3.5.1. Determination of Melanin Content

Before measuring the effect of PFF-A on tyrosinase activity and melanin content, its toxicity to B16F10 cells was measured. B16F10 cells were seeded (2 × 10⁴ cells/mL) in 96-well plates and, after 24 h, the cells were treated with various concentrations of PFF-A and cell viability was determined using the MTT assay, following a previously described method [15,39]. Melanin content was determined by a modified method from Hosoi et al. (1985) [40]. In brief, B16F10 cells were plated in 24-well plates at 2 × 10⁴ cells/mL and treated with PFF-A in the presence or absence of α-MSH for 48 h. Cell pellets were harvested after washing with PBS, liquefied in 1 N NaOH at 60 °C for 1 h, and mixed. Melanin content was determined by measuring the absorbance at 450 nm using a microplate reader (BioTek). For the accurate calculation of melanin content, absorbance values were normalized to total protein absorbance values. Arbutin (200 μM) was selected as a positive control.