Evaluation of Wound Closure Activity of Nigella sativa, Melastoma malabathricum, Pluchea indica, and Piper sarmentosum Extracts on Scratched Monolayer of Human Gingival Fibroblasts

Abstract

Nigella sativa, Melastoma malabathricum, Pluchea indica, and Piper sarmentosum are common Asian traditional medicines to treat minor wounds. This study aimed to investigate the in vitro wound healing properties of aqueous extracts of these plants using human gingival fibroblast (HGF) monolayer as study model. DPPH scavenging activity of the extracts was evaluated and effect on HGF proliferation was determined. Their effect on HGF's function to synthesize collagen was indicated by the level of hydroxyproline produced and effect on wound healing activity was assessed using an in vitro scratch assay. The influence of the extracts on expression of bFGF and TGF-β was also determined. Results revealed all four extracts to exhibit low free radical scavenging activity. The extract from N. sativa (NSSE) compared to the others showed favourable enhancement of HGF proliferation with EC50 of 22.67 ± 3.06 µg/mL (P < 0.05) with accelerated wound closure activity despite its nonsignificant effect on collagen synthesis. In addition to the elevated level of bFGF by up to 15% at 100 µg/mL of NSSE, a slightly better effect was observed on the expression of TGF-β. NSSE thus showed that promising wound healing properties and data obtained may contribute towards validation of its traditional use for the healing of oral wounds.

Figure 1

The proliferative effect of four plants extracts on HGF as indicated by the increase in percentage of HGF population following exposure to the extracts. The rate of cell proliferation of NSSE-treated cell was linear at concentrations below 50 µg/mL but slowed down at higher concentrations above 50 µg/mL. The values plotted were the mean of triplicate tests (n = 3). P values at P < 0.05 were indicated by (∗) and at P < 0.01 by (∗∗).

Figure 2

Micrographs showing the coverage of scratched wounds by HGF under various conditions at 42 h of incubation. (a) Negative control (HGF in basic media); (b) positive control (HGF treated with 10 ng/mL bFGF); and (c) test sample (HGF treated with 25 μg/mL NSSE). The red lines marked the boundaries of the scratched wounds and the arrows indicated the direction of cells movement to cover the wound areas.

Figure 3

Quantitative measurement of cells number migrating in the corresponding scratched wound areas in Figure 2. The control was HGF in basic media, NSSE was the test sample at 25 μg/mL, and bFGF was an enhancer added at 10 ng/mL. The values plotted were means of 3 determinations (n = 3). P values were indicated at P < 0.05 by (∗) and at P < 0.01 by (∗∗).

Figure 4

A bar chart indicating the production of collagen by HGF under various culture conditions. DMEM/0.3% FBS was a basic medium used as a negative control; DMEM/10% FBS was an enriched medium used as a positive control; allantoin (25 µg/mL) is a skin enhancer often used in skincare products which was used for comparative purpose; and NSSE (25 µg/mL) was the test sample. The values plotted were means of 6 determinations (n = 6). P values at P < 0.01 were indicated by (∗∗).

Figure 5

A bar chart showing the effect of NSSE on the production of bFGF by HGF. Two different concentrations at 25 and 100 μg/mL were used and compared to a control. The values plotted were means of 3 determinations (n = 3). P values at P < 0.01 were indicated by (∗∗).

Figure 6

A bar chart showing the effect of NSSE on the production of TGF-β by HGF. Two different concentrations at 25 and 100 μg/mL were used and compared to a control. The values plotted were means of 3 determinations (n = 3). P values at P < 0.05 were indicated by (∗).