Separation and purification of isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze by counter-current chromatography comparing two kinds of solvent systems.

The first preparative separation of a flavonoid sulphate isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze by counter-current chromatography (CCC) was presented. Two kinds of solvent systems were used. A conventional organic/aqueous solvent system n-butanol-ethyl acetate-water (4:1:5, v/v) was used, yielding isorhamnetin 3-sulphate 2.0 mg with a purity of 93.4% from 83 mg of pre-enriched crude extract obtained from 553 mg ethanol extract by macroporous resin. A one-component organic/salt-containing system composed of n-butanol-0.25% sodium chloride aqueous solution (1:1, v/v) was also used, and the LC column packed with macroporous resin has been employed for desalination of the target compound purified from CCC. As a result, 2.1 mg of isorhamnetin 3-sulphate with a purity of over 97% has been isolated from 402 mg of crude extract without pre-enrichment. Compared with the conventional organic/aqueous system, the one-component organic/salt-containing aqueous system was more suitable for the separation of isorhamnetin 3-sulphate, and purer target compound was obtained from the crude extract without pre-enrichment using the new solvent system. The chemical structure was confirmed by ESI-MS and (1)H, (13)C NMR. In summary, our results indicated that CCC using one-component organic/salt-containing aqueous solution is very promising and powerful for high-throughput purification of isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze.