CN1204148C - Separation and purification process of lentinan - Google Patents
Separation and purification process of lentinan Download PDFInfo
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- CN1204148C CN1204148C CN 02138962 CN02138962A CN1204148C CN 1204148 C CN1204148 C CN 1204148C CN 02138962 CN02138962 CN 02138962 CN 02138962 A CN02138962 A CN 02138962A CN 1204148 C CN1204148 C CN 1204148C
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- lentinan
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- 229920001491 Lentinan Polymers 0.000 title claims abstract description 65
- 229940115286 lentinan Drugs 0.000 title claims abstract description 65
- 238000000926 separation method Methods 0.000 title claims abstract description 14
- 238000000746 purification Methods 0.000 title abstract description 3
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 25
- 238000001914 filtration Methods 0.000 claims abstract description 16
- 239000000843 powder Substances 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims abstract description 6
- 230000004151 fermentation Effects 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 239000012528 membrane Substances 0.000 claims description 20
- 239000000284 extract Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 16
- 239000012535 impurity Substances 0.000 claims description 11
- 244000144992 flock Species 0.000 claims description 9
- 229920002521 macromolecule Polymers 0.000 claims description 7
- 239000012141 concentrate Substances 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- 238000003809 water extraction Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 10
- 238000002360 preparation method Methods 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 5
- 239000012043 crude product Substances 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract 1
- 238000012869 ethanol precipitation Methods 0.000 abstract 1
- 238000010255 intramuscular injection Methods 0.000 abstract 1
- 239000007927 intramuscular injection Substances 0.000 abstract 1
- 230000007721 medicinal effect Effects 0.000 abstract 1
- 238000011045 prefiltration Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 102000005606 Activins Human genes 0.000 description 1
- 108010059616 Activins Proteins 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000000488 activin Substances 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003245 working effect Effects 0.000 description 1
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- Separation Using Semi-Permeable Membranes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The present invention discloses the separation and purification technology of lentinan, which mainly solves the defect of insufficient performance of medical effect caused by low lentinan content, water insolubility and indefinite molecular weight of the existing lentinan. Lentinan crude products obtained by submerged fermentation are used as raw materials of the technology. The technology comprises the steps of extraction with hot water, prefiltration by a micrometer-scale filter, the fraction of ultrafiltration films with molecular weight trapped values as 50 thousand to 100 thousand daltons, the concentration of ultrafiltration films with molecular weight trapped values as 6 thousand to 50 thousand daltons, final filtration by a conventional filter, ethanol precipitation, desiccation, etc. As a result, lentinan powder whose average molecular weight is strictly controlled between 40 thousand to 90 thousand is obtained, and the average molecular weight is desired by clinical application; moreover, the lentinan powder has the advantages of stable quality, high content of effective component and water solubility. The obtained lentinan powder can be directly used as an oral preparation, or can be used as raw materials of a preparation for intramuscular injection.
Description
Technical field
The present invention relates to the extractive technique of lentinan, refer to particularly a kind of from the Lentinan crude product that submerged fermentation obtains the technology of separation and purification Lentinan.
Background technology
At present, Lentinan is widely used in the treatment and assisting therapy of viral hepatitis, immunologic hypofunction and malignant tumour etc. and be used as a kind of biological response modifier because it has the T lymphocytes in human body of enhancing activity, regulates characteristics such as human body humoral immune function, human activin anti-tumor factor.But existing be used for clinically the Lentinan preparation particularly oral preparations mostly exist following deficiency: the content of preparation Lentinan is too low, generally be in 15～28% scope, impurity composition wherein is too high, make its effective ingredient both impure also unstable, thereby influence result of treatment; Simultaneously, the specificity of Lentinan is too poor in the preparation, the not strict control of molecular weight, from 10,000 to 1,000,000 dalton do not wait, wherein the molecular weight of quite a few Lentinan needs average 4～90,000 daltonian molecular weight to differ greatly with clinical treatment on 100,000 dalton, and the too small then metabolism of its molecular weight is unfavorable for resident absorption in human body too soon, the excessive then water-soluble variation of its molecular weight also is not easy to be absorption of human body, and these all make the curative effect of its preventing and treating diseases have a greatly reduced quality.In order to address the above problem, Granted publication number discloses the novel technology for extracting of a kind of lentinan or Lentinan for the Chinese invention patent specification sheets of CN1032428C, this process using single-stage ultra-fine filter is held back, ethanol sedimentation extracts lentinan, though can improve the content of Lentinan, but its yield only is 1～1.5%, and the molecular weight of the lentinan particularly lentinan of macromolecule does not add control, invalid element wherein and impurity are all more, and its quality and curative effect are still undesirable.Publication number provides a kind of method of extracting lentinan for injection from the lentinan crude product that submerged fermentation obtains for the Chinese invention patent ublic specification of application of CN1140202A, the alcohol of this method employing different concns is analysed, the alcohol precipitation step makes water-soluble lentinan, though also can improve the content of lentinan, but this method equipment used volume is big, the solvent recuperation amount is big, the molecular weight of gained lentinan does not add control yet, can influence its quality and curative effect equally.
