CN100391982C - Resin separation purification method of lentinan - Google Patents
Resin separation purification method of lentinan Download PDFInfo
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- CN100391982C CN100391982C CNB2006100319101A CN200610031910A CN100391982C CN 100391982 C CN100391982 C CN 100391982C CN B2006100319101 A CNB2006100319101 A CN B2006100319101A CN 200610031910 A CN200610031910 A CN 200610031910A CN 100391982 C CN100391982 C CN 100391982C
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Abstract
The present invention provides a method for purifying and separating lentinan by resin, which comprises: ultrasonic waves are used for assisting boiled water for extraction; proteinase is hydrolyzed; trichloroacetic acid is deproteinized, and ethanol is precipitated for obtaining crude polysaccharide; the crude polysaccharide is dissolved and dialyzed; a D392 resin column is used for adsorbing the crude polysaccharide; the crude polysaccharide is eluted by distilled water and NaCl solutions with different concentration; positive reaction parts of the polysaccharide is fetched out to be dialyzed, and alcohol is deposited; precipitates are dried, and purified polysaccharide is obtained. The total yield of the present invention can reach 1.155/1000, and the purity of lentinan reaches more than 98.3%. Moreover, the present invention has the advantages of simple technology, low cost and easy industrial production.
Description
Technical field
The present invention relates to the extracting method of effective ingredients in plant, specifically, provide a kind of extracting and purifying method of lentinan.
Background technology
Lentinan (Lentinan) is a kind of immunity regulating type antitumour auxiliary drug, is the high-molecular biologic goods that adopt the extraction from mushroom fruiting body of natural product chemistry technological method, separation, purifying to obtain, and its molecular weight is generally at 40-80 ten thousand.Japan in 1986 at first produces and lentinan is made powder injection in domestic and international sale, and it has biological activitys such as antiviral, antitumor, inducing interferon formation, adjusting and raise immunity, has been widely used in clinical.Its main route of administration is intravenous drip or injects.Lentinan is an internationally recognized curative effect immunomodulator preferably, and the clinical treatment that is mainly used in malignant tumours such as cancer of the stomach, lung cancer, intestinal cancer and cancerous thoracoascites also has better action to the immunologic function that improves hepatitis and aids patient.Lentinus edodes polysaccharide injecta is domestic unique up to now a kind of macromolecular polysaccharide lyophilized injectable powder that can injection for intravenous, become the principal item of domestic anti tumor immune response conditioning agent, also be used as simultaneously a kind of important biotechnological formulation, healthcare products raw material and foodstuff additive and be widely used in food and Medical Biology research field.
Lentinan is the polarity macromolecular cpd, easily causes the polysaccharid glucoside chain break under the acidic conditions.Its extraction separation is very complicated, adopts boiling water mostly, and alkaline solution extracts.Free protein in the extracting solution is mainly removed with methods such as Sevage method, Freon 113 method and Tricholroacetic Acid methods, conjugated protein remove the general proteolytic enzyme of using, small molecular weight impurity is removed available dialysis method, the external dialysis apparatus that adopts is dialysed continuously, and also available ultra-filtration method obtains the polysaccharide of certain molecular weight scope.The purification process of polysaccharide is a lot, and step-by-step precipitation method, salting-out process, metal complex method, the quaternary amine precipitator method, DEAE-Mierocrystalline cellulose method and other various dissimilar gel filtration chromatography methods are specifically arranged.Methods such as sulfuric acid-phynol method commonly used are measured polysaccharide content.
The homopolysaccharide that the lentinan system of medicine injection obtains from mushroom fruiting body, its structural formula is:
At present, the extraction and separation process of lentinan is very complicated and yield is very low, and the production cost height causes market value high, and it costs an arm and a leg and is difficult to accept for common patient.The clear 48-6767 of Japanese Patent (1973), clear 49-484 (1974), Chinese patent CN 1153179A (1997) needs to use comparatively expensive consumptive material, and complex process.Also have report to adopt the gel filtration chromatography method, but this method can only obtain to use sample for research on a small quantity, because of gel costs an arm and a leg, production cost is very high, and prior art remains in many shortcomings, is difficult to drop into large-scale production.Target of the present invention is the existing technology of innovation, improves yield, obtains the separating and purifying lentinan technology of a lower production cost.
Summary of the invention
The object of the invention is to provide a kind of resin separation purification processing method of lentinan, verifies this processing step simplification through repeated experiments, and the lentinan yield effectively improves, and production cost significantly reduces.
