CN1789287A - Poly-sulfated chondroitin sulfate and preparation method thereof - Google Patents

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

A kind of polysulfated chondroitin sulfate and preparation method thereof

technical field

The invention belongs to the field of biomedicine, in particular to a polysulfated chondroitin sulfate and a preparation method thereof.

Background technique

Chondroitin sulfate is a kind of sulfated glycosaminoglycan compound. The molecular weight of natural products is generally between 10kD and 50kD, and it widely exists in animal cartilage tissue.

Domestic and foreign studies have shown that chondroitin sulfate has a strong effect of lowering blood triglycerides and cholesterol, and can effectively prevent the formation of atherosclerosis. Petitou et al. have reported sulfated sulfate in the U.S. patent "Preparation of sulfated glycosaminoglycan derivatives of dermatan sulfate and chondroitin sulfate" (Sulfated glycosaminoglycanoid derivatives of the dermatan sulfate and chondroitin sulfate type, Patent No. US5382570) A method for preparing chondroitin, but this method has disadvantages such as complex process, high reagent cost, unfavorable industrial production and different structures and activities of the obtained products, thus limiting its application.

Contents of the invention

In view of the deficiencies in the prior art, the object of the present invention is to provide a polysulfated chondroitin sulfate and a preparation method thereof, which uses a one-step chemical synthesis method to carry out structural modification to the naturally extracted chondroitin sulfate to prepare a Strong anti-inflammatory and analgesic effects of polysulfated chondroitin sulfate.

The polysulfated chondroitin sulfate of the present invention contains glucuronic acid derivatives and acetylgalactosamine derivatives in the molecule, and is characterized in that the molecular structure consists of 2-O-sulfate group-glucuronic acid-(1→3 )-2-N-acetyl-2-deoxy-4 or 6-O-sulfate galactose, its chemical structure is as follows:

The above polysulfated chondroitin sulfate structure, R is SO ₃ or H, n is 1 to 30, the molecular weight of polysulfated chondroitin sulfate is 1kD ~ 30kD, and the ratio of sulfate to carboxylate in its molecular structure is 2 ~ 4.

The preparation method of above-mentioned polysulfated chondroitin sulfate comprises the following steps:

(1) The polysulfation of chondroitin sulfate: chondroitin sulfate is made into the solution that mass percentage concentration is 15%～30%, with chlorosulfonic acid: the mixed acid that concentrated sulfuric acid is 1: 0.05～0.1 joins in the above-mentioned solution, The final concentration of chondroitin sulfate by mass percentage is 10% to 15%, at a temperature of 10°C to 70°C, stirring and reacting for 2 to 10 hours, and then adding an organic solvent to terminate the reaction, so that more sulfated chondroitin sulfate precipitates;

(2) Separation of the required polysulfated chondroitin sulfate fragments: make the above precipitate into an aqueous solution, adjust the pH to 7±0.2, add 4 to 5 times the amount of organic solvent to precipitate it; then make the precipitate into Aqueous solution, adjust pH to 10±0.2 with alkali, heat to 85℃±2℃, and continuously carry out gas exchange in the reactor, keep warm for 3±0.5 hours, then adjust pH to 7 with hydrochloric acid, add 4 to 5 times the amount Precipitate with organic solvent to realize desalination and amine removal;

(3) Collecting and preparing polysulfated chondroitin sulfate: the precipitate finally obtained in step (2) is made into an aqueous solution with a concentration of 10% by mass, and is separated by gel chromatography to a concentration of 0.01 to 1.0 mol/L The sodium chloride solution was used for elution, and the part with high content of polysulfated chondroitin sulfate was collected, desalted with Sephadex G-10, and conventionally freeze-dried polysulfated chondroitin sulfate.

In the above-mentioned preparation method of polysulfated chondroitin sulfate, the ratio of chlorosulfonic acid:concentrated sulfuric acid in step (1) is preferably 1:0.05-0.07.

In the above-mentioned preparation method of polysulfated chondroitin sulfate, the reaction temperature in step (1) is preferably 30°C-40°C, and the stirring time is preferably 4-6 hours.

In the above method for preparing polysulfated chondroitin sulfate, the organic solvent in step (1) or (2) is one of methanol, ethanol, acetone, and isopropanol.

Wherein, the above-mentioned organic solvent is preferably ethanol with a concentration of 95% by volume.

In the preparation method of the above-mentioned polysulfated chondroitin sulfate, in the gel chromatography described in step (3), the gel used is one of Sephadex G-50, Sephadex G-75, Sephadex G-100, Superdex 200 one.

Wherein, the gel is preferably a Sephadex G-75 gel column of 3.5 × 120 cm.

In the above method for preparing polysulfated chondroitin sulfate, the eluent in step (3) is preferably a sodium chloride solution with a concentration of 0.2-0.5 mol/L.

The preparation method of the polysulfated chondroitin sulfate of the present invention has the characteristics of simple process, low reagent cost, less stringent production environment and equipment requirements, and high product purity, which is extremely beneficial to industrial production.

