Preparative isolation and purification of flavonoids and protocatechuic acid from sea buckthorn juice concentrate (Hippophae rhamnoides L. ssp rhamnoides) by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC)-a support free all liquid-liquid chromatography technique-has been successfully used for the preparative isolation of isorhamnetin 3-O-beta-D-glucoside, isorhamnetin 3-O-beta-rutinoside, quercetin 3-O-beta-D-glucoside, syringetin 3-O-beta-D-glucoside and protocatechuic acid from sea buckthorn juice concentrate (Hippophae rhamnoides L. ssp. rhamnoides, Elaeagnaceae). The preparative HSCCC instrument was a multilayer coil planet centrifuge equipped with three preparative coils. Separation was performed with a two phase solvent system (n-hexane-n-butanol-water, 1:1:2 v/v/v) in 'head-to-tail' mode. Each injection of 4.1 g crude ethyl acetate extract yielded isorhamnetin 3-O-beta-D-glucoside (95 mg), isorhamnetin 3-O-beta-rutinoside (10 mg), quercetin 3-O-beta-D-glucoside (5 mg), and protocatechuic acid (34 mg) with purities >98%. The flavonoid syringetin 3-O-beta-D-glucoside (2 mg) was a novel compound for H. rhamnoides. Chemical structures of all compounds were determined by HPLC-ESI-MS-MS, 1D-NMR (H-1, C-13, DEPT 135) spectroscopy and for elucidation of glycosidic linkages 2D-NMR (HMBC) spectroscopy was used.