A high-efficiency extraction process of heparin sodium

Abstract

The invention discloses an efficient extraction process of sodium heparin, belongs to the technical field of sodium heparin extraction, and aims to improve the extraction efficiency and purity of sodium heparin, while reducing production costs and environmental impact. The process includes three main steps: first, mixing pig small intestinal mucosa with water, adding mixed proteases and adjusting the pH value, performing enzymolysis with the assistance of ultrasound, and then adding sodium chloride and heating precipitation; second, using a specially prepared modified adsorption resin for two times of adsorption and elution to enrich and purify sodium heparin; finally, removing impurities by ethanol precipitation and centrifugal separation to obtain a high-purity sodium heparin solution, and then drying to obtain a dry product. Compared with the prior art, the present invention has the advantages of high extraction efficiency, good purity, simple operation, low cost and environmental friendliness, and is suitable for industrial large-scale production of sodium heparin.

Description

Technical Field

[0001] The invention relates to the technical field of efficient extraction of heparin sodium, in particular to a process for efficient extraction of heparin sodium. Background Art

[0002] As an important anticoagulant drug, heparin sodium plays an irreplaceable role in the medical field. It is mainly extracted from animal tissues, especially from the mucosa of the small intestine of pigs. Due to the growing clinical demand for heparin sodium, it is particularly important to develop an efficient, economical and scalable extraction process.

[0003] Traditional methods for extracting sodium heparin have some limitations, mainly including: Traditional methods usually rely on a long salting out or acid hydrolysis process, which has low extraction efficiency and cannot meet the needs of large-scale production. Due to the lack of efficient separation and purification methods, the sodium heparin obtained by traditional processes is not of high purity and may contain a variety of impurities, affecting the safety and effectiveness of the drug. The chemical reagents used in traditional processes and the large amount of water consumption, as well as the wastewater generated, put a certain amount of pressure on the environment.

[0004] In order to overcome the above problems, enzymatic hydrolysis has attracted widespread attention in recent years due to its high efficiency and mildness. Enzymatic hydrolysis uses specific proteases to break down the proteins around heparin sodium, thereby releasing heparin sodium. In addition, the application of adsorption resins also provides new possibilities for the purification of heparin sodium. However, the efficient extraction process of heparin sodium combined with ultrasound-assisted enzymatic hydrolysis and adsorption resin still has the problem of low extraction rate and purity of heparin sodium.

[0005] China's invention patent CN103848929B discloses an efficient extraction process for sodium heparin, which specifically includes enzymolysis of a mixture of intestinal mucosa and water, wherein the ratio of intestinal mucosa to water is 1:4, and then conventional adsorption, incremental adsorption, elution, precipitation purification, and drying are performed to obtain sodium heparin. Since the extraction process of the invention adds secondary enzymolysis and incremental adsorption, the yield of sodium heparin is improved, and the purity of the obtained sodium heparin is also improved. Specifically, the yield of sodium heparin can reach 100 million IU / 1200-1400 pig intestines, which is 30-45% higher than the conventional process; the purity of the produced sodium heparin can reach 110-130 IU / mg; the efficient extraction process of the invention saves about 40% of water and 20-40% of salt, uses less water and salt, has high economic benefits, and is an efficient sodium heparin extraction process. However, the efficient sodium heparin extraction process of the invention has the problem of low extraction rate and purity of sodium heparin. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention solves the deficiencies in the prior art in terms of heparin sodium extraction efficiency, purity, cost, environmental impact and operational complexity through a series of innovative technical means, and provides a more efficient, environmentally friendly and economical heparin sodium extraction process.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] An efficient extraction process of heparin sodium is as follows, in parts by weight:

[0009] Step 1, 8 to 12 parts of small intestinal mucosa and water are mixed at a mass ratio of 1:3 to 5, 3 to 4 parts of mixed proteases are added, the pH value is adjusted to 7.5 to 9 with a 0.5 to 2 mol / L sodium hydroxide aqueous solution, ultrasonic enzymolysis is performed at 40 to 50° C., sodium chloride is added to adjust the salinity to 1.5 to 3 g / kg, and then stirring is continued for 2 to 4 hours, the temperature is raised to 70 to 80° C., and natural precipitation is performed for 5 to 15 hours, and the filtrate is collected by filtration;

