Method for extracting heparinoid from heparin sodium alcohol precipitation alcohol

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a method for extracting heparinoid from heparin sodium alcohol precipitation alcohol, which comprises the following steps: S1, pretreatment: filtering the collected heparin sodium alcohol precipitation alcohol to obtain clear filtrate; s2, concentration: concentrating the clarified filtrate in a reduced pressure distillation manner, and stopping distillation when the clarified filtrate is concentrated to 1 / 5-1 / 3 of the original volume to obtain a concentrated solution; s3, precipitation: slowly adding acetone into the concentrated solution while stirring, and precipitating and separating out the heparinoid to obtain a solid-liquid mixture. According to the method, a large amount of waste alcohol precipitation alcohol generated in the heparin sodium production process is deeply developed, the heparinoid product with the high additional value is extracted, waste of heparinoid resources is greatly reduced, and the economic benefits of enterprises are improved; meanwhile, organic substances in the waste liquid are recycled, so that the chemical oxygen demand of the discharged waste liquid is effectively reduced, the subsequent sewage treatment cost is reduced, and good environmental benefits are achieved.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically a method for extracting heparin-like substances from sodium heparin precipitated in alcohol. Background Technology

[0002] Heparinoids are a class of acidic mucopolysaccharides similar to heparin in chemical structure and biological activity, mainly existing in the form of sodium salts. They have significant pharmacological effects such as anticoagulation, antithrombosis, anti-inflammation, and improvement of microcirculation. In clinical medicine, heparinoids are often used to treat superficial phlebitis, soft tissue injuries, edema, and hematomas. In the cosmetics and daily chemical industry, due to their excellent moisturizing properties and ability to promote skin barrier repair, they are also widely used as active additives in high-end skin care products. Therefore, heparinoids have extremely high economic value and market demand.

[0003] Currently, existing heparin sodium production processes suffer from significant technical deficiencies in handling the high-value byproduct, heparin sodium ethanol precipitation, generated during key purification steps. Traditional treatment methods often focus on simple ethanol recovery, while discharging the dissolved heparin-like resources as wastewater or subjecting them to crude treatment. This approach not only results in a huge waste of valuable bioactive substances but also imposes a heavy environmental and economic burden on enterprises due to the high concentration of organic matter in the residual liquid.

[0004] Furthermore, existing technologies often employ atmospheric pressure and high-temperature evaporation during the recovery process, which may lead to the degradation of heat-sensitive heparin and the loss of its bioactivity. For the small amount of crude product recovered, the complex composition of alcohol precipitation, containing a large amount of inorganic salts and various small molecule impurities, coupled with the lack of efficient and gentle purification methods in current processes, results in a final heparin product with low purity and high salt content, making it difficult to directly meet the stringent requirements for raw materials in pharmaceutical preparations and high-quality cosmetics. Therefore, developing a heparin extraction method that integrates gentle concentration, efficient precipitation, and deep purification is of significant practical importance and an urgent technological need. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting heparin-like substances from heparin sodium ethanol precipitation, comprising the following steps: S1. Pretreatment: The collected heparin sodium precipitate is filtered to obtain a clear filtrate. S2. Concentration: The clarified filtrate is concentrated by vacuum distillation. When the volume is reduced to 1 / 5-1 / 3 of the original volume, distillation is stopped to obtain the concentrated liquid. S3. Precipitation: Slowly add acetone to the concentrated solution while stirring, so that heparin-like substances precipitate out to obtain a solid-liquid mixture; S4. Separation and washing: The solid-liquid mixture is separated by centrifugation, the supernatant is discarded, and the precipitate is collected. The collected precipitate is washed 2-3 times with anhydrous ethanol. Each time, the precipitate is resuspended in anhydrous ethanol, stirred evenly, and then centrifuged again to obtain the washed precipitate. S5. Drying: Place the washed precipitate in a vacuum drying oven and dry it until the precipitate reaches a constant weight to obtain crude heparin. S6. Purification: The crude heparin-like product is dissolved in deionized water and purified by passing it through an ion exchange chromatography column.

[0006] Preferably, in step S2, the distillation temperature is controlled at 40-50℃ and the vacuum degree is controlled at -0.08-0.09MPa.

[0007] Preferably, in step S3, the volume ratio of acetone to the concentrate is 1-2:1.

