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Purification of (+)-dihydromyricetin from leaves extract of Ampelopsis grossedentata using high-speed countercurrent chromatograph with scale-up triple columns

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Abstract

Purification of (+)-dihydromyricetin from an extract (16 g) of leaves of Ampelopsis grossedentata was performed using a preparative triple-column countercurrent chromatograph. With a solvent system composed of n-hexane–ethyl acetate–methanol–water (1:3:2:4, v/v) 11.3 g of (+)-dihydromyricetin was obtained at a high purity of over 99% by HPLC at 254 nm in 9 h.

Introduction

The leaves of Ampelopsis grossedentata are used for beverages in South China. The main component in the leaves is (+)-dihydromyricetin (Fig. 2), also called (+)-ampelopsin, that occupies 15–20% (w/w) of dry tender leaves. (+)-Dihydromyricetin has minimum toxicity and can inhibit the contraction of rabbit aortic strips evoked by noradrenaline and high K+ solution, which implies the effect of inhibiting hypertension [1]. It was also found to show an inhibitory activity on the ethanol-induced muscle relaxation in rats [2]. However, it is not easy to purify (+)-dihydromyricetin though it comprises about 70% of the direct methanol extract of the leaves [3].

This paper describes the preparative purification of (+)-dihydromyricetin from dried leaves of Ampelopsis grossedentata using a high-speed countercurrent chromatograph equipped with three scale-up columns [4], [5].

Section snippets

Apparatus

The present study employed a triplet coil planet centrifuge countercurrent chromatograph manufactured at the Institute of Food and Biological Engineering, Hangzhou University of Commerce, Hangzhou, China. It holds a set of three multilayer coil separation columns each consisting of 42 m long×5 mm I.D. PTFE (polytetrafluoroethylene) tubing wound around a holder hub of 22 cm in length×7 cm in diameter. The three columns were connected in series to make a total capacity of 2460 ml.

The stationary

HS-CCC separation

Fig. 1 shows the HSCCC separation of 16 g of the crude extract of (+)-dihydromyricetin. Three components, I, II and III, were obtained. Although HPLC analysis of the crude sample showed more than five peaks (Fig. 2A), component II (Fig. 1) showed a single peak (Fig. 2B). HPLC analyses also revealed that components I and III were found to be a mixture of two and three compounds, respectively. Except for myricetin present in component III, all other compounds were unknown. It suggested that

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