[0010] Step 2, cooling the filtrate prepared in step 1 to 60-68° C., adding 2-4 parts of modified adsorption resin for stirring and adsorption for 2-4 hours, adding 0.5-2 parts of mixed protease for enzymolysis for 1-5 hours after the modified adsorption resin is adsorbed, and after enzymolysis, using 0.5-2 parts of modified adsorption resin for stirring and adsorption for 3-8 hours, filtering out the modified adsorption resin, and washing the modified adsorption resin with a 6-10wt% sodium chloride aqueous solution for 20-40 minutes, collecting the washing liquid, and then eluting the modified adsorption resin with a saturated sodium chloride aqueous solution for 3-8 hours, collecting the eluate, combining the eluate and the washing liquid to obtain a mixed solution, adding 90-98wt% ethanol aqueous solution until the ethanol concentration of the mixed solution is 35-45 degrees, standing for 5-15 hours, and collecting the precipitate;

[0011] Step 3, adding the precipitate obtained in step 2 to a 3-8wt% sodium chloride aqueous solution with a mass multiple of 15-25 times, centrifuging at 14000-16000 rpm for 3-8 minutes to remove solid impurities to obtain a purified solution, adding 90-98wt% ethanol aqueous solution again until the ethanol concentration of the purified solution is 35-45 degrees, collecting the precipitate after standing for 5-15 hours, and drying the precipitate to obtain purified heparin sodium.

[0012] The small intestinal mucosa is one of pig small intestinal mucosa and sheep small intestinal mucosa.

[0013] The mixed protease is composed of papain and trypsin in a mass ratio of 1:1-3.

[0014] The conditions for ultrasonic enzymolysis are as follows: ultrasonic time is 30 to 50 minutes, frequency is 20 to 60 kHz, and power is 200 to 400 W.

[0015] The preparation method of the modified adsorption resin is as follows, in parts by weight:

[0016] 8 to 12 parts of styrene-divinylbenzene copolymer are dried in an oven at 60 to 75° C. for 3 to 5 hours to remove moisture, 0.5 to 1 part of a hydrophilic modifier is dissolved in 18 to 22 parts of a 40 to 60 wt% ethanol aqueous solution and mixed with the copolymer; 0.2 to 0.4 parts of a negative charge provider are mixed, and in the presence of 0.1 to 0.3 parts of an initiator, the mixture is reacted at 70 to 90° C. for 8 to 12 hours under a nitrogen atmosphere; 1.2 to 1.8 parts of a crosslinking agent are added, the mixture is mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a crosslinking reaction is carried out at 80 to 100° C. for 4 to 8 hours under a nitrogen atmosphere. After the reaction is completed, the solid is collected by filtration, washed with anhydrous ethanol and water for 1 to 3 times respectively, and dried at 30 to 50° C. to a constant weight to obtain a modified adsorption resin.

[0017] The hydrophilic modifier is at least one of maltodextrin, carboxymethyl cellulose, ascorbic acid, glycerol and polyvinyl pyrrolidone.

[0018] The initiator is dibenzoyl peroxide.

[0019] The cross-linking agent is at least one of 1,2,3-trivinylbenzene and divinylbenzene.

[0020] The negative charge providing agent is at least one of maleic anhydride, methacrylic acid and itaconic acid.

[0021] In the efficient heparin sodium extraction process of the present invention, each substance has a specific role:

[0022] Porcine small intestinal mucosa is used as the raw material for the extraction of heparin sodium because it is a natural source of heparin sodium.

[0023] The water is mixed with the pig small intestinal mucosa to form a slurry, which is convenient for the subsequent enzymatic hydrolysis and extraction process.

[0024] A mixed protease (papain and trypsin) is used in the enzymatic hydrolysis step to hydrolyze the proteins in the small intestinal mucosa and release heparin sodium.

[0025] The pH value was adjusted to 8 using 1 mol / L sodium hydroxide aqueous solution, which is the optimal pH range for the catalytic activity of the protease.

[0026] Ultrasound is used during the enzymatic hydrolysis process to help improve the efficiency of enzymatic hydrolysis and help open the tissue structure through mechanical action, making it easier for the enzyme to contact and act on the substrate.

[0027] Sodium chloride is used to adjust salinity, affecting protein solubility and release of heparin sodium.

