CN100484931C - Isolation preparing process for a plurality of isoflavones components in astragalus root - Google Patents
Isolation preparing process for a plurality of isoflavones components in astragalus root Download PDFInfo
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- CN100484931C CN100484931C CNB200510136731XA CN200510136731A CN100484931C CN 100484931 C CN100484931 C CN 100484931C CN B200510136731X A CNB200510136731X A CN B200510136731XA CN 200510136731 A CN200510136731 A CN 200510136731A CN 100484931 C CN100484931 C CN 100484931C
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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
The present invention relates to the separation simultaneously from the gathering isoflavone extract of a kind of employing chromatography and prepare highly purified onocol (Formononetin), 3-hydroxyl-9,10-dimethoxy red sandalwood alkane (3-hydroxy-9,10-dimethoxy pterocarpane) and 2 ', 7-dihydroxyl-3 ', 4 '-dimethoxy isoflavan (2 ', 7-dihydroxy-3 ', 4 '-dimethoxy isoflavane) method of three kinds of principal monomer compositions.
Background technology
Gathering isoflavone is the secondary metabolite of the Radix Astragali, according to the difference of chemical structure, mainly is divided into the glucosides class of isoflavones, isoflavan and the red sandalwood alkane aglycon and the mating type thereof of free type.Modern pharmacological research shows that gathering isoflavone class material has effects such as antisepsis and anti-inflammation, antiviral, reducing blood-fat, anticancer and tool follicular hormone, and therefore separating the highly purified compound monomer of preparation becomes an important research contents.The preparation of onocol mainly contains two kinds of extraction method and chemical synthesiss, and 3-hydroxyl-9,10-dimethoxy red sandalwood alkane and 2 ', 7-dihydroxyl-3 ', the preparation of 4 '-dimethoxy isoflavan has only extraction method.Classical chemical synthesis desired raw material or reagent is difficult to obtain and the reaction product complexity, the separation and purification difficulty.Shortcomings such as traditional extraction method is loaded down with trivial details in steps, cycle length, poor reproducibility.Employed solvent elution system is comparatively complexity and toxicity big (as chloroform) also, and solvent-oil ratio is big.Therefore needs separation method a kind of fast, efficient, favorable reproducibility satisfies the demand to mentioned component.
Summary of the invention
Purpose of the present invention aim to provide a kind of efficient, can repeat, low cost, high-recovery, be suitable for the methods that a large amount of separation prepare gathering isoflavone principal monomer component.
For achieving the above object, the technical solution used in the present invention is:
The method for separating and preparing of multiple isoflavones components in a kind of Radix Astragali, adopt silica gel column chromatography that the plant milk extract that contains gathering isoflavone is carried out enrichment and concentrates after, separate preparation by reversed phase high efficiency liquid phase preparative chromatography again.Thereby guaranteed that this preparation method satisfies the repeatability of gathering isoflavone principal monomer component preparation and the requirement of linear amplification under the situation of lower cost and higher yields.
Comprise following concrete steps:
1) with silica gel column chromatography the enrichment that the extract that contains gathering isoflavone carries out target component is concentrated earlier; Employed eluent is a petroleum ether-ethyl acetate, and their volume ratio is 1:1～1.5;
2) flow out part and detect with thin-layer chromatography, thin layer detects fluorescence under ultraviolet lamp, and developping agent is a petroleum ether-ethyl acetate, and their volume ratio is 1:1～1.5; The target product R that will contain
f〉=0.62 part merges, be condensed into medicinal extract after;
3) with methyl alcohol-chloroformic solution system dissolving, the volume ratio of methyl alcohol-chloroform is 1:2～3, filtering membrane, with high performance liquid preparative chromatography to its separate, purifying and obtain onocol, 9,10-dimethoxy red sandalwood alkane and 2 ', 7-dihydroxyl-3 ', the pure product of three compounds of 4 '-dimethoxy isoflavan.
