CN1104095A - Pilose antler growth-factor preparation and technology for making it - Google Patents
Pilose antler growth-factor preparation and technology for making it Download PDFInfo
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- CN1104095A CN1104095A CN93121547A CN93121547A CN1104095A CN 1104095 A CN1104095 A CN 1104095A CN 93121547 A CN93121547 A CN 93121547A CN 93121547 A CN93121547 A CN 93121547A CN 1104095 A CN1104095 A CN 1104095A
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Abstract
The present invention relates to a pilose antler growth factor preparation and its production technology. It uses pilose antler as raw material and makes it pass through such processes of pulverization, digestion, suction-filtering, sterilization and chromatography etc. of filtration separation, etc. to obtain the invented polypeptide component preparation which mainly contains nerve growth factor, fibroblast growth factor and epithelial cell growth factor, etc. and has no foreign protein antigenicity. The invented product mainly is used for curing retrograde diseases of nerve system, brain trauma, anemia and diabetes. etc..
Description
The antler growth factor formulations is to be raw material with Cornu Cervi Pantotrichum, separates the preparation that mainly contains somatomedin that obtains through diafiltration, and its technology is through pulverizing the method that lixiviate degerming chromatography again goes out growth factor preparation.
Existing cell growth factor, as Liva in 1956, Montol cini isolated nerve growth factor NGF from snake venom, after have under the mice jaw arteries and veins isolate NGF, extract FGF etc. from the Medulla Bovis seu Bubali health.Cell growth factor is a class peptide class bioactive substance, has extremely strong biological activity, and human body is being brought into play cell Growth and Differentiation and regulating and controlling effect, can be used for treating nervous system and moves back the type disease, cerebral trauma, anemia and diabetes etc.The somatomedin that goes out through above-mentioned separation and Extraction also just is used for experiment.Because the growth factor preparation in these sources also has the anti-unit's property of foreign protein and lacks the safety foundation, now also do not make medicine and be used for clinical.The cell growth factor source is to be used for the difficult problem that clinical medicine is produced.Therefore occurred using gene engineering technique in the nineties, implanted in the escherichia coli with people's growth factor gene and separate the NGF preparation method again through increasing the bacterium expression.
The object of the present invention is to provide a kind of be easy to get and have safety, the effect of paying is less, can be used for clinical cell growth factor preparation and preparation method.
Cell growth factor of the present invention is the antler growth factor formulations, and it is as good as the anti-unit's property polypeptide of kind of albumen class preparation for Cornu Cervi Pantotrichum separates contain nerve growth factor (BGF), fibroblast growth factor (FGF), the epithelial cell growth factor (EGF) etc. that obtain through diafiltration.Separate the antler growth factor formulations obtain through diafiltration, its active substance mainly is that the molecular weight polypeptide form with 5000-50000 exists.For main active substances is played a role as much as possible, be main preparation so antler growth factor formulations of the present invention is preferably the polypeptide of 5000-50000 molecular weight.According to the characteristic of somatomedin in the Cornu Cervi Pantotrichum, separated preparation is that pH value is 5-9.5.Also can separate the peptide formulations that mainly contains nerve growth factor (NGF) that obtains through diafiltration with Cornu Cervi Pantotrichum.And to be preferably the 14000-15000 molecular weight peptide be main preparation.Can also be for separating mainly containing fibroblast growth factor (FGF) or mainly containing epithelial cell growth factor (EGF) peptide class preparation of obtaining through diafiltration with Cornu Cervi Pantotrichum.The pharmaceutical products that these preparations are all directly grown and regulated and control as cell.
Cornu Cervi Pantotrichum is the conventional medicament raw material, and being has multiple benefiting action, the medicinal history of existing more than one thousand years, and the medicine that goes out through lixiviate is oral and injects and all used proof to have good safety, has no side effect, and is as good as the anti-elementary reaction of kind of albumen.So isolate preparation through diafiltration based on cell growth factor with Cornu Cervi Pantotrichum, have good safety, there is not the effect of paying, be as good as the anti-elementary reaction of kind of albumen, more do not have the infection of source of disease.Particularly in the growth factor preparation that the diafiltration separation obtains, closed ganglioside in the also natural Lip river of nerve growth factor (NGF), more can promote the drug action of nerve growth factor by Cornu Cervi Pantotrichum.Can form and contain the antler growth factor formulations that close nerve growth factor and the natural Lip river of ganglioside, such preparation is active better, has formed the more efficient drug preparation.
