CN101659706B - Method for purifying and separating X-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus - Google Patents
Method for purifying and separating X-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus Download PDFInfo
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- CN101659706B CN101659706B CN2008101203173A CN200810120317A CN101659706B CN 101659706 B CN101659706 B CN 101659706B CN 2008101203173 A CN2008101203173 A CN 2008101203173A CN 200810120317 A CN200810120317 A CN 200810120317A CN 101659706 B CN101659706 B CN 101659706B
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 41
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 41
- 150000004676 glycans Chemical class 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 22
- 241000222640 Polyporus Species 0.000 title abstract description 7
- 235000013399 edible fruits Nutrition 0.000 title abstract 3
- 150000001875 compounds Chemical class 0.000 claims abstract description 4
- 238000000746 purification Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims abstract 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 112
- 240000001080 Grifola frondosa Species 0.000 claims description 33
- 235000007710 Grifola frondosa Nutrition 0.000 claims description 33
- 230000008021 deposition Effects 0.000 claims description 29
- 239000006228 supernatant Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 6
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 4
- 238000012869 ethanol precipitation Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 239000008280 blood Substances 0.000 abstract description 2
- 210000004369 blood Anatomy 0.000 abstract description 2
- 238000003809 water extraction Methods 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 47
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 238000000605 extraction Methods 0.000 description 7
- 238000004062 sedimentation Methods 0.000 description 5
- 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 4
- 239000008103 glucose Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000009987 spinning Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000222684 Grifola Species 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 240000005995 Laetiporus sulphureus Species 0.000 description 1
- 235000007714 Laetiporus sulphureus Nutrition 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 241000222341 Polyporaceae Species 0.000 description 1
- 241000222383 Polyporales Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 206010047601 Vitamin B1 deficiency Diseases 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 208000002894 beriberi Diseases 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 206010013990 dysuria Diseases 0.000 description 1
- AZHSSKPUVBVXLK-UHFFFAOYSA-N ethane-1,1-diol Chemical compound CC(O)O AZHSSKPUVBVXLK-UHFFFAOYSA-N 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000005554 pickling Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 201000002282 venous insufficiency Diseases 0.000 description 1
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- Polysaccharides And Polysaccharide Derivatives (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a method for purifying and separating X-fraction polysaccharide from the fruit bodies or mycelia of polyporus frondosus, which is characterized in that: crude polysaccharide extracted from the fruit bodies or the mycelia of the polyporus frondosus is subjected to complex reaction and primary purification, and is deproteinized by a sevag method, and then X-fraction pure polysaccharide of the polyporus frondosus is prepared by a graded ethanol precipitation method. The polysaccharide has good effect of reducing blood fat. The crude polysaccharide is obtained by a water extraction and ethanol precipitation method, and is separated and purified according to different natures of a complex compound and the X-fraction polysaccharide to ethanol precipitation concentration. The method has convenient operation, obtains the target product with high purity, and provides a feasible way for producing the X-fraction polysaccharide of the polyporus frondosus on a large scale, and preparing various medicaments and health-care foods.
Description
Technical field
The present invention relates to the extraction and separation method of edible mushrooms effective constituent, be specifically related to the extraction and separation method of a kind of Grifola Frondosa sporophore or mycelium polysaccharides component.
Background technology
Grifola frondosa (Polyporus frondosus) is under the jurisdiction of Mycophyta, Basidiomycotina, Aphyllophorales, polyporaceae, Polyporus.Grifola frondosa is famous edible, medicinal fungi, and is nutritious, tasty, is the macro fungi of " but edible, can mend medicine, the whole body is precious ".Grifola frondosa property is flat, flavor is sweet, can be used for treating diseases such as dysuria, oedema, beriberi, hepatic ascites, mellitus, hypertension and obesity.
Known mycelium or sporophore from the Grifola frondosa Pseudomonas extract by having β-1; The 3-key connects the β-1 of branched glucose; The 6-key connects that the glucose main chain constitutes or by having β-1, the 6-key connects the β-1 of branched glucose, and the polysaccharide that the 3-key connects glucose main chain formation has antitumour activity.
