Preparative Separation and Purification of Cyclosporin D from Fungus Hypoxylon Spp. by Improved Closed-loop Recycling Counter-Current Chromatography

Abstract Improved closed-loop recycling counter-current chromatography (CLR CCC) with a two-phase solvent system composed of n-hexane–acetonitrile (1:1, v/v) was developed for separation, purification and preparation of cyclosporin D from the crude extract of fungus Hypoxylon Spp. (sj18). 28 mg cyclosporin D was successfully purified from 300 mg crude extract sample. The purity was 95.2% after five cycles, determined by HPLC. The structure of cyclosporin D was identified and assigned by 1H NMR, 13C NMR and mass spectrometric analyses. In addition, in the study, we show an interesting phenomenon that cyclosporin D can be prepared by the conventional CCC in n-hexane–ethyl acetate–methanol–water solvent system (2.5:1:2.5:1, v/v/v/v), and can also be prepared by the improved closed-loop recycling CCC in n-hexane–acetonitrile solvent system (1:1, v/v), but the efficiency of preparation varies greatly.