3.7. Mushroom Tyrosinase Inhibitory Assay

Inhibition of mushroom tyrosinase by A. biebersteinii extracts was performed using the method described previously by Uchida and co-investigators [44]. Briefly, 120 μL phosphate buffer (100 mM, pH = 6.8) was mixed with 20 μL sample solution or pure compounds (1 mg/mL) and 20 μL of mushroom tyrosinase (500 U/mL) and pre-incubated at room temperature for 10 min. Following the addition of 40 μL L-DOPA (4 mM), the samples were incubated for another 20 min at RT. The dopachrome formation was measured spectrophotometrically at λ = 450 nm using FilterMax F5 microplate reader (FilterMax F5 Molecular Devices, San Jose, CA, USA). The obtained values were corrected by the absorbance value of the extract without mushroom tyrosinase and L-DOPA. At the same time, DMSO, solubilized in adequate concentrations in phosphate buffer, was added and used as a positive control relevant to the 100% tyrosinase activity. Each sample was analyzed in 3 independent repetitions.