Tyrosinase activity
Cellular tyrosinase activity was estimated by measuring the rate of L-3,4-dihydroxyphenylalanine (ʟ-DOPA) oxidase activity (Kim et al., 2013). B16 cells were seeded in a 6-well plate at a density of 2.5 × 105 cells/well. After 24 h of incubation, the cells were treated with different concentrations of the test compounds for 24 h, 8-MOP was added at 50 µM as a positive control, and 2 µL of DMSO was added to the control group. Cells were then lysed with PBS containing 1% Triton X-100 and 1% sodium deoxycholate for 30 min at −20 °C. The lysates were centrifuged at 12,000 × g for 15 min. A reaction mixture containing 90 μL of each cell lysate and 10 μL of 10 mM ʟ-DOPA was added to each well in a 96-well plate. After incubation for 20–60 min (according to the amount of dopachrome formed) at 37 °C in the dark, the product dopachrome was detected at 490 nm using a multi-plate reader (Spectra Max M5/M5e). The tyrosinase activity of each sample was calculated relative to that of the control group and corrected for the protein concentrations, considering the control group as representing 100% activity.