CN116284499B - Preparation method of sheep-derived low-molecular heparin sodium - Google Patents
Preparation method of sheep-derived low-molecular heparin sodium Download PDFInfo
- Publication number
- CN116284499B CN116284499B CN202210896015.5A CN202210896015A CN116284499B CN 116284499 B CN116284499 B CN 116284499B CN 202210896015 A CN202210896015 A CN 202210896015A CN 116284499 B CN116284499 B CN 116284499B
- Authority
- CN
- China
- Prior art keywords
- sheep
- glycosaminoglycan
- heparin sodium
- sodium
- molecular heparin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920000669 heparin Polymers 0.000 title claims abstract description 68
- 241001494479 Pecora Species 0.000 title claims abstract description 62
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 title claims abstract description 59
- 229960001008 heparin sodium Drugs 0.000 title claims abstract description 57
- 239000003055 low molecular weight heparin Substances 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 229920002683 Glycosaminoglycan Polymers 0.000 claims abstract description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 25
- 239000000243 solution Substances 0.000 claims abstract description 18
- 210000000936 intestine Anatomy 0.000 claims abstract description 16
- 210000004877 mucosa Anatomy 0.000 claims abstract description 12
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 8
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 6
- 238000006731 degradation reaction Methods 0.000 claims abstract description 5
- YRTWNFXASSUQEW-UHFFFAOYSA-M sodium;methanol;acetate Chemical compound [Na+].OC.CC([O-])=O YRTWNFXASSUQEW-UHFFFAOYSA-M 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 49
- 239000000047 product Substances 0.000 claims description 27
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000002244 precipitate Substances 0.000 claims description 15
- 230000003647 oxidation Effects 0.000 claims description 14
- 238000007254 oxidation reaction Methods 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 14
- 150000003839 salts Chemical class 0.000 claims description 13
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 238000012869 ethanol precipitation Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 8
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical group [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 230000001590 oxidative effect Effects 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 239000012043 crude product Substances 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- KCXMKQUNVWSEMD-UHFFFAOYSA-N benzyl chloride Chemical compound ClCC1=CC=CC=C1 KCXMKQUNVWSEMD-UHFFFAOYSA-N 0.000 claims description 6
- 229940073608 benzyl chloride Drugs 0.000 claims description 6
- 239000007800 oxidant agent Substances 0.000 claims description 6
- UREZNYTWGJKWBI-UHFFFAOYSA-M benzethonium chloride Chemical compound [Cl-].C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 UREZNYTWGJKWBI-UHFFFAOYSA-M 0.000 claims description 5
- 229960001950 benzethonium chloride Drugs 0.000 claims description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- 238000002835 absorbance Methods 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 230000001376 precipitating effect Effects 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- 238000005194 fractionation Methods 0.000 claims description 2
- 229940127215 low-molecular weight heparin Drugs 0.000 claims description 2
- 239000012286 potassium permanganate Substances 0.000 claims description 2
- 150000003333 secondary alcohols Chemical class 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 229960005153 enoxaparin sodium Drugs 0.000 abstract description 11
- CIJQTPFWFXOSEO-NDMITSJXSA-J tetrasodium;(2r,3r,4s)-2-[(2r,3s,4r,5r,6s)-5-acetamido-6-[(1r,2r,3r,4r)-4-[(2r,3s,4r,5r,6r)-5-acetamido-6-[(4r,5r,6r)-2-carboxylato-4,5-dihydroxy-6-[[(1r,3r,4r,5r)-3-hydroxy-4-(sulfonatoamino)-6,8-dioxabicyclo[3.2.1]octan-2-yl]oxy]oxan-3-yl]oxy-2-(hydroxy Chemical compound [Na+].[Na+].[Na+].[Na+].O([C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1O)NC(C)=O)O[C@@H]1C(C[C@H]([C@@H]([C@H]1O)O)O[C@@H]1[C@@H](CO)O[C@H](OC2C(O[C@@H](OC3[C@@H]([C@@H](NS([O-])(=O)=O)[C@@H]4OC[C@H]3O4)O)[C@H](O)[C@H]2O)C([O-])=O)[C@H](NC(C)=O)[C@H]1C)C([O-])=O)[C@@H]1OC(C([O-])=O)=C[C@H](O)[C@H]1O CIJQTPFWFXOSEO-NDMITSJXSA-J 0.000 abstract description 11
- 230000032050 esterification Effects 0.000 abstract description 2
- 238000005886 esterification reaction Methods 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000002904 solvent Substances 0.000 abstract description 2
- 239000007858 starting material Substances 0.000 abstract description 2
- 239000010891 toxic waste Substances 0.000 abstract description 2
- 239000002912 waste gas Substances 0.000 abstract description 2
- 239000008213 purified water Substances 0.000 description 12
- 239000003814 drug Substances 0.000 description 9
- 229960002897 heparin Drugs 0.000 description 9
