Abstract
An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v), and (0:1:0:1, v/v). The separation was repeated three times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4′-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%), and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, 1H NMR, and 13C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And, four separation rules of flavonols were established according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.
ACKNOWLEDGMENTS
This work was financially supported by the Chinese Academy of Sciences Innovative Research International Partnership Project and grants from the National Key Technology R&D Program (code: 2007BAI30B00) and the National Natural Science Foundation of China (code: 30873455).