Mostly Cornu Cervi Pantotrichum processing in the past is that drying is deposited with the steaming and decocting in boiled water of bright Cornu Cervi Pantotrichum.How to ooze extraction with steeping in wine in use, or together decoct with other raw materials, so just with the loss of the cell growth factor in the Cornu Cervi Pantotrichum, last may have very micro-cell growth factor.Even the Cornu Cervi Pantotrichum that has is taked lyophilization, but when decocting, most cell growth factor, particularly nerve growth factor, fibroblast growth factor, epithelial cell growth factor has also all lost under high-temperature.The lixiviate of useful organisms technology is isolating, and also just at aminoacid, neurospongium sphingomyelins, prostatitis arteries and veins element, male estrogen, materials such as choline polysaccharide carry out.The medicine that extraction separation goes out also just based on these materials, may only have few cell growth factor.
So the preparation method of antler growth factor formulations of the present invention is that Cornu Cervi Pantotrichum is pulverized lixiviate, active somatomedin is oozed out.Lixiviating solution goes out to contain polypeptide formulations such as nerve growth factor, fibroblast growth factor, epithelial cell growth factor through chromatography after the sucking filtration degerming.In order to make the active ingredient survival in the Cornu Cervi Pantotrichum, need deposit the back for Cornu Cervi Pantotrichum and concentrate the processing preparation, but preferably earlier that bright Cornu Cervi Pantotrichum is freezing when fresh, also lyophilization is preserved, and then pulverizes at low temperatures, generally all pulverizing below 4 ℃.For lixiviate effectively fully goes out active cell growth factor, lixiviate preferably adopts pH value to carry out lixiviate at the buffer of 5-9.5, also can be respectively carries out lixiviate respectively with the multiple pH value buffer of PH5-9.5 scope.As with the 0.05M HAC-NAC buffer of PH5.0, or with the 0.25mml/l phosphate buffer lixiviate of PH6.8, or with the Tris-HCl buffer lixiviate of PH9.0.In order to obtain nerve growth factor, fibroblast growth factor and epithelial cell growth factor, after the degerming of lixiviating solution process sucking filtration, chromatography adopts 5000D and 50000D filter membrane or molecular sieve through twice filtration, isolates the polypeptide formulations that mainly contains cell growth factor 5000-50000 molecular weight.Generally be earlier to filter out material greater than 5000D with 5000D filter membrane or sieve chromatography.Reuse 50000D filter membrane or sieve chromatography filter out the material greater than 50000D.So chromatography goes out the polypeptide formulations of 5000-50000 molecular weight, the antler growth factor formulations based on nerve growth factor, fibroblast growth factor and epithelial cell growth factor just of the present invention simply and easily.
For obtaining the antler growth factor formulations of the ganglioside that closes based on nerve growth factor or nerve growth factor and natural Lip river.Can be with after the Cornu Cervi Pantotrichum process buffer lixiviate of PH5-9.5 of pulverizing, lixiviating solution is collected concentrated solution M through the 5000D membrane filtration
1, the gleanings molecular weight is greater than 5000D.With M
1Concentrated solution upper prop CM leclutose32, the back is with containing the 0.05mol/l Tris-HCl PH9.0 eluent eluting of 0.4molNaCl, and elution process is collected the washing liquid of taking off of active peak III, IV, and reuse is collected takes off washing liquid upper prop SephadexG50.With the Tris-HCl eluent eluting that contains 0.2mol/l NaCl0.05mol/lPH9.0.Elution process is collected active peak II, obtains PH9.0-9.65, molecular weight is the nerve growth factor of 14000-15000.Perhaps, with M
1Concentrated solution upper prop CM-Cellntose32 is with the eluent eluting of 0.05M HAC-NaAC PH5.0.Elution process is collected the eluent of active III V.Upper prop SephadexG50 after the eluent lyophilizing of collecting, reuse 0.05M HAC-NaAC PH 5.0 buffer solution elution, elution process is collected the eluent of active peak II, has promptly obtained molecular weight about 15000, and PH is at 5.8 nerve growth factor preparation.
Reuse M
1The lixiviate concentrated solution is through saltouing upper prop methylol-Sephadex C50.Through the phosphate buffer eluting of 0.05M PH9.0, elution process is collected the eluent of active peak III, through lyophilizing upper prop Sepharose-heparin affinity chromatography again.With 0.05M PH9.0 phosphate buffer washing liquid, elution process is collected the eluent of active peak II, III, and then obtaining β FGF is main preparation.
The present invention compared with the prior art, antler growth factor formulations of the present invention is to the foreign protein antigenicity of human body, do not have other the effect of paying, and do not have other oxious components, has higher safety, can be widely used in clinical.The treatment nervous system is moved back the type disease, cerebral trauma, cerebrovascular injury, anemia and diabetes have better curative effect.With the antler growth factor formulations that preparation method of the present invention makes, the somatomedin loss is few, the extraction ratio height, and the growth factor content height has good active.
