Mushroom tyrosinase assay protocol

The mushroom tyrosinase was used for in vitro bioassays as described previously with some modifications^58,59. Briefly, 140 μl of phosphate buffer (20 mM, pH 6.8), 20 μl of mushroom tyrosinase (30 U/ml), and 20 μl of the inhibitor solution were placed in the wells of a 96-well plate. After pre-incubation for 10 min at room temperature, 20 μl of L-DOPA (3,4-dihydroxyphenylalanine) (0.85 mM) was added and the plate was further incubated at 25 °C for 20 min. Subsequently the absorbance of dopachrome was measured at 492 nm using a micro plate reader (OPTI Max, Tunable). Kojic acid was used as a reference inhibitor. The extent of inhibition by the test compounds was expressed as the percentage of concentration necessary to achieve 50% inhibition (IC₅₀). Each concentration was analyzed in three independent experiments run in triplicate. The IC₅₀ values were determined by the data analysis and graphing software, Origin.