2.4.6 Tyrosinase Inhibition Assay

The tyrosinase inhibitor potential was investigated according to an improved (Morlock et al., 2021b) workflow (Taibon et al., 2015). To prepare the substrate solution, 45 mg levodopa, 25 mg CHAPS, and 75 mg PEG 8000 were dissolved in 10 ml of phosphate buffer (1.4 mg/ml K₂HPO₄, 1.68 mg/ml Na₂HPO₄, pH 6.8) and stored at 4°C until use. The levodopa substrate solution was sprayed onto the chromatogram (1 ml, blue nozzle, level 6) and subsequently dried for 2 min in a stream of cold air. Then, 1 ml of enzyme solution (400 U/ml in phosphate buffer) was sprayed onto the plate (blue nozzle, level 6), followed by incubation at room temperature for 20 min. After incubation, the plate was immediately dried and documented. Tyrosinase inhibition activity was apparent as colorless (white) zones on a greyish-brown background. The positive control was kojic acid (0.1 mg/ml in ethanol, 1, 3, and 6 μl/band).