To evaluate the effect of chalcone-21 on mushroom tyrosinase
activity, mixtures consisting of vehicle or various concentrations of
chalcone-21 (80 mL) in 50 mM potassium phosphate buffer (pH 6.5)
and mushroom tyrosinase (20 mL, 10 mg/mL in phosphate buffer, pH
6.5) were prepared in 96-well plates and incubated for 5 min at
room temperature. The reaction was started by addition of L-DOPA
(100 mL, 10 mM in phosphate buffer, pH 6.5) and further incubated
for 30 min at 37 C. The absorbance at 475 nm was measured using
a microplate reader.