CN1616655A - Method for preparing superoxide dismutase - Google Patents
Method for preparing superoxide dismutase Download PDFInfo
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- CN1616655A CN1616655A CN 200410041723 CN200410041723A CN1616655A CN 1616655 A CN1616655 A CN 1616655A CN 200410041723 CN200410041723 CN 200410041723 CN 200410041723 A CN200410041723 A CN 200410041723A CN 1616655 A CN1616655 A CN 1616655A
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Abstract
The present invention discloses SOD preparing process. SOD is prepared with fresh pig blood, and through preparing coarse SOD and purifying the coarse SOD. The coarse SOD preparing process includes the steps of cold storage, cleaning, extraction, heating to change, extraction, precipitation, sterilization, dialysis to desalt and freeze drying. The coarse SOD purifying process includes dissolving, column chromatography, elution, dialysis and drying to obtain final product light blue and green CU.Zn.SOD, which has specific enzyme activity up to 3600-10000 U/mg.
Description
Affiliated technical field
The invention belongs to zymetology, be specifically related to the preparation method of superoxide-dismutase.
Background technology
SOD is " superoxide-dismutase ", writes a Chinese character in simplified form that " Superoxde dismutase is unique uncontested anti-ageing, the disease-resistance substance of generally acknowledging in the world.The oxide compound dismutase is a metalloid enzyme, extensively is present in the organism, and it is a kind of oxygen free radical scavenger, has the effect that the protection body is avoided damaging, and people, animal are all had no side effect.
The SOD superoxide-dismutase is a single-minded scavenging agent of removing human body excess oxygen free radical.Oxyradical is to quicken aging, produces the root of various diseases.In 20th century, scientists has proposed eight big theories of human body diseases aging, and free radical theory is central unique being proved and the most reliable a kind of theory, the elaboration that it is more clearly the process of organism disease aging.People's every minute all will be breathed, histoorgan is at every moment all carrying out oxygen metabolism, under the normal physiological situation, the generation and the removing of free radical are in running balance, when certain factor generates too much oxyradical or exceeds the removing ability or removing ability when weakening, too much oxygen free radical injury life macromolecule destroys the structure and the function of cell, cause the generation and the development of disease, thereby quicken organism aging process.Modern medicine is verified: the disease that oxyradical causes, surpassed kind more than 100, and the serious health that is threatening us is with long-lived.
SOD is the No.1 killer of oxyradical in the body, is the single-minded scavenging agent of scientific circles' oxyradical of proving, generally acknowledging both at home and abroad.Under the effect of SOD efficient catalytic, free radical is hydrogen peroxide and oxygen by disproportionation, under the coordinative role of associating scavenging agent SOD---CCP, makes it to become water and newborn oxygen, thereby thoroughly removes intravital excess oxygen free radical.At present, be widely used in fields such as medicine, health care, food, makeup.The commodity that gone on the market on the market spend one's remaining years in happiness SOD oral liquid, the anti-ageing wine of Ding Shi, big precious SOD honey etc.
The market that SOD uses mainly concentrates on three aspects, i.e. medicine, food and daily chemical products.Indication as medicinal application aspect SOD is a lot, the tissue fibrosis that causes as sacroiliitis, reperfusion injury syndromes, radiotherapy, burn and scald etc.But as medicinal present the exploitation of having carried out anti-curing arthritis and anti-radiotherapy damage effect aspect.In case as the new drug approval, the SOD preparation of other formulations and medication purpose will obtain exploitation very soon.Therefore, SOD has as medicinal great potential.Aspect foodstuff additive, be applied to milk, beer, beverage, lozenge etc., and it is directly oral to have made capsule.At present, the popularity rate that contains the SOD protective foods is also very low, in case masses have been familiar with its function, will produce bigger market.As the cosmetics additive application facet, consumption was the situation of rapid increase in recent years, because, SOD is used for various forms of makeup, it is sure brightens the welcome that is subjected to consumers in general with effect of dispelling spots deeply.SOD will continue to increase in the application of day chemical industry.SOD in other respects as the agricultural application also research among, each breakthrough can both cause its enormous and latent market.According in market every profession and trade demand and export volume prediction, can consume about 10,000,000,000,000 units of SOD every year.
