CN1789287A - Poly-sulfated chondroitin sulfate and preparation method thereof - Google Patents
Poly-sulfated chondroitin sulfate and preparation method thereof Download PDFInfo
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- CN1789287A CN1789287A CN 200510045393 CN200510045393A CN1789287A CN 1789287 A CN1789287 A CN 1789287A CN 200510045393 CN200510045393 CN 200510045393 CN 200510045393 A CN200510045393 A CN 200510045393A CN 1789287 A CN1789287 A CN 1789287A
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Abstract
The invention discloses a ploysulfated chondroitin sulfate, containing glucuronic acid derivant and acetaminogalactose derivant in molecule, the characteristics of the invention comprising: the molecular structure comprises 2-0-sulfuric acid- glucuronic acid- (1->3)-2-N- acetyl group- 2- deoxidation- 4 or 6-0-sulfuric acid galactose, the molecular weight is 1KD-30KD, and the proportion between sulfate radical and carboxylate is 2-4. The invention also discloses a method for preparing the ploysulfated chondroitin sulfate, that is by sulphonating the chondroitin sulfate. The ploysulfated chondroitin sulfate possesses appreciable action of resisting tumour breeding, inhibiting tumour transferring and inhibiting mastocyte pulling-off granule, and etc, the anticoagulating potency<= 10IU/mg, and also possesses strong antiphlogistic and analgesic action.
Description
Technical field
The invention belongs to biomedicine field, particularly relate to a kind of poly-sulfated chondroitin sulfate and preparation method thereof.
Background technology
Chondroitin sulfate is the Sulfated glycosaminoglycan compounds of a class, and the molecular weight of natural product generally between 10kD～50kD, extensively is present in the animal cartilage tissue.
Studies show that both at home and abroad chondroitin sulfate has the stronger reduction triglyceride in blood and the effect of cholesterol, can prevent atherosclerotic formation effectively.People such as Petitou are at United States Patent (USP) " preparation of the Sulfated glycosaminoglycan analog derivative of dermatan sulfate and chondroitin sulfates " (Sulfated glycosaminoglycanoidderivatives of the dermatan sulfate and chondroitin sulfate type, patent No. US5382570) once reported the preparation method of sulfated chondroitin sulfate, but this method has complex process, reagent cost height, be unfavorable for that suitability for industrialized production and products therefrom have shortcomings such as different structures and activity, thereby limited its application.
Summary of the invention
At the deficiencies in the prior art, the object of the present invention is to provide a kind of poly-sulfated chondroitin sulfate and preparation method thereof, this one step of method employing chemical synthesis is carried out structural modification to the chondroitin sulfate of natural extract, prepares a kind of the have stronger anti-inflammatory and the poly-sulfated chondroitin sulfate of analgesic activity.
Poly-sulfated chondroitin sulfate of the present invention; contain glucal acid derivative and acetylamino galactosamine derivative in the molecule; it is characterized in that: molecular structure is made up of 2-O-sulfate-glucuronic acid-(1 → 3)-2-N-ethanoyl-2-deoxidation-4 or 6-O-sulfate semi-lactosi, and its chemical structure is as follows:
Above-mentioned poly-sulfated chondroitin sulfate structure, R is SO
3Or H, n is 1 to 30, and the molecular weight of poly-sulfated chondroitin sulfate is 1kD～30kD, and the ratio of sulfate radical and carboxylate radical is 2～4 in its molecular structure.
The preparation method of above-mentioned poly-sulfated chondroitin sulfate may further comprise the steps:
(1) chondroitin sulfate is poly-sulfated: it is 15%～30% solution that chondroitin sulfate is made into mass percent concentration, with chlorsulfonic acid: the vitriol oil is that 1: 0.05～0.1 nitration mixture joins in the above-mentioned solution, the final concentration that makes the chondroitin sulfate mass percent is 10%～15%, 10 ℃～70 ℃ of temperature, stirring reaction 2～10 hours, add the organic solvent termination reaction then, get the poly-sulfated chondroitin sulfate throw out;
(2) separate needed poly-sulfated chondroitin sulfate fragment: above-mentioned throw out is made into the aqueous solution, transfers pH to 7 ± 0.2, the organic solvent that adds its 4～5 times of amounts makes it precipitation; Again throw out is made into the aqueous solution,, is heated to 85 ℃ ± 2 ℃ with adjusting PH with base to 10 ± 0.2, and constantly carry out the interior gaseous interchange of reactor, and be incubated 3 ± 0.5 hours, transfer pH to 7 with hydrochloric acid then, add its 4～5 times amount organic solvents and make it precipitation, realize desalination and remove amine;
(3) collect, prepare poly-sulfated chondroitin sulfate: it is 10% aqueous solution that the final throw out that obtains of step (2) is made into mass percent concentration, carrying out gel chromatography separates, with concentration is that the sodium chloride solution of 0.01～1.0mol/L carries out wash-out, collect the high part of poly-sulfated chondroitin sulfate content, with Sephadex G-10 desalination, normal freeze-drying gets poly-sulfated chondroitin sulfate.
