Preparative isolation and purification of four compounds from the Chinese medicinal herb Rhizoma Anemarrhenae by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the separation and purification of mangiferin, neomangiferin, cis-hinkiresinol and (-)-4′-O-methylnyasol from the Chinese medicinal herb Rhizoma Anemarrhenae. Five hundred milligrams of crude extracts were separated by using n-butanol–acetic acid (1%) (1:1, v/v) as the two-phase solvent system and yielded 35.3 mg of neomangiferin and 245.4 mg of mangiferin. During this separation, cis-hinkiresinol and (-)-4′-O-methylnyasol were still maintained in the stationary phase. The stationary phase was collected, evaporated to dryness and separated with light petroleum–ethyl acetate–methanol–water (1:1:1.2:0.8, v/v) and 1:1:1.4:0.6 (v/v) in gradient elution, which yielded 17.2 mg of cis-hinkiresinol and 12.4 mg of (-)-4′-O-methylnyasol. The purities of mangiferin, neomangiferin, cis-hinkiresinol and (-)-4′-O-methylnyasol were 96.3, 98.0, 97.3 and 98.2%, respectively, as determined by HPLC. The chemical structures of these components were identified by 1H NMR and 13C NMR.