CN1020734C - Method for extracting superoxide-dismutase - Google Patents

Abstract

Blood from human and animals is used as a raw material, a method of anticoagulation, hemocyte washing, hemolysis, the addition of ethyl alcohol and chloroform to remove haemoglobin, underpressure distillation to remove the ethyl alcohol and the chloroform, ph value regulation to an isoelectric point, cold acetone precipitation, acetone dehydration and ether evacuation is used for simultaneously extracting superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSHPx) from erythrocytes, leucocytes and platelets. Thus, the total activity loss of the SOD is low, and the SOD maintains various small molecular antioxidants, such as selenium, cystine and cysteine, multiple vitamins, trace elements, amino acids, etc. The SOD compound enzyme not only can be used in medicine, but also can be used as an additive in various foods and cosmetics.

Description

Method for extracting superoxide-dismutase

The present invention relates to a kind of being used for from the technical process and the working method of people and multiple animal blood extraction superoxide-dismutase.

Superoxide-dismutase (Superoxide Dismutase is hereinafter to be referred as SOD) extensively is present in people and multiple animal blood and the tissue, since 1969 are found, has been extracted medicinal.The mechanism of superoxide radical from SOD removing human body, it also needs catalase (CAT), Selenoperoxidase (GSHPx) synergy.We the mixture that contains SOD and CAT, GSHPx simultaneously be called the SOD mixture, experimentation on animals shows, the simple SOD of the result of treatment of SOD compound as antisenility will get well.And the existing both at home and abroad method of extracting SOD, as the isopropanol precipitating method; Mantoquita precipitation foreign protein, ion exchange method and hyperconcentration, gel filtration method; Ethanol-trichloromethane precipitator method etc. remove and have destroyed CAT and GSHPx in leaching process.Therefore, need a kind of method of from blood, extracting the SOD mixture.China's " medicine industry " magazine 1983.(4): 4 and 1985.(16): the method for the 9 pure enzymes of introducing of extraction SOD, product neither the SOD mixture in the middle of it.Through to the retrieval of 1964-1988 world patent document (WPI) and non-patent literature (GAR), Methodsen Enzymolog 1978.108(1): the method for the pure enzyme of multiple extraction SOD introduced in literary compositions such as 51, but do not see the introduction that extraction SOD mixture method is arranged.

Task of the present invention provides a kind of method of extracting the SOD mixture from humans and animals blood.Its extract contains SOD and CAT, GSHPx simultaneously, makes the total activity loss of SOD few, and keeps multiple small molecules oxidation inhibitor (as selenium, Gelucystine, halfcystine) and multivitamin, trace element, amino acid etc.This SOD mixture not only can be done medicinal, also can be used for numerous food and makeup are made additive.It is with short production cycle, operates easy relatively.Can be widely used in industrial production, bird, packing house all can adopt.Simultaneously because processing treatment the blood that went out of use originally, can reduce environmental pollution.

Can solve in order to following method by this task of the present invention:

2. Production Flow Chart 1 used antithrombotics is prepared as using anhydrous citric acid sodium, and concentration is 3.13%; As preparing with the Sodium Citrate that contains 2 molecular crystal water, concentration is 3.8%.All should adjust potential of hydrogen to PH6.4-7.6 before using.

3. Production Flow Chart 2 used concentration of sodium chloride solution are 2%-5%, rather than 0.9% sodium chloride solution.

4. the used deionized water of Production Flow Chart 3 should precooling; Mix to be placed in-5 ℃ of-0 ℃ of refrigeratores with hemocyte and spend the night, the hemocyte complete swelling is broken, to improve the quality product and the yield of SOD mixture.

5. when Production Flow Chart 4 added with pre-cooled ethanol and trichloromethane, by volume ratio added respectively, the vigorous stirring while adding.As add excessive velocities and make partial concn too high, can cause inactivation and other protein denaturation of enzyme.Before feeding intake, Ying Yu starts electric blender.It is enough that churning time and speed are wanted, and prevents that bulk or coarse particles shape thing from forming.