[0008] Preferably, in step S3, the stirring speed is controlled at 100-150 r / min, and the sedimentation time is 2-4 hours.

[0009] Preferably, in step S4, the centrifuge speed is set to 3000-4000 r / min, and the centrifugation time is 10-15 minutes.

[0010] Preferably, in step S5, the drying temperature of the drying oven is controlled at 30-40℃, the vacuum degree is -0.09-0.1MPa, and the drying time is 4-6 hours.

[0011] Preferably, in step S6, the crude heparin-like product is dissolved in deionized water and purified by ion exchange chromatography, including: A strongly basic anion exchange resin was selected. The column was first rinsed with deionized water, and then eluted with sodium chloride solution. The eluent containing heparin was collected. The eluent is dialyzed using a dialysis bag to remove salts and small molecule impurities. The dialysis solution was freeze-dried to obtain a high-purity heparin-like product.

[0012] Preferably, in step S6, the molar concentration of the sodium chloride solution is 0.5-1.5 mol / L.

[0013] Preferably, in step S6, the molecular weight cutoff of the dialysis bag is 1000-3000 Da, and the dialysis time is 12-24 hours.

[0014] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention addresses the deep development of a large amount of waste alcohol precipitation generated during the production of heparin sodium, extracting high-value-added heparin-like products, significantly reducing the waste of heparin-like resources, realizing the high-value utilization of by-products, and improving the economic benefits of enterprises; at the same time, by recovering the organic matter therein, the oxygen demand of the discharged waste liquid is effectively reduced, reducing the load and cost of subsequent sewage treatment, and has good environmental benefits. (2) In view of the heat sensitivity of heparin, the present invention adopts a vacuum distillation process in the concentration step to strictly control the temperature at 40-50℃. This mild concentration method avoids the sugar chain breakage or loss of biological activity that may be caused by traditional high temperature and normal pressure evaporation, and retains the anticoagulant and anti-inflammatory activities of heparin to the maximum extent, ensuring the efficacy and functionality of the final product. (3) In view of the problem of complex impurities and high inorganic salt content in alcohol precipitation, the present invention innovatively adopts a combination strategy of ion exchange chromatography + dialysis. Ion exchange chromatography utilizes the principle of specific adsorption to effectively separate heparin-like substances from impurities such as proteins and pigments. Dialysis treatment specifically solves the problem of sodium chloride residue introduced during the elution process and deeply removes inorganic salts and small molecule impurities. (4) In the precipitation step, the present invention introduces acetone as a precipitant and optimizes its ratio with the concentrate. Compared with ethanol precipitation alone, acetone can more significantly change the polarity of the solution and disrupt the dissolution balance of heparin, thereby making the precipitation of heparin more complete and faster and improving the recovery rate of heparin. Attached Figure Description

[0015] Figure 1 This is a schematic flowchart of the overall method in one embodiment of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] On the one hand, such as Figure 1 As shown, the present invention provides a method for extracting heparin-like substances from heparin sodium ethanol precipitation, comprising the following steps: S1. Pretreatment: The collected heparin sodium precipitate is filtered to obtain a clear filtrate. It should be noted that, for example, using a filter membrane with a pore size of 0.45 μm for vacuum filtration can effectively remove visible impurities, providing a relatively pure starting material for subsequent extraction steps; S2. Concentration: The clarified filtrate is concentrated by vacuum distillation. When the volume is reduced to 1 / 5 to 1 / 3 of the original volume, distillation is stopped to obtain the concentrated liquid. It should be noted that concentrating the filtrate by vacuum distillation can reduce the alcohol content of the solvent and increase the concentration of heparin in the solution. S3. Precipitation: Slowly add acetone to the concentrate while stirring, so that heparin-like substances precipitate out to obtain a solid-liquid mixture; It should be noted that the addition of acetone can change the polarity of the concentrate, disrupt the dissolution environment of heparin, and promote its precipitation. S4. Separation and Washing: The solid-liquid mixture is separated by centrifugation. The supernatant is discarded, and the precipitate is collected. The collected precipitate is washed 2-3 times with anhydrous ethanol. Each time, the precipitate is resuspended in anhydrous ethanol, stirred evenly, and then centrifuged again to obtain the washed precipitate. It should be noted that this step can remove impurities and residual acetone adsorbed on the surface of the solid-liquid mixture; S5. Drying: Place the washed precipitate in a vacuum drying oven and dry it until the precipitate reaches a constant weight to obtain crude heparin. S6. Purification: The crude heparin-like product is dissolved in deionized water and purified by passing it through an ion exchange chromatography column.

