Abstract: The effects of topical application of Benzyl N'-(indol-3-Ylmethylidene)-Hydrazinecarbodithioate derivatives (BHC) on the process of wound healing enclosure and histology of healed wound were assessed. Five groups of adult male Sprague Dawley rats, all animals were experimentally wound in the posterior neck area. About 0.2 mL of 10% Tween 20 solution was applied to the wound of Group 1 animals (negative control). Intrasite gel was applied topically to wounds of Group 2 animals as reference. About 0.2 mL of 25, 50 and 100 mg mL^-1 of compound derivative were applied to the wounds of Group 3, 4 and 5, respectively. Macroscopically, wound dressed with compound derivative significantly healed earlier than those treated with 10% Tween 20 solution. Also wound dressed with 100 mg mL^-1 compound derivative accelerate the rate of wound healing compared to those dressed with Intrasite and 25 and 50 mg mL^-1 compound derivative. Histological analysis of healed wounds dresses with compound derivative showed comparatively less scar width at wound enclosure and healed wound contained less macrophages and large amount of collagen with angiogenesis compared to wounds dressed with 10% Tween 20 solution. In conclusion, wounds dressed with 100 mg mL^-1 of compound derivative significantly enhanced the acceleration of the rate of wound healing enclosure and histologically healed wounds contain comparatively less macrophages and more collagen with angiogenesis.

INTRODUCTION

Indole derivatives constitute an important class of therapeutical agent in medicinal chemistry including antidepressive (Joshi and Chand, 1982), ant allergic (Unangst et al., 1989), antimicrobial (Canoira et al., 1989), antioxidant (Kaneko et al., 2000; Liu and Ng, 2000). On the other hand, antimicrobial (Tarafder et al., 2002), Anticancer (Ali et al., 2002), antibacterial and antifungal (Singh and Varshney, 2006) properties have been reported to be associated with the schiff bases derived from S-alkyl- or S-aryldithiocarbazate.

There are no data available regarding wound healing processing of this derivative compound in rats. Therefore, The present study was undertaken to evaluate the rate of wound healing properties of derivatives (BHC) in experimental rats. The current study revealed that wounds treated with derivatives (BHC) as topical application of wounds significantly accelerate the wound healing process.

MATERIALS AND METHODS

Preparation of S-benzyldithiocarbazate: This compound was synthesized as reported previously (Tarafder and Ali, 1980). A mixture of hydrazine hydrate (10 g, 0.2 moL) and potassium hydroxide (11.4 g, 0.2 moL) in 90% ethanol (70 mL) was cooled in an ice bath. Carbon disulphide (15.2 g, 0.2 moL) was then added drop-wise with vigorous stirring. The temperature of the reaction mixture was not allowed to rise above 5°C during the period of addition of carbon disulfide. To the mixture, 40% ethanol (60 mL) was added and the solution was cooled in ice. Benzyl chloride (25.3 g, 0.2 moL) was then added slowly with vigorous stirring. The white product was separated by filtration, washed with water and dried in air. The crude product was recrystallized from absolute ethanol yield 23 g (58%).

Preparation of benzyl N'-(indol-3-ylmethylidene)- hydrazinecarbodithioate: The Schiff base was synthesized as reported previously (Khaledi et al., 2008). Indole-3-carbaldehyde (4.35 g, 0.03 moL) and S-benzyl dithiocarbazate (5.94 g, 0.03 moL) were heated in methanol (300 mL) for 3 h. The solution was set aside for few hours whereupon a yellow precipitate formed. This precipitate was filtered off, washed with cold ethanol and dried over silica gel yield, 8.3 g (85%) (Fig. 1).

Intrasite gel: Intrasite gel was purchased from University Malaya Medicial center Pharmacy. Intrasite gel is a colorless transparent aqueous gel which contains a modified Carboxy Methyl Cellulose (CMC) polymer together with propylene glycol as a humectants and preservative. Intrasite gel is an amorphous hydrogel which gently re-hydrates necrotic tissue, facilitate autolytic debridemen while being able to loosen and absorb slough and exudates, cleaning the way for effective wound healing.

