3.9. Mushroom Tyrosinase Inhibitory Assay

In order to assess the inhibition of the monophenolase activity of mushroom tyrosinase by Allium sp. extracts, the protocol described by Wang and co-workers was applied [60]. 20 µL of diluted sample was mixed with 80 µL of phosphate buffer (100 mM, pH = 6.8) was mixed with 80 µL of phosphate buffer (100 mM, pH = 6.8) and 20 µL mushroom tyrosinase working solution (500 U). The samples were pre-incubated for 10 min at room temperature, 80 µL 2 mM L-tyrosine was added and the samples were incubated for further 20 min at room temperature, in darkness. The method described by Uchida et al. [61] was implicated to analyze the inhibition of the diphenolase activity of mushroom tyrosinase. 20 µL of diluted sample was mixed with 120 µL of phosphate buffer (100 mM, pH = 6.8) and 20 µL tyrosinase solution (500 U). The samples were pre-incubated for 10 min at room temperature, then 40 µL 4 mM l-DOPA was added and the samples were incubated for a further 20 min at room temperature, in darkness. In both analyses the absorbance of formed dopachrome was measured using FilterMax F5 microplate reader (Molecular Devices, San Jose, CA, USA) at λ = 450 nm. The obtained values were corrected by the absorbance of the extracts without tyrosinase and the substrate (L-tyrosine or l-DOPA). Control sample (100% tyrosinase activity) was composed of phosphate buffer, tyrosinase, substrate and equal volume of the solvent. 100 µg/mL kojic acid was used as a reference tyrosinase inhibitor. Tyrosinase was calculated based on the Equation

where Abs(S)—the absorbance of the sample (extract + tyrosinase + substrate), and Abs(C)—the absorbance of the control sample (solvent + tyrosinase + substrate). Each sample was analyzed in three independent repetitions.