Preparative isolation and purification of two isoflavones from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

doi:10.1016/S0021-9673(03)00315-7

Journal of Chromatography A

Volume 992, Issues 1–2, 11 April 2003, Pages 193-197

https://doi.org/10.1016/S0021-9673(03)00315-7 Get rights and content

Abstract

Two isoflavones, calycosin-7-O-β-d-glycoside and formononetin-7-O-β-d-glycoside, were separated from n-butanol extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography in two steps using two different solvent systems composed of ethyl acetate–ethanol–n-butanol–water (30:10:6:50, v/v) and ethyl acetate–ethanol–water (5:1:5, v/v). From 200 mg of crude extract, calycosin-7-O-β-d-glycoside (12 mg) and formononetin-7-O-β-d-glycoside (10 mg) were isolated at over 95% purity by HPLC analyses, and their structures were identified by MS, ¹H NMR and ¹³C NMR.

Introduction

The roots of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao, and certain species of Astragalus (Leguminosae) have long been used as an anti-perspirant, a diuretic or a tonic in Chinese traditional medicine under the name of Huang-qi in China and Ougi in Japan [1], [2], [3], [4] while the studies on pharmacology and clinical practice have demonstrated the immunostimulating, cardiotonic and antiaging activities. Its isoflavones, including calycosin-7-O-β-d-glycoside and formononetin-7-O-β-d-glycoside, showed antimicrobial and superoxide anion scavenging activities [5], [6], [7].

The flavonoids were well known as one group of the beneficial components [2], [3], [4], [8], [9], [10], and have suitable chromophores for UV detection so that it has been chosen as “marker compounds” for the chemical evaluation or standardization of Huang-qi and its products [4], [11], [12].

The preparative separation and purification of calycosin-7-O-β-d-glycoside and formononetin-7-O-β-d-glycoside from plant materials by conventional methods is tedious and usually requires multiple chromatography steps. High-speed counter-current chromatography (HSCCC), being a unique liquid–liquid partition chromatography which uses no solid matrix, eliminates irreversible adsorption of sample onto the solid support [13] and therefore is considered as a suitable alternative for the separation of phenolic compounds such as flavonoids and hydroxyanthraquinones [14], [15], [16].

The present paper introduces a method for the separation of calycosin-7-O-β-d-glycoside and formononetin-7-O-β-d-glycoside, whose chemical structures are given in Fig. 1A, from a crude extract of A. membranaceus by HSCCC.

Section snippets

Apparatus

The analytical HSCCC instrument employed in the present study is a Model GS20 analytical high-speed counter-current chromatograph designed and constructed in Beijing Institute of New Technology Application (Beijing, China). The apparatus holds a pair of column holders symmetrically on the rotary frame at a distance of 5 cm from the central axis of the centrifuge. The multilayer coil separation column was prepared by winding a 50 m long, 0.85 mm I.D. PTFE (polytetrafluoroethylene) tube directly

Results and discussion

The crude extract of A. membranaceus was first analyzed by HPLC. The result indicates that the crude sample contains several compounds including calycosin-7-O-β-d-glycoside and formononetin-7-O-β-d-glycoside and some unknown compounds (Fig. 1A).

HSCCC separation was carried out using two different solvent systems. In the first separation, we examined the solvent systems composed of ethyl acetate–ethanol–n-butanol–water at different volume ratios such as 50:10:0:50, 50:5:5:50, 30:10:5:50,

Acknowledgments

The authors would like to thank senior engineers Xining Li, Xueli Cao, Guoqing Xu and Xiao Han for their excellent technical assistance in the research work.

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Short communicationPreparative isolation and purification of two isoflavones from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

Abstract

Introduction

Section snippets

Apparatus

Results and discussion

Acknowledgments

Phytochemistry

J. Chromatogr.

J. Chin. Pharm. Sci.

Zhiwu Ziyuan Yu Huanjing

Short communication
Preparative isolation and purification of two isoflavones from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography