CN101407801B - Preparation of earthworm fibrinolytic enzyme from Pheretima guillelmi Michaelsen and lyophilized powder preparation for injection prepared thereby - Google Patents
Preparation of earthworm fibrinolytic enzyme from Pheretima guillelmi Michaelsen and lyophilized powder preparation for injection prepared thereby Download PDFInfo
- Publication number
- CN101407801B CN101407801B CN200810182567XA CN200810182567A CN101407801B CN 101407801 B CN101407801 B CN 101407801B CN 200810182567X A CN200810182567X A CN 200810182567XA CN 200810182567 A CN200810182567 A CN 200810182567A CN 101407801 B CN101407801 B CN 101407801B
- Authority
- CN
- China
- Prior art keywords
- earthworm
- earthworm fibrinolysin
- fibrinolysin
- damping fluid
- preparation
- 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
- 241000361919 Metaphire sieboldi Species 0.000 title claims abstract description 105
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 101710196208 Fibrinolytic enzyme Proteins 0.000 title abstract description 9
- 238000002347 injection Methods 0.000 title abstract 3
- 239000007924 injection Substances 0.000 title abstract 3
- 241000099813 Metaphire guillelmi Species 0.000 title description 2
- 239000008176 lyophilized powder Substances 0.000 title 1
- 230000000694 effects Effects 0.000 claims abstract description 14
- 108010088842 Fibrinolysin Proteins 0.000 claims description 73
- 229940001501 fibrinolysin Drugs 0.000 claims description 72
- 238000000502 dialysis Methods 0.000 claims description 30
- 238000010612 desalination reaction Methods 0.000 claims description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 238000013016 damping Methods 0.000 claims description 17
- 239000012530 fluid Substances 0.000 claims description 17
- 239000012043 crude product Substances 0.000 claims description 14
- 239000012264 purified product Substances 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 229920005654 Sephadex Polymers 0.000 claims description 7
- 239000012507 Sephadex™ Substances 0.000 claims description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 7
- 239000002953 phosphate buffered saline Substances 0.000 claims description 7
- 229920002684 Sepharose Polymers 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000007670 refining Methods 0.000 claims description 2
- 239000007975 buffered saline Substances 0.000 claims 3
- 239000000843 powder Substances 0.000 abstract description 28
- 208000007536 Thrombosis Diseases 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 3
- 238000000034 method Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 2
- 241001208378 Metaphire Species 0.000 abstract 2
- 238000001990 intravenous administration Methods 0.000 abstract 2
- 239000011543 agarose gel Substances 0.000 abstract 1
- 238000004587 chromatography analysis Methods 0.000 abstract 1
- 239000000499 gel Substances 0.000 abstract 1
- 238000010521 absorption reaction Methods 0.000 description 14
- 239000003814 drug Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 210000001772 blood platelet Anatomy 0.000 description 10
- 239000002504 physiological saline solution Substances 0.000 description 9
- 108010058861 Fibrin Fibrinogen Degradation Products Proteins 0.000 description 8
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- 239000000208 fibrin degradation product Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 239000003527 fibrinolytic agent Substances 0.000 description 7
- 238000012856 packing Methods 0.000 description 7
- 230000003480 fibrinolytic effect Effects 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 238000010253 intravenous injection Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000009064 mailuoning Substances 0.000 description 5
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 4
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 229960005356 urokinase Drugs 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 3
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 239000003146 anticoagulant agent Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000004623 platelet-rich plasma Anatomy 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 3
- 238000011146 sterile filtration Methods 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 241000243686 Eisenia fetida Species 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- 102000008946 Fibrinogen Human genes 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 102000005686 Serum Globulins Human genes 0.000 description 2
- 108010045362 Serum Globulins Proteins 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 210000001168 carotid artery common Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000004731 jugular vein Anatomy 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 230000002537 thrombolytic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 241001006778 Amythas Species 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 241001372586 Bimastos Species 0.000 description 1
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 206010019468 Hemiplegia Diseases 0.000 description 1
- 206010020565 Hyperaemia Diseases 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000243679 Lumbricidae Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108010023197 Streptokinase Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 208000001435 Thromboembolism Diseases 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 230000000702 anti-platelet effect Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940099062 chloraseptic Drugs 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000009841 epithelial lesion Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 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 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000622 irritating effect Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000000867 larynx Anatomy 0.000 description 1
- 231100001252 long-term toxicity Toxicity 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 231100000862 numbness Toxicity 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 239000008354 sodium chloride injection Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 229960005202 streptokinase Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a preparation method of an earthworm fibrinolytic enzyme from a Metaphire William and an intravenous freeze-dried powder injection prepared by the earthworm fibrinolytic enzyme. In the invention, the separation and purification of the earthworm fibrinolytic enzyme are realized through the chromatography of a carboxymethyl-agarose gel C1-6B column and a gel column, thereby obtaining the earthworm fibrinolytic enzyme from the Metaphire William and with higher purity; the molecular weight of the earthworm fibrinolytic enzyme obtained by the method is 28,000 to 30,000 Dalton and the specific activity of the earthworm fibrinolytic enzyme achieves 100,000 units/mg. Simultaneously the invention takes the earthworm fibrinolytic enzyme as a material for preparing the intravenous freeze-dried powder injection used for curing thrombotic diseases.
