Investigation of unsaponifiable matter of plant oils and isolation of eight phytosterols by means of high-speed counter-current chromatography

Abstract

Phytosterols are minor components of plant oils. Due to their beneficial effect on human serum cholesterol level, new products supplemented with phytosterols have been marketed. Commercial phytosterol standards are frequently of insufficient purity, very expensive, only available in (semi-) synthetic form or not available at all. For this reason we aimed to explore the unsaponifiable matter of three plant oils (rapeseed oil, linseed oil and olive oil) in order to study their compositions and to purify several phytosterols. We fractionated ∼100mg of the unsaponifiable matter of the plant oils with high-speed counter-current chromatography (HSCCC) by the combination of an enrichment step and a purification step. In the first part (enrichment step) composition of phytosterols, alkanes, fatty alcohols from 14:0 to 30:0 including isomers, 15-nonacosanone and other ketones as well as further minor compounds in the different fractions was studied by GC/MS. By means of the solvent system n-hexane/methanol/aqueous silver nitrate solution (34/24/1, v/v/v) in normal phase mode (tail-to-head) β-sitosterol could be isolated (6.4mg, purity ≥99%) and several phytosterols (e.g. citrostadienol, cycloeucalenol and erythrodiol) could be enriched. Moreover, the fast eluting hydrocarbons squalene and nonacosane as well as the later eluting phytol (pure, 7mg) and geranyl geraniol could also be efficiently enriched.Suited HSCCC fractions from the three plant oils were merged and re-injected into the HSCCC system (purification step). The HSCCC purification step provided 6.9mg campesterol (≥99%), 2.9mg brassicasterol (≥99%), 0.3mg Δ5-avenasterol (≥90%), 9.5mg cycloartenol (≥90%), 3.7mg 24-methylene-cycloartanol (≥99%), and ∼1mg of an unknown compound (∼80%) isolated from rapeseed and linseed oil. Furthermore, the combined HSCCC enrichment and purification of a hydrogenated sterol standard provided two pure phytostanols (campestanol ≥99% and sitostanol ≥99%) and several by-products.