[0028] The modified adsorption resin is used to adsorb and enrich heparin sodium. The modified resin has better hydrophilicity and selectivity, which helps to improve the extraction efficiency.

[0029] Maltodextrin, as a hydrophilic modifier, increases the hydrophilicity and biocompatibility of the resin and helps the adsorption of heparin sodium.

[0030] Itaconic acid, as a negative charge provider, introduces negative charges and increases the negative charge density of the resin, which may indirectly improve the adsorption efficiency of heparin sodium by improving the hydrophilicity and pore structure of the resin.

[0031] Dibenzoyl peroxide was used as an initiator to initiate the polymerization reaction between the resin and the modifier under a nitrogen atmosphere.

[0032] 1,2,3-Trivinylbenzene, as a crosslinking agent, reacts with styrene-divinylbenzene copolymer to form a three-dimensional network structure, thereby optimizing the structure of the modified adsorption resin.

[0033] Anhydrous ethanol is used in the washing step to remove impurities from the resin.

[0034] A 50 wt% ethanol aqueous solution was used as a solvent to dissolve maltodextrin and mix it with the copolymer.

[0035] 95 wt % ethanol aqueous solution is used in the precipitation step to induce precipitation of heparin sodium by increasing the ethanol concentration in the solution, thereby facilitating subsequent collection and purification.

[0036] The combined action of these substances enables the efficient extraction and purification of heparin sodium.

[0037] Compared with the prior art, it has the following beneficial effects:

[0038] 1) The present invention uses ultrasound-assisted enzymolysis to accelerate the release of heparin sodium and improve the extraction efficiency. High-purity heparin sodium can be obtained through two adsorption and elution steps and subsequent precipitation and purification processes.

[0039] 2) The present invention adopts maltodextrin as a hydrophilic modifier to improve the hydrophilicity and biocompatibility of the resin, and optimizes the adsorption structure of the resin through cross-linking through 1,2,3-trivinylbenzene, which is conducive to the adsorption of heparin sodium; the use of the modified adsorption resin improves the adsorption capacity and elution efficiency of heparin sodium, thereby improving the overall yield and potency.

[0040] 3) The present invention may improve the enzymatic hydrolysis efficiency, reduce the use of raw materials and reagents, and reduce costs by using mixed proteases, especially a specific ratio of papain and trypsin. DETAILED DESCRIPTION

[0041] Main sources of substances:

[0042] Papain: Guangzhou Guangyu Biotechnology Co., Ltd., enzyme activity: 100,000 u / g.

[0043] Trypsin: Ningxia Xiasheng Industrial Group Co., Ltd., product number: GDG-2009, enzyme activity: 200,000 u / g.

[0044] Rohm and Haas resin: Shanghai Kaizhi New Material Technology Co., Ltd., item number: 5325.

[0045] Styrene-divinylbenzene copolymer: Chengdu McCarthy Chemical Co., Ltd., reagent number: M021137.

[0046] Maltodextrin: Nanjing Jiayi Xinwei Chemical Co., Ltd., product number: JY6547.

[0047] Carboxymethyl cellulose: Renqiu Litian Chemical Co., Ltd., model: LT-011.

[0048] Polyvinyl pyrrolidone: Xi'an Tianmao Chemical Co., Ltd., model: ar.

[0049] The design concept of the present invention is to develop an efficient, stable and scalable heparin sodium extraction process. The design of this process takes into account several key factors to ensure high extraction rate and high purity of heparin sodium. The following are the main aspects of the design concept:

[0050] Porcine small intestinal mucosa was used as the main source of heparin sodium because it is an animal tissue with a high heparin sodium content.

[0051] Enzymatic hydrolysis is performed using a mixed protease (papain and trypsin) to improve the release efficiency of heparin sodium. The type and ratio of enzymes are carefully selected to ensure effective enzymatic hydrolysis of the proteins surrounding heparin sodium.

[0052] Ultrasound is used during the enzymatic hydrolysis process to improve the penetration ability and efficiency of the enzyme and accelerate the release of heparin sodium.

[0053] Maintain appropriate pH and temperature conditions to ensure enzyme activity and stable release of heparin sodium.

[0054] The salinity was adjusted by sodium chloride to optimize the solubility of sodium heparin and the subsequent precipitation conditions.