In the preparation method of above-mentioned poly-sulfated chondroitin sulfate, the described chlorsulfonic acid of step (1): the vitriol oil is preferably 1: 0.05～and 0.07.
In the preparation method of above-mentioned poly-sulfated chondroitin sulfate, preferably 30 ℃～40 ℃ of the described temperature of reaction of step (1), churning time preferably 4～6 hours.
In the preparation method of above-mentioned poly-sulfated chondroitin sulfate, step (1) or (2) described organic solvent are one of methyl alcohol, ethanol, acetone, Virahol.
Wherein, above-mentioned organic solvent preferably concentration of volume percent be 95% ethanol.
In the preparation method of above-mentioned poly-sulfated chondroitin sulfate, in the described gel chromatography of step (3), the gel of employing is one of Sephadex G-50, Sephadex G-75, Sephadex G-100, Superdex 200.
Wherein, described gel is preferably the Sephadex G-75 gel column of 3.5 * 120cm.
In the preparation method of above-mentioned poly-sulfated chondroitin sulfate, the described elutriant of step (3) preferably concentration is the sodium chloride solution of 0.2～0.5mol/L.
The preparation method of poly-sulfated chondroitin sulfate of the present invention has that technology is simple, reagent cost is low, and production environment and equipment requirements are not harsh, and the high characteristics of product purity, and the utmost point is beneficial to suitability for industrialized production.
The poly-sulfated chondroitin sulfate that the method for the invention makes has stronger anti-inflammatory and analgesic activity.Through verification experimental verification, its anticoagulant efficiency≤10IU/mg, and have tangible antitumor propagation and suppress effects such as metastases, inhibition mast cell degranulation.
Embodiment
Below in conjunction with embodiment content of the present invention is further elaborated.
Embodiment 1
(1) chondroitin sulfate is poly-sulfated: it is 20% solution that chondroitin sulfate elaboration (commercially available) 100g is made into mass percent concentration, with chlorsulfonic acid: the vitriol oil is that 1: 0.05 nitration mixture joins in the above-mentioned solution, the final concentration that makes the chondroitin sulfate mass percent is 10%, 35 ℃ of temperature, stirring reaction 5 hours, add 4000ml dehydrated alcohol termination reaction then, get the poly-sulfated chondroitin sulfate throw out;
(2) separate needed poly-sulfated chondroitin sulfate fragment: with above-mentioned throw out 1000ml dissolved in distilled water, be made into the aqueous solution, transfer pH to 7 with 20% sodium hydroxide, 95% ethanol that adds its 4 times of amounts makes it precipitation; Again throw out is made into 10% aqueous solution, transfers pH to 10, be heated to 85 ℃ with 20% sodium hydroxide, and constantly carry out the interior gaseous interchange of reactor, and be incubated 3 hours, transfer pH to 7 with 6mol/L hydrochloric acid then, add its 4 times amount 95%7 alcohol and make it precipitation, realize desalination and remove amine;
(3) collect, prepare poly-sulfated chondroitin sulfate: it is 10% aqueous solution that the final throw out that obtains of step (2) is made into mass percent concentration, (3.5 * 120cm) separate last Sephadex G-75 gel column, the sodium chloride solution that with concentration is 0.2mol/L carries out wash-out, collect the high part of poly-sulfated chondroitin sulfate content, with Sephadex G-10 desalination, normal freeze-drying gets poly-sulfated chondroitin sulfate.
The poly-sulfated chondroitin sulfate of above-mentioned gained is carried out molecular weight determination, structure determination and anti-inflammatory activity respectively to be measured and obtains a kind of poly-sulfated chondroitin sulfate; contain glucal acid derivative and acetylamino galactosamine derivative in the molecule; molecular structure is made up of 2-O-sulfate-glucuronic acid-(1 → 3)-2-N-ethanoyl-2-deoxidation-4 or 6-O-sulfate semi-lactosi, and its main chemical structure is as follows:
The relative molecular weight of this poly-sulfated chondroitin sulfate is 1kD～30kD, and R is SO
3Or H, n is 1 to 30, the ratio of sulfate radical and carboxylate radical is 2～4, has the obvious suppression mast cell degranulation, anticoagulant efficiency≤10IU/mg.Also have effects such as tangible antitumor propagation and inhibition metastases, stronger anti-inflammatory and analgesic activity are arranged.
