Melanin Inhibition Assay with NNFE

Cells were seeded at a density of 1 × 10⁵ cells/mL into a 24-well culture plate (BD Falcon, Bedford, MA, USA) and left to grow overnight. The old media was substituted with fresh media and treated with various concentrations of NNFE. After incubation for 3 d, cells were rewashed with PBS, lysed using 1 N NaOH. The absorbance of the sample at 405 nm was measured with a microplate reader (VICTOR3, Perkin Elmer)⁹ . Inhibition of melanin biosynthesis (%) was estimated as follows: $$\mathrm{Inhibition}\mathrm{of}\mathrm{melanin}\mathrm{production}(\%)=\left(\mathrm{A}-\mathrm{B}\right)/\mathrm{A}\times \mathrm{100}$$ where A and B is the absorbance of cells lysate treated with and without of NNFE or arbutin (positive control), respectively.