The polysulfated chondroitin sulfate prepared by the method of the invention has strong anti-inflammatory and analgesic effects. It has been verified by experiments that its anticoagulant potency is ≤10IU/mg, and it also has obvious effects of anti-tumor proliferation, inhibition of tumor metastasis, and inhibition of mast cell degranulation.

Detailed ways

The content of the present invention will be further elaborated below in conjunction with the embodiments.

Example 1

(1) Polysulfation of chondroitin sulfate: 100 g of chondroitin sulfate boutique (commercially available) is made into a solution with a mass percent concentration of 20%, and chlorosulfonic acid: concentrated sulfuric acid is 1: 0.05 mixed acid is added in the above solution , so that the final concentration of chondroitin sulfate mass percent is 10%, at a temperature of 35° C., stirring and reacting for 5 hours, then adding 4000 ml of absolute ethanol to terminate the reaction, and there will be more sulfated chondroitin sulfate precipitates;

(2) Separation of the required polysulfated chondroitin sulfate fragments: Dissolve the above precipitate with 1000ml distilled water to make an aqueous solution, adjust the pH to 7 with 20% sodium hydroxide, add 4 times the amount of 95% ethanol to make it Precipitation; then make the precipitate into a 10% aqueous solution, adjust the pH to 10 with 20% sodium hydroxide, heat to 85°C, and continuously perform gas exchange in the reactor, keep it warm for 3 hours, and then adjust the pH with 6mol/L hydrochloric acid To 7, add 4 times the amount of 95% 7 alcohol to precipitate it to achieve desalination and amine removal;

(3) Collection and preparation of polysulfated chondroitin sulfate: the precipitate finally obtained in step (2) is made into a 10% aqueous solution with a concentration of 10% by mass, and separated on a Sephadex G-75 gel column (3.5 × 120cm), and the concentration is It was eluted with 0.2mol/L sodium chloride solution, and the part with high content of polysulfated chondroitin sulfate was collected, desalted with Sephadex G-10, and conventionally freeze-dried polysulfated chondroitin sulfate.

The polysulfated chondroitin sulfate obtained above is subjected to molecular weight determination, structure determination and anti-inflammatory activity determination respectively to obtain a polysulfated chondroitin sulfate, which contains glucuronic acid derivatives and acetylgalactosamine derivatives in the molecule, and the molecular structure Composed of 2-O-sulfate-glucuronic acid-(1→3)-2-N-acetyl-2-deoxy-4 or 6-O-sulfate galactose, its main chemical structure is as follows:

The polysulfated chondroitin sulfate has a relative molecular weight of 1kD to 30kD, R is _SO3 or H, n is 1 to 30, the ratio of sulfate to carboxylate is 2 to 4, and has obvious inhibitory effect on mast cell degranulation, Anticoagulant titer ≤ 10IU/mg. It also has obvious anti-tumor proliferation and inhibition of tumor metastasis, etc., and has strong anti-inflammatory and analgesic effects.

Example 2

(1) Polysulfation of chondroitin sulfate: 200 g of chondroitin sulfate boutique (commercially available) is made into a solution with a mass percentage concentration of 30%, and chlorosulfonic acid: concentrated sulfuric acid is 1: 0.07 mixed acid is added in the above solution , so that the final concentration of chondroitin sulfate mass percent is 10%, at a temperature of 40 ° C, stirring and reacting for 6 hours, then adding 4000ml of absolute ethanol to terminate the reaction, and more sulfated chondroitin sulfate precipitates;

(2) Separation of the required polysulfated chondroitin sulfate fragments: Dissolve the above precipitate with 1000ml distilled water to make an aqueous solution, adjust the pH to 7 with 20% sodium hydroxide, and add 5 times the amount of 95% methanol to make it Precipitation; then make the precipitate into a 10% aqueous solution, adjust the pH to 10 with 20% sodium hydroxide, heat to 85°C, and continuously perform gas exchange in the reactor, keep it warm for 3.5 hours, and then adjust the pH with 6mol/L hydrochloric acid To 7, add 5 times the amount of 95% methanol to precipitate it to achieve desalination and amine removal;

(3) Collecting and preparing polysulfated chondroitin sulfate: the precipitate finally obtained in step (2) is made into a 10% aqueous solution with a concentration of 10% by mass, separated on a Sephadex G-100 gel column (3.5 × 120cm), and separated by concentration It was eluted with 0.5mol/L sodium chloride solution, and the part with high content of polysulfated chondroitin sulfate was collected, desalted with Sephadex G-10, and routinely freeze-dried polysulfated chondroitin sulfate.

