CN103627736A - Method for extracting resveratrol in polygonum cuspidatum fermentation liquor - Google Patents
Method for extracting resveratrol in polygonum cuspidatum fermentation liquor Download PDFInfo
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- CN103627736A CN103627736A CN201310720626.5A CN201310720626A CN103627736A CN 103627736 A CN103627736 A CN 103627736A CN 201310720626 A CN201310720626 A CN 201310720626A CN 103627736 A CN103627736 A CN 103627736A
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- resveratrol
- ethyl acetate
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- macroporous resin
- elutriant
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Abstract
The invention discloses a method for extracting resveratrol in polygonum cuspidatum fermentation liquor. The method comprises the following steps: (1) preparing an endophytic fungi L1 spore suspension liquid; (2) preparing a culture medium; (3) fermenting so as to obtain the polygonum cuspidatum fermentation liquor; (4) extracting the polygonum cuspidatum fermentation liquor with ethyl acetate for four times, combining ethyl acetate phases, concentrating till drying so as to obtain a crude resveratrol extract; (5) carrying out chromatography jointly through macroporous resin and silica gel; (6) refining so as to obtain the resveratrol product. The method is simple, has low production cost, slightly pollutes the environment, and is applicable to industrial production. The purity of the resveratrol purified and separated from the polygonum cuspidatum fermentation liquor by the method reaches 92wt%.
Description
Technical field
The present invention relates to a kind of extracting method of trans-resveratrol, especially relate to the extracting method of trans-resveratrol in a kind of giant knotweed fermented liquid.
Background technology
Trans-resveratrol is a kind of plant antibiotic, and it is mainly present in pericarp, peanut, mulberry fruit, pine tree, polygonaceae plant (giant knotweed) 12 sections of grape, in 72 kind of plant of 31 genus.Trans-resveratrol is the biological chemical substance producing for resisting environmental stress (as awful weather, microbial infection etc.), has significant pharmacology and biological action, as anticancer, neuroprotective, anti-ageing, anti-inflammatory and prolongs life etc.
At present, in giant knotweed fermented liquid, the extracting method of trans-resveratrol is generally macroporous resin, silica gel or ion exchange resin single-column chromatography, and these traditional technology costs are high, and purity is low.As can be seen here, find a kind of low cost, method for extracting resveratrol that purity is high, become the task of top priority of current trans-resveratrol production industry.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes the deficiencies in the prior art, and the extracting method of trans-resveratrol in the giant knotweed fermented liquid that a kind of cost is low is provided, and the trans-resveratrol purity extracting is high.
The technical scheme that the present invention solves its technical problem employing is: the extracting method of trans-resveratrol in a kind of giant knotweed fermented liquid, comprises the following steps:
(1) endogenetic fungus L1 spore suspension preparation: in 10ml physiological saline, fully shake from Shang Tiao five rings, endogenetic fungus L1 PDA inclined-plane thalline;
Described endogenetic fungus L1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is the patent application document that CGMCC NO.4558(is 201110130127.1 referring to application number);
(2) substratum is prepared: fresh giant knotweed is crushed to 180 orders, in mass ratio giant knotweed 1.5wt%, dehydrated alcohol 2 wt%, CMC-Na 2.5 wt%, NH
4nO
30.2 wt%, Na
2hP0
40.06 wt%, CaCl
20.1 wt% preparation fermention medium, adjusting Initial pH is 5.5;
(3) fermentation: first by the substratum in step (2) through 121 ℃, 30min moist heat sterilization; By in the endogenetic fungus L1 spore suspension access 250ml triangular flask of 3ml, the substratum of the initial pH 5.5 of 30mL is housed in described triangular flask, then is under 28 ℃ of temperature, rotating speed 220r/min at culture condition again, fermentation 60h, obtains giant knotweed fermented liquid;
(4) extract: by ethyl acetate, divide extraction giant knotweed fermented liquid four times, combined ethyl acetate phase, is concentrated into dryly, obtains trans-resveratrol crude extract;
(5) macroporous resin-silica gel associating chromatography: step (4) gained trans-resveratrol crude extract is dissolved in to the mixed solution of ethanol and ethyl acetate, in the mixed solution of described ethanol and ethyl acetate, the volume ratio of ethanol and ethyl acetate is 2:3; Then use filter paper filtering, discard filter residue, obtain filtrate; Toward macroporous resin column, add deionized water, when deionized water liquid level flushes with macroporous resin column top surface, gained filtrate is added to macroporous resin column top, when filtrate liquid level did not have macroporous resin column top, with the mixed solution that ethanol and ethyl acetate volume ratio are 2:3, as elutriant, carry out wash-out, macroporous resin chromatography elution speed is lml/mim, collects elutriant; By the elutriant of collection, be the trans-resveratrol mixed solution after macroporous resin chromatography again, join in silica gel column chromatography, treat that trans-resveratrol mixed solution infiltrates in silica gel, use again ethanol: the ethanol that ethyl acetate volume ratio is 4:1 and the mixed solution of ethyl acetate carry out wash-out as silicagel column with elutriant, silica gel column chromatography elution speed is lml/mim, obtains elutriant;
