Sheep-derived high-titer-ratio glycosaminoglycan mixture and preparation method thereof
Through a preparation method including adsorption, washing, elution, degradation and reduction, the problem of single supply of existing low-molecular heparin seed sources and low applicability of preparation processes is solved, and the preparation of high titer-biased glycosaminoglycan mixtures of goat sources is achieved, with high anticoagulation and antithrombotic effects.
Patent Information
- Application Number
- CN202411883362.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-11
AI Technical Summary
The existing low-molecular heparin seed source supply is single, and the preparation process is low, making it difficult to meet the market demand for high-titer glycosaminoglycan mixtures.
A method for preparing a high-titer glycosaminoglycan mixture from goat sources is proposed, including the preparation of a glycosaminoglycan mixture of different weight average molecular weight segments and titer ratios through the steps of adsorption, washing, elution, degradation and reduction of goat intestinal mucosa.
The preparation of a high titer than glucosaminoglycan mixture in goat sources was achieved, solving the problem of single seed source supply and low applicability of preparation processes, and has high anticoagulation and antithrombotic effects.
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Figure CN119591746A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of pharmaceutical biotechnology, and in particular to a sheep-derived high-cost-ratio glycosaminoglycan mixture and a preparation method thereof. Background Art
[0002] Heparin drugs are currently the most widely used anticoagulant and antithrombotic drugs in clinical practice. Unfractionated heparin is a highly sulfated glycosaminoglycan mixture with an average molecular weight of 16kDa, mainly composed of repeating disaccharide structural units of uronic acid and aminoglucose. Low molecular weight heparin has a molecular weight of less than 8000Da and is mainly depolymerized by unfractionated heparin through chemical degradation or enzymatic hydrolysis. Compared with unfractionated heparin, low molecular weight heparin accounts for more than 80% of the heparin market and has a wider range of applications.
[0003] At present, the main source of low molecular weight heparin is pigs. The low molecular weight heparin products included in EP (European Pharmacopoeia), USP (United States Pharmacopoeia) and CP (Chinese Pharmacopoeia) clearly indicate that the source is limited to pig intestinal mucosa. The risk of a single source has caused countries to begin to pay attention to and study other sources to prepare anticoagulant and antithrombotic drugs. my country has the largest number of sheep in stock in the world, but the utilization rate of sheep intestines is low. In order to meet market demand, it is of great significance to develop a sheep-derived high-efficiency glycosaminoglycan mixture that can be used as a clinical anticoagulant and antithrombotic drug.
[0004] In addition, the current low molecular weight heparin market mainly includes products such as enoxaparin sodium, dalteparin sodium, tinzaparin, parparin sodium and bemiparin sodium, and the preparation processes include β elimination degradation, nitrite degradation, heparinase degradation, Cu 2+ / H2O2 oxidative depolymerization, etc. The properties of common low molecular weight heparin market products are shown in the following table:
[0005] The mechanism of action of low molecular weight heparin is mainly through the characteristic pentasaccharide sequence in its structure that specifically binds to antithrombin III (ATIII) to form a dimer, thereby enhancing the activity of ATIII and improving the activity of Xa coagulation factor, thereby effectively inhibiting thrombosis. At the same time, due to the short chain length of low molecular weight heparin, the inhibition of IIa coagulation factor is poor, so the risk of bleeding is also reduced to a great extent. Heparin itself is not an anticoagulant, but it enhances the activity of certain enzymes that inhibit thrombin factors.
[0006] According to the mechanism of action of low molecular weight heparin, its clinical application has little to do with the depolymerization process used. The most relevant factors are its molecular weight and activity ratio. A lower molecular weight gives it a higher bioavailability and half-life, and a higher anti-Xa factor / anti-IIa factor ratio gives it a lower risk of bleeding. Therefore, it is of great significance to develop a low molecular weight and high cost-effective glycosaminoglycan mixture. Summary of the invention
[0007] The present invention provides a sheep-derived high-efficiency glycosaminoglycan mixture and a preparation method thereof, which can realize the preparation of anticoagulant and antithrombotic glycosaminoglycan mixtures with different weight-average molecular weight segments and potency ratios, and solves the problems of single source supply of low molecular weight heparin and low applicability of the preparation process in the related art.
