Preparative isolation and purification of three rotenoids and one isoflavone from the seeds of Millettia pachycarpa Benth by high-speed counter-current chromatography

Abstract

Both analytical and preparative high-speed counter-current chromatography (HSCCC) were used to isolate and separate chemical bioactive constituents from the seeds of Millettia pachycarpa Benth, a famous traditional Chinese medicine. Three rotenoids and one isoflavone were successfully purified for the first time by HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (HEMWat) (1:0.8:1:0.6, v/v/v/v). The separation parameters were first performed on the analytical HSCCC and optimized conditions were then scaled up to preparative HSCCC. The separation produced 160.2 mg tephrosin, 14.6 mg 4′,5′-dimethoxy-6,6-dimethylpyranoisoflavone, 109.4 mg deguelin, 6.7 mg 6a,12a-dehydrodeguelin with respective purities of 95, 93, 95, 95%, in one single run from 400 mg crude extract of the seeds of M. pachycarpa Benth. The purity of the isolated compounds was analyzed by high-performance liquid chromatography (HPLC) and their structures were identified by electrospray ionization mass spectrometry (ESI-MS); 1H nuclear magnetic resonance ( 1H NMR) and 13C nuclear magnetic resonance ( 13C NMR) analysis. This paper is an excellent example of the role that CCC is playing in isolating active compounds for pre-clinical trials of new chemical entities, even when scaling up between centrifuges from different manufacturers.