ORIGINAL ARTICLE

Wound healing effect of Haruan (Channa striatus ) spray

Lia Laila, Febriyenti Febriyenti, Salizawati M Salhimi, Saringat Baie
Laila L, Febriyenti F, Salhimi SM, Baie S. Wound healing effect of Haruan (Channa striatus ) spray. Int Wound J 2011; 8:484491

ABSTRACT
Haruan (Channa striatus ) is a type of fresh water sh in Malaysia that is known to promote wound healing. Haruan water extract has been formulated in an aerosol system which can produce a lm for wound dressing. As topical preparation, Haruan spray needs to be evaluated in terms of the possibility to cause irritation reaction or toxic response. Three experiments were carried out to evaluate the safety of Haruan spray which are Primary Skin Irritation test, Intracutaneous test and Systemic Injection test. The result shows that Haruan spray gave no signicant responses to all the above tests. The investigation of the effect of Haruan spray as wound dressing in the healing process was performed in Sprague-Dawley rats where 6-cm long full-thickness incision wound and burn wound were made on the back of the animals. Haruan spray was tested and compared with blank formula as control. Tensile strength test of treated wound was carried out at the 3rd, 6th, 9th and 12th day after wounding and treatment. The burn wounds contraction was measured daily for 21 days. Results showed that haruan water extract spray formula is not only effective but also safe for application to both incision and burn wounds.
Key words: Aerosol Burn wound Irritation test Tensile strength Wound healing

Key Points
Channa striatus, or commonly
known as Haruan in Malaysia, is a carnivorous fresh water sh that is used by the patients in the postoperative period, in the belief that it promotes wound healing and reduces postoperative pain and discomfort it contains biochemical components such as amino acids and fatty acids which are important for the synthesis of collagen bres during wound healing

INTRODUCTION
Wound can be described as a result of any process which leads to disruption of normal anatomic structure of a tissue and can be classied as closed and open wounds which are characterised by a break in the skin (1,2). In normal condition, wound can heal by itself, but how fast it is can be affected by many factors such as the nutrition and the dressing. Nutritional deciencies can impede wound

Authors: L Laila, BPharm, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia, Faculty of Pharmacy, University of Sumatera Utara, Medan, Indonesia; F Febriyenti, MSc, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia, Faculty of Pharmacy, Andalas University, Padang, Indonesia; SM Salhimi, PhD, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia; S Baie, PhD, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia Address for correspondence: Dr SM Salhimi, PhD, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia E-mail: saliza@usm.my

healing, and several nutritional factors such as amino acids and fatty acids may improve healing time and wound outcome (3). Channa striatus, or commonly known as Haruan in Malaysia, is a carnivorous fresh water sh that is used by the patients in the postoperative period, in the belief that it promotes wound healing and reduces postoperative pain and discomfort. Haruan is regarded as wholesome food by many Asians and is usually consumed in the form of soup. It contains biochemical components such as amino acids and fatty acids which are important for the synthesis of collagen bres during wound healing (4). Apart from nutrition like amino acid and fatty acid, wound dressings play an important role for the healing process of wound. Wound dressing can provide a suitable environment at the surface of the wound to promote the healing process. Even though wound dressings such as plasters and bandages can protect the wound, they may also cause sufferings and pains to the patient during the dressing and

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removal processes. In order to overcome the problem, a development of wound dressing was carried out in the type of lm. Film dressing was produced in the 18th century for the rst time. One concerned area for improvement lay in the method of application. In the majority of lm dressing, the lm had to be removed completely from the backing sheets prior to use. It had a possibility to curl up and stick rmly to itself. It wasted the time and cost because the dressing had to be discarded (5). Aerosol system has been developed to produce a new delivery system of drug, that is, for producing wound dressing as a lm. It is possible to minimize the discomforts especially during the dressing process by spraying the dressing concentrate which forms lm on the wound. For all the above reasons, the study used a combination of the advantageous biochemical components of haruan sh and the comfort ability of aerosol lm dressing for wound healing. The objective of the study was to investigate the healing effect of the Haruan spray in tensile strength measurement and burn wound. In addition, the safety aspect of Haruan spray as a wound dressing also need to be evaluated for the potential to cause irritant reaction or toxic response before conducting the healing evaluation.

medium was purchased from Fisher Scientic (Labourough, UK). Sesame oil was purchased from Fluka (Buchs, Switzerland). All chemicals were used without further purication.