[0018] Furthermore, in step S2, the distillation temperature is controlled at 40-50℃, and the vacuum degree is controlled at -0.08-0.09MPa.

[0019] It should be noted that by controlling the distillation temperature and vacuum level, both rapid evaporation of alcohol can be ensured, while the degradation of heparin at high temperatures can be avoided.

[0020] Furthermore, in step S3, the volume ratio of acetone to concentrate is 1-2:1.

[0021] Furthermore, in step S3, the stirring speed is controlled at 100-150 r / min, and the sedimentation time is 2-4 hours.

[0022] Furthermore, in step S4, the centrifuge speed is set to 3000-4000 r / min, and the centrifugation time is 10-15 minutes.

[0023] Furthermore, in step S5, the drying temperature of the drying oven is controlled at 30-40℃, the vacuum degree is -0.09--0.1MPa, and the drying time is 4-6 hours.

[0024] Further, in step S6, the crude heparin-like product is dissolved in deionized water and purified by ion exchange chromatography, including: A strongly basic anion exchange resin was selected. The column was first rinsed with deionized water, and then eluted with sodium chloride solution. The eluent containing heparin was collected. The eluent is dialyzed using a dialysis bag to remove salts and small molecule impurities. The dialysis solution was freeze-dried to obtain a high-purity heparin-like product.

[0025] Furthermore, in step S6, the molar concentration of the sodium chloride solution is 0.5-1.5 mol / L.

[0026] Furthermore, in step S6, the molecular weight cutoff of the dialysis bag is 1000-3000 Da, and the dialysis time is 12-24 hours.

[0027] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0029] In this invention, the purity of the heparin-like product is determined by the carbazole-sulfuric acid method to determine the uronic acid content, and calculated by the normalization method of impurity peak area detection using high performance liquid chromatography (HPLC); the constant weight of the product refers to the difference between two consecutive drying weighings being less than 0.3 mg.

[0030] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0031] Example 1: A method for extracting heparin-like substances from sodium heparin ethanol precipitation, comprising the following steps: S1. Pretreatment: Take 10L of heparin sodium to precipitate alcohol, and filter under reduced pressure using a filter membrane with a pore size of 0.45μm to obtain a clear filtrate. S2. Concentration: Transfer the filtrate to a rotary evaporator, control the distillation temperature at 40℃ and the vacuum degree at -0.08MPa for reduced pressure distillation, and concentrate it to 1 / 5 of the original volume, i.e. 2L. S3. Precipitation: Slowly add 4L of acetone to the concentrate while stirring at 100r / min. The precipitation time is 2 hours. S4. Separation and washing: Transfer the precipitate to a centrifuge and centrifuge at 3000 r / min for 15 minutes to separate the precipitate; wash the precipitate with 500 mL of anhydrous ethanol, resuspend the precipitate and stir evenly, then centrifuge again at 3000 r / min for 10 minutes. Repeat the above washing operation once, that is, wash twice in total. S5. Drying: Place the washed precipitate in a vacuum drying oven and dry it for 6 hours at 30°C and a vacuum of -0.09 MPa to obtain crude heparin. S6. Purification: Dissolve the crude heparin in 500 mL of deionized water and pass it through a chromatographic column packed with D201 type strong basic anion exchange resin. First, rinse the column with deionized water, then elute with 0.5 mol / L sodium chloride solution and collect the eluent containing heparin. Place the eluent into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze it in deionized water for 12 hours. Finally, freeze-dry the dialyzed solution to obtain 10.5 g of high-purity heparin product, with a purity of 95%.

