Effective two-dimensional counter-current chromatographic method for simultaneous isolation and purification of oridonin and ponicidin from the crude extract of Rabdosia rubescens

A preparative two-dimensional counter-current chromatographic system (2D-CCC) for simultaneous separation and purification of oridonin and ponicidin from the crude extract of Rabdosia rubescens was presented. It was based on the use of a high-speed CCC (HSCCC, total column volume Vc = 146 ml) instrument in the first dimension (1st-D) and a preparative upright CCC (UCCC, Vc = 1500 ml) column in the second dimension (2nd-D). The interface was a homemade column-switching system with a 50-ml sample loop. In this way, almost the whole interested region from the first dimension was transferred on-line to the second dimension for further separation. The use of a pair of two-phase solvent systems composed of n-hexane–ethyl acetate–methanol–water (1:5:1:5 and 3:5:3:5, v/v) in the two dimensions permitted the simultaneous separation of oridonin and ponicidin, making this 2D-CCC system with satisfactory resolution and peak capacity. During about 9 h separation, two injections with about a 250 mg amount of the crude extract for each, was accomplished using this 2D-CCC system, yielding 60 mg of oridonin (1) and 10 mg of ponicidin (2) all at a purity of over 95% as determined by HPLC analysis. Their chemical structures were identified by electrospray ionization (ESI) MS, 1H NMR and 13C NMR.