Tyrosinase Inhibitory Activity

The extracts capacity to inhibit the tyrosinase activity was measured according to the method of Chen et al. [33], using l-3,4-dihydroxyphenylalanine (l-DOPA) as substrate. For each extract, four wells of a 96-well plate were designated as A, B, C, and D, and each one contained a reaction mixture (200 μL) as follows: (A) 66 mM PBS (pH 6.8; 120 μL) and mushroom tyrosinase in PBS (46 U/mL; 40 μL); (B) PBS (160 μL); (C) PBS (80 μL), tyrosinase (40 μL), and aqueous extract solution with 5% DMSO (40 μL); and (D) PBS (120 μL) and aqueous extract solution with 5% DMSO (40 μL). After incubation of the reaction mixtures for 10 min at room temperature, 2.5 mM l-DOPA in PBS (40 μL) was added and the plate was incubated again for 20 min. The absorbance was measured at 475 nm (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). A kojic acid solution (0.10 mg/mL) was used as positive control. The inhibition percentage of the tyrosinase activity was calculated as follows:

$$I(\%) = \frac{(\text{A} - \text{B}) - (\text{C} - \text{D})}{(\text{A} - \text{B})} \times 100\tag{1}$$

IC50 values were then calculated from the obtained inhibition percentage values.
