CN105061628A - Extraction method for coriolus versicolor polysaccharide - Google Patents
Extraction method for coriolus versicolor polysaccharide Download PDFInfo
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- CN105061628A CN105061628A CN201510542537.5A CN201510542537A CN105061628A CN 105061628 A CN105061628 A CN 105061628A CN 201510542537 A CN201510542537 A CN 201510542537A CN 105061628 A CN105061628 A CN 105061628A
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Abstract
The invention discloses an extraction method for coriolus versicolor polysaccharide. The extraction method comprises the following steps that 1, coriolus versicolor is extracted with alcohol, and filter residues are taken for standby application; 2, the filter residues are extracted with water, and filter liquid is collected for standby application; 3, the filter liquid is concentrated into extract, sedimentation is performed through alcohol, and sediment is obtained through centrifugation; 4, the sediment is washed with absolute ethyl alcohol and acetone sequentially, the washed sediment is dissolved with distilled water and permeates through a hollow fibrous membrane with molecular weight cut off one million, concentrated liquid is collected and then permeates through a hollow fibrous membrane with molecular weight cut off one and a half millions, permeate liquid is collected, centrifuged and filtered, and filtered liquid is taken for standby application; 5, spray drying is performed on the filtered liquid, and the coriolus versicolor polysaccharide is obtained. The coriolus versicolor polysaccharide obtained through the extraction method is high in purity, white in color and high in anti-tumor activity.
Description
Technical field
The invention belongs to technical field of biological extraction, be specifically related to the extracting method of krestin.
Background technology
Krestin is one of important chemical composition of rainbow conk, has antitumor, immunostimulant and liver-protective effect.But krestin is due to extraction process backwardness, and foreign matter content is high, and color and luster is orange grey, moreover, ordinary method is extracted the inhibition rate of tumor cell obtaining krestin and is only about 30%.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of extracting method of krestin, and the krestin purity that the method obtains is high, and antitumour activity is good.
Technical scheme provided by the invention is the extracting method of krestin, comprises the following steps:
1) rainbow conk is broken, with 6 ~ 12 times of weight 80 ~ 95v% alcohol reflux 2 ~ 3 times, reflux 2 ~ 3h at every turn, and filtration, gets filter residue for subsequent use;
2) by the water refluxing extraction 2 ~ 3 time of filter residue by 6 ~ 12 times of weight, extract 2 ~ 3h at every turn, filter, it is for subsequent use to collect filtrate;
3) filtrate being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1 ~ 1.3, adds alcohol settling, centrifugally must precipitate;
4) precipitation is washed respectively with the dehydrated alcohol of 2 ~ 4 times of weight, acetone successively, precipitation after washing is dissolved with 2 ~ 4 times of distilled water, cross the hollow-fibre membrane that molecular weight cut-off is 1,000,000, collect concentrated solution, then concentrated solution is crossed the hollow-fibre membrane that molecular weight cut-off is 1,500,000, collection permeate is centrifugal, filters, and it is for subsequent use to get filtrate;
5) get filtrate spraying dry, be krestin.
Step 3) in, ethanol to the ethanol content adding 90 ~ 95v% in medicinal extract is 80 ~ 85v%.
Step 4) in, after precipitation after washing is dissolved in distilled water, first cross the hollow-fibre membrane that molecular weight cut-off is 1,000,000, such molecular weight lower than 1,000,000 polysaccharide and oligose, monose and pigment all along with permeate flow out, the hollow-fibre membrane that molecular weight cut-off is 1,500,000 crossed by the concentrated solution collected, macro-molecular protein and molecular weight retain higher than the glycocalix of 1,500,000, collect permeate.
The present invention crosses hollow-fibre membrane twice, not only can farthest remove impurity and pigment, and to ensure finished product purity and color and luster, and the krestin molecular weight that extraction obtains is between 1,000,000 ~ 1,500,000, and its anti-tumor activity is stronger.
Embodiment
The present invention is further elaborated for following specific embodiment, but not as a limitation of the invention.
Embodiment 1
1) rainbow conk is broken, with 6 times of weight 80v% alcohol reflux 2 times, reflux 2h at every turn, and filtration, gets filter residue for subsequent use;
2) by the water refluxing extraction 2 time of filter residue by 6 times of weight, extract 2h at every turn, filter, it is for subsequent use to collect filtrate;
3) filtrate being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1, and ethanol to the ethanol content adding 90v% in medicinal extract is 80v% precipitation, centrifugal, is precipitated;
4) precipitation is washed respectively with the dehydrated alcohol of 2 times of weight, acetone successively, precipitation after washing is dissolved with 2 times of distilled water, cross the hollow-fibre membrane that molecular weight cut-off is 1,000,000, collect concentrated solution, then concentrated solution is crossed the hollow-fibre membrane that molecular weight cut-off is 1,500,000, collection permeate is centrifugal, filters, and it is for subsequent use to get filtrate;
5) get filtrate spraying dry, be krestin.Measuring polysaccharide content with phend-sulphuric acid is 93.6%, and color is white.