It also is known comprising the following steps to prepare cancer-resisting substance, and said step is: with hot water to Grifola frondosa, tree flower bacterium department resemble ear orchid (Grifola gigantea) (Tonbimai) or Laetiporus sulphureus (Masutake) extract; The concentrating under reduced pressure extracting solution precipitates liquid concentrator with organic solvent, throw out is dialysed removing low-molecular-weight material, and with lipophilic organic solvent extraction impurity from dialyzate, to remove them.
Yet; Those methods of more than describing are for a large amount of medicament of preparation and to come to prepare effectively for the heath food from limited resources be to be not suitable for; Because the purification step of these methods is quite complicated, and contains the material that suppresses immune-enhancing activity in the product of gained.
Summary of the invention
Technical problem to be solved by this invention is to utilize Grifola frondosa X fraction polysaccharide to ethanol alcohol precipitation concentration different properties, makes Grifola Frondosa sporophore or mycelium X component obtain the fractional separation purifying, and a kind of method of effective extraction X fraction polysaccharide is provided.
The present invention realizes that the technical scheme of Grifola Frondosa sporophore or mycelium X fraction polysaccharide fractional separation purifying is:
With raw material pulverizing, hot water extraction, ethanol sedimentation adds the complex compound complexing, pickling, alkali redissolution sevag method deproteinated; Ethanol sedimentation, alkali redissolves, and ethanol precipitates once more, and is centrifugal, gets supernatant; Continue to add the alcohol deposition, collect current pure hypostasis, alcohol is washed, and lyophilize obtains grifolan.
Separating and purifying technology of the present invention is achieved through following steps:
A. the extraction of Crude polysaccharides
With Grifola Frondosa sporophore or mycelium is raw material, is ground into fine powder, adds 10-40 times water; Hot water extracted 1-6 hour for 90-120 ℃, and centrifugal, being evaporated to the liquid concentrator specific density is 1.02-1.15; Slowly ethanol to the final concentration of adding 95% is 30%--80%, stirs 4 ℃ of hold over night; Filter, be deposited in preserve in the ethanol subsequent use.
The classification alcohol precipitation purifying of B.X fraction polysaccharide
(1) the gained Crude polysaccharides is extracted ethanol among the A, redissolves with 5-30 zero(ppm) water doubly;
(2) CTAOH of adding 20%--100% regulates PH >=10,4 ℃ maintenance 24-72h to no longer producing deposition, spinning, and abandoning supernatant must precipitate;
(3) acetum of getting above-mentioned (2) gained deposition adding 1-6 1-60% doubly washs 1-5 time, and is centrifugal, and the washing with alcohol 1-5 of 50%--95% time, centrifugal, the gained deposition is redissolved with the NaOH solution of 0.1-1M, and centrifugal, supernatant is with sevag method deproteinated;
(4) adding of the solution behind the deproteinated in (3) ethanol being made alcohol concn is 50%～70%, leaves standstill, centrifugal; Get deposition and redissolve the back and add ethanol to make alcohol concn be 30%～40%, leave standstill, centrifugal; Get supernatant and add ethanol to make alcohol concn be 70%～90%, leave standstill, centrifugal, the collecting precipitation thing, ethanol with 95% and absolute ethanol washing, lyophilize promptly gets Grifola frondosa X fraction polysaccharide.
Above-mentioned cryodesiccated program is :-47 ℃ of 2h, and-30 ℃ of 4h ,-5 ℃ of 24h, 27 ℃ of 4h, vacuum tightness remains on 0.08MPa～0.09Mpa.
The fractionation method of Grifola Frondosa sporophore or mycelium X fraction polysaccharide is characterized in that wherein said:
1. the polysaccharide of X component be can with the protein-polysaccharide of CTA-group set, aggregation is water insoluble, wherein sugared content is about 80%, protein contnt 6%--10%, molecular weight are 25～450,000 dalton and have good reducing blood lipid.