[0008] The technical solution of the present invention is as follows: The present invention provides a method for preparing a sheep-derived high-efficiency glycosaminoglycan mixture, comprising the following steps: S1, dissolving, adsorbing, washing and eluting the crude heparin from the sheep intestinal mucosa to obtain an eluate, adding ethanol to the eluate, collecting the first precipitate, and drying to obtain a macromolecular sheep-derived glycosaminoglycan; S2, dissolving the macromolecular sheep-derived glycosaminoglycan to obtain a solution, adding a first sodium nitrite to the solution under a first acidic condition, performing a first degradation, and then adding a first sodium borohydride under a first alkaline condition, performing a first reduction, performing ultrafiltration, collecting the drug solution m1 at the filtration end, and collecting the drug solution M1 at the reflux end; S3, under a second acidic condition, adding a second sodium nitrite to the drug solution M1, performing a second degradation, and then under a second alkaline condition, adding a second sodium borohydride, performing a second reduction, to obtain drug solution m2; S4. Mix the drug solution m1 and the drug solution m2, concentrate by ultrafiltration to obtain a concentrated solution, add an oxidant to the concentrated solution for oxidation, add ethanol for precipitation, collect the second precipitate, and obtain a sheep-derived high-efficiency glycosaminoglycan mixture.
[0009] As a further technical solution, in step S1, an anion exchange resin is used during the adsorption.
[0010] As a further technical solution, in step S1, during the washing, a sodium chloride solution with a mass concentration of 3.8% to 4% is used for washing until the absorbance values at A260nm and A280nm are independently less than 0.2.
[0011] As a further technical solution, in step S1, during the elution, a sodium chloride solution with a mass concentration of 12% to 15% is used.
[0012] As a further technical solution, in step S1, when ethanol is added, the volume concentration of ethanol is 25% to 30%.
[0013] As a further technical solution, in step S2, the pH value of the first acidic condition is 2.5~3.0.
[0014] As a further technical solution, in step S2, the amount of the first sodium nitrite added is 1.3% to 2% of the mass of the macromolecular sheep-derived glycosaminoglycan.
[0015] As a further technical solution, in step S2, the first degradation time is 1 to 3 hours.
[0016] As a further technical solution, in step S2, the pH value of the first alkaline condition is 9.5-10.0.
[0017] As a further technical solution, in step S2, the mass ratio of the first sodium borohydride to the first sodium nitrite is 1:1-3.
[0018] As a further technical solution, in step S2, the first reduction time is 8 to 24 hours.
[0019] As a further technical solution, during the ultrafiltration, in step S2, a 5000Da ultrafiltration membrane is used and the amount of water added is 1 to 6 times the volume of the dissolved solution.
[0020] As a further technical solution, in step S3, the pH value of the second acidic condition is 2.5-3.0.
[0021] As a further technical solution, in step S3, the amount of the second sodium nitrite added is 0.6% to 3% of the mass of the macromolecular sheep-derived glycosaminoglycan.
[0022] As a further technical solution, in step S3, the second degradation time is 1 to 3 hours.
[0023] As a further technical solution, in step S3, the pH value of the second alkaline condition is 9.5-10.0.
[0024] As a further technical solution, in step S3, the mass ratio of the second sodium borohydride to the second sodium nitrite is 1:1-3.
[0025] As a further technical solution, in step S3, the second reduction time is 8 to 24 hours.
[0026] As a further technical solution, in step S4, a 1000Da or 3000Da ultrafiltration membrane is used for ultrafiltration concentration.
[0027] As a further technical solution, the oxidant includes one of hydrogen peroxide and peracetic acid.
[0028] As a further technical solution, in step S4, the amount of the oxidant added is 1.5% to 2% of the volume of the concentrated solution.
[0029] As a further technical solution, in step S4, the oxidation time is 23-24 hours and the pH value is 9.5-10.5.
[0030] The present invention also provides a sheep-derived high-cost glycosaminoglycan mixture prepared by the method for preparing the sheep-derived high-cost glycosaminoglycan mixture.
[0031] As a further technical solution, the general structural formula of the sheep-derived high-efficiency glycosaminoglycan mixture is:
[0032] n is 1 to 20, R is —H or —SO3Na, and R' is one of —SO3Na, —CO—CH3, and —H; The reducing end of the glycosaminoglycan includes 6-O-thio-2,5-anhydro-mannitol.
[0033] As a further technical solution, the weight average molecular weight of the glycosaminoglycan mixture is 3000-6500.