[0055] A modified adsorption resin was designed and synthesized. By introducing a hydrophilic modifier (maltodextrin) and a negative charge provider (itaconic acid), the negative charge density of the resin was increased, which may indirectly improve the adsorption efficiency of heparin sodium by improving the hydrophilicity and pore structure of the resin. 1,2,3-Trivinylbenzene was used as a cross-linking agent to form a stable three-dimensional network structure and optimize the adsorption structure of the resin.

[0056] The purity and yield of heparin sodium are improved by two adsorption steps and washing and elution with different sodium chloride solutions.

[0057] Heparin sodium is precipitated with ethanol and impurities are removed by centrifugation to obtain a high-purity heparin sodium solution. The purified heparin sodium solution is dried to obtain a dry heparin sodium product.

[0058] Environmental considerations such as the use of non-toxic solvents and reduced waste generation were taken into account throughout the process design, while optimizing cost effectiveness.

[0059] The design concept of the present invention reflects the careful consideration of each link in the process of sodium heparin extraction, and aims to achieve high efficiency, high purity and industrial production of sodium heparin extraction through scientific methods and process optimization.

[0060] Example 1

[0061] An efficient extraction process of heparin sodium is as follows:

[0062] Step 1, 10 kg of porcine small intestinal mucosa was mixed with water at a mass ratio of 1:4, 3.5 kg of mixed protease was added, the pH value was adjusted to 8 with a 1 mol / L sodium hydroxide aqueous solution, ultrasonic enzymolysis was performed at 45°C, the ultrasonic time was 40 minutes, the frequency was 40 kHz, the power was 320 W, sodium chloride was added to adjust the salinity to 2 g / kg, and then stirring was continued for 3 hours and then the temperature was raised to 75°C, natural precipitation was performed for 10 hours, and the filtrate was collected by filtration;

[0063] Step 2, cooling the filtrate prepared in step 1 to 65°C, adding 3kg of modified adsorption resin for stirring and adsorption for 3 hours, adding 1kg of mixed protease for enzymolysis for 3 hours after the modified adsorption resin is adsorbed, and then using 1kg of modified adsorption resin for stirring and adsorption for 6 hours, filtering out the modified adsorption resin, and washing the modified adsorption resin with 8wt% sodium chloride aqueous solution for 30 minutes, collecting the washing liquid, and then eluting the modified adsorption resin with saturated sodium chloride aqueous solution for 6 hours, collecting the eluate, combining the eluate and the washing liquid to obtain a mixed solution, adding 95wt% ethanol aqueous solution until the ethanol concentration of the mixed solution is 40 degrees, and collecting the precipitate after standing for 10 hours;

[0064] Step 3, adding the precipitate obtained in step 2 to a 5wt% sodium chloride aqueous solution with a mass multiple of 20 times, centrifuging at 15000 rpm for 5 minutes to remove solid impurities to obtain a purified solution, adding 95wt% ethanol aqueous solution again until the ethanol concentration of the purified solution is 40 degrees, collecting the precipitate after standing for 10 hours, and drying the precipitate to obtain purified heparin sodium.

[0065] The mixed protease is composed of papain and trypsin in a mass ratio of 1:2.

[0066] The preparation method of the modified adsorption resin is as follows:

[0067] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70°C for 4 hours to remove moisture, 0.8 kg of maltodextrin was dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; then 0.3 kg of itaconic acid was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80°C for 10 hours under a nitrogen atmosphere; 1.5 kg of 1,2,3-trivinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90°C for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40°C to constant weight to obtain a modified adsorption resin.

[0068] Example 2

[0069] An efficient extraction process of heparin sodium is basically the same as that of Example 1, the only difference being that the preparation method of the modified adsorption resin is different.

[0070] The preparation method of the modified adsorption resin is as follows:

[0071] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70°C for 4 hours to remove moisture, 0.8 kg of carboxymethyl cellulose was dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; then 0.3 kg of itaconic acid was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80°C for 10 hours under a nitrogen atmosphere; 1.5 kg of 1,2,3-trivinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90°C for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40°C to constant weight to obtain a modified adsorption resin.

[0072] Example 3

[0073] An efficient extraction process of heparin sodium is basically the same as that of Example 1, the only difference being that the preparation method of the modified adsorption resin is different.

