CN101254209A - Calf spleen extract injection prepared by inphase opposition column chromatography and preparation thereof - Google Patents
Calf spleen extract injection prepared by inphase opposition column chromatography and preparation thereof Download PDFInfo
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- CN101254209A CN101254209A CNA2008100505392A CN200810050539A CN101254209A CN 101254209 A CN101254209 A CN 101254209A CN A2008100505392 A CNA2008100505392 A CN A2008100505392A CN 200810050539 A CN200810050539 A CN 200810050539A CN 101254209 A CN101254209 A CN 101254209A
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Abstract
The invention discloses a calf spleen extract injection prepared by reversed-phase column chromatography and a preparation method thereof. The original calf spleen extract solution is purified by reversed-phase column chromatography with simple process, so as to remarkably improve the stability of product and the quality standard of pharmacologically active substance. The inventive method utilizes the reversed-phase column chromatography instead of conventional adsorption, elution and ion-exchange column chromatography, so as to reduce production process and meet the requirement of improving product quality standard. Compared with the prior calf spleen extract solution, the inventive product has the advantages of low toxic and side effects and low content of pigment impurities. The No.7 peak area in HPLC chromatogram is improved from 17.66% before reversed-phase chromatography to 51.82% after reversed-phase chromatography, thus indicating high unit content of effective component.
Description
Technical field
The present invention relates to the production technology of calf spleen extract injection, a kind of calf spleen extract injection prepared by inphase opposition column chromatography and preparation method especially are provided, belong to biological pharmacy technical field.
Background technology
Many studies confirm that extensively exists low molecule tumour-inhibitory substance in biological intravital multiple histiocyte and the body fluid, may be the intermediate product of the specific synthetic or substance metabolism of histiocyte.Its structure and physicochemical property differ, but remove malignant conversioning cell in vivo, prevent that tumor from taking place to play an important role in the process with development.
Bibliographical information, the polypeptides matter that extracts from Lien Sus domestica have natural biological and learn actively, not only can suppress growth of tumor, still can stimulating immune system, and impel interferon to discharge, increase the T cytoactive, globality ground human body immunity improving ability and anti-cancer ability.Domestic research to the spleen peptide at present is less, and it is raw material with the Lien Bovis seu Bubali that bibliographical information is arranged, and adopts ultrafiltration and centrifugation technique to prepare the low molecule tumour-inhibitory substance of spleen.
Calf spleen extract injection can be used for human body immunity improving power, obviously improves the leukocyte number.Leukopenia, the various malignant tumor that can cause at treatment aplastic anemia, essential thrombocytopenia, lonizing radiation, be used when improving the tumor patient dyscrasia.Yet the calf spleen extract injection product impurity that existing market is sold is more, the time have anaphylaxis to take place, and pharmacologically active instability has influenced the use curative effect of this medicine.
Summary of the invention
The invention provides a kind of calf spleen extract injection, leukogenic effect is apparently higher than existing product.
The invention also discloses the preparation method of calf spleen extract injection, former calf spleen extract solution is carried out the reversed phase column chromatography purification, purifying process is easy, obviously improves the quality standard of product stability and pharmacologically active.
Calf spleen extract solution of the present invention is characterized in that:
Be faint yellow settled solution; PH value is 5.0～7.0; Molecular weight 4000-6000 dalton;
Add water and make the solution that contains polypeptide 20ug among every 1ml,, absorption maximum is arranged at the wavelength place of 194nm according to spectrophotometry;
Protein: calf spleen extract solution 2.0ml, add 20% sulfosalicylic acid solution 2.0ml, muddiness must not take place in mixing;
Trap: calf spleen extract solution adds water and makes the solution that contains polypeptide 20ug among every 1ml, should be 2.0～2.5 at the ratio of 260nm and 280nm wavelength place trap.
The preparation method of calf spleen extract solution of the present invention may further comprise the steps:
1, gets calf spleen extract injection 200ml, be splined on the anion-exchange chromatography post and carry out reversed phase chromatography (comprising the DEAE cellulose, anionites such as macroreticular weakly base acrylic acid type anion exchange resin);
2, balance liquid is the phosphate buffer of 5～20mmol/ml, pH5～7;
3, collect vigor peak with function of increasing leukocyte.
