Tyrosinase Inhibition Assay with L-Dopa

Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was assayed using L-Dopa as the substrate as reported in our previous studies with some modifications^{30 (link)–32 (link)}. The enzyme diphenolase activity was monitored spectrophotometrically by observing dopachrome formation at 490 nm. All the test samples were first dissolved in DMSO at 10 mM and diluted to the required concentrations. Initially, in a 96-well microplate, 10 µl of test samples were added to 160 µl of 50 mM phosphate buffer (pH = 6.8) and then 10 µl tyrosinase (500 U mL⁻¹) was added. After the mixture was pre-incubated at 28 °C for 20 min, 20 µl of L-Dopa solution (7 mM) was added to the mixture. After 10 min incubation absorbance of samples was measured. DMSO without test compounds was used as the control, and kojic acid was used as a positive control. Each assay was conducted as three separate replicates. The inhibitory activity of the tested compounds was expressed as the concentration that inhibited 50% of the enzyme activity (IC₅₀). The percentage inhibition ratio was calculated according to the following equation: $$\text{Inhibition}\left(\%\right)=100\ast \left({\text{Abs}}_{\text{control}}-{\text{Abs}}_{\text{compound}}\right)/{\text{Abs}}_{\text{control}}$$