PERTANIKA JOURNAL OF TROPICAL AGRICULTURAL SCIENCE

Osteoporosis affects over 200 million people worldwide, with its prevalence expected to rise due to ageing populations. Side effects associated with long-term use of synthetic treatments have driven interest in natural alternatives. Alpha-Asarone, a natural metabolite found in several therapeutic plants, was identified as a major metabolite in Piper sarmentosum Roxb. (kaduk), a local plant known for its bone-protective properties. While Alpha-Asarone is known for its neuroprotective and anti-inflammatory properties, its osteogenic role remains unexplored. This study aimed to assess the cytotoxicity of Alpha-Asarone and its potential to induce osteogenic differentiation in vitro using MC3T3-E1 pre-osteoblast cells. Cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay across Alpha-Asarone concentrations (0–800 μM). For osteogenic evaluation, cells were treated with 1–20 μM Alpha-Asarone over 21 days. Osteogenic differentiation was assessed through ALP activity and von Kossa staining. Untreated cells served as the negative control, while cells treated with ascorbic acid (50 μg/mL) and β-glycerophosphate (10 mM) served as the positive control. Alpha-Asarone exhibited a dose-dependent cytotoxic response in the cytotoxicity assay, with no cytotoxic effects at the concentrations used for osteogenic evaluation. After 21 days, von Kossa staining revealed a significant increase in mineralised matrix formation, indicated by intensified dark brown staining, with a 95.1% increase. ALP activity was also elevated, with the highest increase at 174%. Alpha-Asarone at 1 µM produced the most significant enhancement in both mineralisation and ALP activity. Alpha-Asarone showed its promise as a natural osteogenic agent by effectively and safely inducing MC3T3-E1 cell differentiation.