[0074] The preparation method of the modified adsorption resin is as follows:

[0075] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70°C for 4 hours to remove moisture, 0.8 kg of ascorbic acid was dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; then 0.3 kg of itaconic acid was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80°C for 10 hours under a nitrogen atmosphere; 1.5 kg of 1,2,3-trivinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90°C for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40°C to constant weight to obtain a modified adsorption resin.

[0076] Example 4

[0077] An efficient extraction process of heparin sodium is basically the same as that of Example 1, the only difference being that the preparation method of the modified adsorption resin is different.

[0078] The preparation method of the modified adsorption resin is as follows:

[0079] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70°C for 4 hours to remove moisture, 8 g of glycerol was dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; then 0.3 kg of itaconic acid was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80°C for 10 hours under a nitrogen atmosphere; 1.5 kg of 1,2,3-trivinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90°C for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40°C to constant weight to obtain a modified adsorption resin.

[0080] Example 5

[0081] An efficient extraction process of heparin sodium is basically the same as that of Example 1, the only difference being that the preparation method of the modified adsorption resin is different.

[0082] The preparation method of the modified adsorption resin is as follows:

[0083] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70°C for 4 hours to remove moisture, 0.8 kg of polyvinyl pyrrolidone was dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; then 0.3 kg of itaconic acid was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80°C for 10 hours under a nitrogen atmosphere; 1.5 kg of 1,2,3-trivinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90°C for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40°C to constant weight to obtain a modified adsorption resin.

[0084] Example 6

[0085] An efficient extraction process of heparin sodium is basically the same as that of Example 1, the only difference being that the preparation method of the modified adsorption resin is different.

[0086] The preparation method of the modified adsorption resin is as follows:

[0087] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70° C. for 4 hours to remove moisture, 0.4 kg of maltodextrin and 0.4 kg of carboxymethyl cellulose were dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; 0.3 kg of itaconic acid was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80° C. for 10 hours under a nitrogen atmosphere; 1.5 kg of 1,2,3-trivinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90° C. for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40° C. to constant weight to obtain a modified adsorption resin.

[0088] Example 7

[0089] An efficient extraction process of heparin sodium is basically the same as that of Example 1, the only difference being that the preparation method of the modified adsorption resin is different.

[0090] The preparation method of the modified adsorption resin is as follows:

[0091] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70°C for 4 hours to remove moisture, 0.8 kg of maltodextrin was dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; 0.3 kg of maleic anhydride was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80°C for 10 hours under a nitrogen atmosphere; 1.5 kg of 1,2,3-trivinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90°C for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40°C to constant weight to obtain a modified adsorption resin.

[0092] Example 8

[0093] An efficient extraction process of heparin sodium is basically the same as that of Example 1, the only difference being that the preparation method of the modified adsorption resin is different.

[0094] The preparation method of the modified adsorption resin is as follows:

[0095] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70° C. for 4 hours to remove moisture, 0.8 kg of maltodextrin was dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; 0.3 kg of methacrylic acid was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80° C. for 10 hours under a nitrogen atmosphere; 1.5 kg of 1,2,3-trivinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90° C. for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40° C. to constant weight to obtain a modified adsorption resin.

[0096] Example 9

[0097] An efficient extraction process of heparin sodium is basically the same as that of Example 1, the only difference being that the preparation method of the modified adsorption resin is different.

[0098] The preparation method of the modified adsorption resin is as follows:

[0099] 10 kg of styrene-divinylbenzene copolymer was dried in an oven at 70°C for 4 hours to remove moisture, 0.8 kg of maltodextrin was dissolved in 20 kg of 50 wt% ethanol aqueous solution and mixed with the copolymer; then 0.3 kg of itaconic acid was mixed, and in the presence of 0.2 kg of dibenzoyl peroxide, the mixture was reacted at 80°C for 10 hours under a nitrogen atmosphere; 1.5 kg of divinylbenzene was added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and a cross-linking reaction was carried out at 90°C for 6 hours under a nitrogen atmosphere. After the reaction was completed, the solid was collected by filtration, washed twice with anhydrous ethanol and water respectively, and dried at 40°C to constant weight to obtain a modified adsorption resin.