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 210000004347 intestinal mucosa Anatomy 0.000 description 6
- 241000282898 Sus scrofa Species 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000001858 anti-Xa Effects 0.000 description 4
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 229960002442 glucosamine Drugs 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 241000282849 Ruminantia Species 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- 150000002016 disaccharides Chemical class 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 101000860173 Myxococcus xanthus C-factor Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 206010047249 Venous thrombosis Diseases 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 229920001586 anionic polysaccharide Polymers 0.000 description 1
- 150000004836 anionic polysaccharides Chemical class 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000002682 general surgery Methods 0.000 description 1
- 238000001631 haemodialysis Methods 0.000 description 1
- 230000000322 hemodialysis Effects 0.000 description 1
- -1 heparin quaternary ammonium salt Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 208000004043 venous thromboembolism Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a preparation method of sheep-derived low-molecular heparin sodium, which comprises the following steps: treating the sheep intestine mucosa glycosaminoglycan aqueous solution with anion exchange resin; adding sodium acetate methanol solution to precipitate during the esterification of glycosaminoglycan; adding carbonate into degradation reaction to degrade; and finally obtaining the sheep-derived low-molecular heparin sodium fine product. The method uses sheep intestine mucosa glycosaminoglycan as a starting material, the process route is short, the prepared sheep source low molecular heparin sodium has high yield and good quality, no waste gas and toxic waste water are discharged in the process, the solvent can be recycled, the environment friendliness is high, the product quality completely accords with European pharmacopoeia and United states pharmacopoeia standards of enoxaparin sodium, and the method is suitable for industrial production.
Description
Technical Field
The invention relates to a preparation method of sheep-derived low-molecular heparin sodium, and belongs to the technical field of medical biology.
Background
Heparin is a complex mixture of linear anionic polysaccharides with an average molecular weight of 16000Da. The average molecular weight of the low molecular heparin is 4000-6000Da, and the low molecular heparin can be obtained by degrading glycosaminoglycan through an enzymatic method or a chemical method.
Heparin and low molecular heparin thereof are anticoagulants widely used clinically. Is mainly applied to kidney dialysis, heart and lung bypass and venous thrombosis. Can be used for preventing venous thromboembolic diseases (preventing intravenous thrombosis), especially thrombosis related to orthopedics or general surgery; as modern medical procedures (e.g., hemodialysis, open heart surgery, treatment of thrombosis, etc.) become more common, the global demand for heparin continues to grow.
At present, heparin and low molecular heparin mainly come from pig intestines, and more than half of global heparin raw material medicines are supplied from China. Hog cholera in 2018, resulting in a severe reduction in live pig stock, reliance on a single species makes the supply chain of this key drug very short, and there is a need to find sources of other species for these drugs, including sheep tissue, such as sheep intestines. Sheep intestines are generally used as food materials, and the social utilization value of medicines is low, so that reasonable utilization of sheep intestines is needed.
In addition, in countries with most muslims, it is clearly specified that only ruminant products such as cattle and sheep are allowed to be eaten, and non-ruminant products such as pigs and dogs are forbidden to be eaten, so that development of heparin products from other species is urgent in the halal market to be used as anticoagulants.
CN105131153a discloses a method for preparing enoxaparin sodium in sheep. The method takes sheep intestinal mucosa as a raw material, firstly, the sheep intestinal mucosa is pretreated by sodium chloride aqueous solution, and then ethanol precipitation refining is carried out at room temperature to obtain sheep intestinal mucosa heparin; mixing with benzethonium chloride aqueous solution to obtain sheep intestinal mucosa heparin quaternary ammonium salt; and obtaining the finished product of the sheep intestinal mucosa heparin through conventional esterification, degradation, decoloration and refining. Although the method solves the problem of adopting ruminant animal products as raw materials, the preparation method has low product yield and the product quality is still to be further improved.
