<i>Acorus gramineus</i> ethanol extract suppresses MITF-mediated melanogenesis under oxidative stress conditions

<i>Acorus gramineus</i> 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 <i>Acorus gramineus</i> 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.