Isolation And Purification Of Bioactive Compounds From The Stem Bark Of Erythrina Variegata Via High-speed Counter-current Chromatography

High-speed countercurrent chromatography (HSCCC) has been widely used for separation and purification of chemical compounds from natural products. In this paper, HSCCC was used to separate and purify bioactive compounds from ethyl acetate fraction of the stem bark of Erythrina variegata. The optimal conditions for HSCCC separation, the antioxidant and antibacterial activities of isolated compounds were studied.(1) The optimal conditions for HSCCC separation were investigated. The two-phase solvent system was composed of n-hexane-ethyl acetate-methanol-water (1:4:1:4, v/v). The upper phase was used as stationary phase and the other as mobile phase. The revolution speed was set850rpm and flow rate was2.0mL/min. At this time, the retention of the stationary phase was54%. After one-step HSCCC separation,75mg of protocatechuic acid,32mg of chlorogenic acid and44mg of caffeic acid were purified from420mg of the ethyl acetate fraction. The purity of each compound was over95%and that of protocatechuic acid was even determined up to99.7%by high performance liquid chromatography analysis.(2) The chemical structures of the three isolates were confirmed by UV, HPLC-MS/MS,1H NMR and13C NMR. They were protocatechuic acid, chlorogenic acid and caffeic acid. The results indicated that the three compounds were first obtained from the stem bark of Erythrina variegata.The antioxidant and antibacterial tests were also studied.2,2-diphenyl-1-picrylhydrazyl (DPPH) assay indicated that the three compounds had high antioxidant activities. The order of antioxidant activities was caffeic acid> protocatechuic acid> chlorogemic acid. And their IC50values were22.5(μg/mL (protocatechuic acid),41.9μg/mL (chlorogenic acid),20.9μg/mL (caffeic acid), respectively. Their antibacterial activities against Enterobacter sakazakii, Vibrio parahaemolyticus, Salmonella typhimurium, Staphylo coccus aureus, Enterobacterium were vestigated. Each of the isolates showed more antibacterial than ethyl acetate fraction. Protocatechuic acid inhibited Vibrio parahaemolyticus growth most potently of the three isolates, and had a minimum inhibitory concentration (MIC) of400μg/mL. Chlorogenic acid had the MIC of600μg/mL against Enterobacter sakazakii. MIC for caffeic acid against Salmonella typhimurium was600μg/mL.(3) The effect of inons in the solvent system of n-hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) for salicylic acid was studied. Different kinds of acid and salt on the the partition coefficient of salicylic acid were discussed. And the concentrations of acid and salt were also studied. The results showed the partition coefficient could be modified by adding acid or salt to the solvent system. There were twenty graphs, seventeen tables and one hundred references in this paper.