6. ethanol and the trichloromethane in the extract removed in the underpressure distillation of Production Flow Chart 5, and condition is, extracting speed is 0.8-1.2 liter/second, and distillation temperature is 30 ℃-45 ℃, lasting 2-4 hour.

7. Production Flow Chart 6 used acetone are chilled to-15 ℃ in advance--20 ℃, slowly add in the extract.

8. in whole leaching process, the temperature of extracting solution is controlled at below 10 ℃ except that the link that specially adjusts.If can be controlled at about 0 ℃, then better.

9. in whole leaching process, the potential of hydrogen of extracting solution is controlled at PH6-9 except that the link that specially adjusts.

10. Production Flow Chart 9 adds diethyl ether when draining, and churning time is about 1 minute.Drain speed, as in 1 liter of vacuum vessel, the speed of evacuation is 0.5 liter/second-1.0 liters/second, and highest attainable vacuum is 5 * 10 ^-4Holder.

11. Production Flow Chart 4,7,8,9 adds centrifugal behind the organic solvents, uses refrigerated centrifuge.Temperature in the tophan pot is-5 ℃-0 ℃.Centrifugation time is 2-4 minute, and rotating speed is 1500 rev/mins-2500 rev/mins.

One. Production Flow Chart

1. take hemocyte.Get Freshman or animal blood, add antithrombotics,, discard blood plasma, keep sedimentary hemocyte 0 ℃ of-4 ℃ of standing over night.

2. washing hemocyte.Add 2-5 2% sodium chloride solution doubly, mixing, the protometrocyte suspension is with 1500 rev/mins-3500 rev/mins centrifugal back abandoning supernatant.Repeat once to add chlorination sodium solution, centrifugal, the process of abandoning supernatant liquor again, become clean hemocyte.(comprising red corpuscle, white corpuscle, thrombocyte)

3. haemolysis.Add deionized water, stir, refrigeration is spent the night (or 8-10 hour), and the hemocyte swelling is broken.

4. removal oxyphorase.Add ethanol and trichloromethane, and vigorous stirring, centrifugal afterwards, abandon precipitation, get its supernatant liquid filtering, be weak yellow liquid, add hydrochloric acid, transfer potential of hydrogen near SOD iso-electric point (PH5.6-6.8), metered volume, put preserve in-5 ℃ of-0 ℃ of refrigerators standby.

5. remove ethanol and trichloromethane.The extract of Production Flow Chart 4 preparations is placed the underpressure distillation device, be heated to 30 ℃-45 ℃,, do not contained the SOD mixture extract of ethanol and trichloromethane simultaneously with the vacuum pump decompression.This liquid is concentrated with ultra-fine filter, obtain the SOD mixture and concentrate extract, adjust potential of hydrogen near SOD iso-electric point (PH5.6-6.8) with 4N hydrochloric acid then.

6. sediment composite.Add cold acetone, constantly stir, precipitation occurs, become milky suspension, leave standstill a moment, centrifugal, abandon supernatant liquor, slough part moisture, stay its throw out.

7. preliminary hydro-extraction.It is an amount of to add 75% acetone, centrifugal, abandons supernatant liquor, sloughs part moisture, stays its throw out.

8. dehydration again.It is an amount of to add pure acetone, centrifugal, abandons supernatant liquor, continues dehydration, stays its throw out.

9. drain.It is an amount of to add absolute ether, finishes dehydration, drains, and is the SOD composite product.

Two. production technique

1. the blood source must be fresh, avoids taking place haemolysis and blood coagulation; Must not sneak into animal gastric content and other foreign material in the blood source.

12. when adding SOD mixture suspension in vacuum vessel, amount is wanted suitably at every turn, with do not lump, not agglomerating degree of being.

The method of the formed extraction of above Production Flow Chart and technology SOD mixture is compared with the method for the pure product of extraction SOD of bibliographical information, saves reagent, improves the product yield, reduces environmental pollution, and helps the recovery of acetone and other organic solvent.Its characteristics have:

1. when being raw material with humans and animals blood, can extract the SOD mixture in red corpuscle, white corpuscle, the thrombocyte simultaneously.

