Separation Of Effective Components From Flaveria Bidentis (L.) Kuntze And Mikania Micrantha H. B. Kunth By Ph-Zone-Refining Countercurrent Chromatography

pH-zone-refining counter-current chromatography was developed based on preparative HSCCC. It is generally employed as a large-scale preparative technique for separation according to the pKa and hydrophobility. The method elutes highly concentrated rectangular peaks. It has been widely used in the separation of natural products, including alkaloids, organic acids, dye mixtures, peptides, enantiomers and so on. In this article, effective components had been separated from Flaveria bidentis (L.) Kuntze, Lonicerae Flos and Mikania micrantha H. B. Kunth by pH-zone-refining countercurrent chromatography.Chologenic acid and caffeic acid were selected as test samples for separation by pH-zone-refining CCC. Chologenic acid was successfully separated from Flaveria bidentis (L.) Kuntze and Lonicerae Flos by using the method.1. GS-20A CCC was successfully used for sepration of chlorogenic acid and caffeic acid.The two phase solvent system was composed of methyl tert-butyl ether-acetonitrile-water4:1:5(v/v) where trifluoroacetic acid (TFA; 8mM) was added to the organic stationary phase as a retainer and NH4OH (10mM) to the aqueous mobile phase as an eluter.2. Chologenic acid has been separated from natural products and trace caffeic acid was collected for the futher sepration.Using GS-10A CCC by the solvent system methyl tert-butyl ether-acetonitrile-n-butanol-water4:1:1:5(v/v) where TFA and NH4OH (10mM) decresased to3mM, it could collect16.2mg of chlorogenic acid with the purity of92%from1.4g of F.bidents one time. The same method was used to separate chlorogenic acid from the crude extract of Lonicerae Flos, which134mg of chlorogenic acid was separated with the purity of99%from1.3g of the crude sample.In the study of Mikania micrantha H. B. Kunth, three kinds of effective components were separated. The main results were as follow:1. Optimize the extraction of the effective components. The extraction conditions are as follow:the powder was extracted by petroleum ether, ethyl acetate, and80%ethanol in this order90minutes (1time). The extract of80%ethanol was concentrated under reduced pressure. After that, the crude sample dissvoled by the lower phase of the solvent system methyl tert-butyl ether-acetonitrile-water2:2:3(v/v), then extracted by the same volume of the upper phase with10mM TFA (3times). The crude sample was dried by the rotary evaporator under60℃.2. pH-zone-refining CCC was used for the separation of the effective components from Mikania micrantha H. B. K. The two phase solvent system was methyl tert-butyl ether-acetonitrile-water2:2:3(v/v) with3mM TFA in the upper phase and3mM NH4OH in the lower phase. It could separate three kinds of components from the crude sample one time.