CN1445245A - Dermatan sulfate with low molecule and its preparing method - Google Patents
Dermatan sulfate with low molecule and its preparing method Download PDFInfo
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- CN1445245A CN1445245A CN 03112180 CN03112180A CN1445245A CN 1445245 A CN1445245 A CN 1445245A CN 03112180 CN03112180 CN 03112180 CN 03112180 A CN03112180 A CN 03112180A CN 1445245 A CN1445245 A CN 1445245A
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Abstract
A low-molecular (3000-8000) dermatan sulfate is prepared from the dermatan sulfate through degradation by hydrogen peroxide, ultrafilter, and gel chromatography. Its advantages are sure thrombus-resistant and thrombolytic functions, easy absorption, long semi-life and high biologic utilization rate.
Description
Technical field
The invention belongs to biomedicine field, particularly relate to a kind of low molecular weight dermatan sulfate and preparation method thereof.
Background technology
Dermatan sulfate is a class formation complexity, Sulfated glycosaminoglycan, and the natural product molecular weight extensively is present in the animal tissues generally at 15kD～5kD.Studies show that both at home and abroad the dermatan sulfate anticoagulating active is lower than unfraction heparin, its antithrombotic acitivity is but obviously than unfraction heparin height, and hemorrhage side effect is obviously little than unfraction heparin, so dermatan sulfate has become a kind of novel antithrombotic reagent.Yet there are two main pharmacokinetics problems in dermatan sulfate: one is that the transformation period is shorter, and another is subcutaneous and during the intramuscular injection path administration, and bioavailability is low.Along with the rising of dermatan sulfate molecular weight, its bioavailability reduces.People such as Mascellani had once reported a kind of preparation method of low molecular weight dermatan sulfate at United States Patent (USP) " dermatan sulfate and pharmaceutical preparation composition thereof with degraded of anti-thrombosis function, fibrinolysis and anti-inflammatory activity " (patent No. US4973580), this method is to make the catalyst degradation dermatan sulfate with heavy metal ion, have complex process, yield is low, and has introduced harmful shortcomings such as heavy metal ion.
Summary of the invention
The objective of the invention is to overcome above-mentioned the deficiencies in the prior art, single use the hydrogen peroxide degradation dermatan sulfate, a kind of low molecular weight dermatan sulfate with stronger anti thrombotic action and certain thrombolytic effect and preparation method thereof is provided.
The present invention realizes by following approach:
A kind of low molecular weight dermatan sulfate; a kind of low molecular weight dermatan sulfate; contain iduronic acid derivative and acetylamino galactosamine derivative in the molecular structure; it is characterized in that: molecular structure is made up of 2-O-sulfate-iduronic acid-(1 → 3)-2-N-ethanoyl-2-deoxidation-4-0-sulfate semi-lactosi; also contain 2-N-ethanoyl-2-deoxidation-4 in the sugar chain; 6-0-two sulfate semi-lactosis, its chemical structure is as follows:
Above-mentioned low molecular weight dermatan sulfate is characterized in that: molecular weight is 3000～8000 dalton, and the ratio of sulfate radical and carboxylate radical is 0.8～1.2, and activating heparin cofactor II activity is 100～200U/mg, anticoagulant efficiency≤10 IU/mg.
The hydrogen peroxide degradation method that the present invention sets up causes the fracture of dermatan sulfate main chain (1 → 4) glycosidic link, obtain low molecular weight dermatan sulfate, do not introduce other impurity, improve temperature of reaction and increase the molecular weight that concentration of hydrogen peroxide helps reducing dermatan sulfate, controlling reaction time can obtain the product of desired molecule amount.
