CN112279936B

CN112279936B - Preparation method of heparinoids

Info

Publication number: CN112279936B
Application number: CN202011258271.9A
Authority: CN
Inventors: 骆峰; 李向宇; 杨升平; 董婷婷
Original assignee: Shanghai Huiwen Biotech Corp ltd
Current assignee: Shanghai Huiwen Biotech Corp ltd
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2023-06-30
Anticipated expiration: 2040-11-12
Also published as: CN112279936A

Abstract

The invention provides a preparation method of heparinoids, which comprises the following specific steps: chondroitin sulfate is used as a raw material, and refined heparan is obtained through polysulfation, nanofiltration impurity removal, column treatment, hydrogen peroxide, nanofiltration desalination and freeze drying. The preparation method of the heparinoid sodium has the advantages of simple synthesis process, easy control of production operation, no special requirement on production equipment, less use of organic reagents, environmental protection, easy treatment, stable molecular weight range of products, physical and chemical indexes controlled within the quality standard range, normal product yield, high purity and less impurities, and is suitable for large-scale industrial production.

Description

Preparation method of heparinoids

Technical Field

The invention relates to the technical field of chemical industry, in particular to a preparation method of heparinoids.

Background

Heparinoids, i.e. heparinoids, are acidic mucopolysaccharides with anticoagulant activity, which are similar to heparin in chemical structure to some extent. The heparinoids can promote the release of endogenous aminopolysaccharides with antithrombotic activity by combining with the surface of vascular endothelium, can improve local microcirculation, increase blood flow and blood flow speed, thereby improving intermediate metabolism, promoting the supply of skin nutrition and the excretion of wastes, and has the effects of resisting thrombosis, diminishing inflammation, relieving pain, improving the blood circulation of affected parts, absorbing exudates, curing edema and edema, promoting tissue repair and the like; under the synergistic effect of the cutin softening agent and the natural humectant, the excessive growth of collagen fibers can be inhibited, and the scar formation is lightened; and can retain skin surface moisture, soften collagen fiber, enhance elasticity, and soften hypertrophic scar. Is clinically applicable to vascular embolism, varicose vein, superficial phlebitis, scar softening and the like.

Meanwhile, the heparan prepared by using the chondroitin sulfate is used as sulfated polysaccharide, has wide antibacterial spectrum, has good inhibition effect on a plurality of pathogenic bacteria such as suppurative bacteria (staphylococcus aureus), intestinal flora (escherichia coli), pathogenic fungi (candida albicans), viruses (poliovirus) and the like, can effectively control inflammatory diseases, improve the blood circulation of affected parts, absorb seepage and cure edema and edema.

Petitou et al have disclosed a method for preparing sulfated chondroitin sulfate in U.S. patent "preparation of chondroitin sulfate and dermatan sulfate sulfated chondroitin sulfate derivatives" (US 5382570), but the method has the characteristics of complex process, high reagent cost, being unfavorable for industrial production, and different structures and activities of the obtained products, thereby limiting the application thereof.

Patent specification CN1789287a discloses a polysulfated chondroitin sulfate and a preparation method thereof, wherein a method for preparing the polysulfated chondroitin sulfate by using chlorosulfonic acid and concentrated sulfuric acid is disclosed, but the chlorosulfonic acid and the concentrated sulfuric acid have high risk, are difficult to operate, have high requirements on equipment in industrial production, and are limited in application. In addition, the applicant repeats the above documents, and the yield of the product is low, and the operation difficulty is high, so that the method is not suitable for large-scale industrial production.

Patent specification with publication number CN104877042a discloses a method for preparing heparinoids by using different sulfonating agents, but there is not much data about heparinoids, and when the applicant repeats the above-mentioned literature method, the product has more impurities and uneven molecular weight distribution, which is not beneficial to the use of the product, and more organic reagents are introduced, which is not suitable for large-scale industrial production.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provide a preparation method of heparinoids, which has the advantages of low cost, environmental protection, few organic reagents, easy grasp, stable molecular weight, low impurity content, safety, effectiveness and stable sulfonation degree.

