CN117362476B - Method for preparing refined heparin by using bovine lung and corresponding refined heparin - Google Patents
Method for preparing refined heparin by using bovine lung and corresponding refined heparin Download PDFInfo
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- 229920000669 heparin Polymers 0.000 title claims abstract description 103
- 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 title claims abstract description 76
- 229960002897 heparin Drugs 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 45
- 241000283690 Bos taurus Species 0.000 title claims abstract description 30
- 210000004072 lung Anatomy 0.000 title claims abstract description 30
- 239000011347 resin Substances 0.000 claims abstract description 85
- 229920005989 resin Polymers 0.000 claims abstract description 85
- 238000001179 sorption measurement Methods 0.000 claims abstract description 34
- 238000001556 precipitation Methods 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 69
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 61
- 239000000243 solution Substances 0.000 claims description 56
- 238000005406 washing Methods 0.000 claims description 46
- 238000001914 filtration Methods 0.000 claims description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- 238000002791 soaking Methods 0.000 claims description 31
- 239000011780 sodium chloride Substances 0.000 claims description 30
- 239000012153 distilled water Substances 0.000 claims description 27
- 238000001035 drying Methods 0.000 claims description 27
- 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 claims description 27
- 229960001008 heparin sodium Drugs 0.000 claims description 27
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 238000002156 mixing Methods 0.000 claims description 20
- 230000001105 regulatory effect Effects 0.000 claims description 18
- 239000002002 slurry Substances 0.000 claims description 17
- 238000001994 activation Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 14
- 230000004913 activation Effects 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 10
- 230000001590 oxidative effect Effects 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 229940088598 enzyme Drugs 0.000 claims description 6
- 230000003311 flocculating effect Effects 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims description 5
- 230000001054 cortical effect Effects 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 210000003437 trachea Anatomy 0.000 claims description 5
- 108090000526 Papain Proteins 0.000 claims description 4
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 229940055729 papain Drugs 0.000 claims description 4
- 235000019834 papain Nutrition 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 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 claims description 3
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 229910052744 lithium Inorganic materials 0.000 claims description 3
- UMPKMCDVBZFQOK-UHFFFAOYSA-N potassium;iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[K+].[Fe+3] UMPKMCDVBZFQOK-UHFFFAOYSA-N 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000012588 trypsin Substances 0.000 claims description 3
- 108091005658 Basic proteases Proteins 0.000 claims description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 2
- 108010059712 Pronase Proteins 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 7
- 239000002253 acid Substances 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 239000000805 composite resin Substances 0.000 abstract description 4
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 abstract description 3
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 abstract description 3
- 229920001287 Chondroitin sulfate Polymers 0.000 abstract description 3
- 229940059329 chondroitin sulfate Drugs 0.000 abstract description 3
- 229920002674 hyaluronan Polymers 0.000 abstract description 3
- 229960003160 hyaluronic acid Drugs 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 11
- 229940064551 bovine heparin Drugs 0.000 description 10
- 235000019441 ethanol Nutrition 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 239000012286 potassium permanganate Substances 0.000 description 9
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 description 8
- 230000003647 oxidation Effects 0.000 description 8
- 238000007254 oxidation reaction Methods 0.000 description 8
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 5
- 238000004042 decolorization Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000012043 crude product Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 210000000813 small intestine Anatomy 0.000 description 4
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000007670 refining Methods 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 238000005189 flocculation Methods 0.000 description 2
- 230000016615 flocculation Effects 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 229940127217 antithrombotic drug Drugs 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical compound N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 1
- 150000001728 carbonyl compounds Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 125000000600 disaccharide group Chemical group 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- AEMOLEFTQBMNLQ-CLQWQSTFSA-N l-iduronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@@H]1O AEMOLEFTQBMNLQ-CLQWQSTFSA-N 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- 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)
Abstract
The invention discloses a method for preparing refined heparin by using bovine lungs and corresponding refined heparin, and relates to the technical field of heparin preparation. The invention takes bovine lung as raw material, crude heparin is obtained through enzymolysis, resin adsorption, elution and precipitation, the crude heparin is redissolved, oxidized and flocculated, precipitated and dried to obtain refined heparin, the resin is adsorbed and eluted by using composite resin, and the resin is activated before adsorption, so that the resin adsorption efficiency is improved, the impurity removal rate of protein, hyaluronic acid, chondroitin sulfate and the like is improved, the purity of the crude heparin is improved, ferrate is used as an oxidized flocculant, and acid is adopted to activate the ferrate, so that the crude heparin can be effectively purified, and the high-titer and high-purity refined heparin can be prepared, and the titer of the high-titer and high-purity refined heparin can reach more than 180 IU/mg.
Description
Technical Field
The invention belongs to the technical field of heparin preparation, and particularly relates to a method for preparing refined heparin by using bovine lungs and corresponding refined heparin.
Background
Heparin is known from the liver, and is mostly extracted from porcine intestinal mucosa or porcine and bovine lungs. The mucopolysaccharide sulfate consists of D-glucosamine, L-iduronic acid and glucuronic acid alternately, has a molecular weight of 3000-40000 and an average molecular weight of about 15000, has a large amount of negative charges, and is widely applied to clinic as an anticoagulant and an antithrombotic drug.
Compared with pig hepatins, bovine heparin has certain disadvantages, such as less than 75% of pig hepatins in potency and darker color. As the color of the substance is mainly a result of the selective absorption of light by the substance, the color of heparin is mainly related to the chemical structure of the heparin itself and to the residual colored impurities. In addition, during the placing process, the color of heparin gradually deepens, and because amino compounds and carbonyl compounds in heparin undergo condensation and polymerization to generate brown substances such as melanoidin and the like. Therefore, it is particularly important to select a proper decoloring process to improve the quality of bovine heparin products.
Journal paper ("research on heparin sodium extraction and refining technology in pig small intestine mucosa", ren Gongyuan et al, food research and development, 1 st 2007) uses pig small intestine mucosa as raw material to extract and purify heparin sodium, and two methods are adopted: one is ethanol precipitation and the other is macroporous anion resin exchange. Extracting the crude heparin sodium by a resin exchange method, removing impurities, purifying and refining the crude heparin by a potassium permanganate and hydrogen peroxide two-step oxidation method, and finally centrifuging at a low temperature to obtain the refined heparin sodium. The traditional extraction process conditions are improved, and the average yield of the crude product is 2.257%, the average yield of the fine product is 0.741%, wherein the effective value of the crude product is 73.19U/mg, and the effective value of the fine product is 146.38U/mg. However, the crude product of the technical proposal has low purity and low titer, and the oxidation of hydrogen peroxide is easy to lead to the degradation of heparin, and the oxidation of potassium permanganate is easy to bring impurities.
Journal paper (research on bovine heparin decolorization process, liu Libo, etc., chemical and biological engineering, 2022, 11-year) is to treat crude bovine heparin by deproteinizing, and then refining bovine heparin by adopting 3 oxidation processes such as hydrogen peroxide process, peracetic acid process, potassium permanganate process, etc., so as to study the influence of different oxidation processes on the decolorization effect and structure of bovine heparin. The results show that the decolorization rates of the hydrogen peroxide process, the peracetic acid process and the potassium permanganate process are 92.0%, 96.0% and 95.9%, respectively, and the recovery rates are 96.0%, 91.3% and 95.3% respectively; after being oxidized by hydrogen peroxide, the disaccharide structure of bovine heparin has little change; after oxidation by oxyacetic acid, Δ Glyser is oxidized to Δ Glyser ox1; after oxidation by potassium permanganate, Δ Glyser is oxidized to Δ Glyser ox1 and Δ Glyser ox2; after the refined bovine heparin is prepared into injection, the color darkening speed of the hydrogen peroxide process bovine heparin injection is the fastest, and then the peroxyacetic acid process and the potassium permanganate process are sequentially adopted. Therefore, the bovine heparin or structure oxidized by the hydrogen peroxide process, the peracetic acid process, the potassium permanganate process and other processes can be changed, or the prepared injection can be deep in color, and the comprehensive performance is still not good.
