CN102180934A - Preparation method of tubeimoside I - Google Patents
Preparation method of tubeimoside I Download PDFInfo
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- CN102180934A CN102180934A CN2011100291272A CN201110029127A CN102180934A CN 102180934 A CN102180934 A CN 102180934A CN 2011100291272 A CN2011100291272 A CN 2011100291272A CN 201110029127 A CN201110029127 A CN 201110029127A CN 102180934 A CN102180934 A CN 102180934A
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Abstract
The invention provides a preparation method of tubeimoside I. The method comprises the following steps of: soaking tubeimu tuber bulbs into an appropriate quantity of organic solvent; extracting by heating, diacolation or by adopting an ultrasonic method; filtering to obtain a tubeimu tuber extracting solution; collecting eluent by adopting one or more processes of polyamide column chromatography, Sephadex LH-20 column chromatography, medium-pressure aluminum oxide column chromatography and the like; recovering a reagent from the eluent at reduced pressure; and cleaning acetone repeatedly to obtain tubeimoside I. The purity of the tubeimoside I prepared with the method is over 90 percent; the method is simple and practical and is suitable for large-scale production.
Description
Technical field:
The invention belongs to active ingredient of Chinese herbs and separate preparation field, particularly, the present invention relates to a kind of method for preparing Tubeimoside I of from Rhizome of Paniculate Bolbostemma, separating.
Background technology:
Tubeimoside I (Tubeimoside A) is the saponin(e that obtains from cucurbitaceous plant Rhizome of Paniculate Bolbostemma (Bolbostemma paniculatum) extraction separation, is one of main effective constituent of Rhizome of Paniculate Bolbostemma.Molecular formula: C
63H
98O
29, molecular weight: 1319.43.Show that through pharmacological testing it has various active such as antitumor, antiviral, immunosuppression.Tubeimoside I content in plant is higher, and employing HPLC methods such as Wei Youliang are measured Tubeimoside I content in the Rhizome of Paniculate Bolbostemma of the different places of production, and moving phase is 65% methyl alcohol, and flow velocity 1.0ml/min detects wavelength 214nm; The content that obtains under the pseudo-wild condition Tubeimoside I in plantation Rhizome of Paniculate Bolbostemma and the commercially available medicine materical crude slice Rhizome of Paniculate Bolbostemma is respectively 1.776% and 1.009%.Employing HPLC method research such as Li Xiaojing and set up each place of production Rhizome of Paniculate Bolbostemma medicinal material finger printing (the HPLC finger printing research of Rhizome of Paniculate Bolbostemma. herbal medicine .2007,38 (6): 926-929.), adopt 70% alcohol heating reflux to extract, ethyl acetate and water-saturated n-butanol extraction preparation need testing solution.Have not yet to see the patent report of Tubeimoside I preparation and study in great detail, so the invention provides a kind of preparation method of Tubeimoside I.
Summary of the invention:
The present invention aims to provide a kind of preparation method of Tubeimoside I.This method avoids using propyl carbinol, has guaranteed the yield and the purity of Tubeimoside I.
The technical solution used in the present invention is as follows:
1) with the Rhizome of Paniculate Bolbostemma bulb of commercially available Rhizome of Paniculate Bolbostemma medicine materical crude slice or fresh collection as raw material, select for use water, methyl alcohol, ethanol or two kinds of mixed solvents in them to extract;
2) with 1) in extract the extract obtain and separate through one or more modes of polyamide column chromatography, Sephadex LH-20 column chromatography, middle pressure alumina column chromatography, water, acetonitrile, ethanol, methylene dichloride, methyl alcohol, chloroform or their at least two kinds of mixtures carry out wash-out as eluent;
3) elutriant merging 2) reclaims reagent, drying;
4) with 3) in the dry thing of gained with washing with acetone repeatedly, be drying to obtain product.
The 4-20 that extracts solvent load in the described step 1) and be material quantity doubly.
Extracting in the described step 1) is behind solvent soaking, adopts a kind of in extracting of heating extraction, supersound extraction, diacolation, extracts 1-3 time.
Described step 2) optional 60-80% aqueous ethanolic solution of polyamide column chromatography eluent or 20-50% acetonitrile solution in.
Described step 2) Sephadex LH-20 column chromatography is normal pressure or medium pressure column chromatography in, optional 60-80% aqueous ethanolic solution of eluent or 20-50% acetonitrile solution.