It is also designed for wounds that are granulating and epithelialising. It can also be used to provide the optimum moist wound management environment during the later stages of wound closure. It is non-adherent and does not harm viable tissue or the skin surrounding the wound. This makes Intrasite gel ideal for every stage in the wound management process. (Intrasite gel is a trademark for Smith and Nephew Ltd) (Williams, 1994).

Fig. 1:	Benzyl N'-(indol-3-ylmethylidene)-hydrazinecar-bodithioate

Lignocaine HCl (2%, 100 mg 5 mL^-1): The local anesthesia was purchased from experimental animal house, Faculty of Medicine, University Malaya. About 1 mL of Lignocaine was injected subcutaneous.

Experimental animals: Sprague Dawley adult male rats were obtained from the experimental animal house, Faculty of Medicine, University of Malaya and Ethic No. PM/27/07/2009/MAA (R). The rats were divided randomly into 5 groups of 6 rats each. Each rat that weighted between 200-220 g was housed separately (one rat per cage). The animals were maintained on standard pellet diet and tap water. The study was approved by the ethics Committee for animal experimentation, Faculty of Medicine, University of Malaya, Malaysia. Throughout the experiments, all animals received human care according to the criteria outlined in the Guide for the Care and Use of laboratory Animals prepared by the National Academy of Sciences and published by the national Institute of health.

Experimentally induced wounds: The animals were anesthetized by diethyl ether. The skin shaved by electrical shaver, disinfected with 70% alcohol and injected with 1 mL of Lignocaine HCl (2%, 100 mg 5 mL^-1). An area of uniform wound 2.00 cm in diameter was excised from the nape of the dorsal neck of all rats with the aid of round seal as described by Morton and Malone (1972), (Fig. 2) with slight modification. Avoid incision of the muscle layer and tension of skin was kept constant during the procedure. The wound area was measured immediately under light diethyl ether anesthesia as described by Nayak and Pinto Pereira (2006) with slight modification.

Fig. 2:	About 2 cm diameter excision skin wound on day 0 before application of vehicle

Topical application of vehicles: Wounds of Group 1 animals were dressed with 0.2 mL of 10% Tween 20 solution as a negative control twice daily. Wounds of Group 2 rats were dresses topically with Intrasite gel as a reference, twice daily. About 0.2 mL of 25, 50 and 100 mg mL^-1 of compound derivative were applied topically twice daily to the wound of Group 3, 4 and 5 (experimental animals), respectively as described by Chah et al. (2006) with slight modification. The wound was observed daily until complete epithelization.

The wound closure rate was assessed by tracing the wound on days 1, 5, 10 and 15 post-wounding using transparency papers and permanent marker as described by Nayak and Pinto Pereira (2006) with slight modification. The wound areas recorded were measured using a graph paper. Number of days required for falling of scar without any residual raw wound gave the period of epithelization. The percent wounds healing on these days are determined.

Histological evaluation of healed wounds: The skin specimen from wounds healed areas were fixed in 10% buffered formalin and processed by paraffin tissue processing machine. The healed skin was assessed by taking a 5 μ section stained with hematoxylin and eosin.

Statistical analysis: All values are reported as mean±SEM and the statistical significance of differences among groups were assessed using one-way ANOVA. A value of p<0.05 was considered significant.

RESULTS AND DISCUSSION

Wounds dressed with compound derivative showed considerable signs of dermal healing and significantly (p<0.05) healed earlier compared to wounds dressed with 10% Tween 20 (negative control) (Table 1, Fig. 3 and 4). Animals in the 100 mg mL^-1 of compound derivative treated group showed significantly better healing and healed faster compared to animals in the 25 and 50 mg mL^-1 compound-treated groups.

