CN101338305A - Process for extracting superoxide dismutase and reduction glutathion from animal blood - Google Patents
Process for extracting superoxide dismutase and reduction glutathion from animal blood Download PDFInfo
- Publication number
- CN101338305A CN101338305A CNA2008103042551A CN200810304255A CN101338305A CN 101338305 A CN101338305 A CN 101338305A CN A2008103042551 A CNA2008103042551 A CN A2008103042551A CN 200810304255 A CN200810304255 A CN 200810304255A CN 101338305 A CN101338305 A CN 101338305A
- Authority
- CN
- China
- Prior art keywords
- reduced glutathion
- dismutase
- superoxide
- time
- minutes
- 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.)
- Granted
Links
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 146
- 229960003180 glutathione Drugs 0.000 title claims abstract description 139
- 210000004369 blood Anatomy 0.000 title claims abstract description 79
- 239000008280 blood Substances 0.000 title claims abstract description 79
- 102000019197 Superoxide Dismutase Human genes 0.000 title claims abstract description 78
- 108010012715 Superoxide dismutase Proteins 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 57
- 241001465754 Metazoa Species 0.000 title claims abstract description 34
- 235000003969 glutathione Nutrition 0.000 title claims description 95
- 230000008569 process Effects 0.000 title description 9
- 239000000284 extract Substances 0.000 claims abstract description 11
- 238000012545 processing Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 106
- 229940032362 superoxide dismutase Drugs 0.000 claims description 71
- 238000000108 ultra-filtration Methods 0.000 claims description 41
- 239000006228 supernatant Substances 0.000 claims description 37
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 32
- 241001494479 Pecora Species 0.000 claims description 24
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 24
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 24
- 229960001763 zinc sulfate Drugs 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 21
- 239000011347 resin Substances 0.000 claims description 21
- 229920005989 resin Polymers 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000000835 fiber Substances 0.000 claims description 19
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 18
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 18
- 210000003743 erythrocyte Anatomy 0.000 claims description 18
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 16
- 239000012141 concentrate Substances 0.000 claims description 15
- 238000000502 dialysis Methods 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- 238000001556 precipitation Methods 0.000 claims description 10
- 238000012423 maintenance Methods 0.000 claims description 9
- 229910021529 ammonia Inorganic materials 0.000 claims description 8
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000003957 anion exchange resin Substances 0.000 claims description 7
- 239000003729 cation exchange resin Substances 0.000 claims description 7
- 230000001186 cumulative effect Effects 0.000 claims description 7
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008676 import Effects 0.000 claims description 7
- 239000008176 lyophilized powder Substances 0.000 claims description 7
- 206010018910 Haemolysis Diseases 0.000 claims description 6
- 150000001450 anions Chemical class 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 230000008588 hemolysis Effects 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 6
- 229940038773 trisodium citrate Drugs 0.000 claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 5
- 238000012856 packing Methods 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 241000272525 Anas platyrhynchos Species 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 4
- 150000001768 cations Chemical class 0.000 claims description 4
- 238000010612 desalination reaction Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 235000008504 concentrate Nutrition 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000010419 fine particle Substances 0.000 claims description 3
- 235000014666 liquid concentrate Nutrition 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 108010024636 Glutathione Proteins 0.000 abstract 5
- 230000031877 prophase Effects 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 20
- 230000000694 effects Effects 0.000 description 18
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 14
- 239000013558 reference substance Substances 0.000 description 11
- 238000006701 autoxidation reaction Methods 0.000 description 9
- 229940079877 pyrogallol Drugs 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000010949 copper Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 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 2
- 208000030507 AIDS Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- UXTMROKLAAOEQO-UHFFFAOYSA-N chloroform;ethanol Chemical compound CCO.ClC(Cl)Cl UXTMROKLAAOEQO-UHFFFAOYSA-N 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000007667 floating Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 239000012716 precipitator Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 208000002177 Cataract Diseases 0.000 description 1
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 208000037319 Hepatitis infectious Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 208000017442 Retinal disease Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- TVZPLCNGKSPOJA-UHFFFAOYSA-N copper zinc Chemical compound [Cu].[Zn] TVZPLCNGKSPOJA-UHFFFAOYSA-N 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 208000021921 corneal disease Diseases 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 208000019995 familial amyotrophic lateral sclerosis Diseases 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 208000005252 hepatitis A Diseases 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000019988 mead Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000004223 radioprotective effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- -1 superoxide anion free radical Chemical class 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- TUGDLVFMIQZYPA-UHFFFAOYSA-N tetracopper;tetrazinc Chemical compound [Cu+2].[Cu+2].[Cu+2].[Cu+2].[Zn+2].[Zn+2].[Zn+2].[Zn+2] TUGDLVFMIQZYPA-UHFFFAOYSA-N 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a method for obtaining superoxide dismutase and glutathione from the blood of an animal which is carried out according to the following steps: prophase processing, extracting the superoxide dismutase and extracting the glutathione. The method of the invention for obtaining superoxide dismutase and glutathione from the blood of the animal fully utilizes resources, can simultaneously extract the superoxide dismutase SOD and the glutathione GSH, simplifies the production technique, reduces the production cost and improves the product quality for the glutathione GSH.
Description
One, technical field
(Superoxide Dismutase SOD) and the method for reduced glutathion (GSH), is a kind of superoxide-dismutase and reduced glutathion method of obtaining from animal blood to the present invention relates to extract from animal blood superoxide-dismutase.
Two, background technology
Superoxide-dismutase (be Superoxide Dismutase, be called for short SOD) is a kind of intravital metalloenzyme of various biologies that extensively is present in.SOD is first antioxidase that plays a role in the oxygen scavenging activity reaction process, can specificity remove the superoxide anion free radical (O that organism produces
2 .-), the various damages that body internal cause free radical is caused all have protection and therapeutic action.Be used for the treatment of aspects such as osteoarthritis, rheumatoid arthritis, radiation and chemotherapy, burn, familial amyotrophic lateral sclerosis clinically.This external radioprotective, antitumor and pre-many-side such as anti-aging discover that the prevention and the treatment of SOD and multiple disease have substantial connection.Clinical diagnosis, the therapeutic value of SOD constantly are familiar with by people, have been widely used in other fields such as healthcare products, foodstuff additive and makeup.
Gsh is the tripeptide compound that is formed by L-glutamic acid, halfcystine and glycine, extensively is present in the organism with reduced form (being GSH) and two kinds of forms of oxidized form (GSSG), and be main antioxidant, pair cell has multiple biochemical action.The clinical treatment that is used for toxic hepatitis and infectious hepatitis; have certain effect to suppressing cataract and cornea and retinal diseases; to the protection of detoxifcation, cancer radiation and the chemotherapy of organism and heavy metal, to pulmonary fibrosis, liver cancer, acquired immune deficiency syndrome (AIDS) also is useful drug combination; also be widely used in food-processing industry, cosmetic industry etc.
Since McCord and Fridovich at first extracted SOD in 1969 from the red corpuscle of ox since, Chinese scholar was purified into Cu, Zn Rising-moon OD from materials such as ox blood, pig blood, duck blood, pork liver, Plantula Brassicae chinensis, human red cell and sheep blood.Its method of extracting purifying is improved on the basis of McCord and Fridovich method and is come.Document shows that by the Cu that extracts purifying in the animal body, Zn-SOD, its enzyme compare the Cu apparently higher than purifying from Plantula Brassicae chinensis leaf cell solute and chloroplast(id) alive, Zn-SOD.
The extracting and purifying method of SOD:
Reference ecology of domestic animals newspaper (2006,27 (5): 46) openly reported the ox blood copper-zinc superoxide dismutase scale production process research of Wuyun Dalai, Sun Zhenjun, Wang Chong etc., as shown in Figure 1.
As far back as 1888, GSH is just by Rey-Pail-hade, and J. at first separates from yeast, and nineteen twenty-one, Hopkins obtained crystallization, proposition GSH such as nineteen twenty-nine Hopkins, Kendall are tripeptides, and nineteen thirty-five illustrates its chemical structure by Harington and Mead and synthesized.In the last few years, people successfully obtained GSH by methods such as direx process, chemical synthesis, fermentation method and enzyme transforming process.(1999,20 (5): 246) reported openly that Liu Xiaoying, Ding Yiyi purify from pig blood has obtained GSH to reference China biochemical drug magazine, as shown in Figure 2.Reference medicine biotechnology (1999,6 (4): 203) openly reported Tao Rui, Wu Wutong, permitted the research that inspired enzyme process prepares gsh technology, as shown in Figure 3.
At present, preparation SOD mainly contains the precipitator method, thermally denature method, chromatography and ultrafiltration process.Precipitator method solvent consumption is big, cost is high; The thermally denature method heats inhomogeneous difficult amplification when extensive separation and purification; Though ultrafiltration process can be removed a large amount of small molecular weight impurities, a small amount of SOD still can see through the PAN film, and cycles of concentration is big more, loses also manyly more, and concentration polarization is serious, makes the very fast decline of the saturating liquid measure of film.Yan Jiaqi adopts after the first thermally denature method separation and purification SOD with the PS membrane ultrafiltration, but in containing the enzyme liquid of organic solvent heating SOD instability, and heating to be difficult for even energy consumption big, the technology more complicated.
In sum, existing known technology all is to extract purifying SOD and GSH respectively, and this had both caused the waste of resource, had polluted environment again.
Three, summary of the invention
The invention provides a kind of superoxide-dismutase and reduced glutathion method of from animal blood, obtaining, the deficiencies in the prior art have been overcome, can from animal blood, extract superoxide-dismutase and reduced glutathion simultaneously, not only improved the utilization ratio of resource but also reduced pollution environment, and method is simple, production cost is lower, has improved the quality product of GSH.
Technical scheme of the present invention is achieved like this: a kind of superoxide-dismutase and reduced glutathion method of from animal blood, obtaining, and it carries out according to the following steps:
Handle early stage: obtain solution of red blood cells through centrifugation after adding Trisodium Citrate earlier in fresh animal blood, secondly in solution of red blood cells, add deionized water and obtain hemolysate, in hemolysate, add the centrifugal supernatant liquor that obtains behind the solution of copper sulfate and zinc sulfate then, at last supernatant liquor is obtained concentrated solution with the hollow-fibre ultrafiltration device ultrafiltration and through liquid;
Extract superoxide-dismutase: centrifugally after handling the solution that adds copper sulfate and zinc sulfate in the gained concentrated solution above-mentioned early stage obtain containing the superoxide-dismutase supernatant liquor, will contain the superoxide-dismutase supernatant liquor and obtain containing the superoxide-dismutase concentrated solution through the hollow-fibre ultrafiltration device ultrafiltration;
Extract reduced glutathion: see through and obtain containing the reduced glutathion elutriant through 732 cation exchange resin columns and 717 anion-exchange resin columns after liquid concentrates handling gained above-mentioned early stage, will contain the reduced glutathion elutriant and concentrate and obtain containing the reduced glutathion concentrated solution.
Be further optimization and/or selection below to technique scheme:
Handle and carry out as follows early stage: it is 3.8% to 4.2% Trisodium Citrate that every liter of fresh animal blood adds 125 milliliters to 170 milliliters, concentration, leave 10 minutes to 20 minutes separating red corpuscles of the heart with per minute 3000, add volume again and be the physiological saline Washed Red Blood Cells solution 1 time to 3 times of 1 times to 2 times of solution of red blood cells volume; Then with solution of red blood cells at 0 ℃ of following ice bath, and to add volume be the deionized water of 2 times to 4 times of solution of red blood cells, stirs haemolysis and obtained hemolysate in 30 minutes; Add and account for the copper sulfate of hemolysate cumulative volume 1.0% to 10.0% and the aqueous solution of zinc sulfate, the concentration of copper-bath is 5% to 0.5%, the concentration of solution of zinc sulfate is 7% to 0.7%, the consumption of copper sulfate and solution of zinc sulfate is 1: 2 to 2: 1 by volume, temperature is controlled between 55 ℃ to 80 ℃, heats 10 minutes to 20 minutes, is cooled to room temperature, leave the heart 10 minutes to 30 minutes with per minute 3000, obtain supernatant liquor; Supernatant liquid filtering is removed fine particle, be the hollow-fibre ultrafiltration device ultrafiltration of 5KD to 15KD again through molecular weight cut-off, the control import is pressed between the 15psi to 30psi, the maintenance reflux pressure is 10psi, promptly keeping pressure difference is 5psi to 20psi, be concentrated into 10% to 20% of supernatant liquor volume, obtain concentrated solution and see through liquid.
Extracting superoxide-dismutase carries out as follows: add 1 times of deionized water dilution to 3 times of volumes in the gained concentrated solution with handling above-mentioned early stage, adding volume again is the copper sulfate of diluent cumulative volume 1% to 20% and the aqueous solution of zinc sulfate, the concentration of copper-bath is 5% to 0.5%, solution of zinc sulfate concentration is 7% to 0.7%, the consumption volume ratio of copper sulfate and solution of zinc sulfate is 1: 2 to 2: 1, stir evenly, water bath heat preservation is 10 minutes to 20 minutes between 55 ℃ to 80 ℃, be cooled to room temperature, left the heart 10 minutes to 30 minutes with per minute 3000, collection contains the superoxide-dismutase supernatant liquor; To contain the superoxide-dismutase supernatant liquor is the hollow-fibre ultrafiltration device ultrafiltration of 5KD to 15KD through molecular weight cut-off, the control import is pressed between the 15psi to 30psi, the maintenance reflux pressure is 10psi, promptly keeping pressure difference is 5psi to 20psi, be concentrated into and contain 10% to 20% of superoxide-dismutase supernatant liquor volume, obtain containing for the first time the superoxide-dismutase concentrated solution; Get and contain for the first time the superoxide-dismutase concentrated solution, through the hollow-fibre ultrafiltration device dynamic dialysis of 5KD to 15KD 12 hours, per minute 3000 left the heart 10 minutes, removed insoluble protein, must contain the superoxide-dismutase dialyzate; To contain the superoxide-dismutase dialyzate is the hollow-fibre ultrafiltration device ultrafiltration of 5KD to 15KD through molecular weight cut-off, the control intake pressure is between 15psi to 30psi, the maintenance return pressure is 10psi, promptly keeping pressure difference is 5psi to 20psi, be concentrated into and contain 10% to 20% of superoxide-dismutase dialysate volumes, obtain containing for the second time the superoxide-dismutase concentrated solution; Add 1 times to the 2 times pre-cold acetone of volume with containing the superoxide-dismutase concentrated solution second time, placed 1 hour to 2 hours, per minute 3000 leaves the heart and must precipitate in 10 minutes to 20 minutes; Precipitate the green lyophilized powder of dry light green or pale blue and contain superoxide-dismutase.