The detailed process of described step 1) is, with Radix Astragali total flavones crude extract dissolve with methanol, with silica gel mixed sample, the mass ratio of sample and silica gel is 1:2～3, mix sample evenly after, volatilize solvent, grind evenly, fill chromatography column with silica gel and carry out column chromatography; The boiling spread of the used sherwood oil of column chromatography is 60～90 ℃, and silica gel is 100～200 orders;
Used filter membrane 0.2～0.5 μ m in the described step 3); High performance liquid preparative chromatography is a reversed phase high efficiency liquid phase preparative chromatography, and packing material is reverse phase silica gel C18, particle diameter 5～10 μ m; Moving phase (being eluent) is methyl alcohol and the aqueous solution two-phase system that contains formic acid, and two-phase solvent is that 45:55～55:45 carries out wash-out with the volume fraction ratio, and adding the formic acid concn scope is 0.1～0.5%; The control flow velocity is 19～300ml/min; Adopt UV-detector 230nm on-line monitoring elution curve simultaneously.
The present invention has following advantage:
1. the compound purity height that is separated to.The invention discloses separation, the purification process of gathering isoflavone principal monomer component, used is reversed-phased high performace liquid chromatographic, have can guarantee higher peak type resolution, favorable reproducibility, the time is short, fractional dose is big, the rate of recovery is high, isolating environment relaxes, save characteristics such as solvent, can directly separate the product that slightly get sample in a large number, the compound purity that is separated to can reach more than 98%, can be used for the quality control of Milkvetch Root and preparation.
2. low-cost, high, the good reproducibility of yield.The inventive method rate of recovery reaches more than 80%, and the solvent for use system is simple, and its eluting solvent is a usual vehicle, and therefore low price is a kind of method of extraction gathering isoflavone principal monomer component of economy; The production that can be various isoflavones monomer components in the Radix Astragali provides novel process.
Description of drawings
Fig. 1 is the high-efficient liquid phase analysis figure of Radix Astragali flavone crude product among the embodiment 1;
Fig. 2 contains onocol, 9,10-dimethoxy red sandalwood alkane and 2 ', 7-dihydroxyl-3 ', the high-efficient liquid phase analysis figure of the component Fr2 of 4 '-dimethoxy isoflavan among the embodiment 1;
Fig. 3 is an onocol, 9 among the embodiment 1,10-dimethoxy red sandalwood alkane and 2 ', 7-dihydroxyl-3 ', the high performance liquid preparative chromatography figure of 4 '-dimethoxy isoflavan;
Fig. 4 is the high-efficient liquid phase analysis figure of the compound (being followed successively by onocol, 9,10-dimethoxy red sandalwood alkane and 2 ', 7-dihydroxyl-3 ', 4 '-dimethoxy isoflavan) of purifying among the embodiment 1.
The invention will be further described below in conjunction with embodiment and accompanying drawing.
Embodiment 1
1. take by weighing Radix Astragali total flavones crude extract medicinal extract 20g (efficient liquid phase chromatographic analysis is seen Fig. 1), add methyl alcohol and make its dissolving, with silica gel be that sample is mixed in 1:2～3 (w/w) with the mass ratio, mix sample evenly after, volatilize solvent, grind evenly.
2. fill the chromatography column column chromatography with 100～200 purpose silica gel (1300g), eluting solvent is that petroleum ether-ethyl acetate (volume ratio is 1:1) is with 4 times of column volume wash-outs.
3. flow out part and detect with thin-layer chromatography, developping agent is petroleum ether-ethyl acetate (volume ratio is 1:1), and developer is collected and contained target product (R for containing 5% vitriolic methanol solution
f〉=0.62) cut will contain onocol, 9,10-dimethoxy red sandalwood alkane and 2 ', and 7-dihydroxyl-3 ', the part of 4 '-dimethoxy isoflavan is merged into component Fr2, is condensed into medicinal extract 757mg.The efficient liquid phase chromatographic analysis of component Fr2 (as Fig. 2).
