Melanin Content Quantification in B16 Cells

The intracellular melanin levels were determined as previously described [19 (link),67 (link)] with some modifications. B16 cells were seeded in 6-well plates at a concentration of 2.5 × 10⁵ cells/well and allowed to attach for 24 h. This assay was divided into 7 groups as follows: control, non-treatment; IBMX: 50 µM IBMX (PanReac AppliChem, Barcelona, Spain); theophylline: IBMX + 0.01 mg/mL theophylline (Sigma Chemical, St. Louis, MO, USA); arbutin: IBMX + 0.01 mg/mL arbutin (Sigma Chemical, St. Louis, MO, USA); PES1CMU-RBO: IBMX + 0.01 mg/mL rice bran oil; PES1CMU-DFRB: IBMX + 0.01 mg/mL de-oiled rice bran extract; and PES1CMU-H: IBMX + 0.01 mg/mL husk extract. After 48 h of incubation, cell pellets were collected and lysed with 1 N NaOH containing 10% DMSO at 80 °C for 30 min. The intracellular melanin release was measured at 405 nm using a microplate reader. The results were expressed as a fold change in melanin content compared to the control.