[0032] Example 2, a method for extracting heparin-like substances from sodium heparin ethanol precipitation, includes the following steps: S1. Pretreatment: Take 15L of heparin sodium to precipitate alcohol, and filter under reduced pressure using a filter membrane with a pore size of 0.45μm to obtain a clear filtrate. S2. Concentration: Transfer the filtrate to a rotary evaporator, control the distillation temperature at 45℃ and the vacuum degree at -0.085MPa, and carry out vacuum distillation to concentrate it to 1 / 3 of the original volume, i.e. 5L. S3, Precipitation: Add 10L of acetone to the concentrate while stirring at 120r / min. The precipitation time is 3 hours. S4. Separation and washing: Transfer the precipitate to a centrifuge and centrifuge at 4000 r / min for 12 minutes to separate the precipitate; wash the precipitate with 800 mL of anhydrous ethanol, resuspend the precipitate and stir evenly, then centrifuge again at 4000 r / min for 10 minutes. Repeat the above washing operation twice, for a total of 3 washes. S5. Drying: Place the washed precipitate in a vacuum drying oven and dry it for 6 hours at 35°C and a vacuum of -0.095MPa to obtain crude heparin. S6. Purification: The crude heparin was dissolved in 800 mL of deionized water and passed through a chromatographic column packed with D201 type strong basic anion exchange resin. The column was first rinsed with deionized water, and then eluted with 1 mol / L sodium chloride solution. The eluent containing heparin was collected. The eluent was placed in a dialysis bag with a molecular weight cutoff of 2000 Da and dialyzed in deionized water for 18 hours. Finally, the dialyzed solution was freeze-dried to obtain 16.8 g of high-purity heparin product, with a purity of 96%.

[0033] Example 3, a method for extracting heparin-like substances from sodium heparin ethanol precipitation, includes the following steps: S1. Pretreatment: Take 20L of heparin sodium to precipitate alcohol, and filter under reduced pressure using a filter membrane with a pore size of 0.45μm to obtain a clear filtrate; S2. Concentration: Transfer the filtrate to a rotary evaporator, control the distillation temperature at 50℃ and the vacuum degree at -0.09MPa, and perform vacuum distillation to concentrate to 6.6L; S3, Precipitation: Add 13L of acetone to the concentrate while stirring at 150r / min. The precipitation time is 4 hours. S4. Separation and washing: Transfer the precipitate to a centrifuge and centrifuge at 4000 r / min for 10 minutes to separate the precipitate; wash the precipitate with 1000 mL of anhydrous ethanol, resuspend the precipitate and stir evenly, then centrifuge again at 4000 r / min for 10 minutes. Repeat the above washing operation twice, for a total of 3 washes. S5. Drying: Place the washed precipitate in a vacuum drying oven and dry it for 4 hours at 40°C and a vacuum of -0.1MPa to obtain crude heparin. S6. Purification: The crude heparin was dissolved in 1000 mL of deionized water and passed through a chromatographic column packed with D201 type strong basic anion exchange resin. The column was first rinsed with deionized water, and then eluted with 1.5 mol / L sodium chloride solution. The eluent containing heparin was collected. The eluent was placed in a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water for 24 hours. Finally, the dialyzed solution was freeze-dried to obtain 22.3 g of high-purity heparin product, with a purity of 97%.

[0034] Comparative Example 1 differs from Example 2 only in that, in step S3, anhydrous ethanol of the same volume is used instead of acetone for precipitation. All other steps and parameters remain consistent with Example 2. A final product of 12.1g heparin-like substance was obtained, with a purity of 88%.

[0035] Comparative Example 2 differs from Example 2 only in that the ion-exchange chromatography column purification step in step S6 is omitted, and the crude product obtained in step S5 is directly dissolved and then dialyzed and lyophilized. Other steps and parameters remain consistent with Example 2. A final product of 15.5g heparin-like substance was obtained, with a purity of 82%, and the product had a yellowish appearance and high ash content.

[0036] To more intuitively illustrate the beneficial effects of the present invention, the relevant data of Examples 1-3 and Comparative Examples 1-2 are summarized in the following table: Table 1: Group Raw material volume (L) Precipitator Refining methods Product weight (g) Product purity (%) Remark Example 1 10 acetone Column chromatography + dialysis 10.5 95% High purity, white color Example 2 15 acetone Column chromatography + dialysis 16.8 96% High purity, white color Example 3 20 acetone Column chromatography + dialysis 22.3 97% High purity, white color Comparative Example 1 15 ethanol Column chromatography + dialysis 12.1 88% The yield decreased significantly. Comparative Example 2 15 acetone Dialysis only 15.5 82% Low purity, incomplete impurity removal As shown in the table above, the acetone precipitation method of the present invention significantly increases the product weight obtained with the same amount of raw materials compared with the traditional ethanol precipitation method, indicating that acetone has a higher precipitation efficiency for heparin-like substances. At the same time, comparing Example 2 and Comparative Example 2, it can be seen that the lack of the ion exchange chromatography step leads to a significant decrease in product purity, proving the necessity of the combined purification process of the present invention.

