Abstract: The ethanolic extract of Lantana camara leaf was evaluated for their wound healing potential in rats. Four groups of adult male Wistar albino rats were experimentally wound in the posterior neck area. A thin layer of blank placebo was applied topically to wounds of Group 1. Wound of Group 2 and 3 animals were dressed with a thin layer of placebo containing 5 and 10% L. camara extract, respectively. A thin layer of intrasite gel was applied topically to wounds of Group 4 animals as reference. The effects of these topical applicants on the rate of wound healing and histology were assessed. Wound dressed with placebo containing plant extracts significantly healed earlier than those treated with blank placebo. Wounds dressed with placebo containing 10% extract significantly accelerate wound healing activity compared to wounds dressed with placebo containing 5% extract. Histological analyses of healed wounds confirmed the results. Wounds dressed with placebo containing extracts showed markedly less scar width at the wound enclosure and wounds contained large amounts of fibroblast proliferation and more mature and densely packed collagen with accompanying angiogenesis compared to wounds dressed only with blank placebo. We conclude that L. camara extract significantly enhanced the acceleration rate of wound enclosure in rats.

INTRODUCTION

Lantana camara L. (Verbenaceae) is listed as one of the important medicinal plants of the world (Ross, 1999). Lantana plant has been reported to possess a number of medicinal properties (Ghisalberti, 2000). Various parts of the plant are used in the treatment of itches, cuts, ulcers, swellings, bilious fever, catarrh, eczema, dysentery and chest complaints of children, fistula, pustules and rheumatism (Agarwal, 1997). Some metabolites isolated from their leaves have antitumor activity (Mahato et al., 1994); inhibitor of protein kinase C (Herbert et al., 1991) antiviral (Inada et al., 1995); antinociceptive and antipyretic activity (Uzcategui et al., 2004); antithrombin activity (O’ Neill et al., 1998); antimicrobial (Kumar et al., 2006); antimutagenic (Barre et al., 1997); antimotility (Sagar et al., 2005); antiinflammatory (Basu and Hazra, 2006); insecticidal and termicidal effects (Bouda et al., 2001; Verma and Verma, 2006) and antioxidant activity (Saini et al., 2007). There are no data available regarding its wound healing potential of L. camara leaves. Therefore, the present study was undertaken to evaluate the wound healing property of ethanolic leaf extract in rats.

MATERIALS AND METHODS

Placebo: An aqueous cream placebo was obtained from Department of Pharmacy, Faculty of Medicine, University of Malaya (Sunward Pharmaceutical SDN BHD. MAL 19920890X).

Intrasite gel: Intrasite gel was purchased from University Malaya Medicial center Pharmacy. Intrasite gel is a colorless transparent aqueous gel, which Contains a Modified Carboxymethylcellulose (CMC) polymer together with propylene glycol as a mumectant 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).

Lignocaine HCl (2%, 100 mg/5 mL): The local anesthesia was purchased for Animal House, Faculty of Medicine, University Malaya.

Plant materials: The leaf of Lantana camara were collected from University Malaya garden and identified by comparison with specimens available at the Herbarium of Rimba Ilmu, Institute of Science Biology University Malaya. The plants were, wash with water and dried in incubator at 50°C for 5-7 days. The dried materials were ground to powder using a grinder.

Preparation of L. camara alcoholic extract: The dried powder was extracted by maceration in ethanol (100 g/1500 mL, w v^-1) in a conical flask for 5 days at 37°C. Afterwards, the solvent was filtered using a filter funnel and the solvents were distilled under reduced pressure in EYELA rotary evaporator until excess solvent evaporated. The alcoholic extract of L. camara was mixed homoge-neously with blank Vaseline in the concentration of 5% and 10% (w w^-1) each. The extracts were kept in fridge at 4°C until used.

Experimental animals: Wistar albino rats were obtained from the animal house, Faculty of Medicine, University of Malaya and Ethic No. PM 28/-9/2006 MAA (R). The rats were divided randomly into 4 groups of 6 rats each. Each rat that weighted between 200-250 g was housed separately. The animals were maintained on standard pellet diet and tap water.

Experimentally induced wounds: The animals were anesthetized by diethyl ether. The skin shaved, disinfected with 70% alcohol and injected with 1 mL of Lignocaine HCl (2%, 100 mg/5 mL). An circle 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 Melone (1972) with slight modification (Fig. 1). Avoid incision of the muscle layer and tension of skin was kept constant during the procedure.