Embodiment of the present invention are: a kind of resin separation purification method of lentinan, it is characterized in that, and may further comprise the steps:
(1) get mushroom dry powder, add 20 times of water logging bubbles and spend the night, ultrasonic disruption 15min boils 2-3h, coarse filtration, and filtrate is centrifugal, collects supernatant and is extracting solution, and filter residue repeats to extract once united extraction liquid at least;
(2) extracting solution is concentrated into small volume, adds papain hydrolysis 8h under 55 ℃, pH5.5-6.5 condition, slowly adds the 3-5% trichoroacetic acid(TCA) under the 5-15 ℃ of stirring, stirs 10min, spends the night the centrifugal precipitation of removing, the neutralization of NaOH solution under 5-15 ℃;
(3) slowly adding ethanol to determining alcohol under the stirring is 75%, places 4h for 5-15 ℃, the supernatant that inclines, centrifugal collecting precipitation, throw out dehydrated alcohol, and acetone, ether washing, drying promptly gets Crude polysaccharides;
(4) use the water dissolution Crude polysaccharides, making polysaccharide liquid concentration is 1-2%, goes up resin column behind the polysaccharide soln dialysis 48h, is washed to sugar-free, and the NaCl eluant solution is collected elutriant.Elutriant is concentrated into small volume, dialysis 48h, and alcohol precipitation is got precipitation, washing, the dry purified polysaccharide that gets.
In the described step (4), during with the NaCl eluant solution, use 0.2,0.5 successively, 1mol/L NaCl eluant solution, collect elutriant respectively.
In the described step (4), used resin is a D392 negatively charged ion macroporous adsorbent resin.This resin can use with 1NNaOH regeneration.
The present invention adopts ultrasonic wave to assist the mushroom boiling water extraction, papain hydrolysis, and the trichoroacetic acid(TCA) deproteinated, ethanol sedimentation gets Crude polysaccharides, Crude polysaccharides dissolving back dialysis, the absorption of D392 resin column, different concns NaCl eluant solution, get sugared reacting positive and partly dialyse, alcohol precipitation, the dry purified polysaccharide that gets.Technology total recovery of the present invention can reach 1.155 ‰, and lentinan purity reaches more than 98.3%, and work simplification, and cost reduces, and helps suitability for industrialized production.
Embodiment
(1) extract: get dried thin mushroom 2kg, pulverize, place pot, add 20 times of distilled water of raw materials quality, soaked overnight, boil 3h behind the supersound process 15min next day, filters, and residue extracts 1-2 time with aforesaid method again, merging filtrate.
(2) deproteinated: filtrate decompression is concentrated into 50% of former filtrate volume, add papain hydrolysis 8h under 55 ℃, pH5.5-6.5 condition, the add-on of papoid is extracted 2 ‰ of solution weight after adding papoid, slowly add the 3-5% trichoroacetic acid(TCA) under 10 ℃ of stirrings, stir 10min, centrifugal the removing in back of spending the night under 10 ℃ precipitated, and gets supernatant and neutralizes with NaOH solution.
(3) alcohol precipitation: stir that slowly to add ethanol to solution alcohol concn down be 75%, leave standstill, treat that precipitation produces hypsokinesis and removes supernatant, lower sediment is through the centrifugal precipitation that obtains, and precipitation is through ethanol, acetone, ether Xian Di in succession, drying, weigh mushroom Crude polysaccharides 101.37 grams.
(4) purifying: the mushroom Crude polysaccharides is dissolved in the 900g water, and application of sample is in the absorption of D392 resin column, water Xian behind the dialysis 48h, 0.2,0.5,1mol/L NaCl eluant solution, Fractional Collections is got sugared reacting positive and partly is concentrated into small volume, carries out alcohol precipitation after the dialysis, taking precipitate, the washing, drying, weigh purified polysaccharide 2.31g, total recovery is .1.155 ‰, lentinan purity 98.3%.