Key Points
the study used a combination of
the advantageous biochemical components of haruan sh and the comfort ability of aerosol lm dressing for wound healing the objective of the study was to investigate the healing effect of the Haruan spray in tensile strength measurement and burn wound in addition, the safety aspect of Haruan spray as a wound dressing also needs to be evaluated for the potential to cause irritant reaction or toxic response before conducting the healing evaluation the Haruan spray was prepared in two formulae: formula P1 contains only haruan water extract as active ingredient and formula P2 contains haruan extract and fusidic acid as the additional ingredient the animal studies were conducted using rabbits, mice and rats

Methods Preparation of Haruan spray

The Haruan spray was prepared in two formulae: formula 1 (P1) contains only haruan water extract as active ingredient and formula 2 (P2) contains haruan extract and fusidic acid as the additional ingredient. The preparation of the concentrates followed the method that was described by Febriyenti et al. (6). The concentrate was lled into the can and then crimped. The crimped can was lled with butane gas as the propellant.

Animals
The animal studies were conducted using the facility of Universiti Sains Malaysia, Penang, Malaysia. The animals (rabbits, mice and rats) were housed in standard environmental conditions under a 12-h lightdark cycle lighting for rat and mice; 14-h light, 10-h dark for rabbit (7) in solid bottom cage with top-ventilated stainless steel cover. All animals were allowed to move freely and were provided access to food and water. Animals welfare guidelines were adopted from Guide to the Care and Use of Experimental Animals (8). The handling of mice and rats followed the protocol described by Deacon (9). All the methods procedures included for the animals have been approved by Animal Ethic Committee Universiti Sains Malaysia with Ref. No. USM/PPSF/50 (028) Jld.2.

MATERIALS AND METHODS Materials

Haruan water extract was obtained from Major Interest (M) Sdn. Bhd. (Kedah, Malaysia). Hydroxypropyl methylcellulose (HPMC) and -tocopherol (Vitamin E) were purchased from Sigma Chemical Co. (St. Louis, MO). Polyethylene glycol (PEG) 400 was purchased from Merck-Schuchardt (Hohenbrunn, Germany). Glycerine, methyl paraben and propyl paraben were purchased from R&M Chemicals, Essex, UK. Fusidic acid was supplied by CM Meditech, Sdn. Bhd. (Penang, Malaysia). Ethanol 96% was bought from Systerm (Penang, Malaysia). Propane gas was purchased from MOX Sdn. Bhd. (Penang, Malaysia). Nembutal 6% was purchased from CEVA (Barcelona, Spain). Sprague-Dawley (SD) rats were provided by the Animal House at Universiti Sains Malaysia (Penang, Malaysia). Normal saline 09% was purchased from Thai Otsuka Pharmaceutical Co, Ltd. (Bangkok, Thailand). The PEG 400 as a
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Primary irritation test

The aerosols were assessed for skin irritation using the test method from A.S.T.M. (10,11) with slight modications. Two preparations were evaluated, namely, formula P1 and formula P2. The test sites were observed for erythema and oedema at 1, 24 and 48 hours after removal of the test preparation and given the scores according to Table 1. The scores for both erythema and oedema of the abraded and intact skin at three time intervals for each formula were totalled and divided by 6 to obtain the Primary Irritation Index (PII) for each test rabbit. The average of PII was
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Table 1 Grading values for the primary skin irritation and intracutaneous tests Skin responses Erythema and eschar formation No erythema Very slight erythema (barely perceptible) Well-dened erythema Moderate to severe erythema Severe erythema (beet-redness) to slight eschar formation (injuries in depth) Oedema formation No oedema Very slight oedema (barely perceptible) Slight oedema (edges of area well dened by denite raising Moderate oedema (raised approximately 10 mm) Severe oedema (raised more than 10 mm and extending beyond exposure area) Total possible score for irritation Score

Table 3 Classication of toxic symptoms for systemic injection test Numerical no. 0 1 2 3 4 5 Symptoms No symptoms Respiratory depth a = increased, b = decreased Motor activity a = increased, b = decreased Convulsion a = tonic, b = clonic, c = mixed Ataxia Death

0 1 2 3 4

0 1 2 3 4 8

after injections according to the classication of toxic symptoms given in Table 3.