Extracting reduced glutathion carries out as follows: see through liquid 40 ℃ to 50 ℃ of bath temperatures with handling gained above-mentioned early stage, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, cycles of concentration is 5 times to 15 times, obtains containing for the first time the reduced glutathion concentrated solution; To contain the reduced glutathion concentrated solution uses sulfuric acid adjust pH to 2 to 4, last 732 cation exchange resin columns, 732 resin cation (R.C.) order numbers are 60 order to 80 orders, the elutriant ammonia concn is 0.25 mol to 1.0 mol, the elutriant ammonia flow rate be 0.5 to 1.5 times of resin volume/hour, obtain containing for the first time the reduced glutathion elutriant; To contain the reduced glutathion elutriant first time 40 ℃ to 50 ℃ of bath temperatures, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, and cycles of concentration is 5 times to 15 times, obtains containing for the second time the reduced glutathion concentrated solution; Use sulfuric acid adjust pH to 4 to 5 with containing the reduced glutathion concentrated solution second time, last 717 anion-exchange resin columns, 717 resin anion(R.A) order numbers are 60 order to 80 orders; The elutriant sulfuric acid concentration is 0.25 mol to 1.0 mol; Elutriant sulfuric acid flow velocity is 0.5 to 2.0 times of resin volume/h, obtains containing for the second time the reduced glutathion elutriant; With containing for the second time the reduced glutathion elutriant molecular weight of packing into is 12 hours desalinations of dialysis in 100 the dialysis tubing, obtains containing the reduced glutathion dialyzate; To contain the reduced glutathion dialyzate 40 ℃ to 50 ℃ of bath temperatures, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, and cycles of concentration is 5 times to 15 times, is contained the reduced glutathion concentrated solution for the third time; To contain the reduced glutathion concentrated solution for the third time and add 1 times to 2 times pre-cooled ethanol, place 1 hour to 2 hours, and leave the heart with per minute 3000 and obtained precipitation in 10 minutes to 20 minutes; The precipitation drying obtains reduced glutathion white lyophilized powder.
The dry freeze-drying that adopts, drying chamber pressure is 0.8Mpa to 0.9Mpa, and bed thickness is 1cm, and freezing temp is-40 ℃.
Fresh animal blood can adopt fresh sheep blood or fresh ox blood or fresh cattle and sheep to mix blood or fresh pig blood or fresh duck blood or fresh human blood.
The present invention obtains superoxide-dismutase and reduced glutathion method from animal blood, it has made full use of resource, can extract SOD and GSH simultaneously, has simplified production technique, has reduced production cost, has improved the quality product of GSH.
Four, description of drawings
Accompanying drawing 1 is existing extraction purifying Cu, the technical process of one of Zn-SOD method.
Accompanying drawing 2 is existing technical process of extracting one of purifying GSH method.
Accompanying drawing 3 is existing technical process of two of extracting purifying GSH method.
Accompanying drawing 4 is technological process of productions of the embodiment of the invention.
Accompanying drawing 5 is the reduced glutathion typical curve.
Five, embodiment the present invention is not subjected to the restriction of following embodiment, can determine concrete embodiment according to the technical scheme and the practical situation of the invention described above.
Below in conjunction with embodiment the present invention is done further argumentation:
This obtain superoxide-dismutase from animal blood and the reduced glutathion method is carried out according to the following steps:
Handle early stage: obtain solution of red blood cells through centrifugation after adding Trisodium Citrate earlier in fresh animal blood, secondly in solution of red blood cells, add deionized water and obtain hemolysate, in hemolysate, add the centrifugal supernatant liquor that obtains behind the solution of copper sulfate and zinc sulfate then, at last supernatant liquor is obtained concentrated solution with the hollow-fibre ultrafiltration device ultrafiltration and through liquid;
Extract superoxide-dismutase: handle the back centrifugal superoxide-dismutase supernatant liquor that obtains containing of the solution (being the copper zinc solution) that adds copper sulfate and zinc sulfate in the gained concentrated solution in above-mentioned early stage, will contain the superoxide-dismutase supernatant liquor and obtain containing the superoxide-dismutase concentrated solution through the hollow-fibre ultrafiltration device ultrafiltration;
Extract reduced glutathion: see through and obtain containing the reduced glutathion elutriant through 732 cation exchange resin columns and 717 anion-exchange resin columns after liquid concentrates handling gained above-mentioned early stage, will contain the reduced glutathion elutriant and concentrate and obtain containing the reduced glutathion concentrated solution.
Can from animal blood, obtain superoxide-dismutase to this according to actual needs below and the reduced glutathion method makes further optimization and/or improvements:
Above-mentioned in earlier stage handle and can carry out as follows: every liter of fresh animal blood adds 125 milliliters or 150 milliliters or 170 milliliters, concentration is 3.8% or 4.2% Trisodium Citrate, leave the heart 10 minutes or 15 minutes or 20 minutes separating red corpuscles with per minute 3000, add volume again and be the physiological saline Washed Red Blood Cells solution 1 time or 2 times or 3 times of 1 times of solution of red blood cells volume or 2 times; Then with solution of red blood cells at 0 ℃ of following ice bath, and add the deionized water that volume is 2 times of solution of red blood cells or 3 times or 4 times, stir haemolysis and obtained hemolysate in 30 minutes; Add and account for the copper sulfate of hemolysate cumulative volume 1.0% or 5.0% or 10.0% and the aqueous solution of zinc sulfate, the concentration of copper-bath is 5% or 3% or 0.5%, the concentration of solution of zinc sulfate is 7% or 3% or 0.7%, the consumption of copper sulfate and solution of zinc sulfate is 1: 2 or 2: 1 by volume, temperature is controlled between 55 ℃ or 65 ℃ or 80 ℃, heats 10 minutes or 15 minutes or 20 minutes, is cooled to room temperature, leave the heart 10 minutes or 20 minutes or 30 minutes with per minute 3000, obtain supernatant liquor; Supernatant liquid filtering is removed fine particle, be the hollow-fibre ultrafiltration device ultrafiltration of 5KD or 10KD or 15KD again through molecular weight cut-off, the control import is pressed between 15psi or 22psi or the 30psi, the maintenance reflux pressure is 10psi, promptly keeping pressure difference is 5psi or 12psi or 20psi, be concentrated into 10% or 20% of supernatant liquor volume, obtain concentrated solution and see through liquid.