[0100] Comparative Example 1

[0101] An efficient extraction process of heparin sodium is as follows:

[0102] Step 1, 10 kg of small intestinal mucosa was mixed with water at a mass ratio of 1:4, 3.5 kg of mixed protease was added, the pH value was adjusted to 8 with a 1 mol / L sodium hydroxide aqueous solution, ultrasonic enzymolysis was performed at 45°C, the ultrasonic time was 40 minutes, the frequency was 40 kHz, the power was 320 W, sodium chloride was added to adjust the salinity to 2 g / kg, and then stirring was continued for 3 hours, the temperature was raised to 75°C, natural precipitation was performed for 10 hours, and the filtrate was collected by filtration;

[0103] Step 2, cooling the filtrate prepared in step 1 to 65°C, adding 3kg of Rohm and Haas resin for stirring and adsorption for 3 hours, adding 1kg of mixed protease for enzymolysis for 3 hours after the Rohm and Haas resin is adsorbed, and then using 1kg of Rohm and Haas resin for stirring and adsorption for 6 hours again after the enzymolysis, filtering out the Rohm and Haas resin, and washing the Rohm and Haas resin with 8wt% sodium chloride aqueous solution for 30 minutes, collecting the washing liquid, and then eluting the Rohm and Haas resin with saturated sodium chloride aqueous solution for 6 hours, collecting the eluate, combining the eluate and the washing liquid to obtain a mixed solution, adding 95wt% ethanol aqueous solution until the ethanol concentration of the mixed solution is 40 degrees, and collecting the precipitate after standing for 10 hours;

[0104] Step 3, adding the precipitate obtained in step 2 to a 5wt% sodium chloride aqueous solution with a mass multiple of 20 times, centrifuging at 15000 rpm for 5 minutes to remove solid impurities to obtain a purified solution, adding 95wt% ethanol aqueous solution again until the ethanol concentration of the purified solution is 40 degrees, collecting the precipitate after standing for 10 hours, and drying the precipitate to obtain purified heparin sodium.

[0105] The mixed protease papain and trypsin are composed in a mass ratio of 1:2.

[0106] Test Example 1

[0107] Heparin sodium extraction rate test

[0108] The extraction process according to the embodiments of the present invention and the comparative examples is operated, including steps such as enzymatic hydrolysis, precipitation, adsorption, elution, and purification. The content of heparin sodium in the extract is quantitatively determined by the absorbance of heparin sodium at a specific wavelength. The extraction rate is calculated based on the measured heparin sodium content and the amount of raw materials used. The calculation formula for the extraction rate is:

[0109] Extraction rate (%) = (measured heparin sodium content / theoretical heparin sodium content) × 100%

[0110] In order to ensure the accuracy and repeatability of the test results, each experimental condition was repeated at least three times and the average value was taken. The test results are shown in Table 1.

[0111] Table 1

[0112] Experimental protocol Heparin sodium extraction rate (%) Example 1 98.9 Example 2 99.1 Example 3 98.2 Example 4 98.3 Example 5 98.0 Example 6 99.4 Example 7 98.4 Example 8 98.2 Example 9 98.1 Comparative Example 1 96.1

[0113] Test Example 2

[0114] Heparin Sodium Potency Test

[0115] The heparin sodium purified in the examples of the present invention and the comparative examples, the heparin sodium titer test generally refers to the determination of the anticoagulant activity of heparin sodium, because its titer is directly related to its effectiveness in clinical application. A series of heparin sodium standard solutions with known concentrations are prepared for establishing a standard curve.

[0116] The purified heparin sodium of the present invention and the comparative example is dissolved in a solvent to prepare a test solution. The titer of the heparin sodium is evaluated by measuring the change in plasma coagulation time after the addition of the heparin sodium. The sample solution and the standard solution are added to a test system containing a coagulation system. The reaction time of each sample and standard is recorded, such as the coagulation time or other related indicators. A standard curve is drawn according to the reaction time of the standard. The titer of the heparin sodium is calculated according to the reaction time of the sample and the standard curve.

[0117] To ensure the accuracy and repeatability of the results, each sample should be tested at least three times independently. The test results are shown in Table 2.