Incision wound model

The SD rats were anaesthetized with pentobarbitone sodium (50 mg/kg body weight) administered intraperitoneally. Incision wound was created by a 6-cm long full-thickness incision with a scalpel blade. Wounds were closed with equally spaced interrupted silk sutures (4,1517) as seen in Figure 1. After wound creation, experimental animals were randomly divided into three groups. Group 1: wounds treated with blank formula P, group 2: wounds treated with formula P1 and group 3: wounds treated with formula P2. Each group consisted of 24 animals. The wounds were medicated with a local

calculated by dividing the sum of PII for each preparation of the entire animals with the number of animals tested.

Intracutaneous test
The intracutaneous test was performed according to the method from A.S.T.M. and USP (12,13). All the injection sites on the extract and blank were examined at 24, 48 and 72 hours intervals for gross evidence of tissue reactivity and scoring according to Table 1.

Systemic injection test

The systemic injection test was performed using the method of A.S.T.M. and USP (13,14). Each mouse was weighed prior to the injection and a calculated dose of the extract was administered intraperitoneally or intravenously to the mice in accordance with Table 2. The mice were observed for gross signs of toxicity at 0 (immediately after injection), 4, 24, 48 and 72 hours
Table 2 Amount and routes of systemic injection of extracts or blank Rate (ml/seconds) 01 01

Extract or blank Normal saline Ethanol:normal saline (1:20) PEG 400 Sesame oil

Dose/kg

Route

50 ml Intravenous 50 ml Intravenous 10 g Intraperitoneal 50 ml Intraperitoneal

Figure 1. Incision wound closed with interrupted catgut.

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Figure 2. Texture Analyzer applying force to break the healed wound.

application (enough to cover the wound) of each aerosol. Rats were euthanised at intervals of 3, 6, 9 and 12 days after wounds creation and tensile strength analysis was performed on the same day.

Tensile strength test

Animals were euthanised and 6-cm long strips of skin having incision wound in the centre were cut out. Skin was xed in between two clamps of TA.XT2 Texture Analyzer (Figure 2), which was used to measure the tensile strength. Sutures were removed before analyzing the tensile strength of healed wounds (4). Crosssectional area of the wound was determined and force applied to break the wound was given automatically by a computer software of texture analyzer. Tensile strength of wounds was calculated using the following formula (18): Tensile strength = Breaking load(force)/ Cross-sectional area, where Cross-sectional area = Thickness Widthof skin strip.

Papp et al. (19,20) with slight modications. The rats were anaesthetized with pentobarbitone sodium (50 mg/kg body weight) that was administered intraperitoneally. Dorsal part of the animal was clipped with electric clippers followed by scrubbing the skin with 70% ethanol and normal saline. Burn wounds were created using a metal rod (15 cm diameter) heated to 95 2 C in water and exposed for 10 seconds. Control rats were untreated, while experimental rats were treated with formula P (without active ingredient), formula P1 and formula P2. The aerosols were placed in such a way that the wounds could be completely covered. Animals were randomly divided into four groups with six rats for each group. Experiment was conducted for 21 days to see the wound contraction. The wound size was determined using the tracing method (21,22). The wound outlines were drawn onto a clear plastic sheet with a marking pen and the areas were determined planimetrically. The condition of the wounds was examined and photographs were taken from 0 (before aerosols application) up to postwounding observation time. Wound closure was calculated as the percentage of wound area contraction using the formula: Percentage of wound closure = Area (0 day) Area (x day)/Area (0 day) 100%.

Statistical analysis
Results of tensile strength test and wound contraction were represented as mean SD. Oneway ANOVA followed by Duncan post hoc test was used to identify differences between groups. It showed a statistical difference if P < 005 was obtained.