(6) refining: by step (5) gained elutriant, concentrating under reduced pressure, to just there being precipitation to occur, when solution becomes muddy, stops concentrating, and by concentrated solution above crystallization of standing 24h under 4 ℃ of conditions, obtains yellow crystals, through lyophilize, obtains trans-resveratrol product.
Further, in step (5), described filter paper is accurate filter paper.
The extracting method of the present invention's trans-resveratrol, first adopt ethyl acetate to divide extraction giant knotweed fermented liquid four times, to ensure extraction yield and the yield of trans-resveratrol, then adopt macroporous resin-silica gel associating chromatography that trans-resveratrol is refining, guarantee the up-to-standard of trans-resveratrol product, final refining, in elutriant from veratryl alcohol part, concentrating under reduced pressure, again by the standing crystallization of concentrated solution, obtain yellow crystals, through lyophilize, obtain trans-resveratrol product.
Technique of the present invention is simple, and production cost is low, and environmental pollution is little, and suitability for industrialized is produced.Utilize the present invention to purify to giant knotweed fermented liquid separated, the trans-resveratrol after purifying can reach 92wt%, far away higher than using separately the trans-resveratrol purity of macroporous resin and silica gel single-column chromatography gained.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
The present embodiment comprises the following steps:
(1) endogenetic fungus L1 spore suspension preparation: in 10ml physiological saline, fully shake from Shang Tiao five rings, endogenetic fungus L1 PDA inclined-plane thalline;
Described endogenetic fungus L1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is the patent application document that CGMCC NO.4558(is 201110130127.1 referring to application number);
(2) substratum preparation: fresh giant knotweed is crushed to 180 orders, gets the fresh Giant Knotweed Rhizome 0.45g(of 180 order giant knotweed 1.5wt%), dehydrated alcohol 0.6g(2%), CMC-Na0.75g(2.5%), NH
4nO
30.06g(0.2%), Na
2hP0
40.018g(0.06%), CaCl
20.03g(0.1%) be mixed with fermention medium, pH value is 5.5;
(3) fermentation: first by the substratum in step (2) through 121 ℃, 30min moist heat sterilization; By in the endogenetic fungus L1 spore suspension access 250ml triangular flask of 3ml, the substratum of the initial pH 5.5 of 30mL is housed in described triangular flask, then is under 28 ℃ of temperature, rotating speed 220r/min at culture condition again, fermentation 60h, obtains giant knotweed fermented liquid;
(4) extract: get 200ml giant knotweed fermented liquid, by four extractions of 800ml ethyl acetate average mark, combined ethyl acetate phase, be concentrated into dryly, obtain trans-resveratrol crude extract;
(5) macroporous resin one silica gel associating chromatography: step (4) gained trans-resveratrol crude extract is dissolved in to the mixed solution of ethanol and ethyl acetate, in the mixed solution of described ethanol and ethyl acetate, the volume ratio of ethanol and ethyl acetate is 2:3; Then use filter paper filtering, discard filter residue, obtain filtrate; Toward macroporous resin column, add deionized water, when deionized water liquid level flushes with macroporous resin column top surface, gained filtrate is added to macroporous resin column top, when filtrate liquid level did not have macroporous resin column top, with the mixed solution that ethanol and ethyl acetate volume ratio are 2:3, as elutriant, carry out wash-out, macroporous resin chromatography elution speed is lml/mim, collects elutriant; By the elutriant of collection, be the trans-resveratrol mixed solution after macroporous resin chromatography again, join in silica gel column chromatography, treat that trans-resveratrol mixed solution infiltrates in silica gel, use again ethanol: the ethanol that ethyl acetate volume ratio is 4:1 and the mixed solution of ethyl acetate carry out wash-out as silicagel column with elutriant, silica gel column chromatography elution speed is lml/mim, obtains elutriant;
(6) refining: by step (5) gained elutriant, concentrating under reduced pressure, to just there being precipitation to occur, when solution becomes muddy, stops concentrating, and by concentrated solution above crystallization of standing 24h under 4 ℃ of conditions, obtains yellow crystals, through lyophilize, obtains trans-resveratrol product.