There were no significant differences between wounds dressed with 25 and 50 mg mL^-1 of compound derivative in the term of duration of wound healing enclosure (Table 1). Histologically, wound dressed with compound derivative contained comparably less scar at wound enclosure and healed wound contained few macrophages and more collagen and proliferating blood capillaries compared with wound dressed with 10% Tween 20 solution (Fig. 5-8).

Table 1:	Time required for wound healing by compound derivative in experimental animals
All values were expressed as mean and ±standard error mean. Mean with different superscripts were significantly different (p<0.05)

Fig. 3:	Complete wound healing on day 12 with 0.2 mL of 100 mg kg-1 compound derivative on day 12

Fig. 4:	Histological section of healed wound dressed with 0.2 mL of 100 mg kg-1 compound derivative showng comparatively narrow scar at the wound closure (H and E stain x4)

In the present study, topical application of compound derivative significantly enhanced the rate of wound healing and histologically, healed wound contain less macrophages, more collagen and angiogenesis. Wound healing effects may be due to up-regulation of collagen expression (Bonte et al., 1993) and an increase in tensile strength of the wounds (Suguna et al., 1996).

Fig. 5:	Histological section of healed wound dressed with 0.2 mL of Tween 80 (negative control) showing comparatively wide scar at the wound closure (H and E stain x4)

Fig. 6:	Histological section of healed wound dressed with 0.2 mL of 100 mg k-1 compound derivative. Granulation tissue contains comparatively more collagen, fibroblast and bloog capillaries and few or absence of inflammatory cells (macrophages) (H and E stain x4)

Similarly, enhanced healing activity has been attributed to increased collagen formation and angiogenesis (Trabucchi et al., 1986; Shukla et al., 1999). Collagen played a central role in the healing of wounds and it is a principal component of connective tissue and provides a structural framework for the regenerating tissue (Cohen et al., 1992). Angiogenesis in granulation tissues improves circulation to the wound site thus providing oxygen and nutrients essential for the healing process (Szabo et al., 1995) that include re-epithelization.

Stimulate epithelial cell proliferation and angiogenesis are important for wound healing process (Buntrock et al., 1982). With the consistence of the present study, Habibipour et al. (2003) showed that histological analysis of the treated healed wound group contained a large amount of fibroblast proliferation, collagen synthesis and neovascularization which resulted in an increased wound tensile strength and accelerated healing wound.

Fig. 7:	Incomplete wound healing on day 17 in animal treated with 0.2 mL of 10% Tween 80 (negative control)

Fig. 8:	Histological section of healed wound dressed with 10% Tween 20 (negative control). Granulation tissue contains less collagen, fibroblast and blood capillaries and more inflammatory cells (macrophages) (H and E stain x40)

Wound healing mechanisms may be contributed to stimulate the production of antioxidants in wound site and provides a favorable environment for tissue healing (Shukla et al., 1999).

Compound derivative have shown antioxidant activity (Kaneko et al., 2000; Liu and Ng, 2000). Antioxidants have been reported to play a significant role in the wound healing process. It appears that antioxidant may be an important contributory factor in the wound healing property (Shukla et al., 1999). Topical applications of compounds with antioxidant properties significantly improve wound healing and protect tissues from oxidative damage (Martin, 1996).

CONCLUSION

In conclusion, the current study revealed that the dressed with compound derivative, as topical application of wounds enhanced significantly wound healing process.

ACKNOWLEDGEMENTS

The researchers would like to express their gratitudes to the staff of the Faculty of Medicine Animal House for the care and supply of rats. The researchers would also like to acknowledge the Universiti Teknologi MARA for financial support under the DANA (600-RMI/ST/DANA 5/3/Dst (289/2009) and FRGS(600-RMI/ST/FRGS 5/3/ Fst (34/2010) grants. In addition the authors would also like to thank the University of Malaya for its contribution to this research.