Example 3

(1) Polysulfation of chondroitin sulfate: 100 g of chondroitin sulfate boutique (commercially available) is made into a solution with a mass percentage concentration of 25%, and chlorosulfonic acid: concentrated sulfuric acid is 1: 0.09 mixed acid is added in the above solution , making the final concentration of chondroitin sulfate mass percent 10%, at a temperature of 30° C., stirring and reacting for 10 hours, then adding 4000 ml of acetone to terminate the reaction, resulting in more sulfated chondroitin sulfate precipitates;

(2) Separation of the required polysulfated chondroitin sulfate fragments: dissolve the above precipitate with 1000ml of distilled water to make an aqueous solution, adjust the pH to 7 with 20% sodium hydroxide, add 4 times the amount of acetone to make it precipitate; Then make the precipitate into 10% aqueous solution, adjust the pH to 10 with 20% sodium hydroxide, heat to 85°C, and continuously carry out gas exchange in the reactor, keep it warm for 3 hours, then adjust the pH to 7 with 6mol/L hydrochloric acid , add 4 times the amount of acetone to precipitate it, to achieve desalination and amine removal;

(3) Collecting and preparing polysulfated chondroitin sulfate: the precipitate finally obtained in step (2) is made into a 10% aqueous solution with a concentration of 10% by mass, and separated on a Sephadex G-50 gel column (3.5 × 120cm) to determine the concentration It was eluted with 0.8mol/L sodium chloride solution, and the part with high content of polysulfated chondroitin sulfate was collected, desalted with Sephadex G-10, and conventionally freeze-dried polysulfated chondroitin sulfate.

Claims (10)

1. A polysulfated chondroitin sulfate, containing glucuronic acid derivatives and acetylgalactosamine derivatives in the molecule, is characterized in that: molecular structure is by 2-O-sulfate group-glucuronic acid-(1→3) -2-N-acetyl-2-deoxy-4 or 6-O-sulfate galactose, its chemical structure is as follows:

Wherein: R is SO ₃ or H, and n is 1 to 30. 2. polysulfated chondroitin sulfate according to claim 1 is characterized in that: the molecular weight of described polysulfated chondroitin sulfate is 1kD～30kD, and the ratio of sulfate and carboxylate in its molecular structure is 2～4 . 3. the preparation method of polysulfated chondroitin sulfate described in claim 1 or 2, comprises the following steps: (1) The polysulfation of chondroitin sulfate: chondroitin sulfate is made into the solution that mass percentage concentration is 15%～30%, with chlorosulfonic acid: the mixed acid that concentrated sulfuric acid is 1: 0.05～0.1 joins in the above-mentioned solution, The final concentration of chondroitin sulfate by mass percentage is 10% to 15%, at a temperature of 10°C to 70°C, stirring and reacting for 2 to 10 hours, and then adding an organic solvent to terminate the reaction, so that more sulfated chondroitin sulfate precipitates; (2) Separation of the required polysulfated chondroitin sulfate fragments: make the above precipitate into an aqueous solution, adjust the pH to 7±0.2, add 4 to 5 times the amount of organic solvent to precipitate it; then make the precipitate into Aqueous solution, adjust pH to 10±0.2 with alkali, heat to 85℃±2℃, and continuously carry out gas exchange in the reactor, keep warm for 3±0.5 hours, then adjust pH to 7 with hydrochloric acid, add 4 to 5 times the amount Precipitate with organic solvent to realize desalination and amine removal; (3) Collecting and preparing polysulfated chondroitin sulfate: the precipitate finally obtained in step (2) is made into an aqueous solution with a concentration of 10% by mass, and is separated by gel chromatography to a concentration of 0.01 to 1.0 mol/L The sodium chloride solution was used for elution, and the part with high content of polysulfated chondroitin sulfate was collected, desalted with Sephadex G-10, and conventionally freeze-dried polysulfated chondroitin sulfate. 4. the preparation method of polysulfated chondroitin sulfate according to claim 3 is characterized in that: the chlorosulfonic acid described in step (1): concentrated sulfuric acid is 1: 0.05～0.07. 5. The preparation method of polysulfated chondroitin sulfate according to claim 3, characterized in that: the reaction temperature in step (1) is 30°C to 40°C, and the stirring time is 4 to 6 hours. 6. the preparation method of polysulfated chondroitin sulfate according to claim 3 is characterized in that: the organic solvent described in step (1) or (2) is one of methanol, ethanol, acetone, Virahol. 7. the preparation method of polysulfated chondroitin sulfate according to claim 6 is characterized in that: described organic solvent is the ethanol that volume percent concentration is 95%. 8. the preparation method of polysulfated chondroitin sulfate according to claim 3 is characterized in that: in the gel chromatography described in step (3), the gel that adopts is Sephadex G-50, Sephadex G-75 , Sephadex G-100, one of Superdex200. 9. the preparation method of polysulfated chondroitin sulfate according to claim 8 is characterized in that: the gel is a Sephadex G-75 gel column of 3.5 * 120cm. 10. The preparation method of polysulfated chondroitin sulfate according to claim 3, characterized in that: the eluent in step (3) is a sodium chloride solution with a concentration of 0.2 to 0.5 mol/L.