Folin ciocalteu reagent

The levels of total polyphenols in perilla pomace were determined using the Folin-Ciocalteu method with a slight modification 21) . In brief, 100 μL of the sample was added to 500 μL of Folin-Ciocalteu reagent (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) and incubated for 30 min at room temperature. The mixture was then added 400 μL of 7.5％ sodium carbonate solution. After incubation for 1 h, 200 μL of the reaction mixture was transferred to a 96-well plate, and absorbance was measured at 765 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) . Gallic acid was used as the standard compound, and the total polyphenol content was expressed as gallic acid equivalents (mg GE/g dry weight) . To measure the radical oxygen species scavenging activity of perilla pomace polyphenols, 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity was measured according to the method described by Oki et al. 22) . Briefly, the sample solution (360 μL) , 200 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0, 180 μL pH 6.0) , and 400 μM DPPH in ethanol (180 μL) were mixed in a microtube. After incubation for 20 min at room temperature, 200 μL of the reaction mixture was transferred to a 96-well plate, and the absorbance was measured at 520 nm using a spectrophotometer.