A method for fractionally extracting chondroitin sulfate b, heparan sulfate and heparin sodium

Abstract

This invention belongs to the field of heparin sodium extraction technology, specifically involving a method for graded extraction of chondroitin sulfate B, heparan sulfate and heparin sodium. It consists of the following steps: (1) dissolving crude heparin in deionized water, centrifuging to remove insoluble matter from the heparin solution, and taking the supernatant to obtain crude heparin solution; (2) adjusting the pH of the crude heparin solution from step (1) to 8-9, then adding A98 resin, and adsorbing at 60-62℃; (3) using NaCl solution to elute the resin adsorbed in step (2) twice, then adding anhydrous ethanol three times for precipitation, collecting the precipitate, adjusting the pH to 9-10, adding potassium permanganate solution at 75-80℃ for oxidization for 30-40 min, filtering with diatomaceous earth, taking the filtrate, adding anhydrous ethanol to the filtrate for precipitation, and drying to obtain heparan sulfate; this invention has a high extraction rate and good extraction effect, and can also extract heparan sulfate and heparin sodium during the extraction of chondroitin sulfate B.

Description

Technical Field

[0001] This invention belongs to the field of heparin sodium extraction technology, specifically relating to a method for graded extraction of chondroitin sulfate B, heparan sulfate and heparin sodium. Background Technology

[0002] Glycosaminoglycans are a class of heterogeneous, negatively charged, unbranched carbohydrate macromolecules formed by the cross-linking of repeating disaccharide units composed of alternating amino sugars and hexuronic acids. Based on the different types of disaccharides in the glycan, glycosaminoglycans are classified as: heparin and heparan sulfate, chondroitin sulfate and dermatan sulfate, and keratin sulfate and hyaluronic acid. Different glycounit structures lead to different functions and distributions. Heparin and heparan sulfate have anticoagulant effects both in vivo and in vitro, and are widely used in clinical applications such as renal dialysis, extracorporeal circulation, and the treatment of venous thromboembolism. A common extraction method involves hydrolyzing cartilage tissue with an alkaline solution. The alkaline conditions disrupt the bonds between proteins and chondroitin sulfate in the cartilage tissue, releasing chondroitin sulfate in a free state. Then, trypsin, papain, alkaline protease, and a chondroitin hydrolysis-specific complex enzyme are added for enzymatic hydrolysis. Trichloroacetic acid is then added to the enzymatic or alkaline hydrolysis solution, stirred, and allowed to stand to denature and precipitate the protein. The precipitate is removed to obtain a clear chondroitin sulfate solution. Ethanol is then added, stirred, and allowed to stand to precipitate chondroitin sulfate. The precipitate is collected by centrifugation or filtration to obtain chondroitin sulfate. However, existing methods have difficulty extracting heparan sulfate and heparin sodium during the chondroitin sulfate extraction process; therefore, the current technology needs further improvement. Summary of the Invention

[0003] The purpose of this invention is to provide a method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium with high extraction rate and good extraction effect.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A method for graded extraction of chondroitin sulfate B, heparin sulfate and sodium heparin, comprising the following steps: (1) dissolving crude heparin in deionized water, centrifuging to remove insoluble matter from the heparin solution, and taking the supernatant to obtain crude heparin solution. (2) Adjust the pH of the crude heparin solution from step (1) to 8-9, then add A98 resin and complete the adsorption at 60-62℃; (3) Use NaCl solution to elute the resin that has been adsorbed in step (2) twice, then add anhydrous ethanol three times to precipitate, collect the precipitate, adjust the pH to 9-10, add potassium permanganate solution at 75-80℃ for 30-40 min, filter with diatomaceous earth and take the filtrate, then add anhydrous ethanol to the filtrate to precipitate and dry to obtain heparan sulfate. (4) Use NaCl solution to elute the resin that has been adsorbed in step (2). Take the eluent and add anhydrous ethanol to precipitate the precipitate for the first time. Dissolve the precipitate in deionized water, centrifuge, take the supernatant, add anhydrous ethanol to precipitate the precipitate for the second time, add potassium permanganate solution to oxidize for 30-40 min, filter with diatomaceous earth pad, take the filtrate and add anhydrous ethanol to precipitate the precipitate for the third time. (5) After dissolving the precipitate from step (4) in deionized water, add sodium nitrite solution, adjust the pH to 1.8-2, react for 80-90 min, then adjust the pH to 6-7, add anhydrous ethanol to precipitate, take the precipitate, and use a 10KD ultrafiltration membrane for ultrafiltration to obtain high-purity chondroitin sulfate B. (6) Use NaCl solution to elute the resin that has been adsorbed in step (2), collect the eluent, add anhydrous ethanol to precipitate for the first time, take the precipitate and add deionized water, then add chondroitin sulfate B degrading enzyme solution, and enzymatically hydrolyze at 35-37℃ for 6-8 hours. Then add anhydrous ethanol to precipitate for 5-7 hours and take the precipitate. (7) After dissolving the precipitate from step (6) in deionized water, adjust the pH to 9-10.0. Then, oxidize it for 8-10 hours after adding hydrogen peroxide solution at 25-30℃. After filtering with a 0.2-0.3μm filter membrane, freeze dry to obtain sodium heparin.