The present embodiment products obtained therefrom reaches 92% through HPLC and mass spectrometric detection trans-resveratrol purity.
Embodiment 2
The present embodiment comprises the following steps:
(1) endogenetic fungus L1 spore suspension preparation: in 10ml physiological saline, fully shake from Shang Tiao five rings, endogenetic fungus L1 PDA inclined-plane thalline;
Described endogenetic fungus L1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is the patent application document that CGMCC NO.4558(is 201110130127.1 referring to application number);
(2) substratum preparation: fresh giant knotweed is crushed to 180 orders, gets the fresh Giant Knotweed Rhizome 0.45g(of 180 order giant knotweed 1.5wt%), dehydrated alcohol 0.6g(2%), CMC-Na0.75g(2.5%), NH
4nO
30.06g(0.2%), Na
2hP0
40.018g(0.06%), CaCl
20.03g(0.1%) be mixed with fermention medium, pH value is 5.5;
(3) fermentation: first by the substratum in step (2) through 121 ℃, 30min moist heat sterilization; By in the endogenetic fungus L1 spore suspension access 250ml triangular flask of 3ml, the substratum of the initial pH 5.5 of 30mL is housed in described triangular flask, then is under 28 ℃ of temperature, rotating speed 220r/min at culture condition again, fermentation 60h, obtains giant knotweed fermented liquid;
(4) extract: get 200ml giant knotweed fermented liquid, by four extractions of 800ml ethyl acetate average mark, combined ethyl acetate phase, be concentrated into dryly, obtain trans-resveratrol crude extract;
(5) macroporous resin-silica gel associating chromatography: step (4) gained trans-resveratrol crude extract is dissolved in to the mixed solution of ethanol and ethyl acetate, in the mixed solution of described ethanol and ethyl acetate, the volume ratio of ethanol and ethyl acetate is 2:3; Then use filter paper filtering, discard filter residue, obtain filtrate; Toward macroporous resin column, add deionized water, when deionized water liquid level flushes with macroporous resin column top surface, gained filtrate is added to macroporous resin column top, when filtrate liquid level did not have macroporous resin column top, with the mixed solution that ethanol and ethyl acetate volume ratio are 2:3, as elutriant, carry out wash-out, macroporous resin chromatography elution speed is lml/mim, collects elutriant; By the elutriant of collection, be the trans-resveratrol mixed solution after macroporous resin chromatography again, join in silica gel column chromatography, treat that trans-resveratrol mixed solution infiltrates in silica gel, use again ethanol: the ethanol that ethyl acetate volume ratio is 4:1 and the mixed solution of ethyl acetate carry out wash-out as silicagel column with elutriant, silica gel column chromatography elution speed is lml/mim, obtains elutriant;
(6) refining: by step (5) gained elutriant, concentrating under reduced pressure, to just there being precipitation to occur, when solution becomes muddy, stops concentrating, and by concentrated solution above crystallization of standing 24h under 4 ℃ of conditions, obtains yellow crystals, through lyophilize, obtains trans-resveratrol product.
