Preparative separation of structural isomeric pentacyclic triterpene oleanolic acid and ursolic acid from natural products by pH-zone-refining countercurrent chromatography.

In this work, pH-zone-refining countercurrent chromatography was investigated in the preparative separation of two bioactive components, oleanolic acid and ursolic acid, from three different natural products, , apple peels and Thunb. Oleanolic acid and ursolic acid are structurally isomeric pentacyclic triterpene acids that are widely distributed in many natural products. However, it was difficult to separate these components with high purity by conventional methods. A biphasic solvent system composed of -hexane-dichloromethane-methanol-water (7 : 3 : 2 : 8, v/v) was selected, in which an optimized concentration of 10 mmol L trifluoroacetic acid was added in the upper phase as the retainer and 10 mmol L ammonia (with 25-28% NH) was added in the aqueous phase as the eluter. Consequently, 38.56 mg of oleanolic acid with 99.01% purity was separated from 100 mg of the crude extract of , while 65.6 mg of a mixture of ursolic acid (90.98%) and oleanolic acid (6.51%) and 46.6 mg of a mixture of ursolic acid (74.35%) and oleanolic acid (23.61%) were separated from 100 mg of the crude extract of apple peels and 100 mg of the crude extract of Thunb., respectively, by pH-zone-refining countercurrent chromatography using the above selected biphasic solvent system. The results showed that pH-zone-refining countercurrent chromatography is an efficient method for the preparative separation of pentacyclic triterpene acids from natural products.