[0005] Furthermore, in step (1), after dissolving crude heparin in deionized water, the pH is adjusted to 0.5-1 using HCl solution. After centrifugation at -5℃ to -1℃ at a speed of 4000-8000 rpm for 4-5 minutes, the precipitate after centrifugation is the insoluble matter in the heparin solution.

[0006] Furthermore, in step (3), NaCl solution is used for two elutions. The first elution is performed using NaCl solution, and the eluent is not collected until the resin is washed clean and drained. The second elution is performed by adding NaCl solution to continue elution, and the eluent is collected. In step (3), anhydrous ethanol is added to the eluent twice. The first time, after adding anhydrous ethanol to the eluent, the precipitate is collected after precipitation for 1-2 hours. The second time, after adding anhydrous ethanol to the precipitate, the precipitate is dissolved in deionized water, the pH is adjusted to 1-2, and then centrifuged at 4000-8000 rpm for 4-5 minutes at 0-5℃. The supernatant is collected and the pH is adjusted to 6-8. The third time, anhydrous ethanol is added to the supernatant and the precipitate is collected after precipitation for 4-6 hours.

[0007] Furthermore, in step (4), the mass percentage of the NaCl solution is 5-6%, and the precipitate is dissolved in deionized water by adding HCl solution to the solution after dissolving the precipitate in deionized water to adjust the pH to 1-2, and centrifuging at 4000-8000 rpm for 4-5 minutes at 0-5℃; the second addition of anhydrous ethanol for precipitation is to adjust the pH of the supernatant after centrifugation to 6-7 with NaOH solution, and then add anhydrous ethanol for precipitation for 4-6 hours; the potassium permanganate oxidation is to adjust the pH of the precipitate to 9-10 with NaOH solution, and then add potassium permanganate solution at 75-80℃, where the mass percentage of the NaOH solution is 20%-30% and the mass percentage of the potassium permanganate solution is 1%-3%; the third addition of anhydrous ethanol for precipitation is to adjust the pH of the filtrate to 6-7 with HCl solution.

[0008] Furthermore, in step (5), after the precipitate is dissolved in deionized water, HCl solution is added to adjust the pH to 1.8-2, and then sodium nitrite solution is added at 20-25℃, wherein the mass percentage of the sodium nitrite solution is 1%-3%; after adding the sodium nitrite solution, HCl solution is used to adjust the pH to 1.8-2, and the reaction is carried out for 90-100 minutes to adjust the pH to 6-7.

[0009] Furthermore, the pH adjustment is performed using NaOH solution or HCl solution, with NaOH solution accounting for 20%-30% by mass, HCl solution accounting for 10%-25% by mass, and potassium permanganate solution accounting for 2%-5% by mass.

[0010] Furthermore, in steps (1) and (2), the mass-to-volume ratio of crude heparin, deionized water, and A98 resin is 10-30g: 100-150ml: 50-60g; in step (3), the mass percentage of NaCl solution used in the first elution is 2%-3%; the mass percentage of NaCl solution used in the second elution is 3.8-4.0%, and the mass-to-volume ratio of A98 resin to NaCl solution used in the first elution is 50-60g: 100-200ml; the volume ratio of NaCl solution used in the first elution to NaCl solution used in the second elution is 100-200ml: 200-300ml; the volume ratio of the eluent to the first added anhydrous ethanol in step (3) is 200-400ml: 350-850ml; the mass-volume ratio of the precipitate to anhydrous ethanol in the second addition of anhydrous ethanol to the precipitate is 10-20g: 30-50ml; the volume ratio of the supernatant to the third added anhydrous ethanol is 50-80ml: 100-120ml; the mass-volume ratio of the precipitate after precipitation with anhydrous ethanol in the supernatant to the potassium permanganate solution is 30-40g: 50-60ml; and the volume ratio of the filtrate to anhydrous ethanol is 40-50ml: 80-100ml.

[0011] In step (4), the NaCl solution has a mass percentage of 5-6%. The precipitate is dissolved in deionized water by adding HCl solution to the solution after dissolving the precipitate in deionized water to adjust the pH to 1-2. The centrifugation conditions are as follows: [The text abruptly ends here, so the translation stops as well.] Furthermore, in step (4), the volume ratio of the NaCl solution to the NaCl solution used in the second elution in step (3) is 300-500ml: 200-300ml; the volume ratio of the eluent to the first added anhydrous ethanol is 200-300ml: 400-600ml; the mass-volume ratio of the precipitate after the first ethanol precipitation, deionized water, and the second added anhydrous ethanol is 10-20g: 100-150ml: 200-300ml; the mass-volume ratio of the precipitate after the second anhydrous ethanol precipitation to the potassium permanganate solution is 30-40g: 200-300ml; and the volume ratio of the filtrate to the third added anhydrous ethanol precipitate is 200ml-300ml: 400-600ml.

[0012] Furthermore, in step (5), the mass-to-volume ratio of the precipitate dissolved in deionized water, deionized water, sodium nitrite solution, and wastewater ethanol is: 50-80g: 100-200ml: 100-150ml: 150-200ml.