Description
Technical field
The present invention relates to the biological chemistry pharmaceutical technology, specifically, relate to a kind of preparation method and used for intravenous injection freeze-dried powder prepared therefrom that encircles the earthworm fibrinolysin of earthworm from the William chamber.
Background technology
Thrombotic diseases such as cerebral thrombosis, myocardial infarction, coronary heart disease etc. are the principal diseases of serious harm human health and life, its treatment usually needs to use thrombolytic drug to treat, at present, the antithrombotic reagent of Ying Yonging has urokinase, streptokinase, sense of organization plasminogen activator etc. clinically, but in the use, hemorrhage side effect often appears, though the thrombus of new formation there is restraining effect, very poor to outmoded thrombus effect.And, the medicine material source difficulty that has, complicated process of preparation costs an arm and a leg.In recent years, people more and more pay close attention to from earthworm and to extract earthworm fibrinolysin and be used for the treatment of thrombotic diseases.
Earthworm is called earthworm on the Chinese medicine, its property is salty, cold, it is fanatical hot-tempered to cure mainly height, convulsion with spasms, headache due to pathogenic wind-heat, hot eyes, apoplexy and hemiplegia, pant larynx numbness, arthralgia, sores etc., modern study show that fresh earthworm body contains the multiple protein enzyme, and earthworm fibrinolysin be the class in the earthworm body, extracted can fibrinolytic enzyme, its molecular weight is 15000～60000 dalton, it is present in the digestive tube of earthworm.Earthworm fibrinolysin has dual-use function, except energy direct hydrolysis scleroproein, can also activate profibr(in)olysin and become plasminogen, thereby have the effect of indirect hydrolysis of fibrin.Earthworm fibrinolysin is not single a kind of enzyme, but has the general name of the multiple protein lytic enzyme of identical function.Report through document, it contains enzyme amount difference different earthworm kinds, fibrinolytic is also different, and in present disclosed document and the patent, mostly earthworm fibrinolysin is from Eisenia foetida (Eisenia Foetida Sarigny), Lumbricidae Bimostos (Bimastos), Amythas dancatala and ginseng hair earthworm etc. separate extraction, do not see about extract the report of the active earthworm plasmin of certain molecular weight from William chamber ring earthworm (Metaphire guillelmi).
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method who encircles the earthworm fibrinolysin of earthworm from the William chamber.
It is feedstock production low toxicity, used for intravenous injection freeze-dried powder efficiently with described earthworm fibrinolysin that the technical problem that the present invention further will solve provides a kind of.
For solving the problems of the technologies described above, technical scheme provided by the present invention is: the preparation method of described earthworm fibrinolysin comprises the steps:
(1) the earthworm fibrinolysin crude product extracts: get fresh earthworm and wash, under 4 ℃ ± 1 ℃ low temperature, 0.01～0.1mol/L the damping fluid that contains 0.1mol/L NaCl that adds pH6～8 of 1-5 times of weight part of earthworm, smash homogenate to pieces, extracting 2～5 hours, 4000～6000 rev/mins then, centrifugal 10～30 minutes, get supernatant liquor, add ammonium sulfate to 60%～80% saturation ratio, spend the night in 4 ℃ ± 1 ℃ placement, 4000～6000 rev/mins, centrifugal 10～30 minutes, taking precipitate, the dialysis desalination, lyophilize obtains the earthworm fibrinolysin crude product;
(2) purifying of earthworm fibrinolysin crude product: with 0.01～0.1mol/L damping fluid dissolving of the earthworm fibrinolysin crude product in the step (1) with pH6～8,4000～6000 rev/mins, centrifugal 10～30 minutes, getting supernatant liquor separates with carboxymethyl-sepharose Cl-6B post, with sodium-chlor straight line gradient mode wash-out, the concentration of this sodium chloride solution from the 0mol/L linear increment to 1mol/L, collect the elutriant of each elution peak, the dialysis desalination, lyophilize, detect through activity, obtain the earthworm fibrinolysin purified product;
(3) the earthworm fibrinolysin purified product is refining: with the 0.01～0.1mol/L damping fluid dissolving of the earthworm fibrinolysin purified product in the step (2) with pH6～8, separate with gel column, 0.01～0.1mol/L buffer solution elution with pH6～8, collect the elutriant of active elution peak, the dialysis desalination, lyophilize obtains the earthworm fibrinolysin highly finished product, its molecular weight is 28000～30000 dalton, and the ratio work of described earthworm fibrinolysin reaches every milligram of protein 10 ten thousand units.
