Tyrosinase inhibition assay

Tyrosinase inhibition was determined using the DOPA-chrome formation method as described previously with slight modifications¹³. Briefly, in 96-well plates, 20 μL aliquots of DMSO (control) or test compounds at varying concentrations were mixed with 40 μL aliquots of 30 U/mL mushroom tyrosinase (Sigma Aldrich) and 100-μL of 0.1-M phosphate buffer (pH 6.8). They were preincubated for 10 min at room temperature. Reactions were initiated by adding 4 μL aliquots of 10 mM L-DOPA to each well and incubating at 37 °C for 20 min. Tyrosinase activity was then determined by measuring absorbance at 475 nm. Kojic acid was used as a positive control. Experiments were performed in triplicate. Percentage of tyrosinase inhibition was calculated using the following equation:

where E is the absorbance of the enzyme reaction, Eb is the absorbance of the enzyme blank, T is the absorbance of the test sample, and Tb is the absorbance of the test blank. IC₅₀ values were determined from a linear graph of percent elastase inhibition against concentration (250, 125, 62.5, 31.25, 15.6 µg/mL).