[0013] Furthermore, in step (6), the mass percentage of the NaCl solution is 10%-15%, and the volume ratio of the NaCl solution used in step (6) to the NaCl solution in step (4) is 300-350:300-500ml. The volume ratio of the eluent, the first added anhydrous ethanol, the deionized water, the chondroitin sulfate B degrading enzyme solution, and the second added anhydrous ethanol is 100-200ml:200-300ml:100-200ml:50-80ml:200-300ml. In step (7), the mass-volume ratio of the precipitate, the deionized water, and the sodium peroxide solution is 20-30g:80-100ml:50-100ml, and the mass percentage of the sodium peroxide solution is 1%-5%.

[0014] This invention can extract chondroitin sulfate B, heparin sulfate, and heparin sodium in stages. It can also extract heparin sulfate and heparin sodium during the extraction of chondroitin sulfate B, with high extraction yield and good extraction efficiency. Detailed Implementation Example

[0015] A method for graded extraction of chondroitin sulfate B, heparin sulfate and sodium heparin, comprising the following steps: (1) dissolving crude heparin in deionized water, centrifuging to remove insoluble matter from the heparin solution, and taking the supernatant to obtain crude heparin solution. In step (1), crude heparin is dissolved in deionized water and the pH is adjusted to 0.5 using HCl solution. After centrifugation at -5℃ for 4 minutes at 4000 rpm, the precipitate after centrifugation is the insoluble matter in the heparin solution.

[0016] Adjust the pH of the crude heparin solution from step (1) to 8, then add A98 resin and complete the adsorption at 60°C. In steps (1) and (2), the mass-to-volume ratio of crude heparin, deionized water and A98 resin is 10g:100ml:50g. The resin that had been adsorbed in step (2) was eluted twice with NaCl solution, and then anhydrous ethanol was added three times to precipitate it. The precipitate was collected, the pH was adjusted to 9, and potassium permanganate solution was added at 75°C for 30 min to oxidize it. After filtration with diatomaceous earth, the filtrate was collected, and anhydrous ethanol was added to the filtrate to precipitate it. After drying, heparan sulfate was obtained. In step (3), NaCl solution is used for two elutions. The first elution is performed using NaCl solution, and the eluent is not collected until the resin is washed clean and drained. The second elution is performed by adding NaCl solution to continue elution, and the eluent is collected. In step (3), anhydrous ethanol is added to the eluent twice. The first time, after adding anhydrous ethanol to the eluent, the precipitate is collected after 1 hour of precipitation. The second time, after adding anhydrous ethanol to the precipitate, the precipitate is dissolved in deionized water, the pH is adjusted to 1, and then centrifuged at 4000 rpm for 4 minutes at 0°C. The supernatant is collected and the pH is adjusted to 6. The third time, anhydrous ethanol is added to the supernatant and the precipitate is collected after 4 hours of precipitation.

[0017] In step (3), the mass percentage of NaCl solution used in the first elution is 2%; the mass percentage of NaCl solution used in the second elution is 3.8%; the mass-volume ratio of A98 resin to the NaCl solution used in the first elution is 50g:100ml; the volume ratio of the NaCl solution used in the first elution to the NaCl solution used in the second elution is 100ml:200ml. In step (3), the volume ratio of the eluent to the first added anhydrous ethanol is 200ml:350ml; the mass-volume ratio of the precipitate to anhydrous ethanol in the second addition of anhydrous ethanol to the precipitate is 10g:30ml; the volume ratio of the supernatant to the third added anhydrous ethanol is 50ml:100ml; the mass-volume ratio of the precipitate taken after precipitation with anhydrous ethanol in the supernatant to the potassium permanganate solution is 30g:50ml; and the volume ratio of the filtrate to anhydrous ethanol is 40ml:80ml.

[0018] Then use NaCl solution to elute the resin that has been adsorbed in step (2). Take the eluent and add anhydrous ethanol to precipitate the precipitate for the first time. Dissolve the precipitate in deionized water, centrifuge, take the supernatant and add anhydrous ethanol to precipitate the precipitate for the second time. Add potassium permanganate solution to oxidize for 30 min, filter with diatomaceous earth pad, take the filtrate and add anhydrous ethanol to precipitate the precipitate for the third time. In step (4), the mass percentage of the NaCl solution is 5%. The precipitate is dissolved in deionized water by adding HCl solution to the solution after dissolving the precipitate in deionized water to adjust the pH to 1. The centrifugation conditions are 4000 rpm for 4 min at 0°C. The second addition of anhydrous ethanol for precipitation is to adjust the pH of the supernatant after centrifugation to 6 with NaOH solution and then add anhydrous ethanol for precipitation for 4 h. The potassium permanganate solution oxidation is to adjust the pH of the precipitate to 9.5 with NaOH solution and then add potassium permanganate solution at 75°C. The mass percentage of the potassium permanganate solution is 1%. The third addition of anhydrous ethanol for precipitation is to adjust the pH of the filtrate to 6 with HCl solution. In step (4), the volume ratio of NaCl solution to NaCl solution used in the second elution in step (3) is 300ml:200ml; the volume ratio of eluent to anhydrous ethanol added in the first step is 200ml:400ml; the mass-volume ratio of precipitate after the first ethanol precipitation, deionized water, and anhydrous ethanol added in the second step is 10g:100ml:200ml; the mass-volume ratio of precipitate after the second anhydrous ethanol precipitation to potassium permanganate solution is 30g:200ml; and the volume ratio of filtrate to anhydrous ethanol precipitate added in the third step is 200ml:400ml.