Fig. 1:	2.00 cm diameter excision skin wound on day 0 before application of vehicle

Topical application of vehicles: Group 1 animals were dressed topically twice daily with thin layer of blank placebo. Thin layer of placebo containing 5 and 10% Lantana camara extracts each were applied topically twice daily to the wounds of Group 2 and 3 rats, respectively. Thin layer of Intrasite gel were topically applied twice daily to Group 4 animals as reference. The wound was observed daily until complete wound-healing process occurs and then the animals were sacrificed by overdose of diethyl ether.

Histological evaluation of healed wounds: Specimens of skin from healed wounds from each rat were fixed in 10% buffered formalin solution for histopathological studies. Sections of the healed skin were made at a thickness of 5 μL, stained with hematoxylin and eosin (H and E), were assessed for histopathological changes. The microscopic slides were photographed.

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

Wounds treated with alcoholic extracts of L. camara or with Intrasite gel showed considerable signs of dermal healing and significantly (p<0.05) decrease mean wound healing time than those treated with blank placebo (Table 1, Fig. 2 and 3). Also wounds treated with 10% extract or Intrasite gel significantly possesses better healing and healed faster compared to wounds treated with 5% extract in placebo. There were no significant differences between wounds treated with 10% L. camara extract and Intrasite gel in the term of duration of wound healing (Table 1).

Table 1:	Time required for wound healing by L. camara in experimental animals
All values were expressed as mean and ±Standard Error Mean; Mean with different superscripts were significantly different (p<0.05)

Fig. 2:	Complete wound healing with 10% L. camara extract on day 11

Fig. 3:	Wound healing on day 19 in animal treated with blank placebo

Fig. 4:	Histological section of a healed wound treated with 10% L. camara extract showing an intermediate scar region (H and E stain x40)

Histologically, wounds dressed with methanolic extracts of L. camara or Intrasite gel contained markedly fewer inflammatory cells, less scarring at the wound enclosure, more proliferating blood capillaries (angiogenesis) and more collagen fibres compared to wounds dressed only with blank placebo (Fig. 4-7).

Fig. 5:	Histological section of a negative control wound showing a very broad scar region (H and E stain x40)

Fig. 6:	Histological section of a healed wound dressed with placebo containing 10% L. camara extract. Granulation tissue contains more collagen, fibroblast and blood capillaries and there is an absence of inflammatory cells (H and E stain x80)

Fig. 7:	Histological section of a healed wound dressed only with blank placebo. Granulation tissue con-tains less collagen, fibroblast and blood capillaries and more inflammatory cells (H and E stain x80)

DISCUSSION

In the present study, topical application of L. camara extracts significantly enhanced the rate of wound healing. Wound healing effects may be due to up-regulation of human collagen I expression (Bonte et al., 1993) and an increase in tensile strength of the wounds (Suguna et al., 1996). Enhanced healing activity has been attributed to increased collagen formation and angiogenesis (Shukla et al., 1999). Collagen occupies a central role in the healing of wounds, as it is a principal component of connective tissue and provides a structural frame-work, strength and milieu 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. Growth factors such as basic fibroblast growth factor stimulate epithelial cell proliferation and angiogenesis thus are important to accelerate wound healing activity (Buntrock et al., 1982). Oxidative stress plays a major role in the pathogenesis of various diseases including wounds and ulcers. L. camara leaf extract has shown to contain flavonoids (Pan et al., 1993) and terpens (Begum et al., 2003), which may mediate the wound healing process. L. camara prevented lipid peroxidation that may suggest that it may be acting as antioxidant (Saini et al., 2007). 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). Studies have shown the topical application of compounds with antioxidant properties on patients or animals to significantly improve wound healing and protect tissues from oxidative damage (Martin, 1996). L. camara leaf possesses antitumor, anti-viral, antibacterial and anti-inflammatory activities and thus the wound healing activity could be attributed to these properties (Xu et al., 1996). Wound healing properties of L. camara also could be due to antimicrobial activity (Deena and Thoppil, 2000). L. camara extract exhibited significant antimicrobial activity and properties that support folkloric use in the treatment of some diseases as broad spectrum antimicrobial agents. This probably explains the use of these plants by the indigenous people against a number of infections since generation (Kumar et al., 2006).

CONCLUSION

In conclusion, the current study revealed that methanolic extracts of). L. camara were suitable as topical application for wounds as indicated by the significantly faster rate of healing and reduced scarring at the wound enclosure. Histologically, there were also comparably fewer inflammatory cells, more angiogenesis and collagen fibres in the granulation tissue.

ACKNOWLEDGEMENT

The authors express gratitude to the staff of the Faculty of Medicine Animal House for the care and supply of rats and to the University of Malaya for financial support through the Science fund grant 12-02-03-2051.