[0034] As a further technical solution, the anti-factor Xa potency of the glycosaminoglycan mixture is ≥90 IU / mg.
[0035] As a further technical solution, the potency ratio of the glycosaminoglycan mixture against factor Xa and factor IIa is 1.9-10.0.
[0036] As a further technical solution, the monosaccharide structure of the glycosaminoglycan mixture includes IdoA2S (sulfated iduronic acid at position 2), IdoA (iduronic acid), GlcA (glucuronic acid), GlcNS (sulfated glucosamine at position N), GlcNAc (acetylated glucosamine at position N), Glc3S (sulfated glucosamine at position 3), Glc6OH (hydroxyglucosamine at position 6), Glc6S (sulfated glucosamine at position 6), AM-ol6S (sulfated dehydrated mannitol at position 6) and AM-ol6OH (hydroxyl dehydrated mannitol at position 6).
[0037] As a further technical solution, in terms of relative content, in the monosaccharide structure of the glycosaminoglycan mixture, IdoA2S accounts for 78.5% to 80.5% of uronic acid, and Glc6S accounts for 89% to 91% of glucosamine.
[0038] As a further technical solution, the disaccharide and other enzymatically hydrolyzed sugar unit structures of the glycosaminoglycan mixture include △IS (unsaturated 2-sulfated uronic acid-N-sulfated 6-sulfated glucosamine), △IA (unsaturated 2-sulfated uronic acid-N-acetylated 6-sulfated glucosamine), △IIA-IISglu (unsaturated uronic acid-N-acetylated 6-sulfated glucosamine-uronic acid-N-sulfated 3-sulfated 6-sulfated glucosamine), △IM (unsaturated 2-sulfated uronic acid-6-sulfated 2,5-dehydrated mannitol), △IIM (unsaturated uronic acid-6-sulfated 2,5-dehydrated mannitol), and △IS-IM (unsaturated 2-sulfated uronic acid-N-sulfated 6-sulfated glucosamine-2-sulfated uronic acid-6-sulfated 2,5-dehydrated mannitol).
[0039] As a further technical solution, in terms of relative content, among the disaccharides and other enzymatically hydrolyzed sugar unit structures, △IA accounts for 0.6% to 0.75% of the glycosaminoglycan mixture, △IIA-IISglu accounts for 2.45% to 2.55% of the glycosaminoglycan mixture, △IS-IM accounts for 7% to 18% of the glycosaminoglycan mixture, and △IS accounts for 42% to 62% of the glycosaminoglycan mixture.
[0040] In the present invention, the sheep-derived high-efficiency glycosaminoglycan mixture has a higher IdoA2S, accounting for about 78.5% to 80.5% of the entire uronic acid; has a stable Glc6S, accounting for about 89% to 91% of glucosamine; has a stable △IA, accounting for about 0.6% to 0.75% of the mixture, and △IIA-IISglu accounts for about 2.45% to 2.55% of the mixture. The reducing end includes a 6-O-thio-2,5-anhydro-mannitol structure, which is significantly different from the heparin products currently on the market and has a stronger anticoagulant and antithrombotic effect.
[0041] The working principle and beneficial effects of the present invention are: In the present invention, the method for preparing a sheep-derived high-efficiency and high-cost glycosaminoglycan mixture not only effectively utilizes sheep intestines and reduces the risk of single-source supply of low-molecular-weight heparin in the market, but can also be generally used to prepare a glycosaminoglycan mixture with a weight-average low molecular weight segment of 3000-6500 and a potency ratio of 1.9-10.0, so that the glycosaminoglycan mixture has both low molecular weight and high cost performance. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.
