3.2. In Vitro Tyrosinase Inhibitory Assay

The tyrosinase inhibitory activities of the EOs and isolated compounds were investigated in vitro using a colorimetric readout-based enzyme assay optimized by Zengh et al. [25], with slight modifications. The tyrosinase inhibitory activities of EOs, as well as their respective hydrocarbon and oxygenated fractions and pure compounds were investigated in vitro using a colorimetric readout-based enzyme assay that was optimized by Zengh et al. [25], with slight modifications: the assay was carried out at room temperature and tyrosinase inhibition was measured considering control and sample absorbance after 6 min of incubation, rather than after 20 min, so as to operate under the linear portion of the enzymatic reaction, which provides more accurate inhibition results [26,27]. Mushroom tyrosinase from Agaricus bisporus (J.E. Lange) Imbach was selected for this study. L-Tyrosine was used as the substrate, meaning that the overall tyrosinase inhibitory activity was investigated without distinguishing between tyrosinase monophenolase and diphenolase activity. Photometric measurements at 475 nm were performed on a Thermo spectronic Genesys 6 and kojic acid was used as the positive control inhibitor. The solutions of the investigated potential inhibitors (EOs, EO isolated fractions, EO individual compounds, and kojic acid) were prepared in DMSO. Table 7 reports the tested concentrations for each investigated potential inhibitor. The mushroom tyrosinase solution 200 U/mL (27.9 μg/mL) was prepared in sodium phosphate buffer (pH 6.8) and aliquots of 9 mL were stored at −18 °C and thawed just before the experiments. Tyrosine solution 0.1 mg/mL was prepared in sodium phosphate buffer (pH 6.8) and renewed daily. The reaction mixture components were placed in the vial in the following order: 1 mL of mushroom tyrosinase solution 200 U/mL; 1 mL of sodium phosphate buffer solution; 10 μL of EO/single compound/kojic acid solution; and, finally, 1 mL of tyrosine solution 0.1 mg/mL. The final DMSO percentage in the reaction mixture was 0.3%. The assay was performed in a sealed 4 mL vial to avoid the loss of any EO components into the surrounding environment and to minimize their release into the head space above the reaction mixture. The reaction mixture was incubated in a thermostatic water bath at 25 °C for 6 min. Subsequently, the absorbance at 475 nm was registered, as this wavelength allows the identification of dopachrome. The absorbance corresponding to 100% of tyrosinase activity was measured by replacing the EOs/individual compound/kojic acid solution with 10 μL of pure DMSO. Blank solutions were prepared as follows: 2 mL of sodium phosphate buffer solution, 10 μL of EO/individual compound/kojic acid/DMSO solution, and 1 mL of tyrosine solution 0.1 mg/mL. The percentage of tyrosinase inhibition was measured according to the equation below:

ΔA (Control) or (Sample) = A₄₇₅ (Control) or (Sample) − A₄₇₅ (Control Blank) or (Sample Blank).

Tested concentrations for investigated essential oils and for both the relative isolated hydrocarbon and oxygenated fractions.

The concentration-response curve for each inhibitor was determined by plotting the inhibitory activity as a function of inhibitor concentration in the reaction mixture. IC₅₀ values for the inhibitors were obtained by interpolation from the concentration–response curve.