Summary of the invention
Purpose of the present invention is exactly the separation purifying technique that a kind of Lentinan will be provided, and making the Lentinan steady quality, effective component content height and the molecular weight that adopt this technology to make be strictly controlled at clinical treatment needs in the scope.
For achieving the above object, the separation purifying technique of Lentinan proposed by the invention may further comprise the steps:
1) learn from else's experience weight content that submerged fermentation obtains is 15～28% Lentinan dry powder, use hot-water extraction, standing demix then, and removal lower sediment thing obtains tawny upper strata extract.In this step, the most of water-fast impurity that content is on the low side, the unsteered Lentinan dry powder of molecular-weight average is interior is eliminated away.
2) be that 5～15 microns strainer filters in advance to gained upper strata extract with the filter membrane aperture, further remove water-fast impurity, obtain clarifying pre-flock extract.In this step, the water-fast impurity of minute quantity that remains in the extract of upper strata is thoroughly removed, and helps the purifying of Lentinan, can prevent that also these remaining impurity from producing infringement to the ultra-filtration membrane in the subsequent step, prolongs the work-ing life of ultra-filtration membrane.
3) be that 5～100,000 daltonian ultra-filtration membranes carry out fraction to gained pre-flock extract with the molecular weight cutoff value, remove trapped fluid, obtain the ultrafiltration level separatory of most Lentinan molecular weight below 100,000 dalton.In this step, in the pre-flock extract most of molecular weight more than 100,000 dalton, Lentinan and other impurity of poorly water-soluble has been trapped.
4) be that 0.6～50,000 daltonian ultra-filtration membrane concentrates gained ultrafiltration level separatory with the molecular weight cutoff value, remove ultrafiltrated, obtain most Lentinan molecular weight and between 0.6～100,000 dalton, hold back concentrated solution.In this step, most of molecular weight too short Lentinan of residence time below 0.6 ten thousand dalton, in human body also has been filtered in the ultrafiltration level separatory.
5) gained is held back concentrated solution and leave standstill for some time, do last filtration with the conventional filtration device again, remove the only a few macromolecule Lentinan dregs that remains in wherein, obtain transparent concentrated solution.Because the rejection of general ultra-filtration membrane is between 85～90%, through top ultrafiltration fraction and spissated hold back in the concentrated solution still can remaining a small amount of macromolecule dregs, if do not remove, can make the molecular-weight average of the finished product higher, water-soluble variation, the effect of disease preventing and treating reduces.And in this step, the macromolecule impurity of holding back in the concentrated solution can thoroughly be removed clean.
6) be that 20～50% ethanol carries out alcohol and analyses with the transparent concentrated solution concentration of gained, the throw out that alcohol is analysed is that 80～99.5% ethanol washs with concentration again, at last through being drying to obtain the purifying lentinan powder that molecular-weight average is controlled between 4～90,000 dalton, meets clinical needs fully.The weight content of its Lentinan is between 50～80% after measured, and recovery rate is 5～8%, and is soluble in water as clear as crystal.
The invention has the advantages that: after adopting fraction and concentrated two-stage special film ultrafiltration membrane treatment technology directly that raw material Lentinan dry powder water extract is handled, the molecular-weight average of gained purifying lentinan can stably be controlled in the scope that clinical treatment needs, before and after this two-stage ultrafiltration membrane treatment technology, adopt filter in advance and last filtration treatment technology then wherein the lentinan dregs of water-fast impurity and macromolecule thoroughly remove totally.Thereby make gained finished product purity height, quality good, soluble in water, detect and standard substance seminose, the apparent same color spot of glucose through tlc, measuring its molecular-weight average by two appendix VII of Chinese Pharmacopoeia version in 2000 is 4～90,000 dalton, and this molecular weight of clinical needs just.The present invention simultaneously also has characteristics such as separation purifying technique is brief, organic solvent is single, nuisanceless pollution, recovery rate height, can directly be used as oral preparations, also can be used as the raw material of intramuscular injectable formulations.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment:
The separation purifying technique of Lentinan of the present invention may further comprise the steps:
1) learn from else's experience weight content that submerged fermentation obtains is 15～28% Lentinan dry powder, mix with distilled water, the weight ratio of Lentinan dry powder and distilled water is 1: 20～100, weight ratio is 1: 30～50 preferably, the temperature of distilled water is controlled at 60～70 ℃, under whipped state, extracted 4～5 hours, Lentinan fully is dissolved in the water, improve the extraction efficiency of raw material.Stop to stir mixed solution is placed room temperature earlier, move into standing demix under 1～10 ℃ the temperature condition then, can remove the lower sediment thing fast like this, and obtain tawny upper strata extract.