2. when hemocyte is washed, not with 0.9% sodium-chlor (physiological saline) washing 3 times, but wash 1 time with the 2%-5% sodium chloride solution.

4. the haemolysis method is not to use the hypotonic swelling method of deionized water merely, but comprehensively uses hypotonic, low temperature, merges the mechanical means that ice crystal punctures, and makes the molten of hemocyte break once journey fast, fully.

4. adding organic solvent, make the oxyphorase sex change, when removing oxyphorase, the method for the pure product of extraction SOD of bibliographical information is at room temperature to carry out, and present method is to stir down at low temperature (5 ℃-0 ℃), and the oxyphorase denaturation process is accelerated.

5. saved and in extract, added potassium primary phosphate, the operation that the layering post is analysed.

6. the application of reduced pressure distillation method is removed ethanol and the trichloromethane in the extract, is that the method for the pure product of extraction SOD of bibliographical information is unexistent.

7. add acetone and make the sedimentary operation of enzyme, the method for the pure product of extraction SOD of bibliographical information is to carry out about 0 ℃, and present method is at-20 ℃--10 ℃ of conditions are carried out; And before adding acetone, adjust near the potential of hydrogen (PH5.6-6.8) iso-electric point of extract earlier.

8. add 75% acetone and slough part moisture and add pure acetone continuation dehydration again, drain with ether, by present method is initiated.

Embodiment

Extract the production technique of SOD mixture and the example of flow process from ox blood

1. get 50 kilograms of fresh ox bloods, add 6600 milliliters of 3.8% liquor sodii citratises immediately, stir gently, note preventing haemolysis and blood coagulation, get fresh anti-freezing ox blood.

2. anticoagulation is added a cover and place 2L4,2L3 upright freezer, transfer to 2 ℃ of-4 ℃ of standing over night, the hemocyte natural sedimentation.Inhale with YB-DX23 type hand-push electrical suction pump then and abandon the faint yellow blood plasma in upper strata, keep throw out.

3. slowly add 3 times of 2% sodium chloride solution along the bucket wall, stir gently, become uniform blood cell suspension.

4. blood cell suspension is added in the tophan pot, by 3500 rev/mins centrifugal 8 minutes, inhale and to abandon supernatant liquor.

5. by above-mentioned 3,4 liang of step repeated washings once, get hemocyte totally.

6. the blood cell volume after metering is washed, by the deionized water of equal volume amounts adding precooling, vigorous stirring 30 minutes is destroyed hemocyte.Place-5 ℃ of-0 ℃ of refrigeratores to spend the night then.Make the hemocyte completely destroy by refrigeration, get hemolysate.

7. the precooling hemolysate is taken out, by volume add precooling 95% ethanol, vigorous stirring, mixing adds the precooling trichloromethane again, places the electronic agitation vat vigorous stirring of stainless steel 30 minutes, becomes the chocolate mashed prod.Proportioning is:

Hemolysate: 95% ethanol: trichloromethane=1: 0.25: 0.15

8. above-mentioned mashed prod is added in the tophan pot, with 3500 rev/mins of centrifugations 15 minutes, mashed prod promptly was divided into three layers.Be respectively from top to bottom: SOD mixture extract, denatured hemoglobin, the reacted trichloromethane of participation.

9. supernatant liquor part (being SOD mixture extract) in the tophan pot is used the B decompress filter, get faint yellow limpid transparent SOD mixture extract.Behind sampling censorship SOD vigor and protein concentration, accurately measure SOD mixture extract volume, place-15 ℃ of refrigeratores to spend the night, standby.

10. will refrigerate standby extract and place the underpressure distillation device, being heated to the extract temperature with the 600W electric furnace is 40 ℃, and with the vacuum pump decompression, extracting speed is 1 liter/second, continues 4 hours, is not contained the SOD mixture extract of ethanol and trichloromethane simultaneously.This liquid is concentrated with ultra-fine filter, obtain the SOD mixture and concentrate extract, adjust potential of hydrogen to PH6.6 with 4N hydrochloric acid then.

