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

• Published in: Journal of chromatographic science Vol. 59; no. 5; pp. 412 - 418
• Main Authors: Pan, Conglei, Wang, Hao, Shan, Hu, Lü, Haitao
• Format: Journal Article
• Language: English
• Published: United States 21-04-2021
• ISSN: 0021-9665; 1945-239X
• DOI: 10.1093/chromsci/bmab021

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.