Patent (CN 112194740 a) proposes a method for extracting heparin sodium from pig small intestine, comprising the following steps: enzymatic salt decomposition; extracting in PEG2000-K 2SO4-H2 O or PEG2000-K 2SO4-H2 O double water phase system; carrying out back extraction on K 2SO4 or Na 2SO4; ultrafiltration with hollow fiber ultrafiltration membrane to remove PEG2000 in heparin sodium enrichment solution; precipitating with ethanol to obtain heparin sodium crude product; dehydrating the crude heparin sodium product by potassium permanganate, and then performing secondary ethanol precipitation, dehydration and drying to obtain refined heparin sodium product. The method for extracting heparin sodium from the small intestines of pigs utilizes a two-aqueous phase system to extract and back extract the heparin sodium to realize the extraction of the heparin sodium, but the defect that impurities are introduced cannot be avoided by adopting potassium permanganate for purification.
Based on the above, it is necessary to provide a method for preparing refined heparin by using bovine lung as raw material, obtaining crude heparin by enzymolysis, resin adsorption, elution and precipitation, re-dissolving the crude heparin, oxidizing flocculation, precipitation and drying to obtain refined heparin, adsorbing and eluting with composite resin, and activating the resin before adsorption to improve the purity of the crude heparin, wherein ferrate is used as an oxidizing flocculant, and acid is used to activate ferrate, so that the crude heparin can be effectively purified, and the high-titer high-purity refined heparin can be prepared, and the titer of the high-titer high-purity refined heparin can reach more than 180 IU/mg.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a method for preparing refined heparin by using bovine lungs and corresponding refined heparin, which takes bovine lungs as raw materials, crude heparin is obtained through enzymolysis, resin adsorption, elution and precipitation, the crude heparin is redissolved, oxidized and flocculated, precipitated and dried to obtain the refined heparin, the composite resin is used for adsorption-elution, and the resin is activated before adsorption, so that the resin adsorption efficiency is improved, the impurity removal rate of proteins, hyaluronic acid, chondroitin sulfate and the like is improved, the purity of the crude heparin is improved, ferrate is used as an oxidation flocculant, and acid is used for activating the ferrate, so that the crude heparin can be effectively purified, and the high-titer and high-purity refined heparin can be prepared, wherein the titer of the high-purity refined heparin can reach more than 180 IU/mg.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a method for preparing refined heparin from bovine lungs, comprising the steps of:
Taking bovine lung as a raw material, and obtaining crude heparin through enzymolysis, resin adsorption, elution, precipitation and drying; and re-dissolving, oxidizing, flocculating, precipitating and drying the crude heparin product to obtain refined heparin product.
In a preferred embodiment, the resin is a mixture of D254 macroporous resin and D208 macroporous resin in a mass ratio of 1:0.5-2.
In a preferred embodiment, the enzyme employed for the enzymatic hydrolysis is selected from at least one of trypsin, papain, pronase and alkaline protease.
In a preferred embodiment, the method for preparing refined heparin by using bovine lung specifically comprises the following steps:
1) Pretreatment: removing large trachea and external cortical fat in the bovine lung, mincing, and homogenizing into slurry according to the bovine lung and water;
2) Enzymolysis: regulating the pH value of the slurry obtained in the step 1) to 7-9, mixing with enzyme, heating for enzymolysis, and filtering to obtain an enzymolysis solution;
3) Resin adsorption and elution: mixing the enzymolysis liquid with resin, adsorbing and filtering to obtain adsorption resin; eluting the adsorption resin with sodium chloride aqueous solution, and collecting eluent;
4) The precipitation drying process comprises the following steps: adjusting pH of the eluent to 5-8, mixing with ethanol, standing the mixed solution, filtering to obtain precipitate, and drying to obtain crude heparin;
5) And (3) re-dissolving crude heparin: dissolving crude heparin sodium in sodium chloride solution with the mass concentration of 1-3%;
6) Oxidizing and flocculating: adding ferrate accounting for 1-5% of the crude heparin sodium, regulating the pH to 3-4, stirring for reaction, and regulating the pH to 6-7 after the reaction is completed to obtain a reaction solution;
7) And (3) precipitation and drying: and (3) centrifugally separating the reaction liquid obtained in the step (6), mixing the supernatant with ethanol, standing the mixed liquid, filtering to obtain precipitate, washing and drying to obtain refined heparin sodium.
In a preferred embodiment, the mass ratio of bovine lung to water in step 1) is 1:2-6.
