twice daily. Treatment with these drug continued up to 14 consecutive days. One milliliter of blood samples were collected at 7th and 14th day. The 0.1 mL of blood sample from total volume was diluted 10 times in chilled distilled water for the estimation of antioxidant enzyme levels in burn wound rat model. Serum was separated by centrifugation at 3,000 rpm for 10 min from remaining blood samples. All samples were stored in plastic containers and frozen until the time of analysis. Serum was separated out for analysis of myeloperoxidase activity, xanthine oxidase activity and total protein. All enzyme parameters were measured at 0-4°C.

Experimental Burn Model
The burn model was prepared according to the method of Holla et al. (1998) and Rao et al. (2000). Partial thickness burn wounds were inflicted on overnight-starved animals under ketamine (10 mg kg^-1, i.m.) anesthesia by pouring hot molten wax at 80°C. The wax was poured on the shaven back of the animal through a cylinder of 350 mm² circular. The wax was allowed to stay on the skin till it gets solidified. Instantly, after the injury and on subsequent days, the drugs were applied topically.

Antioxidant Parameters
Superoxide Dismutase (SOD) Determination
The reaction mixture composed of 1.0 mL carbonate buffer (0.2 M, pH 10.2), 0.8 mL KCl (0.015 M), 0.1 mL of diluted blood and water to make the final volume to 3.0 mL. The reaction was started by adding 0.2 mL of epinephrine (0.025 M). Change in absorbance was recorded at 480 nm at 15 sec interval for 1 min at 25°C (UV-1800 SHIMADZU). Suitable control lacking enzyme preparation was run simultaneously. One unit of enzyme activity is defined as the amount of enzyme causing 50% inhibition of auto oxidation of epinephrine (Misra and Fridovich, 1972).

Catalase (CAT) Activity
Catalase activity was measured by the method of Luck (1965). The reaction mixture consisted of 0.3 mL phosphate buffer, (0.2 M pH 6.8), 0.1 mL H₂O₂ (1 M) and water to make the final volume to 3.0 mL. The reaction was started by adding the suitable aliquot of enzyme preparation. The change in the absorbance was recorded at 15 sec. interval for 1 min at 240 nm at 25°C. Suitable control was run simultaneously. One Unit of enzyme activity was defined as the amount of enzyme that liberates half of the peroxide oxygen from H₂O₂ in 100 sec at 25°C.

Free Radical Mediated Damage Assay
Malondialdehyde (MDA) Determination
Free radical mediated damage was assessed by measurement of the extent of lipid peroxidation in the term of malonaldialdehyde (MDA). It was determined by thiobarbituric acid reaction. The reaction mixture consisted of 0.1 mL of diluted blood, 0.20 mL of 8.1% sodium dodecyl sulphate (SDS), 1.5 mL of 20% acetic acid (pH 3.5), 1.5 mL of 0.8% Thiobarbituric Acid (TBA) and water to make up the volume to 4.0 mL. The tubes were boiled in water bath at 95°C for 1 h and cooled immediately under running tap water. Then 1.0 mL of water and 5.0 mL of mixture of n-butanol and pyridine (15:1 v/v) was added and vortexed. The tubes were centrifuged at 3500 rpm for 30 min. The upper layer was aspirated out and optical density was measured at 532 nm. The reference standard used was 1,1, 3,3 tetraethoxypropane (Ohkawa et al., 1979).

Myeloperoxidase (MPO) Activity Determination
MPO activity was determined by using 4-amino-antipyrine/phenol solution as the substrate for MPO-mediated oxidation by H₂O₂ and changes in absorbance were recorded at 510 nm. One unit of MPO activity is defined as that which degrades 1 μmol H₂O₂ min^-1 at 25°C. Data are presented as U g^-1 protein (Wei and Frenkel, 1993).

Oxidant Enzyme Assay
Xanthine Oxidase (XO) Activity Determination
XO activity was assayed spectrophotometrically at 293 nm and 37°C with xanthine as substrate followed by protocol proposed by Prajda and Weber’s (1975). The formation of uric acid from xanthine increases absorbance. One unit of activity was defined as 1 μmol of uric acid formed min^-1 at 37°C, pH 7.5 and expressed in units g^-1 tissue protein.

Protein Assay
Protein assay in the sample was determined by the method of Lowry et al. (1951).

Statistical Analysis
The results are expressed in Mean±SD. Statistical evaluation of the data was performed by one way-ANOVA followed by student Newman- Keuls using InStat software (Instat Biostatistics, Version 3.0 for Windows, Inc. 1990-98). The statistical difference was analyzed between burn untreated, ampucare and other burn medicine treated groups. The p<0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Wound Measurement
Wound measurement was monitored by the progress of healing through changes in the length, width, area or volume of a wound. The wound surface area was measured with the help of vernier caliper.