Embodiment 2
(1) chondroitin sulfate is poly-sulfated: it is 30% solution that chondroitin sulfate elaboration (commercially available) 200g is made into mass percent concentration, with chlorsulfonic acid: the vitriol oil is that 1: 0.07 nitration mixture joins in the above-mentioned solution, the final concentration that makes the chondroitin sulfate mass percent is 10%, 40 ℃ of temperature, stirring reaction 6 hours, add 4000ml dehydrated alcohol termination reaction then, get the poly-sulfated chondroitin sulfate throw out;
(2) separate needed poly-sulfated chondroitin sulfate fragment: with above-mentioned throw out 1000ml dissolved in distilled water, be made into the aqueous solution, transfer pH to 7 with 20% sodium hydroxide, 95% methyl alcohol that adds its 5 times of amounts makes it precipitation; Again throw out is made into 10% aqueous solution, transfers pH to 10, be heated to 85 ℃ with 20% sodium hydroxide, and constantly carry out the interior gaseous interchange of reactor, and be incubated 3.5 hours, transfer pH to 7 with 6mol/L hydrochloric acid then, add its 5 times amount 95% methyl alcohol and make it precipitation, realize desalination and remove amine;
(3) collect, prepare poly-sulfated chondroitin sulfate: it is 10% aqueous solution that the final throw out that obtains of step (2) is made into mass percent concentration, (3.5 * 120cm) separate last Sephadex G-100 gel column, the sodium chloride solution that with concentration is 0.5mol/L carries out wash-out, collect the high part of poly-sulfated chondroitin sulfate content, with Sephadex G-10 desalination, normal freeze-drying gets poly-sulfated chondroitin sulfate.
Embodiment 3
(1) chondroitin sulfate is poly-sulfated: it is 25% solution that chondroitin sulfate elaboration (commercially available) 100g is made into mass percent concentration, with chlorsulfonic acid: the vitriol oil is that 1: 0.09 nitration mixture joins in the above-mentioned solution, the final concentration that makes the chondroitin sulfate mass percent is 10%, 30 ℃ of temperature, stirring reaction 10 hours, add 4000ml acetone termination reaction then, get the poly-sulfated chondroitin sulfate throw out;
(2) separate needed poly-sulfated chondroitin sulfate fragment: with above-mentioned throw out 1000ml dissolved in distilled water, be made into the aqueous solution, transfer pH to 7 with 20% sodium hydroxide, the acetone that adds its 4 times of amounts makes it precipitation; Again throw out is made into 10% aqueous solution, transfers pH to 10, be heated to 85 ℃ with 20% sodium hydroxide, and constantly carry out the interior gaseous interchange of reactor, and be incubated 3 hours, transfer pH to 7 with 6mol/L hydrochloric acid then, add its 4 times amount acetone and make it precipitation, realize desalination and remove amine;
(3) collect, prepare poly-sulfated chondroitin sulfate: it is 10% aqueous solution that the final throw out that obtains of step (2) is made into mass percent concentration, (3.5 * 120cm) separate last Sephadex G-50 gel column, the sodium chloride solution that with concentration is 0.8mol/L carries out wash-out, collect the high part of poly-sulfated chondroitin sulfate content, with Sephadex G-10 desalination, normal freeze-drying gets poly-sulfated chondroitin sulfate.
Claims (10)
1. poly-sulfated chondroitin sulfate; contain glucal acid derivative and acetylamino galactosamine derivative in the molecule; it is characterized in that: molecular structure is made up of 2-O-sulfate-glucuronic acid-(1 → 3)-2-N-ethanoyl-2-deoxidation-4 or 6-O-sulfate semi-lactosi, and its chemical structure is as follows:
Wherein: R is SO
3Or H, n is 1 to 30.
2. poly-sulfated chondroitin sulfate according to claim 1 is characterized in that: the molecular weight of described poly-sulfated chondroitin sulfate is 1kD～30kD, and the ratio of sulfate radical and carboxylate radical is 2～4 in its molecular structure.
3. the preparation method of claim 1 or 2 described poly-sulfated chondroitin sulfates may further comprise the steps:
(1) chondroitin sulfate is poly-sulfated: it is 15%～30% solution that chondroitin sulfate is made into mass percent concentration, with chlorsulfonic acid: the vitriol oil is that 1: 0.05～0.1 nitration mixture joins in the above-mentioned solution, the final concentration that makes the chondroitin sulfate mass percent is 10%～15%, 10 ℃～70 ℃ of temperature, stirring reaction 2～10 hours, add the organic solvent termination reaction then, get the poly-sulfated chondroitin sulfate throw out;
(2) separate needed poly-sulfated chondroitin sulfate fragment: above-mentioned throw out is made into the aqueous solution, transfers pH to 7 ± 0.2, the organic solvent that adds its 4～5 times of amounts makes it precipitation; Again throw out is made into the aqueous solution,, is heated to 85 ℃ ± 2 ℃ with adjusting PH with base to 10 ± 0.2, and constantly carry out the interior gaseous interchange of reactor, and be incubated 3 ± 0.5 hours, transfer pH to 7 with hydrochloric acid then, add its 4～5 times amount organic solvents and make it precipitation, realize desalination and remove amine;
(3) collect, prepare poly-sulfated chondroitin sulfate: it is 10% aqueous solution that the final throw out that obtains of step (2) is made into mass percent concentration, carrying out gel chromatography separates, with concentration is that the sodium chloride solution of 0.01～1.0mol/L carries out wash-out, collect the high part of poly-sulfated chondroitin sulfate content, with Sephadex G-10 desalination, normal freeze-drying gets poly-sulfated chondroitin sulfate.
