In the past few years, the aqueous three-phase system (A3PS) has attracted a lot of attention due to its effectiveness in the efficient separation. Polysaccharides, carotenoids and polyphenols were separated simultaneously from Lycium barbarum by an aqueous three-phase system (A3PS) formed by acetonitrile/betaine/K2HPO4 as a novel extraction technique. The system is characterized by small amount of machine reagents, few consumables and simple extraction steps. The effects of acetonitrile, betaine and K2HPO4 concentrations on the extraction efficiency were investigated by Response Surface Methodology. The optimum composition of A3PS were 26.2 % (w/w) acetonitrile, 10.2 % (w/w) betaine and 27.6 % (w/w) K2HPO4. Under this condition, the extraction efficiencies of carotenoids, polyphenols, and Lycium barbarum polysaccharides (LBP) were 42.12±0.10 %, 88.61±0.95 % and 84.63±0.46 %, respectively. The obtained LBP was characterized by several technologies. The LBP was proved to consist of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a proportion of 4.14:0.80:4.00:4.43:22.20:39.9:12.44:3.22:8.46:0.41. The molecular weight of LBP was 12554 Da. In addition, LBP exhibited excellent antioxidant activity. In general, as a rapid developing method of extraction and separation, A3PS had potential application in the simultaneous separation of multiple substances.
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