The said extracted superoxide-dismutase can carry out as follows: will handle the deionized water dilution that adds 1 times or 2 times or 3 times volume in the gained concentrated solution above-mentioned early stage, adding volume again is the copper sulfate of diluent cumulative volume 1% or 6% or 10% or 15% or 20% and the aqueous solution of zinc sulfate, the concentration of copper-bath is 5% or 2% or 0.5%, solution of zinc sulfate concentration is 7% or 3.5% or 0.7%, the consumption volume ratio of copper sulfate and solution of zinc sulfate is 1: 2 or 2: 1, stir evenly, water bath heat preservation is 10 minutes or 15 minutes or 20 minutes between 55 ℃ or 60 ℃ or 80 ℃, be cooled to room temperature, leave the heart 10 minutes or 20 minutes or 30 minutes with per minute 3000, collect and contain the superoxide-dismutase supernatant liquor; To contain the superoxide-dismutase supernatant liquor is the hollow-fibre ultrafiltration device ultrafiltration of 5KD or 10KD or 15KD through molecular weight cut-off, the control import is pressed between 15psi or 22psi or the 30psi, the maintenance reflux pressure is 10psi, promptly keeping pressure difference is 5psi or 12psi or 20psi, be concentrated into and contain 10% or 20% of superoxide-dismutase supernatant liquor volume, obtain containing for the first time the superoxide-dismutase concentrated solution; Get and contain for the first time the superoxide-dismutase concentrated solution, through the hollow-fibre ultrafiltration device dynamic dialysis of 5KD or 10KD or 15KD 12 hours, per minute 3000 left the heart 10 minutes, removed insoluble protein, must contain the superoxide-dismutase dialyzate; To contain the superoxide-dismutase dialyzate is the hollow-fibre ultrafiltration device ultrafiltration of 5KD or 10KD or 15KD through molecular weight cut-off, the control intake pressure is between 15psi or 22psi or 30psi, the maintenance return pressure is 10psi, promptly keeping pressure difference is 5psi or 12psi or 20psi, be concentrated into and contain 10% or 20% of superoxide-dismutase dialysate volumes, obtain containing for the second time the superoxide-dismutase concentrated solution; Add 1 times or 2 times of pre-cold acetones of volume with containing the superoxide-dismutase concentrated solution second time, placed 1 hour or 2 hours, per minute 3000 leaves the heart 10 minutes or must precipitate in 15 minutes or 20 minutes; Precipitate the green lyophilized powder of dry light green or pale blue and contain superoxide-dismutase.
The said extracted reduced glutathion can carry out as follows: see through liquid 40 ℃ of bath temperatures or 50 ℃ with handling gained above-mentioned early stage, vacuum tightness is to concentrate under the condition of 0.07Mpa or 0.08Mpa, cycles of concentration is 5 times or 10 times or 15 times, obtains containing for the first time the reduced glutathion concentrated solution; To contain reduced glutathion concentrated solution sulfuric acid adjust pH to 2 or 4, last 732 cation exchange resin columns, 732 resin cation (R.C.) order numbers are 60 orders or 70 orders or 80 orders, the elutriant ammonia concn is 0.25 mol or 0.5 mol or 1.0 mol, the elutriant ammonia flow rate be 0.5 or 1.0 or 1.5 times of resin volume/hour, obtain containing for the first time the reduced glutathion elutriant; To contain the reduced glutathion elutriant first time 40 ℃ of bath temperatures or 50 ℃, vacuum tightness is to concentrate under the condition of 0.07Mpa or 0.08Mpa, and cycles of concentration is 5 times or 10 times or 15 times, obtains containing for the second time the reduced glutathion concentrated solution; To contain reduced glutathion concentrated solution sulfuric acid adjust pH to 4 or 5 second time, last 717 anion-exchange resin columns, 717 resin anion(R.A) order numbers are 60 orders or 70 orders or 80 orders; The elutriant sulfuric acid concentration is 0.25 mol or 0.50 mol or 1.0 mol; Elutriant sulfuric acid flow velocity is 0.5 or 1.0 or 1.5 or 2.0 times of resin volume/h, obtains containing for the second time the reduced glutathion elutriant; With containing for the second time the reduced glutathion elutriant molecular weight of packing into is 12 hours desalinations of dialysis in 100 the dialysis tubing, obtains containing the reduced glutathion dialyzate; To contain the reduced glutathion dialyzate 40 ℃ of bath temperatures or 50 ℃, vacuum tightness is to concentrate under the condition of 0.07Mpa or 0.08Mpa, and cycles of concentration is 5 times or 10 times or 15 times, is contained the reduced glutathion concentrated solution for the third time; To contain the reduced glutathion concentrated solution for the third time and add 1 times or 2 times of pre-cooled ethanols, place 1 hour or 2 hours, and leave the heart 10 minutes or obtained precipitation in 15 minutes or 20 minutes with per minute 3000; The precipitation drying obtains reduced glutathion white lyophilized powder.
Above-mentioned drying means can adopt freeze-drying, and drying chamber pressure is 0.8 Mpa to 0.9Mpa, and bed thickness is 1cm, and freezing temp is 40 ℃.
Above-mentioned fresh animal blood can adopt fresh sheep blood or fresh ox blood or fresh cattle and sheep to mix blood or fresh pig blood or fresh duck blood or fresh human blood.
Above technical characterictic has constituted most preferred embodiment of the present invention, and it has stronger adaptability and best implementation result, can increase and decrease non-essential technical characterictic according to actual needs, satisfies the demand of different situations.
Below the foregoing description is carried out following test:
Reference substance, reagent: (Sigma company produces the reduced glutathion reference substance, the import packing), methyl alcohol, acetonitrile are that U.S. Fisher company produces chromatographically pure, water is redistilled water, 732 Zeo-karbs and 717 anionite-exchange resin (Shanghai San Pu chemical industry Instr Ltd.), all the other reagent are homemade analytical pure, and experimental water is a deionized water.
Instrument and equipment: LC-10AVP high performance liquid chromatograph (institute is made in a day island proper Tianjin) comprises LC-10ATVP binary high-pressure pump, the SPD-10AVP detector, the CTO-10ASVP column oven, the SCL-10AVP central controller, Shim-Pack CLC-ODS post (150mm * 6.0mm, 5 μ m); UV-2501PC type ultraviolet spectrophotometer (institute is made in a day island proper Tianjin); The automatic pure water distiller of SZ-96 (Shanghai Yarong Biochemical Instrument Plant); BS110S electronic balance (German Sartorius company), PB-10pH meter (German Sartorius company).