Claims (3)
1. the resin separation purification method of a lentinan is characterized in that, may further comprise the steps:
(1) get mushroom dry powder, add 20 times of water logging bubbles and spend the night, ultrasonic disruption 15min boils 2-3h, coarse filtration, and filtrate is centrifugal, collects supernatant and is extracting solution, and filtrate repeats to extract once united extraction liquid at least;
(2) extracting solution is concentrated into small volume, adds papain hydrolysis 8h under 55 ℃ of temperature, pH5.5-6.5 condition, slowly adds the 3-5% trichoroacetic acid(TCA) under the 5-15 ℃ of stirring, and stirs 10min, spends the night the centrifugal precipitation of removing, the neutralization of NaOH solution under 5-15 ℃;
(3) slowly adding ethanol to determining alcohol under the stirring is 75%, places 4h for 5-15 ℃, the supernatant that inclines, and centrifugal collecting precipitation, throw out washs with dehydrated alcohol, acetone, ether, and drying promptly gets Crude polysaccharides;
(4) use the water dissolution Crude polysaccharides, making polysaccharide liquid concentration is 1-2%, goes up resin column behind the polysaccharide soln dialysis 48h, is washed to sugar-free, and the NaCl eluant solution is collected elutriant, and elutriant is concentrated into small volume, dialysis 48h, and alcohol precipitation is got precipitation, washing, dry purified polysaccharide.
2. the resin separation purification method of lentinan according to claim 1 is characterized in that, in the described step (4), during with the NaCl eluant solution, uses 0.2,0.5 successively, the 1mol/LNaCl eluant solution, collects elutriant respectively.
3. the resin separation purification method of lentinan according to claim 1 is characterized in that, in the described step (4), used resin is a D392 negatively charged ion macroporous adsorbent resin.
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| CNB2006100319101A CN100391982C (en) | 2006-07-03 | 2006-07-03 | Resin separation purification method of lentinan |
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| CNB2006100319101A CN100391982C (en) | 2006-07-03 | 2006-07-03 | Resin separation purification method of lentinan |
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| CN100391982C true CN100391982C (en) | 2008-06-04 |
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Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100447250C (en) * | 2006-12-26 | 2008-12-31 | 江南大学 | A kind of separation and purification method of rice bran polysaccharide |
| CN101613418A (en) * | 2009-07-22 | 2009-12-30 | 柏林 | A kind of method of extracting agaricus lentinan |
| CN101891836B (en) * | 2010-07-27 | 2012-05-02 | 乔德亮 | Ultrasonic-assisted extraction method of agaricus bisporus polysaccharide |
| CN102731666B (en) * | 2011-04-07 | 2016-05-11 | 大连工业大学 | A kind of method of extracting polysaccharide from Chinese caterpillar fungus culture medium |
| CN102311510A (en) * | 2011-10-21 | 2012-01-11 | 黑龙江大学 | Method for extracting perillafrutescens L. polysaccharide from perillafrutescens L. seeds and purifying perillafrutescens L. polysaccharide |
| CN102977218B (en) * | 2012-10-13 | 2015-09-09 | 内蒙古清谷新禾有机食品集团有限责任公司 | A kind of method extracting polysaccharide from buckwheat bran |
| CN103980379A (en) * | 2014-05-20 | 2014-08-13 | 河南中烟工业有限责任公司 | Lentinan, its extraction and purification method and its application as tobacco humectant |
| CN104311684A (en) * | 2014-10-17 | 2015-01-28 | 华南理工大学 | Method for extracting lentinan by virtue of ultrasonic coupling subcritical water |
| CN105367681A (en) * | 2015-12-16 | 2016-03-02 | 黑龙江众生生物工程有限公司 | Edible and medicinal fungal polysaccharide low-temperature normal-pressure plasma extraction method |
| CN106035876A (en) * | 2016-07-08 | 2016-10-26 | 肇庆医学高等专科学校 | A kind of herbal tea with effervescent tablets for nourishing stomach and health care and preparation method thereof |
| CN106616125A (en) * | 2016-12-27 | 2017-05-10 | 苏州祥林生物科技有限公司 | Beverage containing blueberry and fructus momordicae composition and preparation method thereof |
| CN108142923A (en) * | 2018-03-14 | 2018-06-12 | 广东省微生物研究所（广东省微生物分析检测中心） | Purposes of the mushroom dietary fiber extract in the preparation for preparing treatment and/or prevention intestinal bacilli illness relevant disease |
| CN110541016A (en) * | 2019-09-10 | 2019-12-06 | 贵州好菇粮农业科技有限公司 | Extraction and purification method of lentinan |
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| CN1765934A (en) * | 2004-10-27 | 2006-05-03 | 山西亚宝药业集团股份有限公司 | Nanometer lentinan and preparation method of injection thereof |
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| CN1765934A (en) * | 2004-10-27 | 2006-05-03 | 山西亚宝药业集团股份有限公司 | Nanometer lentinan and preparation method of injection thereof |
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