Disclosure of Invention
The invention aims to provide a preparation method of sheep-derived low-molecular heparin sodium, so as to effectively improve the yield and the product quality of the sheep-derived low-molecular heparin sodium.
The preparation method of the sheep-source low-molecular heparin sodium provided by the invention comprises the following steps:
a. Washing the sheep intestine mucosa glycosaminoglycan aqueous solution with 2% -4% sodium chloride through anion exchange resin until the absorbance values of A 260nm and A 280nm are less than 0.5; eluting with 10-15% sodium chloride solution, adding ethanol into the eluate until the ethanol precipitation degree is 25% -30%, precipitating with ethanol to 4-6 h, and collecting precipitate;
b. adding water to dissolve the precipitate obtained in the step (a), adding an oxidant solution with the volume ratio of 0.5-2% into the water solution for oxidation and decoloration, wherein the oxidation temperature is 20-30 ℃, the oxidation pH is 9.0-11.0, adding ethanol after oxidation for 6-24 hours until the ethanol precipitation degree is more than or equal to 60%, and carrying out secondary alcohol precipitation to obtain the heparin sodium;
c. dissolving the heparin sodium obtained in the step (b) in water, and adding a benzethonium chloride solution to prepare a glycosaminoglycan salt;
d. Adding N, N-dimethylformamide into the glycosaminoglycan salt obtained in the step (c) according to the mass-volume ratio of 1:5-10, stirring and dissolving, adding benzyl chloride according to the mass-volume ratio of the glycosaminoglycan salt to the benzyl chloride of 1:0.5-3, stirring and reacting for 5-24 hours at the temperature of 30-45 ℃, adding sodium acetate methanol solution with the mass ratio concentration of 8-10% after the reaction is finished, precipitating, washing the precipitate with ethanol, and drying to obtain the glycosaminoglycan esterified product;
e. Dissolving the glycosaminoglycan esterified product obtained in the step (d) in water, and adding carbonate to carry out degradation reaction at 50-65 ℃; the mass ratio of the glycosaminoglycan esterified substance to the carbonate is 1:0.1-2, the reaction is carried out for 2-8 hours, the temperature is reduced to 15-45 ℃, the pH is adjusted to be neutral, the organic solvent with the volume of 1.5-2.5 times is directly added for fractionation, and the supernatant is separated, thus obtaining the sheep-source low molecular heparin sodium crude product;
f. adding 0.5-2% of oxidant into the crude water solution to oxidize, wherein the oxidation temperature is 20-30 ℃, the oxidation time is 6-24 h, the oxidation pH is 9.0-11.0, 4 times of ethanol is used for alcohol precipitation, and the sheep-source low-molecular heparin sodium fine product is obtained by freeze vacuum drying.
The method comprises the following steps:
(a) In the step, the mass ratio of the sheep intestine mucosa glycosaminoglycan to the water of the sheep intestine mucosa glycosaminoglycan aqueous solution is preferably 1:10-15; the relevant substances in the precipitate are retained, preferably with a content of < 2%.
The carbonate is preferably sodium carbonate or sodium bicarbonate.
The anion exchange resin is preferably FPA98Cl.
The oxidant can be one of hydrogen peroxide, potassium permanganate and peracetic acid.
The organic solvent can be one of methanol, ethanol and acetone.
According to the method, the sheep-derived low-molecular heparin sodium is prepared by taking sheep intestinal mucosa glycosaminoglycan as a starting material, using carbonate as a degradation agent, and is short in process route, the prepared sheep-derived low-molecular heparin sodium is high in yield and good in quality, waste gas and toxic waste water are not discharged in the process, the solvent can be recycled after being recycled, the environment friendliness is high, and the product quality completely accords with European pharmacopoeia and United states pharmacopoeia standards of enoxaparin sodium, so that the method is suitable for industrial production. The sheep intestine mucosa glycosaminoglycan used in the invention is obtained by enzymolysis, salt hydrolysis and ethanol precipitation of sheep intestine mucosa.
The sheep-derived low molecular heparin sodium prepared by the method of the invention is composed of a polydisperse system, and the weight average molecular weight of the sheep-derived low molecular heparin sodium is 4000-4500Da; the proportion of the molecular weight less than 2000 is 15.0-19.0%; the ratio of 2000-8000 parts is 70.0-75.0%; the proportion of more than 8000 parts is 8.0-11.0%; the anti-Xa factor drying titer is 100-120 IU/mg, the anti-II factor drying titer is 25-35 IU/mg, and the titer ratio is 3.5-4.0; the delta IS content of disaccharide IS 65% -70%, the percentage of reduced glucosamine forming 1,6 dehydration structure IS 17-25%, and the peak of methyl C signal of glucosamine 2-position connected acetyl near C spectrum 24 ppm IS obviously lower than standard substance (pig source).