Damping fluid described in the above-mentioned steps is tris-HCI buffer, phosphate buffered saline buffer or sodium chloride solution; Described dialysis desalination condition is 8000～10000 dalton for the dialysis tubing aperture, will collect the liquid dialysis tubing of packing into, puts into the distilled water of collecting 20～50 times of liquid and stirs dialysis, changes first water, dialyses 18～32 hours in 4～8 hours.
The separation of process described carboxymethyl-sepharose Cl-6B post in the step (2), under the wavelength of 280nm, collected elutriant is carried out UV spectrophotometer measuring, obtain wavelength 280nm and absorb collection of illustrative plates, obtain a plurality of albumen absorption peaks, with the elutriant of each absorption peak, the dialysis desalination, lyophilize, detect through activity, the III absorption peak has very high fibrinolytic, obtains the earthworm fibrinolysin purified product.
Gel column described in the step (3) is sephadex G-75 type or sephadex g-100 type.Further separation and purification through described gel column, under the wavelength of 280nm, collected elutriant is carried out UV spectrophotometer measuring, obtain wavelength 280nm and absorb collection of illustrative plates, it is an albumen absorption peak, with this sample dialysis desalination, lyophilize obtains the earthworm fibrinolysin highly finished product, it is 28000～30000 dalton that this sample is measured its molecular weight through the SDS-polyacrylamide gel electrophoresis, and the ratio work of described earthworm fibrinolysin reaches every milligram of protein 10 ten thousand units.
The present invention also provides a kind of lyophilized injectable powder of being made by the earthworm fibrinolysin and the acceptable accessories combination from William chamber ring earthworm of above-mentioned preparation method's preparation.Described auxiliary material comprises thinner, vehicle, weighting agent, tackiness agent, absorption enhancer, stablizer of pharmaceutical field routine etc.This lyophilized injectable powder can be used for treating thrombotic diseases.
Advantage of the present invention: the present invention has extracted molecular weight at 28000～30000 daltonian earthworm fibrinolysins from the ring earthworm of William chamber, and it is prepared into lyophilized injectable powder can be used for intravenous injection, directly act on the thromboembolism position, in treatment time, can change patient's illness rapidly, reduce mortality ratio.This stable preparation process, quality controllable.
In order further to set forth the result of treatment of earthworm fibrinolysin of the present invention, the earthworm fibrinolysin lyophilized injectable powder that adopts the embodiment of the invention 2 to make carries out following pharmacodynamic experiment, the present invention is further elaborated, but the present invention is not limited to the content that comprises in the following experiment.
Test example 1: the influence that the earthworm fibrinolysin freeze-dried powder forms rabbit arteriovenous shut thrombus in vivo
[test drug]
1. the earthworm fibrinolysin lyophilized injectable powder that adopts the embodiment of the invention 2 to make.It is standby to be diluted to desired concn with physiological saline before the experiment, and 0.25mg/ props up.
2. urokinase: 100,000 units/, white swan pharmaceutcal corporation, Ltd of Harbin high-tech group.
3. heparin sodium: 100u/mg.Changzhou bright biochemical research institute.
[experimental animal]
Rabbit, body weight 2-3kg, male and female dual-purpose.
[test method and result]
Get 48 of rabbit, be divided into 6 groups at random, with 20% urethane auricular vein injecting anesthetic, dosage is 5ml/kg, and back of the body position is fixing, cuts neck, separates right common carotid artery and left external jugular vein.After an end that is full of the polyethylene tube (divide three sections connections, put into surgical thread in the stage casing) of heparin-saline in advance inserted left external jugular vein, will manage the other end and insert right common carotid artery, open the open blood flow of hemostatic clamp.Control group after 15 minutes in Herba Clinopodii, get blood 2ml and measure euglobulin lysis time (ELT), fibrinogen content (FIB) and fibrin degradation product (FDP) (FDP), and take out silk thread rapidly and weigh, then claimed dry weight in 24 hours at 37 ℃ of freeze-day with constant temperature with thrombus.Then after 15 minutes, vein gives physiological saline, earthworm fibrinolysin freeze-dried powder (medicine to be measured), urokinase to other test group respectively, after dosage sees Table 1,3 hour, gets blood and thrombus as stated above, and claims wet, dry weight.
