CN1117766C - Preparation method of mannuronic acid with single degree of polymerization and its application - Google Patents
Preparation method of mannuronic acid with single degree of polymerization and its application Download PDFInfo
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- CN1117766C CN1117766C CN 00111368 CN00111368A CN1117766C CN 1117766 C CN1117766 C CN 1117766C CN 00111368 CN00111368 CN 00111368 CN 00111368 A CN00111368 A CN 00111368A CN 1117766 C CN1117766 C CN 1117766C
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- mannuronic acid
- polymerization degree
- lyase
- single polymerization
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Abstract
The present invention relates to a preparation method of guluronic acid with single polymerization degree, which comprises steps: sodium alginate is dissolved in water, algin lyase is added for a reaction, a boiling water bath is used to heat to make the lyase deactivated; after centrifuging, deposit impurities are removed, the pH of solution is regulated; after centrifuging, generated sediment is removed, the sediment is dissolved by alkaline solution, and the pH is regulated, ethanol is added to generate deposit; after dried, the mixture is separated by an ion exchange chromatography column, and eluent is used to elute the mixture. The present invention is characterized in that the pH regulation range of the solution is from 2.5 to 3.2, the eluent is gradient elution of sodium acetate, and the lyase is special algin lyase generated by vibrio. The product has antineoplastic activity, and is used for developing antineoplastic medicine.
Description
Technical field
The present invention relates to a kind of organic polymer, particularly relate to a kind of mannuronic acid with single polymerization degree of anti-tumor activity.
Technical background
At a Japanese Patent: disclose in (06172375) and a kind ofly prepare the method for mannuronic acid, contained the mannuronic acid lyase in this abalone acetone powder extract with the abalone acetone powder extract.This is a kind of method that acid hydrolysis and enzymolysis prepare the oligomerization mannuronic acid of the polymerization degree 2～7 of using, and its separating effect is not good.
Summary of the invention
It is simple to the purpose of this invention is to provide a kind of preparation process, the preparation method of the mannuronic acid of the single polymerization degree of good separating effect, and this product has anti-tumor activity, can be used for developing anti-tumor medicaments.
A kind of preparation method of mannuronic acid of the single polymerization degree, comprise that to make sodium alginate water-soluble, adding alginate lyase reacts, make enzyme deactivation with the boiling water bath heating, remove precipitated impurities after centrifugal, solution is transferred pH, the centrifugal throw out of removing generation, with alkali lye dissolving and accent pH, add ethanol and produce precipitation, the mannuronic acid mixture of each single polymerization degree is separated with ion-exchange chromatography in dry back, with mannuronic acid mixture elutriant wash-out, substep is collected eluant component, add ethanol respectively and precipitate, freeze-drying after the desalination is characterized in that described solution is transferred the pH scope is 2.5～3.2, described elutriant is the gradient eluent of sodium-acetate, and used alginate lyase is produced by vibrios.
Preparation method's separation efficiency height of the present invention, the mannuronic acid of the various single polymerization degree of making has anti-tumor activity in various degree, can be used for being developed to antitumor drug.
Embodiment
Below by embodiment the present invention is described.
Get the 5g sodium alginate and be dissolved in 100ml water, (enzyme activity unit of alginate lyase is that to make 0.2% alginate solution absorbancy increase by 1 required enzyme amount be the enzyme unit that lives to every min to add 5 units.) by the alginate lyase liquid that vibrios produces, the production method of this enzyme had been narrated in first to file at No. 00111178.7.28 ℃ of reactions down, no longer change up to the 230nm light absorption value, the boiling water bath heating makes enzyme deactivation then, the centrifugal decon in cooling back, supernatant liquor is transferred pH2.85, and the centrifugal precipitation of removing is with the NaOH accent pH to 7 of 4mol/L, add ethanol and make the product precipitation, obtain the mannuronic acid mixture of different polymerization degree after the drying.Separate the mannuronic acid of each single polymerization degree with Q-sepharose fast flow anion-exchange column.The chromatographic column initial elutriant balance of sodium-acetate buffer with 0.1mol/L pH7.5 earlier is good, mannuronic acid with the above-mentioned preparation of 1g is dissolved in chromatographic column on the initial damping fluid again, sodium-acetate gradient eluent wash-out with 0.1～1, UV-detector with 230nm detects the mannuronic acid-content that elutes, substep is collected each elution fraction, add ethanol sedimentation after the desalination respectively and go out mannuronic acid, obtain the mannuronic acid of 2～12 different polymerization degrees after the drying.