Control group
1) rainbow conk is broken, with 6 times of weight 80v% alcohol reflux 2 times, reflux 2h at every turn, and filtration, gets filter residue for subsequent use;
2) by the water refluxing extraction 2 time of filter residue by 6 times of weight, extract 2h at every turn, filter, it is for subsequent use to collect filtrate;
3) filtrate being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1, and ethanol to the ethanol content adding 90v% in medicinal extract is 80v% precipitation, centrifugal, is precipitated;
4) precipitation is washed respectively with the dehydrated alcohol of 2 times of weight, acetone successively, the precipitation after washing is dissolved with 2 times of distilled water, upper D303 type macroporous resin, wash with water to effluent liquid water white transparency, collect effluent liquid, concentrated, spraying dry, is krestin.Measuring polysaccharide content with phend-sulphuric acid is 85.3%, and color is orange grey.
Embodiment 2
1) rainbow conk is broken, with 12 times of weight 95v% alcohol reflux 3 times, reflux 3h at every turn, and filtration, gets filter residue for subsequent use;
2) by the water refluxing extraction 3 time of filter residue by 12 times of weight, extract 3h at every turn, filter, it is for subsequent use to collect filtrate;
3) filtrate being concentrated into relative density at 60 DEG C is the medicinal extract of 1.3, and ethanol to the ethanol content adding 95v% in medicinal extract is 85v% precipitation, centrifugal, is precipitated;
4) precipitation is washed respectively with the dehydrated alcohol of 4 times of weight, acetone successively, precipitation after washing is dissolved with 4 times of distilled water, cross the hollow-fibre membrane that molecular weight cut-off is 1,000,000, collect concentrated solution, then concentrated solution is crossed the hollow-fibre membrane that molecular weight cut-off is 1,500,000, collection permeate is centrifugal, filters, and it is for subsequent use to get filtrate;
5) get filtrate spraying dry, be krestin.Measuring polysaccharide content with phend-sulphuric acid is 93.8%, and color is white.
Embodiment 3
1) rainbow conk is broken, with 10 times of weight 90v% alcohol reflux 2 times, reflux 2h at every turn, and filtration, gets filter residue for subsequent use;
2) by the water refluxing extraction 2 time of filter residue by 10 times of weight, extract 2h at every turn, filter, it is for subsequent use to collect filtrate;
3) filtrate being concentrated into relative density at 60 DEG C is the medicinal extract of 1.2, and ethanol to the ethanol content adding 95v% in medicinal extract is 85v% precipitation, centrifugal, is precipitated;
4) precipitation is washed respectively with the dehydrated alcohol of 3 times of weight, acetone successively, precipitation after washing is dissolved with 3 times of distilled water, cross the hollow-fibre membrane that molecular weight cut-off is 1,000,000, collect concentrated solution, then concentrated solution is crossed the hollow-fibre membrane that molecular weight cut-off is 1,500,000, collection permeate is centrifugal, filters, and it is for subsequent use to get filtrate;
5) get filtrate spraying dry, be krestin.Measuring polysaccharide content with phend-sulphuric acid is 94.1%, and color is white.
For verifying that krestin of the present invention has stronger immunologic enhancement, now carry out following experimentation on animals.
1, anti-Lewis lung cancer activity experiment
Get cleaning grade C57BL/6 mouse 30, Mouse Weight 18 ~ 20g, be divided into 3 groups at random, often organize 10, be respectively experimental group, negative control group and positive controls.
Get eugonic Mice Bearing Lewis Lung Cancer tumour cell and make homogenate about 1 ~ 2 × 10 in conventional manner
7the cancer cell suspension of cfu/ml, in the right armpit subcutaneous vaccination 0.2ml cancer cell suspension of every mouse, after 24h, each group start every day oral administration once, the krestin of experimental group administration embodiment 1, negative control group administration physiological saline, the krestin of positive controls administration reference examples 1, dosage sees the following form 1, successive administration 10 days.After drug withdrawal, next day puts to death all animals, intercepts toes and weighs and calculate tumor control rate.Inhibition rate of tumor growth is by following formulae discovery, and inhibiting rate (%)=(negative control group knurl weight-experimental group/positive controls knurl weight)/negative control group knurl weighs × 100.
Table 1
| Dosage (mg/kg) | Animal weightening finish (g) | Knurl heavy (g) | Inhibiting rate (%) | |
| Negative control group | 1ml | 3.0 | 2.80±0.33 | |
| Positive controls | 2000 | 2.9 | 1.79±0.07 | 36.07* |
| Experimental group | 2000 | 1.8 | 1.06±0.16 | 62.14** |
Note: compared with negative control group, * p < 0.05, * * p < 0.01.
As seen from the above table, adopt ordinary method extract the inhibiting rate of krestin to Mice Bearing Lewis Lung Cancer reach 36.07%, and the present invention extract krestin to the inhibiting rate of Mice Bearing Lewis Lung Cancer up to 62.14%, be obviously better than conventional krestin.
