An Efficient Method for the Preparative Isolation and Purification of Flavonoids from Leaves of Crataegus pinnatifida by HSCCC and Pre-HPLC

Abstract

In this work, flavonoid fraction from the leaves of <i>Crataegus pinnatifida</i> was separated into its seven main constituents using a combination of HSCCC coupled with pre-HPLC. In the first step, the total flavonoid extract was subjected to HSCCC with a two-solvent system of chloroform/methanol/water/<i>n</i>-butanol (4:3:2:1.5, <i>v/v</i>), yielding four pure compounds, namely (-)-epicatechin (<b>1</b>), quercetin-3-<i>O</i>-(2,6-di-α-l-rhamnopyranosyl)-β-d-galactopyranoside (<b>2</b>), 4''-<i>O</i>-glucosylvitexin (<b>3</b>) and 2''-<i>O</i>-rhamnosylvitexin (<b>4</b>) as well as a mixture of three further flavonoids. An extrusion mode was used to rapidly separate quercetin-3-<i>O</i>-(2,6-di-α-l-rhamnopyranosyl)-β-d-galactopyranoside with a big <i>K</i>_D-value. In the second step, the mixture that resulted from HSCCC was separated by pre-HPLC, resulting in three pure compounds including: vitexin (<b>5</b>), hyperoside (<b>6</b>) and isoquercitrin (<b>7</b>). The purities of the isolated compounds were established to be over 98%, as determined by HPLC. The structures of these seven flavonoids were elucidated by ESI-MS and NMR spectroscopic analyses.