Further specify preparation method of the present invention and preparation below in conjunction with example.
After the Cornu Cervi Pantotrichum lyophilization with aquatic foods, pulverize at low temperatures.The Cornu Cervi Pantotrichum slurry of pulverizing is joined 4 ℃ twoly steamed in the sterilized water lixiviating solution lixiviate 24 hours.Twice of lixiviate.For the first time with the lixiviating solution that is five times in the Cornu Cervi Pantotrichum slurry.Use for the second time than primary filtering residue and Duo one times lixiviating solution.Merge twice lixiviate filtrate.Lixiviate filtrate is crossed the 5000D hollow fiber ultrafiltration membrane, collect filtering concentrated solution.The filtering and concentrating liquid of collecting this moment is for containing the gleanings of molecular weight＞5000D.Again this filtrate has been collected with the hollow fiber ultrafiltration membrane filtration of 50000D and contained the polypeptide antler growth factor formulations M of molecular weight at 5000-50000
1Can be mixed with plurality of Chinese injection, oral agents and external agent with this preparation.This diafiltration separates the preparation that obtains, and its total nitrogen content is 13.6%, than traditional preparation that adds heat extraction (total nitrogen content is 7%) to get 6.6% more.Through determination of activity, specific activity adds heat extraction and increases by 500 above.Use M
1Preparation upper prop CM-Cellulose32, the 0.05mol/L Tris-HCl buffer of reuse 0.4molNaCl carries out towards the post eluting, and buffer solution ph is 9.0.Collect the eluent of active peak III IV in elution process.With the eluent upper prop SephadesG50 that collects, the Tris-HCl buffer that reuse contains 0.2mol/L NaCl 0.05mol/L PH9.0 carries out towards the post eluting.Collect the eluent of active peak II in the elution process.This loss of thick fluid liquid then is PH9.1-9.7, mainly contains the antler growth factor formulations of molecular weight at the nerve growth factor of 14000-15000.Used active testing instrument is the 280mm Ultraviolet Detector.Said preparation obtains three bands through the electrophoretic analysis system.Through contrast, be respectively PH9.15,9.45 and 9.65, molecular weight is (NGF) nerve growth factor of about 14500.
Use M
1Preparation upper prop CM-Cellulose32, the eluent of reuse 0.05M HAC-NAC PH 5.0 is towards the post eluting.With the outer detection instrument detection of 280mm bavin, and collect the eluent of active peak IV V.The eluent of collecting after lyophilizing concentrates, upper prop SehadexG 50, reuse 0.05M HAC-NaACPH5.0 buffer solution elution is collected the eluent of active peak II.The eluent of collecting is just for containing the NGF(nerve growth factor) the antler growth factor formulations.Analyze after tested, the molecular weight of the NGF of said preparation is about 15000, and pH value is 5.8, contains 122 aminoacid.
Use M
1Preparation is analysed through sulfuric acid amine salt, upper prop methylol-Sephadex C50 again, and with 0.05M PH9.0 phosphate buffer eluting.Collect the eluent of active peak III.The eluent of collecting after lyophilizing concentrates, upper prop Sepharose-heparin affinity chromatography, and, collect the eluent of active peak II III with 0.05M PH9.0 phosphate buffer eluting.This has just obtained containing the FGF(fibroblast growth factor) Cornu Cervi Pantotrichum factor growth preparation.Said preparation is analyzed after tested, contains the fibroblast growth factor of β FGF.
Another example carries out lixiviate for bright Cornu Cervi Pantotrichum lyophilization is ground into pulpous state again with two steamings or PH9.0Tris-HCl buffer 0.05mol/L.With lixiviating solution through the degerming of G6 funnel sucking filtration, upper prop CM-Cellulose32 again.With the 0.05mol/L PH9.0Tris-HCl buffer solution elution that contains 0.4mol NaCl.The eluent of collecting concentrates or filters with the 1000D hollow fiber ultrafiltration membrane through lyophilizing, obtains the antler growth factor formulations.Said preparation is through the test of SDS-polyacrylamide gel electric ice, and visible the district more than 4 is with, and through this is analyzed, between the PH9.1-9.7 is NGF, and between the PH9.5-10.0 is bFGF.
Claims (10)
1, the antler growth factor formulations is the pharmaceutical products of a kind of cell growth and regulation and control, it is characterized by by Cornu Cervi Pantotrichum to separate the preparation that nerve growth factor (NGF), fibroblast growth factor (FGF), epithelial cell growth factor (EGF) etc. are as good as the anti-unit's property polypeptide of kind of albumen composition that contains that obtains through diafiltration.
