CN1210398C - Superoxide dismutase conjugated substance and preparation method thereof - Google Patents
Superoxide dismutase conjugated substance and preparation method thereof Download PDFInfo
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- CN1210398C CN1210398C CN 02110099 CN02110099A CN1210398C CN 1210398 C CN1210398 C CN 1210398C CN 02110099 CN02110099 CN 02110099 CN 02110099 A CN02110099 A CN 02110099A CN 1210398 C CN1210398 C CN 1210398C
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- low molecular
- molecular heparin
- superoxide dismutase
- superoxide
- conjugated substance
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Abstract
The present invention belongs to the field of biologic medicine, and particularly relates to a superoxide dismutase conjugate and a preparation method thereof. The modification rate of free amino groups of superoxide dismutase on the conjugate is from 11.1% to 88.8%. The preparation method comprises: low molecular heparin activated by sodium periodate is used for modifying the free amino groups on the superoxide dismutase. The prepared conjugate has the characteristics of low antigenicity, long half life, high stability, etc.; the proper characteristics of thrombus resistance, inflammation relieving, microcirculatory promotion, etc. of the low molecular heparin can be fully exerted. The conjugate has the functions of preventing and curing reperfusion injury in ischemic cerebral apoplexy and can be used as an additive in cosmetics.
Description
1. technical field
The invention belongs to biomedicine field, particularly relate to a kind of Low molecular heparin-superoxide dismutase conjugated substance and preparation method thereof.
2. background technology
Superoxide-dismutase (SOD) is the scavenging agent of ultra-oxygen anion free radical, can be used for the disease that caused by excessive ultra-oxygen anion free radical or the control of body injury, as rheumatoid arthritis, radiation injury, blood reperfusion injury etc.But the natural origin superoxide-dismutase has shortcoming such as transformation period weak point and poor stability in certain antigenicity, the body, has limited its application.The method that overcomes the superoxide-dismutase shortcoming mainly is to carry out chemically modified with macromolecular complex its molecule of verifying, and the modifier of employing has polyoxyethylene glycol (PEG), dextran, starch, homologous albumin and polyene to belong to oxy-compound etc.As fill in Buddhist people such as (saifer) at medical biotechnology experiment progress magazine [Adv Exp MedBiol, 1994,366:377～387] the employing polyoxyethylene glycol delivered carries out chemically modified gained superoxide dismutase conjugated substance (PEG-SOD) to superoxide-dismutase, and its immunogenicity reduces, the transformation period prolongs in the body, stability increases.But these binding substancess are for treatment and ultra-oxygen anion free radical diseases associated, and as the cerebral infarction reperfusion injury, curative effect is not very good.
3. summary of the invention
The objective of the invention is to overcome above-mentioned the deficiencies in the prior art, adopt Low molecular heparin (LMWH) to modify superoxide-dismutase, a kind of new superoxide dismutase conjugated substance (LMWH-SOD) and preparation method thereof is provided.
The present invention realizes by following approach:
A kind of new superoxide dismutase conjugated substance is characterized in that free amino group on the superoxide-dismutase is in conjunction with Low molecular heparin, the modification rate of free amino group is that 11.1%～88.8%, superoxide-dismutase molecule can be in conjunction with 1-8 molecular-weight average less than 8000 Low molecular heparin molecule.
A kind of method for preparing above-mentioned superoxide dismutase conjugated substance is characterized in that following steps:
1. the activation of Low molecular heparin: available different activator carries out, as sodium periodate, cyanogen bromide, cyanuric chloride etc., Low molecular heparin concentration is 1%10%, the concentration of activator is 1%～5%, be reflected under 0～4 ℃ of pH4.0～7.0 and the temperature and carry out, continuously stirring reaction 15-20 hour, and with 2%～10% sodium sulfite solution titration to variable color.
2. modification reaction: be reflected under 0～4 ℃ of pH8～10 and the temperature and carry out, stirring reaction 20-30 hour, adopt ultrafiltration process or gel chromatography separate low molecular heparin-superoxide dismutase conjugated substance then, the more Powdered Low molecular heparin-superoxide dismutase conjugated substance of obtained by freeze drying.