Significant wound healing effect was observed in case of ampucare treated group at 7th and 14th days of treatment as compared to group I (untreated) and other burn medicines treated groups (Fig. 1).

Malondialdehyde Levels
The MDA level in group I (untreated) were significantly higher (p<0.001) at 7th and 14th days as compared to ampucare and other treated group. It was observed that these level were significantly lowered (p<0.01) at 7th day and became normalized (p>0.05) at 14th day of the treatment in ampucare treated group as compared to other burn medicine treated groups (Fig. 2).

Superoxide Dismutase and Catalase Activity
It was observed that SOD and CAT activities in group I (untreated) significantly decreased (p<0.01) at 7th and 14th day as compared to ampucare and other treated group.

This decrease went up significantly at 7th day and became normalized (p<0.001) up to 14th day of treatment in case of ampucare treated group as compared to other burn medicine treated groups (Fig. 3, 4).

Myeloperoxidase and Xanthine Oxidase Activity
XO and MPO activities in serum were found to be significantly higher (p<0.001) at 7th and 14th day in group I (untreated). The activity decreased significantly at 7th day and become normalized (p<0.001) up to 14th day of treatment in case of ampucare treated group as compared to other burn medicine treated groups (Fig. 5, 6).

Protein Level Test
Protein level was significantly decreased (p<0.01) in the group I (untreated) as compared to other treated groups. The protein level was found to be statistically significantly (p<0.001) increased after 14 day of treatment in ampucare treated group when compared to other burn medicine treated groups (Fig. 7).

Wound healing is the restoration of physical integrity to internal and external structures and involves complex interactions between the cells and various factors. The generation of reactive oxygen species (ROS) is crucial process in the pathogenesis of wound tissue damage. Thus, the examples of attack of biomolecules by ROS, such as lipid peroxidation, could thereby results in an alteration of the structure of biological molecules. It has been observed that there is a close relationship between the lipid peroxidative reaction and secondary pathological changes following thermal injury (Bertin et al., 2000). A local burn produces oxidant-induced organ changes as evidenced by increased lipid peroxidation in distant organs (Youn et al., 1992). In the present study, the levels of MDA found to be significantly increased in burned rat model. These results are in agreement with previous studies in various tissues (Dubick et al., 2002; Sener et al., 2002).

Dwivedi et al. (2008) reported that ampucare significantly decreases level of MDA in wound tissues of burned rat model. In this study, the tissue-associated myeloperoxidase activity which is an index of neutrophil infiltration, were increased in untreated burn rat model. MPO plays an important role in the production of oxidants by neutrophils, which are responsible for origin of ROS and are reasoned to be the leading effector cells in distant organ damage (Dib et al., 2002). According to present results, ampucare decreases MPO and MDA levels caused by thermal injury as compared to other treated groups. These effects may prevent damage to the cell membranes partly caused by oxygen-free radicals released from polymorphonuclear leukocytes.

Xanthine oxidase is the last enzyme in the pathway of degradation of purine derivatives of nucleic acid the best documented biological product (Parks and Granger, 1986). The XO plays an important role in the pathogenesis of burn injury by producing ROS that causes oxidative damage. Thermally injured rats showed increased level of XO activity which is the source of oxygen radicals participating in edema formation (Till et al., 1989). Burton (1995) reported that XO may impart in lung injury and burn cases. Past studies have reported that XO inhibits post burn oxidative tissue impairment (Demling and LaLonde, 1990). In our study it has been observed that XO activity was suppressed by ampucare in wound tissue of infected rats. Such an effect of ampucare may be an important factor in decreased oxidative damage in the rat burn model as compared to other treated groups.

The antioxidant defense system is noted to suppress lipid peroxidation in mammalian tissues by destroying some ROS that has an important role in induction of the lipid peroxidation process. The antioxidant defense system operates through enzymatic and non-enzymatic components. The system is impressed by burns. It has been reported that non-enzymatic antioxidants, such as glutathione, β-tocopherol and selenium are decreased in the serum and tissue after burn injury (Cetinkale et al., 1997; Bekyarova and Yankova, 1998). Some authors have reported that SOD and CAT activity become changed after burn injury (Saitoh et al., 2001; Lalonde et al., 1996; Youn et al., 1998). Gotoh et al. (2003) reported that SOD synthesis was inhibited in severe burn injuries despite a strong mRNA expression. The tissue enzyme activities were decreased in burn group when compared to other treated groups. This decrease may be related to the consumption of activated enzyme against oxidative stress. The ampucare treatment resulted in improved enzyme activities.

CONCLUSION

In conclusion, ampucare scavenges free oxygen radicals as well as decreases MDA and MPO level in wound tissue or directly increases the antioxidant defense system that prevents inhibition of antioxidant enzymes activity. Considering our result, ampucare would be a beneficial for human who suffer from burn injury.

REFERENCES