The method of the invention takes the sheep heparin sodium obtained in the step b as an initial feeding amount, and the weight yield of the sheep low molecular heparin sodium refined product is up to more than 70%.
Drawings
Fig. 1 is a schematic representation of the molecular weight distribution of enoxaparin sodium standards.
FIG. 2 is a schematic diagram of molecular weight distribution of sheep-derived low molecular weight heparin sodium prepared in example 2 of the present invention.
Fig. 3 is a disaccharide profile of enoxaparin sodium standard.
FIG. 4 is a disaccharide profile of sheep-derived low-molecular heparin sodium prepared in example 2 of the present invention.
Fig. 5 is a C spectrum of enoxaparin sodium standard.
FIG. 6 is a C-spectrum of sheep-derived low molecular heparin sodium prepared in example 2 of the present invention.
Detailed Description
The invention will be described in detail with reference to specific embodiments and accompanying drawings
Example 1:
The preparation method of the sheep-source low-molecular heparin sodium comprises the following specific steps:
(a) Glycosaminoglycan purification
Weighing 100g of sheep intestine mucosa glycosaminoglycan, adding 1000 mL of purified water, stirring for dissolution, adding the water solution into an anion exchange column for adsorption, washing with 2% NaCl after the adsorption is finished until the absorbance values of A 260nm and A 280nm are less than 0.5, and stopping washing; then, elution was performed with 12% NaCl, and the eluate was collected. Ethanol is added into the eluent to ensure that the ethanol precipitation degree of the ethanol reaches 30 percent, and after 4 h of ethanol precipitation, the precipitate is separated and the content of related substances is detected to be 1.1 percent.
(B) Decoloring glycosaminoglycans
Dissolving the precipitate in step (a) in water, fixing the volume of the liquid medicine to 1000 mL, regulating the pH value with 3M sodium hydroxide to 10.0, adding 10mL of 30% hydrogen peroxide, oxidizing 24h in a constant temperature water bath at 20 ℃, and regulating the pH value with 3M hydrochloric acid to 6.0-8.0. Filtering, adding 4000 mL ethanol into the filtrate, standing for 16h, separating supernatant, collecting precipitate, and drying to obtain 51.0g of heparosan.
(C) Preparation of glycosaminoglycan salt
Adding the obtained heparin sodium into 500 mL purified water, stirring and dissolving, weighing 125 g benzethonium chloride, and adding 750 mL of purified water at 50-60 ℃ for dissolving. Mixing the components, stirring and reacting for 2 hours, then standing, and filtering to obtain the dry glycosaminoglycan salt.
(D) Preparation of glycosaminoglycan esterified product
The glycosaminoglycan salt obtained above was added into 1200 mL of N, N-dimethylformamide and dissolved under stirring, 150 mL of benzyl chloride was added, the reaction was stopped after 8 hours of stirring at 35 ℃, and 4050 mL of 8% sodium acetate in methanol was added to precipitate a solid. The obtained solid is washed by ethanol, dehydrated and dried to prepare the glycosaminoglycan esterified substance.
(E) Preparation of sheep-source low-molecular heparin sodium crude product
The glycosaminoglycan esterified substance obtained above was added to 1000 mL of purified water at 65℃and dissolved by stirring, 5.0 g of sodium carbonate was weighed and added to 25 mL of purified water and dissolved by stirring. Adding sodium carbonate solution into glycosaminoglycan esterified substance solution, controlling the temperature to react for 2 hours, cooling to 45 ℃, adjusting the pH to be neutral, adding 2000 mL ethanol for grading, and separating the supernatant to obtain the sheep-source low-molecular heparin sodium crude product.
(F) Preparation of sheep-source low-molecular heparin sodium fine product
Adding 500 mL purified water into the obtained sheep-source low-molecular heparin sodium crude product to dissolve, adding 5 mL of 30% hydrogen peroxide, adjusting the pH of the liquid medicine to 10.0, reacting for 24 hours at the temperature of 20 ℃, adjusting the pH of the liquid medicine to 6.0-8.0 after the reaction is finished, filtering, adding 10 g sodium chloride into the filtrate to stir and dissolve, adding 2000 mL ethanol for alcohol precipitation after dissolving, separating the supernatant, freezing and drying the precipitate, and obtaining 36.5 g of sheep-source low-molecular heparin sodium refined product.
