2.6.2. Tyrosinase Inhibition

The tyrosinase assay was conducted according to the method described by Curto et al. [19] with slight modifications. The enzymatic rate of mushroom tyrosinase (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) was evaluated spectrophotometrically. In a 96-well plate, 70 μL of the stock solution (600 μg/mL in 0.38 mM DMSO and pH 6.5 potassium phosphate buffer) was combined with 30 μL of tyrosinase (333 Units/mL in potassium phosphate buffer) and incubated for 5 min at room temperature. Following incubation, 110 μL substrate (2 mM L-tyrosine) was added to each well. Final concentrations of the test sample and the positive control, kojic acid (Sigma-Aldrich Co. LLC, St. Louis, MO, USA), ranged between 1.5 and 200 μg/mL. The negative control consisted of 0.01% DMSO in potassium phosphate buffer. The rate of tyrosinase was determined for the sample (Abssample), positive control (Abspositive) and the negative control wells (Abscontrol), by measuring the absorbance over a period of 30 min at 492 nm using the BIO-TEK Power-Wave XS multi-well plate reader (A.D.P., Weltevreden

Park, RSA). The percentage tyrosinase inhibition for the sample was calculated as follows and the IC50 value was then calculated.

$$\text{Percentage inhibition } (\%) = 100\% - \left[ \left( \frac{\text{Abs}\_{\text{sample}/\text{positive}}}{\text{Abs}\_{\text{control}}} \right) \times 100 \right]$$