In a preferred embodiment, the conditions for the thermal enzymolysis in step 2) are: heating to 30-50deg.C, adding enzyme 1.5-3.5% of the slurry mass, and performing enzymolysis for 2-8 hr.
In a preferred embodiment, in step 3), the adsorption conditions are 40-60 ℃ for 4-10 hours; the conditions for eluting the adsorption resin with aqueous sodium chloride solution were: washing the adsorption resin with 1-2mol/L sodium chloride solution, stopping washing when the volume of the column bed is 10-15 times, and eluting with 4-5mol/L sodium chloride solution with 5-8 times of the volume of the column bed.
Further, step 3) further comprises a step of resin activation before resin adsorption; the resin activation comprises the following steps: soaking in 1-2 times of absolute ethanol for 20-30 hr, washing with distilled water, and filtering; adding 2-4 times of NaOH solution with mass concentration of 3-5% for soaking for 20-30h, washing with distilled water, and filtering; adding 2-4 times of NaCl solution with mass concentration of 8-12% for soaking for 20-30 hr, washing with distilled water, and filtering; adding 2-4 times of HCl solution with mass concentration of 8-12% for soaking for 20-30 hr, washing with distilled water to neutrality, and filtering to obtain the final product.
In a preferred embodiment, the amount of resin used in step 3) is: 4-15g of resin is added to each 1kg of slurry.
In a preferred embodiment, the ethanol in step 4) is anhydrous ethanol, the volume ratio of ethanol to eluent being 1-2:1; adding ethanol, and standing for 24-48 hr.
In a preferred embodiment, the volume to mass ratio of sodium chloride solution to crude heparin in step 5) is 8-12mL/g.
In a preferred embodiment, in step 6), the reaction conditions are: reacting for 10-20h at 20-40 ℃. In a preferred embodiment, the ferrate in step 6) is selected from at least one of lithium ferrate, sodium ferrate, and potassium ferrate; the pH was adjusted with HCl, H 2SO4、HNO3, naOH and KOH.
In a preferred embodiment, the ethanol in step 7) is anhydrous ethanol, the volume ratio of ethanol to supernatant being 1-2:1; adding ethanol, and standing for 24-48h; the washing conditions are as follows: washing with absolute ethanol for 2-5 times.
In a preferred embodiment, the drying in steps 4) and 8) is selected from drying or freeze drying.
In a second aspect, the invention provides the refined heparin prepared by the method for preparing the refined heparin by using the bovine lung.
Compared with the prior art, the invention has the following beneficial effects:
1. The invention takes bovine lung as raw material, crude heparin is obtained through enzymolysis, resin adsorption, elution and precipitation, the crude heparin is redissolved, oxidized and flocculated, precipitated and dried to obtain refined heparin, the resin is adsorbed and eluted by using composite resin, and the resin is activated before adsorption, so that the resin adsorption efficiency is improved, the impurity removal rate of protein, hyaluronic acid, chondroitin sulfate and the like is improved, the purity of the crude heparin is improved, ferrate is used as an oxidized flocculant, and acid is adopted to activate the ferrate, so that the crude heparin can be effectively purified, and the refined heparin with high titer and high purity is prepared.
2. The titer of the refined heparin prepared by the invention can reach 180IU/mg.
Detailed Description
It is to be noted that the raw materials used in the present invention are all common commercial products, and the sources thereof are not particularly limited.