Best process flow is as follows:
1, gets calf spleen extract injection 200ml, be splined on 400ml macroreticular weakly base acrylic acid type anion exchange resin chromatographic column and carry out reversed phase chromatography;
2, balance liquid is the phosphate buffer of 10mmol/ml, pH5～7;
3, collect vigor peak with function of increasing leukocyte.
Good effect of the present invention is: broken conventional absorption, eluting ion-exchange chromatography method, adopted reversed phase ion displacement chromatography method, reduced the technological process of production, reached the requirement of the standard of improving the quality of products; Product of the present invention is compared with former Calf Spleen extracting solution has that toxic and side effects is little, pigment impurity content is low, and impurity content reduces, thereby toxic and side effects is reduced.No. 7 peak area percentage ratios of high performance liquid chromatography 17.66% before by reversed phase chromatography brought up to 51.82% behind the chromatography, unit active constituent content height.
Experimental example 1
Refining calf spleen extract injection vigor pharmacological evaluation
Adopt numeration of leukocyte to investigate the function of increasing leukocyte of this Calf Spleen refined liquid
1.1 vitality test
This product is added water make the solution that contains polypeptide 2.5mg among every 1ml, as need testing solution.Select 3 of the healthy rabbits of 2～3kg, check before the injection and then carry out intramuscular injection by leukocyte count, dosage was checked leukocyte count by rabbit body weight 1kg intramuscular injection need testing solution 1ml in back 24 hours in injection.
1.3 experimental result
1.3.1 this refining calf spleen extract injection (peptide concentration is 2.5mg/ml), by after the body weight administration, the leukocyte number on average raises 1.36 times before and after the injection
1.3.2 experimental data table
Administered intramuscular is investigated this refined soln and is risen white effect and refining preceding contrast.
Contrast with blank group before the injection:
* * * *P＜0.000001.Referring to Fig. 1, Fig. 2.
Description of drawings
Fig. 1 analyses preceding high-efficient liquid phase chromatogram for the calf spleen extract injection anion exchange resin layer;
Fig. 2 analyses the back high-efficient liquid phase chromatogram for the calf spleen extract injection anion exchange resin layer.
The specific embodiment
The present invention is raw material with the calf spleen extract injection, and concrete implementation step is as follows, but and does not mean that limitation of the scope of the invention.
Embodiment 1
1, gets calf spleen extract injection 200ml, be splined on the anion-exchange chromatography post and carry out reversed phase chromatography (DEAE cellulose);
2, balance liquid is the phosphate buffer of 5～20mmol/ml, pH5～7;
3, collect vigor peak with function of increasing leukocyte.
Embodiment 2
1, gets calf spleen extract injection 200ml, be splined on 400ml macroreticular weakly base acrylic acid type anion exchange resin chromatographic column (D311) and carry out reversed phase chromatography;
2, balance liquid is the phosphate buffer of 10mmol/ml, pH5～7;
3, collect vigor peak with function of increasing leukocyte.See Fig. 2.
Embodiment 3
1, gets calf spleen extract injection 100ml, be splined on 250ml 330 alkalescence epoxy type anion exchange resin chromatographic columns;
2, balance liquid is the phosphate buffer of 5～20mmol/ml, pH5～7;
3, collect vigor peak with function of increasing leukocyte.See Fig. 2.
Embodiment 4
1, gets calf spleen extract injection 200ml, be splined on 400ml macroreticular weakly base acrylic acid type anion exchange resin chromatographic column (DE51, DE52, DE53, QA52) and carry out reversed phase chromatography;
2, balance liquid is the water buffer of the concrete pH6.3 in 10mmol/ml, pH5～7;
3, collect vigor peak with function of increasing leukocyte.See Fig. 2.
