CN102977165A - Optimized purification preparation method for momordica grosvenori flavine - Google Patents
Optimized purification preparation method for momordica grosvenori flavine Download PDFInfo
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- CN102977165A CN102977165A CN2012105263678A CN201210526367A CN102977165A CN 102977165 A CN102977165 A CN 102977165A CN 2012105263678 A CN2012105263678 A CN 2012105263678A CN 201210526367 A CN201210526367 A CN 201210526367A CN 102977165 A CN102977165 A CN 102977165A
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Abstract
The invention discloses an optimized purification preparation method for momordica grosvenori flavine in the technical field of biological medicines. The momordica grosvenori flavine with the purity of over 95 percent is obtained by adsorbing momordica grosvenori boiled liquid with large-aperture resin and diluting the momordica grosvenori boiled liquid to obtain momordica grosvenori flavine extract, performing Sephadex LH-20 column chromatography on the momordica grosvenori flavine extract, diluting and collecting fluid components, and repeatedly performing column chromatography and dilution for at least once. The momordica grosvenori flavine has the advantages of high purity, high yield, simple process, low cost and the like.
Description
Technical field
What the present invention relates to is the method in a kind of biological medicine technology field, specifically a kind of optimization purification preparation method of grosvenorin.
Background technology
Grosvenorin is flavonoid glycoside compound, finds that at present this composition only limits in the mature fruit that is distributed in cucurbitaceous plant Grosvenor Momordica SiraitiagrosvenoriiSwingl (MomordicagrosvenoriiSwingle).Its structural formula as:
Grosvenor Momordica is Chinese peculiar economy, medicinal plant.Grosvenorin is the characteristic chemical constituent of Grosvenor Momordica, has anti-oxidant, antibiotic, anticomplement isoreactivity, can be used as the tinting material that desirable natural yellow pigment is used for food, beverage, and colourity is high, safe without toxic side effect; Highly purified grosvenorin can be used as chemical reference substance and is used for Grosvenor Momordica medicinal material, the qualitative identification that contains the Grosvenor Momordica preparation and quantitative analysis.Grosvenorin content in Grosvenor Momordica is lower.Therefore, it is significant how to prepare grosvenorin from Grosvenor Momordica.
" Acta Pharmaceutica Sinica " is in February, 1994 (the 2nd phase of 29 volumes), 158 pages-160 pages, name is called " separation of flavonoid glycoside and structure determination in the fresh Fructus Momordicae " and discloses a kind of method of separating grosvenorin from fresh Fructus Momordicae, that is: new fresh Fructus Momordicae water extracts, the aqueous solution is behind macroporous resin column chromatography, silica gel column chromatography, and recrystallization obtains grosvenorin.This method requires to use new fresh Fructus Momordicae, and the commercial goods Grosvenor Momordica is dry fruit; Simultaneously, adopt the method to prepare the grosvenorin yield and be lower than 2/100000ths, complex operation, cost is high, is unfavorable for preparing in a large number grosvenorin.
Summary of the invention
The present invention is directed to the prior art above shortcomings, propose a kind of optimization purification preparation method of grosvenorin, have purity height, yield height, simple, the low cost and other advantages of technique.
The present invention is achieved by the following technical solutions: the present invention is by adopting the Grosvenor Momordica water cooking liquid macroporous resin adsorption and obtaining grosvenorin medicinal extract through wash-out, collection is wherein flowed part and is repeated the operation of chromatography wash-out at least one times after grosvenorin medicinal extract being carried out Sephadex LH-20 column chromatography and wash-out again, obtain purity greater than the grosvenorin highly finished product more than 95%, concrete steps are as follows:
1) first Grosvenor Momordica is pulverized, added decocting and boil, centrifugal, get supernatant liquor;
2) supernatant liquor in the step 1) is crossed macroporous resin, 50%(v/v) adopts 60-80%(v/v behind the alcohol flush away impurity) the alcohol wash-out, reclaim solvent, collect wherein stream part, i.e. grosvenorin medicinal extract;
3) with step 2) the grosvenorin medicinal extract of gained, carry out Sephadex LH-20 column chromatography, methyl alcohol 60%-100%(v/v) or its aqueous solution wash-out reclaim methyl alcohol and collect wherein stream part, obtain the grosvenorin crude product;
4) get the grosvenorin crude product and carry out Sephadex LH-20 column chromatography, collecting wherein, stream part obtains purity greater than the grosvenorin highly finished product more than 95%.
