Tyrosinase activity was measured based on Dopa oxidase activity by estimating the dopachrome production according to the method by Qiao et al., 2012 [54 (link)], with some modifications. B16F10 cells were seeded at 5 × 106 cells/mL in 24-well plates. Cells were incubated for 24 h with 70 μM of hdTIPs and 100 μg/mL of kojic acid or arbutin as positive controls. Each well was then washed and replaced with PBS 600 μL and induced with UVA (314–400 nm, 3.0 W) and UVB (280–315 nm, 13.6 W) using OSRAM (Ultra-Vitalux, Germany) for 22 s (0.32 J/cm2). After that, the cells were incubated with medium 600 μL/well for a further 24 h. Then, the cells were trypsinized and the harvested cells were lysed in cell extraction buffer. After normalized protein concentration, 10 μL of each lysate supernatant was aliquoted into a 96-well plate and shaking incubated in dark conditions with 1 mM L-DOPA 90 μL in PBS at 37 °C at 280 rpm for 10 min. The absorbance was measured at 475 nm and all experiments were performed in triplicate to determine the IC50 of the samples.
The cellular tyrosinase inhibitory activity was calculated with the following equation:
where, C are defined as in the previous section.
The cellular tyrosinase inhibitory activity was calculated with the following equation:
where, C are defined as in the previous section.
Free full text: Click here
