2.3. Cell-Free Mushroom Tyrosinase Activity Assay

Cell-free mushroom tyrosinase activity assay was measured using L-tyrosine as a substrate according to a previously described method [32]. Briefly, 1 μL of brassinin (5, 15, and 30 μM) and 94 μL of an assay mixture buffer (1 mM L-tyrosine solution, 50 mM sodium phosphate buffer (pH 6.5), and distilled water (10:10:9, v/v/v), respectively) were added to the 96-well plates. Then, 5 μL of mushroom tyrosinase (1000 units/mL) was added to each well. Following incubation for 30 min at 37 °C, absorbance was determined at 490 nm using a microplate reader.