4. with reversed phase high efficiency liquid phase preparative chromatography separated portion Fr2.Component Fr292.5mg is dissolved in 5mL methyl alcohol-chloroform (volume ratio is 1:2) solution system, crosses 0.45 μ m filter membrane, as preparative chromatography sample introduction solution.The preparative chromatography column internal diameter is 7.7cm, and column length 20cm, column packing are C18 (particle diameter 10 μ m), sample introduction 20mL.Sample is injected the injection annulus of preparative chromatography, make wash-out mutually with methyl alcohol with the aqueous solution that contains 0.5% formic acid, two-phase solvent is 50:50 with the volume fraction ratio, carries out isocratic elution with the flow velocity of 150mL/min, and be 55min working time.UV-detector monitoring flow point by online detects wavelength 230nm (see figure 3), presses the peak cutting and collects effluent, obtain onocol 31.4mg, 9,10-dimethoxy red sandalwood alkane 14.8mg, 2 ', 7-dihydroxyl-3 ', 4 '-dimethoxy isoflavan 24.3mg.The purity of three compounds〉98%, the rate of recovery〉87%, see Fig. 4.
Onocol, 9 wherein, 10-dimethoxy red sandalwood alkane and 2 ', 7-dihydroxyl-3 ', the chemical structural drawing of 4 '-dimethoxy isoflavan is as follows;
Onocol 9,10-dimethoxy red sandalwood alkane
2 ', 7-dihydroxyl-3 ', 4 '-dimethoxy isoflavan
Embodiment 2
The preparation method of component Fr2 is with embodiment 1.Take by weighing component Fr2 287.5mg and be dissolved in 10mL methyl alcohol-chloroform (volume ratio is 1:2) solution system, cross 0.45 μ m filter membrane, as preparative chromatography sample introduction solution.The preparative chromatography column internal diameter is 10cm, and column length 20cm, column packing are C18 (particle diameter 10 μ m), injection annulus 20mL.Sample is injected the injection annulus of preparative chromatography, make wash-out mutually with methyl alcohol with the aqueous solution that contains 0.5% formic acid, the two-phase solvent volume ratio is 50:50, carries out isocratic elution with the flow velocity of 300mL/min, working time 55min.By UV-detector on-line monitoring flow point, detect wavelength 230nm, press the peak cutting and collect effluent, obtain onocol 96mg, 9,10-dimethoxy red sandalwood alkane 41.6mg, 2 ', 7-dihydroxyl-3 ', 4 '-dimethoxy isoflavan 68.2mg.The purity of three compounds〉98%, yield is greater than 80%.
Embodiment 3
The preparation method of component Fr2 is with embodiment 1.Take by weighing component Fr2 11mg and be dissolved in 5mL methyl alcohol-chloroform (volume ratio is 1:2) solution system, cross 0.45 μ m filter membrane, as preparative chromatography sample introduction solution.The preparative chromatography column internal diameter is 2cm, and column length 25cm, column packing are C18 (particle diameter 10 μ m), injection annulus 10mL.Sample is injected the injection annulus of preparative chromatography, make wash-out mutually with methyl alcohol with the aqueous solution that contains 0.5% formic acid, two-phase solvent is that 50:50 carries out isocratic elution with the volume fraction, and flow velocity is 19mL/min, working time 55min.UV-detector monitoring flow point by online detects wavelength 230nm, presses the peak cutting and collects effluent, obtains onocol 3.9mg, 9,10-dimethoxy red sandalwood alkane 1.76mg, 2 ', 7-dihydroxyl-3 ', 4 '-dimethoxy isoflavan 2.84mg.The purity purity of three compounds〉98%, yield is greater than 85%.
In order to confirm products therefrom, above-mentioned crystalline powder has been carried out thin-layer chromatography and mass spectrum evaluation, and and standard control.
Adopt silica gel G F
254Thin layer plate is respectively to each product qualitative detection, and with reference substance R
fValue relatively.
The molecular weight of mass spectrometry results explanation products therefrom is respectively 430,300,302, and the corresponding molecular weight with reference substance of these data is consistent.