At present, because the method for separation and Extraction is not suitable for scale operation, the production cost height, operational difficulty, SOD output can not be met the need of market far away.
Summary of the invention
The objective of the invention is to: the preparation method of superoxide-dismutase is provided, and this preparation method is easy, and is with low cost, suitable for mass production SOD.
Technical solution of the present invention is: this preparation method adopts the fresh pig clot to extract SOD, at first makes crude product SOD by fresh pig blood, and the purifying crude product makes elaboration SOD then.
The preparation process of crude product SOD includes: refrigeration, cleaning, extraction, thermal change, extraction, precipitation, degerming, dialysis desalination, lyophilize.
1, refrigeration: in fresh blood impouring container, place freezer refrigeration that it is lumpd naturally.
2, cleaning: clot is blended, place the flushing of filter screen water, clean filter and do, get the serum-free clot.
3, extract: the serum-free clot is put into container, add the triton x-100 solution of 0.2 times of amount 20% of serum-free clot, stirred 20-40 minute, suction filtration is collected blood plasma.Wherein, it is formulated that 20% triton x-100 solution adds 200 milliliters of triton x-100s by the 800ml pure water, triton x-100 another name emulsifying agent 0P, octyl group benzene Soxylat A 25-7.
4, thermal change: in blood plasma impouring retort, be heated to 65 ℃, under stirring condition isothermal reaction 20-40 minute, be cooled to room temperature, centrifugal collection supernatant liquor; Supernatant liquor is carried out the secondary thermal change, be heated to 65 ℃, constant temperature 20 minutes adds the 4g cupric chloride in the constant temperature phase by the former blood volume of 1kg, is cooled to 10-15 ℃, centrifugal collection supernatant liquor.
5, extraction: in supernatant liquor impouring container, the ethanol one chloroform mixed solution that adds supernatant liquor volume 2/5, wherein ethanol and chloroform volume ratio are 5: 3, stir several minutes, centrifugal collection filtrate is in another container, adjusting pH value is 7.6, under stirring condition, in the 43% ratio adding K of filtrate volume
2HP
4, adjusting pH value once more is 7.6, adds the ethanol one chloroform mixed solution of filtrate volume 1/5 again, after stirring, supernatant liquid is collected in layering in the impouring separating funnel.
6, precipitation: in above-mentioned clear liquid impouring container, add the cold acetone of 1 times of amount of clear liquid, stir, under 4-10 ℃ condition, left standstill centrifugal collecting precipitation 5-6 hour.
7, degerming: the trichloromethane with 2 times of amounts of throw out repeatedly washes throw out;
8, dialysis desalination: add 2g acetone preparation scavenging solution with 500ml distilled water, with scavenging solution to throw out towards Xian Duoci;
9, lyophilize: the throw out lyophilize behind the desalination of will dialysing must be crude product SOD.
The preparation of elaboration SOD includes: dissolving, post layer folding, wash-out, dialysis, drying:
1, dissolving: the SOD crude product of gained is dissolved in PH7.6,2.5 μ mmol/l, K
2HP
4-KH
2PO
4In the buffer reagent, get clear liquor through ultrafiltration.
2, post layer folding: with DEAEsepnadexA-50 chromatography on the clear liquor, last sample flow rate control the 100-200 milliliter/hour.
3, wash-out: go up sample after saturated, carry out gradient elution with same buffer reagent, control flow velocity and be the 100-200 milliliter/hour, sampling monitoring has the active protein peak of SOD.
4, dialysis: in the dialysis tubing of will above-mentioned collected SOD elutriant packing into, the desalination of in distilled water, dialysing, collection dialyzate.
5, drying: with the dialyzate ultrafiltration and concentration, lyophilize then, get final product the CuZn-SOD product of the little band pale bluish green of outward appearance.
The present invention gathers materials on the spot, and turns waste into wealth, and technology is simple, and is with low cost, product yield height, suitability for mass industrialized production.
