Tyrosinase inhibition assay

Tyrosinase plays a major role in melanin synthesis, and tyrosinase inhibition assay has been considered as a universal method to inhibit melanin production [45]. L-tyrosine and L-dopa were selected as substrate respectively to determine the inhibition of tyrosinase monophenolase and diphenolase activity with arbutin as a positive control. First, 50 μL of 1.5 mM L-tyrosine or L-dopa, 100 μL of phosphate buffer (PBS, pH 6.8), and 60 μL of PBS with or without the sample, were mixed. The mixture was preincubated at 37°C for 10 min before 40 μL of 250 units/mL mushroom tyrosinase was added, and the reaction was performed at 37°C for 25 min. Enzyme activity in different concentration of fresh sample was measured at 490 nm. The percentage of inhibition was calculated as follows:

where C was the absorbance at 490 nm with tyrosinase, but without the test sample; C₀ was the absorbance at 490 nm without the test sample and tyrosinase; T was the absorbance at 490nm with the test sample and tyrosinase; and T₀ was the absorbance at 490 nm with the test sample, but without tyrosinase.

According to the linear formula, we calculated the IC50 values of DPPH scavenging activity and tyrosinase inhibition effect. All the data was sorted and used to determine the significance differences of comparative groups Pl-P vs. Pl-SP and Pl-S vs. Pl-SP via T test (P<0.05).