2.6.3. Melanin Inhibition

The effect of the ethanolic extract of *S. perfoliata* on melanin production in UCT-MEL-1 cells was determined as described by Matsuda et al. [20]. Following culturing, as described in Section 2.7.1, the cells were plated in 96-well plates and incubated for 24 h at 37 ◦C in the CO2 incubator. Stock solutions of the ethanolic extract of *S. perfoliata* and kojic acid (400 μg/mL), were prepared in 1.25% DMSO in phosphate buffered saline (PBS). The stock solutions were serial diluted in Dulbecco's Modified Eagle's Medium (DMEM) and added to the 96-well plates, such that the final concentrations in the plates ranged between 6.25 and 200 μg/mL and the final concentration of DMSO present was 0.6%. The treated cells were incubated for 24 h at 37 ◦C in the CO2 incubator. Colour controls, without cells, were prepared in the same manner. An untreated cell control was also included where media was used instead of the sample. Following incubation, the supernatant was removed, combined with HEPES buffer (0.4 M, pH 6.8) at a 1:1 ratio and assayed for extracellular melanin at 475 nm. The attached cells were washed with 100 mL of PBS and lysed following the addition of 40 μL of 1 M NaOH and 10 μL trypsin, for 16 h at room temperature. The optical density of the intracellular melanin was measured at 475 nm.