2. adopt the Different concentrations of alcohol deposition can obtain the different various polysaccharide of molecular weight behind the Grifola frondosa Crude polysaccharides deproteinated; The alcohol precipitating method of Grifola frondosa X component and alcohol concn are: adding ethanol behind the Grifola frondosa Crude polysaccharides deproteinated, to make alcohol concn be 50%～70%; Leave standstill, centrifugal; Getting deposition redissolution back alcohol precipitation alcohol concn is 30%～40%, leaves standstill, centrifugal; Get supernatant and add ethanol to make alcohol concn be 70%～90%, leave standstill, centrifugal; Promptly get grifolan X component (molecular weight is 25～450,000 dalton)
The present invention utilizes water to carry ethanol precipitation and obtains Crude polysaccharides, utilizes complex compound and X fraction polysaccharide to make it obtain separation and purification to PH condition different properties again.The present invention is easy to operate, and gained title product purity is high, is the scale operation of Grifola frondosa X fraction polysaccharide, and preparing various medicines and protective foods provides feasible approach.
Embodiment
The extraction of embodiment 1 Grifola frondosa X component (the about 28W dalton of molecular weight)
Take by weighing Grifola Frondosa sporophore fine powder 15Kg, add 500L water, 100 ℃ are extracted 1h, the centrifugal 20min of 8000rpm; Supernatant concentration to density is 1.04, adds 95% ethanol of 3 times of amounts, and 4 ℃ of hold over night are filtered; The gained deposition adds 30 times hot water redissolution, and is centrifugal, and the CTAOH of adding 45% is to no longer producing deposition, adjusting PH=11; 4 ℃ keep 48h, spinning, and abandoning supernatant must precipitate; Deposition is with 20% the acetic acid washing of 2 times of volumes 2 times, and 90% washing with alcohol 2 times is with 0.5 NaOH solution redissolution, centrifugal again, and supernatant is with sevag method deproteinated 3 times; It is 65% that supernatant adding ethanol makes alcohol concn, deposition, and deposition is redissolved with the NaOH solution of 0.2M, and it is 38% that the adding ethanol sedimentation makes alcohol concn; Deposition, centrifugal, get supernatant, continuing to add ethanol to final concentration is 90%; Deposition is washed respectively with absolute ethyl alcohol with 95%, and lyophilize obtains 2.5g Grifola frondosa X fraction polysaccharide; Polysaccharide content is 80%, and protein content is 7.67%, and molecular weight is 28W dalton.
The extraction of embodiment 2 Grifola frondosa X components (the about 35W dalton of molecular weight)
Take by weighing Grifola frondosa according to limbs fine powder 15Kg, add 500L water, 100 ℃ are extracted 1h, the centrifugal 20min of 8000rpm; Supernatant concentration to density is 1.04, adds 95% ethanol of 3 times of amounts, and 4 ℃ of hold over night are filtered; The gained deposition adds 30 times hot water redissolution, and is centrifugal, and the CTAOH of adding 60% is to no longer producing deposition, adjusting PH=12; 4 ℃ keep 48h, spinning, and abandoning supernatant must precipitate; Deposition is with 20% the acetic acid washing of 2 times of volumes 2 times, and 90% washing with alcohol 2 times is with 0.5 NaOH solution redissolution, centrifugal again, and supernatant is with sevag method deproteinated 3 times; It is 60% that supernatant adding ethanol makes alcohol concn, deposition, and deposition is redissolved with the NaOH solution of 0.2M, and it is 34% that the adding ethanol sedimentation makes alcohol concn; Deposition, centrifugal, get supernatant, continuing to add ethanol to final concentration is 80%; Deposition is washed respectively with absolute ethyl alcohol with 95%, and lyophilize obtains 2.5g Grifola frondosa X fraction polysaccharide; Polysaccharide content is 82%, and protein content is 7.02%, and molecular weight is 35W dalton.
