3.2. Mushroom Tyrosinase Inhibition Assay

Mushroom tyrosinase inhibitory activity was determined using both l-tyrosine and l-DOPA as substrates, based on the procedure described by Jung et al. [23]. Briefly, 190 µL of tyrosinase enzyme (1000 U diluted with mushroom tyrosinase buffer, including 1 mM l-tyrosine and l-DOPA solution) was added, in the presence or absence of compounds (final concentration ranging from 1 to 20 µM, dissolved in 100% DMSO), to each well of a 96-well plate, to provide a final volume of 200 μL. The plate was incubated at 37 °C for 30 min. Tyrosinase activity was quantified by measuring the absorbance at 492 nm using a microplate reader (TECAN, Salzburg, Austria) and the percentage inhibition (%) was obtained from the following equation:

where Ac is the absorbance of the control and As is the absorbance of the sample. The IC₅₀ values were calculated from the log-linear curves and their equations. Average results for three determinations are shown. Kojic acid was used as a positive control.