Abstract
A novel and effective chromatographic approach to the separation and purification of isoflavone compounds from Pueraria lobata is described. The method is based on flash chromatography (FC), coupled to preparative high performance liquid chromatography (prep-HPLC) via a six-way valve. The FC step comprised tandem reversed phase columns, pre-packed with MCI gel (Mitsubishi Chemical Corp., Tokyo, Japan) and C18 (Fuji Silysia Chemical Ltd, Osaka, Japan) resin, respectively, and was designed to separate a crude Pueraria lobata extract into several preliminary fractions. Fractions containing the target compounds were then directly injected via the six-way valve into prep-HPLC columns, without further treatment, for final isolation and purification. Nine isoflavonoids were successfully isolated, three through an online mode and the other six through an offline mode. The purities of all compounds exceeded 95.0%, as determined by HPLC with an UV-vis photodiode array detector. The convenience, low solvent consumption, and time-saving advantages of this method offer an attractive and promising approach to the isolation of natural products.
Highlights
The root of Pueraria lobata (Wild.) Ohwi is an important Chinese herb used in China as an herbal medicine, named “Ge-Gen”, to treat hypertension and anginapectoris [1]
This is the first report of the separation and purification of isoflavones from Pueraria lobata by a coupled method, and this is the first time that flash chromatography (FC) coupled with prep-HPLC has been described as a powerful method in separation and purification technology
C-NMR (150 MHz, DMSO-d6) δ ppm: 153.8 (C-2), 122.8 (C-3), 175.2 (C-4), 127.4 (C-5), 116.1 (C-6), 161.9 (C-7), 103.8 (C-8), 157.7 (C-9), 118.9 (C-10), 124.2 (C-1'), 130.6 (C-2',6'), 115.5 (C-3',5'), 157.5 (C-4'), 100.4 (C-1''), 73.6 (C-2''), 77.0 (C-3''), 70.1 (C-4''), 77.7 (C-5''), 61.1 (C-6''). These data are in agreement with those reported in the literature [16]. This pioneering research has developed an ovelap proach combining FC and prep-HPLC for the efficient isolation and purification of nine isoflavonoid compounds from Pueraria lobata
Summary
The root of Pueraria lobata (Wild.) Ohwi is an important Chinese herb used in China as an herbal medicine, named “Ge-Gen”, to treat hypertension and anginapectoris [1]. We used FC, coupled to two sets of prep-HPLC, connected via a six-way valve (Figure 1) With this combined separation approach, nine isoflavones of Pueraria lobata were isolated and purified, namely 3'-hydroxypuerarin-6''-O-glucoside (1), puerarin-6''-O-glucoside (2), daidzein-4',7-Odiglucoside (3), 3'-hydroxypuerarin (4), 3'-hydroxypuerarin-6''-O-apioside (5), puerarin (6), 3'-methoxypuerarin (7), daidzein8-C-apiosyl(1→6) glucoside (8) and daidzin (9). FC coupled with prep-HPLC has the following distinct advantages over other methods: rapid separation, high recovery, low solvent consumption and elimination of the need for pretreatment. This is the first report of the separation and purification of isoflavones from Pueraria lobata by a coupled method, and this is the first time that FC coupled with prep-HPLC has been described as a powerful method in separation and purification technology
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