The influence that table 1 medicine to be measured forms the rabbit thrombus in vivo
*With relatively relatively p＜0.05 △ and the relatively p＜0.01 T-check of physiological saline group of the large, medium and small dosage group of p＜0.01 ☆ of control group
Table 2 fibrinogen content (FIB)
*Compare p with the physiological saline group〉0.05 SNK check
Table 3 euglobulin lysis time (ELR)
*With relatively p＜0.01 rank test of physiological saline group
Table 4 fibrin degradation product (FDP) (FDP)
*With relatively p＜0.01 rank test of physiological saline group
[conclusion]
Test-results shows, the earthworm fibrinolysin freeze-dried powder through little, in, heavy dose of group relatively has significant difference (p＜0.01) with the physiological saline group, and the further effect of formation of thrombus that suppresses be described; And in, heavy dose of group is remarkable with the control group comparing difference.This medicine is described except that the thrombosis of inhibition is arranged, also has certain thrombolytic effect, between each dosage group dose-dependence is arranged.Blood FIB, ELT and FDP measurement show that the earthworm fibrinolysin freeze-dried powder is not obvious to the influence of FIB, but middle dosage and heavy dose can make ELT significantly shorten, and large, medium and small dosage can make FDP significantly increase, and show that it has the effect that activates fibrinolytic.
Test example 2 earthworm fibrinolysin freeze-dried powders influence laboratory report to rabbit platelet
[test drug]
1, the earthworm fibrinolysin lyophilized injectable powder that adopts the embodiment of the invention 2 to make.It is standby to be diluted to desired concn with physiological saline before the experiment, and 0.25mg/ props up.
2, MAILUONING ZHUSHEYE: Jinling Pharmaceutical Co., Ltd., Jinling Pharmaceutical Factory.
3, sodium chloride injection: the Harbin No.6 Pharmaceutical Factory provides.
4, ADP (adenosine diphosphate (ADP)): U.S. biopool company.
5, Sodium Citrate: Harbin City's chemical reagent factory.
[experimental animal]
White big ear rabbit, body weight 2.0～3.0kg, male and female dual-purpose.
[test method]
Select 40 of healthy rabbits, be divided into 5 groups at random, 8 every group, be respectively the large, medium and small dosage group of earthworm fibrinolysin freeze-dried powder (dosage is respectively 4mg/kg, 2mg/kg, 1mg/kg), blank group and positive control drug Mailuoning group.Get rabbit and weigh, auricular vein is injected earthworm fibrinolysin freeze-dried powder 4mg/kg respectively, 2mg/kg, and 1mg/kg, Mailuoning 1.1ml/kg, blank is organized not injectable drug.Behind the injectable drug 8min, use 5ml syringe heart extracting blood 2.7ml respectively, join in the test tube that is added with the 0.3ml3.8% Sodium Citrate in advance, shake up stand-by.
(1) mensuration of platelet count and platelet adhesion rate:
Get anticoagulation 750 μ l, the adding capacity is in the glass pin of 5ml, places on the platelet adhesion reaction instrument rotating disk, with 3 commentaries on classics/min speed rotation 15min.From anticoagulant tube and glass pin, get blood 20 μ l then respectively, respectively be added to and carry out platelet count in the test tube that fills 0.38ml thrombocyte diluent, calculate adhesion rate.
(2) mensuration of platelet aggregation rate:
With anticoagulation with the centrifugal 5min of 500 commentaries on classics/min, preparation platelet rich plasma (PRP), residue blood plasma is through the centrifugal 10min of 3000 commentaries on classics/min, preparation platelet poor plasma (PPP) is transferred platelet count to 4～5 * 1012/L among the PRP with PPP, gets PRP200 μ l, behind 37 ℃ of incubation 30min, add inductor ADP2 μ mol/L, assemble instrument by the turbidimetry for Determination platelet aggregation rate with blood, platelet aggregation rate is measured and is all finished in 3h.
[test-results]
Table 5 earthworm fibrinolysin freeze-dried powder is to hematoblastic influence
Compare with the blank group,
*P＜0.05,
*P＜0.01 is compared with the Mailuoning group, #P＜0.01
[conclusion]
The earthworm fibrinolysin freeze-dried powder does not have influence to number of platelets, but heavy dose of earthworm fibrinolysin freeze-dried powder (4mg/kg) obviously suppresses platelet adhesion reaction, aggregation capability, and its antiplatelet adhesive attraction is better than Mailuoning.