TDL-5 type whizzer (Anting Scientific Instrument Factory, Shanghai); VV-2000 Rotary Evaporators (German Heidolph company); SHB-circulation ability of swimming is used vacuum pump (Zhengzhou Greatwall Scientific Industrial ﹠ Trading Co., Ltd.) more; WS2-261-79 type digital display temperature control water temperature case (Shanghai revive into plant and instrument factory); DZF-6050 type vacuum drying oven (going up the grand experimental installation of Nereid company limited); RO-40 membrane ultrafiltration device is hollow-fibre ultrafiltration device (Tianjin MoTian Membrane Engineering Technology Co., Ltd).
(1) index determines and measuring method
The extraction yield of superoxide-dismutase (being SOD) and reduced glutathion (being GSH) adopts comprehensive grading (score value is respectively 0.5,0.5).
Comprehensive grading=(SOD total activity/maximum SOD total activity) * 50+ (GSH content/maximum GSH content) * 50
1.SOD vitality test
Micro-pyrogallol autoxidation method according to improvement is that 325nm method (" CNS " GB/T 5009.171-003) is measured its vigor.
1.1 SOD sample liquid is got in the preparation of need testing solution, thin up shakes up, make to survey periodic autoxidation rate-controlling at 0.030 (± 0.002) O.D./min, with the SOD of this concentration as need testing solution.
1.2 assay method
The mensuration of pyrogallol autoxidation speed
Under 25 ℃, press table 1 and add the Tris-HCl damping fluid, add pyrogallol solution then, shake up rapidly and put in the 1cm cuvette, compare with blank pipe, under the 325nm wavelength, read the A value 1 time every 0.5min, the A value of continuous recording 7.0min is calculated its autoxidation speed according to autoxidation rate calculations formula, requires control pyrogallol autoxidation speed at 0.060 (± 0.002) O.D./min.Can be controlled by the add-on of fine setting pyrogallol solution.Pyrogallol autoxidation rate calculations formula:
1.3 the mensuration of trial-product
Get the SOD need testing solution, 25 ℃, press table 1 successively and add Tris-HCl damping fluid, pyrogallol solution and sample liquid, shake up rapidly and put in the 1cm cuvette, compare with blank pipe, measure under the 325nm wavelength with ultraviolet spectrophotometer, require the autoxidation rate-controlling at 0.030 (± 0.002) O.D./min.Press the unit of activity formula and calculate, promptly.
SOD unit of activity definition: under certain condition, in the reaction solution of 1ml, per minute suppresses the enzyme amount that pyrogallol autoxidation speed reaches at 50% o'clock and is defined as a unit of activity.
The unit of activity calculation formula:
Total activity (u)=unit volume vigor (u/ml) * stoste cumulative volume
2.GSH assay
Measure according to high performance liquid chromatography (" two ones of Chinese pharmacopoeia versions in 2000, appendix VD).
2.1 chromatographic condition and system suitability test
Chromatographic column: Shim-Pack CLC-ODS post (150mm * 6.0mm); Moving phase: 0.025mol/L sodium dihydrogen phosphate buffer (with phosphorus acid for adjusting pH value to 3.15); Detect wavelength: 220nm.Theoretical plate number is pressed the reduced glutathion peak and is calculated, and should be not less than 2000, with the resolution of adjacent peak greater than 2.0.
2.2 the preparation precision of reference substance solution takes by weighing reduced glutathion reference substance 15mg, puts in the brown measuring bottle of 25ml, adds moving phase and makes the reference substance solution that every 1ml contains 0.6mg, promptly.
2.3 the preparation of need testing solution is got GSH sample liquid and is filtered through millipore filtration (0.45 μ m), discards filtrate just, gets subsequent filtrate as need testing solution.
2.4 assay method precision is respectively drawn reference substance solution and each 10 μ l of need testing solution, injects liquid chromatograph, the record color atlas is pressed external standard method with calculated by peak area, promptly.
2.5 the investigation of linearity and scope is by the reference substance solution of 2.2 following method preparations 0.16,0.32,0.48,0.64,5 different concns of 0.80mg/ml, the accurate reference substance solution 10 μ l that draw inject liquid chromatograph respectively, the record peak area.Experimental result sees Table 2, and typical curve is seen Fig. 5.
With reference substance concentration (mg/ml) is X-coordinate, and peak area is an ordinate zou, carries out linear regression with method of least squares.Regression equation is: y=301.362x+3.6659 r=0.9997.The result shows that the gsh reference substance is good linear relationship with peak area between 0.16mg/ml～0.8mg/ml concentration range.
2.1 ox blood and sheep blood are compared test:
Get each two parts of fresh anti-freezing ox blood, sheep blood respectively, every part of 300ml, the centrifugal blood plasma that removes, physiological saline gets the about 100ml of clean red corpuscle after floating and washing, under identical experiment condition, by getting supernatant liquor after traditional extraction process haemolysis, ethanol chloroform precipitation, the thermally denature, measure supernatant liquor SOD vigor and GSH content respectively, the results are shown in Table 3.
Data are as can be seen from table 3: when from ox blood and sheep blood, extracting SOD and GSH with Same Way, and the SOD total activity of gained supernatant liquor and GSH total amount, ox blood is slightly higher than sheep blood, but all more approaching.
2.2 the source to sheep blood compares test:
Get each two parts of fresh anti-freezing sheep blood, Sanguis Naemorhedi respectively, every part of 300ml, the centrifugal blood plasma that removes, physiological saline gets the about 100ml of clean red corpuscle after floating and washing, under identical experiment condition, by getting supernatant liquor after traditional extraction process haemolysis, ethanol chloroform precipitation, the thermally denature, measure its SOD vigor and GSH content respectively, the results are shown in Table 4.
Data as can be seen from table 4: when extracting SOD and GSH with Same Way from sheep blood and Sanguis Naemorhedi, the SOD total activity and the GSH total amount of gained supernatant liquor do not have notable difference.
(2) lab scale test-results
The foregoing description gained superoxide-dismutase and reduced glutathion are carried out following test: measure the SOD vigor with the 325nm method, measure protein content with the Lowry method, measure GSH content with the HPLC method, the laboratory lab scale the results are shown in Table 5, table 6.
(3) pilot plant test
Three batch samples of the foregoing description gained superoxide-dismutase and reduced glutathion are tested, and three batches of pilot scales the results are shown in Table 7, table 8, table 9.
Data as can be seen from table 9: extract SOD and GSH simultaneously with present method from sheep blood, the yield and the quality of three batch samples are more stable, wherein three batches of GSH samples all can reach 92%, than reference Tao Rui, Wu Wutong, permitted that inspired (1999,6 (4): the enzyme process of 203) delivering prepares increasing of reporting in the research of gsh technology in the medicine biotechnology.