[0043] Figure 1 This is a two-dimensional nuclear magnetic resonance spectrum of the sheep-derived high-efficiency glycosaminoglycan mixture prepared in Example 1 of Experimental Example 2; Figure 2This is a two-dimensional nuclear magnetic resonance spectrum of the sheep-derived high-cost glycosaminoglycan mixture prepared in Example 2 of Experimental Example 2; Figure 3 This is a two-dimensional nuclear magnetic resonance spectrum of the sheep-derived high-efficiency glycosaminoglycan mixture prepared in Example 3 of Experimental Example 2; Figure 4 This is the two-dimensional nuclear magnetic resonance spectrum of Experimental Example 2; Figure 5 This is the two-dimensional nuclear magnetic resonance spectrum of Kesai in Experimental Example 2; Figure 6 This is the two-dimensional nuclear magnetic resonance spectrum of Fragmin in Experimental Example 2. DETAILED DESCRIPTION
[0044] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0045] In the following embodiments and comparative examples, unless otherwise specified, the crude heparin from sheep intestinal mucosa comes from sheep intestinal mucosa; the model of the anion exchange resin is Rohm and Haas FPA98; the production batch number of Xibao is 3031754; the production batch number of Kesai is BRLG002A; the production batch number of Fragrance is GX9275; heparinase I, heparinase II and heparinase III are all purchased from Beijing Aidehawk International Technology Co., Ltd.
[0046] Example 1 A method for preparing a sheep-derived high-efficiency glycosaminoglycan mixture comprises the following steps: S1. Dissolve 200.0 g of crude sheep intestinal mucosa heparin in 200 mL of purified water, adsorb it through anion exchange resin, wash it with a sodium chloride solution with a mass concentration of 3.8% until the absorbance values at A260nm and A280nm are both less than 0.2, and then elute it with a sodium chloride solution with a mass concentration of 12% to obtain an eluate, add ethanol to the eluate until the volume concentration of ethanol is 30%, collect the precipitate, and dry it to obtain 89.0 g of macromolecular sheep-derived glycosaminoglycan; S2, dissolving all the above-mentioned macromolecular sheep-derived glycosaminoglycans in purified water to a volume of 1780 mL to obtain a solution, adjusting the pH value of the solution to 2.5, adding 1.78 g of sodium nitrite, degrading for 2 h, then adjusting the pH value to 10.0, adding 0.59 g of sodium borohydride, reducing for 16 h, using a 5000Da ultrafiltration membrane for ultrafiltration, collecting the drug solution m1 at the filtration end, collecting the drug solution M1 at the reflux end, and replenishing 10680 mL of water during ultrafiltration; S3, adjust the pH value of the liquid M1 to 2.5, add 2.58g of sodium nitrite, degrade for 2h, then adjust the pH value to 9.5, add 0.86g of sodium borohydride, reduce for 16h, and obtain liquid m2; S4. Mix the drug solutions m1 and m2, and concentrate them through ultrafiltration using a 1000Da ultrafiltration membrane to obtain 1780mL of concentrated solution. Add 33.8mL of hydrogen peroxide to the concentrated solution, and adjust the pH value to 9.5. Oxidize for 24 hours, and adjust the pH value to 6.8. Add ethanol until the volume concentration of ethanol is 85%, separate the precipitate, collect the precipitate, and dry it to obtain 53.4g of a high-efficiency glycosaminoglycan mixture from sheep.
[0047] Example 2 A method for preparing a sheep-derived high-efficiency glycosaminoglycan mixture comprises the following steps: S1. Dissolve 500.0 g of crude sheep intestinal mucosa heparin in 5000 mL of purified water, adsorb it through anion exchange resin, wash it with a sodium chloride solution with a mass concentration of 4.0% until the absorbance values at A260nm and A280nm are both less than 0.15, and then elute it with a sodium chloride solution with a mass concentration of 12% to obtain an eluate, add ethanol to the eluate until the volume concentration of ethanol is 28%, collect the precipitate, and dry it to obtain 230.0 g of macromolecular sheep-derived glycosaminoglycan; S2, dissolving all the above-mentioned macromolecular sheep-derived glycosaminoglycans in purified water to a volume of 4600 mL to obtain a solution, adjusting the pH value of the solution to 2.8, adding 3.68 g of sodium nitrite, degrading for 2 h, then adjusting the pH value to 9.5, adding 3.68 g of sodium borohydride, reducing for 8 h, using a 5000Da ultrafiltration membrane for ultrafiltration, collecting the drug solution m1 at the filtration end, collecting the drug solution M1 at the reflux end, and replenishing 14 L of water during ultrafiltration; S3, adjust the pH value of the liquid M1 to 3.0, add 4.60g of sodium nitrite, degrade for 2h, then adjust the pH value to 9.5, add 4.60g of sodium borohydride, reduce for 8h, and obtain liquid m2; S4. Mix the drug solutions m1 and m2, and concentrate them by ultrafiltration using a 3000Da ultrafiltration membrane to obtain 4600mL of concentrated solution. Add 69mL of peracetic acid to the concentrated solution, adjust the pH value to 10.5, oxidize for 23h, adjust the pH value to 6.8, add ethanol to a volume concentration of 85%, separate the precipitate, collect the precipitate, and dry it to obtain 163.3g of a high-efficiency glycosaminoglycan mixture from sheep.