Described step 2) presses optional methylene chloride-methanol of alumina column eluent or chloroform-methanol in, by 100: 1,50: 1,20: 1,5: 1 gradient elutions.
Characteristics of the present invention are the invention solves owing to use propyl carbinol, silica gel column chromatography to cause the problem that purity is not high, yield is low of Tubeimoside I, and the purity of the Tubeimoside I of the present invention's preparation is higher than 90%, and simple to operate, can enlarge suitability for industrialized production.
Embodiment:
Embodiment 1:
Rhizome of Paniculate Bolbostemma medicine materical crude slice 5kg pulverized 20 mesh sieves, added the 15L50% alcohol immersion after 5 hours, added 50% ethanol to the 30L heating and refluxing extraction, filtered, and the same again refluxing extraction of the dregs of a decoction 2 times is filtered, united extraction liquid, and decompression recycling ethanol is to 25L; Extracting solution is fed (8L, 20-50 order) in the good polyamide column of pre-treatment, use the 3BV water elution earlier, use 80% ethanol elution again, decompression recycling ethanol is concentrated into small volume, gets sample.This sample is added the oven dry of 200g aluminum oxide, press in the alumina column in the adding, first water is eluted to colourless, uses the methylene chloride-methanol gradient elution more successively, from high to low, by 100: 1,50: 1,20: 1 and 5: 1, Fractional Collections was collected the effective constituent section successively, concentrate and produce crystallization, again with washing with acetone repeatedly, lyophilize gets Tubeimoside I 35g, and it is 90.9% that HPLC detects its purity.
Embodiment 2:
Rhizome of Paniculate Bolbostemma medicine materical crude slice 8kg pulverized 20 mesh sieves, added the 32L60% alcohol immersion after 3 hours, added 60% ethanol and extracted to the 80L diacolation, and the same again diacolation of the dregs of a decoction extracts 1 time, filter, and united extraction liquid, decompression recycling ethanol is to 40L; Extracting solution is fed in the good Sephadex LH-20 chromatography column of pre-treatment (15L, 20-50 order), use the 3BV water elution earlier, use 65% ethanol elution again, decompression recycling ethanol is concentrated into small volume, gets sample.This sample is added the oven dry of 300g aluminum oxide, press in the alumina column in the adding, first water is eluted to colourless, uses the methylene chloride-methanol gradient elution more successively, from high to low, by 100: 1,50: 1,20: 1 and 5: 1, Fractional Collections was collected the effective constituent section successively, concentrate and produce crystallization, again with washing with acetone repeatedly, lyophilize gets Tubeimoside I 58g, and it is 92.5% that HPLC detects its purity.
Embodiment 3:
The fresh bulb 5kg of Rhizome of Paniculate Bolbostemma pulverized 20 mesh sieves, added 13L60% methyl alcohol and soaked after 5 hours, added 60% methyl alcohol to the 35L supersound extraction, and each 30min filters, and the same again supersound extraction of the dregs of a decoction 2 times is filtered, united extraction liquid, and reclaim under reduced pressure methyl alcohol is to 28L; Extracting solution is fed (8L, 20-50 order) in the good polyamide column of pre-treatment, use the 3BV water elution earlier, use 75% ethanol elution again, decompression recycling ethanol is concentrated into small volume, gets sample.This sample is added 200g dextrane gel Sephadex LH-20 mix the sample oven dry, add Sephadex LH-20 chromatography column, elder generation's water wash-out, use 23% acetonitrile wash-out again, collect elutriant, concentrate and produce crystallization, more repeatedly with washing with acetone, lyophilize gets Tubeimoside I 37g, and it is 92.1% that HPLC detects its purity.
Embodiment 4:
The fresh bulb 4kg of Rhizome of Paniculate Bolbostemma pulverized 20 mesh sieves, added 12L70% methyl alcohol and soaked after 3 hours, added 70% methyl alcohol to the 48L heating and refluxing extraction, filtered, and the same again refluxing extraction of the dregs of a decoction 1 time is filtered, united extraction liquid, and reclaim under reduced pressure methyl alcohol is to 15L; Extracting solution is fed in the good Sephadex LH-20 medium pressure chromatography post of pre-treatment (8L, 20-50 order), use the 3BV water elution earlier, use 60% ethanol elution again, decompression recycling ethanol is concentrated into small volume, gets sample.This sample is added the oven dry of 300g aluminum oxide, press in the alumina column in the adding, first water is eluted to colourless, uses the chloroform-methanol gradient elution more successively, from high to low, by 100: 1,50: 1,20: 1 and 5: 1, Fractional Collections was collected the effective constituent section successively, concentrate and produce crystallization, again with washing with acetone repeatedly, lyophilize gets Tubeimoside I 27g, and it is 91.3% that HPLC detects its purity.
