Preparative separation of quercetin, ombuin and kaempferide from Gynostemma pentaphyllum by high-speed countercurrent chromatography.

Abstract

An efficient method for the preparative separation of quercetin, ombuin and kaempferide from Gynostemma pentaphyllum by high-speed countercurrent chromatography (HSCCC) was successfully developed for the first time. The extraction conditions were optimized by an orthogonal array design L9 (34), while quercetin, ombuin and kaempferide were efficiently released from rutin, ombuoside and kaempferide glucoside by hydrochloric acid hydrolysis, respectively. Quercetin was purified by HSCCC with two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v) in a single run. From 164.7 mg of the crude extract, 90.5 mg quercetin at 99.23% purity was obtained. Ombuin and kaempferide were purified by HSCCC with two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (8:2:5:5, v/v) in a single run. From 50 mg of the crude extract, 0.4 mg of ombuin at 95.46% purity and 1.1 mg of kaempferide at 98.78% purity were obtained, respectively. Their structures were identified by ultraviolet, Fourier transform infrared, electrospray ionization mass spectrometry (ESI-MS), 1H nuclear magnetic resonance (NMR) and 13C NMR spectra. The purified quercetin had the strongest 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radical scavenging activities.