Acorus gramineus ethanol extract suppresses MITF-mediated melanogenesis under oxidative stress conditions

Acorus gramineus has long been used in East Asia and exhibits diverse biological activities, yet its effects on melanogenesis under oxidative stress remain unclear. This study evaluated the antioxidant capacity of Acorus gramineus ethanol extract (AGEE) and its regulatory effects on melanogenesis in B16F1 melanoma cells under normal and oxidative stress conditions. AGEE showed concentration-dependent antioxidant activity in DPPH radical scavenging, reducing power, hydroxyl radical scavenging, and lipid peroxidation assays, while maintaining low cytotoxicity up to 25 µg/mL. In cell-free systems, AGEE directly inhibited mushroom tyrosinase activity and L-DOPA oxidation. In cellular models, AGEE significantly reduced melanin production under both basal and H₂O₂-induced oxidative stress conditions, with stronger inhibition observed in oxidatively damaged cells, even during α-MSH stimulation. Western blot analysis indicated that oxidative stress decreased melanogenesis-related proteins, including MITF, TYR, TRP-1, and TRP-2. Although α-MSH partially restored MITF expression, AGEE co-treatment attenuated α-MSH-induced MITF upregulation and suppressed downstream melanogenic enzymes in a concentration-dependent manner. Immunofluorescence analysis confirmed reduced MITF and TYR signals following AGEE treatment. Collectively, AGEE exerts multi-level inhibitory effects on melanogenesis via direct enzymatic inhibition, modulation of MITF-dependent pathways, and attenuation of oxidative stress. AGEE effectively suppresses melanogenic activation under both normal and oxidative stress conditions, with enhanced efficacy in redox-imbalanced environments. These findings suggest AGEE as a promising functional ingredient for managing oxidative stress-associated hyperpigmentation.