Description of drawings
Fig. 1 is a crude product SOD preparation flow sketch of the present invention
Fig. 2 is an elaboration SOD preparation flow sketch of the present invention
Embodiment
Adopt fresh pig blood at first to make crude product SOD, the purifying crude product makes elaboration SOD then.
Crude product SOD preparation process is as follows:
1, refrigeration: get in the fresh blood 100kg impouring stainless steel cask, place 10 ℃ of freezer refrigerations 3-4 hour, it is lumpd naturally.
2, clean: clot is blended, place double-layer nylon cloth, elder generation cleans filter with distilled water again and does with tap water flushing 3 times, gets the serum-free clot.
3, extract: the serum-free clot is put into stainless steel cask, adds 20% triton x-100 solution of 0.2 times of amount of serum-free clot, and under 3000 rev/mins condition vigorous stirring 30 minutes, suction filtration is collected blood plasma.Wherein, adding 200 milliliters of triton x-100s with the 800ml pure water, to make concentration be 20% triton x-100 solution, and song draws the logical another name of X-100 polyoxyethylene nonylphenol ether, octyl phenyl Soxylat A 25-7.
4, thermal change: in blood plasma impouring stainless steel interlayer retort, be heated to 65 ℃, isothermal reaction is 30 minutes under stirring condition, is cooled to 25 ℃ of room temperatures then, centrifugal collection supernatant liquor; Supernatant liquor is carried out the secondary thermal change, 65 ℃ of constant temperature of temperature 20 minutes, the constant temperature phase adds the 400g cupric chloride, is cooled to 10 ℃, centrifugal collection supernatant liquor.
5, extraction: in supernatant liquor impouring stainless steel cask, the ethanol one chloroform mixed solution that adds filtrate volume 2/5, ethanol wherein: chloroform (V: V)=5: 3, stirred 5 minutes, centrifugal collection filtrate is in another stainless steel cask, adjusting pH value is 7.6, under stirring condition, in the 43% ratio adding K of filtrate volume
2HP
4, adjusting pH value once more is 7.6, adds the ethanol one chloroform mixed solution of filtrate volume 1/5 again, after stirring, supernatant liquid is collected in layering in the impouring separating funnel.
6, precipitation: in above-mentioned clear liquid impouring Enamel jar, add the cold acetone of 1 times of amount of clear liquid, stir, under 4-10 ℃ condition, left standstill centrifugal collecting precipitation 5-6 hour.
7, degerming: with twice flushing of trichloromethane throw out of 2 times of amounts of above-mentioned throw out;
8, dialysis desalination: add 2g acetone preparation scavenging solution with 500ml distilled water, with scavenging solution to throw out towards Xian Sanci;
9, lyophilize: get 50g crude product SOD with moisture eliminator 0-4 ℃ of above-mentioned throw out of lyophilize in freezer, than vigor 3000u/mg, protein concn 40%.
The preparation of elaboration SOD:
1, dissolving: the SOD crude product of above-mentioned gained is dissolved in PH7.6,2.5 μ mmol/lK
2HP
4-KH
2PO
4In the buffer reagent, get clear liquor through ultrafiltration.Wherein, buffer reagent is by K
2HP
4, PKH
2PO
4The equal-volume preparation.
2, post layer folding: with DEAEsepnadexA-50 chromatography on the clear liquor, post Ф 7.5-40cm, this post is earlier steady with buffer reagent, last sample flow rate control the 100-200 milliliter/hour.
3, wash-out: after last sample is extremely saturated, carry out gradient elution with same buffer reagent, control flow velocity be the 100-200 milliliter/hour, careful sampling monitoring has the active protein peak of SOD, its " optical density(OD) maximum value " with the active maximum protein peak ultraviolet absorption of SOD of CuZn-SOD in different blood source there are differences: ox blood is the 258nm place, and pig blood is the 265nm place.
4, dialysis: with above-mentioned collected SOD elutriant, in the dialysis tubing of packing into, the desalination of dialysing in distilled water is collected dialyzate.