Among the present invention:
Described Grosvenor Momordica is dry, mature fruit.
Described solvent is water, extracts 1-2 time.
Described macroporous resin is HZ-801 or D101 type.
Described collection is wherein flowed part and referred to: adopt TLC to detect, wherein: it is the volume ratio configuration of 60:35:5 that developping agent adopts chloroform-methanol-water; Developer adopts 10% (v/v) sulfuric acid-ethanol; Detect by the heating colour developing, merge all stream parts that contain grosvenorin.
Beneficial effect
(1) boils, then decocting liquid can be obtained the high-purity Momordica grosvenori flavine by macroporous resin, Sephadex LH-20 post by Grosvenor Momordica dry fruit decocting.Adopt the grosvenorin of aforesaid method preparation, purity is high, more than 95%; Simple process, yield height can reach more than 0.1%.
(2) method that adopts decocting to extract has been removed most of fat-soluble component in the Grosvenor Momordica (such as grease, wax); Economic security has also improved the efficient of extracting simultaneously.
(3) adopt macroporous resin purification, use first the ethanol elution of lower concentration, the interference of having removed the water-soluble impurities such as most of saponin(e, sugar; Simultaneously, ethanol is recyclable Reusability again, has saved cost.
(4) grosvenorin medicinal extract carries out first Sephadex LH-20 column chromatography 1 time, has removed remaining triterpene glucoside, the convenient grosvenorin crude product that obtains; Crude product carries out Sephadex LH-20 column chromatography again, can obtain highly purified grosvenorin.Sample loss is few in the whole process, and the again renewable rear use of Sephadex LH-20 filler, the recyclable rear Reusability of eluting solvent methyl alcohol greatly reduce cost.
Description of drawings
Fig. 1 is the grosvenorin nuclear magnetic resonance map:
1H-NMR(500MHZ, DMSO-d6)
Fig. 2 is the grosvenorin nuclear magnetic resonance map:
13C-NMR(125MHZ, DMSO-d6)
Embodiment
The below elaborates to embodiments of the invention, and present embodiment is implemented under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Present embodiment is implemented under following implementation condition and technical requirements condition:
1) ripe Grosvenor Momordica dry fruit 200g is ground into meal, adds the 1000mL decocting and boils 0.5h, and 4800 rev/mins of centrifugal 20min get the aqueous solution.
2) water intaking solution slowly flows through HZ-801 macroporous resin (6.5cm * 80cm, flow velocity 5mL/min), uses first 50%(v/v) (flow velocity 10mL/min) is colourless to elutriant for the alcohol wash-out, collects ethanol eluate, and decompression recycling ethanol is for next time; Continue and use 60%(v/v) alcohol wash-out (flow velocity 10mL/min) colourless to elutriant, collect elutriant, decompression recycling ethanol gets grosvenorin medicinal extract 4.2g.
3) grosvenorin medicinal extract is carried out SephadexLH-20 column chromatography (4.0cm * 100cm), 60% methanol-eluted fractions (flow velocity 3mL/min, 30mL/ flows part) colourless to elutriant, reclaim under reduced pressure methyl alcohol is for next time, and TLC detects (developping agent: chloroform-methanol-water 60:35:5; Developer: 10% sulfuric acid-ethanol; Coloration method: the heating colour developing), merge and mainly contain grosvenorin stream part, decompression and solvent recovery gets grosvenorin crude product 351mg.
4) grosvenorin crude product dissolve with methanol, again add Sephadex LH-20 post (among the 2.5cm * 60cm), with 60% (v/v) methanol aqueous solution wash-out (flow velocity 1mL/min, 10mL/ flows part) colourless to elutriant, the above-mentioned condition of TLC detects, merge and mainly contain grosvenorin stream part, decompression and solvent recovery gets grosvenorin highly finished product 219mg, and checking its purity through HPLC is 97.1%.
Embodiment 2
Present embodiment is implemented under following implementation condition and technical requirements condition:
1) ripe Grosvenor Momordica dry fruit 200g is ground into meal, adds the 1000mL decocting and boils 0.5h, and 4800 rev/mins of centrifugal 20min get supernatant liquor; The dregs of a decoction add water 800mL again, again decoct 0.5h, and are centrifugal, get supernatant liquor.Merge supernatant liquor and get the Grosvenor Momordica aqueous solution.
