4.5. Anti-Melanogenesis Activity Assays

The anti-melanogenesis activities of compounds 2b and 2f were assessed using a standard melanin content assay with minor modifications [51]. Briefly, B16F10 cells were seeded at a density of 5 × 10⁴/well in a 24-well plate and allowed to adhere to wells in a DMEM solution containing penicillin/streptomycin (100 IU/100 µg/mL) and 10% heat-inactivated FBS in a standard humidified 5% CO₂ atmosphere at 37 °C. After culture for 24 h, cells were exposed to α-MSH (0.5 µM for 2b and 1.0 µM for 2f) and various concentrations (0, 5, 10, or 25 µM) of 2b or 2f or kojic acid at 25 µM in a standard humidified 5% CO₂ atmosphere at 37 °C for 48 h. Cells were then rinsed twice with PBS and lysed in 200 µL of 1N NaOH containing 10% dimethyl sulfoxide (DMSO) for 1 h at 60 °C. Lysates were transferred to the wells of a 96-well plate, and melanin absorbances were measured at 405 nm using a microplate reader (VersaMax^TM, Molecular Devices, Sunnyvale, CA, USA). All experiments were conducted three times in triplicate. Kojic acid, 1 N NaOH, DMSO, and α-MSH were purchased from Sigma-Aldrich (St. Louis, MO, USA).