Claims (4)
1, a kind of calf spleen extract solution is characterized in that:
Be faint yellow settled solution; PH value is 5.0～7.0; Molecular weight 4000-6000 dalton;
Add water and make the solution that contains polypeptide 20ug among every 1ml,, absorption maximum is arranged at the wavelength place of 194nm according to spectrophotometry;
Protein: calf spleen extract solution 2.0ml, add 20% sulfosalicylic acid solution 2.0ml, muddiness must not take place in mixing;
Trap: calf spleen extract solution adds water and makes the solution that contains polypeptide 20ug among every 1ml, should be 2.0～2.5 at the ratio of 260nm and 280nm wavelength place trap.
2, the preparation method of calf spleen extract solution according to claim 1 is characterized in that with the Calf Spleen being the aqueous solution that raw material makes, through anti-phase anion exchange resin layer analyse, the technical process of distilling under reduced pressure, step is as follows:
Said anion exchange resin layer is analysed and is, water extract is analysed post through anion exchange resin layer; Balance liquid is the phosphate buffer or the distilled water of 5～20mmol/ml, pH5～7, collects the vigor peak with function of increasing leukocyte;
Described anionite: be selected from DEAE cellulose, the macroreticular weakly base acrylic acid type anion exchange resin etc. any one.
3, the described calf spleen extract solution of claim 1 improves the application of exempting from the body epidemic disease power medicine in preparation.
4, the application of the leukopenia that causes at preparation treatment aplastic anemia, essential thrombocytopenia, lonizing radiation of the described calf spleen extract solution of claim 1, various malignant tumor, disease medicament.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100505392A CN101254209B (en) | 2008-03-26 | 2008-03-26 | Calf spleen extract injection prepared by inphase opposition column chromatography and preparation thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008100505392A CN101254209B (en) | 2008-03-26 | 2008-03-26 | Calf spleen extract injection prepared by inphase opposition column chromatography and preparation thereof |
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| CN101254209A true CN101254209A (en) | 2008-09-03 |
| CN101254209B CN101254209B (en) | 2011-02-16 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108129549A (en) * | 2017-12-19 | 2018-06-08 | 浙江丰安生物制药有限公司 | The extracting method of polypeptide in a kind of spleen aminopeptide |
| CN108148111A (en) * | 2017-12-19 | 2018-06-12 | 浙江丰安生物制药有限公司 | The extracting method of polypeptide in a kind of spleen aminopeptide |
| CN113975296A (en) * | 2021-12-30 | 2022-01-28 | 北京第一生物化学药业有限公司 | Application of animal spleen extract in preparation of medicine for treating Alzheimer's disease |
| CN114940698A (en) * | 2022-07-22 | 2022-08-26 | 北京第一生物化学药业有限公司 | Method for extracting acidic polypeptide from spleen aminopeptide stock solution |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1253008A (en) * | 1998-10-30 | 2000-05-17 | 任天保 | Osteophyte pill and osteophyte-eliminating ointment for treating hyperosteogeny |
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2008
- 2008-03-26 CN CN2008100505392A patent/CN101254209B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108129549A (en) * | 2017-12-19 | 2018-06-08 | 浙江丰安生物制药有限公司 | The extracting method of polypeptide in a kind of spleen aminopeptide |
| CN108148111A (en) * | 2017-12-19 | 2018-06-12 | 浙江丰安生物制药有限公司 | The extracting method of polypeptide in a kind of spleen aminopeptide |
| CN108129549B (en) * | 2017-12-19 | 2020-06-05 | 浙江丰安生物制药有限公司 | Method for extracting polypeptide in spleen aminopeptide |
| CN108148111B (en) * | 2017-12-19 | 2020-06-05 | 浙江丰安生物制药有限公司 | Method for extracting polypeptide in spleen aminopeptide |
| CN113975296A (en) * | 2021-12-30 | 2022-01-28 | 北京第一生物化学药业有限公司 | Application of animal spleen extract in preparation of medicine for treating Alzheimer's disease |
| CN113975296B (en) * | 2021-12-30 | 2022-04-12 | 北京第一生物化学药业有限公司 | Application of animal spleen extract in preparation of medicine for treating Alzheimer's disease |
| CN114940698A (en) * | 2022-07-22 | 2022-08-26 | 北京第一生物化学药业有限公司 | Method for extracting acidic polypeptide from spleen aminopeptide stock solution |
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| CN101254209B (en) | 2011-02-16 |
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