2) be that 5～15 microns strainer filters in advance to gained upper strata extract with the filter membrane aperture.This strainer can be selected cell filter for use, and the filter membrane aperture is got 10 microns for good, and filtration area is 0.1 square metre, and permeable amount is per hour 20 liters.Can further remove water-fast impurity like this, obtain clarifying pre-flock extract.
3) be that 5～100,000 daltonian hyperfiltration membrane assemblies carry out fraction to gained pre-flock extract with the molecular weight cutoff value.Fraction is 0.1 square metre with the filtration area of hyperfiltration membrane assembly, and permeable amount is per hour 10 liters.The temperature of pre-flock extract is controlled at 10～45 ℃, and pressure-controlling is at 0.1～0.2Mpa.Remove trapped fluid, just can obtain the ultrafiltration level separatory of most Lentinan molecular weight below 100,000 dalton.
4) be that 0.6～50,000 daltonian hyperfiltration membrane assembly concentrates gained ultrafiltration level separatory with the molecular weight cutoff value.The filtration area that concentrates with hyperfiltration membrane assembly is 0.1 square metre, and permeable amount is per hour 10 liters.The temperature of ultrafiltration level separatory is controlled at 10～45 ℃, and pressure-controlling is at 0.1～0.2Mpa.Remove ultrafiltrated, just can obtain most Lentinan molecular weight and between 0.6～100,000 dalton, hold back concentrated solution.Keep above-mentioned suitable ultrafiltration fraction and ultrafiltration and concentration parameter, can stablize filtering velocity, guarantee to be met the best Lentinan that requires of molecular-weight average.
5) gained being held back concentrated solution is to leave standstill for some time under 1～10 ℃ the condition in temperature, does last filtration with conventional middling speed filter paper again, can thoroughly remove the only a few macromolecule Lentinan dregs that remains in wherein, obtains transparent concentrated solution.
6) under whipped state, be that 40% ethanol carries out alcohol and analyses with the transparent concentrated solution of gained with concentration, it is fully precipitated, filtration alcohol is analysed throw out and is drained, be that 99% ethanol carries out washing by soaking with concentration then, row filters and drains again, at last through 30～50 ℃ of dryings, promptly get molecular-weight average and be controlled at Lentinan powder finished product between 4～90,000 dalton.
Claims (6)
1. the separation purifying technique of a Lentinan may further comprise the steps:
1) learn from else's experience weight content that submerged fermentation obtains is 15～28% Lentinan dry powder, use hot-water extraction, standing demix then, and removal lower sediment thing obtains tawny upper strata extract;
2) be that 5～15 microns strainer filters in advance to gained upper strata extract with the filter membrane aperture, further remove water-fast impurity, obtain clarifying pre-flock extract;
3) be that 5～100,000 daltonian ultra-filtration membranes carry out fraction to gained pre-flock extract with the molecular weight cutoff value, remove trapped fluid, obtain the ultrafiltration level separatory of most Lentinan molecular weight below 100,000 dalton;
4) be that 0.6～50,000 daltonian ultra-filtration membrane concentrates gained ultrafiltration level separatory with the molecular weight cutoff value, remove ultrafiltrated, obtain most Lentinan molecular weight and between 0.6～100,000 dalton, hold back concentrated solution;
5) gained is held back concentrated solution and leave standstill for some time, do last filtration with the conventional filtration device again, remove the only a few macromolecule Lentinan dregs that remains in wherein, obtain transparent concentrated solution;
6) be that 20～50% ethanol carries out alcohol and analyses with the transparent concentrated solution concentration of gained, the throw out that alcohol is analysed is that 80～99.5% ethanol washs with concentration again, is controlled at Lentinan powder between 4～90,000 dalton through being drying to obtain molecular-weight average at last.
