2.2. Tyrosinase inhibition assay

Monophenolase and diphenolase activity was measured as described previously by monitoring the dopachrome absorbance at 475 nm¹⁷. l-Tyrosine and l-DOPA were used as reaction substrate for monophenolase and diphenolase assays, respectively. In the reaction, l-tyrosine (50 µL, 2 mM) or l-DOPA (50 µL, 0.5 mM), 90 µL of pH 6.8 phosphate buffer and 5 µL volume of different concentrations of final compounds (1a–1o) in DMSO were mixed and incubated at 30 °C. Finally, 20 units of enzyme was quickly added to the reaction mixture and incubated at 30 °C for 10 min. The absorbance at 475 nm was recorded with a microplate reader. During these incubations, the final concentration of DMSO was limited to 3% by volume. The assay was carried out in triplicate and DMSO was used as control¹⁷.

The same experimental protocol described above was adapted for inhibition kinetics, with different inhibitor concentration. However the absorbance was measured at 1 min reaction intervals for twelve minutes¹⁶.

Measurement method was substantially the same to that described above, changing the added concentration of the enzyme solution, different concentrations of inhibitor on mushroom tyrosinase l-DOPA as reaction substrate¹⁶.