Preparative Isolation And Purification Of Flavones From Glycyrrhiza Uralensis And Semen Sojae Praeparatum By High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC) is a unique liquid-liquid partition chromatography that uses no solid support matrix. This technique has recently been used to effectively separate a large variety of natural products. In this paper, we have reviewed the rationale, operational approach and the main influential factors of high-speed counter-current chromatography. Furthermore, we summarized several phase systems that universally used in HSCCC separation.Glycyrrhiza Uralensis and Semen Sojae Praeparatum are traditional Chinese herbs that recorded in Chinese Pharmacopoeia. There are many active ingredients including flavones, triterpenes and alkaloids in Glycyrrhiza Uralensis and the research demonstrates that the licorice flavones display notable effects in antioxidant, antibiosis and anticancer. In this paper, we have utilized HSCCC to separate Licoflavonol, Liquiritigenin, Formononetin, and Licoisoflavone A these four flavones from Glycyrrhiza Uralensis crude extracts and optimized the phase system, sample concentration and the flow rate of mobile phase. The two-phase solvent system was composed of n-hexane: ethyl acetate: methanol: water (1:2:1:1). The upper phase was stationary phase and the lower phase was mobile phase. Rotational direction was clockwise and rotational speed was 850r/min. Sample concentration was 20mg/ml and the flow rate was 3.0ml/min. The effluent was detected at 260nm. There were 26mg Licoflavonol, 8mg Liquiritigenin, 12mg Formononetin and 10mg Licoisoflavone A through one step separation from 400mg glycyrrhiza uralensis crude extracts, and the purity was separately: 96.3ï¼…, 95.7ï¼…, 96.5ï¼…and 96.8ï¼…. MP, UV, MS, ¹H-NMR and ¹³C-NMR identified the structures of the four fractions.The main active composition in Semen Sojae Praeparatum are soybean isoflavones. For the structures are similar to estrogen, they have displayed much physiological activity in prophylaxis and therapy in cancer, cardiovascular diseases, and osteoporosis. HSCCC has been generally used in separation and purification of soybean isoflavones, however, those separation phase systems most composed of chloroform. In this paper, we refined the hypotoxicity phase system of n-hexane: ethyl acetate: methanol: water (5:6:5:6) to separate daidzein and genistein from Semen Sojae Praeparatum crude extracts. Using this new solvent system, we got daidzein 11mg and genistein 6.8mg from 200mg crude soybean extracts through one step isolation, and the purity of two fractions was separately: 96.3ï¼…and 93.7ï¼…. The upper phase was mobile phase and the lower phase was stationary phase. Rotational direction was clockwise and rotational speed was 850r/min. Sample concentration was 10mg/ml and the flow rate was 2.0ml/min. The effluent was detected at 260nm.