Tyrosinase Inhibition Assay Protocol

Tyrosinase inhibition activity was evaluated using the method used by Peyrot et al. [43 (link)]. In a 96-well microplate, 60 µL of ammonium formate buffer (50 mM, pH 6.4) were mixed with 10 µL of inhibitor solution at different concentrations: 10,000, 5000, 1000, 500, 100, 50, 10, 5, 1 and 0.5 µM in DMSO. Then, 20 µL of tyrosine at 4.42 mM in ammonium formate buffer was added. After 10 s of shaking, 10 µL of mushroom tyrosinase (5000 U.mL⁻¹ in ammonium formate buffer) were added and the mixture was incubated for 10 min at 37 °C. The amount of dopachrome produced during incubation was determined by absorbance readings at 420 nm every 15 s in a microplate Biotek (Winooski, VT, USA), Epoch 2 spectrophotometer. IC₅₀ values, corresponding to the concentration required to inhibit 50% of tyrosinase activity, were obtained using GraphPad Prism^® software version 6.01. Kojic acid was used as a reference and all measurements were performed at least in duplicate.