Example 1
A method for preparing refined heparin by using bovine lung specifically comprises the following steps:
1) Pretreatment: removing large trachea and external cortical fat in the bovine lung, mincing, mixing the bovine lung (200 g) with water according to a mass ratio of 1:6, and homogenizing to obtain slurry;
2) Enzymolysis: regulating the pH value of the slurry obtained in the step 1) to 7, heating to 37 ℃, adding papain accounting for 3.5% of the mass of the slurry, carrying out enzymolysis for 2 hours, and filtering to obtain enzymolysis liquid;
3) Resin adsorption and elution:
15g of the mixed resin (D254 macroporous resin 5g and D208 macroporous resin 10 g) was taken for activation: soaking in 2 times of absolute ethanol for 30h, washing with distilled water, and filtering; adding NaOH solution with mass concentration of 5% and mass concentration of 3 times, soaking for 30 hours, washing with distilled water, and filtering; adding NaCl solution with the mass concentration of 12% and 3 times of the mass, soaking for 30 hours, washing with distilled water, and filtering; adding HCl solution with mass concentration of 12% and mass concentration of 3 times, soaking for 30 hours, washing with distilled water to neutrality, and filtering to obtain filtrate;
Mixing the enzymolysis liquid with the activated resin, adsorbing for 10 hours at 40 ℃, and filtering to obtain adsorption resin; washing the adsorption resin with 1.5mol/L sodium chloride solution, stopping washing when the volume of the bed is 15 times, eluting with 4mol/L sodium chloride solution with 8 times of the volume of the bed, and collecting eluent;
4) The precipitation drying process comprises the following steps: regulating the pH value of the eluent to 7, mixing with absolute ethyl alcohol with the same volume, standing the mixed solution for 24 hours, filtering to obtain a precipitate, and freeze-drying to obtain crude heparin;
5) And (3) re-dissolving crude heparin: dissolving crude heparin sodium in sodium chloride solution with the mass concentration of 1%; the volume-mass ratio of the sodium chloride solution to the crude heparin is 8mL/g;
6) Oxidizing and flocculating: adding sodium ferrate with the mass of 5% of the crude heparin sodium, regulating the pH value to 4 by hydrochloric acid, stirring, reacting at 25 ℃ for 10 hours, and regulating the pH value to 6.8 after the reaction is completed to obtain a reaction solution;
7) And (3) precipitation and drying: and (3) centrifugally separating the reaction liquid obtained in the step (6), taking supernatant, mixing with absolute ethyl alcohol in equal volume, standing the mixed liquid for 24 hours, filtering to obtain precipitate, washing with absolute ethyl alcohol for 3 times, and freeze-drying to obtain refined heparin sodium.
Example 2
A method for preparing refined heparin by using bovine lung specifically comprises the following steps:
1) Pretreatment: removing large trachea and external cortical fat in the bovine lung, mincing, mixing the bovine lung (200 g) with water according to the mass ratio of 1:3, and homogenizing to obtain slurry;
2) Enzymolysis: regulating the pH value of the slurry obtained in the step 1) to 8, heating to 40 ℃, adding trypsin accounting for 2.5% of the mass of the slurry, carrying out enzymolysis for 6 hours, and filtering to obtain an enzymolysis liquid;
3) Resin adsorption and elution:
6g of the mixed resin (D254 macroporous resin 4g and D208 macroporous resin 2 g) was taken for activation: soaking in 1 times of absolute ethanol for 24 hours, washing with distilled water, and filtering; adding NaOH solution with the mass concentration of 4% and the mass concentration of 4 times, soaking for 20 hours, and washing and filtering with distilled water; adding NaCl solution with the mass concentration of 10% and the mass concentration of 4 times, soaking for 20 hours, and washing and filtering by using distilled water; adding 4 times of 10% HCl solution with mass concentration, soaking for 20 hr, washing with distilled water to neutrality, and filtering to obtain filtrate;
Mixing the enzymolysis liquid with the activated resin, adsorbing for 6 hours at 60 ℃, and filtering to obtain adsorption resin; washing the adsorption resin with 1.5mol/L sodium chloride solution, stopping washing when the volume of the bed is 12 times, eluting with 4mol/L sodium chloride solution with 6 times of the volume of the bed, and collecting eluent;
4) The precipitation drying process comprises the following steps: regulating the pH value of the eluent to 7, mixing with absolute ethyl alcohol with the same volume, standing the mixed solution for 24 hours, filtering to obtain a precipitate, and freeze-drying to obtain crude heparin;
5) And (3) re-dissolving crude heparin: dissolving crude heparin sodium in sodium chloride solution with mass concentration of 2%; the volume-mass ratio of the sodium chloride solution to the crude heparin is 12mL/g;
6) Oxidizing and flocculating: adding lithium ferrate with the mass of 1% of the crude heparin sodium, regulating the pH value to 3 by using hydrochloric acid, stirring, reacting for 20 hours at 40 ℃, and regulating the pH value to 6.8 after the reaction is completed to obtain a reaction solution;
7) And (3) precipitation and drying: and (3) centrifugally separating the reaction liquid obtained in the step (6), taking supernatant, mixing with absolute ethyl alcohol in equal volume, standing the mixed liquid for 24 hours, filtering to obtain precipitate, washing with absolute ethyl alcohol for 3 times, and freeze-drying to obtain refined heparin sodium.
