Acidified ethanolic extract of Sembada Hitam black rice bran protects cell cycle and cellular senescence of H2O2-induced NIH3T3 fibroblasts

Hydrogen peroxide (H2O2) a reactive oxygen species formed by the body, which in high concentrations leads to cellular senescence. Bran of Sembada Hitam black rice is rich in antioxidants that may protect cells from oxidative damage. This research evaluates the potential of Sembada Hitam Bran Acidified Ethanolic Extract (SHB-AEE) in preserving cell cycle progression and mitigating senescence in NIH3T3 fibroblasts exposed to H2O2. SHB-AEE was obtained through maceration using a mixture of 85 mL ethanol absolute and 15 mL HCl 1N as an acid modifier. The cytotoxicity of SHB-AEE was determined using the MTT assay. Based on cytotoxicity results, NIH3T3 were sybsequenly treated with specific concentrations of H2O2 and SHB-AEE for cell cycle analysis via flow cytometry and senescence-associated-β-galactosidase (SA-β-Gal) staining. Data were analyzed with ANOVA followed by Tukey HSD for cytotoxicity, cytoprotective, and SA-β-Gal assay or Duncan Test for cell cycle data (p ¤ 0.05). Results showed that SHB-AEE was non-cytotoxic, maintaining cell viability above 80. At low concentrations, SHB-AEE promoted normal cell cycle progression and reduced senescence in NIH3T3 fibroblasts under oxidative stress. However, at high concentrations, it exhibited the opposite effect, indicating a strong dose-dependent response. These findings suggest that SHB-AEE has potential antioxidant and cytoprotective properties against oxidative stress-induced senescence, but further studies are required to elucidate its molecular mechanisms and optimize treatment concentration for maximum cytoprotection. © 2026 Marmara University Press.