[0019] (5) After dissolving the precipitate from step (4) in deionized water, add sodium nitrite solution, adjust the pH to 1.8, react for 80 min, then adjust the pH to 6, add anhydrous ethanol to precipitate, take the precipitate, and use a 10KD ultrafiltration membrane for ultrafiltration to obtain high-purity chondroitin sulfate B; the mass-volume ratio of the precipitate dissolved in deionized water, deionized water, sodium nitrite solution and sewage ethanol in step (5) is: 50g: 100ml: 100ml: 150ml.

[0020] In step (5), after the precipitate is dissolved in deionized water, HCl solution is added to adjust the pH to 1.8, and then sodium nitrite solution is added at 20°C. The mass percentage of the sodium nitrite solution is 1%. After adding the sodium nitrite solution, HCl solution is used to adjust the pH to 1.8, and the reaction is carried out for 90 minutes to adjust the pH to 6.

[0021] The resin adsorbed in step (2) is then eluted with NaCl solution, the eluent is collected, anhydrous ethanol is added for precipitation for the first time, deionized water is added to the precipitate, chondroitin sulfate B degrading enzyme solution is added, and enzymatic hydrolysis is carried out at 35°C for 6 hours. Anhydrous ethanol is added for precipitation for 5 hours, and the precipitate is collected. The mass percentage of NaCl solution in step (6) is 10%, the volume ratio of NaCl solution used in step (6) to NaCl solution in step (4) is 300:300 ml, and the volume ratio of the eluent, the first added anhydrous ethanol, the deionized water, the chondroitin sulfate B degrading enzyme solution, and the second added anhydrous ethanol is 300 ml: 200 ml: 100 ml: 50 ml: 200 ml. (7) After dissolving the precipitate from step (6) in deionized water, the pH was adjusted to 9.5. Hydrogen peroxide solution was added at 25°C and oxidized for 8 hours. Then, the precipitate was filtered through a 0.2 μm filter membrane and freeze-dried to obtain sodium heparin.

[0022] In step (7), the mass-volume ratio of the precipitate, deionized water, and sodium peroxide solution is 20g:80ml:50ml, and the mass percentage of the sodium peroxide solution is 1%.

[0023] The pH adjustment is performed using NaOH solution or HCl solution, with NaOH solution accounting for 20% by mass, HCl solution accounting for 10% by mass, and potassium permanganate solution accounting for 2% by mass. Example

[0024] A method for graded extraction of chondroitin sulfate B, heparin sulfate and sodium heparin, comprising the following steps: (1) dissolving crude heparin in deionized water, centrifuging to remove insoluble matter from the heparin solution, and taking the supernatant to obtain crude heparin solution. In step (1), crude heparin is dissolved in deionized water and the pH is adjusted to 0.8 using HCl solution. After centrifugation at -3°C at 6000 rpm for 4 minutes, the precipitate after centrifugation is the insoluble matter in the heparin solution.

[0025] (2) Adjust the pH of the crude heparin solution from step (1) to 7.5, then add A98 resin and complete the adsorption at 61°C. In steps (1) and (2), the mass-to-volume ratio of crude heparin, deionized water and A98 resin is 20g:130ml:55g. (3) Use NaCl solution to elute the resin that has been adsorbed in step (2) twice, then add anhydrous ethanol three times to precipitate, collect the precipitate, adjust the pH to 9, add potassium permanganate solution at 78℃ for 35 min, filter with diatomaceous earth and take the filtrate, then add anhydrous ethanol to the filtrate to precipitate and dry to obtain heparan sulfate. In step (3), NaCl solution is used for two elutions. The first elution is performed using NaCl solution, and the eluent is not collected until the resin is washed clean and drained. The second elution is performed by adding NaCl solution to continue elution, and the eluent is collected. In step (3), anhydrous ethanol is added to the eluent twice. The first time, after adding anhydrous ethanol to the eluent, the precipitate is collected after precipitation for 1.5 hours. The second time, after adding anhydrous ethanol to the precipitate, the precipitate is dissolved in deionized water, the pH is adjusted to 1.5, and then centrifuged at 6000 rpm for 4.5 minutes at 3°C. The supernatant is collected and the pH is adjusted to 7. The third time, anhydrous ethanol is added to the supernatant and the precipitate is collected after precipitation for 5 hours.

[0026] In step (3), the mass percentage of NaCl solution used in the first elution is 2.5%; the mass percentage of NaCl solution used in the second elution is 3.9%; the mass-volume ratio of A98 resin to the NaCl solution used in the first elution is 55g:150ml; the volume ratio of the NaCl solution used in the first elution to the NaCl solution used in the second elution is 150ml:250ml. In step (3), the volume ratio of the eluent to the first added anhydrous ethanol is 300ml:500ml; the mass-volume ratio of the precipitate to anhydrous ethanol in the second addition of anhydrous ethanol to the precipitate is 15g:35ml; the volume ratio of the supernatant to the third added anhydrous ethanol is 70ml:110ml; the mass-volume ratio of the precipitate after precipitation with anhydrous ethanol in the supernatant to the potassium permanganate solution is 35:55ml; and the volume ratio of the filtrate to anhydrous ethanol is 45ml:90ml.

