A novel therapeutic effect of mannitol-rich extract from the brown seaweed Sargassum ilicifolium using in vitro and in vivo models

Abstract

Abstract Background Wound healing is an active, complex, integrated series of cellular, physiological, and biochemical changes initiated by the stimulus of injury in a tissue. The present study was performed to investigate the potential wound healing abilities of Sargassum ilicifolium crude extracts (CE) that were characterized by 1H NMR and FTIR Spectrometric measurements. Materials and methods Seaweed samples were collected from southern coastal sites of Sri Lanka. To determine the cytotoxicity and proliferation of S. ilicifolium CE were used for the MTT and alamarBlue assays respectively. The scratch and exclusion wound models were used to HaCaT and HDF cells to assess the cell proliferation and migration. RAW 264.7 cells (macrophages) were used to evaluate Nitric Oxide (NO) production and phagocytosis activities. Moreover, Fifteen, 8-week-old, female, New Zealand rabbits were selected and divided into five groups: excision skin wounds (10.40 ± 0.60 mm) were induced in groups I, II, and III. Rabbits in groups I and IV were given S. ilicifolium CE (orally, 100 mg/kg day, two weeks), whereas groups II and V were given equal amounts of distilled water. Wound healing properties were measured and wound tissue samples were collated, formalin-fixed, wax-embedded, stained (Hematoxylin and Eosin; Van Gieson) and examined for the healing process. Results Anti-inflammatory and wound healing activities were observed in RAW 264.7, HDF and HaCaT cells treated with S. ilicifolium aqueous extracts when compared to the control groups. S. ilicifolium extracts concentration 8 - 4 μg/μL, (P<0.05) had remarkable the highest proliferative and migratory effects on RAW 264.7, HDF and HaCaT cells when compared with the control. RAW 264.7 cell proliferation and/or migration were higher in S. ilicifolium extracts (4 μg/μL, 232.8 ± 10.07%) compared with the control (100 %). Scratch wound healing were remarkably enhanced in 24 h, 48 h (P<0.05) when treated with S. ilicifolium on HaCaT cells. Rabbits treated with the CE of S. ilicifolium showed a significantly increased wound healing activities (P<0.05) within three days with a close wound area of 57.21 ± 0.77 % compared with control group (26.63 ± 1.09 %). Histopathology, aspartate aminotransferase and alanine aminotransferase levels evidenced no toxic effects on seaweed treated groups. Histopathological results also revealed that the healing process was significantly faster in the rabbit groups which were as treated with CE of S. ilicifolium orally with the evidence of enhanced early granulation tissue (connective tissue and angiogenesis) and significant epithelization compared to the control. Conclusions Cell proliferation and migration are significantly faster when treated with S. ilicifolium aqueous extracts. Moreover, there are no toxic effect of S. ilicifolium aqueous extracts on RAW 264.7, HDF and HaCaT cell lines. In this study, it is revealed that S. ilicifolium has potential remedial agent; D-Mannitol for skin wound healing properties that by promote keratinocyte and fibroblast proliferation and migration. These findings show that S. ilicifolium have promising wound healing properties.

Keywords

• Sargassum ilicifolium
• D-mannitol
• HDF
• HaCaT
• RAW 264.7
• Toxicity