4. the preparation method of poly-sulfated chondroitin sulfate according to claim 3, it is characterized in that: the described chlorsulfonic acid of step (1): the vitriol oil is 1: 0.05～0.07.
5. the preparation method of poly-sulfated chondroitin sulfate according to claim 3, it is characterized in that: the described temperature of reaction of step (1) is 30 ℃～40 ℃, and churning time is 4～6 hours.
6. the preparation method of poly-sulfated chondroitin sulfate according to claim 3, it is characterized in that: step (1) or (2) described organic solvent are one of methyl alcohol, ethanol, acetone, Virahol.
7. the preparation method of poly-sulfated chondroitin sulfate according to claim 6, it is characterized in that: described organic solvent is that concentration of volume percent is 95% ethanol.
8. the preparation method of poly-sulfated chondroitin sulfate according to claim 3, it is characterized in that: in the described gel chromatography of step (3), the gel of employing is Sephadex G-50, Sephadex G-75, one of Sephadex G-100, Superdex200.
9. the preparation method of poly-sulfated chondroitin sulfate according to claim 8, it is characterized in that: described gel is the Sephadex G-75 gel column of 3.5 * 120cm.
10. the preparation method of poly-sulfated chondroitin sulfate according to claim 3, it is characterized in that: the described elutriant of step (3) is that concentration is the sodium chloride solution of 0.2～0.5mol/L.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102125543A (en) * | 2011-02-28 | 2011-07-20 | 王啸龙 | Heparin-free anticoagulant infection inhibitor for hemodialysis and preparation method thereof |
| CN103582653A (en) * | 2011-05-12 | 2014-02-12 | 诺西斯有限公司 | Biotechnological sulphated chondroitin sulphate at position 4 or 6 on the same polysaccharide chain, and process for the preparation thereof |
| WO2016197799A1 (en) * | 2015-06-10 | 2016-12-15 | 浙江三门恒康制药有限公司 | Method for preparing heparinoid |
| CN108752500A (en) * | 2018-07-03 | 2018-11-06 | 江南大学 | A method of preparing curdlan sulfuric ester |
| CN109970882A (en) * | 2019-03-08 | 2019-07-05 | 河北常山生化药业股份有限公司 | A kind of preparation method of poly-sulfated chondroitin sulfate |
| CN113631584A (en) * | 2019-03-05 | 2021-11-09 | 中国医学科学院药物研究所 | Chondroitin sulfate polysaccharide, semi-synthesis preparation method and application thereof |
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2005
- 2005-12-20 CN CN 200510045393 patent/CN1789287A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102125543A (en) * | 2011-02-28 | 2011-07-20 | 王啸龙 | Heparin-free anticoagulant infection inhibitor for hemodialysis and preparation method thereof |
| CN103582653A (en) * | 2011-05-12 | 2014-02-12 | 诺西斯有限公司 | Biotechnological sulphated chondroitin sulphate at position 4 or 6 on the same polysaccharide chain, and process for the preparation thereof |
| CN103582653B (en) * | 2011-05-12 | 2016-08-17 | 诺西斯有限公司 | 4 or 6 Sulfated chondroitin sulfates of biotechnology at same polysaccharide chain and preparation method thereof |
| WO2016197799A1 (en) * | 2015-06-10 | 2016-12-15 | 浙江三门恒康制药有限公司 | Method for preparing heparinoid |
| CN108752500A (en) * | 2018-07-03 | 2018-11-06 | 江南大学 | A method of preparing curdlan sulfuric ester |
| CN113631584A (en) * | 2019-03-05 | 2021-11-09 | 中国医学科学院药物研究所 | Chondroitin sulfate polysaccharide, semi-synthesis preparation method and application thereof |
| CN113631584B (en) * | 2019-03-05 | 2023-09-29 | 中国医学科学院药物研究所 | Chondroitin sulfate polysaccharide, semisynthesis preparation method and application thereof |
| CN109970882A (en) * | 2019-03-08 | 2019-07-05 | 河北常山生化药业股份有限公司 | A kind of preparation method of poly-sulfated chondroitin sulfate |
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