Table 1 325nm method application of sample table (Tab1 325nm operation method)
Table 2 linear relationship is investigated experimental result
| Reference substance concentration mg/ml | 0.16 | 0.32 | 0.48 | 0.64 | 0.80 |
| Peak area | 485672 | 972322 | 1459629 | 1901321 | 2432066 |
Table 3 ox, the comparison test of sheep blood
| Supernatant liquor volume (ml) | Vigor (U/ml) | Total activity (U) | GSH content (μ g/ml) | GSH total amount (mg) | |
| Ox blood 1 | 108 | 367.2 | 39657.6 | 346.7 | 37.4 |
| Ox blood 2 | 104 | 384.5 | 39988.0 | 353.5 | 36.8 |
| Sheep blood 1 | 109 | 349.3 | 38073.7 | 335.4 | 36.6 |
| Sheep blood 2 | 106 | 362.8 | 38456.8 | 336.7 | 35.7 |
Table 4 sheep, Sanguis Naemorhedi comparison test
| Supernatant liquor volume (ml) | Vigor (U/ml) | Total activity (U) | GSH content (μ g/ml) | GSH total amount (mg) | |
| Sheep blood 1 | 104 | 357.8 | 37211.2 | 349.8 | 36.4 |
| Sheep blood 2 | 106 | 347.3 | 36813.8 | 334.6 | 35.5 |
| Sanguis Naemorhedi 1 | 102 | 357.6 | 36475.2 | 346.5 | 35.3 |
| Sanguis Naemorhedi 2 | 107 | 351.4 | 37599.8 | 326.2 | 34.9 |
Table 5 lab scale testing data is summed up
| Testing sequence | Liquor capacity (ml) | SOD total activity (U) | The SOD rate of recovery (%) | GSH total amount (mg) | The GSH rate of recovery (%) |
| Behind the haemolysis | 400ml | 45820 | - | 53.1 | - |
| Behind the thermal change | 197ml | 42682 | 93.15 | 48.7 | 91.71 |
| After the ultrafiltration | 40ml | 35773 | 78.07 | 42.6 | 80.23 |
| Behind the secondary thermal change | 60ml | 34015 | 74.24 | - | - |
| After the cationic resin column | 60ml | - | - | 26.1 | 49.15 |
| Behind the resin anion(R.A) post | 60ml | - | - | 14.4 | 27.12 |
Table 6 lab scale test final data
| SOD total activity (U) | SOD Tot Prot (mg) | SOD weight (mg) | Protein content (%) | SOD is than live (U/mg) | GSH weight (mg) | GSH content (%) |
| 33979 | 7.2 | 8.3 | 86.75 | 4719 | 8.9 | 91.52 |
Table 7 pilot plant test SOD data
| Lot number | Sample one | Sample two | Sample three |
| Sheep blood charging capacity (L) | 50 | 50 | 50 |
| Hemolysate total activity (ten thousand U) | 3179.52 | 3084.02 | 3166.19 |
| Total activity behind the thermal change (ten thousand U) | 2717.21 | 2771.30 | 2734.04 |
| Yield (%) | 85.46 | 89.86 | 86.35 |
| Total activity after the ultrafiltration (ten thousand U) | 2202.77 | 2259.64 | 2254.32 |
| Yield (%) | 69.28 | 73.27 | 71.20 |
| Total activity behind the secondary thermal change (ten thousand U) | 1959.54 | 2043.16 | 2049.16 |
| Yield (%) | 61.63 | 66.25 | 64.72 |
Table 8 pilot plant test GSH data
| Lot number | Sample one | Sample two | Sample three |
| Hemolysate total amount (g) | 10.2713 | 10.0880 | 9.9565 |
| Total amount behind the thermal change (g) | 9.1488 | 8.7268 | 8.7858 |
| Yield (%) | 89.07 | 86.51 | 88.24 |
| Total amount after the ultrafiltration (g) | 7.6440 | 7.4913 | 7.2433 |
| Yield (%) | 74.42 | 74.26 | 72.75 |
| Total amount after the cationic resin column (g) | 4.4183 | 4.5758 | 4.4235 |
| Yield (%) | 43.02 | 45.36 | 44.43 |
| Total amount behind the resin anion(R.A) post (g) | 2.4785 | 2.5603 | 2.5898 |
| Yield (%) | 24.13 | 25.38 | 26.01 |
Test agent censorship data in the table 9
| Lot number | Sample one | Sample two | Sample three |
| SOD example weight (g) | 4.6765 | 4.3570 | 4.5418 |
| Protein content (%) | 85.3 | 89.1 | 89.6 |
| SOD is than live (U/mg) | 3839 | 4161 | 4179 |
| GSH example weight (g) | 1.4035 | 1.5203 | 1.5673 |
| GSH content (%) | 92.1 | 93.5 | 92.2 |
Claims (10)
1, a kind of superoxide-dismutase and reduced glutathion method of obtaining from animal blood is characterized in that carrying out according to the following steps:
Handle early stage: obtain solution of red blood cells through centrifugation after adding Trisodium Citrate earlier in fresh animal blood, secondly in solution of red blood cells, add deionized water and obtain hemolysate, in hemolysate, add the centrifugal supernatant liquor that obtains behind the solution of copper sulfate and zinc sulfate then, at last supernatant liquor is obtained concentrated solution with the hollow-fibre ultrafiltration device ultrafiltration and through liquid;
Extract superoxide-dismutase: centrifugally after handling the solution that adds copper sulfate and zinc sulfate in the gained concentrated solution above-mentioned early stage obtain containing the superoxide-dismutase supernatant liquor, will contain the superoxide-dismutase supernatant liquor and obtain containing the superoxide-dismutase concentrated solution through the hollow-fibre ultrafiltration device ultrafiltration;
Extract reduced glutathion: see through and obtain containing the reduced glutathion elutriant through 732 cation exchange resin columns and 717 anion-exchange resin columns after liquid concentrates handling gained above-mentioned early stage, will contain the reduced glutathion elutriant and concentrate and obtain containing the reduced glutathion concentrated solution.
2, superoxide-dismutase and the reduced glutathion method of obtaining from animal blood according to claim 1 is characterized in that early stage, processing was carried out as follows:
It is 3.8% to 4.2% Trisodium Citrate that every liter of fresh animal blood adds 125 milliliters to 170 milliliters, concentration, leave 10 minutes to 20 minutes separating red corpuscles of the heart with per minute 3000, add volume again and be the physiological saline Washed Red Blood Cells solution 1 time to 3 times of 1 times to 2 times of solution of red blood cells volume;
Then with solution of red blood cells at 0 ℃ of following ice bath, and to add volume be the deionized water of 2 times to 4 times of solution of red blood cells, stirs haemolysis and obtained hemolysate in 30 minutes;
Add and account for the copper sulfate of hemolysate cumulative volume 1.0% to 10.0% and the aqueous solution of zinc sulfate, the concentration of copper-bath is 5% to 0.5%, the concentration of solution of zinc sulfate is 7% to 0.7%, the consumption of copper sulfate and solution of zinc sulfate is 1: 2 to 2: 1 by volume, temperature is controlled between 55 ℃ to 80 ℃, heats 10 minutes to 20 minutes, is cooled to room temperature, leave the heart 10 minutes to 30 minutes with per minute 3000, obtain supernatant liquor;
Supernatant liquid filtering is removed fine particle, be the hollow-fibre ultrafiltration device ultrafiltration of 5KD to 15KD again through molecular weight cut-off, the control import is pressed between the 15psi to 30psi, the maintenance reflux pressure is 10psi, promptly keeping pressure difference is 5psi to 20psi, be concentrated into 10% to 20% of supernatant liquor volume, obtain concentrated solution and see through liquid.