11. SOD mixture extract is placed the electronic agitation vat of stainless steel, actuating motor, 1.2 times that press the extract volume slowly add-20 ℃ of industrial acetones, occur a large amount of white depositions in the bucket.

12. leave standstill a moment, with the freezing low speed centrifuge of FL.O6D large vol,, centrifugal 4 minutes with 2000 rev/mins at-5 ℃.

13. supernatant liquor is collected in the porcelain enamel barrel, makes acetone and reclaim usefulness.Rapidly 50 milliliters in 75% acetone is added in each tophan pot with quantitative charger, start the combination motor stirrer and constantly stir, make it to become the even muddy thing of fine particulate, place-5 ℃ of tophan potes, centrifugal 4 minutes with 2000 rev/mins.

14. supernatant liquor is collected in the porcelain enamel barrel, makes acetone and reclaim usefulness.Add 50 milliliters in 100% acetone rapidly with quantitative charger, start the combination motor stirrer and stir, progressively dehydration becomes the even muddy thing of white fine particulate, places-5 ℃ of tophan potes, and is centrifugal 4 minutes with 2000 rev/mins.

15. supernatant liquor is collected in the porcelain enamel barrel, makes acetone and reclaim usefulness.Fast with quantitative charger with 50 milliliters of absolute ethers, start the combination motor stirrer and stir rapidly, progressively dehydration becomes the even muddy thing of fine particulate, places-5 ℃ of tophan potes, and is centrifugal 4 minutes with 2000 rev/mins.

16. supernatant liquor is collected in the porcelain enamel barrel, makes ether and reclaim usefulness.Add 80 milliliters of ether, stir rapidly, promptly become the fine particulate thing, pour into immediately in 2500 milliliters of vacuum vessels, place on the Kang Shi shaker mixer, use 2XZ-1 type (or 2XZ-0.5 type and 2XZ-2 type) rotary-vane vaccum pump, the decompressing and extracting while vibrating is SOD mixture finished product.

Claims (6)

1, a kind of from humans and animals blood extraction superoxide-dismutase, the method that comprises superoxide-dismutase (SOD) in red corpuscle, white corpuscle, the thrombocyte, catalase (CAT), Selenoperoxidase (GSHPx), it is characterized in that, employed technical process is that (1) uses the 3.13%-3.8% liquor sodii citratis to blood anticoagulant; (2) use 2%-5% sodium chloride solution washing hemocyte; (3) comprehensively use hypotonic, low temperature, vigorous stirring, merge the mechanical means that ice crystal punctures and finish haemolysis; (4) add ethanol and trichloromethane and remove oxyphorase; (5) ethanol and trichloromethane are removed in underpressure distillation; (6) ultrafiltration and concentration; (7) near the potential of hydrogen (PH5.6-6.8) iso-electric point of adjustment extract, at-20 ℃--add acetone under 10 ℃ of conditions, make the enzyme precipitation; (8) dewater with acetone; (9) ether is drained.

2, according to claim 1, it is characterized in that, before to blood anticoagulant, adjust the potential of hydrogen of antithrombotics earlier to PH6.4-7.6.

3, according to claim 1, it is characterized in that, adding organic solvent, make the oxyphorase sex change, when removing oxyphorase, is continuously stirring under-5 ℃-0 ℃ low temperature.

According to claim 1, it is characterized in that 4, application of reduced pressure distillation method, extracting speed are 0.8-1.2 liter/second, distillation temperature is 30 ℃-45 ℃, continues 2-4 hour, removes ethanol and trichloromethane.

5, according to claim 1, it is characterized in that, slough part moisture, add the product that pure acetone continues dehydration, the method drained with ether is obtained the SOD mixture again with 75% acetone.

According to claim 1, it is characterized in that 6, in the whole production flow process, the temperature of extracting solution all remains on below 10 ℃.