Product toxic side effect of the present invention is little, harmless to body, described product earthworm fibrinolysin freeze-dried powder is in the dog long term toxicity test, establish four groups altogether, every group of 6 domestic hybrid dogs, intravenous injection every day physiological saline 20ml, the earthworm fibrinolysin freeze-dried powder is respectively 150ug/kg, 750ug/kg, 3750ug/kg.12 weeks of successive administration, its result shows, intravenous injection earthworm fibrinolysin freeze-dried powder is little, in, big three dosage groups and blank group relatively, similar substantially in weight increase, acropetal coefficient, hematological indices, blood parameters, blood coagulation, no difference of science of statistics.The tissue abnormalities phenomenon is not found in the histopathology report yet, and cardiac diagnosis lead is checked also no abnormality seen simultaneously.Therefore prove this freeze-dried powder when dosage is 150ug/kg to 3750ug/kg, in 12 weeks of continuous use, no overt toxicity reaction after two all recovery stages observed, is not seen the delayed toxicity reaction yet.
The product of prepared of the present invention and scleroproein have special avidity, through highly purified, check through Physiology and biochemistry, only can guarantee under the situation that scleroproein exists, just to play thrombolytic effect, hemorrhage, the haemolysis that can not use urokinase etc. existing medicine produced, cause side effect such as cohesion.And test-results proves that product of the present invention does not have allergen, heat source response.The irritative response of epithelial lesion, subcutaneous connective tissue hyperemia, checking cellular infiltration is not seen by test exhausted animal tissues, can use safely.Specific embodiments:
In order to understand the present invention better, further illustrate content of the present invention below in conjunction with embodiment, but content of the present invention is not limited to the following examples.
Embodiment 1
(1) gets fresh earthworm 2400g, wash, under 4 ℃ ± 1 ℃ low temperature, the 0.01mol/L Tris-HCl damping fluid that contains 0.1mol/L NaCl that adds the pH6 of 1 times of weight part, homogenate, and extracting 2 hours, 4000 rev/mins then, centrifugal 10 minutes, get supernatant liquor, add ammonium sulfate to 60% saturation ratio again, spend the night, saltout in 4 ℃ ± 1 ℃ placement, 4000 rev/mins, centrifugal 10 minutes, get precipitation, the dialysis desalination, lyophilize obtains the earthworm fibrinolysin crude product;
(2) with of the 0.01mol/LTris-HCl damping fluid dissolving of this earthworm fibrinolysin crude product with pH6,4000 rev/mins, centrifugal 10 minutes, getting supernatant liquor separates with carboxymethyl-sepharose Cl-6B post, with sodium-chlor straight line gradient mode wash-out, the concentration of this sodium chloride solution from the 0mol/L linear increment to 1mol/L, under the wavelength of 280nm, collected elutriant is carried out UV spectrophotometer measuring, obtain wavelength 280nm and absorb collection of illustrative plates, collect each absorption peak, the dialysis desalination, lyophilize, detect through activity, the III absorption peak has very high fibrinolytic, promptly obtains the earthworm fibrinolysin purified product;
(3) this earthworm fibrinolysin purified product is dissolved in the 0.01mol/L Tris-HCl damping fluid of pH6, separate with sephadex G-75 type (SephadexG-75) gel column, 0.01mol/L Tris-HCl buffer solution elution with pH6, under the wavelength of 280nm, collected elutriant is carried out UV spectrophotometer measuring, obtain wavelength 280nm and absorb collection of illustrative plates, it is an albumen absorption peak, with this sample dialysis desalination, lyophilize, obtain the earthworm fibrinolysin highly finished product, it is 28000～30000 dalton that this sample is measured its molecular weight through the SDS-polyacrylamide gel electrophoresis, and the ratio work of described earthworm fibrinolysin reaches every milligram of protein 10 ten thousand units.
The condition of dialysis desalination is 8000～10000 dalton for the dialysis tubing aperture among the above-mentioned preparation method, will collect the liquid dialysis tubing of packing into, puts into 20 times distilled water and stirs and dialyse, and changes first water, dialyses 18 hours in 4 hours.
Get the earthworm fibrinolysin 250mg that makes, add low-molecular-weight dextran aseptic aqueous solution 1000ml, Sterile Filtration, 1000 of packing, every 1ml, lyophilized injectable powder is made in lyophilize.