[0048] Example 3 A method for preparing a sheep-derived high-efficiency glycosaminoglycan mixture comprises the following steps: S1. Dissolve 1.0 kg of crude sheep intestinal mucosa heparin in 10.0 L of purified water, adsorb it through anion exchange resin, wash it with a sodium chloride solution with a mass concentration of 3.8% until the absorbance values at A260nm and A280nm are both less than 0.18, and then elute it with a sodium chloride solution with a mass concentration of 15% to obtain an eluate, add ethanol to the eluate until the volume concentration of ethanol is 26%, collect the precipitate, and dry it to obtain 395.0 g of macromolecular sheep-derived glycosaminoglycan; S2, dissolving all the above-mentioned macromolecular sheep-derived glycosaminoglycans in purified water to a volume of 7.9 L to obtain a solution, adjusting the pH value of the solution to 3.0, adding 5.14 g of sodium nitrite, degrading for 2 h, then adjusting the pH value to 9.8, adding 1.71 g of sodium borohydride, reducing for 24 h, using a 5000Da ultrafiltration membrane for ultrafiltration, collecting the drug solution m1 at the filtration end, collecting the drug solution M1 at the reflux end, and replenishing 7.9 L of water during ultrafiltration; S3, adjust the pH value of the liquid M1 to 2.5, add 2.37g of sodium nitrite, degrade for 2h, then adjust the pH value to 9.5, add 0.79g of sodium borohydride, reduce for 24h, and obtain liquid m2; S4. Mix the drug solutions m1 and m2, and concentrate them by ultrafiltration using a 3000Da ultrafiltration membrane to obtain 7.9L of concentrated solution. Add 158mL of hydrogen peroxide to the concentrated solution, and adjust the pH value to 10. Oxidize for 24 hours and adjust the pH value to 6.8. Add ethanol until the volume concentration of ethanol is 85%, separate the precipitate, collect the precipitate, and dry it to obtain 259.5g of a high-efficiency glycosaminoglycan mixture from sheep.
[0049] Experimental Example 1 The molecular weight and distribution of the sheep-derived high-efficiency glycosaminoglycan mixture prepared in Examples 1 to 3 were determined by referring to the molecular exclusion chromatography method of Part IV of the 2020 edition of the Chinese Pharmacopoeia 0514. The potency detection method of the European Pharmacopoeia 11.2 General Rules for Low Molecular Weight Heparin was used to determine the potency of the sheep-derived high-efficiency glycosaminoglycan mixture prepared in Examples 1 to 3, and the potency ratio of anti-factor Xa and anti-factor IIa was calculated. The test results are shown in Table 1 below.
[0050] Table 1 Molecular weight and titer results
[0051] The sheep-derived high-cost glycosaminoglycan mixtures prepared in Examples 1 to 3 were compared with commercially available products of bemiparin sodium, enoxaparin sodium and dalteparin sodium, respectively. The results are shown in Tables 2 to 4 below.
[0052] Table 2 Comparison of sheep-derived high-efficiency glycosaminoglycan mixture and bemiparin sodium in Example 1
[0053] Table 3 Comparison of sheep-derived high-efficiency glycosaminoglycan mixture and enoxaparin sodium in Example 2
[0054] Table 4 Comparison of sheep-derived high-efficiency glycosaminoglycan mixture and dalteparin sodium in Example 3
[0055] As shown in Tables 2 to 4, the preparation method of the present invention can realize the preparation of anticoagulant and antithrombotic glycosaminoglycan mixtures of different molecular weight segments and potency ratios, and can meet the physical and chemical indicators of bemiparin sodium, enoxaparin sodium and dalteparin sodium required for different molecular weight segments and potency ratios.