RESULTS Primary skin irritation test

This test was performed to evaluate the potential of Haruan spray to cause irritation or allergic reactions through contact with intact and abraded skin of rabbits. The average values of primary dermal irritation index (PDII) for each formula and control are shown in Table 4. It is observed that Haruan spray for formula P1 and formula P2 gave values 022 and 014, respectively, which was below 1. Compared to Table 1, it showed that the index that
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Burn wound model

Adult, female SD rats, weight ranging between 200 and 300 g were selected in this study. The aerosols were assessed for burn wound model by using the test method of Priya et al. and
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Key Points
primary skin irritation testthe
ability of Haruan spray to cause irritation to the skin of rabbits was very small intra cutaneous testHaruan spray is safe and non irritant to the skin of rabbits injection testnone of the mice died during the observation period and no symptom related to toxicity was observed in these mice

Table 4 Average primary skin irritation index values, mean SD, N = 6 Formula P1 P2 Control Average primary skin irritation index values 022 04 014 016 0

Table 6 Classication of systemic injection test result, N = 5 Classication number of systemic injection response Extracting media Normal saline Ethanol:normal saline (1:20) PEG 400 Sesame oil 2b is decrease in motor activity. Control 0 2b 0 0 P1 0 2b 0 0 P2 0 2b 0 0

we got was below than that of very slight erythema, which indicates that the ability of Haruan spray to cause irritation to the skin of rabbits was very small. Comparison between the categories of PDII is as follows: 00 non irritant, 0005 negligible irritant, 0520 mild irritant, 2050 moderate irritant and 5080 severe irritant (23). Both responses can be regarded as negligible, indicating that both of the Haruan spray formulae are suitable to be used in wound dressing. However, there is a possibility for Haruan spray to cause allergic reactions to highly sensitive skin.

Systemic injection test

The components of Haruan spray need to be evaluated for its ability to cause toxicity reaction when applied to the wound which may penetrate into the blood stream. This test was conducted to evaluate the systemic response of mice to the extract of Haruan spray in order to ensure that there was no unexpectable systemic reaction from Haruan spray. Table 6 showed that none of the mice died during the observation period and no symptom related to toxicity was observed in these mice. For the formula which was extracted using ethanol:normal saline (1:20), all the mice showed the decrease in motor activity. It is suggested that injection of ethanol could decrease the amount of acetylcholine released into cortical cups (24) and the lack of acetylcholine decreased the motor system of the rat. This result was also observed in the control group, which strongly suggests that the response was not given by the Haruan spray component but by the ethanol as the media.

Intracutaneous test
This test was performed to evaluate the response of rabbits to the Haruan spray extracts following intracutaneous injections. Table 5 showed the irritation scores of response for each formula and control. It was observed that the response from rabbits to each extracts from four different media were similar to those of the control. These results supported the results of primary skin irritation test, indicating that Haruan spray is safe and non irritant to the skin of rabbits.
Table 5 Intracutaneous skin response of the extracted sample, mean SD, N = 10 Intracutaneous skin response 0 0 0 0 0 0 0 0 0 0 0 0

Tensile strength test

Figure 3 shows the condition of incision wounds that was observed in the 3rd, 6th, 9th and 12th day. There was no signicant
day 3 day 6

Formula P1

Extracting media Normal saline Ethanol:normal saline (1:20) PEG 400 Sesame oil Normal saline Ethanol:normal saline (1:20) PEG 400 Sesame oil Normal saline Ethanol:normal saline (1:20) PEG 400 Sesame oil

P2

day 9

day 12

Blank

Figure 3. Pictures of incision wound healing at days 3, 6, 9 and 12.

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However, in the last 10 days, there was no signicant difference between the groups.

DISCUSSIONS
Tensile strength of wounded skin will increase with time even though it is left untreated. Collagen is one of the major component that is mainly responsible for tensile strength of the skin (25). Collagen provides strength, integrity and structure in normal tissue. Collagen is needed to repair the injured part and bring back the structure and function (26). Collagen levels in a wound peak 23 weeks after injury but the tensile strength of the scar tissue continues to increase. The increase in strength relates to changes in the collagen structure from the early phase to the second phase that are more stable (5). Therefore, tensile strength increases at a slower pace and has been documented to increase up to 1 year after wounding in animal studies (27). A sufcient nutrition is required to enhance the proliferation of broblast cell to produce collagen bre network in wound healing. Cellular proliferation and protein synthesis in the process of wound healing occur at the wound site (28). The presence of amino and fatty acids in the lm produced by Haruan spray were suggested to promote the proliferation of broblast cells. It means that the production of collagen will increase thereby increasing the tensile strength. Some interventions can make the healing process faster or delay the healing time of the wound. In this study, the wounds were

