*2.8. Tyrosinase Inhibitory Activity*

The tyrosinase inhibitory activity of each sample was determined via a method previously described [34] using a SPECTROstar Nano Multi-Detection Microplate Reader with 96-well plates (BMG Labtech, Ortenberg, Germany). Samples were disolved in water containing 5% dimethylsulfoxide (DMSO). For each sample, four wells were designated as A, B, C, and D, where each one contained a reaction mixture (200 μL) as follows: (A) 140 μL of 66 mM phosphate buffer solution (pH = 6.6) (PBS), 40 μL of mushroom tyrosinase in PBS (23 U/mL) (Tyr); (B) 160 μL PBS; (C) 80 μL PBS, 40 μL Tyr, 40 μL sample, and 80 μL PBS; and (D) 120 μL PBS and 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 the tyrosinase activity was calculated using the following Equation (1):

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

The results were expressed as mg kojic acid equivalents per gram of dry weight of extract (mg KAE/g extract) using a calibration curve between 0.01–0.10 mg kojic acid/mL of solution.
