3.4.3. Tyrosinase Inhibition Assay

Tyrosinase inhibitor assays were conducted in 96-well plates using a modified method originally described by Wróbel-Biedrawa [51]. The process involves the conversion of L-tyrosinase to L-DOPA, then to DOPA-quinone, facilitated by the tyrosinase enzyme. This reaction results in the solution turning brown. In brief, 10 μL of the sample (concentrations of 10.0, 1.0, and 0.1 mg/mL in 10% DMSO) was combined with 150 μL of a phosphoric buffer containing mushroom tyrosinase (pH = 6.88, 100 U/mL). This mixture was then incubated for 10 min at room temperature. A control (A_C) was also prepared that did not contain any inhibitor. Following incubation, L-tyrosine (0.3 mg/mL) was added to each well, and the absorbance was measured at 492 nm (using a kinetic model every 5 min). Two time points (t₁ and t₂) were selected within the linear range of the graph. All samples were tested in triplicate. The inhibition of tyrosinase was calculated using a specific equation, with kojic acid used as a standard.

A_S—the difference in absorbance between times t₂ and t₁ for the sample;

A_C—the difference in absorbance between times t₂ and t₁ for the positive control.