Mechanism of Wound-Healing Activity of Hippophae rhamnoides L. Leaf Extract in Experimental Burns

Abstract

The present investigation was undertaken to evaluate the healing efficacy of lyophilized aqueous leaf extract of Sea buckthorn (Hippophae rhamnoides L., family Elaeagnaceae) (SBT) and to explore its possible mechanism of action on experimental burn wounds in rats. The SBT extract, at various concentrations, was applied topically, twice daily for 7 days. Treatment with silver sulfadiazine (SSD) ointment was used as reference control. The most effective concentration of the extract was found to be 5.0% (w/w) for burn wound healing and this was further used for detailed study. The SBT-treated group showed faster reduction in wound area in comparison with control and SSD-treated groups. The topical application of SBT increased collagen synthesis and stabilization at the wound site, as evidenced by increase in hydroxyproline, hexosamine levels and up-regulated expression of collagen type-III. The histological examinations and matrix metalloproteinases (MMP-2 and -9) expression also confirmed the healing efficacy of SBT leaf extract. Furthermore, there was significant increase in levels of endogenous enzymatic and non-enzymatic antioxidants and decrease in lipid peroxide levels in SBT-treated burn wound granulation tissue. The SBT also promoted angiogenesis as evidenced by an in vitro chick chorioallantoic membrane model and in vivo up-regulated vascular endothelial growth factor (VEGF) expression. The SBT leaf extract had no cytotoxic effect on BHK-21 cell line. In conclusion, SBT aqueous leaf extract possesses significant healing potential in burn wounds and has a positive influence on the different phases of wound repair.

Figure 1

Density of blood vessels in in vitro CAM assays of chick eggs (12-days-old) for neovascularization. Control eggs were loaded with sterile methylcellulose disks and experimental eggs were treated with 80 μg SBT leaf extract impregnated in methylcellulose disks. Blood vessels were analyzed in terms of number of red pixel per unit area using NIH Image J software (v1.38) and AngioQuant software. Values are mean ± SE of six eggs. *P < .05 compared with control untreated eggs.

Figure 2

Histopathological changes on eighth post-wounding day in skin wound section of (a) control rats showing non-epithelialized wound surface with the presence of inflammatory cells, (b) SBT leaf extract (5.0%) and (c) SSD treated rats showing wound surface with well-organized thick epithelium. Collagen alignment is well developed in SBT leaf extract treated burn wounds. Scale bar, 100 μm.

Figure 3

Masson's trichome staining for collagen on eighth-day postwounding in skin wound section of (a) control rats showing less and irregularly arranged collagen, (b) SBT leaf extract (5.0%) treated rats showing compact and well-aligned collagen fibers. Scale bar, 20 μm.

Figure 4

(a) Matrix metalloproteinase expression by gelatin zymography (10% SDS-PAGE, 1 g L⁻¹ gelatin) in SBT leaf extract (5.0%) treated and untreated burn wound tissue of experimental rats after 4 and 7 days of treatment. (b) Expression of standard marker proteins (200–6.9 kDa, BIO RAD, 10% SDS–PAGE). (c) VEGF and collagen type-III analyzed by western blot in SBT leaf extract (5.0%) treated and untreated wound tissue of experimental rats after 4 and 7 days of topical treatment.

Figure 5

The schematic diagram showing the possible effect of the SBT leaf extract in promoting wound-healing activity.