A kind of method for preparing above-mentioned low molecular weight dermatan sulfate is characterized in that may further comprise the steps:
(1) degraded of dermatan sulfate: the concentration of the dermatan sulfate aqueous solution is 1%～10%, the pH value of solution is 4～10, the concentration of hydrogen peroxide is 1%～20%, temperature is 20～80 ℃, reaction times is 2～18 hours, degraded finishes and uses organic solvent deposit, organic solvent commonly used such as methyl alcohol, ethanol, acetone, Virahol etc.;
(2) separate needed low molecular weight dermatan sulfate fragment: after above-mentioned degraded product is made into the aqueous solution, separate needed fragment with gel chromatography with ultrafiltration process, gel commonly used is Sephadex G-50, Sephadex G-75, Sephadex G-100, Superdex 200 etc., carry out wash-out with sodium chloride solution, the concentration of sodium-chlor is 0.01～1.0mol/L.Collect the high part of low molecular weight dermatan sulfate content, with Sephadex G-10 desalination, the lyophilize sub-dermatan sulfate that makes low score.
The low molecular weight dermatan sulfate that the present invention makes has stronger anti thrombotic action and certain thrombolytic effect, also has oral easy absorption, long half time and bioavailability advantages of higher.
Embodiment
Provide a preferred examples of the invention process below.
The key of low molecular weight dermatan sulfate preparation is the control molecular weight, and this method adopts the hydrogen peroxide degradation method, obtains the controllable low molecular weight dermatan sulfate of palliating degradation degree.
(1) preparation of low molecular weight dermatan sulfate: dermatan sulfate elaboration 1g is made into 1.0% solution with distilled water, regulate pH4～5, drip 30% hydrogen peroxide and make final concentration reach 2%, in 70 ℃ of stirring in water bath 15 hours, transfer pH7 with sodium hydroxide solution, 3 times of amount methanol extractions;
(2) purifying of low molecular weight dermatan sulfate: the throw out of step (1) is made into 0.5% solution, with the molecular retention value is the membrane ultrafiltration of 8kD, collect and see through liquid, after concentrating with Rotary Evaporators, and last Sephadex G-100 preparative chromatography post (2.6 * 82cm), with 0.3mol/L sodium chloride solution wash-out, the 206nm ultraviolet detection, collect the high part of low molecular weight dermatan sulfate content, with Sephadex G-10 desalination, lyophilize gets required low molecular weight dermatan sulfate.
The low molecular weight dermatan sulfate of above-mentioned gained is carried out molecular weight determination, structure determination and anticoagulant efficiency respectively to be measured and obtains a kind of low molecular weight dermatan sulfate; contain iduronic acid derivative and acetylamino galactosamine derivative in the molecular structure; molecular structure is made up of 2-O-sulfate-iduronic acid-(1 → 3)-2-N-ethanoyl-2-deoxidation-4-O-sulfate semi-lactosi; also contain 2-N-ethanoyl-2-deoxidation-4 in the sugar chain; 6-O-two sulfate semi-lactosis, its chemical structure is as follows:
The relative molecular weight of this low molecular weight dermatan sulfate is 3000～8000 dalton, and the ratio of sulfate radical and carboxylate radical is 0.8～1.2, and activating heparin cofactor II activity is 100～200U/mg, anticoagulant efficiency≤10IU/mg.
Claims (3)
1. low molecular weight dermatan sulfate; contain iduronic acid derivative and acetylamino galactosamine derivative in the molecular structure; it is characterized in that: molecular structure is made up of 2-O-sulfate-iduronic acid-(1 → 3)-2-N-ethanoyl-2-deoxidation-4-O-sulfate semi-lactosi; also contain 2-N-ethanoyl-2-deoxidation-4 in the sugar chain; 6-O-two sulfate semi-lactosis, its chemical structure is as follows:
2. low molecular weight dermatan sulfate according to claim 1 is characterized in that: molecular weight is 3000～8000 dalton, and the ratio of sulfate radical and carboxylate radical is 0.8～1.2, and activating heparin cofactor II activity is 100～200U/mg, anticoagulant efficiency≤10IU/mg.
