3.4. Tyrosinase Inhibition Assay

In our experiments, we investigated the ability of plant extracts and natural compounds to inhibit the oxidation of l-DOPA (l-3,4-dihydroxyphenylalanine) to dopaquinone and subsequently to dopachrome by the enzyme tyrosinase employing a protocol from Masuda et al. [37] with slight modifications. Test samples were dissolved in DMSO to stock solutions of 10 mg/mL and were diluted in the proper concentration in phosphate buffer 1/15 M (NaH₂PO₄/Na₂HPO₄), pH 6.8; final concentrations of DMSO in the well did not exceeded 3%. In 96-well plates, 80 μL of phosphate buffer, 40 μL of sample in the same buffer and 40 μL mushroom tyrosinase (92 Units/mL), in the same buffer, were mixed. The contents of each well were incubated for 10 min at 25 °C, before the addition of 40 μL of 2.5 mM L-DOPA in phosphate buffer. After incubation at 25 °C for 5 min, the absorbance at 475 nm of each well was measured using a microplate reader. Blanks for every sample w/o tyrosinase were also performed, while kojic acid was used as positive control. The percentage inhibition of the tyrosinase activity was calculated by the following equation: [(A − B) − (C − D)]/(A − B) × 100, where A: Control (w/o sample), B: Blank (w/o sample, w/o tyrosinase), C: Sample, D: Blank sample (w/o tyrosinase).