4.2.1. Mushroom Tyrosinase Assay

Mushroom tyrosinase inhibitory assays were performed on compounds 1a–1j and 2a–2i as previously described with slight modification [73]. Briefly, a 200 µL mixture [170 µL of substrate solution (potassium phosphate buffer 14.7 mM + 293 µM L-tyrosine solution), 20 µL of tyrosinase, and 10 µL of 1a–1j or 2a–2i (final concentration: 50 μM) or kojic acid (50 μM)] was added to a 96-well microplate and incubated at 37 °C for 30 min. Absorbances were measured at 475 nm using a microplate reader (VersaMaxTM, Molecular Devices, Sunnyvale, CA, USA). Kojic acid was used as the positive control. The experiment was repeated three times. Mushroom tyrosinase inhibitions % were calculated using:

where A is the absorbance of the test compound and B is the absorbance of the blank.

To determine the IC₅₀ values of tyrosinase inhibition by 1c, 1h, 2a, or kojic acid, dose-dependent inhibition experiments were performed in triplicate at 3–5 different concentrations. Log-linear curves were plotted, and their equations were determined. IC₅₀ was defined as the concentration that inhibited tyrosinase activity by 50%.