[0056] Experimental Example 2 The characteristic uronic acid, characteristic glucosamine and characteristic structure 2,5-anhydro-mannitol of the sheep-derived high-cost glycosaminoglycan mixture prepared in Examples 1 to 3 of the present invention were analyzed by 2D-HSQC-NMR analysis technology, and compared with commercially available low molecular weight heparin products. The specific experimental method is: 0.100g of sample was added with 0.5mL of heavy water, and transferred to a nuclear magnetic resonance test tube with a diameter of 5mm, and the two-dimensional nuclear magnetic resonance spectra of each sample solution at 298K were collected using an Avance III HD 500MHZ NMR spectrometer (such as Figures 1 to 6 The cross peaks were integrated and the relative percentage of monosaccharides was calculated after normalization. The results are shown in Table 5 below.
[0057] Table 5 2D-HSQC-NMR data comparison
[0058] As shown in Table 5, the sheep-derived high-efficiency glycosaminoglycan mixture prepared by the present invention has a high IdoA2S, which accounts for about 78.5% to 80.5% of the total uronic acid in terms of relative content. It has a stable Glc6S that accounts for about 89% to 91% of glucosamine, which is higher than Xibao and Kesai. Other uronic acid and glucosamine monosaccharide components are also different from commercially available low molecular weight heparin varieties.
[0059] The preparation method uses sodium nitrite degradation. Under acidic conditions, sodium nitrite forms nitrosyl cations, which attack the amino nitrogen atom at the second position of glucosamine as an electrophilic reagent, and then forms a diazonium salt through tautomerism to generate N2, which triggers the cleavage of the glycosidic bond and the inner ether ring to generate a 2,5-anhydro-mannitol structure. As the weight-average molecular weight of the product decreases, the cleavage increases, the corresponding GlcNS content gradually decreases, and the AM-ol6S and AM-ol6OH contents gradually increase. However, when the sheep-derived high-cost glycosaminoglycan mixture prepared by the present invention is close to the molecular weight of the commercially available low-molecular-weight heparin product, the GlcNS content is generally higher than the commercially available low-molecular-weight heparin product.
[0060] Experimental Example 3 Heparinase (composed of heparinase I, heparinase II and heparinase III in a mass ratio of 1:1:1) was used to enzymatically hydrolyze the sheep-derived high-cost glycosaminoglycan mixture prepared in Examples 1 to 3 and the commercially available low-molecular-weight heparin product at 25°C for 72 hours to obtain disaccharides and other enzymatic fragments, which were detected by referring to the detection method for identifying B-1.6 dehydrate derivatives under the monograph of enoxaparin sodium in the European Pharmacopoeia, wherein the optimized injection volume was 10 μL. The test results are shown in Table 6 below.
[0061] Table 6 Disaccharide data comparison
[0062] As shown in Table 6, the sheep-derived high-efficiency glycosaminoglycan mixture prepared by the present invention has stable △IA and △IIA-IISglu components. In terms of relative content, △IA accounts for about 0.6% to 0.75% of the mixture, which is significantly lower than the commercially available low molecular weight heparin products, and △IA-IISglu accounts for about 2.45% to 2.55% of the mixture, which is significantly lower than Fragmin.
[0063] When the preparation method using sodium nitrite degradation is used, the chain breaking is initiated by the glucosamine NS site, so as the weight-average molecular weight of the product decreases, △IS, △IIS (unsaturated uronic acid-N-sulfated 6-position sulfated glucosamine) and △IIIS (unsaturated 2-position sulfated uronic acid-N-sulfated glucosamine) gradually decrease, and △IM, △IIM and △IS-IM gradually increase.
[0064] According to the different weight average molecular weights of the target products, in terms of relative content, ΔIS-IM in the sheep-derived high-efficiency glycosaminoglycan mixture prepared by the present invention accounts for 7% to 18% of the mixture, and ΔIS accounts for about 42% to 62% of the mixture.
[0065] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing a sheep-derived high-efficiency glycosaminoglycan mixture, characterized in that: The following steps are involved: S1, dissolving, adsorbing, washing and eluting the crude heparin from the sheep intestinal mucosa to obtain an eluate, adding ethanol to the eluate, collecting the first precipitate, and drying to obtain a macromolecular sheep-derived glycosaminoglycan; S2, dissolving the macromolecular sheep-derived glycosaminoglycan to obtain a solution, adding a first sodium nitrite to the solution under a first acidic condition, performing a first degradation, and then adding a first sodium borohydride under a first alkaline condition, performing a first reduction, performing ultrafiltration, collecting the drug solution m1 at the filtration end, and collecting the drug solution M1 at the reflux end; S3, under a second acidic condition, adding a second sodium nitrite to the drug solution M1, performing a second degradation, and then under a second alkaline condition, adding a second sodium borohydride, performing a second reduction, to obtain drug solution m2; S4. Mix the drug solution m1 and the drug solution m2, concentrate by ultrafiltration to obtain a concentrated solution, add an oxidant to the concentrated solution for oxidation, add ethanol for precipitation, collect the second precipitate, and obtain a sheep-derived high-efficiency glycosaminoglycan mixture.
