Mushroom tyrosinase ec 1.14.18.1

The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was used for in vitro bioevaluation as described previously with some modifications [40 (link)–41 (link)]. Briefly, 140μL of phosphate buffer (20mM, pH 6.8), 20μL of mushroom tyrosinase (30U/mL) and 20μL of the inhibitor solution were placed in the wells of a 96-well micro plate. After pre-incubation for 10 min at room temperature, 20μL of L-DOPA (3,4-dihydroxyphenylalanine) (0.85mM) was added and the plate was further incubated at 25°C for 20min. Subsequently the absorbance of dopachrome was measured at 492nm using a micro plate reader (OPTI_Max, Tunable). Kojic acid was used as a reference inhibitor and for negative tyrosinase inhibitor phosphate buffer was used instead of the inhibitor solution. The extent of inhibition by the synthesized carvacrol derivatives 4a-f and 6a-d was expressed as the percentage of concentration necessary to achieve 50% inhibition (IC₅₀). Each concentration was analyzed in three independent experiments run in triplicate. The IC₅₀ values were determined by the data analysis and graphing software Origin 8.6, 64-bit.

Mushroom tyrosinase (EC 1.14.18.1) was purchased from Sigma-Aldrich. Tyrosinase inhibition assays were conducted as described in [52 (link)] with slight modifications. First, in order to characterize the Michaelis-Menten parameters of the enzyme kinetics, 100 μl of a mushroom tyrosinase solution (25 U) in sodium phosphate buffer (50 mM, pH 6.5) were pipetted in each well of a flat-bottom microtiter plate (Nunc 167008). Two-fold dilutions of L-Dopa, from 4.8 to 0.075 mM (final concentrations) were mixed (100 μl) with the enzyme solution. The enzymatic reaction was followed by measuring the absorbance at 475 nm every 30 s over 15 min, with shaking (150 rpm) between measurements. Temperature was kept constant at 30ºC. The values of K_M and v_max under these conditions were obtained from Lineweaver-Burk plots. For tyrosinase inhibition assays, each well of the microtiter plate was filled with 100 μl of a mushroom tyrosinase solution and the appropriate amount of the test substance dissolved in DMSO. The compounds were pre-incubated with the enzyme at 30ºC for 10 min. Subsequently, the enzymatic reaction was triggered by addition of 100 μl of L-Dopa (1.2 mM test concentration). Formation of dopachrome was followed by measuring the absorbance at 475 nm as described above. Kojic acid (Sigma-Aldrich) was used as positive control.

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: $$\text{Inhibition}\left(\%\right)=100\ast \left({\text{Abs}}_{\text{control}}-{\text{Abs}}_{\text{compound}}\right)/{\text{Abs}}_{\text{control}}$$