3, superoxide-dismutase and the reduced glutathion method of obtaining from animal blood according to claim 1 and 2 is characterized in that extracting superoxide-dismutase and carries out as follows:
Add 1 times of deionized water dilution in the gained concentrated solution with handling above-mentioned early stage to 3 times of volumes, adding volume again is the copper sulfate of diluent cumulative volume 1% to 20% and the aqueous solution of zinc sulfate, the concentration of copper-bath is 5% to 0.5%, solution of zinc sulfate concentration is 7% to 0.7%, the consumption volume ratio of copper sulfate and solution of zinc sulfate is 1: 2 to 2: 1, stir evenly, water bath heat preservation is 10 minutes to 20 minutes between 55 ℃ to 80 ℃, be cooled to room temperature, left the heart 10 minutes to 30 minutes with per minute 3000, collection contains the superoxide-dismutase supernatant liquor;
To contain the superoxide-dismutase supernatant liquor is the hollow-fibre ultrafiltration device ultrafiltration of 5KD to 15KD through molecular weight cut-off, the control import is pressed between the 15psi to 30psi, the maintenance reflux pressure is 10psi, promptly keeping pressure difference is 5psi to 20psi, be concentrated into and contain 10% to 20% of superoxide-dismutase supernatant liquor volume, obtain containing for the first time the superoxide-dismutase concentrated solution;
Get and contain for the first time the superoxide-dismutase concentrated solution, through the hollow-fibre ultrafiltration device dynamic dialysis of 5KD to 15KD 12 hours, per minute 3000 left the heart 10 minutes, removed insoluble protein, must contain the superoxide-dismutase dialyzate;
To contain the superoxide-dismutase dialyzate is the hollow-fibre ultrafiltration device ultrafiltration of 5KD to 15KD through molecular weight cut-off, the control intake pressure is between 15psi to 30psi, the maintenance return pressure is 10psi, promptly keeping pressure difference is 5psi to 20psi, be concentrated into and contain 10% to 20% of superoxide-dismutase dialysate volumes, obtain containing for the second time the superoxide-dismutase concentrated solution;
Add 1 times to the 2 times pre-cold acetone of volume with containing the superoxide-dismutase concentrated solution second time, placed 1 hour to 2 hours, per minute 3000 leaves the heart and must precipitate in 10 minutes to 20 minutes;
Precipitate the green lyophilized powder of dry light green or pale blue and contain superoxide-dismutase.
4, superoxide-dismutase and the reduced glutathion method of obtaining from animal blood according to claim 1 and 2 is characterized in that extracting reduced glutathion and carries out as follows:
See through liquid 40 ℃ to 50 ℃ of bath temperatures with handling gained above-mentioned early stage, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, and cycles of concentration is 5 times to 15 times, obtains containing for the first time the reduced glutathion concentrated solution;
Use sulfuric acid adjust pH to 2 to 4 with containing the reduced glutathion concentrated solution first time, last 732 cation exchange resin columns, 732 resin cation (R.C.) order numbers are 60 order to 80 orders, the elutriant ammonia concn is 0.25 mol to 1.0 mol, the elutriant ammonia flow rate be 0.5 to 1.5 times of resin volume/hour, obtain containing for the first time the reduced glutathion elutriant;
To contain the reduced glutathion elutriant first time 40 ℃ to 50 ℃ of bath temperatures, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, and cycles of concentration is 5 times to 15 times, obtains containing for the second time the reduced glutathion concentrated solution;
Use sulfuric acid adjust pH to 4 to 5 with containing the reduced glutathion concentrated solution second time, last 717 anion-exchange resin columns, 717 resin anion(R.A) order numbers are 60 order to 80 orders; The elutriant sulfuric acid concentration is 0.25 mol to 1.0 mol; Elutriant sulfuric acid flow velocity is 0.5 to 2.0 times of resin volume/h, obtains containing for the second time the reduced glutathion elutriant;
With containing for the second time the reduced glutathion elutriant molecular weight of packing into is 12 hours desalinations of dialysis in 100 the dialysis tubing, obtains containing the reduced glutathion dialyzate;
To contain the reduced glutathion dialyzate 40 ℃ to 50 ℃ of bath temperatures, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, and cycles of concentration is 5 times to 15 times, is contained the reduced glutathion concentrated solution for the third time;
To contain the reduced glutathion concentrated solution for the third time and add 1 times to 2 times pre-cooled ethanol, place 1 hour to 2 hours, and leave the heart with per minute 3000 and obtained precipitation in 10 minutes to 20 minutes;
The precipitation drying obtains reduced glutathion white lyophilized powder.
5, superoxide-dismutase and the reduced glutathion method of obtaining from animal blood according to claim 3 is characterized in that extracting reduced glutathion and carries out as follows:
See through liquid 40 ℃ to 50 ℃ of bath temperatures with handling gained above-mentioned early stage, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, and cycles of concentration is 5 times to 15 times, obtains containing for the first time the reduced glutathion concentrated solution;
Use sulfuric acid adjust pH to 2 to 4 with containing the reduced glutathion concentrated solution first time, last 732 cation exchange resin columns, 732 resin cation (R.C.) order numbers are 60 order to 80 orders, the elutriant ammonia concn is 0.25 mol to 1.0 mol, the elutriant ammonia flow rate be 0.5 to 1.5 times of resin volume/hour, obtain containing for the first time the reduced glutathion elutriant;
To contain the reduced glutathion elutriant first time 40 ℃ to 50 ℃ of bath temperatures, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, and cycles of concentration is 5 times to 15 times, obtains containing for the second time the reduced glutathion concentrated solution;
Use sulfuric acid adjust pH to 4 to 5 with containing the reduced glutathion concentrated solution second time, last 717 anion-exchange resin columns, 717 resin anion(R.A) order numbers are 60 order to 80 orders; The elutriant sulfuric acid concentration is 0.25 mol to 1.0 mol; Elutriant sulfuric acid flow velocity is 0.5 to 2.0 times of resin volume/h, obtains containing for the second time the reduced glutathion elutriant;
With containing for the second time the reduced glutathion elutriant molecular weight of packing into is 12 hours desalinations of dialysis in 100 the dialysis tubing, obtains containing the reduced glutathion dialyzate;
To contain the reduced glutathion dialyzate 40 ℃ to 50 ℃ of bath temperatures, vacuum tightness is to concentrate under the condition of 0.07Mpa to 0.08Mpa, and cycles of concentration is 5 times to 15 times, is contained the reduced glutathion concentrated solution for the third time;
To contain the reduced glutathion concentrated solution for the third time and add 1 times to 2 times pre-cooled ethanol, place 1 hour to 2 hours, and leave the heart with per minute 3000 and obtained precipitation in 10 minutes to 20 minutes;
The precipitation drying obtains reduced glutathion white lyophilized powder.