2. The method for preparing a sheep-derived high-cost glycosaminoglycan mixture according to claim 1, characterized in that: In step S1, an anion exchange resin is used during the adsorption; and / or During the washing, a sodium chloride solution with a mass concentration of 3.8% to 4% is used for washing until the absorbance values at A260nm and A280nm are independently less than 0.2; and / or During the elution, a sodium chloride solution with a mass concentration of 12% to 15% is used; and / or When ethanol is added, the volume concentration of ethanol is 25% to 30%.
3. The method for preparing a sheep-derived high-cost glycosaminoglycan mixture according to claim 1, characterized in that: In step S2, the pH value of the first acidic condition is 2.5-3.0; and / or The amount of the first sodium nitrite added is 1.3% to 2% of the mass of the macromolecular sheep-derived glycosaminoglycan; and / or The first degradation period is 1 to 3 hours; and / or The pH value of the first alkaline condition is 9.5-10.0; and / or The mass ratio of the first sodium borohydride to the first sodium nitrite is 1:1-3; and / or The first reduction time is 8 to 24 hours; and / or During the ultrafiltration, a 5000Da ultrafiltration membrane is used, and the amount of water replenished is 1 to 6 times the volume of the dissolved solution.
4. The method for preparing a sheep-derived high-cost glycosaminoglycan mixture according to claim 1, characterized in that: In step S3, the pH value of the second acidic condition is 2.5-3.0; and / or The amount of the second sodium nitrite added is 0.6% to 3% of the mass of the macromolecular sheep-derived glycosaminoglycan; and / or The second degradation period is 1 to 3 hours; and / or The pH value of the second alkaline condition is 9.5-10.0; and / or The mass ratio of the second sodium borohydride to the second sodium nitrite is 1:1-3; and / or The second reduction time is 8 to 24 hours.
5. The method for preparing a sheep-derived high-cost glycosaminoglycan mixture according to claim 1, characterized in that: In step S4, during the ultrafiltration concentration, a 1000Da or 3000Da ultrafiltration membrane is used; and / or The oxidant comprises one of hydrogen peroxide and peracetic acid; and / or The amount of the oxidant added is 1.5% to 2% of the volume of the concentrated solution; and / or During the oxidation, the time is 23-24 hours and the pH value is 9.5-10.
5.
6. A sheep-derived high-efficiency glycosaminoglycan mixture, characterized in that: The mixture is prepared by the method for preparing a sheep-derived high-cost glycosaminoglycan mixture as described in any one of claims 1 to 5.
7. The sheep-derived high-cost glycosaminoglycan mixture according to claim 6, characterized in that: The weight average molecular weight of the glycosaminoglycan mixture is 3000-6500.
8. The sheep-derived high-cost glycosaminoglycan mixture according to claim 6, characterized in that: The anti-factor Xa potency of the glycosaminoglycan mixture is ≥90 IU / mg.
9. The sheep-derived high-cost glycosaminoglycan mixture according to claim 6, characterized in that: The potency ratio of the glycosaminoglycan mixture against factor Xa and factor IIa is 1.9-10.
0.
10. The sheep-derived high-cost glycosaminoglycan mixture according to claim 6, characterized in that: In terms of relative content, in the monosaccharide structure of the glycosaminoglycan mixture, IdoA2S is 78.5% to 80.5% of uronic acid, and Glc6S is 89% to 91% of glucosamine; and / or In terms of relative content, among the disaccharides and other enzymatically hydrolyzed sugar unit structures, △IA accounts for 0.6% to 0.75% of the glycosaminoglycan mixture, △IIA-IISglu accounts for 2.45% to 2.55% of the glycosaminoglycan mixture, △IS-IM accounts for 7% to 18% of the glycosaminoglycan mixture, and △IS accounts for 42% to 62% of the glycosaminoglycan mixture.