[0118] Table 2

[0119] Experimental protocol Heparin sodium titer (IU / mg) Example 1 139 Example 2 141 Example 3 134 Example 4 135 Example 5 128 Example 6 148 Example 7 135 Example 8 137 Example 9 127 Comparative Example 1 122

[0120] It can be seen from Test Examples 1 to 2 that the purified heparin sodium obtained in Example 6 of the present invention has a good extraction rate and potency.

[0121] The hydrophilicity, pore structure and mechanical stability of the modified adsorption resin are all key factors affecting the extraction rate and potency of heparin sodium. In Example 1, maltodextrin is used as a hydrophilic modifier, and maltodextrin is a polysaccharide composed of multiple glucose units. This polysaccharide structure provides abundant hydroxyl groups (-OH), which increases the hydrophilicity of the resin. The hydrophilic modifier helps to improve the dispersibility and stability of the resin in the aqueous phase system, thereby improving the adsorption efficiency of heparin sodium. Itaconic acid, as a negative charge provider, can react with the benzene ring structure of the resin, introduce a carboxyl group (-COOH) to form a negative charge. Increasing the negative charge density of the resin may indirectly improve the adsorption efficiency of heparin sodium by improving the hydrophilicity and pore structure of the resin. 1,2,3-trivinylbenzene is used as a cross-linking agent to form a three-dimensional network structure in combination with styrene-divinylbenzene copolymer. This structure provides more adsorption sites and channels for heparin sodium molecules, which helps to improve adsorption capacity and efficiency. In the final precipitation step, the addition of a high concentration of ethanol aqueous solution helped to induce the aggregation and precipitation of heparin sodium molecules, while the hydrophilicity and negative charge properties of the modified resin may promote this process and further improve the precipitation efficiency and purity.

[0122] The maltodextrin in Example 1 and the carboxymethyl cellulose in Example 2 are both polysaccharide derivatives with more hydrophilic functional groups, such as hydroxyl (-OH). These functional groups can form hydrogen bonds with water molecules, increase the solubility and stability of the resin in aqueous solution, and thus improve the adsorption efficiency of heparin sodium. Although the ascorbic acid in Example 3, the glycerol in Example 4, and the polyvinyl pyrrolidone in Example 5 are also hydrophilic, their molecular structures and the distribution of hydroxyl groups are different from those of maltodextrin and carboxymethyl cellulose, which may affect their interaction with heparin sodium. The high molecular weight of maltodextrin and carboxymethyl cellulose may cause the modified resin to have a better pore structure, providing more adsorption sites and channels for heparin sodium molecules. In contrast, ascorbic acid, glycerol, and polyvinyl pyrrolidone may not be able to form the same favorable pore structure, thereby affecting the adsorption efficiency.

[0123] The mode of adopting maltodextrin and carboxymethyl cellulose compound in embodiment 6 may produce synergistic effect, thereby improving the extraction rate and titer of heparin sodium. Maltodextrin and carboxymethyl cellulose are both hydrophilic polymers, and their compounding may form more hydrophilic sites in resin, increase the interaction between resin and heparin sodium molecule, thereby improving adsorption efficiency. The compounding of two kinds of modifiers may help to form a more optimized pore structure, which not only increases the adsorption site of heparin sodium, but also may improve the release of heparin sodium in the elution process, thereby improving the extraction rate. The two kinds of modifiers may also form additional interactions between molecules, such as hydrogen bonds, which may enhance the overall structure of the resin and improve its adsorption capacity to heparin sodium. In summary, the compounding of maltodextrin and carboxymethyl cellulose in embodiment 6 may produce synergistic effect on multiple levels, thereby improving the extraction rate and titer of heparin sodium.

[0124] Compared with itaconic acid in Example 1, maleic anhydride and methacrylic acid in Example 7 and Example 8 are less effective. Both maleic anhydride and methacrylic acid contain a double bond and can undergo copolymerization with the resin. However, the number of negative charges they provide may be different from that of itaconic acid. Itaconic acid has two carboxyl groups, which can introduce more negative charges, while maleic anhydride and methacrylic acid each have only one carboxyl group. Increasing the negative charge density of the resin may indirectly improve the adsorption efficiency of heparin sodium by improving the hydrophilicity and pore structure of the resin.