In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:

a method for preparing heparinoids, comprising the steps of:

1) Pretreatment: formamide and polar organic solvent according to the weight ratio of 1: mixing at a ratio of 1-10;

2) Sulfonation reaction: dissolving chondroitin sulfate in the solvent obtained in the step 1) with the volume of 6-10 times, uniformly mixing, adding pyridine sulfur trioxide, and reacting for 2-10 hours at the temperature of 50-90 ℃;

3) Nanofiltration amine: cooling the mixed solution in the step 2) to room temperature, regulating the pH value to 7, carrying out nanofiltration operation by using a nanofiltration membrane with a cutoff molecular weight of 300-3000, continuously adding deionized water during the nanofiltration operation, and stopping adding the deionized water when the conductivity of the permeate is measured to be 300-600; stopping nanofiltration operation when the ratio of the total weight of the solid to the volume of the intercepting liquid is 10-50% W/V, and collecting the liquid;

4) Deproteinizing the ion exchange resin: sequentially passing the liquid obtained in the step 3) through a strong acid cation exchange resin and a strong alkaline anion exchange resin, carrying out macromolecular protein treatment, collecting a part with pH less than 5, and then regulating the pH to 6.5-7.8 by alkali liquor;

5) Hydrogen peroxide decolorization: filtering and concentrating the solution obtained in the step 4), regulating the pH value to 9-11, and adding hydrogen peroxide for decoloring for 3-12h;

6) Nanofiltration for desalting and freeze drying: filtering and concentrating the solution obtained in the step 5), regulating the pH value to 6-7, carrying out nanofiltration operation by using a nanofiltration membrane, continuously adding deionized water during the nanofiltration operation, and stopping adding the deionized water when the conductivity of the permeate is measured to be 1-300; stopping nanofiltration when the ratio of the total weight of the solid to the volume of the intercepting liquid is 10-80% W/V, collecting the liquid, and freeze-drying to obtain the heparinoid.

Further, in the preparation method of heparinoids as described above, in step 1), the polar organic solvent is N, N-dimethylformamide or pyridine.

Further, in the preparation method of heparinoids as described above, in step 1), formamide and a polar organic solvent are mixed according to the following ratio 1: mixing at a ratio of 1-5.

Further, in the preparation method of heparinoids as described above, in step 1), formamide and a polar organic solvent are mixed according to the following ratio 1: mixing in proportion of 1.

Further, in the preparation method of heparinoids as described above, in step 3), the nanofiltration membrane has a cutoff molecular weight of 300-3000.

Further, according to the preparation method of the heparinoids, in the step 4), the alkali liquor is a NaOH solution with the mass percent concentration of 30%.

Further, in the preparation method of heparinoids as described above, in the step 5), the concentration of the hydrogen peroxide is 30% by mass.

Further, in the preparation method of heparinoids as described above, in step 6), the nanofiltration membrane has a cutoff molecular weight of 300-3000.

The beneficial effects of the invention are as follows:

the preparation method of the heparinoid sodium provided by the invention has the advantages of simple synthesis process, easiness in control of production operation, no special requirement on production equipment, less use of organic reagents, environment friendliness, easiness in treatment, stable molecular weight range of products, physical and chemical indexes controlled within the quality standard range, normal product yield, high purity and few impurities, and is suitable for large-scale industrial production.

Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a molecular weight GPC chart of the first embodiment of the present invention.

FIG. 2 is a molecular weight GPC chart of comparative example one of the present invention.

FIG. 3 is a molecular weight GPC chart of comparative example II of the present invention.

FIG. 4 is a molecular weight GPC chart of comparative example III of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

A method for preparing heparinoids, comprising the steps of:

1) Pretreatment: formamide and N, N-dimethylformamide are mixed according to the proportion of 1:1, and 1000ml of the mixed solvent was prepared.

2) Sulfonation reaction: 100g of chondroitin sulfate is dissolved in 1000ml of the solvent in the step 1), and pyridine sulfur trioxide is added after uniform mixing, and the mixture is heated to 70 ℃ and stirred for reaction for 5 hours.

3) Nanofiltration amine: cooling the mixed solution in the step 2) to room temperature, regulating the pH value to 8+/-0.3 by using a NaOH solution with the mass percent concentration of 30%, adding 3L of deionized water, performing nanofiltration operation by using a nanofiltration membrane with the cutoff molecular weight of 300, continuously adding deionized water during the nanofiltration operation, measuring the conductivity of the permeate to be 350, stopping adding water, and discharging the liquid when the volume of the cutoff liquid is 500 ml.