Adopting present method can prepare the polymerization degree is 2～12 mannuronic acid.These oligosaccharides detect to unimodal with high performance anion exchange chromatography (high performance anion exchange chromatography HPAEC), are illustrated as the single polymerization degree.
With tetrazolium bromide (TTM) determination of experimental method with the anti-tumor activity of the mannuronic acid of each single polymerization degree of method of the present invention preparation.During experiment cancer cells is made into 10 with the RPMI1640 substratum of 10% calf serum (FCS)
4Individual/the ml suspension, in 37 ℃, 5%CO
2, 24h is fostered in 100 μ l/ holes (96 orifice plate), and each is organized correspondence and adds manninotriose aldehydic acid, make its concentration be respectively 1,10,100ug/ml, blank group correspondence adds substratum, every group of 5 parallel holes, add the TTM 20ul of 5mg/ml behind the cultivation 24h, cultivate 4h, go supernatant liquor to add 150ul dimethyl sulfoxide (DMSO) (DMSO), measure 570nm with microplate reader, (630nm is for setting wavelength) light absorption value.Used JEG-3 is the Ovcar-3 Proliferation of Human Ovarian Cell.Tumour inhibiting rate when recording the dosage of manninotriose aldehydic acid 100ug/ml with this method is 18.6%.The mannuronic acid of other various polymerization degree has also been measured tumour inhibiting rate with this method, and its result is similar.
Claims (2)
1. the preparation method of the mannuronic acid of a single polymerization degree, comprise that to make sodium alginate water-soluble, adding alginate lyase reacts, make enzyme deactivation with the boiling water bath heating, remove precipitated impurities after centrifugal, solution is transferred pH, the centrifugal throw out of removing generation, with alkali lye dissolving and accent pH, add ethanol and produce precipitation, the mannuronic acid mixture of each single polymerization degree is separated with ion-exchange chromatography in dry back, with mannuronic acid mixture elutriant wash-out, substep is collected eluant component, add ethanol respectively and precipitate, freeze-drying after the desalination is characterized in that described solution is transferred the scope of pH is 2.5～3.2, described elutriant is the gradient eluent of sodium-acetate, and described enzyme is the specificity alginate lyase that is produced by vibrios.
2. the method for claim 1 is characterized in that the single polymerization degree of described mannuronic acid is respectively 2～12.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00111368 CN1117766C (en) | 2000-09-07 | 2000-09-07 | Preparation method of mannuronic acid with single degree of polymerization and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00111368 CN1117766C (en) | 2000-09-07 | 2000-09-07 | Preparation method of mannuronic acid with single degree of polymerization and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1341665A CN1341665A (en) | 2002-03-27 |
| CN1117766C true CN1117766C (en) | 2003-08-13 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 00111368 Expired - Lifetime CN1117766C (en) | 2000-09-07 | 2000-09-07 | Preparation method of mannuronic acid with single degree of polymerization and its application |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7090856B2 (en) | 2003-06-19 | 2006-08-15 | Hong Kong University Of Science And Technology | Anti-fouling exopolysaccharides isolated from cultures of Vibrio alginolyticus and Vibrio proteolyticus |
| CN1562050A (en) | 2004-03-24 | 2005-01-12 | 中国海洋大学 | Use of oligose alginate in anti-dementia and anti-diabetes |
| CN103961365B (en) * | 2014-03-25 | 2016-06-29 | 青岛海洋生物医药研究院股份有限公司 | Oligomannuronic acid salt is in the application of preparation preventing and treating hepatic injury and various hepatitis, hepatic fibrosis or liver cirrhosis medicine |
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2000
- 2000-09-07 CN CN 00111368 patent/CN1117766C/en not_active Expired - Lifetime
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| CN1341665A (en) | 2002-03-27 |
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