CN1153179A - Method for separation and purification of lentinan - Google Patents
Method for separation and purification of lentinan Download PDFInfo
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- CN1153179A CN1153179A CN 95113378 CN95113378A CN1153179A CN 1153179 A CN1153179 A CN 1153179A CN 95113378 CN95113378 CN 95113378 CN 95113378 A CN95113378 A CN 95113378A CN 1153179 A CN1153179 A CN 1153179A
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- lentinan
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Abstract
A process for extracting, separating and purifying lentinan comprises hot water extraction of champignon, concentration, alcohol precipitation, dissolving precipitate, dialysis, EDAE-cellulose adsorption, eluting with alkali solutionin different concentrations, merging eluents showing positive in saccharide reaction, neutralizing with acid, ultrafiltration, concentration, and freeze drying. Its advantages are low cost and high quality of the product.
Description
The present invention relates to natural product chemistry, more specifically relate to the separation of polysaccharides purification process.
Lentinan, trade(brand)name power are carried energy, and English name Lentinan is a kind of immunity regulating type antitumour auxiliary drug.
The lentinan system polysaccharide that the extraction separation purifying obtains from the umbrella mushroom section fungi mushroom fruiting body of artificial culture.The various countries scientist just carries out extensive studies to all kinds of polysaccharide as far back as early seventies, the discovery polysaccharide not only can be treated the cancer that body immune system is subjected to major injury, can treat the damaged disease of panimmunity again, as chronic viral hepatitis and some drug-resistant bacteria or the viral chronic disease that causes, can also treat the autoimmune disease such as rheumatosis.The sugar chain of discovered in recent years polysaccharide has conclusive effect in molecular biology, it can control cell fission and differentiation, regulates the growth and the aging of cell, and in addition, polysaccharide pharmaceutically is being a kind of good adjuvant again.
Because polysaccharide is the polarity macromolecular cpd, can cause 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 three kinds of methods such as Sevage method, Freon 113 method and Tricholroacetic Acid method, the first two kind method is applied in the microbial polysaccharide more, the latter is applied in the vegetable polysaccharides more, conjugated protein remove available proteolytic enzyme, small molecular weight impurity is removed available dialysis method, the continuous dialysis apparatus dialysis of external employing, also available ultra-filtration method obtains the polysaccharide of certain molecular weight scope.The purification process of polysaccharide is a lot, and branch's precipitator method, salting-out process, metal complex method, the quaternary amine precipitator method are arranged.DEAE-Mierocrystalline cellulose method and other various dissimilar gel filtration chromatography methods.Measure polysaccharide with colorimetric methods such as sulfuric acid-phynol methods.
The one-component (I) that lentinan system obtains from mushroom fruiting body
Its extraction separation is very complicated and yield is very low, the cost height.The clear 48-6767 of Japanese Patent (1973); Clear 49-484 (1974).Recently once had report to adopt the gel filtration chromatography method, but this method can only obtain use sample for research on a small quantity, can't be applied to produce, and since gel cost an arm and a leg and make cost very high, owing to prior art remains in many shortcomings.The object of the invention is produced the separation purification method of the lentinan that cost is low in order to seek a kind of new comparatively easy being easy to.
The present invention implements through the following steps:
Concentrate---→ the enriched material lyophilize.
Get the raw material mushroom, use hot water extraction, extracting solution is evaporated to small volume, stirs slowly to add ethanol down, make it produce precipitation, precipitation is placed in the dialysis tubing and dialyses through water-soluble Jie, and dialyzate concentrates, ethanol sedimentation, after washing with alcohol, vacuum-drying gets raw product to precipitation again.The thick lentinan of gained is soluble in water, after the DEAE-cellulose adsorption, washes with water, uses the dilute alkaline soln wash-out, and used alkali can be Na
2CO
3, K
2CO
3, NaHCO
3, KHCO
3, NaOH, KOH, the concentration of alkali elutriant can be 1～2mole/L, the elutriant fraction collection is got sugared reacting positive partly, with the hydrochloric acid neutralization, places on the ultra-fine filter and filters, and does not get to see through liquid and concentrate, lyophilize promptly gets the single component of lentinan.