[0027] (4) Use NaCl solution to elute the resin that has been adsorbed in step (2). Take the eluent and add anhydrous ethanol to precipitate the precipitate for the first time. Dissolve the precipitate in deionized water, centrifuge, take the supernatant and add anhydrous ethanol to precipitate the precipitate for the second time. Add potassium permanganate solution to oxidize for 35 min, filter with diatomaceous earth pad, take the filtrate and add anhydrous ethanol to precipitate the precipitate for the third time. In step (4), the mass percentage of NaCl solution is 5.5%. The precipitate is dissolved in deionized water by adding HCl solution to the solution after dissolving the precipitate in deionized water to adjust the pH to 1.5. The centrifugation conditions are 5000 rpm for 4.5 min at 3°C. The second addition of anhydrous ethanol for precipitation is to adjust the pH of the supernatant after centrifugation to 6.5 with NaOH solution and then add anhydrous ethanol for precipitation for 5 h. The potassium permanganate solution oxidation is to adjust the pH of the precipitate to 9.5 with NaOH solution and then add potassium permanganate solution at 78°C. The mass percentage of potassium permanganate solution is 2%. The third addition of anhydrous ethanol for precipitation is to adjust the pH of the filtrate to 6.5 with HCl solution. In step (4), the volume ratio of NaCl solution to NaCl solution used in the second elution in step (3) is 400ml:250ml; the volume ratio of eluent to anhydrous ethanol added in the first step is 250ml:500ml; the mass-volume ratio of precipitate after the first ethanol precipitation, deionized water, and anhydrous ethanol added in the second step is 15g:125ml:250ml; the mass-volume ratio of precipitate after the second anhydrous ethanol precipitation to potassium permanganate solution is 35g:250ml; and the volume ratio of filtrate to anhydrous ethanol precipitate added in the third step is 250ml:500ml.

[0028] (5) After dissolving the precipitate from step (4) in deionized water, add sodium nitrite solution, adjust the pH to 1.9, react for 85 min, then adjust the pH to 6.5, add anhydrous ethanol to precipitate, take the precipitate, and use a 10KD ultrafiltration membrane for ultrafiltration to obtain high-purity chondroitin sulfate B; the mass-volume ratio of the precipitate dissolved in deionized water, deionized water, sodium nitrite solution and sewage ethanol in step (5) is: 70g: 150ml: 120ml: 180ml.

[0029] In step (5), after the precipitate is dissolved in deionized water, HCl solution is added to adjust the pH to 1.9, and then sodium nitrite solution is added at 23°C. The mass percentage of the sodium nitrite solution is 2%. After adding the sodium nitrite solution, HCl solution is used to adjust the pH to 1.9, and the reaction is carried out for 95 minutes to adjust the pH to 6.5.

[0030] The resin adsorbed in step (2) is then eluted with NaCl solution, the eluent is collected, anhydrous ethanol is added for precipitation for the first time, deionized water is added to the precipitate, chondroitin sulfate B degrading enzyme solution is added, and enzymatic hydrolysis is carried out at 36°C for 7 hours. Then anhydrous ethanol is added for precipitation for 6 hours, and the precipitate is collected. The mass percentage of NaCl solution in step (6) is 13%, the volume ratio of NaCl solution used in step (6) to NaCl solution in step (4) is 320:400 ml, and the volume ratio of the eluent, the first added anhydrous ethanol, the deionized water, the chondroitin sulfate B degrading enzyme solution, and the second added anhydrous ethanol is 320 ml: 250 ml: 150 ml: 70 ml: 250 ml. (7) After dissolving the precipitate from step (6) in deionized water, adjust the pH to 9.5, add hydrogen peroxide solution at 28°C and oxidize for 9 hours. Then filter with a 0.25 μm filter membrane and freeze dry to obtain sodium heparin.

[0031] In step (7), the mass-volume ratio of the precipitate, deionized water, and sodium peroxide solution is 25g:90ml:80ml, and the mass percentage of the sodium peroxide solution is 4%.

[0032] The pH adjustment is performed using NaOH solution or HCl solution, with NaOH solution accounting for 25% by mass, HCl solution accounting for 20% by mass, and potassium permanganate solution accounting for 3% by mass. Example

[0033] A method for graded extraction of chondroitin sulfate B, heparin sulfate and sodium heparin, comprising the following steps: (1) dissolving crude heparin in deionized water, centrifuging to remove insoluble matter from the heparin solution, and taking the supernatant to obtain crude heparin solution. In step (1), crude heparin is dissolved in deionized water and the pH is adjusted to 1 using HCl solution. After centrifugation at -1℃ and 8000 rpm for 5 minutes, the precipitate after centrifugation is the insoluble matter in the heparin solution.