2) water intaking solution, the D101 macroporous resin column of slowly flowing through (6.5cm * 80cm, flow velocity 5mL/min) is used first 50%(v/v) (flow velocity 10mL/min) is colourless to elutriant for the alcohol wash-out, collects ethanol eluate, decompression recycling ethanol is for next time; Continue and use 80%(v/v) alcohol wash-out (flow velocity 10mL/min) colourless to elutriant, collect elutriant, Recycled ethanol gets grosvenorin medicinal extract 6.7g.
The medicinal extract that 3) will contain grosvenorin carries out the Sephadex LH-20 post (chromatography of 4.0cm * 100cm), 100% methanol-eluted fractions (flow velocity 3mL/min, 30mL/ flows part) colourless colourless to elutriant to elutriant, reclaim under reduced pressure methyl alcohol is for next time, and TLC detects and mainly contains grosvenorin stream part (developping agent: chloroform-methanol-water 60:35:5; Developer: 10% sulfuric acid-ethanol; Coloration method: the heating colour developing), merge these stream parts, decompression and solvent recovery gets grosvenorin crude product 508mg.
4) grosvenorin crude product dissolve with methanol, again add Sephadex LH-20 post (among the 2.5cm * 60cm), methanol-eluted fractions with 100% (flow velocity 1mL/min, 10mL/ flows part) colourless to elutriant, the above-mentioned condition of TLC detects and mainly contains grosvenorin stream part, decompression and solvent recovery gets grosvenorin highly finished product 340mg, and checking its purity through HPLC is 96.2%.
Embodiment 3
Present embodiment is implemented under following implementation condition and technical requirements condition:
1) ripe Grosvenor Momordica dry fruit 200g is ground into meal, adds the 1000mL decocting and boils 0.5h, and 4800 rev/mins of centrifugal 20min get the aqueous solution.
2) water intaking solution, the D101 macroporous resin column of slowly flowing through (6.5cm * 80cm, flow velocity 5mL/min) is used first 50%(v/v) (flow velocity 10mL/min) is colourless to elutriant for the alcohol wash-out, collects ethanol eluate, decompression recycling ethanol is for next time; Continue and use 70% ethanol elution, collect elutriant, decompression recycling ethanol gets pure medicinal extract 5.1g.
The medicinal extract that 3) will contain grosvenorin carries out Sephadex LH-20 column chromatography (4cm * 100cm), methanol aqueous solution wash-out (flow velocity 3mL/min 80%(v/v), 30mL/ flows part) colourless to elutriant, reclaim methyl alcohol for next time, TLC detects and mainly contains grosvenorin stream part (developping agent: chloroform-methanol-water 60:35:5; Developer: 10% sulfuric acid-ethanol; Coloration method: the heating colour developing), merge these stream parts, decompression and solvent recovery gets grosvenorin crude product 422mg.
4) grosvenorin crude product dissolve with methanol, again add Sephadex LH-20 post (among the 2.5cm * 60cm), use 80%(v/v) methanol aqueous solution wash-out (flow velocity 1mL/min, 10mL/ flows part) colourless to elutriant, the above-mentioned condition of TLC detects and mainly contains grosvenorin stream part, merge these stream parts, decompression and solvent recovery gets grosvenorin 273mg, and checking its purity through HPLC is 96.7%.
Claims (9)
1. the optimization purification preparation method of a grosvenorin, it is characterized in that, by the Grosvenor Momordica water cooking liquid being adopted macroporous resin adsorption and obtaining grosvenorin medicinal extract through wash-out, collection is wherein flowed part and is repeated the operation of chromatography wash-out at least one times after grosvenorin medicinal extract being carried out Sephadex LH-20 column chromatography and wash-out again, obtains purity greater than the grosvenorin highly finished product more than 95%.
2. method according to claim 1 is characterized in that, described method specifically comprises the steps:
1) first Grosvenor Momordica is pulverized, added decocting and boil, centrifugal, get supernatant liquor;
2) supernatant liquor in the step 1) is crossed macroporous resin, 50%(v/v) adopts 60-80%(v/v behind the alcohol flush away impurity) the alcohol wash-out, reclaim solvent, collect wherein stream part, i.e. grosvenorin medicinal extract;
3) with step 2) the grosvenorin medicinal extract of gained, carry out Sephadex LH-20 column chromatography, methyl alcohol 60%-100%(v/v) or its aqueous solution wash-out reclaim methyl alcohol and collect wherein stream part, obtain the grosvenorin crude product;
4) get the grosvenorin crude product and carry out Sephadex LH-20 column chromatography, collecting wherein, stream part obtains purity greater than the grosvenorin highly finished product more than 95%.
