CN102757511A - Grifola frondosa galactan and preparation method thereof - Google Patents
Grifola frondosa galactan and preparation method thereof Download PDFInfo
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- CN102757511A CN102757511A CN2012102019516A CN201210201951A CN102757511A CN 102757511 A CN102757511 A CN 102757511A CN 2012102019516 A CN2012102019516 A CN 2012102019516A CN 201210201951 A CN201210201951 A CN 201210201951A CN 102757511 A CN102757511 A CN 102757511A
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- grifola frondosa
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Abstract
The invention provides grifola frondosa galactan and a preparation method thereof. The grifola frondosa galactan is obtained by the steps of hot water leaching, ultrafiltration, alcohol precipitation, ion column chromatography, re-ultrafiltration and freeze-drying. The grifola frondosa galactan has high purity up to more than 90%, contains more than 90% of components with a molecular weight of more than 5.9 million Daltons, and serves the effect of inhibiting the tumor growth.
Description
Technical field
The present invention relates to the edible medicinal fungus field, relate in particular to a kind of Grifola frondosa Polygalactan and preparation method thereof.
Background technology
Grifola frondosa is as a kind of edible medicinal fungus, and its bioactive ingredients mainly is a polysaccharose substance.At present, its separate study achievement is shown that the activeconstituents of Grifola frondosa mainly is 3 VISOSEs, albumen VISOSE, mixed polysaccharide etc. both at home and abroad, molecular weight has immunocompetence and suppresses tumor promotion within 2,000,000 dalton.But not seeing both at home and abroad has the research report that separates the Polygalactan that obtains biologically active.
Summary of the invention:
The present invention at first provides a kind of Grifola frondosa Polygalactan; Its preparation principle is: use earlier the hot water extraction Grifola Frondosa sporophore; Thereafter integrated use HMW (greater than 100KD) membrane ultrafiltration, lower concentration alcohol precipitation (being lower than 30%), ion-exchange chromatography divide dried up extract; Purifying obtains the Grifola frondosa Polygalactan solution of HMW (greater than 5900KD), obtains the Grifola frondosa Polygalactan through concentrated, drying (lyophilize).
Specifically, a kind of Grifola frondosa Polygalactan of the present invention prepares through following method:
1. the water logging of 10～50 times of volumes there is not Grifola Frondosa sporophore, 80～100 ℃ of lixiviates 1～5 hour;
2. vat liquor carries out ultrafiltration with 10～1,000,000 daltonian hollow fiber ultrafiltration membrane after centrifugal, collects the part that is trapped, i.e. trapped fluid;
3. trapped fluid adds 10～50% aqueous ethanolic solution, collecting precipitation then;
4. precipitate behind dissolved in distilled water, the sodium chloride aqueous solution with～50% carries out ion exchange chromatography;
5. first salt peak part of ion exchange chromatography is carried out ultrafiltration with 10～1,000,000 daltonian hollow fiber ultrafiltration membrane, collects the solution that is trapped;
6. above-mentioned solution concentration is dry, promptly obtain the Grifola frondosa Polygalactan.
Preferred Grifola frondosa Polygalactan of the present invention prepares through following method:
1. the water logging of 10～30 times of volumes there is not Grifola Frondosa sporophore, 90～100 ℃ of lixiviates 1～3 hour;
2. soak body fluid and after centrifugal, carry out ultrafiltration, collect the part that is trapped, i.e. trapped fluid with 50～1,000,000 daltonian hollow fiber ultrafiltration membrane;
3. trapped fluid adds 10～30% aqueous ethanolic solution, collecting precipitation then;
4. precipitate behind dissolved in distilled water, the sodium chloride aqueous solution with～30% carries out ion exchange chromatography;
5. first salt peak part of ion exchange chromatography is carried out ultrafiltration with 50～1,000,000 daltonian hollow fiber ultrafiltration membrane, collects the solution that is trapped;
6. above-mentioned solution concentration is dry, promptly obtain the Grifola frondosa Polygalactan.
The present invention also provides the method for preparing above-mentioned Grifola frondosa Polygalactan, and this method comprises the steps:
2. 80～100 ℃ of floodings;
2. vat liquor carries out ultrafiltration with 10～1,000,000 daltonian hollow fiber ultrafiltration membrane;
3. ultrafiltrated is with 10～50% aqueous ethanolic solution deposition;
4. after precipitating water dissolution, carry out ion exchange chromatography;
5. first salt peak part of ion exchange chromatography is carried out ultrafiltration with 10～1,000,000 daltonian hollow fiber ultrafiltration membrane;
6. the ultrafiltrated concentrate drying promptly gets the Grifola frondosa Polygalactan.
The present invention also provides the application of above-mentioned Grifola frondosa Polygalactan in the medicine of preparation prevention or treatment tumour.Above-mentioned Grifola frondosa Polygalactan sea can be used for prevention or treatment tumour.