Example 3
A method for preparing refined heparin by using bovine lung specifically comprises the following steps:
1) Pretreatment: removing large trachea and external cortical fat in the bovine lung, mincing, mixing the bovine lung (200 g) with water according to a mass ratio of 1:4, and homogenizing to obtain slurry;
2) Enzymolysis: regulating the pH value of the slurry obtained in the step 1) to 7, heating to 37 ℃, adding papain accounting for 2.5% of the mass of the slurry, carrying out enzymolysis for 4 hours, and filtering to obtain enzymolysis liquid;
3) Resin adsorption and elution:
10g of the mixed resin (5 g of D254 macroporous resin and 5g of D208 macroporous resin) was taken for activation: soaking in 1 times of absolute ethanol for 24 hours, washing with distilled water, and filtering; adding 2 times of NaOH solution with the mass concentration of 4% for soaking for 24 hours, and washing with distilled water for drying; adding 2 times of NaCl solution with the mass concentration of 10% for soaking for 24 hours, and washing with distilled water for drying; adding 2 times of 10% HCl solution with mass concentration, soaking for 24 hr, washing with distilled water to neutrality, and filtering to obtain filtrate;
Mixing the enzymolysis liquid with the activated resin, adsorbing for 6 hours at 50 ℃, and filtering to obtain adsorption resin; washing the adsorption resin with 1.5mol/L sodium chloride solution, stopping washing when the volume of the bed is 12 times, eluting with 4mol/L sodium chloride solution with 6 times of the volume of the bed, and collecting eluent;
4) The precipitation drying process comprises the following steps: regulating the pH value of the eluent to 7, mixing with absolute ethyl alcohol with the same volume, standing the mixed solution for 24 hours, filtering to obtain a precipitate, and freeze-drying to obtain crude heparin;
5) And (3) re-dissolving crude heparin: dissolving crude heparin sodium in sodium chloride solution with mass concentration of 2%; the volume-mass ratio of the sodium chloride solution to the crude heparin is 10mL/g;
6) Oxidizing and flocculating: adding potassium ferrate with the mass of 3% of the crude heparin sodium, regulating the pH value to 3 by using hydrochloric acid, stirring, reacting for 15 hours at 30 ℃, and regulating the pH value to 6.8 after the reaction is completed to obtain a reaction solution;
7) And (3) precipitation and drying: and (3) centrifugally separating the reaction liquid obtained in the step (6), taking supernatant, mixing with absolute ethyl alcohol in equal volume, standing the mixed liquid for 24 hours, filtering to obtain precipitate, washing with absolute ethyl alcohol for 3 times, and freeze-drying to obtain refined heparin sodium.
Comparative example 1
Example 3 was repeated except that 10g of macroporous resin D254 was used instead of the mixed resin.
Comparative example 2
Example 3 was repeated except that 10g of D208 macroporous resin was used instead of the mixed resin.
Comparative example 3
Example 3 is followed except that the pH is adjusted to 5 with hydrochloric acid in step 6).
Comparative example 4
The procedure of example 3 was followed except that the resin activation process of step 3) was different, as follows:
10g of the mixed resin (5 g of D254 macroporous resin and 5g of D208 macroporous resin) was taken for activation: soaking in 1 times of absolute ethanol for 24 hours, washing with distilled water, and filtering; adding 2 times of NaOH solution with the mass concentration of 4% for soaking for 24 hours, and washing with distilled water for drying; adding 2 times of 10% HCl solution, soaking for 24 hr, washing with distilled water to neutrality, repeating for 1 time, and filtering.