2, as the said antler growth factor formulations of claim 1, the molecular weight that it is characterized by the polypeptide composition is 5000-50000.
3, as claim 1 or 2 said antler growth factor formulations, it is characterized by PH is 5-9.5.
4, the antler growth factor formulations is the pharmaceutical products of a kind of cell growth and regulation and control, it is characterized by by Cornu Cervi Pantotrichum and separates the preparation that contains nerve growth factor (NGF) peptide that obtains through diafiltration.
5,, it is characterized by and contain the ganglioside that closes with the nerve growth factor Lip river as the said antler growth factor formulations of claim 4.
6, as claim 4 or 5 said antler growth factor formulations, the molecular weight that it is characterized by somatomedin is 14000-15000.
7, the antler growth factor formulations is the pharmaceutical products of a kind of cell growth and regulation and control, it is characterized by by Cornu Cervi Pantotrichum and separates preparation that contains fibroblast growth factor (FGF) peptide that obtains or the preparation that contains epithelial cell growth factor (EGF) peptide through diafiltration.
8, the preparation method of antler growth factor formulations is that Cornu Cervi Pantotrichum is pulverized lixiviate, and lixiviating solution is degerming after filtration, goes out to contain the preparation of polypeptide such as nerve growth factor, fibroblast growth factor, epithelial cell growth factor again through chromatography.
9,, it is characterized in that Cornu Cervi Pantotrichum earlier through lyophilization, at low temperatures pulverizing as the said preparation method of claim 8.
10, as claim 8 or 9 said preparation methoies, it is characterized in that: lixiviate is with the lixiviate respectively of the buffer of PH5-9.5, and chromatography filters out the polypeptide antler growth factor formulations of molecular weight at 5000-50000 for twice with 5000D and 50000D filter membrane or molecular sieve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93121547A CN1104095A (en) | 1993-12-24 | 1993-12-24 | Pilose antler growth-factor preparation and technology for making it |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93121547A CN1104095A (en) | 1993-12-24 | 1993-12-24 | Pilose antler growth-factor preparation and technology for making it |
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| CN1104095A true CN1104095A (en) | 1995-06-28 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN93121547A Pending CN1104095A (en) | 1993-12-24 | 1993-12-24 | Pilose antler growth-factor preparation and technology for making it |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004112806A1 (en) * | 2003-06-26 | 2004-12-29 | Canada Changmin Nutraceutique Co. Ltd | Composition for treating or preventing neural disorders |
| CN100339387C (en) * | 2004-12-01 | 2007-09-26 | 焦安龙 | Method for preparing antler polypeptide and medicine with the antler polypeptide as main active component |
| CN101020715B (en) * | 2007-03-15 | 2010-05-26 | 王利忠 | Process of extracting and preparing deer nerve growth factor (DEER NGF) |
| CN104288180A (en) * | 2014-09-10 | 2015-01-21 | 河南辅仁堂制药有限公司 | Preparation method of pilose antler pure powder |
| CN104987358A (en) * | 2015-08-10 | 2015-10-21 | 吉林玉参医药科技有限公司 | Method for preparing velvet antler protein extracts |
| CN104987359A (en) * | 2015-08-10 | 2015-10-21 | 吉林玉参医药科技有限公司 | Velvet antler protein extract and application and medicinal preparation thereof |
-
1993
- 1993-12-24 CN CN93121547A patent/CN1104095A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004112806A1 (en) * | 2003-06-26 | 2004-12-29 | Canada Changmin Nutraceutique Co. Ltd | Composition for treating or preventing neural disorders |
| CN100339387C (en) * | 2004-12-01 | 2007-09-26 | 焦安龙 | Method for preparing antler polypeptide and medicine with the antler polypeptide as main active component |
| CN101020715B (en) * | 2007-03-15 | 2010-05-26 | 王利忠 | Process of extracting and preparing deer nerve growth factor (DEER NGF) |
| CN104288180A (en) * | 2014-09-10 | 2015-01-21 | 河南辅仁堂制药有限公司 | Preparation method of pilose antler pure powder |
| CN104987358A (en) * | 2015-08-10 | 2015-10-21 | 吉林玉参医药科技有限公司 | Method for preparing velvet antler protein extracts |
| CN104987359A (en) * | 2015-08-10 | 2015-10-21 | 吉林玉参医药科技有限公司 | Velvet antler protein extract and application and medicinal preparation thereof |
| CN104987359B (en) * | 2015-08-10 | 2018-11-20 | 吉林玉参医药科技有限公司 | A kind of pilose antler protein extract and its application, pharmaceutical preparation |
| CN104987358B (en) * | 2015-08-10 | 2018-11-20 | 吉林玉参医药科技有限公司 | A kind of preparation method of pilose antler protein extract |
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