The weight yield of the sheep-derived low-molecular heparin sodium fine product in the embodiment is 71.5%.
Example 2:
(a) Glycosaminoglycan purification
Weighing 2kg of sheep intestine mucosa glycosaminoglycan, adding 20L of purified water, stirring and dissolving, adding the aqueous solution into an FPA98Cl ion exchange column for adsorption, washing with 4% NaCl after the adsorption is finished until the absorbance values of A 260nm and A 280nm are less than 0.5, and stopping washing; then, elution was performed with 15% NaCl, and the eluate was collected. Ethanol is added into the eluent to ensure that the ethanol precipitation degree of the ethanol reaches 25 percent, and after the ethanol precipitation is 6 h, the precipitate is separated and the content of related substances is detected to be 0.9 percent.
(B) Decoloring glycosaminoglycans
Separating supernatant, dissolving precipitate in water, fixing volume to 20L, regulating pH with 3M sodium hydroxide to 11.0, adding 200mL peracetic acid, oxidizing in a water bath at 30deg.C for 6h, and regulating pH to 6.0-8.0 with 3M hydrochloric acid. Filtering, adding 80L ethanol into the filtrate, standing for 16h, separating supernatant, collecting precipitate, and drying to obtain 1018g of heparan.
(C) Preparation of glycosaminoglycan salt
Adding 10L of purified water into the obtained heparin sodium, stirring to dissolve, weighing 2.5 kg of benzethonium chloride, and adding 15L of purified water at 50-60 ℃ to dissolve. Mixing the components, stirring and reacting for 2 hours, then standing, and filtering to obtain the dry glycosaminoglycan salt.
(D) Preparation of glycosaminoglycan esterified product
The glycosaminoglycan salt obtained above was dissolved by adding 24L of N, N-dimethylformamide and 3L of benzyl chloride, the reaction was stopped after stirring at 35℃for 8 hours, and 81L of 8% sodium acetate in methanol was added to precipitate a solid. The obtained solid is washed by ethanol, dehydrated and dried to prepare the glycosaminoglycan esterified substance.
(E) Preparation of sheep-source low-molecular heparin sodium crude product
Adding purified water at 10L and 55 ℃ into the glycosaminoglycan esterified substance obtained in the above way, stirring and dissolving, weighing sodium bicarbonate of 2 kg, adding purified water at 10L and 55 ℃ and stirring and dissolving. Adding the glycosaminoglycan esterified substance solution into sodium bicarbonate solution, controlling the temperature to react for 8 hours, reducing the temperature to 15-45 ℃, adjusting the pH to be neutral, and adding 40L ethanol for alcohol precipitation. Separating the supernatant to obtain a sheep-derived low-molecular heparin sodium crude product.
(F) Preparation of sheep-source low-molecular heparin sodium fine product
Adding 10L of purified water into the obtained crude sheep-source low-molecular heparin sodium product to dissolve, adding 100 mL of peracetic acid, adjusting the pH value of the liquid medicine to 11.0, reacting for 6 hours at the temperature of 30 ℃, adjusting the pH value of the liquid medicine to 6.0-8.0 after the reaction is finished, filtering, adding 40L of methanol into the filtrate for alcohol precipitation, separating the supernatant, and drying to obtain the sheep-source low-molecular heparin sodium fine product 750 g.
The weight yield of the sheep-derived low-molecular heparin sodium refined product in the embodiment is 73.6%.
The detection results of the sheep-derived low-molecular heparin sodium fine products prepared by the embodiment of the invention are shown in tables 1, 2, 3 and 4 in detail; fig. 2, 4 and 6.
Wherein, the detection methods of sodium content, nitrogen content, sulphur-to-carboxylic ratio, 1.6-anhydride, molecular weight, distribution, potency and the like refer to European pharmacopoeia EP10.5.
The sheep-derived low-molecular heparin sodium prepared by the method has high yield (the weight yield of the sheep-derived low-molecular heparin sodium refined product prepared by taking the sheep-derived low-molecular heparin sodium obtained in the step b as an initial feeding amount is more than 70%) and high cost performance value (more than 3.7).
