Comprehensive evaluation of the antioxidant capacity of Perilla frutescens leaves extract and isolation of free radical scavengers using step-wise HSCCC guided by DPPH-HPLC

To comprehensively examine the antioxidant capacity of Perilla frutescens leaves extract, eight assays including hydroxyl radical, hydrogen peroxide, ferrous ion-chelating, total antioxidant capacity, nitric oxide, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,2'-diphenyl-1-picrylhydrazyl, and ferrous reducing antioxidant assays were employed. The results indicated that the ethyl acetate-soluble fraction of P. frutescens leaves extract displayed the highest antioxidant activities. The antioxidant constituents from ethyl acetate-soluble fraction of P. frutescens leaves extract were screened by 2,2'-diphenyl-1-picrylhydrazyl high-performance liquid chromatography, and 11 compounds were identified after isolation using step-wise high-speed countercurrent chromatography. Protocatechuic acid, chlorogenic acid, caffeic acid, rosmarinic acid, luteolin, methyl-rosmarinic acid, apigenin, and 4',5,7-trimethoxyflavone are the major antioxidant constituents in ethyl acetate-soluble fraction of P. frutescens leaves extract.