The present embodiment products obtained therefrom reaches 30% through HPLC and mass spectrometric detection trans-resveratrol purity.
Embodiment 3
The present embodiment comprises the following steps:
(1) endogenetic fungus L1 spore suspension preparation: in 10ml physiological saline, fully shake from Shang Tiao five rings, endogenetic fungus L1 PDA inclined-plane thalline;
Described endogenetic fungus L1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is the patent application document that CGMCC NO.4558(is 201110130127.1 referring to application number);
(2) substratum preparation: fresh giant knotweed is crushed to 180 orders, gets the fresh Giant Knotweed Rhizome 0.45g(of 180 order giant knotweed 1.5wt%), dehydrated alcohol 0.6g(2%), CMC-Na0.75g(2.5%), NH
4nO
30.06g(0.2%), Na
2hP0
40.018g(0.06%), CaCl
20.03g(0.1%) be mixed with fermention medium, pH value is 5.5;
(3) fermentation: first by the substratum in step (2) through 121 ℃, 30min moist heat sterilization; By in the endogenetic fungus L1 spore suspension access 250ml triangular flask of 3ml, the substratum of the initial pH 5.5 of 30mL is housed in described triangular flask, then is under 28 ℃ of temperature, rotating speed 220r/min at culture condition again, fermentation 60h, obtains giant knotweed fermented liquid;
(4) extract: get 200ml giant knotweed fermented liquid, by four extractions of 800ml ethyl acetate average mark, combined ethyl acetate phase, be concentrated into dryly, obtain trans-resveratrol crude extract;
(5) macroporous resin-silica gel associating chromatography: step (4) gained trans-resveratrol crude extract is dissolved in to the mixed solution of ethanol and ethyl acetate, in the mixed solution of described ethanol and ethyl acetate, the volume ratio of ethanol and ethyl acetate is 2:3; Then use filter paper filtering, discard filter residue, obtain filtrate; Toward macroporous resin column, add deionized water, when deionized water liquid level flushes with macroporous resin column top surface, gained filtrate is added to macroporous resin column top, when filtrate liquid level did not have macroporous resin column top, with the mixed solution that ethanol and ethyl acetate volume ratio are 2:3, as elutriant, carry out wash-out, macroporous resin chromatography elution speed is lml/mim, collects elutriant; By the elutriant of collection, be the trans-resveratrol mixed solution after macroporous resin chromatography again, join in silica gel column chromatography, treat that trans-resveratrol mixed solution infiltrates in silica gel, use again ethanol: the ethanol that ethyl acetate volume ratio is 4:1 and the mixed solution of ethyl acetate carry out wash-out as silicagel column with elutriant, silica gel column chromatography elution speed is lml/mim, obtains elutriant;
(6) refining: by step (5) gained elutriant, concentrating under reduced pressure, to just there being precipitation to occur, when solution becomes muddy, stops concentrating, and by concentrated solution above crystallization of standing 24h under 4 ℃ of conditions, obtains yellow crystals, through lyophilize, obtains trans-resveratrol product.
The present embodiment products obtained therefrom reaches 87% through HPLC and mass spectrometric detection trans-resveratrol purity.