[0034] (2) Adjust the pH of the crude heparin solution from step (1) to 9, then add A98 resin and complete the adsorption at 62°C. In steps (1) and (2), the mass-to-volume ratio of crude heparin, deionized water and A98 resin is 30g:150ml:60g. (3) Use NaCl solution to elute the resin that has been adsorbed in step (2) twice, then add anhydrous ethanol three times to precipitate, collect the precipitate, adjust the pH to 10, add potassium permanganate solution at 80℃ for 40 min, filter with diatomaceous earth and take the filtrate, then add anhydrous ethanol to the filtrate to precipitate and dry to obtain heparan sulfate. In step (3), NaCl solution is used for two elutions. The first elution is performed using NaCl solution, and the eluent is not collected until the resin is washed clean and drained. The second elution is performed by adding NaCl solution to continue elution, and the eluent is collected. In step (3), anhydrous ethanol is added to the eluent twice. The first time, after adding anhydrous ethanol to the eluent, the precipitate is collected after 2 hours of precipitation. The second time, after adding anhydrous ethanol to the precipitate, the precipitate is dissolved in deionized water, the pH is adjusted to 2, and then centrifuged at 8000 rpm for 5 minutes at 5°C. The supernatant is collected and the pH is adjusted to 8. The third time, anhydrous ethanol is added to the supernatant, and after 6 hours of precipitation, the precipitate is collected.

[0035] In step (3), the mass percentage of NaCl solution used in the first elution is 3%; the mass percentage of NaCl solution used in the second elution is 4.0%; the mass-volume ratio of A98 resin to the NaCl solution used in the first elution is 60g:200ml; the volume ratio of the NaCl solution used in the first elution to the NaCl solution used in the second elution is 200ml:300ml. In step (3), the volume ratio of the eluent to the first added anhydrous ethanol is 400ml:850ml; the mass-volume ratio of the precipitate to anhydrous ethanol in the second addition of anhydrous ethanol to the precipitate is 20g:50ml; the volume ratio of the supernatant to the third added anhydrous ethanol is 80ml:120ml; the mass-volume ratio of the precipitate taken after precipitation with anhydrous ethanol in the supernatant to the potassium permanganate solution is 40g:60ml; and the volume ratio of the filtrate to anhydrous ethanol is 50ml:100ml.

[0036] (4) Use NaCl solution to elute the resin that has been adsorbed in step (2). Take the eluent and add anhydrous ethanol to precipitate the precipitate for the first time. Dissolve the precipitate in deionized water, centrifuge, take the supernatant and add anhydrous ethanol to precipitate the precipitate for the second time. Add potassium permanganate solution to oxidize for 40 min, filter with diatomaceous earth pad, take the filtrate and add anhydrous ethanol to precipitate the precipitate for the third time. In step (4), the mass percentage of the NaCl solution is 6%. The precipitate is dissolved in deionized water by adding HCl solution to the solution after dissolving the precipitate in deionized water to adjust the pH to 2. The centrifugation is carried out at 5°C at a speed of 8000 rpm for 5 minutes. The second addition of anhydrous ethanol for precipitation is to adjust the pH of the supernatant after centrifugation to 7 with NaOH solution and then add anhydrous ethanol for precipitation for 6 hours. The potassium permanganate solution oxidation is to adjust the pH of the precipitate to 10 with NaOH solution and then add potassium permanganate solution at 80°C. The mass percentage of the potassium permanganate solution is 3%. The third addition of anhydrous ethanol for precipitation is to adjust the pH of the filtrate to 7 with HCl solution. In step (4), the volume ratio of NaCl solution to NaCl solution used in the second elution in step (3) is 500ml:300ml; the volume ratio of eluent to anhydrous ethanol added in the first step is 300ml:600ml; the mass-volume ratio of precipitate after the first ethanol precipitation, deionized water, and anhydrous ethanol added in the second step is 20g:150ml:300ml; the mass-volume ratio of precipitate after the second anhydrous ethanol precipitation to potassium permanganate solution is 40g:300ml; and the volume ratio of filtrate to anhydrous ethanol precipitate added in the third step is 300ml:600ml.

[0037] (5) After dissolving the precipitate from step (4) in deionized water, add sodium nitrite solution, adjust the pH to 2, react for 90 min, then adjust the pH to 7, add anhydrous ethanol to precipitate, take the precipitate, and use a 10KD ultrafiltration membrane for ultrafiltration to obtain high-purity chondroitin sulfate B; the mass-volume ratio of the precipitate dissolved in deionized water, deionized water, sodium nitrite solution and sewage ethanol in step (5) is: 80g: 200ml: 150ml: 200ml.

[0038] In step (5), after the precipitate is dissolved in deionized water, HCl solution is added to adjust the pH to 2, and then sodium nitrite solution is added at 25°C. The mass percentage of the sodium nitrite solution is 3%. After adding the sodium nitrite solution, HCl solution is used to adjust the pH to 2, and the reaction is carried out for 100 minutes to adjust the pH to 7.