4) Deproteinizing the ion exchange resin: the liquid obtained in the step 3) sequentially passes through 500ml of strong acid cation exchange resin and 500ml of strong alkaline anion exchange resin, the part with the PH less than 5 is collected, and then NaOH solution with the mass percent concentration of 30% is used for regulating the PH of the solution to 7+/-0.5.

5) Hydrogen peroxide decolorization: filtering and concentrating the solution obtained in the step 4) to 200ml, regulating the pH value to 10+/-0.3, and adding 15ml of 30% hydrogen peroxide solution by mass concentration for decolorization for 8 hours.

6) Nanofiltration for desalting and freeze drying: and 5) filtering and concentrating the solution obtained in the step 5), regulating the pH value to 7+/-0.3, carrying out nanofiltration operation by using a nanofiltration membrane with a cutoff molecular weight of 2000, adding deionized water all the time during the nanofiltration operation, and stopping adding water when the conductivity of the permeate is measured to be 200. Stopping adding water when the volume of the intercepting liquid is 500ml, collecting liquid, and freeze drying to obtain heparinoid.

The molecular weight GPC chart of this example can be seen in FIG. 1, and shows that the molecular weight distribution is uniform, only one peak is present in the chart, no other molecular size is substantially present, and the molecular weight is moderate.

The relevant safety and efficacy data are shown in table 2.

Comparative example one

A method for preparing heparinoids, comprising the steps of:

2) Sulfonation reaction: 100g of chondroitin sulfate is dissolved in 1000ml of the solvent in the step 1), pyridine sulfur trioxide is added after uniform mixing, heating is carried out to 70 ℃, stirring reaction is carried out for 5h, and NaOH solution with the mass percent concentration of 30% is used for adjusting the PH value to 8+/-0.3.

3) Deproteinizing the ion exchange resin: the liquid obtained in the step 2) sequentially passes through 500ml of strong acid cation exchange resin and 500ml of strong alkaline anion exchange resin, the part with the PH less than 5 is collected, and then NaOH solution with the mass percent concentration of 30% is used for regulating the PH of the solution to 7+/-0.5.

4) Hydrogen peroxide decolorization: filtering and concentrating the solution obtained in the step 3) to 200ml, regulating the pH value to 10+/-0.3, and adding 15ml of 30% hydrogen peroxide solution by mass concentration for decolorization for 8 hours.

5) Nanofiltration for desalting and freeze drying: and 4) filtering and concentrating the solution obtained in the step 4), regulating the pH value to 7+/-0.3, carrying out nanofiltration operation by using a nanofiltration membrane with a cutoff molecular weight of 2000, adding deionized water all the time during the nanofiltration operation, and stopping adding water when the conductivity of the permeate is measured to be 200. Stopping adding water when the volume of the intercepting liquid is 500ml, collecting liquid, and freeze drying to obtain heparinoid.

Compared to embodiment one: comparative example one did not have a nanofiltration amine step, and the subsequent steps were followed in the same manner, and the nitrogen content was far higher than in example one of the present invention when the same test was finally performed, and specific tests were compared with table 1 below.

The comparative example can be seen in FIG. 2, which shows that the molecular weight distribution is not uniform and that there are various molecular weight products.

Comparative example two

A method for preparing heparinoids, comprising the steps of:

2) Sulfonation reaction: 100g of chondroitin sulfate is dissolved in 1000ml of the solvent in the step (1), and pyridine sulfur trioxide is added after uniform mixing, and the mixture is heated to 70 ℃ and stirred for reaction for 5 hours.

3) Nanofiltration amine: cooling the mixed solution in the step (2) to room temperature, regulating the PH value to 8+/-0.3 by using a NaOH solution with the mass percent concentration of 30%, adding 3L of deionized water, carrying out nanofiltration operation by using a nanofiltration membrane with the cutoff molecular weight of 300, continuously supplementing deionized water during the nanofiltration operation, measuring the conductivity of the permeate to be 350, stopping adding water, and discharging liquid when the volume of the cutoff liquid is 500 ml.

4) Hydrogen peroxide decolorization: filtering and concentrating the solution obtained in the step (3) to 200ml, regulating the pH value to 10+/-0.3, and adding 15ml of 30% hydrogen peroxide solution by mass concentration for decolorization for 8 hours.