Embodiment 1:
Get 10 kilograms of qualified raw material dried thin mushrooms, after the pulverizing, place the reactor of strap clamp cover, add clear water, the add-on of water is about 1.5 times of raw material volume, jacket steam is heated to boiling, kept 6 hours under boiling then, filter, residue extracts three times with aforesaid method again, merge whole filtrates, be evaporated to small volume, stir the ethanol that slowly adds isopyknic 95% above concentration down, leave standstill and placed 4 hours, precipitation produces, the supernatant liquor that inclines, lower sediment is through the centrifugal precipitation that obtains, and precipitation is behind ethanol Xian Di, soluble in water, place dialysis tubing to tap water dialysis 3 days, dialyzate is evaporated to small volume, and the concentration that adds equal-volume under stirring is the ethanol more than 95%, after static 4 hours, centrifugal must the precipitation, precipitation is through ethanol, after ether washs in succession, dry in 60 ℃ vacuum drier, get about 100 grams of lentinan crude product.
Crude product 100g is soluble in water, and is injected in the 1500g DEAE-Mierocrystalline cellulose at first water Xian Di, uses NaOH 1mole/L eluant solution then, collects the elutriant HCl neutralization of the sugared reacting positive part of NaOH wash-out.Ultra-filtration on amicon, the fenestra of ultra-filtration is got not permeation parts through being 100,000, is concentrated into small volume, and lyophilize gets single component lentinan 2g, and total recovery is 0.02%, lentinan content 97.3%.
Embodiment 2:
Get qualified raw material dried thin mushroom 12Kg, after the pulverizing, place the reactor of strap clamp cover, add clear water, the add-on of water is about 1.8 times of raw material volume, jacket steam is heated to boiling, under boiling, kept 7 hours then, filter, residue is pressed said process again and is extracted 2 times, merges all filtrates, be evaporated to small volume, stirring the concentration that adds equal-volume down is 95% above ethanol, and supernatant liquor is toppled in static placement 5 hours, lower sediment is through the centrifugal precipitation that obtains, precipitation is after washing with alcohol, and is soluble in water, places dialysis tubing to water dialysis 3 days, dialyzate is evaporated to small volume, stir the ethanol that adds equal-volume down, static 4 hours, topple over supernatant liquor, lower sediment is through centrifugal, obtain precipitation, precipitation is again through ethanol, and ether washs in succession, get the about 126g of lentinan crude product 60 ℃ of following vacuum-dryings, crude product 126g is soluble in water, is added to (1500g) in the DEAE-Mierocrystalline cellulose, and waste liquid discards, the DEAE-Mierocrystalline cellulose that contains lentinan washes with water earlier, collect the alkali elutriant and do sugared reaction test with the dilute NaOH solution wash-out then, collect the part of sugared reacting positive, sugared reacting positive portion is done ultra-filtration.The resistance filter value molecular weight of filtering membrane is 100,000, gets and is not excluded part, is concentrated to the lentinan 2.64g that the small volume lyophilize gets single component, total recovery is 0.022%, product is through Congo red reaction, and ultraviolet determination is found to be maximum absorption band at 480～495nm place then.Survey ultraviolet after the neutralization again, its maximum absorption is moved to 505-520nm, and two scopes of this reaction uv-absorbing are peculiar by β (1 → 3) dextran.Lentinan belongs to β (1 → 3) dextran, so two obtained the maximum absorption also should be arranged.
Infrared spectra has 3500～3300cm
-1(the O-H stretching vibration that the sugar ring is gone up OH); 2920～2800cm
-1(the sugar ring is gone up the vibration of C-H angle); 1100～1000cm
-1(the sugar ring is gone up the C-O stretching vibration); 890cm
-1(the anomeric carbon characteristic absorbance of beta comfiguration) waits four charateristic avsorption bands,
13C NMR (Nuclear Magnetic Resonance) spectrum tool 104.66ppm (the C-1 signal of β-1,3 dextran); (88.04ppm C-3 signal); (77.92ppm C-5 signal); (75.19ppm C-2 signal); (70.13ppm C-4 signal) and; (62.17ppm C-6 signal).
