2.8. In vitro tyrosinase inhibitory activity

Assays were performed as previously described by Chiocchio et al. (2018) with slight modifications. Mushroom tyrosinase solution was freshly prepared from 1.73 mg of 500 U/mL mushroom tyrosinase diluted in 10.0 mL of 0.05 M phosphate buffer solution, pH 6.5. Substrate solution was also freshly prepared by diluting 1.81 mg of L-tyrosine in 10.0 mL of 0.05 M phosphate buffer solution. Each of the solutions and sample were mixed at certain volumes according to Table 2.

Composition of solution in tyrosinase inhibitor assay.

All of the mixtures were incubated at 25 °C for 30 min, and the reaction was monitored using a microplate reader at 475 nm. The percentage inhibition of tyrosinase activity was calculated using the following equation:

where A is solution a absorbance; B is solution b absorbance; C is solution c absorbance; and D is solution d absorbance. Analyses were performed in triplicate.