Embodiment 2
(1) gets fresh earthworm 2400g, wash, under 4 ℃ ± 1 ℃ low temperature, the 0.1mol/L phosphate buffered saline buffer that contains 0.1mol/L NaCl that adds the pH8 of 3 times of weight parts, homogenate, and extracting 5 hours, 6000 rev/mins then, centrifugal 30 minutes, get supernatant liquor, add ammonium sulfate to 80% saturation ratio again, spend the night, saltout in 4 ℃ ± 1 ℃ placement, 6000 rev/mins, centrifugal 30 minutes, get precipitation, the dialysis desalination, lyophilize obtains the earthworm fibrinolysin crude product;
(2) with of the 0.1mol/LTris-HCl damping fluid dissolving of this earthworm fibrinolysin crude product with pH8,6000 rev/mins, centrifugal 30 minutes, getting supernatant liquor separates with carboxymethyl-sepharose Cl-6B post, with sodium-chlor straight line gradient mode wash-out, the concentration of this sodium chloride solution from the 0mol/L linear increment to 1mol/L, under the wavelength of 280nm, collected elutriant is carried out UV spectrophotometer measuring, obtain wavelength 280nm and absorb collection of illustrative plates, collect each absorption peak, the dialysis desalination, lyophilize, detect through activity, the III absorption peak has very high fibrinolytic, promptly obtains the earthworm fibrinolysin purified product;
(3) this earthworm fibrinolysin purified product is dissolved in the 0.1mol/L phosphate buffered saline buffer of pH8, separate with sephadex g-100 type (SephadexG-100) gel column, 0.1mol/L phosphate buffered saline buffer wash-out with pH8, under the wavelength of 280nm, collected elutriant is carried out UV spectrophotometer measuring, obtain wavelength 280nm and absorb collection of illustrative plates, it is an albumen absorption peak, with this sample dialysis desalination, lyophilize, obtain the earthworm fibrinolysin highly finished product, it is 28000～30000 dalton that this sample is measured its molecular weight through the SDS-polyacrylamide gel electrophoresis, and the ratio work of described earthworm fibrinolysin reaches every milligram of protein 10 ten thousand units.
The condition of dialysis desalination is 8000～10000 dalton for the dialysis tubing aperture among the above-mentioned preparation method, will collect the liquid dialysis tubing of packing into, puts into 50 times distilled water and stirs and dialyse, and changes first water, dialyses 32 hours in 8 hours.
Get the earthworm fibrinolysin 250mg that makes, add starch, lactose etc. and be mixed with aseptic aqueous solution 1000ml, Sterile Filtration, 1000 of packing, every 1ml, lyophilized injectable powder is made in lyophilize.
Embodiment 3
(1) gets fresh earthworm 2400g, wash, under 4 ℃ ± 1 ℃ low temperature, the 0.05mol/L sodium-chlor damping fluid that contains 0.1mol/L NaCl that adds the pH7.8 of 5 times of weight parts, homogenate, and extracting 5 hours, 5000 rev/mins then, centrifugal 20 minutes, get supernatant liquor, add ammonium sulfate to 70% saturation ratio again, spend the night, saltout in 4 ℃ ± 1 ℃ placement, 4000 rev/mins, centrifugal 10 minutes, get precipitation, the dialysis desalination, lyophilize obtains the earthworm fibrinolysin crude product;
(2) with of the 0.05mol/LTris-HCl damping fluid dissolving of this earthworm fibrinolysin crude product with pH7.8,5000 rev/mins, centrifugal 20 minutes, getting supernatant liquor separates with carboxymethyl-sepharose Cl-6B post, with sodium-chlor straight line gradient mode wash-out, the concentration of this sodium chloride solution from the 0mol/L linear increment to 1mol/L, under the wavelength of 280nm, collected elutriant is carried out UV spectrophotometer measuring, obtain wavelength 280nm and absorb collection of illustrative plates, collect each absorption peak, the dialysis desalination, lyophilize, detect through activity, the III absorption peak has very high fibrinolytic, promptly obtains the earthworm fibrinolysin purified product;
(3) this earthworm fibrinolysin purified product is dissolved in the 0.05mol/L sodium-chlor damping fluid of pH7.8, separate with sephadex g-100 type (SephadexG-100) gel column, 0.05mol/L sodium-chlor buffer solution elution with pH7.8, under the wavelength of 280nm, collected elutriant is carried out UV spectrophotometer measuring, obtain wavelength 280nm and absorb collection of illustrative plates, it is an albumen absorption peak, with this sample dialysis desalination, lyophilize, obtain the earthworm fibrinolysin highly finished product, it is 28000～30000 dalton that this sample is measured its molecular weight through the SDS-polyacrylamide gel electrophoresis, and the ratio work of described earthworm fibrinolysin reaches every milligram of protein 10 ten thousand units.
The condition of dialysis desalination is 8000～10000 dalton for the dialysis tubing aperture among the above-mentioned preparation method, will collect the liquid dialysis tubing of packing into, puts into 30 times distilled water and stirs and dialyse, and changes first water, dialyses 24 hours in 6 hours.
Get the earthworm fibrinolysin 250mg that makes, add sodium-chlor aseptic aqueous solution 1000ml, Sterile Filtration, 1000 of packing, every 1ml, lyophilized injectable powder is made in lyophilize.
