Preparative separation of phytosterol analogues from green alga Chlorella vulgaris using recycling counter-current chromatography.

The unicellular alga Chlorella vulgaris is a well-known health food. It has been proven that the minor phytosterols, ergosterol and its analogue, are an important class of bioactive substances in C. vulgaris. In this work, a recycling counter-current chromatographic approach was proposed for preparative separation of two analogue sterols from crude extract of C. vulgaris. The separation unit was set up with a type-J instrument coupled with a column switching valve. A two-phase solvent system composed of n-hexane/dichloromethane/acetonitrile (10:3:7, v/v/v) was selected and optimized. After five cycles of separation, two analogue sterols were baseline separated, producing 11.7 mg 26-nor-25-isopropyl-5,7,22-trien-3β-ol and 20.3 mg ergosterol from 300 mg of C. vulgaris extract. Their purities were both above 95%. The structures of two sterols were identified by using NMR spectroscopy.