3. method for preparing claim 1 or 2 described low molecular weight dermatan sulfates is characterized in that may further comprise the steps:
(1) preparation of low molecular weight dermatan sulfate: the dermatan sulfate elaboration is made into 1.0%～10% solution with distilled water, regulate pH4～10, the hydrogen peroxide of dropping 30% makes final concentration reach 1%～20%, in temperature is 20～70 ℃ of stirring in water bath 2～18 hours, transfer pH7 with sodium hydroxide solution, 3 times of amount methanol extractions.
(2) purifying of low molecular weight dermatan sulfate: the throw out of step (1) is made into 0.5% solution, with the molecular retention value is the membrane ultrafiltration of 8kD, collect and see through liquid, after concentrating with Rotary Evaporators, last SephadexG-75 preparative chromatography post with 0.01～1.0mol/L sodium chloride solution wash-out, is collected the high part of low molecular weight dermatan sulfate content, with Sephadex G-10 desalination, lyophilize gets required low molecular weight dermatan sulfate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031121802A CN100345868C (en) | 2003-04-18 | 2003-04-18 | Dermatan sulfate with low molecule and its preparing method |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB031121802A CN100345868C (en) | 2003-04-18 | 2003-04-18 | Dermatan sulfate with low molecule and its preparing method |
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| CN1445245A true CN1445245A (en) | 2003-10-01 |
| CN100345868C CN100345868C (en) | 2007-10-31 |
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| CNB031121802A Expired - Fee Related CN100345868C (en) | 2003-04-18 | 2003-04-18 | Dermatan sulfate with low molecule and its preparing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168570B (en) * | 2007-11-15 | 2010-05-26 | 暨南大学 | Method for degrading kelp polysaccharide sulfate |
| CN106146688A (en) * | 2016-07-27 | 2016-11-23 | 河北常山生化药业股份有限公司 | Heparinoid technique prepared by heparin sodium leftover bits and pieces |
| CN106188341A (en) * | 2016-07-27 | 2016-12-07 | 河北常山生化药业股份有限公司 | High-purity sulfuric acid dermatan technique prepared by heparin sodium leftover bits and pieces |
| CN111040047A (en) * | 2019-12-11 | 2020-04-21 | 东营天东制药有限公司 | Process and application for refining low-molecular dermatan sulfate by enzyme-ultrafiltration method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9711443D0 (en) * | 1997-06-03 | 1997-07-30 | Leo Pharm Prod Ltd | Chemical suppositions |
-
2003
- 2003-04-18 CN CNB031121802A patent/CN100345868C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168570B (en) * | 2007-11-15 | 2010-05-26 | 暨南大学 | Method for degrading kelp polysaccharide sulfate |
| CN106146688A (en) * | 2016-07-27 | 2016-11-23 | 河北常山生化药业股份有限公司 | Heparinoid technique prepared by heparin sodium leftover bits and pieces |
| CN106188341A (en) * | 2016-07-27 | 2016-12-07 | 河北常山生化药业股份有限公司 | High-purity sulfuric acid dermatan technique prepared by heparin sodium leftover bits and pieces |
| CN106188341B (en) * | 2016-07-27 | 2019-09-17 | 河北常山生化药业股份有限公司 | Heparin sodium leftover bits and pieces prepares high-purity sulfuric acid dermatan technique |
| CN111040047A (en) * | 2019-12-11 | 2020-04-21 | 东营天东制药有限公司 | Process and application for refining low-molecular dermatan sulfate by enzyme-ultrafiltration method |
| WO2021114941A1 (en) * | 2019-12-11 | 2021-06-17 | 东营天东制药有限公司 | Low molecular weight dermatan sulfate, refining process therefor and application thereof |
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| Publication number | Publication date |
|---|---|
| CN100345868C (en) | 2007-10-31 |
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Granted publication date: 20071031 Termination date: 20100418 |