Claims (2)
1. encircle the preparation method of the earthworm fibrinolysin of earthworm from the William chamber, it is characterized in that, may further comprise the steps:
(1) the earthworm fibrinolysin crude product extracts: get fresh earthworm and wash, under 4 ℃ ± 1 ℃ low temperature, 0.01～0.1mol/L the damping fluid that contains 0.1mol/LNaCl that adds pH6～8 of 1-5 times of weight part of earthworm, smash homogenate to pieces, extracting 2～5 hours, 4000～6000 rev/mins then, centrifugal 10～30 minutes, get supernatant liquor, add ammonium sulfate to 60%～80% saturation ratio, spend the night 4000～6000 rev/mins in 4 ℃ ± 1 ℃ placement, centrifugal 10～30 minutes, taking precipitate, dialysis desalination, lyophilize, obtain the earthworm fibrinolysin crude product, wherein said damping fluid is tris-HCI buffer or phosphate buffered saline buffer;
(2) purifying of earthworm fibrinolysin crude product: with 0.01～0.1mol/L damping fluid dissolving of the earthworm fibrinolysin crude product in the step (1) with pH6～8,4000～6000 rev/mins, centrifugal 10～30 minutes, getting supernatant liquor separates with carboxymethyl-sepharose C1-6B post, with sodium-chlor straight line gradient mode wash-out, the concentration of this sodium chloride solution from the 0mol/L linear increment to 1mol/L, collect the elutriant of each elution peak, the dialysis desalination, lyophilize, detect through activity, obtain the earthworm fibrinolysin purified product, wherein said damping fluid is tris-HCI buffer or phosphate buffered saline buffer;
(3) the earthworm fibrinolysin purified product is refining: with the 0.01～0.1mol/L damping fluid dissolving of the earthworm fibrinolysin purified product in the step (2) with pH6～8, separate with gel column, 0.01～0.1mol/L buffer solution elution with pH6～8, collect the elutriant of active elution peak, the dialysis desalination, lyophilize, obtain the earthworm fibrinolysin highly finished product, its molecular weight is 28000～30000 dalton, the ratio work of described earthworm fibrinolysin reaches every milligram of protein 10 ten thousand units, and wherein said damping fluid is tris-HCI buffer or phosphate buffered saline buffer.
2. the preparation method of the earthworm fibrinolysin from William chamber ring earthworm according to claim 1, it is characterized in that: the gel column described in the step (3) is sephadex G-75 type or sephadex g-100 type.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810182567XA CN101407801B (en) | 2008-12-05 | 2008-12-05 | Preparation of earthworm fibrinolytic enzyme from Pheretima guillelmi Michaelsen and lyophilized powder preparation for injection prepared thereby |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810182567XA CN101407801B (en) | 2008-12-05 | 2008-12-05 | Preparation of earthworm fibrinolytic enzyme from Pheretima guillelmi Michaelsen and lyophilized powder preparation for injection prepared thereby |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101407801A CN101407801A (en) | 2009-04-15 |
| CN101407801B true CN101407801B (en) | 2010-11-03 |
Family
ID=40571013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810182567XA Active CN101407801B (en) | 2008-12-05 | 2008-12-05 | Preparation of earthworm fibrinolytic enzyme from Pheretima guillelmi Michaelsen and lyophilized powder preparation for injection prepared thereby |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101407801B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2011338109B2 (en) * | 2011-04-11 | 2013-04-18 | Well Stone Co. | Manufacturing method for dried earthworm powder |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119928A (en) * | 2011-02-21 | 2011-07-13 | 江中药业股份有限公司 | Preparation method of lumbrokinase enteric-coated pellets |
| CN103239706B (en) * | 2013-05-13 | 2014-11-26 | 肖梅芬 | Earthworm protein for reducing blood glucose, improving microcirculation and eliminating diabetic complication and application thereof |
| CN103289981A (en) * | 2013-06-24 | 2013-09-11 | 广州润虹医药科技有限公司 | A preparation method for lyophilized powder of earthworm fibrinolytic enzymes |
| CN109260231B (en) * | 2017-07-18 | 2021-09-03 | 首都儿科研究所 | Preparation method of earthworm extract with cough stopping, phlegm eliminating, anti-inflammatory and antimicrobial functions |
| CN107362126A (en) * | 2017-09-04 | 2017-11-21 | 银川凤仪堂生物工程有限公司 | Power simulation extract freeze-drying powder and preparation technology |
-
2008
- 2008-12-05 CN CN200810182567XA patent/CN101407801B/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2011338109B2 (en) * | 2011-04-11 | 2013-04-18 | Well Stone Co. | Manufacturing method for dried earthworm powder |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101407801A (en) | 2009-04-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101407801B (en) | Preparation of earthworm fibrinolytic enzyme from Pheretima guillelmi Michaelsen and lyophilized powder preparation for injection prepared thereby | |