Comparative example 5
The procedure of example 3 was followed except that the resin activation process of step 3) was different, as follows:
10g of the mixed resin (5 g of D254 macroporous resin and 5g of D208 macroporous resin) was taken for activation: adding 2 times of NaOH solution with the mass concentration of 4% for soaking for 24 hours, and washing with distilled water for drying; soaking in 1 times of absolute ethanol for 24 hours, washing with distilled water, and filtering; adding 2 times of NaCl solution with the mass concentration of 10% for soaking for 24 hours, and washing with distilled water for drying; adding 2 times of 10% HCl solution, soaking for 24 hr, washing with distilled water to neutrality, and filtering.
Test case
Potency: sheep plasma method is adopted, and the unit is IU/mg:
Decoloring rate: spectrophotometry measurement, wherein the detection wavelength of the pigment is 400nm, and the decoloring rate is calculated according to the following calculation formula, and the unit is percent:
wherein: a Front part 、A Rear part (S) absorbance of sample before and after decolorization
Nucleic acid: spectrophotometric measurement, wherein the detection wavelength is set to 260nm, and the absorbance at 260nm is measured;
protein: the content of protein is measured by trichloroacetic acid method, and the unit is calculated by dry product;
molecular weight: by high performance liquid gel permeation chromatography;
heavy metal: measured by inductively coupled plasma mass spectrometry, in ppm;
Ethanol residue: GC chromatography assay, in mg;
the physicochemical data of examples 1-3 and comparative examples 1-5 are shown in Table 1.
TABLE 1
As can be seen from Table 1, the high-potency and high-purity heparin products prepared in examples 1-3 of the present invention have a potency greater than 180 and a potency and purity significantly higher than those of comparative examples 1-5. Comparing example 3 with comparative examples 1-2, it can be seen that, in example 3, the D254 macroporous resin and the D208 macroporous resin are used in combination, and compared with comparative examples 1-2, the purity and the potency of the refined heparin can be significantly improved by using an equal amount of single macroporous resin (D254 macroporous resin or D208 macroporous resin), and thus, the D254 macroporous resin and the D208 macroporous resin can be used in combination to synergistically improve the purification effect. Comparing examples 1-3 with comparative example 3, it can be seen that adjusting the pH to 3-4 during oxidative flocculation can better activate ferrate, improving its decolorization and purification effects. Comparing example 3 with comparative examples 4-5, it can be seen that the resin activation process has a great influence on the purification effect, and the invention adopts the activation process of ethanol soaking activation, naOH solution soaking activation, naCl solution soaking activation and HCl solution soaking activation, so that the resin can be activated to the maximum extent, and the purification effect is improved.
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.
Claims (6)
1. A method for preparing refined heparin by using bovine lung, which is characterized by comprising the following steps:
1) Pretreatment: removing large trachea and external cortical fat in the bovine lung, mincing, and homogenizing into slurry according to the bovine lung and water;
2) Enzymolysis: regulating the pH value of the slurry obtained in the step 1) to 7-9, mixing with enzyme, heating for enzymolysis, and filtering to obtain an enzymolysis solution;
3) Resin adsorption and elution: mixing the enzymolysis liquid with resin, adsorbing and filtering to obtain adsorption resin; eluting the adsorption resin with sodium chloride aqueous solution, and collecting eluent;
4) The precipitation drying process comprises the following steps: adjusting pH of the eluent to 5-8, mixing with ethanol, standing the mixed solution, filtering to obtain precipitate, and drying to obtain crude heparin;
5) And (3) re-dissolving crude heparin: dissolving crude heparin sodium in sodium chloride solution with the mass concentration of 1-3%;
6) Oxidizing and flocculating: adding ferrate accounting for 1-5% of the crude heparin sodium, regulating the pH to 3-4, stirring for reaction, and regulating the pH to 6-7 after the reaction is completed to obtain a reaction solution;
7) And (3) precipitation and drying: centrifugally separating the reaction liquid obtained in the step (6), mixing the supernatant with ethanol, standing the mixed liquid, filtering to obtain precipitate, washing and drying to obtain refined heparin sodium;
the resin is a mixture of D254 macroporous resin and D208 macroporous resin, and the mass ratio of the two is 1:0.5-2;
Step 3) the resin is activated before being adsorbed; the resin activation comprises the following steps: soaking in 1-2 times of absolute ethanol for 20-30 hr, washing with distilled water, and filtering; adding 2-4 times of NaOH solution with mass concentration of 3-5% for soaking for 20-30h, washing with distilled water, and filtering; adding 2-4 times of NaCl solution with mass concentration of 8-12% for soaking for 20-30 hr, washing with distilled water, and filtering; adding 2-4 times of HCl solution with mass concentration of 8-12% for soaking for 20-30 hr, washing with distilled water to neutrality, and filtering to obtain filtrate;
The ferrate is selected from at least one of lithium ferrate, sodium ferrate, and potassium ferrate.
