One-Step Preparative Separation of Phytosterols from Edible Brown Seaweed Sargassum horneri by High-Speed Countercurrent Chromatography

Sargassum horneri, a sargassaceae brown alga, is one of the main species in the subtidal seaweeds flora extensively distributed in the Yellow and East China Sea. It has been proven that the phytosterols are an important class of bioactive substances in S. horneri. In this work, a counter-current chromatography approach is proposed for preparative separation of phytol and two analogue sterols from a crude extract of S. horneri. A two-phase solvent system composed of n-hexane-acetonitrile-methanol (5:5:6, v/v) was selected and optimized. The effects of rotary speed and flow rate on the retention of the stationary phase were carefully studied. Under the optimum conditions, phytol and two analogue sterols, fucosterol and saringosterol, were baseline separated, producing 19.8 mg phytol, 23.7 mg fucosterol, and 3.1 mg saringosterol from 300 mg of crude S. horneri extract in one-step separation. The purities of three target compounds were all above 85%. The structures of phytol and two sterols were identified by nuclear magnetic resonance spectroscopy.