Figure 4. Tensile strength of wounded skin at 3rd, 6th, 9th and 12th day after treatment with different formula. P, blank; P1, formula with haruan extract; P2, formula with haruan extract and fusidic acid. Data presented as mean SD, N = 6.

difference between the groups treated with formula P, P1 or P2 visually. In Figure 4, all groups show that an increase in tensile strength started from day 6 until day 9, but after day 9 there was no signicant increase in tensile strength. As in Figure 3, visually the conditions of all wounds were free of infection.

Key Points
collagen is one of the major
components that is mainly responsible for tensile strength of the skin collagen provides strength, integrity and structure in normal tissue collagen is needed to repair the injured part and bring back the structure and function collagen levels in a wound peak 23 weeks after injury but the tensile strength of the scar tissue continues to increase

Wound contraction
The graph in Figure 5 shows that the area of each wound contracted with time. Until day 3 there was no signicant difference between each group. From day 4, P1 treatment signicantly showed a higher percentage of wound closure compared with the control group. In the rst 10 days, the graph showed that the group treated with P1 gave a higher percentage of wound closure compared with other groups. At day 4 the percentage of closure for the group treated with P1 was nearly two times than that of the untreated group.

Figure 5. Percentage of wound closure for each group in different days. Control, (untreated); P, blank; P1, formula with Haruan extract; P2, formula with Haruan extract and fusidic acid. Data presented as mean SD, N = 6. *Signicance P < 005 compared with control.
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Key Points
in this study, the wounds were
exposed to the wound dressing which is a lm that was made from polymer with haruan extract as active ingredient the acceleration of wound closure for group treated with formula P2 was slow compared with the group treated by formula P1 this might be due to the presence of fusidic acid in formula P2 which inhibited the process of scarring and delayed the healing process the approach of aerosol system for wound dressing with nutrition gained from haruan water extract to accelerate incision and burn wound healing could provide an effective and safe application to reduce patient suffering, simplify therapeutic procedure and improve the quality of life

exposed to the wound dressing which is a lm that was made from polymer with haruan extract as active ingredient (6). The evaluation of the lm properties for the mechanical strength and water vapour permeability had been done in another study (29). As a wound dressing, Haruan spray can be a good intervention which can help to protect the wound from outside inuence (dust, bacteria) to enter the wound. It can reduce the risk of infection which can delay healing (30). The acceleration of wound closure for group treated with formula P2 was slow compared with the group treated by formula P1. This might be due to the presence of fusidic acid in formula P2 which inhibited the process of scarring and delayed the healing process. Zimmermann and Trust reported that antibiotics like gentamycin, tetracycline and ampicillin which induced disruption of protein synthesis affected the collagen construction and consequently the healing process (31). Therefore, we hypothesised that fusidic acid has the similar mechanism of action with the reported antibiotics. The use of topical antimicrobials which had succeeded in killing bacteria was thought to be harmful to the cells involved in wound healing, especially broblast cells (32,33). Emergency treatment as rst aid is important to bring down the temperature of the burn site. The cooling effect from the propellant of aerosol could cool down the temperature of burn site to prevent the progression of the burn. It can help to stop the burning process by cooling down the burn site below the temperature required to cause an injury. Prolonged inammation caused by the high progression of burn can be prevented by this cooling effect. The increase of inammation beyond certain level will lead to healing impairment like destruction of early migratory effect and delay healing progression. This could probably be the reason why the untreated group in the rst week observation showed a slow progress of closure.

simplify therapeutic procedure and improve the quality of life.

ACKNOWLEDGEMENT
The authors would like to thank the staff of the animal facility for their invaluable assistance. This research was supported by a Grant number 1001/PFarmasi/815041 from Universiti Sains Malaysia.

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3 4

5 6

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CONCLUSION
The approach of aerosol system for wound dressing with nutrition gained from haruan water extract to accelerate incision and burn wound healing could provide an effective and safe application to reduce patient suffering,
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