Preparing process for Isolation of a plurality of isoflavones components in astragalus root

Abstract

The invention relates to a method for separating various isoflavone components from astragalus. It comprises two steps: (1) enriching plant extrant containing astragalus saponins with silica gel column; (2) separating the target component by reversed phase preparative liquid chromatography.The invention is characterized by low cost, high production and good repeatness, and it can provide new processes for preparing isoflavone components.

Description

technical field

[0001] The present invention relates to a method for simultaneously separating and preparing high-purity formononetin (Formononetin), 3-hydroxyl-9,10-dimethoxy pterostane (3-hydroxy-9) from astragalus isoflavone extracts by chromatography , 10-dimethoxy pterocarpane) and 2', 7-dihydroxy-3', 4'-dimethoxy isoflavane (2', 7-dihydroxy-3', 4'-dimethoxy isoflavane) three main monomers method of ingredients. Background technique

[0002] Astragalus isoflavones are secondary metabolites of Astragalus membranaceus. According to different chemical structures, they are mainly divided into free isoflavones, isoflavanes, pterostalanoids and their combined glycosides. Modern pharmacological studies have shown that astragalus isoflavones have anti-bacterial, anti-inflammatory, anti-viral, hypolipidemic, anti-cancer, and follicle hormone functions. Therefore, the separation and preparation of high-purity compound monomers has become an important research content. The prepa...

Examples

Embodiment 1

[0021] 1. Weigh 20 g of the crude extract of Astragalus total flavonoids (see Figure 1 for HPLC analysis), add methanol to dissolve it, mix the sample with silica gel at a mass ratio of 1:2 to 3 (w / w), and mix After the sample is uniform, evaporate the solvent and grind evenly.

[0022] 2. Use 100-200 mesh silica gel (1300g) to fill the chromatography column and perform column chromatography. The elution solvent is petroleum ether-ethyl acetate (1:1 by volume) and eluted with 4 times the column volume.

[0023] 3. the outflow part detects with thin-layer chromatography, developing agent is sherwood oil-ethyl acetate (volume ratio is 1: 1), and chromogen is the methanol solution containing 5% sulfuric acid, collects and contains target product (R f ≥0.62), the fractions containing formononetin, 9,10-dimethoxypurantane and 2',7-dihydroxy-3',4'-dimethoxyisoflavane were combined into a group Divided into Fr2, concentrated into extract 757mg. High-performance liquid chromatograph...

Embodiment 2

[0031] The preparation mode of component Fr2 is the same as embodiment 1. 287.5 mg of component Fr2 was weighed and dissolved in 10 mL of methanol-chloroform (volume ratio 1:2) solution system, and passed through a 0.45 μm filter membrane as a preparative chromatographic injection solution. The inner diameter of the preparative chromatographic column is 10 cm, the column length is 20 cm, the column filler is C18 (particle size 10 μm), and the injection volume is 20 mL. Inject the sample into the injection loop of the preparative chromatography, use methanol and 0.5% formic acid as the eluting phase, the volume ratio of the two-phase solvents is 50:50, carry out isocratic elution at a flow rate of 300mL / min, and run for 55min . On-line monitoring of the fractions by a UV detector, the detection wavelength is 230nm, cut according to the peak and collect the effluent to obtain 96 mg of formononetin, 41.6 mg of 9,10-dimethoxy pterocarpine, 2',7-dihydroxy-3 ', 4'-Dimethoxyisoflav...

Embodiment 3

[0033] The preparation mode of component Fr2 is the same as embodiment 1. 11 mg of component Fr2 was weighed and dissolved in 5 mL of methanol-chloroform (volume ratio 1:2) solution system, and passed through a 0.45 μm filter membrane to prepare a chromatographic injection solution. The inner diameter of the preparative chromatographic column is 2 cm, the column length is 25 cm, the column filler is C18 (particle size 10 μm), and the injection loop is 10 mL. Inject the sample into the injection loop of the preparative chromatography, use methanol and an aqueous solution containing 0.5% formic acid as the eluting phase, carry out isocratic elution with the volume fraction of the two-phase solvent at 50:50, the flow rate is 19mL / min, and the running time is 55min . The fraction was monitored by an online UV detector, the detection wavelength was 230nm, the effluent was cut according to the peak and the effluent was collected to obtain 3.9mg of formononetin, 1.76mg of 9,10-dimet...