The invention has the advantages that:
1. combination utilization water carry, ultrafiltration, alcohol precipitation and ion column chromatography, separate the Polygalactan of acquisition HMW (molecular weight is greater than 5,900,000 dalton, high effective liquid chromatography for measuring) first;
3. step is easy to operate, weak point consuming time, purity high (Polygalactan content more than 90%, high effective liquid chromatography for measuring);
4. the Polygalactan that obtains has the effect that improves the organism immunocompetence, suppresses tumour, can be used for preparing antitumor, improve immunocompetent preparation.
Embodiment
Material source:
Grifola Frondosa sporophore: purchase Qingyuan's grid pharmaceutcal corporation, Ltd in Zhejiang
Ultra-filtration membrane: hollow-fibre membrane (molecular weight that dams be respectively 10,000,100,000,500,000,750,000,1,000,000 etc.), available from Shanghai Fu Lite Industrial Co., Ltd.
The pillar of ion column chromatography: (26 * 100cm), filler is Sephacryl S-200, S300, S400HR series (available from Amersham company) to gel column
Embodiment one:
Adopt 10 times water logging not have Grifola Frondosa sporophore 1kg, under 80 ℃ of conditions, be incubated 2 hours, repeat 4 times, extracting solution ultra-filtration membrane with 100,000 after 5000rpm/min is centrifugal carries out ultrafiltration, collects the part that is trapped; With 10% ethanol sedimentation trapped fluid; Throw out with dissolved in distilled water after, through ion column chromatography (moving phase is 50% sodium-chlor), collect first main peak part of salt face; The ultra-filtration membrane ultrafiltration desalination of warp 1,000,000 again, concentrated; Last lyophilize promptly obtains about Grifola frondosa Polygalactan 10g, measure through high-efficient liquid phase technique, wherein molecular weight at the Grifola frondosa Polygalactan more than 5,900,000 more than 90%.
Embodiment two:
Adopt 20 times water logging not have Grifola Frondosa sporophore 1kg, under 90 ℃ of conditions, be incubated 1 hour, repeat 3 times, extracting solution ultra-filtration membrane with 500,000 after 5000rpm/min is centrifugal carries out ultrafiltration, collects the part that is trapped; With 20% ethanol sedimentation trapped fluid; Throw out with dissolved in distilled water after; Through ion column chromatography (moving phase is 30% sodium-chlor), collect first main peak part of salt face, the ultra-filtration membrane ultrafiltration desalination of warp 750,000 again, concentrated; Last lyophilize promptly obtains about Grifola frondosa Polygalactan 15g, wherein molecular weight at the Grifola frondosa Polygalactan more than 5,900,000 more than 90%.
Embodiment three:
Adopt 40 times water logging not have Grifola Frondosa sporophore, under 100 ℃ of conditions, be incubated 1 hour, repeat 2 times, extracting solution ultra-filtration membrane with 750,000 after 5000rpm/min is centrifugal carries out ultrafiltration, collects the part that is trapped; With 30% ethanol sedimentation trapped fluid; Throw out with dissolved in distilled water after; Through ion column chromatography (moving phase is 10% sodium-chlor), collect first main peak part of salt face, the ultra-filtration membrane ultrafiltration desalination of warp 500,000 again, concentrated; Last lyophilize promptly obtains about Grifola frondosa Polygalactan 15g, wherein molecular weight at the Grifola frondosa Polygalactan more than 5,900,000 more than 90%.
Embodiment 4 presses down the experiment of knurl active animal:
1 given the test agent
The Grifola frondosa Polygalactan that embodiment 1 prepares is prepared to desired concn with saline water, and the intraperitoneal administration volume is the 0.2ml/20g mouse.With the endoxan is control sample, Hengrui Medicine Co., Ltd., Jiangsu Prov.'s (lot number: 11031921), get injection and be diluted to desired concn (3mg/ml) with saline water and get final product.
2 laboratory animal
ICR mouse (SPF level), Shanghai Si Laike laboratory animal responsibility ltd, animal conformity certification number: SCXK (Shanghai) 2007-0005; Animal quality conformity certification number: 2007000530457.Age in body weight: 18-20g/6 week.
3 knurl sources: the S180 solid tumor is gone down to posterity by Shanghai Institute of Pharmaceutical Industry pharmacology evaluation study center and keeps.
4 TPs
Extract an amount of seroperitoneum with aseptic empty needle, press 1:8 dilution, every right side of mice armpit subcutaneous vaccination tumour cell suspension 0.2ml in the 0.5h with 0.9% sodium chloride solution of sterilization.The inferior random packet administration in the future of inoculation after the last administration, is put to death the dislocation of mouse cervical vertebra, dissects taking-up knurl piece rapidly and weighs, and calculates tumor control rate by following formula:
Tumor control rate %=［ (the average knurl of the average knurl weight-administration of control group group is heavy)/average knurl of control group is heavy ］ * 100%
Heavy (the g)/administration group mean body weight (10g) of the average spleen of spleen index=administration group
Heavy (the g)/administration group mean body weight (10g) of the average thymus gland of thymus index=administration group
The grouping situation:
1) Grifola frondosa Polygalactan (1mg/kg, ip * 10qd)
2) Grifola frondosa Polygalactan (5mg/kg, ip * 10qd)
3) Grifola frondosa Polygalactan (25mg/kg, ip * 10qd)
4) endoxan (30mg/kg, ip * 7qd)
5) contrast (ip * 10qd)
5 experiment conclusion
Each dosage of Grifola frondosa Polygalactan is according to ip * 10qd scheme administration, and the growth of mouse S180 solid tumor is had certain restraining effect, and immune organ is had promoter action.Experimental result and related data see table 1 for details.