The extraction of embodiment 3 Grifola frondosa X components (the about 43W dalton of molecular weight)
Take by weighing Grifola Frondosa sporophore fine powder 15Kg, add 500L water, 120 ℃ are extracted 1h, the centrifugal 20min of 8000rpm; Supernatant concentration to density is 1.04, adds 95% ethanol of 3 times of amounts, and 4 ℃ of hold over night are filtered; The gained deposition adds 30 times hot water redissolution, and is centrifugal, and the CTAOH of adding 80% is to no longer producing deposition, adjusting PH=11.5; 4 ℃ keep 48h, spinning, and abandoning supernatant must precipitate; Deposition is with 20% the acetic acid washing of 2 times of volumes 2 times, and 90% washing with alcohol 2 times is with 0.5 NaOH solution redissolution, centrifugal again, and supernatant is with sevag method deproteinated 3 times; It is 55% that supernatant adding ethanol makes alcohol concn, deposition, and deposition is redissolved with the NaOH solution of 0.2M, and it is 32% that the adding ethanol sedimentation makes alcohol concn; Deposition, centrifugal, get supernatant, continuing to add ethanol to final concentration is 73%; Deposition is washed respectively with absolute ethyl alcohol with 95%, and lyophilize obtains 2.5g Grifola frondosa X fraction polysaccharide; Polysaccharide content is 85%, and protein content is 6.91%, and molecular weight is 43W dalton.
Claims (2)
1. the method for purifying and separating of Grifola Frondosa sporophore or mycelium X fraction polysaccharide is characterized in that this method is Grifola Frondosa sporophore or mycelium to be extracted the Crude polysaccharides that obtains carry out the complex reaction preliminary purification and make Grifola frondosa X component holosaccharide with the method for employing classification alcohol precipitation behind the sevag method deproteinated; The separation of wherein said X fraction polysaccharide is purified and is comprised the following steps:
1. with the Grifola frondosa Crude polysaccharides, add CTA-OH and regulate PH >=10 and make grifolan X component form complex compound sediment;
2. adopt the classification alcohol precipitation to carry out purifying throw out dissolving back: adding ethanol behind resolution of precipitate that 1. makes and the deproteinated, to make alcohol concn be 50%～70%, leaves standstill, centrifugal; Get deposition and redissolve the back and add ethanol to make alcohol concn be 30%～40%, leave standstill, centrifugal; Get supernatant and add ethanol to make alcohol concn be 70%～90%, leave standstill, centrifugal, the collecting precipitation thing;
3. throw out in getting 2. with 95% ethanol washing and precipitating, redissolves, lyophilize, i.e. and lyophilize program is :-47 ℃ of 2h ,-30 ℃ of 4h ,-5 ℃ of 24h, 27 ℃ of 4h, vacuum tightness remains on 0.08MPa～0.09MPa, obtains Grifola frondosa X component holosaccharide.
2. the method for purifying and separating of Grifola Frondosa sporophore as claimed in claim 1 or mycelium X fraction polysaccharide is characterized in that wherein said:
Grifola frondosa X component holosaccharide be can with the protein-polysaccharide of CTA-group set, this polysaccharide is water insoluble with the protein-polysaccharide that the CTA-group is gathered, wherein sugared content is 80%, protein contnt 6%--10%, molecular weight are 25～450,000 dalton.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008101203173A CN101659706B (en) | 2008-08-25 | 2008-08-25 | Method for purifying and separating X-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus |
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| CN2008101203173A CN101659706B (en) | 2008-08-25 | 2008-08-25 | Method for purifying and separating X-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus |
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| CN101659706A CN101659706A (en) | 2010-03-03 |
| CN101659706B true CN101659706B (en) | 2012-06-06 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102408494B (en) * | 2011-11-30 | 2013-11-20 | 杭州众芝康菇生物技术有限公司 | Grifola frondosa polysaccharide ZZK component and preparation method thereof |
| CN102757511B (en) * | 2012-06-17 | 2016-12-21 | 上海市农业科学院 | A kind of Grifola frondosa galactan and preparation method thereof |
| CN103833866B (en) * | 2013-12-23 | 2015-10-28 | 浙江老艺人生物科技有限公司 | A kind of method extracting grifolan based on ultrasonic wave-vacuum impregnation assist efficient |
| CN113651900B (en) * | 2021-10-10 | 2022-11-15 | 吉林农业大学 | Grifola frondosa purified polysaccharide with blood lipid regulating function and preparation method thereof |
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Effective date of registration: 20121017 Address after: 323800 Zhejiang city of Lishui province Qingyuan County Star Road Garden Bridge Patentee after: Zhejiang Fangge Pharmaceutical Co., Ltd. Address before: 323800 Zhejiang province Qingyuan County Star Road Garden Bridge No. 98 Patentee before: Xu Caiquan |