6, according to described superoxide-dismutase and the reduced glutathion method of from animal blood, obtaining of claim 3, it is characterized in that the dry freeze-drying that adopts, drying chamber pressure is 0.8Mpa to 0.9Mpa, and bed thickness is 1cm, and freezing temp is-40 ℃.
7, according to described superoxide-dismutase and the reduced glutathion method of from animal blood, obtaining of claim 4, it is characterized in that the dry freeze-drying that adopts, drying chamber pressure is 0.8Mpa to 0.9Mpa, and bed thickness is 1cm, and freezing temp is-40 ℃.
8, according to described superoxide-dismutase and the reduced glutathion method of from animal blood, obtaining of claim 5, it is characterized in that the dry freeze-drying that adopts, drying chamber pressure is 0.8Mpa to 0.9Mpa, and bed thickness is 1cm, and freezing temp is-40 ℃.
9,, it is characterized in that fresh animal blood adopts fresh sheep blood or fresh ox blood or fresh cattle and sheep to mix blood according to claim 1 or 2 described superoxide-dismutase and the reduced glutathion methods of from animal blood, obtaining.
10, described according to Claim 8 superoxide-dismutase and the reduced glutathion method of obtaining from animal blood is characterized in that fresh animal blood adopts fresh sheep blood or fresh ox blood or fresh cattle and sheep to mix blood or fresh pig blood or fresh duck blood or fresh human blood.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008103042551A CN101338305B (en) | 2008-08-28 | 2008-08-28 | Process for extracting superoxide dismutase and reduction glutathion from animal blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008103042551A CN101338305B (en) | 2008-08-28 | 2008-08-28 | Process for extracting superoxide dismutase and reduction glutathion from animal blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101338305A true CN101338305A (en) | 2009-01-07 |
| CN101338305B CN101338305B (en) | 2010-12-22 |
Family
ID=40212475
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008103042551A Expired - Fee Related CN101338305B (en) | 2008-08-28 | 2008-08-28 | Process for extracting superoxide dismutase and reduction glutathion from animal blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101338305B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250851A (en) * | 2011-08-10 | 2011-11-23 | 天津宝迪农业科技股份有限公司 | Novel process for extracting superoxide dismutase SOD from animal blood cell liquid |
| CN103361320A (en) * | 2013-07-25 | 2013-10-23 | 天津核生科技生物工程有限公司 | Method of extracting SOD from milk |
| CN105506060A (en) * | 2015-12-30 | 2016-04-20 | 海口奇力制药股份有限公司 | Method for measuring content of rhCNB (Recombinant Human Calcineurin B) |
| CN113564265A (en) * | 2021-08-27 | 2021-10-29 | 浙江省农业科学院 | Method for identifying age of duck based on quantitative PCR |
-
2008
- 2008-08-28 CN CN2008103042551A patent/CN101338305B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250851A (en) * | 2011-08-10 | 2011-11-23 | 天津宝迪农业科技股份有限公司 | Novel process for extracting superoxide dismutase SOD from animal blood cell liquid |
| CN103361320A (en) * | 2013-07-25 | 2013-10-23 | 天津核生科技生物工程有限公司 | Method of extracting SOD from milk |
| CN103361320B (en) * | 2013-07-25 | 2014-06-18 | 天津核生科技生物工程有限公司 | Method of extracting SOD from milk |
| CN105506060A (en) * | 2015-12-30 | 2016-04-20 | 海口奇力制药股份有限公司 | Method for measuring content of rhCNB (Recombinant Human Calcineurin B) |
| CN105506060B (en) * | 2015-12-30 | 2017-05-24 | 海口奇力制药股份有限公司 | Method for measuring content of rhCNB (Recombinant Human Calcineurin B) |
| CN113564265A (en) * | 2021-08-27 | 2021-10-29 | 浙江省农业科学院 | Method for identifying age of duck based on quantitative PCR |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101338305B (en) | 2010-12-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11111317B2 (en) | Cordyceps militaris medium polysaccharide, method for separating and purifying same, and use thereof | |
| Himmelhoch et al. | Purification of myeloperoxidases from bone marrow | |
| CN101508715B (en) | Extraction and purification process for cordycepin in cordyceps militaris link | |
| CN101338305B (en) | Process for extracting superoxide dismutase and reduction glutathion from animal blood | |
| CN103288953B (en) | Method for separating and purifying functional protein in plasma | |
| CN108524929A (en) | A kind of production method of rabies vacciness | |
| CN104447892A (en) | Arginine-fructosyl-glucose detection method and medical application thereof | |
| CN107011433A (en) | A kind of phycocyanin of the extraction of nostoc phycobniliprotein, purification process and purifying | |
| CN102838684A (en) | Separating and purifying process of isochrysis galbana exopolysaccharide | |
| CN1749274B (en) | Separating and purifying method for new recombinant human interferon alpha2b | |
| CN102495169B (en) | Purifying and analyzing identification method for anti-oxidative peptide after controlled-enzymatic hydrolysis of laver | |
| CN117512031A (en) | Purification method of pneumococcal capsular polysaccharide | |
| CN102453087B (en) | Method for purification and preparation of mono-substituted PEG-EPO (polyethylene glycol-erythropoietin) | |
| CN101914511B (en) | Method for preparing high-purity pancreatic kininogenase | |
| CN113398251B (en) | Recombinant human chorionic gonadotropin freeze-dried powder injection and preparation method thereof | |
| CN113480632B (en) | Purification process of recombinant protein rhCG expressed in CHO (Chinese hamster ovary) cells | |
| CN102319420B (en) | The application of turtle peptide in pharmacy | |
| CN101481403A (en) | Double aqueous phase solid-liquid separation method for yeast origin recombinant protein fermentation liquor | |
| CN111763243B (en) | Gorgon fish immune active peptide and preparation method and application thereof | |
| CN100528175C (en) | Deporteinnized calf serum injection and its preparing method | |
| CN104450848A (en) | Milk micro-molecular biological active peptide and preparation method thereof | |
| CN106635986B (en) | Ginseng polysaccharide for promoting proliferation and differentiation of CIK cells, culture medium, culture method and application | |
| CN100417390C (en) | Effective part of sweet wormwood, its preparation method and application thereof in pharmacy | |
| CN109824561B (en) | Extraction and detection method of L-cysteine in rice seedling tissue | |
| EP0175267A2 (en) | Testing agent for neutrophil function |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20101222 |