2.3. Sulforhodamine B (SRB) Assay

This assay was conducted as previously described [21], A375 and HDF-a cells were seeded in a 96-well microtiter plate at a density of 10,000 cells per well to allow the cells to attach to the plate. Then, cells were treated with different concentrations of isolated compounds with vehicle control (DMSO) which had been previously prepared in 10% culture medium. The cells were incubated in the incubator for 24, 48, and 72 h and periodically checked using an inverted microscope. Later, the cells were fixed with cold 40% trichloroacetic acid (TCA) solution, to achieve the final concentration of 10%. The plates were incubated at 4 °C for 1 h and then rinsed five times with water. The TCA-fixed cells were stained by adding Sulforhodamine B solution (0.4% SRB in 0.1% acetic acid) and left at room temperature for 1 h. Afterwards, the plates were quickly rinsed four times with 1% acetic acid and flicked to remove the unbound dye and then left to air-dry overnight. The bounded stain was solubilised by adding 10 mM Tris base buffer solution to each well. The optical density was measured at 510 nm by using a microtiter plate reader (Infinite^® M200, Tecan, Switzerland). The data was normalized to untreated wells, GI₅₀ value was calculated as the concentration that results in 50% cell growth inhibition and graphs were drawn on OriginPro software.