Claims (1)
1. an extracting method for trans-resveratrol in giant knotweed fermented liquid, is characterized in that, comprises the following steps:
(1) endogenetic fungus L1 spore suspension preparation: in 10ml physiological saline, fully shake from Shang Tiao five rings, endogenetic fungus L1 PDA inclined-plane thalline;
Described endogenetic fungus L1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC NO.4558;
(2) substratum is prepared: fresh giant knotweed is crushed to 180 orders, in mass ratio giant knotweed 1.5wt%, dehydrated alcohol 2 wt%, CMC-Na 2.5 wt%, NH
4nO
30.2 wt%, Na
2hP0
40.06 wt%, CaCl
20.1 wt% preparation fermention medium; Adjusting Initial pH is 5.5;
(3) fermentation: first by the substratum in step (2) through 121 ℃, 30min moist heat sterilization; By in the endogenetic fungus L1 spore suspension access 250ml triangular flask of 3ml, the substratum of the initial pH 5.5 of 30mL is housed in described triangular flask, then is under 28 ℃ of temperature, rotating speed 220r/min at culture condition again, fermentation 60h, obtains giant knotweed fermented liquid;
(4) extract: by ethyl acetate, divide extraction giant knotweed fermented liquid four times, combined ethyl acetate phase, is concentrated into dryly, obtains trans-resveratrol crude extract;
(5) macroporous resin-silica gel associating chromatography: step (4) gained trans-resveratrol crude extract is dissolved in to the mixed solution of ethanol and ethyl acetate, in the mixed solution of described ethanol and ethyl acetate, the volume ratio of ethanol and ethyl acetate is 2:3; Then use filter paper filtering, discard filter residue, obtain filtrate; Toward macroporous resin column, add deionized water, when deionized water liquid level flushes with macroporous resin column top surface, gained filtrate is added to macroporous resin column top, when filtrate liquid level did not have macroporous resin column top, with the mixed solution that ethanol and ethyl acetate volume ratio are 2:3, as elutriant, carry out wash-out, macroporous resin chromatography elution speed is lml/mim, collects elutriant; By the elutriant of collection, be the trans-resveratrol mixed solution after macroporous resin chromatography again, join in silica gel column chromatography, treat that trans-resveratrol mixed solution infiltrates in silica gel, use again ethanol: the ethanol that ethyl acetate volume ratio is 4:1 and the mixed solution of ethyl acetate carry out wash-out as silicagel column with elutriant, silica gel column chromatography elution speed is lml/mim, obtains elutriant;
(6) refining: by step (5) gained elutriant, concentrating under reduced pressure, to just there being precipitation to occur, when solution becomes muddy, stops concentrating, and by concentrated solution above crystallization of standing 24h under 4 ℃ of conditions, obtains yellow crystals, through lyophilize, obtains trans-resveratrol product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104313061A (en) * | 2014-09-05 | 2015-01-28 | 潍坊学院 | Culture medium capable of increasing resveratrol content of gentrin knotweed and preparation method thereof |
| CN105901738A (en) * | 2016-06-14 | 2016-08-31 | 益倍（天津）生物科技有限公司 | Fermentation grape extract, preparing method of fermentation grape extract and preparation containing fermentation grape extract |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102199548A (en) * | 2011-05-18 | 2011-09-28 | 湖南农业大学 | Polygonum cuspidatum root endophytic strain G3 for efficiently transforming polydatin into resveratrol |
| CN103408402A (en) * | 2013-08-13 | 2013-11-27 | 甘肃农业大学 | Method for extracting high-purity resveratrol from grape seed |
-
2013
- 2013-12-24 CN CN201310720626.5A patent/CN103627736A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102199548A (en) * | 2011-05-18 | 2011-09-28 | 湖南农业大学 | Polygonum cuspidatum root endophytic strain G3 for efficiently transforming polydatin into resveratrol |
| CN103408402A (en) * | 2013-08-13 | 2013-11-27 | 甘肃农业大学 | Method for extracting high-purity resveratrol from grape seed |
Non-Patent Citations (2)
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| 刘华金: "虎杖内生菌高效转化白藜芦醇苷的研究", 《中国优秀硕士学位论文全文数据库》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313061A (en) * | 2014-09-05 | 2015-01-28 | 潍坊学院 | Culture medium capable of increasing resveratrol content of gentrin knotweed and preparation method thereof |
| CN104313061B (en) * | 2014-09-05 | 2018-04-27 | 潍坊学院 | A kind of culture medium for improving Resveratrol in Rhizoma Polygoni Cuspidati content and preparation method thereof |
| CN105901738A (en) * | 2016-06-14 | 2016-08-31 | 益倍（天津）生物科技有限公司 | Fermentation grape extract, preparing method of fermentation grape extract and preparation containing fermentation grape extract |
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Application publication date: 20140312 |