2. the separation purifying technique of Lentinan according to claim 1, it is characterized in that: the weight ratio of Lentinan dry powder and water is 1: 20～100 in the said step 1), water temperature is controlled at 60～70 ℃, extracts under whipped state 4～5 hours, at 1～10 ℃ of standing demix.
3. the separation purifying technique of Lentinan according to claim 1 is characterized in that: selecting the filter membrane aperture said step 2) for use is 10 microns cell filter, and filtration area is 0.1 square metre, and permeable amount is per hour 20 liters.
4. the separation purifying technique of Lentinan according to claim 1, it is characterized in that: fraction is 0.1 square metre with the filtration area of ultra-filtration membrane in the said step 3), permeable amount is per hour 10 liters; The temperature of pre-flock extract is 10～45 ℃, and pressure is 0.1～0.2Mpa.
5. the separation purifying technique of Lentinan according to claim 1 is characterized in that: the filtration area that concentrates with ultra-filtration membrane in the said step 4) is 0.1 square metre, and permeable amount is per hour 10 liters; The temperature of ultrafiltration level separatory is 10～45 ℃, and pressure is 0.1～0.2Mpa.
6. the separation purifying technique of Lentinan according to claim 1 is characterized in that: will hold back concentrated solution in the said step 5) and in temperature be under 1～10 ℃ the condition and leave standstill for some time, and use the middling speed filter paper filtering again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02138962 CN1204148C (en) | 2002-08-22 | 2002-08-22 | Separation and purification process of lentinan |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02138962 CN1204148C (en) | 2002-08-22 | 2002-08-22 | Separation and purification process of lentinan |
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| Publication Number | Publication Date |
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| CN1477130A CN1477130A (en) | 2004-02-25 |
| CN1204148C true CN1204148C (en) | 2005-06-01 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 02138962 Expired - Fee Related CN1204148C (en) | 2002-08-22 | 2002-08-22 | Separation and purification process of lentinan |
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Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NO20014256D0 (en) | 2001-09-03 | 2001-09-03 | Bjoern Kristiansen | Preparation of immunostimulatory compound |
| US7514085B2 (en) | 2004-07-16 | 2009-04-07 | Medimush A/S | Immune modulating compounds from fungi |
| EP1896600A2 (en) | 2005-06-15 | 2008-03-12 | Medimush A/S | Anti-cancer combination treatment and kit-of-part |
| CN100453559C (en) * | 2005-11-04 | 2009-01-21 | 杨勇杰 | A kind of method of extracting 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 |
| CN101265303B (en) * | 2008-04-24 | 2010-06-02 | 吉林大学 | A method for extracting lentinan from waste mushroom culture medium |
| CN102086234B (en) * | 2009-12-07 | 2012-10-03 | 北大国际医院集团重庆大新药业股份有限公司 | Preparation method for improving quality of medium molecular weight hydroxyethyl starch |
| CN101775422B (en) * | 2010-02-05 | 2012-04-04 | 武汉迪奥药业有限公司 | A kind of mushroom polysaccharide and preparation method thereof |
| CN101921346B (en) * | 2010-08-31 | 2011-12-21 | 浙江省林业科学研究院 | Radial flow chromatography for polysaccharides of mushroom hyphae |
| CN101991091B (en) * | 2010-09-30 | 2012-10-17 | 福建省宏顺食品饮料有限公司 | Process for producing mushroom concentrated juice |
| CN102863547A (en) * | 2012-09-25 | 2013-01-09 | 福建惠泽生物科技有限公司 | Method for extracting active polysaccharides from pleurotus eryngii and preparing functional beverage of active polysaccharides |
| CN104119456B (en) * | 2014-07-04 | 2017-03-22 | 中国科学院过程工程研究所 | Method for separation and purification of edible (medicinal) mushroom active components by multistage membrane process |
| CN105861590A (en) * | 2016-06-23 | 2016-08-17 | 洛阳伊龙药业有限公司 | Production technology of lentinus edodes mycelia polysaccharide bulk drug |
| WO2019215073A1 (en) * | 2018-05-07 | 2019-11-14 | Jennewein Biotechnologie Gmbh | A SIMPLE METHOD FOR THE PURIFICATION OF LACTO-N-NEOTETRAOSE (LNnT) FROM CARBOHYDRATES OBTAINED BY MICROBIAL FERMENTATION |
| CN110483653B (en) * | 2019-07-16 | 2022-03-18 | 南昌大学 | A kind of preparation method, product and application of lentinan component with immune activity |
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2002
- 2002-08-22 CN CN 02138962 patent/CN1204148C/en not_active Expired - Fee Related
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