3.7. Assay of Intracellular Tyrosinase Activity

The intracellular tyrosinase activity was determined as described in an article with some modifications [32,34]. B16-F10 cells (1 × 10⁵/well) were treated with 70% E-EA (60, 30 µg/mL), three compounds (20, 10 µM), or arbutin (4 mM) for 2 h, followed by the addition of α-MSH (200 nM) for 72 h. The cells were lysed in a lysis buffer (Promega, Madison, WI, USA) with protease inhibitor cocktail (PIC) for 30 min at 4 °C and then centrifuged at 13,000 RPM for 10 min at 4 °C. The lysates (50 μg) were dissolved in 100 mM phosphate buffer (pH 6.8) and treated with L-dopa (2 mg/ml) in a 96-well plate at 37 °C for 2 h. The production amount of dopachrome was measured using an ELISA microplate reader (ELx808) at an absorbance of 490 nm.