Separation of Anthocyanins in Echinacea Purpurea Flower by High Speed Counter current Chromatography and the Research on the Antioxidant Activity of the Anthocyanins

KeyWord:high speed counter-current chromatography  Echinacea purpurea  anthocyanins  DPPH?  antioxidant activity

Abstract:

      An efficient method for the isolation and preparation of two anthocyanins£¬cyaniding 3-O-£¨¦Â-D-giucopyranoside£©£¨compound 1£© and cyaniding 3-O-£¨6¡å-O-malonyl-¦Â-D-glucopyranoside£©£¨compound 2£©£¬from Echinacea purpurea flower by high speed counter-current chromatography£¨HSCCC£© was established.The fresh flower was extracted with 60% ethanol£¨containing 0.1% HCl£© at low temperature and dark conditions.2.1 g of crude sample for HSCCC was prepared after extracted with ethyl acetate and passed through an adsorbing column£¨2 cm¡Á 30 cm£¬containing 100 mL D101 macroporous resin£©.Then water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid£¨6¡Ã3¡Ã2¡Ã1¡Ã0.001£© was used as the two-phase solvent system£¬in which the upper phase was used as the stationary phase£¬while the lower phase was used as the mobile phase£¬the flow rate was set at 2.0 mL/min£¬and the revolution speed was 850 r/min.9.8 mg compound 1 with purity of 95.1% and 14.3 mg compound 2 with purity of 98.2% were obtained from 160 mg crude sample by one-step£¬as determined by high-performance liquid chromatography£¨HPLC£©.The chemical structures were identified by mass spectrometry£¨MS£© and nuclear magnetic resonance imaging£¨NMR£©.Additionally£¬the antioxidant activities of the two compounds were also evaluated by the method of 1,1-diphenyl-2-picrylhydrazyl£¨DPPH?£© radical scavenging assay with Vc as the control group.The results of antioxidative activity test showed that the two compounds have strong capacity of scavenging DPPH£¨EC50£¼10 mg/L£©.Both of their EC50 were lower than that of Vc£¬and the scavenging ability of compound 1 was stronger than that of compound 2.