[0125] The main difference in chemical structure between the divinylbenzene used in Example 9 and the 1,2,3-trivinylbenzene used in Example 1 is the number of their functional groups and cross-linking ability. 1,2,3-trivinylbenzene contains three double bonds, which can provide more cross-linking points and form a denser three-dimensional network structure with more uniform and more suitable characteristics. This structure may increase the porosity and specific surface area of ​​the resin and provide more adsorption sites for heparin sodium. In contrast, divinylbenzene has only two double bonds, and the cross-linked network that may be formed is relatively sparse, and the pore structure is not optimized enough, resulting in reduced porosity and a reduction in the number of adsorption sites.

[0126] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. An efficient extraction process of heparin sodium, characterized in that: The process is as follows, in parts by weight: Step 1, 8-12 parts of small intestinal mucosa and water are mixed at a mass ratio of 1:3-5, 3-4 parts of mixed protease are added, the pH value is adjusted to 7.5-9 with 0.5-2 mol / L sodium hydroxide aqueous solution, ultrasonic enzymolysis is performed at 40-50°C, sodium chloride is added to adjust the salinity to 1.5-3 g / kg, and then stirring is continued for 2-4 hours, the temperature is raised to 70-80°C, and natural precipitation is performed for 5-15 hours, and the filtrate is collected by filtration; Step 2, cooling the filtrate prepared in step 1 to 60-68° C., adding 2-4 parts of modified adsorption resin for stirring and adsorption for 2-4 hours, adding 0.5-2 parts of mixed protease for enzymolysis for 1-5 hours after the modified adsorption resin is adsorbed, and after enzymolysis, using 0.5-2 parts of modified adsorption resin for stirring and adsorption for 3-8 hours, filtering out the modified adsorption resin, and washing the modified adsorption resin with a 6-10wt% sodium chloride aqueous solution for 20-40 minutes, collecting the washing liquid, and then eluting the modified adsorption resin with a saturated sodium chloride aqueous solution for 3-8 hours, collecting the eluate, combining the eluate and the washing liquid to obtain a mixed solution, adding 90-98wt% ethanol aqueous solution until the ethanol concentration of the mixed solution is 35-45 degrees, standing for 5-15 hours and collecting the precipitate; Step 3, adding the precipitate obtained in step 2 to a 3-8wt% sodium chloride aqueous solution with a mass multiple of 15-25 times, centrifuging at 14000-16000 rpm for 3-8 minutes to remove solid impurities to obtain a purified solution, adding 90-98wt% ethanol aqueous solution again until the ethanol concentration of the purified solution is 35-45 degrees, standing for 5-15 hours, collecting the precipitate, and drying the precipitate to obtain purified heparin sodium; The mixed protease is composed of papain and trypsin in a mass ratio of 1:1-3; The conditions of ultrasonic enzymolysis are as follows: ultrasonic time is 30-50 minutes, frequency is 20-60kHz, and power is 200-400W; The preparation method of the modified adsorption resin is as follows, in parts by weight: 8-12 parts of styrene-divinylbenzene copolymer are dried in an oven at 60-75°C for 3-5 hours to remove moisture, 0.5-1 parts of a hydrophilic modifier are dissolved in 18-22 parts of a 40-60 wt% ethanol aqueous solution and mixed with the copolymer; Then, 0.2-0.4 parts of a negative charge provider are mixed, and in the presence of 0.1-0.3 parts of an initiator, the mixture is reacted at 70-90° C. for 8-12 hours under a nitrogen atmosphere; 1.2-1.8 parts of a crosslinking agent are added, mixed with the copolymer, the hydrophilic modifier and the negative charge provider, and the crosslinking reaction is carried out at 80-100° C. for 4-8 hours under a nitrogen atmosphere. After the reaction is completed, the solid is collected by filtration, washed with anhydrous ethanol and water for 1-3 times respectively, and dried at 30-50° C. to constant weight to obtain a modified adsorption resin; The hydrophilic modifier is at least one of maltodextrin, carboxymethyl cellulose, ascorbic acid, glycerol, and polyvinyl pyrrolidone; The initiator is dibenzoyl peroxide; The cross-linking agent is at least one of 1,2,3-trivinylbenzene and divinylbenzene; The negative charge providing agent is at least one of maleic anhydride, methacrylic acid and itaconic acid.

2. The efficient extraction process of heparin sodium as claimed in claim 1, characterized in that: The small intestinal mucosa is one of pig small intestinal mucosa and sheep small intestinal mucosa.