[0039] The resin adsorbed in step (2) is then eluted with NaCl solution, the eluent is collected, anhydrous ethanol is added for precipitation for the first time, deionized water is added to the precipitate, chondroitin sulfate B degrading enzyme solution is added, and enzymatic hydrolysis is carried out at 37°C for 8 hours. Anhydrous ethanol is added for precipitation for 7 hours, and the precipitate is collected. The mass percentage of NaCl solution in step (6) is 15%, the volume ratio of NaCl solution used in step (6) to NaCl solution in step (4) is 350:500 ml, and the volume ratio of the eluent, the first added anhydrous ethanol, the deionized water, the chondroitin sulfate B degrading enzyme solution, and the second added anhydrous ethanol is 350 ml: 300 ml: 200 ml: 80 ml: 300 ml. (7) After dissolving the precipitate from step (6) in deionized water, the pH was adjusted to 10.0. Hydrogen peroxide solution was added at 30°C and oxidized for 10 hours. Then, the precipitate was filtered through a 0.3 μm filter membrane and freeze-dried to obtain sodium heparin.

[0040] In step (7), the mass-volume ratio of the precipitate, deionized water, and sodium peroxide solution is 30g:100ml:100ml, and the mass percentage of the sodium peroxide solution is 5%.

[0041] The pH adjustment is performed using NaOH solution or HCl solution, with NaOH solution accounting for 30% by mass, HCl solution accounting for 25% by mass, and potassium permanganate solution accounting for 5% by mass.

[0042] Take the intestinal mucosa of the same pig, divide it into three groups, each group is 100g, grind it into a paste, add NaCl solution, adjust the pH to 7-9, add hydrolytic protease, trypsin, chondroitin sulfate AC, and nuclease at 32℃-35℃, and then hydrolyze at a constant temperature for 2-4h to obtain the enzymatic hydrolysate; then add anhydrous ethanol for the first precipitation for 2-3h, remove the supernatant, take the precipitate; then add anhydrous ethanol again to grind, filter, and dry in an oven at 50-60℃ for 3-4h to obtain crude heparin.

[0043] The crude heparin obtained from the above three groups of porcine intestinal mucosa was used with heparan sulfate separated in Examples 1, 2, and 3 of this invention. The heparan sulfate was detected by high performance liquid chromatography, and the detection results are shown below: As can be seen from the above, the content of heparan sulfate, chondroitin B sulfate, and heparin sodium extracted using the separation method of the present invention is relatively high, and it can effectively separate heparan sulfate, chondroitin B sulfate, and heparin sodium from crude heparin step by step.

Claims

1. A method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium, characterized in that, It consists of the following steps: (1) dissolving crude heparin in deionized water, centrifuging to remove insoluble matter from the heparin solution, and taking the supernatant to obtain crude heparin solution; (2) Adjust the pH of the crude heparin solution from step (1) to 8-9, then add A98 resin and complete the adsorption at 60-62℃; (3) Use NaCl solution to elute the resin that has been adsorbed in step (2) twice, then add anhydrous ethanol three times to precipitate, collect the precipitate, adjust the pH to 9-10, add potassium permanganate solution at 75-80℃ for 30-40 min, filter with diatomaceous earth and take the filtrate, then add anhydrous ethanol to the filtrate to precipitate and dry to obtain heparan sulfate. (4) Use NaCl solution to elute the resin that has been adsorbed in step (2). Take the eluent and add anhydrous ethanol to precipitate the precipitate for the first time. Dissolve the precipitate in deionized water, centrifuge, take the supernatant, add anhydrous ethanol to precipitate the precipitate for the second time, add potassium permanganate solution to oxidize for 30-40 min, filter with diatomaceous earth pad, take the filtrate and add anhydrous ethanol to precipitate the precipitate for the third time. (5) After dissolving the precipitate from step (4) in deionized water, add sodium nitrite solution, adjust the pH to 1.8-2, react for 80-90 min, then adjust the pH to 6-7, add anhydrous ethanol to precipitate, take the precipitate, and use a 10KD ultrafiltration membrane for ultrafiltration to obtain high-purity chondroitin sulfate B. (6) Use NaCl solution to elute the resin that has been adsorbed in step (2), collect the eluent, add anhydrous ethanol to precipitate for the first time, take the precipitate and add deionized water, then add chondroitin sulfate B degrading enzyme solution, and enzymatically hydrolyze at 35-37℃ for 6-8 hours. Then add anhydrous ethanol to precipitate for 5-7 hours and take the precipitate. (7) After dissolving the precipitate from step (6) in deionized water, adjust the pH to 9-10.

0. Then, oxidize it for 8-10 hours after adding hydrogen peroxide solution at 25-30℃. After filtering with a 0.2-0.3μm filter membrane, freeze dry to obtain sodium heparin.

2. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, In step (1), crude heparin is dissolved in deionized water and the pH is adjusted to 0.5-1 using HCl solution. After centrifugation at -5℃ to -1℃ for 4-5 minutes at a speed of 4000-8000 rpm, the precipitate after centrifugation is the insoluble matter in the heparin solution.

3. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, In step (3), NaCl solution is used for two elutions. The first elution is performed using NaCl solution. The eluent is not collected until the resin is washed clean and drained. The second elution involves adding NaCl solution to continue elution, and then collecting the eluent. In step (3), anhydrous ethanol is added to the eluent twice. The first time, after adding anhydrous ethanol to the eluent, the precipitate is collected after precipitation for 1-2 hours. The second time, after adding anhydrous ethanol to the precipitate, the precipitate is dissolved in deionized water, the pH is adjusted to 1-2, and then centrifuged at 4000-8000 rpm for 4-5 minutes at 0-5℃. The supernatant is collected and the pH is adjusted to 6-8. The third time, anhydrous ethanol is added to the supernatant and the precipitate is collected after precipitation for 4-6 hours.

4. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, In step (4), the mass percentage of NaCl solution is 5-6%. The precipitate is dissolved in deionized water by adding HCl solution to the solution after dissolving the precipitate in deionized water to adjust the pH to 1-2. The centrifugation is carried out at 0-5℃ at a speed of 4000-8000 rpm for 4-5 minutes. The second addition of anhydrous ethanol for precipitation is to adjust the pH of the supernatant after centrifugation to 6-7 with NaOH solution and then add anhydrous ethanol for precipitation for 4-6 hours. The potassium permanganate oxidation is to adjust the pH of the precipitate to 9-10 with NaOH solution and then add potassium permanganate solution at 75-80℃. The mass percentage of NaOH solution is 20%-30% and the mass percentage of potassium permanganate solution is 1%-3%. The third addition of anhydrous ethanol for precipitation is to adjust the pH of the filtrate to 6-7 with HCl solution.

5. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, In step (5), after the precipitate is dissolved in deionized water, HCl solution is added to adjust the pH to 1.8-2, and then sodium nitrite solution is added at 20-25℃. The mass percentage of the sodium nitrite solution is 1%-3%. After adding the sodium nitrite solution, HCl solution is used to adjust the pH to 1.8-2, and the reaction is carried out for 90-100 minutes to adjust the pH to 6-7.

6. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, The pH adjustment is achieved using NaOH solution or HCl solution, with NaOH solution comprising 20%-30% by mass, HCl solution comprising 10%-25% by mass, and potassium permanganate solution comprising 2%-5% by mass.

7. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, In steps (1) and (2), the mass-to-volume ratio of crude heparin, deionized water, and A98 resin is 10-30g: 100-150ml: 50-60g; in step (3), the mass percentage of NaCl solution used in the first elution is 2%-3%; the mass percentage of NaCl solution used in the second elution is 3.8-4.0%, and the mass-to-volume ratio of A98 resin to NaCl solution used in the first elution is 50-60g: 100-200ml; the volume ratio of NaCl solution used in the first elution to NaCl solution used in the second elution is 100-200ml: 200-300ml; the volume ratio of the eluent to the first added anhydrous ethanol in step (3) is 200-400ml: 350-850ml; the mass-volume ratio of the precipitate to anhydrous ethanol in the second addition of anhydrous ethanol to the precipitate is 10-20g: 30-50ml; the volume ratio of the supernatant to the third added anhydrous ethanol is 50-80ml: 100-120ml; the mass-volume ratio of the precipitate after precipitation with anhydrous ethanol in the supernatant to the potassium permanganate solution is 30-40g: 50-60ml; the volume ratio of the filtrate to anhydrous ethanol is 40-50ml: 80-100ml. In step (4), the mass percentage of NaCl solution is 5-6%. The precipitate is dissolved in deionized water by adding HCl solution to the solution after dissolving the precipitate in deionized water to adjust the pH to 1-2. The centrifugation conditions are 4000-8000 rpm for 4-5 minutes at 0-5℃.

8. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, In step (4), the volume ratio of NaCl solution to NaCl solution used in the second elution in step (3) is 300-500ml: 200-300ml; the volume ratio of eluent to anhydrous ethanol added for the first time is 200-300ml: 400-600ml; the mass-volume ratio of precipitate after the first ethanol precipitation, deionized water, and anhydrous ethanol added for the second time is 10-20g: 100-150ml: 200-300ml; the mass-volume ratio of precipitate after the second anhydrous ethanol precipitation to potassium permanganate solution is 30-40g: 200-300ml; and the volume ratio of filtrate to anhydrous ethanol precipitate added for the third time is 200ml-300ml: 400-600ml.

9. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, In step (5), the mass-to-volume ratio of the precipitate dissolved in deionized water, deionized water, sodium nitrite solution, and wastewater ethanol is: 50-80g: 100-200ml: 100-150ml: 150-200ml.

10. The method for graded extraction of chondroitin sulfate B, heparin sulfate, and heparin sodium according to claim 1, characterized in that, In step (6), the mass percentage of the NaCl solution is 10%-15%, and the volume ratio of the NaCl solution used in step (6) to the NaCl solution in step (4) is 300-350:300-500ml. The volume ratio of the eluent, the first added anhydrous ethanol, the deionized water, the chondroitin sulfate B degrading enzyme solution, and the second added anhydrous ethanol is 100-200ml:200-300ml:100-200ml:50-80ml:200-300ml. In step (7), the mass-volume ratio of the precipitate, the deionized water, and the sodium peroxide solution is 20-30g:80-100ml:50-100ml, and the mass percentage of the sodium peroxide solution is 1%-5%.