Preparative Isolation and Purification of Calycosin and Formononetin from Astragali Radix using Hydrolytic Extraction Combined with High Speed Countercurrent Chromatography.

Calycosin and formononetin were efficiently extracted from Astragali Radix and purified by high-speed countercurrent chromatography. Calycosin and formononetin could be hydrolyzed from calycosin-7-glucoside and ononin, respectively. The best extraction conditions were realized by single factor and orthogonal experiments, which were 100% ethanol, 2.5 mol/L hydrochloric acid, 1:40 ratio of solid to liquid, extracted 2 h and one time. The two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (3:5:3:5, v/v) was selected for the purification of calycosin, and 1.3 mg calycosin (the purity was 95.8% and the recovery was 85.9%) was obtained from 264.9-mg crude extraction. The two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (4:5:4:5, v/v) was selected for the purification of formononetin, and 2.0 mg formononetin (the purity was 98.9% and the recovery was 84.4%) was obtained from 248.9-mg crude extraction. Their structures were identified by HPLC, melting points, UV, FTIR, ESI-MS, 1H NMR and 13C NMR spectrum. According to the antioxidant activity assay, the scavenging abilities of calycosin to 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radicals (·OH) were stronger. The scavenging effect of formononetin was not demonstrated.

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

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.