Combined application of macroporous resin and high speed counter-current chromatography for preparative separation of three flavonoid triglycosides from the leaves of Actinidia valvata Dunn

In this paper, the combined techniques of macroporous resin column chromatography and high speed counter-current chromatography were applied for preparative separation of flavonoid triglycosides from the leaves of Actinidia valvata Dunn, a famous Chinese medicinal herb. Twelve kinds of macroporous resins were investigated by adsorption and desorption tests. HPD-300 resin showed the maximum effectiveness and thus was selected for the first cleaning-up, in which 20% ethanol was used to remove the undesired constituents and 60% ethanol to elute the targets. The crude extract was then purified by high speed counter-current chromatography with the solvent system composed of ethyl acetate?n-butanol?water (2:1:3 and 4:1:5, v/v). Three flavonoid triglycosides, namely, kaempferol 3-O-a-l-rhamnopyranosyl-(1?3)-a-l-rhamnopyranosyl-(1?6)-beta-d-galactopyranoside, kaempferol 3-O-a-l-rhamnopyranosyl-(1?3)-(4-O-acetyl-a-l-rhamnopyranosyl)-(1?6)-beta-d-galactopyranoside and kaempferol 3-O-a-l-rhamnopyranosyl-(1?3)-(2,4-di-O-acetyl-a-l-rhamnopyranosyl)-(1?6)-beta-d-galactopyranoside, were obtained. The purities of the separated compounds were all over 95% as determined by HPLC area normalization method. Their chemical structures were confirmed by UV, MS, NMR, and the standards.