Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave-assisted extraction coupled with high-speed counter-current chromatography

摘要

A rapid method combining microwave-assisted extraction and high-speed counter-current chromatography was applied for preparative separation of six bioactive compounds including loganic acid (I), isoorientin-4'-O-glucoside (II), 6'-O-β-d-glucopyranosyl gentiopicroside (III), swertiamarin (IV), gentiopicroside (V), sweroside (VI) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. Microwave-assisted extraction parameters were predicted by central composite design-response surface methodology. That is, 5.0 g dried roots of G. Crassicaulis was extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by high-speed counter-current chromatography with n-butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail-to-head elution mode. 16.3 mg, 8.8 mg, 12.8 mg, 25.1 mg, 40.7 mg and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPCL with photo diode array detection and quadrupole time-of-flight MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, microwave-assisted extraction coupled with high-speed counter-current chromatography could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.