Claims (6)
1. the preparation method of a Tubeimoside I is characterized in that may further comprise the steps:
1) with the Rhizome of Paniculate Bolbostemma bulb of commercially available Rhizome of Paniculate Bolbostemma medicine materical crude slice or fresh collection as raw material, select for use water, methyl alcohol, ethanol or two kinds of mixed solvents in them to extract;
2) with 1) in extract the extract obtain and separate through one or more modes of polyamide column chromatography, Sephadex LH-20 column chromatography, middle pressure alumina column chromatography, water, acetonitrile, ethanol, methylene dichloride, methyl alcohol, chloroform or their at least two kinds of mixtures carry out wash-out as eluent;
3) elutriant merging 2) reclaims reagent, drying;
4) with 3) in the dry thing of gained with washing with acetone repeatedly, be drying to obtain product.
2. according to the described preparation method of claim 1, it is characterized in that: the 4-20 that extracts solvent load in the described step 1) and be material quantity doubly.
3. according to the described preparation method of claim 1, it is characterized in that: extracting in the described step 1) is behind solvent soaking, adopts a kind of in extracting of heating extraction, supersound extraction, diacolation, extracts 1-3 time.
4. according to the described preparation method of claim 1, it is characterized in that: optional 60-80% aqueous ethanolic solution of polyamide column chromatography eluent or 20-50% acetonitrile solution described step 2).
5. according to the described preparation method of claim 1, it is characterized in that: Sephadex LH-20 column chromatography is normal pressure or medium pressure column chromatography described step 2), optional 60-80% aqueous ethanolic solution of eluent or 20-50% acetonitrile solution.
6. according to the described preparation method of claim 1, it is characterized in that: press optional methylene chloride-methanol of alumina column eluent or chloroform-methanol in described step 2), by 100: 1,50: 1,20: 1,5: 1 gradient elutions.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011100291272A CN102180934A (en) | 2011-01-24 | 2011-01-24 | Preparation method of tubeimoside I |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011100291272A CN102180934A (en) | 2011-01-24 | 2011-01-24 | Preparation method of tubeimoside I |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104940214A (en) * | 2015-05-22 | 2015-09-30 | 德清奥丽芙生物科技有限公司 | Application of peimine in preparation of novel immunosuppressive agent |
| CN109336943A (en) * | 2018-10-26 | 2019-02-15 | 宁县恒瑞康生物科技有限公司 | A kind of Bolbostemma paniculatum glucoside A purification process |
| CN111007190A (en) * | 2019-12-03 | 2020-04-14 | 广东一方制药有限公司 | Method for constructing UPLC (ultra performance liquid chromatography) characteristic spectrum of rhizoma bolbostemmae medicinal material and method for measuring component content of rhizoma bolbostemmae medicinal material |
| CN114028555A (en) * | 2021-12-10 | 2022-02-11 | 浙江大学 | Tubeimoside with immunological adjuvant effect, and preparation method and application thereof |
-
2011
- 2011-01-24 CN CN2011100291272A patent/CN102180934A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104940214A (en) * | 2015-05-22 | 2015-09-30 | 德清奥丽芙生物科技有限公司 | Application of peimine in preparation of novel immunosuppressive agent |
| CN104940214B (en) * | 2015-05-22 | 2018-07-24 | 德清奥丽芙生物科技有限公司 | Peimine is used to prepare the application of immunosuppressor |
| CN109336943A (en) * | 2018-10-26 | 2019-02-15 | 宁县恒瑞康生物科技有限公司 | A kind of Bolbostemma paniculatum glucoside A purification process |
| CN111007190A (en) * | 2019-12-03 | 2020-04-14 | 广东一方制药有限公司 | Method for constructing UPLC (ultra performance liquid chromatography) characteristic spectrum of rhizoma bolbostemmae medicinal material and method for measuring component content of rhizoma bolbostemmae medicinal material |
| CN114028555A (en) * | 2021-12-10 | 2022-02-11 | 浙江大学 | Tubeimoside with immunological adjuvant effect, and preparation method and application thereof |
| CN114028555B (en) * | 2021-12-10 | 2024-02-20 | 浙江大学 | Tubeimoside with immunoadjuvant function, preparation method and application thereof |
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Application publication date: 20110914 |