Preparative Isolation and Purification of Four Flavonoids from the Petals of Nelumbo nucifera by High-speed Counter-current Chromatography

Introduction - Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective - To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high-speed counter-current chromatography (HSCCC). Methodology - Following an initial clean-up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC-PAD, ESI-MS, H-1-NMR and C-13-NMR. Results - The separation was performed using a two-phase solvent system composed of ethyl acetate-methanol-water-acetic acid (4:1:5:0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow-rate of 1.0 mL/min in the head-to-tail elution mode. Ultimately, 5.0 mg syringetin-3-O-beta-d-glucoside, 6.5 mg quercetin-3-O-beta-d-glucoside, 12.8 mg isorhamnetin-3-O-beta-d-glucoside and 32.5 mg kaempferol-3-O-beta-d-glucoside were obtained from 125 mg crude sample. Conclusion - The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright (C) 2009 John Wiley & Sons, Ltd.