technical field

[0001] The present invention relates to a method for simultaneously separating and preparing high-purity formononetin (Formononetin), 3-hydroxyl-9,10-dimethoxy pterostane (3-hydroxy-9) from astragalus isoflavone extracts by chromatography , 10-dimethoxy pterocarpane) and 2', 7-dihydroxy-3', 4'-dimethoxy isoflavane (2', 7-dihydroxy-3', 4'-dimethoxy isoflavane) three main monomers method of ingredients. Background technique

[0002] Astragalus isoflavones are secondary metabolites of Astragalus membranaceus. According to different chemical structures, they are mainly divided into free isoflavones, isoflavanes, pterostalanoids and their combined glycosides. Modern pharmacological studies have shown that astragalus isoflavones have anti-bacterial, anti-inflammatory, anti-viral, hypolipidemic, anti-cancer, and follicle hormone functions. Therefore, the separation and preparation of high-purity compound monomers has become an important research content. The prepa...

Examples

Embodiment 1

[0021] 1. Weigh 20 g of the crude extract of Astragalus total flavonoids (see Figure 1 for HPLC analysis), add methanol to dissolve it, mix the sample with silica gel at a mass ratio of 1:2 to 3 (w / w), and mix After the sample is uniform, evaporate the solvent and grind evenly.

[0022] 2. Use 100-200 mesh silica gel (1300g) to fill the chromatography column and perform column chromatography. The elution solvent is petroleum ether-ethyl acetate (1:1 by volume) and eluted with 4 times the column volume.

[0023] 3. the outflow part detects with thin-layer chromatography, developing agent is sherwood oil-ethyl acetate (volume ratio is 1: 1), and chromogen is the methanol solution containing 5% sulfuric acid, collects and contains target product (R f ≥0.62), the fractions containing formononetin, 9,10-dimethoxypurantane and 2',7-dihydroxy-3',4'-dimethoxyisoflavane were combined into a group Divided into Fr2, concentrated into extract 757mg. High-performance liquid chromatograph...

Embodiment 2

[0031] The preparation mode of component Fr2 is the same as embodiment 1. 287.5 mg of component Fr2 was weighed and dissolved in 10 mL of methanol-chloroform (volume ratio 1:2) solution system, and passed through a 0.45 μm filter membrane as a preparative chromatographic injection solution. The inner diameter of the preparative chromatographic column is 10 cm, the column length is 20 cm, the column filler is C18 (particle size 10 μm), and the injection volume is 20 mL. Inject the sample into the injection loop of the preparative chromatography, use methanol and 0.5% formic acid as the eluting phase, the volume ratio of the two-phase solvents is 50:50, carry out isocratic elution at a flow rate of 300mL / min, and run for 55min . On-line monitoring of the fractions by a UV detector, the detection wavelength is 230nm, cut according to the peak and collect the effluent to obtain 96 mg of formononetin, 41.6 mg of 9,10-dimethoxy pterocarpine, 2',7-dihydroxy-3 ', 4'-Dimethoxyisoflav...

Embodiment 3

[0033] The preparation mode of component Fr2 is the same as embodiment 1. 11 mg of component Fr2 was weighed and dissolved in 5 mL of methanol-chloroform (volume ratio 1:2) solution system, and passed through a 0.45 μm filter membrane to prepare a chromatographic injection solution. The inner diameter of the preparative chromatographic column is 2 cm, the column length is 25 cm, the column filler is C18 (particle size 10 μm), and the injection loop is 10 mL. Inject the sample into the injection loop of the preparative chromatography, use methanol and an aqueous solution containing 0.5% formic acid as the eluting phase, carry out isocratic elution with the volume fraction of the two-phase solvent at 50:50, the flow rate is 19mL / min, and the running time is 55min . The fraction was monitored by an online UV detector, the detection wavelength was 230nm, the effluent was cut according to the peak and the effluent was collected to obtain 3.9mg of formononetin, 1.76mg of 9,10-dimet...

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