*2.8. Tyrosinase Inhibitory Activity*

Tyrosinase inhibitory activity of each sample was determined by method previously described by Chen et al. [22] using a SPECTROstar Nano Multi-Detection Microplate Reader with 96-well plates (BMG Labtech, Ortenberg, Germany). Samples were dissolved in water containing 5% DMSO; for each sample four wells were designated as A, B, C, D; each one contained a reaction mixture (200 μL) as follows: (A) 120 μL of 66 mM phosphate buffer solution (pH = 6.8) (PBS), 40 μL of mushroom tyrosinase in PBS (46 U/mL) (Tyr), (B) 160 μL PBS, (C) 80 μL PBS, 40 μL Tyr, 40 μL sample, and (D) 120 μL PBS, 40 μL sample. The plate was then incubated at room temperature for 10 min; after incubation, 40 μL of 2.5 mM L-DOPA in PBS solution were added in each well and the mixtures were incubated again at room temperature for 20 min. The absorbance of each well was measured at 475 nm, and the inhibition percentage of tyrosinase activity was calculated by the following equation, using a kojic acid solution (0.10 mg/mL) as a positive control:

$$\% \text{I} = \frac{(\text{A} - \text{B}) - (\text{C} - \text{D})}{(\text{A} - \text{B})} \times 100 \tag{2}$$