| Milani Júnior et al. | Snake bites by the jararacuçu (Bothrops jararacussu): clinicopathological studies of 29 proven cases in São Paulo State, Brazil. | |
| JP5048673B2 (en) | A kind of extract that prevents or treats thrombotic diseases | |
| CN104004806A (en) | Lumbricus polypeptide having anticoagulant and thrombolytic effects, and enzymatic hydrolysis preparation method and application thereof | |
| CN103289981A (en) | A preparation method for lyophilized powder of earthworm fibrinolytic enzymes | |
| US4532129A (en) | Composition containing and method of using a fibrinolytic active principle | |
| CN102002488B (en) | Phascolosoma esculenta plasmin and preparation method thereof | |
| EP0020780A1 (en) | Fibrinolytic material and process for producing same | |
| Dieter et al. | Studies on an undetermined circulating anticoagulant. Case report and laboratory findings | |
| CN101892210B (en) | Sipunculus nudus plasmin and preparation method thereof | |
| CN106563120B (en) | A kind of antithrombotic biological agent prepared using clam worm active peptide | |
| US4479937A (en) | Process of treating inflammation with human urinary thiol protease inhibitor | |
| Ueyama et al. | Progressing ischemic stroke in a homozygote with variant antithrombin III. | |
| Silberman et al. | Effects of ancrod (Arvin) in mice: studies of plasma fibrinogen and fibrinolytic activity | |
| KR101780643B1 (en) | Method for purifying heparin using enzymolysis | |
| CN102242102A (en) | Preparation method of X factor activator from bothrops atrox venom | |
| CN101880656B (en) | Agkistrodon halys venom thrombin and preparation method and application thereof | |
| CN113755476A (en) | Preparation method and application of maggot kinase | |
| CN1128220C (en) | Process for separating worm kinase | |
| CN1548534B (en) | Reptilase and its production process and application | |
| CN1044560C (en) | Method of preparation of soluble earthworm cellvibrio | |
| CN101812436B (en) | Agkistrodon acutus venom thrombin-like enzyme, preparation method and application thereof | |
| CN106632602A (en) | Perinereis aibuhitensis anticoagulation peptide and use thereof | |
| KR100261657B1 (en) | Process for manufacturing rumbrokinase liquid and use thereof | |
| Racanelli et al. | Neutralization of the antithrombotic effects of heparin and fraxiparinR by protamine sulfate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160817 Address after: 150090 D block, 17 floor, No. 17 Li Shun Street, Nangang District, Heilongjiang, Harbin Patentee after: Heilongjiang Tian Yong Kun Pharmaceutical Co.,Ltd. Address before: 3612, room 150090, long Kun garden, 289 Hongqi Avenue, Heilongjiang, Harbin Patentee before: Guo Xiuhua Patentee before: Shang Zhiguo |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160922 Address after: 150090 D block, 17 floor, No. 17 Li Shun Street, Nangang District, Heilongjiang, Harbin Patentee after: Heilongjiang Tian Yong Shen Pharmaceutical Co.,Ltd. Address before: 3612, room 150090, long Kun garden, 289 Hongqi Avenue, Heilongjiang, Harbin Patentee before: Guo Xiuhua Patentee before: Shang Zhiguo |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200730 Address after: Room 102, unit 6, building 3, Longkun community, No. 289, Hongqi Street, Nangang District, Harbin City, Heilongjiang Province Patentee after: Guo Xiuhua Address before: 150090 D block, 17 floor, No. 17 Li Shun Street, Nangang District, Heilongjiang, Harbin Patentee before: Heilongjiang Tian Yong Shen Pharmaceutical Co.,Ltd. |
|
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 150000 floor 3, unit 2, building 5, accelerator Park, No. 16155, Zhongyuan Avenue, Songbei District, Harbin City, Heilongjiang Province Patentee after: Guo Xiuhua Address before: Room 102, unit 6, building 3, Longkun community, 289 Hongqi Street, Nangang District, Harbin City, Heilongjiang Province Patentee before: Guo Xiuhua |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231026 Address after: Room C201, Building 2, Innovation and Entrepreneurship Plaza, Science and Technology Innovation City, High tech Industrial Development Zone, Harbin City, Heilongjiang Province, 150000 yuan (No. 669, Science and Technology First Street) Patentee after: Tiande Yongkun Pharmaceutical Co.,Ltd. Address before: 150000 floor 3, unit 2, building 5, accelerator Park, No. 16155, Zhongyuan Avenue, Songbei District, Harbin City, Heilongjiang Province Patentee before: Guo Xiuhua |