And then:
As can be seen from fig. 1 and fig. 2, the molecular weight and distribution of the sheep-derived low molecular heparin sodium prepared by the method of the invention are similar to those of enoxaparin sodium standard.
From table 2 it can be seen that: the weight average molecular weight of the sheep-derived low molecular heparin sodium prepared by the invention is 4250Da and 4263Da respectively, the proportion of the molecular weight less than 2000 is 17.5% and 17.2% respectively, the proportion of the 2000-8000 is 73.0% and 73.5% respectively, and the proportion of the part more than 8000 is 9.5% and 9.3% respectively. Meets the self-determined sheep-derived low molecular heparin sodium standard of the invention, and also meets the molecular weight and distribution standard specified by the EP and USP of enoxaparin sodium.
As can be seen from table 3: the sheep-derived low molecular heparin sodium prepared by the method has the anti-Xa factor folding titer of 107IU/mg, 116 IU/mg and the anti-IIa factor folding titer of 29.1 IU/mg and 31.0IU/mg respectively, the titer ratio is 3.7, and the anti-Xa factor, anti-IIa factor and cost performance value standards specified by enoxaparin sodium EP and USP are met.
The anti-Xa factor and anti-IIa factor activities of the sheep-derived low molecular heparin sodium prepared by the method are higher than those of the swine-derived enoxaparin sodium standard, and the side surface of the sheep-derived low molecular heparin sodium sample also reflects that the sheep-derived low molecular heparin sodium sample has more sulfonated modified sites.
As can be seen from Table 3, FIG. 3 and FIG. 4, the reduced end glucosamine of sheep-derived low molecular heparin sodium prepared by the method of the invention has a1, 6 dehydration structure percentage of 21.7 percent, and meets the 1, 6-anhydride content (15-25 percent) specified by the EP and USP of enoxaparin sodium.
As can be seen from fig. 5 and fig. 6, the signal peak of methyl C of glucosamine 2-linked acetyl near the C spectrum 24 ppm of the sheep-derived low molecular heparin sodium sample is significantly lower than that of the standard (swine source), which can be used as the basis for judging different sources.
Claims (5)
1. The preparation method of the sheep-derived low-molecular heparin sodium is characterized by comprising the following steps of:
a. Washing the sheep intestine mucosa glycosaminoglycan aqueous solution with 2% -4% sodium chloride through anion exchange resin until the absorbance values of A 260nm and A 280nm are less than 0.5; eluting with 10-15% sodium chloride solution, adding ethanol into the eluate until the ethanol precipitation degree is 25% -30%, precipitating with ethanol to 4-6 h, and collecting precipitate;
b. adding water to dissolve the precipitate obtained in the step (a), adding an oxidant solution with the volume ratio of 0.5-2% into the water solution for oxidation and decoloration, wherein the oxidation temperature is 20-30 ℃, the oxidation pH is 9.0-11.0, adding ethanol after oxidation for 6-24 hours until the ethanol precipitation degree is more than or equal to 60%, and carrying out secondary alcohol precipitation to obtain the heparin sodium;
c. dissolving the heparin sodium obtained in the step (b) in water, and adding a benzethonium chloride solution to prepare a glycosaminoglycan salt;
d. adding N, N-dimethylformamide into the glycosaminoglycan salt obtained in the step (c) according to the mass-volume ratio of 1:5-10, stirring and dissolving, adding benzyl chloride according to the mass-volume ratio of the glycosaminoglycan salt to the benzyl chloride of 1:0.5-3, stirring and reacting for 5-24 hours at the temperature of 30-45 ℃, adding sodium acetate methanol solution with the mass ratio concentration of 8-10% after the reaction is finished, precipitating, washing the precipitate with ethanol, and drying to obtain the glycosaminoglycan esterified product;
e. Dissolving the glycosaminoglycan esterified product obtained in the step (d) in water, and adding carbonate to carry out degradation reaction at 50-65 ℃; the mass ratio of the glycosaminoglycan esterified substance to the carbonate is 1:0.1-2, the reaction is carried out for 2-8 hours, the temperature is reduced to 15-45 ℃, the pH is adjusted to be neutral, the organic solvent with the volume of 1.5-2.5 times is directly added for fractionation, and the supernatant is separated to obtain the sheep-source low molecular heparin sodium crude product; the carbonate is sodium carbonate or sodium bicarbonate;
f. adding 0.5-2% of oxidant into the crude water solution to oxidize, wherein the oxidation temperature is 20-30 ℃, the oxidation time is 6-24 h, the oxidation pH is 9.0-11.0, 4 times of ethanol is used for alcohol precipitation, and the sheep-source low-molecular heparin sodium fine product is obtained by freeze vacuum drying.
