3.2.2. Tyrosinase Assay

The L-tyrosine substrate and the mushroom tyrosinase were purchased from Sigma Aldrich. The test samples were prepared by first dissolving mushroom tyrosinase (250 U/mL) and l-Tyrosine (0.1 mg/mL) in DMSO and diluting the stock to different concentrations using phosphate buffer (66.7 mM at pH 6.8) to obtain a final DMSO concentration of 1%. The 96-well plate was divided into four groups: Blank control (BC), experimental control (EC), sample control (SC) and sample experiment (SE). All wells contained 40 μL of tyrosinase and had a total volume of 200 μL. On top of

the enzyme solution, the BC group had phosphate buffer (160 μL) in the wells; EC contained phosphate buffer (80 μL) and l-Tyrosine (80 μL) in each well; SC wells consisted of phosphate buffer (120 μL) and sample solution (40 μL); SE wells, on the other hand, had phosphate buffer (40 μL), l-Tyrosine (80 μL) and sample solution (40 μL) in them. These assay mixtures were incubated at 37 ◦C for 30 min and measured at 475 nm using the microplate reader (Molecular Devices, CA, US). Percentages of tyrosinase activity were calculated using this formula:

% Tyrosinase activity = [(SE − SC) ÷ (EC − BC)] × 100% (1)
