Determination of Mushroom Tyrosinase Inhibition Activity

The tyrosinase inhibitory activity was determined according to a slightly improved version of the method previously reported by Morais et al.25 A series of 50 μL TSAT (0.01–1 mg/mL, in 50% DMSO) solutions were added to the reaction system containing 100 μL of PBS buffer (0.1 M, pH 6.8) and 50 μL tyrosinase (100 U/mL), respectively, and reacted at 25 °C for 15 min. Then, 50 μL L L-DOPA (3.5 mM) solution was added to the reaction system and incubated at 37 °C for 10 min. The absorbance (475 nm) was determined. VC was used as the positive control. The inhibition activity of TSAT was calculated by the following formula:

where A₀ represents the absorbance of the negative control with 50% DMSO instead of the sample; the system contains L-DOPA and tyrosinase; A₁ represents the test system containing the sample and contains L-DOPA and tyrosinase; and A₂ is the blank system (sample plus L-DOPA) absorbance. The IC₅₀ value was calculated to indicate the tyrosinase inhibitory activity of TSAT. All operations were performed in parallel three times.