Mushroom Tyrosinase Inhibition Assay

The measurement of Mushroom tyrosinase activity was modified from the published method [65 (link),66 (link)]. l-Tyrosine (Bio Basic, Ontario, Canada) or l-DOPA (Sigma Chemical, St. Louis, MO, USA) were used as substrates for monophenolase and diphenolase activity, respectively. Each sample was diluted to a series of concentrations (0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, and 5 mg/mL). Mushroom tyrosinase (Sigma Chemical, St. Louis, MO, USA) was dissolved in 0.1 M phosphate buffer (pH = 6.8) to obtain the final concentration of 100 units/mL. The reaction was started by adding 40 µL of sample solution, 80 µL of phosphate buffer, 40 µL of Mushroom tyrosinase, and 40 µL of substrates (1.5 mM l-tyrosine or 2.5 mM l-DOPA solution). After incubation at room temperature for 15 min, the l-dopachrome formation was measured at 475 nm. The percentage of Mushroom tyrosinase inhibition was calculated using the following equation:
where A = vehicle control, B: = vehicle control without Mushroom tyrosinase, C = sample mixed with Mushroom tyrosinase, and D = sample without Mushroom tyrosinase. The results were expressed as IC₅₀ values (the concentration that caused 50% Mushroom tyrosinase inhibition).