Table 1 Grifola frondosa Polygalactan is studied mouse S180 solid tumor models antitumor curative effect tested number effect relationship
Compare with control group: * P 0.05, * * P < 0.01.
Claims (5)
1. Grifola frondosa Polygalactan is characterized in that it prepares through following method:
1. the water logging of 10～50 times of volumes there is not Grifola Frondosa sporophore, 80～100 ℃ of lixiviates 1～5 hour;
2. vat liquor carries out ultrafiltration with 10～1,000,000 daltonian hollow fiber ultrafiltration membrane after centrifugal, collects the part that is trapped, i.e. trapped fluid;
3. trapped fluid adds 10～50% aqueous ethanolic solution, collecting precipitation then;
4. precipitate behind dissolved in distilled water, the sodium chloride aqueous solution with～50% carries out ion exchange chromatography;
5. first salt peak part of ion exchange chromatography is carried out ultrafiltration with 10～1,000,000 daltonian hollow fiber ultrafiltration membrane, collects the solution that is trapped;
6. above-mentioned solution concentration is dry, promptly obtain the Grifola frondosa Polygalactan.
2. Grifola frondosa Polygalactan according to claim 1 is characterized in that it prepares through following method:
1. the water logging of 10～30 times of volumes there is not Grifola Frondosa sporophore, 90～100 ℃ of lixiviates 1～3 hour;
2. soak body fluid and after centrifugal, carry out ultrafiltration, collect the part that is trapped, i.e. trapped fluid with 50～1,000,000 daltonian hollow fiber ultrafiltration membrane;
3. trapped fluid adds 10～30% aqueous ethanolic solution, collecting precipitation then;
4. precipitate behind dissolved in distilled water, the sodium chloride aqueous solution with～30% carries out ion exchange chromatography;
5. first salt peak part of ion exchange chromatography is carried out ultrafiltration with 50～1,000,000 daltonian hollow fiber ultrafiltration membrane, collects the solution that is trapped;
6. above-mentioned solution concentration is dry, promptly obtain the Grifola frondosa Polygalactan.
3. a method for preparing claim 1 or 2 each said Grifola frondosa Polygalactans is characterized in that this method comprises the steps:
1. 80～100 ℃ of floodings;
2. vat liquor carries out ultrafiltration with 10～1,000,000 daltonian hollow fiber ultrafiltration membrane;
3. ultrafiltrated is with 10～50% aqueous ethanolic solution deposition;
4. after precipitating water dissolution, carry out ion exchange chromatography;
5. first salt peak part of ion exchange chromatography is carried out ultrafiltration with 10～1,000,000 daltonian hollow fiber ultrafiltration membrane;
6. the ultrafiltrated concentrate drying promptly gets the Grifola frondosa Polygalactan.
4. claim 1 or 2 each said Grifola frondosa Polygalactans application in the medicine of preparation prevention or treatment tumour.
5. claim 1 or 2 each said Grifola frondosa Polygalactans are used for prevention or treatment tumour.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754005A (en) * | 2016-03-04 | 2016-07-13 | 江苏江大源生态生物科技股份有限公司 | Method for extracting grifola frondosa polysaccharase with high yield |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1634983A (en) * | 2004-10-20 | 2005-07-06 | 上海市农业科学院 | Method for preparing Griflola frondosa proteoglycan |
| CN101659706A (en) * | 2008-08-25 | 2010-03-03 | 徐财泉 | Method for purifying and separating X-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus |
| CN101736053A (en) * | 2010-02-01 | 2010-06-16 | 南京泽朗医药科技有限公司 | Technique for extracting Grifola frondosa water-soluble polysaccharide |
| CN102408494A (en) * | 2011-11-30 | 2012-04-11 | 杭州众芝康菇生物技术有限公司 | Grifola frondosa polysaccharide ZZK component and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1634983A (en) * | 2004-10-20 | 2005-07-06 | 上海市农业科学院 | Method for preparing Griflola frondosa proteoglycan |
| CN101659706A (en) * | 2008-08-25 | 2010-03-03 | 徐财泉 | Method for purifying and separating X-fraction polysaccharide from fruit bodies or mycelia of polyporus frondosus |
| CN101736053A (en) * | 2010-02-01 | 2010-06-16 | 南京泽朗医药科技有限公司 | Technique for extracting Grifola frondosa water-soluble polysaccharide |
| CN102408494A (en) * | 2011-11-30 | 2012-04-11 | 杭州众芝康菇生物技术有限公司 | Grifola frondosa polysaccharide ZZK component and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754005A (en) * | 2016-03-04 | 2016-07-13 | 江苏江大源生态生物科技股份有限公司 | Method for extracting grifola frondosa polysaccharase with high yield |
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