Claims (7)
1. the method for separating and preparing of multiple isoflavones components in the Radix Astragali, it is characterized in that: after adopting silica gel column chromatography that the plant milk extract that contains gathering isoflavone is carried out enrichment and concentrates, separate preparation onocol, 9 by reversed phase high efficiency liquid phase preparative chromatography again, 10-dimethoxy red sandalwood alkane and 2 ', 7-dihydroxyl-3 ', three compounds of 4 '-dimethoxy isoflavan;
1) with silica gel column chromatography the enrichment that the extract that contains gathering isoflavone carries out target component is concentrated earlier; Employed eluent is a petroleum ether-ethyl acetate, and their volume ratio is 1:1～1.5;
2) flow out part and detect with thin-layer chromatography, thin layer detects fluorescence under ultraviolet lamp, and developping agent is a petroleum ether-ethyl acetate, and their volume ratio is 1:1～1.5; The target product R that will contain
r〉=0.62 part merges, be condensed into medicinal extract after;
3) with methyl alcohol-chloroformic solution system dissolving, the volume ratio of methyl alcohol-chloroform is 1:2～3, filtering membrane, with reversed phase high efficiency liquid phase preparative chromatography to its separate, purifying and obtain onocol, 9,10-dimethoxy red sandalwood alkane and 2 ', 7-dihydroxyl-3 ', the pure product of three compounds of 4 '-dimethoxy isoflavan.
2. according to the method for separating and preparing of multiple isoflavones components in the described Radix Astragali of claim 1, it is characterized in that: the detailed process of described step 1) is, with Radix Astragali total flavones crude extract dissolve with methanol, with silica gel mixed sample, the mass ratio of sample and silica gel is 1:2～3, mix sample evenly after, volatilize solvent, grind evenly, fill chromatography column with silica gel and carry out column chromatography.
3. according to the method for separating and preparing of multiple isoflavones components in the described Radix Astragali of claim 1, it is characterized in that: reversed phase high efficiency liquid phase preparative chromatography in the described step 3), packing material is reverse phase silica gel C18, particle diameter 5～10 μ m.
4. according to the method for separating and preparing of multiple isoflavones components in the described Radix Astragali of claim 1, it is characterized in that: to separate the moving phase of preparation be methyl alcohol to reversed phase high efficiency liquid phase preparative chromatography and contain the aqueous solution two-phase system of formic acid in the described step 3), and adding the formic acid concn scope is 0.1～0.5%; Two-phase solvent is that 45:55～55:45 carries out wash-out with the volume fraction ratio.
5. according to the method for separating and preparing of multiple isoflavones components in the described Radix Astragali of claim 1, it is characterized in that: in the described step 3) reversed phase high efficiency liquid phase preparative chromatography separate the preparation flow velocity be 19～300ml/mi.
6. according to the method for separating and preparing of multiple isoflavones components in the described Radix Astragali of claim 1, it is characterized in that: described step 3) adopts the filter membrane of 0.2～0.5 μ m.
7. according to the method for separating and preparing of multiple isoflavones components in the described Radix Astragali of claim 1, it is characterized in that: the boiling spread of the used sherwood oil of column chromatography is 60～90 ℃ in the described step 1), and silica gel is 100～200 orders.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102357092B (en) * | 2011-09-01 | 2012-11-28 | 兰州大学 | Use of flavanone natural product 7, 3'-dyhydroxy-2', 4'-dimethoxy-isoflavane (DDIF) |
| CN102628839B (en) * | 2012-02-22 | 2013-10-30 | 广西壮族自治区中医药研究院 | Preparation of high-purity camellianin and its quality control method |
| CN104510752B (en) * | 2014-12-25 | 2017-11-14 | 上海中医药大学 | The medical usage of red sandalwood alkane glycosides |
| CN108948038B (en) * | 2018-07-09 | 2020-09-22 | 江西中医药大学 | Neopteridine flavonoid compound and application thereof |
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Non-Patent Citations (2)
| Title |
|---|
| 膜荚黄芪毛状根中异黄酮成分的反相高效液相色谱分析. 郑志仁等.药学学报，第33卷第2期. 1998 * |
| 蒙古黄芪中黄酮类成分的研究. 马晓丰等.中草药，第36卷第9期. 2005 * |
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