Low molecular heparin-superoxide dismutase conjugated substance that the present invention makes compare with natural superoxide-dismutase have not only that antigenicity is low, long half time, stable high characteristic, and can give full play to characteristics such as the distinctive antithrombotic of Low molecular heparin, anti-inflammatory, promotion microcirculation, and binding substances greatly increases by the hemato encephalic barrier ability, can play a role from many aspects for control cerebral infarction reperfusion injury, will better effect be arranged than natural superoxide-dismutase and other binding substances thereof.In addition,, also have more superiority, obviously increase as stability, thereby can in makeup, keep greater activity for a long time, and have Low molecular heparin to promote the effect of skin microcirculation than natural superoxide-dismutase as cosmetics additive.
4. embodiment
The key of Low molecular heparin-superoxide dismutase conjugated substance preparation is the activation of Low molecular heparin.Low molecular heparin activatory method has multiple, as sodium periodate activation method, cyanogen bromide activation, cyanuric chloride activation method etc.At this only for sodium periodate activation Low molecular heparin, further prepare the example of Low molecular heparin-superoxide dismutase conjugated substance.
(1) activation of Low molecular heparin: take by weighing Low molecular heparin 0.6g and be dissolved among the distilled water 10ml, add 12% sodium periodate solution 1ml, transfer pH4.0～6.0,4 ℃ following stirring reaction 20h, the sodium sulfite solution of Dropwise 5 % gets activatory Low molecular heparin solution to variable color.
(2) modification reaction: take by weighing superoxide-dismutase 100mg, be dissolved among the carbonate buffer solution 3ml of 0.3mol/L, pH9.5, add activatory Low molecular heparin solution 3ml, 4 ℃ of following induction stirring reaction 24h.Reacting liquid filtering is collected active eluant after the sephadex g-100 post separates, and lyophilize gets Powdered Low molecular heparin-superoxide dismutase conjugated substance.
Claims (2)
1. superoxide dismutase conjugated substance, it is characterized in that free amino group on the superoxide-dismutase is in conjunction with Low molecular heparin, the modification rate of free amino group is that 11.1%～88.8%, superoxide-dismutase molecule can be in conjunction with 1-8 molecular-weight average less than 8000 Low molecular heparin molecule.
2. method for preparing above-mentioned superoxide dismutase conjugated substance is characterized in that following steps:
(1) activation of Low molecular heparin: use sodium periodate as activator, Low molecular heparin concentration is 5%～10%, the concentration of activator is 1%～5%, being reflected at pH4.0～7.0 and temperature descends for 0～4 ℃, continuously stirring reaction 15-20 hour, and with 5%～10% sodium sulfite solution titration to variable color;
(2) modification reaction: be reflected under 0～4 ℃ of pH8～10 and the temperature and carry out, stirring reaction 20-30 hour, adopt ultrafiltration process or gel chromatography separate low molecular heparin-superoxide dismutase conjugated substance then, the more Powdered ultra-low molecular heparin-superoxide dismutase conjugated substance of obtained by freeze drying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02110099 CN1210398C (en) | 2002-03-07 | 2002-03-07 | Superoxide dismutase conjugated substance and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02110099 CN1210398C (en) | 2002-03-07 | 2002-03-07 | Superoxide dismutase conjugated substance and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1371993A CN1371993A (en) | 2002-10-02 |
| CN1210398C true CN1210398C (en) | 2005-07-13 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 02110099 Expired - Fee Related CN1210398C (en) | 2002-03-07 | 2002-03-07 | Superoxide dismutase conjugated substance and preparation method thereof |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103674862B (en) * | 2012-09-14 | 2015-11-18 | 中国科学院长春应用化学研究所 | A kind of method of screening superoxide anion scavenger |
| CN103674870B (en) * | 2012-09-14 | 2015-11-18 | 中国科学院长春应用化学研究所 | A kind of method measuring superoxide dismutase activity |
| CN103805587A (en) * | 2014-01-13 | 2014-05-21 | 李健 | Superoxide dismutase fatty acid clathrate compound and preparation method thereof |
| CN111544567B (en) * | 2020-05-14 | 2021-01-26 | 扬子江药业集团广州海瑞药业有限公司 | Atosiban injection pharmaceutical composition and preparation method thereof |
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- 2002-03-07 CN CN 02110099 patent/CN1210398C/en not_active Expired - Fee Related
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| CN1371993A (en) | 2002-10-02 |
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Granted publication date: 20050713 Termination date: 20180307 |