CN1453355A - Method of extracting superoxide dismutase from earthworm - Google Patents
Method of extracting superoxide dismutase from earthworm Download PDFInfo
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- CN1453355A CN1453355A CN 02109527 CN02109527A CN1453355A CN 1453355 A CN1453355 A CN 1453355A CN 02109527 CN02109527 CN 02109527 CN 02109527 A CN02109527 A CN 02109527A CN 1453355 A CN1453355 A CN 1453355A
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- earthworm
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Abstract
The process of extracting superoxide dismutase (SOD) from earthworm body includes the steps: washing live earthworm, wet milling to obtain earthworm juice, freezing, homogenating in buffering liquid, centrifugation to obtain the supernatant; heating, cooling, filtering to obtain filtrate, mixing with acetone, dissolving the precipitate with buffering liquid to obtain supernatant; mixing the supernatant with Mg, Cu and Zn via stirring, centrifugation to obtain supernatant; adding ammonium sulfate solution to the supernatant and centrifugation to collect precipitate; dissolving the precipitate in buffering solution, dialysis up to no sulfate radical, chromatography to collect eluted liquid, concentration, cooling and drying to obtain green blue Cu and Zn SOD.
Description
1, technical field:
The present invention relates to a kind of its preparation process that extracts superoxide-dismutase in the organism.
2, background technology:
Superoxide-dismutase (SuperOxide dismutase is called for short SOD) belongs to metalloenzyme, is main oxygen free radical scavenger, and it can the intravital superoxide anion of single-minded removing people
As medicinal enzyme and toolenzyme that great medical value is arranged, biochemical at home and abroad boundary and the world of medicine cause great concern.At present SOD is widely used in rheumatoid arthritis, some autoimmune disorder, and the illnesss such as aging, blood embolism, blood oxygen and other material poisonings of the generation of tumour, tissue are all had certain curative effect.
Domestic-developed SOD resource has ox blood, pig blood and Rosa roxburghii etc. at present, is raw material with these materials, and the resource of consumption is very big, does not find also that by the retrieval pertinent literature with earthworm (being commonly called as earthworm) be the method that resource is extracted SOD.
3, summary of the invention:
(1) goal of the invention: the purpose of this invention is to provide a kind of method of extracting superoxide-dismutase in the body of earthworm, to solve the problem that aspects such as consumption of natural resource is big exist.
(2) technical scheme:
1, extract the method for superoxide-dismutase in the earthworm body, its step is as follows:
A, the earthworm that will live are cleaned, and place fresh water or contain the organic acid sodium salt that is no more than 0.3% (by weight) and clean, and the dirt in its digestive tube is thoroughly removed, then at all dirts of removing on its surperficial body;
B, the earthworm wet-milling of will living, the earthworm juice that obtains freezing back under-60 ℃～-10 ℃ temperature stores;
C, fresh earthworm juice or its frozen material homogenate 2L/kg in pH value 7.6,25mmol/LTris-Gly damping fluid stirred 1～2 hour, and centrifugal 30 minutes of 15000r/min abandons throw out and gets the stillness of night;
D, the above-mentioned stillness of night are cooled to 4 ℃ in 65 ℃ of heating 10～20 minutes, filter the acetone that adds 1.25 times of volumes in the rear filtrate, collecting precipitation, and throw out is abandoned throw out and is got supernatant liquor with a little P H value 7.6, the dissolving of 25mmol/LTris-Gly damping fluid;
E, supernatant liquor under agitation add and include 10molmg, 10molCu, 10molZn, and saturation ratio reaches 45%, continue to stir 15 minutes, centrifugal 30 minutes then, discard precipitation, to the continuous ammoniumsulphate soln that adds of supernatant liquor relaying at 2000r/min, make its saturation ratio reach 65%, centrifugal collecting precipitation;
F, with above-mentioned resolution of precipitate in pH value 7.6,25mmol/LTris-Gly damping fluid, then dialysis is to there not being SO
2- 4, dialyzate is gone up DEAE-Sephadex A-50 chromatography column at last, and (column volume is 2.5 * 50cm), collects elutriant, concentrates, cooling, drying, promptly gets glaucous Cu, ZnSOD.
(3), advantage and effect:
Utilize earthworm to extract superoxide-dismutase (SOD), its major advantage is that raw material easily gets, and is with low cost, do not contend over raw materials with other industry, and it be than will saving 5～6 times with extraction such as pig, ox bloods, and extracts SOD composition and same measuring than high 2～3 times.
4, embodiment:
1, extract the method for superoxide-dismutase in the earthworm body, its step is as follows:
A, the earthworm that will live are cleaned, and place fresh water or contain the organic acid sodium salt that is no more than 0.3% (by weight) and clean, and the dirt in its digestive tube is thoroughly removed, then at all dirts of removing on its surperficial body;
B, the earthworm wet-milling of will living, the earthworm juice that obtains freezing back under-30 ℃ temperature stores;
C, fresh earthworm juice or its frozen material homogenate 2L/kg in pH value 7.6,25mmol/LTris-Gly damping fluid stirred 1 hour, and centrifugal 30 minutes of 15000r/min abandons throw out and gets the stillness of night;
D, the above-mentioned stillness of night are cooled to 4 ℃ in 65 ℃ of heating 20 minutes, filter the acetone that adds 1.25 times of volumes in the rear filtrate, collecting precipitation, and throw out is abandoned throw out and is got supernatant liquor with a little P H value 7.6, the dissolving of 25mmol/LTris-Gly damping fluid;
E, supernatant liquor under agitation add and include 10molmg, 10molCu, 10molZn, and saturation ratio reaches 45%, continue to stir 15 minutes, centrifugal 30 minutes then, discard precipitation, to the continuous ammoniumsulphate soln that adds of supernatant liquor relaying at 2000r/min, make its saturation ratio reach 65%, centrifugal collecting precipitation;
F, with above-mentioned resolution of precipitate in pH value 7.6,25mmol/LTris-Gly damping fluid, then dialysis is to there not being SO
2- 4, dialyzate is gone up DEAE-Sephadex A-50 chromatography column at last, and (column volume is 2.5 * 50cm), collects elutriant, concentrates, cooling, drying, promptly gets glaucous Cu, ZnSOD.