2. The method for preparing sheep-derived low molecular weight heparin sodium according to claim 1, wherein (a) the sheep intestine mucosa glycosaminoglycan aqueous solution has a mass ratio of sheep intestine mucosa glycosaminoglycan to water of 1:10-15; the precipitate obtained has a content of substances of interest of < 2%.
3. The method for preparing sheep-derived low molecular heparin sodium according to claim 1 or 2, wherein the anion exchange resin is FPA98Cl anion exchange resin.
4. The method for preparing sheep-derived low molecular heparin sodium according to claim 1 or 2, wherein the oxidant is one of hydrogen peroxide, potassium permanganate and peracetic acid.
5. The method for preparing sheep-derived low molecular heparin sodium according to claim 1 or 2, wherein the organic solvent is one of methanol, ethanol and acetone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210896015.5A CN116284499B (en) | 2022-07-28 | 2022-07-28 | Preparation method of sheep-derived low-molecular heparin sodium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210896015.5A CN116284499B (en) | 2022-07-28 | 2022-07-28 | Preparation method of sheep-derived low-molecular heparin sodium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116284499A CN116284499A (en) | 2023-06-23 |
| CN116284499B true CN116284499B (en) | 2024-07-23 |
Family
ID=86831053
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210896015.5A Active CN116284499B (en) | 2022-07-28 | 2022-07-28 | Preparation method of sheep-derived low-molecular heparin sodium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116284499B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117946294A (en) * | 2023-11-13 | 2024-04-30 | 河北常山凯库得生物技术有限公司 | Preparation method of energy-saving and environment-friendly enoxaparin sodium |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2912070A1 (en) * | 2012-10-24 | 2015-09-02 | Celanese Acetate LLC | Polysaccharide ester microspheres and methods and articles relating thereto |
| EP3724235A2 (en) * | 2017-12-11 | 2020-10-21 | Biological E Limited | Process for the preparation of low molecular weight heparin |
Family Cites Families (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816255B (en) * | 2011-06-10 | 2015-11-25 | 王芃 | A kind of preparation method of ultra-low molecular heparin sodium |
| CN102585038A (en) * | 2012-03-13 | 2012-07-18 | 麦科罗夫（南通）生物制药有限公司 | Islamic enoxaparin sodium and method for producing and purifying same |
| CN102603925B (en) * | 2012-03-21 | 2013-12-11 | 东营天东制药有限公司 | Method for directly producing enoxaparin sodium from crude product heparin sodium |
| CN102618614B (en) * | 2012-04-13 | 2013-07-31 | 天津高蜚生物科技发展有限公司 | Method for preparing urechis unicinctus glycosaminoglycan and antibacterial peptide simultaneously |
| CN103342761B (en) * | 2013-07-15 | 2016-01-06 | 河北常山生化药业股份有限公司 | A kind of membrane sepn prepares Enoxaparin Sodium technique |
| CN104086674B (en) * | 2014-07-28 | 2016-08-17 | 常州千红生化制药股份有限公司 | A kind of technique preparing Enoxaparin Sodium |
| CN104558252B (en) * | 2015-02-03 | 2017-06-20 | 华北制药华坤河北生物技术有限公司 | A kind of method that Enoxaparin Sodium is produced by heparin sodium crude |
| CN105131153A (en) * | 2015-08-21 | 2015-12-09 | 苏州融析生物科技有限公司 | Sheep enoxaparin sodium compound preparation method, compound and application of compound |
| CN105237657A (en) * | 2015-10-30 | 2016-01-13 | 山东大学 | Preparation method for low-molecular heparin originated from new species |
| CN107759712B (en) * | 2016-08-19 | 2020-03-24 | 苏州融析生物科技有限公司 | Sheep-derived low-molecular-weight heparin and preparation method and application thereof |
| CN106432547A (en) * | 2016-09-14 | 2017-02-22 | 苏州天马精细化学品股份有限公司 | Method for preparing enoxaparin sodium through heparin benzyl ester |
| CN108219030A (en) * | 2016-12-21 | 2018-06-29 | 鲁南制药集团股份有限公司 | A kind of preparation method of Enoxaparin Sodium crude product |
| ES2888148B2 (en) * | 2019-04-26 | 2022-11-14 | Farm Rovi Lab Sa | PROCEDURE FOR OBTAINING LOW MOLECULAR WEIGHT HEPARINS BY TANGENTIAL FLOW FILTRATION |