2. The method of claim 1, wherein the enzyme used for enzymolysis is at least one selected from the group consisting of trypsin, papain, pronase and alkaline protease.
3. The method of claim 1, wherein the mass ratio of bovine lung to water in step 1) is 1:2-6.
4. The method according to claim 1, wherein the conditions for the thermal enzymolysis in step 2) are: heating to 30-50deg.C, adding enzyme 1.5-3.5% of the slurry mass, and performing enzymolysis for 2-8 hr.
5. The method according to claim 1, wherein in step 3), the adsorption conditions are 40-60 ℃ for 4-10 hours; the conditions for eluting the adsorption resin with aqueous sodium chloride solution were: washing the adsorption resin with 1-2mol/L sodium chloride solution, stopping washing when the volume of the column bed is 10-15 times, and eluting with 4-5mol/L sodium chloride solution with 5-8 times of the volume of the column bed.
6. The method of claim 1, wherein the resin is used in step 3) in an amount of: adding 4-15g of resin into each 1kg of slurry; the ethanol in the step 4) is absolute ethanol, and the volume ratio of the ethanol to the eluent is 1-2:1; adding ethanol, and standing for 24-48h; the volume-mass ratio of the sodium chloride solution to the crude heparin in the step 5) is 8-12mL/g; in the step 6), the reaction condition is 20-40 ℃ for 10-20h; adjusting the pH by adopting HCl, H 2SO4、HNO3, naOH and KOH; the ethanol in the step 7) is absolute ethanol, and the volume ratio of the ethanol to the supernatant is 1-2:1; adding ethanol, and standing for 24-48h; the washing conditions are as follows: washing with absolute ethanol for 2-5 times; the drying in steps 4) and 8) is selected from drying or freeze drying.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952204A (en) * | 2012-10-09 | 2013-03-06 | 江苏联众肠衣有限公司 | Novel production technique of heparin sodium |
| CN106519077A (en) * | 2016-11-17 | 2017-03-22 | 江苏联众肠衣有限公司 | Preparation process of high-potency heparin sodium |
| CN108329405A (en) * | 2018-02-08 | 2018-07-27 | 黄石市典雅生物科技有限公司 | It is protected under a kind of resin adsorption state and the method for purified heparin sodium |
| CN109731137A (en) * | 2019-03-13 | 2019-05-10 | 成都氢润医疗科技有限公司 | The preparation method of albumin coating with biological functions and material with biological functions |
| CN111393542A (en) * | 2020-05-16 | 2020-07-10 | 青岛科技大学 | Novel method for refining crude heparin sodium |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952204A (en) * | 2012-10-09 | 2013-03-06 | 江苏联众肠衣有限公司 | Novel production technique of heparin sodium |
| CN106519077A (en) * | 2016-11-17 | 2017-03-22 | 江苏联众肠衣有限公司 | Preparation process of high-potency heparin sodium |
| CN108329405A (en) * | 2018-02-08 | 2018-07-27 | 黄石市典雅生物科技有限公司 | It is protected under a kind of resin adsorption state and the method for purified heparin sodium |
| CN109731137A (en) * | 2019-03-13 | 2019-05-10 | 成都氢润医疗科技有限公司 | The preparation method of albumin coating with biological functions and material with biological functions |
| CN111393542A (en) * | 2020-05-16 | 2020-07-10 | 青岛科技大学 | Novel method for refining crude heparin sodium |
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Denomination of invention: A method for preparing high-quality heparin using bovine lung and corresponding high-quality heparin Granted publication date: 20240517 Pledgee: Tonghua Huinan sub branch of Bank of Jilin Co.,Ltd. Pledgor: Haotai Health Jisheng (Jilin) Biotechnology Co.,Ltd. Registration number: Y2024220000127 |