Abstract: In this study, anthocyanins from mulberry fruit were separated and purified by sequential macroporous resin adsorption chromatography and high-speed counter-current chromatography (HSCCC), and were identified by UV-Vis spectroscopy, high performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance. We further evaluated the inhibitory effects of the crude extract and the purified components on lipid peroxidation and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical. The HSCCC was performed using a two-phase solvent system composed of n-butanol, methyl tert-butyl ether, acetonitrile, water and trifluoroacetic acid (2:2:1:5:0.01, V/V) with the upper phase as the stationary phase and the lower phase as the mobile phase under the conditions of rotation speed of 850 r/min, flow rate of 2 mL/min and detection wavelength of 254 nm. Finally, four peaks (components I, II, III and IV) were displayed on the HSCCC chromatogram. Component IV was identified as non-anthocyanin, while components I, II and III as delphinidin-3-glucoside, cyanidin-3-glucoside and pelargonidin-3-glucoside with purities of and 92.27%, 94.05% and 90.82%, respectively and their contents in the crude extract were 17.4, 33.7, 9.8 mg/100 mg, respectively. The 50% inhibiting concentration of the crude extract, and components I, II and III were (0.77 ± 0.02), (0.34 ± 0.02), (0.55 ± 0.04) and (0.68 ± 0.01) g/L for inhibiting lipid peroxidation and (0.40 ± 0.01), (0.16 ± 0.01), (0.22 ± 0.01) and (0.35 ± 0.03) g/L for scavenging DPPH radical, respectively.

Key words: high-speed counter-current chromatography, mulberry, anthocyanins, structure identification, antioxidant activity