Cellular tyrosinase activity assay

The oxidation of DOPA to DOPA chrome was analyzed by spectrophotometry as indicator of tyrosinase activity(18). 10⁶ cells of B16F10 melanoma were plated in each well of 96-well plate overnight. After treating of cell with different concentrations (0.2-200 μg/mL) of P. atlantica subsp. mutica extracts and essential oil for 24 h, The cells were detached using trypsin; washed with PBS, and lysed with 50 μL sodium phosphate buffer (pH 6.8, 100 mM) containing 1% triton X-100 and 0.1 mM phenylmethylsulfonyl fluoride. The lysates were centrifuged at 10,000 rpm for 20 min at 4 °C. 100 μL of each lysate (each containing 100 mg protein) was mixed with 30 μL of 5 mM DOPA in 96 well plate and incubated at 37 °C for 2 h, the absorbance was measured at 475 nm with Synergy H4 Hybrid Multi-Mode microplate reader (BioTek, Winooski, USA).