5) Nanofiltration for desalting and freeze drying: and (3) filtering and concentrating the solution obtained in the step (4), regulating the pH value to 7+/-0.3, carrying out nanofiltration operation by using a nanofiltration membrane with a cutoff molecular weight of 2000, adding deionized water all the time during the nanofiltration operation, and stopping adding water when the conductivity of the permeate is measured to be 200. Stopping adding water when the volume of the intercepting liquid is 500ml, collecting liquid, and freeze drying to obtain heparinoid.

Compared to embodiment one: comparative example II did not have the step of removing protein by ion resin, and the following steps were carried out according to the same steps, and the nitrogen content and the protein content were far higher than those of the first example of the present invention when the same detection was carried out finally, and the specific detection can be compared with the following table 1.

The comparative example can be seen in the molecular weight GPC chart of FIG. 3, which shows that the molecular weight distribution is not uniform, and that there are various molecular weight products.

The relevant safety and efficacy data are shown in table 2.

Comparative example three

A method for preparing heparinoids, comprising the steps of:

4) Deproteinizing the ion exchange resin: and (3) sequentially passing the liquid obtained in the step (3) through 500ml of strong acid cation exchange resin and 500ml of strong alkaline anion exchange resin, collecting a part with the PH less than 5, and then regulating the PH of the solution to 7+/-0.5 by using a NaOH solution with the mass percent concentration of 30%.

5) Hydrogen peroxide decolorization and freeze drying: filtering and concentrating the solution obtained in the step (4) to 200ml, regulating the pH value to 10+/-0.3, adding 15ml of 30% hydrogen peroxide solution by mass concentration for decolorization for 8 hours, and freeze-drying to obtain the heparinoid sample.

Compared to embodiment one: the third comparative example does not have a nanofiltration salt removal step, the subsequent steps are carried out according to the same steps, and when the same detection is carried out finally, the content of partial salt ions is far higher than that of the first embodiment of the invention, and the specific detection can be compared with the following table 1:

table 1 comparison table of test results

The comparative example can be seen in FIG. 4 for molecular weight GPC chart, which shows that the molecular weight distribution is not uniform, while there are various molecular weight products.

Relevant safety and efficacy data are shown in table 2:

table 2 related safety and efficacy data vs. table

The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.

Claims

1. A method for preparing heparinoids, comprising the steps of:

1) Pretreatment: formamide and N, N-dimethylformamide are mixed according to the proportion of 1:1, mixing in proportion, and preparing 1000ml of mixed solvent;

2) Sulfonation reaction: dissolving 100g of chondroitin sulfate in 1000ml of the solvent in the step 1), uniformly mixing, adding pyridine sulfur trioxide, heating to 70 ℃, and stirring for reaction for 5 hours;

3) Nanofiltration amine: cooling the mixed solution in the step 2) to room temperature, regulating the PH value to 8+/-0.3 by using a NaOH solution with the mass percent concentration of 30%, adding 3L of deionized water, carrying out nanofiltration operation by using a nanofiltration membrane with the cutoff molecular weight of 300, continuously adding deionized water during the nanofiltration operation, measuring the conductivity of the permeate to be 350, stopping adding water, and discharging liquid when the volume of the cutoff liquid is 500 ml;

4) Deproteinizing the ion exchange resin: sequentially passing the liquid obtained in the step 3) through 500ml of strong acid cation exchange resin and 500ml of strong alkaline anion exchange resin, collecting a part with the PH less than 5, and then regulating the PH of the solution to 7+/-0.5 by using a NaOH solution with the mass percent concentration of 30%;

5) Hydrogen peroxide decolorization: filtering and concentrating the solution obtained in the step 4) to 200ml, regulating the pH value to 10+/-0.3, and adding 15ml of 30% hydrogen peroxide solution by mass concentration for decolorization for 8 hours;

6) Nanofiltration for desalting and freeze drying: filtering and concentrating the solution obtained in the step 5), regulating the pH value to 7+/-0.3, carrying out nanofiltration operation by using a nanofiltration membrane with a cutoff molecular weight of 2000, adding deionized water all the time during the nanofiltration operation, and stopping adding water when the conductivity of the permeate is measured to be 200; stopping adding water when the volume of the intercepting liquid is 500ml, collecting liquid, freeze drying to obtain heparinoid,

the protein content of the heparinoids is <3%.