5, drying: with the dialyzate ultrafiltration and concentration, lyophilize then, get final product the CuZn-SOD product 30g of the little band pale bluish green of outward appearance, the ratio vigor of its enzyme can reach 3600-10000u/mg, protein concn 36%.
Store: go into warehouse temperature 0-5 ℃, the quality guaranteed period can reach more than 3 years.
Detection method:
One, UV detection:
The SOD elaboration that takes a morsel is placed on a blank sheet of paper, and under the irradiation of fluorescent lamp, color does not change then of poor quality, and color slowly becomes white and illustrates that then quality is good.
Two, chemical detection:
1, the elaboration SOD that takes a morsel is placed in the beaker, pours a certain amount of pyrogallol into, emits steam bubble, and the beginning oxidation is after 2 minutes, and SOD changes color, is turned black by light blue.2, be poured on the sheet glass, flow and to walk to a direction, sinuous flow not, the sticky shape of shape illustrates that quality reaches a standard.
Claims (5)
1, the preparation method of superoxide-dismutase is characterized in that: this preparation method adopts the fresh pig clot to extract SOD, at first makes crude product SOD by fresh pig blood, and the purifying crude product makes elaboration SOD then.
2, the preparation method of superoxide-dismutase according to claim 1 is characterized in that: the preparation process of crude product SOD includes: refrigeration, cleaning, extraction, thermal change, extraction, precipitation, degerming, dialysis desalination, lyophilize:
(1), refrigeration: in fresh blood impouring container, place freezer refrigeration that it is lumpd naturally.
(2), cleaning: clot is blended, place the flushing of filter screen water, clean filter and do, get the serum-free clot.
(3), extract: the serum-free clot is put into container, add the triton x-100 solution of the concentration 20% of 0.2 times of amount of serum-free clot, stirred 20-40 minute, suction filtration is collected blood plasma.
(4), thermal change: in blood plasma impouring retort, be heated to 65 ℃, under stirring condition isothermal reaction 20-40 minute, be cooled to room temperature, centrifugal collection supernatant liquor; Supernatant liquor is carried out the secondary thermal change, 65 ℃ of constant temperature of temperature 20 minutes, the constant temperature phase adds the 4g cupric chloride by the former blood volume of 1kg, is cooled to 10-15 ℃, centrifugal collection supernatant liquor.。
(5), extraction: in supernatant liquor impouring container, the ethanol one chloroform mixed solution that adds supernatant liquor volume 2/5, ethanol wherein: chloroform (V: V)=5: 3, stir several minutes, centrifugal collection filtrate is in another container, adjusting pH value is 7.6, under stirring condition, in the 43% ratio adding K of filtrate volume
2HP
4, adjusting pH value once more is 7.6, adds the ethanol one chloroform mixed solution of filtrate volume 1/5 again, after stirring, supernatant liquid is collected in layering in the impouring separating funnel.
(6), precipitation: in above-mentioned clear liquid impouring container, add the cold acetone of 1 times of amount of clear liquid, stir, under 4-10 ℃ condition, left standstill centrifugal collecting precipitation 5-6 hour.
(7), degerming: with the trichloromethane flushing throw out of 2 times of amounts of above-mentioned throw out;
(8), the dialysis desalination: add 2g acetone preparation scavenging solution with 500ml distilled water, with scavenging solution to throw out towards Xian;
(9), lyophilize: get crude product SOD50g with moisture eliminator 0-4 ℃ of above-mentioned throw out of lyophilize in freezer, than vigor 3000u/mg, protein concn 40%.
3, the preparation method of superoxide-dismutase according to claim 2 is characterized in that: to make concentration be 20% triton x-100 solution to add the 200ml triton x-100 in the 800ml pure water.
4, the preparation method of superoxide-dismutase according to claim 1 is characterized in that: the preparation of elaboration SOD includes: dissolving, post layer folding, wash-out, dialysis, drying:
(1), dissolving: the SOD crude product of gained is dissolved in PH7.6,2.5 μ mmol/l, K
2HP
4-KH
2PO
4In the buffer reagent, get clear liquor through ultrafiltration.