Assay:
Lentinan peak and the impurity peaks that obtains with high performance liquid chromatography, be converted into concentration, can try to achieve the content of lentinan, or adopt anthrone colorimetry (contrast is the standard lentinan) to measure content by peak area.
Claims (3)
1. a separating and purifying lentinan method is characterized in that perfume (or spice) is eaten through hot water extraction and concentrates, add ethanol and produce precipitation, water-soluble Jie of throw out back dialysis, dialyzate concentrates, ethanol, ether wash crude product, the water-soluble again DEAE-cellulose adsorption of crude product, washing, Xian takes off with different concns alkali lye, and the elutriant fraction collection merges sugared reacting positive Xian and takes off liquid, in the hydrochloric acid and ultra-filtration, get that to see through liquid not concentrated freeze-dried.
2. the method according to claim 1 is characterized in that: used alkali is Na
2CO
3, KCO
3, NaHCO
3, KHCO
3, NaOH, KOH.
3. the method according to claim 1 is characterized in that: the concentration that Xian takes off alkali lye is 1～2mole/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95113378A CN1069650C (en) | 1995-12-29 | 1995-12-29 | Method for separation and purification of lentinan |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95113378A CN1069650C (en) | 1995-12-29 | 1995-12-29 | Method for separation and purification of lentinan |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1153179A true CN1153179A (en) | 1997-07-02 |
| CN1069650C CN1069650C (en) | 2001-08-15 |
Family
ID=5079989
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95113378A Expired - Lifetime CN1069650C (en) | 1995-12-29 | 1995-12-29 | Method for separation and purification of lentinan |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1069650C (en) |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001044263A1 (en) * | 1999-12-16 | 2001-06-21 | Beijing Zhong Ke Zhi Chuang Science & Technology Ltd. | Oligosaccharides, a process for preparation thereof and pharmaceutical combination containing the same compounds |
| CN1095471C (en) * | 1998-11-06 | 2002-12-04 | 陈哲超 | Method for processing lentinan protein |
| CN1100792C (en) * | 2000-05-18 | 2003-02-05 | 厦门中卫多糖研究所 | Longan polysaccharide extracting process |
| CN1100793C (en) * | 2000-09-25 | 2003-02-05 | 黄寿祺 | Loquat polysaccharide extracting process |
| CN1305908C (en) * | 2002-11-29 | 2007-03-21 | 湖北大学 | Extracting technology for mountain ophiopogon root polysaccharide |
| EP1774012A2 (en) * | 2004-07-16 | 2007-04-18 | Medimush A/S | Immune modulating compounds from fungi |
| CN100346796C (en) * | 2000-01-12 | 2007-11-07 | 有限会社生命科学研究所 | Phy siologically active substance EEM-S originating in mushrooms, process for producing same and drugs |
| CN100349922C (en) * | 2004-10-27 | 2007-11-21 | 山西亚宝药业集团股份有限公司 | Nanometer mushroom amylose and preparation method of its injection |
| CN1978467B (en) * | 2005-12-06 | 2011-07-13 | 成都三康药物研究所 | Method for separating and purifying lentinan |
| CN102190739A (en) * | 2010-03-12 | 2011-09-21 | 南京易亨制药有限公司 | Process for extracting lentinan and method for measuring molecular weight distribution of lentinan |
| CN101161112B (en) * | 2006-10-10 | 2011-11-23 | 上海慈瑞医药科技有限公司 | A method for separating and purifying lentinan |
| CN101261203B (en) * | 2007-03-09 | 2012-01-18 | 金文准 | Alkaline soluble lentinan extraction, separation, purification and molecular weight determination |
| CN107550945A (en) * | 2017-10-12 | 2018-01-09 | 北京市农林科学院 | Protect Pleurotus citrinopileatus extract and its application of wounded hepatocytes |