2, anti-C-26 colon cancer reactive experiment
Get cleaning grade C57BL/6 mouse 30, Mouse Weight 18 ~ 20g, be divided into 3 groups at random, often organize 10, be respectively experimental group, negative control group and positive controls.
Get eugonic mouse C-26 colon cancer tumours cell and make homogenate about 1 ~ 2 × 10 in conventional manner
7the cancer cell suspension of cfu/ml, in the right armpit subcutaneous vaccination 0.2ml cancer cell suspension of every mouse, after 24h, each group start every day oral administration once, the krestin of experimental group administration embodiment 1, negative control group administration physiological saline, the krestin of positive controls administration reference examples 1, dosage sees the following form 2, successive administration 10 days.After drug withdrawal, next day puts to death all animals, intercepts toes and weighs and calculate tumor control rate.Inhibition rate of tumor growth is by following formulae discovery, and inhibiting rate (%)=(negative control group knurl weight-experimental group/positive controls knurl weight)/negative control group knurl weighs × 100.
Table 2
| Dosage (mg/kg) | Animal weightening finish (g) | Knurl heavy (g) | Inhibiting rate (%) | |
| Negative control group | 1ml | 3.8 | 2.99±0.26 | |
| Positive controls | 2000 | 3.8 | 1.92±0.23 | 35.79** |
| Experimental group | 2000 | 1.2 | 1.19±0.06 | 60.20** |
Note: compared with negative control group, * p < 0.05, * * p < 0.01.
As seen from the above table, adopt ordinary method extract the inhibiting rate of krestin to mouse C-26 colorectal carcinoma reach 35.79%, and the present invention extract krestin to the inhibiting rate of Mice Bearing Lewis Lung Cancer up to 60.20%, be obviously better than conventional krestin.
Claims (2)
1. the extracting method of krestin, is characterized in that: comprise the following steps:
1) rainbow conk is broken, with 6 ~ 12 times of weight 80 ~ 95v% alcohol reflux 2 ~ 3 times, reflux 2 ~ 3h at every turn, and filtration, gets filter residue for subsequent use;
2) by the water refluxing extraction 2 ~ 3 time of filter residue by 6 ~ 12 times of weight, extract 2 ~ 3h at every turn, filter, it is for subsequent use to collect filtrate;
3) filtrate being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1 ~ 1.3, adds alcohol settling, centrifugally must precipitate;
4) precipitation is washed respectively with the dehydrated alcohol of 2 ~ 4 times of weight, acetone successively, precipitation after washing is dissolved with 2 ~ 4 times of distilled water, cross the hollow-fibre membrane that molecular weight cut-off is 1,000,000, collect concentrated solution, then concentrated solution is crossed the hollow-fibre membrane that molecular weight cut-off is 1,500,000, collection permeate is centrifugal, filters, and it is for subsequent use to get filtrate;
5) get filtrate spraying dry, be krestin.
2. the extracting method of krestin according to claim 1, is characterized in that: step 3) in, ethanol to the ethanol content adding 90 ~ 95v% in medicinal extract is 80 ~ 85v%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109106731A (en) * | 2018-11-13 | 2019-01-01 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of preparation method and its anti-trioxypurine application of Coriolus Versicolor P.E. |
| CN114805626A (en) * | 2022-05-10 | 2022-07-29 | 湖南中药谷集团研究院有限公司 | Polysaccharide with anticancer activity, its preparation method and application in preparing anticancer drugs |
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| CN102911279A (en) * | 2012-10-23 | 2013-02-06 | 江苏神华药业有限公司 | Polysaccharide-peptide and liposome thereof |
| CN104231100A (en) * | 2014-09-04 | 2014-12-24 | 中山安荞生物科技有限公司 | Method for extracting coriolus versicolor polysaccharides |
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2015
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| CN1651076A (en) * | 2004-12-29 | 2005-08-10 | 江苏华源药业有限公司 | Method of extracting rainbow conk saccharide peptide for injection in rainbow conk saccharided peptide crude product obtained from submerged fermentation |
| CN102911279A (en) * | 2012-10-23 | 2013-02-06 | 江苏神华药业有限公司 | Polysaccharide-peptide and liposome thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109106731A (en) * | 2018-11-13 | 2019-01-01 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of preparation method and its anti-trioxypurine application of Coriolus Versicolor P.E. |
| CN109106731B (en) * | 2018-11-13 | 2021-07-20 | 广东省微生物研究所（广东省微生物分析检测中心） | Preparation method of coriolus versicolor extract and application of coriolus versicolor extract in reducing uric acid |
| CN114805626A (en) * | 2022-05-10 | 2022-07-29 | 湖南中药谷集团研究院有限公司 | Polysaccharide with anticancer activity, its preparation method and application in preparing anticancer drugs |
| CN114805626B (en) * | 2022-05-10 | 2023-02-03 | 湖南中药谷集团研究院有限公司 | Polysaccharide with anticancer activity, its preparation method and application in preparing anticancer drugs |
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