(2), post layer folding: with DEAEsepnadexA-50 chromatography on the clear liquor, last sample flow rate control the 100-200 milliliter/hour.
(3), wash-out: go up sample after saturated, carry out gradient elution with same buffer reagent, control flow velocity and be the 100-200 milliliter/hour, sampling monitoring has the active protein peak of SOD.
(4), dialysis: with above-mentioned collected SOD elutriant, in the dialysis tubing of packing into, the desalination of dialysing in distilled water is collected dialyzate.
(5), drying: with the dialyzate ultrafiltration and concentration, lyophilize then can get the CuZn-SOD product of the little band pale bluish green of outward appearance.
5, the preparation method of superoxide-dismutase according to claim 4 is characterized in that: with isopyknic K
2HPO
4, KH
2PO
4The preparation buffer reagent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410041723 CN1616655A (en) | 2004-08-14 | 2004-08-14 | Method for preparing superoxide dismutase |
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|---|---|---|---|
| CN 200410041723 CN1616655A (en) | 2004-08-14 | 2004-08-14 | Method for preparing superoxide dismutase |
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|---|---|
| CN1616655A true CN1616655A (en) | 2005-05-18 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100392076C (en) * | 2005-07-27 | 2008-06-04 | 周兴和 | Extraction and modification method for superoxide dismutase |
| CN102181411A (en) * | 2011-04-01 | 2011-09-14 | 黑龙江宝迪肉类食品有限公司 | Method for extracting superoxide dismutase (SOD) from porcine blood red cells |
| CN101461543B (en) * | 2007-12-19 | 2012-02-08 | 中国科学院大连化学物理研究所 | Deep-processing method of deer blood |
| CN101717756B (en) * | 2008-10-09 | 2012-05-09 | 哈尔滨仁皇药业股份有限公司 | Method for preparing superoxide dismutase |
| CN102925416A (en) * | 2012-11-19 | 2013-02-13 | 景志刚 | Method for extracting superoxide dismutase from blood of mammal |
| CN103275943A (en) * | 2012-10-19 | 2013-09-04 | 刘爽 | Method for extracting superoxide dismutase from pig spleen |
| CN105543185A (en) * | 2016-03-10 | 2016-05-04 | 甘肃养泰和生物科技有限公司 | Method for producing SOD (superoxidase dismutase) lyophilized powder through extraction from animal blood clots and purification |
-
2004
- 2004-08-14 CN CN 200410041723 patent/CN1616655A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100392076C (en) * | 2005-07-27 | 2008-06-04 | 周兴和 | Extraction and modification method for superoxide dismutase |
| CN101461543B (en) * | 2007-12-19 | 2012-02-08 | 中国科学院大连化学物理研究所 | Deep-processing method of deer blood |
| CN101717756B (en) * | 2008-10-09 | 2012-05-09 | 哈尔滨仁皇药业股份有限公司 | Method for preparing superoxide dismutase |
| CN102181411A (en) * | 2011-04-01 | 2011-09-14 | 黑龙江宝迪肉类食品有限公司 | Method for extracting superoxide dismutase (SOD) from porcine blood red cells |
| CN103275943A (en) * | 2012-10-19 | 2013-09-04 | 刘爽 | Method for extracting superoxide dismutase from pig spleen |
| CN103275943B (en) * | 2012-10-19 | 2014-09-17 | 吉林省金梓源生物科技有限公司 | Method for extracting superoxide dismutase from pig spleen |
| CN102925416A (en) * | 2012-11-19 | 2013-02-13 | 景志刚 | Method for extracting superoxide dismutase from blood of mammal |
| CN105543185A (en) * | 2016-03-10 | 2016-05-04 | 甘肃养泰和生物科技有限公司 | Method for producing SOD (superoxidase dismutase) lyophilized powder through extraction from animal blood clots and purification |
| CN105543185B (en) * | 2016-03-10 | 2019-01-11 | 甘肃养泰和生物科技有限公司 | A kind of production method extracted from animal clot, purify SOD freeze-dried powder |
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