The activity determination method of SOD is to adopt benzenetriol or suprarenin method among the present invention.
The content assaying method of SOD is to utilize colored immune plate to measure Cu, ZnSOD content among the present invention, or adopts radioimmunology and isotope labeling method.
Earthworm mainly perches in 5 ℃～25 ℃ soil, and its feed is septic organic substance, and breeding cycle is short, ripe fast, and the breeding potential height is the expert who handles domestic refuse.
The earthworm body contains 18 seed amino acids and the medicinal enzyme of kind more than 30, and its great medical value is arranged.
Claims (1)
1, a kind of method of extracting superoxide-dismutase in the earthworm body, it is characterized in that: the preparation process of this method is as follows:
A, the earthworm that will live are cleaned, and place fresh water or contain the organic acid sodium salt that is no more than 0.3% (by weight) and clean, and the dirt in its digestive tube is thoroughly removed, then at all dirts of removing on its surperficial body;
B, the earthworm wet-milling of will living, the earthworm juice that obtains freezing back under-60 ℃～-10 ℃ temperature stores;
C, fresh earthworm juice or its frozen material homogenate 2L/kg in pH value 7.6,25mmol/LTris-Gly damping fluid stirred 1～2 hour, and centrifugal 30 minutes of 15000r/min abandons throw out and gets the stillness of night;
D, the above-mentioned stillness of night are cooled to 4 ℃ in 65 ℃ of heating 10～20 minutes, filter the acetone that adds 1.25 times of volumes in the rear filtrate, collecting precipitation, and throw out is abandoned throw out and is got supernatant liquor with a little P H value 7.6, the dissolving of 25mmol/LTris-Gly damping fluid;
E, supernatant liquor under agitation add and include 10molmg, 10molCu, 10molZn, and saturation ratio reaches 45%, continue to stir 15 minutes, centrifugal 30 minutes then, discard precipitation, to the continuous ammoniumsulphate soln that adds of supernatant liquor relaying at 2000r/min, make its saturation ratio reach 65%, centrifugal collecting precipitation;
F, with above-mentioned resolution of precipitate in pH value 7.6,25mmol/LTris-Gly damping fluid, then dialysis is to there not being SO
2- 4Dialyzate is gone up DEAE-Sephadex A-50 chromatography column at last, and (column volume is 2.5 * 50cm), collects elutriant, concentrates, cooling, drying, promptly gets glaucous Cu, ZnSOD.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02109527 CN1453355A (en) | 2002-04-23 | 2002-04-23 | Method of extracting superoxide dismutase from earthworm |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02109527 CN1453355A (en) | 2002-04-23 | 2002-04-23 | Method of extracting superoxide dismutase from earthworm |
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| CN1453355A true CN1453355A (en) | 2003-11-05 |
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| CN 02109527 Pending CN1453355A (en) | 2002-04-23 | 2002-04-23 | Method of extracting superoxide dismutase from earthworm |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100453641C (en) * | 2006-02-28 | 2009-01-21 | 中国海洋大学 | Method for extracting SOD emzyme from tilapia mossambica viscus |
| CN101297672B (en) * | 2008-06-12 | 2010-09-01 | 武汉凯丽金生物科技有限公司 | Method for producing active peptides from enzymolysis of fish proteins using active enzymes in angleworm intestinal canal |
| CN101245342B (en) * | 2007-02-15 | 2012-01-25 | 四平华科生物技术有限责任公司 | Technique for producing gene rHu(Cu/Zn-SOD) with one-step method |
| CN104593338A (en) * | 2014-12-31 | 2015-05-06 | 唯美度科技（北京）有限公司 | Method of extracting SOD (superoxide dismutase) from earthworms |
| CN105462938A (en) * | 2014-08-06 | 2016-04-06 | 天津贾立明蚯蚓养殖有限公司 | Processing process and formula of Chinese wolfberry-earthworm SOD nutrition oral liquid |
-
2002
- 2002-04-23 CN CN 02109527 patent/CN1453355A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100453641C (en) * | 2006-02-28 | 2009-01-21 | 中国海洋大学 | Method for extracting SOD emzyme from tilapia mossambica viscus |
| CN101245342B (en) * | 2007-02-15 | 2012-01-25 | 四平华科生物技术有限责任公司 | Technique for producing gene rHu(Cu/Zn-SOD) with one-step method |
| CN101297672B (en) * | 2008-06-12 | 2010-09-01 | 武汉凯丽金生物科技有限公司 | Method for producing active peptides from enzymolysis of fish proteins using active enzymes in angleworm intestinal canal |
| CN105462938A (en) * | 2014-08-06 | 2016-04-06 | 天津贾立明蚯蚓养殖有限公司 | Processing process and formula of Chinese wolfberry-earthworm SOD nutrition oral liquid |
| CN104593338A (en) * | 2014-12-31 | 2015-05-06 | 唯美度科技（北京）有限公司 | Method of extracting SOD (superoxide dismutase) from earthworms |
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