| CN114287630A (en) * | 2021-12-30 | 2022-04-08 | 湖北鑫祥顺生物工程有限公司 | Immunoregulation preparation and preparation method thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60243019A (en) * | 1984-05-17 | 1985-12-03 | Mitsubishi Chem Ind Ltd | Hepatic cell proliferation factor |
| JP2646361B2 (en) * | 1987-09-07 | 1997-08-27 | 株式会社ニチレイ | Method for producing acidic polysaccharide having antitumor activity |
| JP2630783B2 (en) * | 1987-09-07 | 1997-07-16 | 株式会社ニチレイ | Method for producing neutral polysaccharide having antitumor activity |
| JPH05345725A (en) * | 1991-06-05 | 1993-12-27 | Taku Mizuno | Water-soluble polysaccharide originated from mushroom, its production and antitumor agent containing the polysaccharide as main ingredient |
| CN1032428C (en) * | 1992-08-15 | 1996-07-31 | 樊菊芬 | Technology for extracting Xianggu mushroom saccharide and Xianggu mushroom fungus saccharide |
-
1995
- 1995-12-29 CN CN95113378A patent/CN1069650C/en not_active Expired - Lifetime
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1095471C (en) * | 1998-11-06 | 2002-12-04 | 陈哲超 | Method for processing lentinan protein |
| WO2001044263A1 (en) * | 1999-12-16 | 2001-06-21 | Beijing Zhong Ke Zhi Chuang Science & Technology Ltd. | Oligosaccharides, a process for preparation thereof and pharmaceutical combination containing the same compounds |
| CN100346796C (en) * | 2000-01-12 | 2007-11-07 | 有限会社生命科学研究所 | Phy siologically active substance EEM-S originating in mushrooms, process for producing same and drugs |
| CN1100792C (en) * | 2000-05-18 | 2003-02-05 | 厦门中卫多糖研究所 | Longan polysaccharide extracting process |
| CN1100793C (en) * | 2000-09-25 | 2003-02-05 | 黄寿祺 | Loquat polysaccharide extracting process |
| CN1305908C (en) * | 2002-11-29 | 2007-03-21 | 湖北大学 | Extracting technology for mountain ophiopogon root polysaccharide |
| EP1774012A2 (en) * | 2004-07-16 | 2007-04-18 | Medimush A/S | Immune modulating compounds from fungi |
| CN100349922C (en) * | 2004-10-27 | 2007-11-21 | 山西亚宝药业集团股份有限公司 | Nanometer mushroom amylose and preparation method of its injection |
| CN1978467B (en) * | 2005-12-06 | 2011-07-13 | 成都三康药物研究所 | Method for separating and purifying lentinan |
| CN101161112B (en) * | 2006-10-10 | 2011-11-23 | 上海慈瑞医药科技有限公司 | A method for separating and purifying lentinan |
| CN101261203B (en) * | 2007-03-09 | 2012-01-18 | 金文准 | Alkaline soluble lentinan extraction, separation, purification and molecular weight determination |
| CN102190739A (en) * | 2010-03-12 | 2011-09-21 | 南京易亨制药有限公司 | Process for extracting lentinan and method for measuring molecular weight distribution of lentinan |
| CN107550945A (en) * | 2017-10-12 | 2018-01-09 | 北京市农林科学院 | Protect Pleurotus citrinopileatus extract and its application of wounded hepatocytes |
| CN114287630A (en) * | 2021-12-30 | 2022-04-08 | 湖北鑫祥顺生物工程有限公司 | Immunoregulation preparation and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1069650C (en) | 2001-08-15 |
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Owner name: JINDING UNITED FIBER TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: SHANGHAI PHARMACEUTICAL INST., CHINESE ACADEMY OF SCIENCES Effective date: 20030425 |
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Effective date of registration: 20030425 Address after: 210009 No. 238 Central Road, Nanjing Patentee after: Technology Centre of Jinling Pharmaceutical Co., Ltd. Address before: 200031 No. 294, Taiyuan Road, Shanghai Patentee before: Shanghai Institute of Materia Medica, Chinese Academy of Sciences |
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Granted publication date: 20010815 |
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