A simple and efficient linear gradient coupled with inner-recycling high-speed counter-current chromatography mode for the preparative separation of flavonoid glycosides from leaves of custard apple.

Abstract

In general, the simultaneous separation and isolation of compounds with a broad polarity range from natural products is a challenge by ordinary high-speed counter-current chromatography (HSCCC). Indeed, the complex solvent system screening methods limit the broader application of HSCCC. We describe herein a rapid and efficient linear gradient CCC (LGCCC) method that enables the separation of flavonoid glycosides with a broad range of KD values from custard apple leaves. Inner-recycling CCC (IRCCC) mode has been further applied for the separation of compounds with similar KD values. Similarly to binary gradient HPLC, the LGCCC mode is achieved by adjustment of the proportion between ethyl acetate (pump A) and n-butanol (pump B) in an ethyl acetate/n-butanol/water solvent system. Various separation factors have been investigated, including separation mode, rotation speed, flow rate, and sample loading. The IRCCC mode has been used for the secondary separation of two epimers with a simple ethyl acetate/water (1:1, v/v) solvent system. Finally, five main flavonoid glycosides have been successfully separated, namely quercetin-3-O-robinobioside (1, 4.8\xa0mg) and rutin (2, 12.1\xa0mg), quercetin-3-O-β-d-glucoside (3, 4.2\xa0mg), kaempferol-3-O-robinobioside (4, 9.6\xa0mg), and kaempferol-3-O-rutinoside (5, 24.6\xa0mg). The purities of the separated flavonoid glycosides were over 98%, as determined by HPLC. Our study indicates that a suitable combination of LGCCC and IRCCC modes is an effective strategy for separating flavonoid glycosides from custard apple leaves. The mathematical expression of the LGCCC was deduced to illuminate the separation mechanism. It may also be applied to obtain component fractions for the further screening of active compounds from complex natural products.