[0037] In summary, this invention proposes a method for extracting heparin-like substances from sodium heparin ethanol precipitation. By employing a mild vacuum concentration process to avoid activity loss, initial enrichment is achieved through acetone precipitation and ethanol washing, followed by deep purification using ion exchange chromatography and dialysis. This successfully recovers heparin-like products with a purity of 95%–97%, low salt content, and complete retention of biological activity from industrial wastewater. This method not only achieves high-value utilization of byproducts, reducing environmental pressure and production costs for enterprises, but also solves the problems of high impurities and low activity in existing technologies, demonstrating significant economic, environmental, and industrial application value.

[0038] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited thereto. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A method for extracting heparin-like substances from sodium heparin ethanol precipitation, characterized in that, Includes the following steps: S1. Pretreatment: The collected heparin sodium precipitate is filtered to obtain a clear filtrate. S2. Concentration: The clarified filtrate is concentrated by vacuum distillation. When the volume is reduced to 1 / 5-1 / 3 of the original volume, distillation is stopped to obtain the concentrated liquid. S3. Precipitation: Slowly add acetone to the concentrated solution while stirring, so that heparin-like substances precipitate out to obtain a solid-liquid mixture; S4. Separation and washing: The solid-liquid mixture is separated by centrifugation, the supernatant is discarded, and the precipitate is collected. The collected precipitate is washed 2-3 times with anhydrous ethanol. Each time, the precipitate is resuspended in anhydrous ethanol, stirred evenly, and then centrifuged again to obtain the washed precipitate. S5. Drying: Place the washed precipitate in a vacuum drying oven and dry it until the precipitate reaches a constant weight to obtain crude heparin. S6. Purification: The crude heparin-like product is dissolved in deionized water and purified by passing it through an ion exchange chromatography column.

2. The method for extracting heparin-like substances from heparin sodium ethanol precipitation according to claim 1, characterized in that, In step S2, the distillation temperature is controlled at 40-50℃ and the vacuum degree is controlled at -0.08-0.09MPa.

3. The method for extracting heparin-like substances from heparin sodium ethanol precipitation according to claim 1, characterized in that, In step S3, the volume ratio of acetone to the concentrate is 1-2:

1.

4. The method for extracting heparin-like substances from sodium heparin ethanol precipitation according to claim 1, characterized in that, In step S3, the stirring speed is controlled at 100-150 r / min, and the sedimentation time is 2-4 hours.

5. The method for extracting heparin-like substances from heparin sodium ethanol precipitation according to claim 1, characterized in that, In step S4, the centrifuge speed is set to 3000-4000 r / min, and the centrifugation time is 10-15 minutes.

6. The method for extracting heparin-like substances from heparin sodium ethanol precipitation according to claim 1, characterized in that, In step S5, the drying temperature of the drying oven is controlled at 30-40℃, the vacuum degree is -0.09-0.1MPa, and the drying time is 4-6 hours.

7. The method for extracting heparin-like substances from heparin sodium ethanol precipitation according to claim 1, characterized in that, In step S6, the crude heparin-like product is dissolved in deionized water and purified by ion exchange chromatography, including: A strongly basic anion exchange resin was selected. The column was first rinsed with deionized water, and then eluted with sodium chloride solution. The eluent containing heparin was collected. The eluent is dialyzed using a dialysis bag to remove salts and small molecule impurities. The dialysis solution was freeze-dried to obtain a high-purity heparin-like product.

8. The method for extracting heparin-like substances from heparin sodium ethanol precipitation according to claim 7, characterized in that, In step S6, the molar concentration of the sodium chloride solution is 0.5-1.5 mol / L.

9. The method for extracting heparin-like substances from heparin sodium ethanol precipitation according to claim 7, characterized in that, In step S6, the molecular weight cutoff of the dialysis bag is 1000-3000 Da, and the dialysis time is 12-24 hours.

Images