3. method according to claim 1 and 2 is characterized in that, described Grosvenor Momordica is dry, mature fruit.
4. method according to claim 2 is characterized in that, described solvent is water, extracts 1-2 time.
5. method according to claim 1 and 2 is characterized in that, described macroporous resin is HZ-801 or D101 type.
6. method according to claim 1 and 2 is characterized in that, the amount of filler of described Sephadex LH-20 column chromatography is 2.5cm * 60cm.
7. method according to claim 2 is characterized in that, described alcohol wash-out refers to: it is colourless to be eluted to elutriant with the 10mL/min flow velocity, collects ethanol eluate, and decompression recycling ethanol is for next time.
8. method according to claim 2 is characterized in that, described methyl alcohol or its aqueous solution wash-out refer to: it is colourless part to be eluted to elutriant with 3mL/min flow velocity, 30mL/ stream, and reclaim under reduced pressure methyl alcohol is for next.
9. method according to claim 1 and 2 is characterized in that, described collection is wherein flowed part and referred to: adopt TLC to detect, wherein: it is the volume ratio configuration of 60:35:5 that developping agent adopts chloroform-methanol-water; Developer adopts 10% (v/v) sulfuric acid-ethanol; Detect by the heating colour developing, merge all stream parts that contain grosvenorin.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105708879A (en) * | 2016-04-26 | 2016-06-29 | 上海交通大学 | Maca extract as well as preparation method and application thereof |
| CN106866759A (en) * | 2017-03-28 | 2017-06-20 | 广西甙元植物制品有限公司 | The technique that grosvenor momordica flavonoid is produced from the waste liquid of Momordica-Glycosides decolorizing resin post discharge |
| CN111233958A (en) * | 2020-03-23 | 2020-06-05 | 湖南华诚生物资源股份有限公司 | Momordica grosvenori flavin metal zinc complex and preparation method thereof |
| CN111848708A (en) * | 2020-07-27 | 2020-10-30 | 湖南华诚生物资源股份有限公司 | Method for separating squalene and other active ingredients from fructus Siraitiae Grosvenorii extraction residue |
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| US4084010A (en) * | 1976-01-01 | 1978-04-11 | Tsunematsu Takemoto | Glycosides having sweetness |
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| CN1611498A (en) * | 2003-10-28 | 2005-05-04 | 广西师范大学 | Grosvenor momordical flavone and its preparing prcess |
| KR20050078386A (en) * | 2004-01-29 | 2005-08-05 | 전라남도 | Method for extracting vitexin and isovitexin from vigna radiata (l.) and extract thereof |
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Patent Citations (4)
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| US4084010A (en) * | 1976-01-01 | 1978-04-11 | Tsunematsu Takemoto | Glycosides having sweetness |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105708879A (en) * | 2016-04-26 | 2016-06-29 | 上海交通大学 | Maca extract as well as preparation method and application thereof |
| CN106866759A (en) * | 2017-03-28 | 2017-06-20 | 广西甙元植物制品有限公司 | The technique that grosvenor momordica flavonoid is produced from the waste liquid of Momordica-Glycosides decolorizing resin post discharge |
| CN106866759B (en) * | 2017-03-28 | 2019-09-10 | 湖南华诚生物资源股份有限公司 | The technique of grosvenor momordica flavonoid is produced from the waste liquid that Momordica-Glycosides decolorizing resin column is discharged |
| CN111233958A (en) * | 2020-03-23 | 2020-06-05 | 湖南华诚生物资源股份有限公司 | Momordica grosvenori flavin metal zinc complex and preparation method thereof |
| CN111848708A (en) * | 2020-07-27 | 2020-10-30 | 湖南华诚生物资源股份有限公司 | Method for separating squalene and other active ingredients from fructus Siraitiae Grosvenorii extraction residue |
| CN111848708B (en) * | 2020-07-27 | 2021-07-20 | 湖南华诚生物资源股份有限公司 | Method for separating squalene and other active ingredients from fructus Siraitiae Grosvenorii extraction residue |
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