| CN112076211A (en) * | 2019-06-13 | 2020-12-15 | 苏州融析生物科技有限公司 | Glycosaminoglycan composition and preparation method and application thereof |
| EP3997238A1 (en) * | 2019-07-09 | 2022-05-18 | Optimvia, LLC | Methods for synthesizing anticoagulant polysaccharides |
| EP4022485A1 (en) * | 2019-10-31 | 2022-07-06 | Siemens Industry Software Netherlands B.V. | Computer implemented method for simulation of tire performance |
| CN113484262B (en) * | 2021-07-06 | 2023-04-07 | 青岛科技大学 | Method for measuring content of heparin sodium |
| CN113999330B (en) * | 2021-12-07 | 2022-08-19 | 潢川县鹏升畜产品有限公司 | Separation and co-production process of low-salt-concentration heparin sodium and active intestinal protein peptide |
| CN116515013A (en) * | 2023-05-15 | 2023-08-01 | 苏州融析生物科技有限公司 | Ultralow molecular heparin and preparation method and application thereof |
-
2022
- 2022-07-28 CN CN202210896015.5A patent/CN116284499B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2912070A1 (en) * | 2012-10-24 | 2015-09-02 | Celanese Acetate LLC | Polysaccharide ester microspheres and methods and articles relating thereto |
| EP3724235A2 (en) * | 2017-12-11 | 2020-10-21 | Biological E Limited | Process for the preparation of low molecular weight heparin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116284499A (en) | 2023-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4500519A (en) | Mucopolysaccharides having biological properties, preparation and method of use | |
| US4981955A (en) | Depolymerization method of heparin | |
| US4804652A (en) | Mucopolysaccharides having biological properties, preparation and application thereof as drugs | |
| US3451996A (en) | Method for the preparation of heparin | |
| EP3590971B1 (en) | Pentosan polysulfate, pharmaceutical composition and anticoagulant | |
| IE52951B1 (en) | Mucopolysaccharides possessing biological properties, their preparation and their application as medicaments | |
| CN116284499B (en) | Preparation method of sheep-derived low-molecular heparin sodium | |
| CN108329405B (en) | Method for protecting and purifying heparin sodium in resin adsorption state | |
| NZ200688A (en) | Antithrombotically-effective polysaccharide containing compositions | |
| IE53633B1 (en) | Process for the preparation of an injectable chondroitin polysulfate a product which can be obtained by this process and a pharmaceutical composition | |
| JP2006291028A (en) | Low-molecular heparin or salt thereof, and manufacturing method thereof | |
| EP0392396A2 (en) | Complexes of iron or other metals with sulphonated derivatives of chitosan | |
| CA2914256C (en) | New processes for the production of chemically-modified heparins | |
| WO2024046048A1 (en) | Anticoagulant heparin-derived oligosaccharide biphenyl dimer, and preparation method therefor and use thereof | |
| CA2489862C (en) | Epimerized derivatives of k5 polysaccharide with a very high degree of sulfation | |
| KR102055628B1 (en) | Method for producing heparin sodium using pig by-products | |
| US4810695A (en) | Chitosan derivatives in the form of coordinated complexes with ferrous ions | |
| AU2011369262B2 (en) | Shark-like chondroitin sulphate and process for the preparation thereof | |
| JP7167039B2 (en) | Pentosan polysulfate and method for producing pentosan polysulfate | |
| JP4186460B2 (en) | Process for producing purified heparin, salt thereof or heparin derivative and process for producing purified low molecular weight heparin, salt or heparin derivative | |
| JP2005501930A (en) | Highly branched amylopectin for use in methods of surgical or therapeutic treatment of mammals, or in diagnostic methods, particularly for use in plasma expanders | |
| CN113461835B (en) | Purification method of hydroxybutyl chitosan | |
| CN114015737A (en) | Preparation method and application of corn glutamine peptide with intestinal barrier protection function | |
| CN104725532B (en) | A kind of method of chondroitin sulfate and dermatan sulfate content in accurate quantification control heparin/heparan | |
| CN112076211A (en) | Glycosaminoglycan composition and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |