CN1698653A - Injection of calf blood de-protein extract and preparation method thereof - Google Patents
Injection of calf blood de-protein extract and preparation method thereof Download PDFInfo
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- CN1698653A CN1698653A CNA2005100769147A CN200510076914A CN1698653A CN 1698653 A CN1698653 A CN 1698653A CN A2005100769147 A CNA2005100769147 A CN A2005100769147A CN 200510076914 A CN200510076914 A CN 200510076914A CN 1698653 A CN1698653 A CN 1698653A
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Abstract
The invention discloses an injection of calf blood de-protein extract and preparation method, wherein 1ml of the injection comprises solid 41-60mg, 55-65wt% of inorganics, 35-45wt% of organic active compound. The making process comprises the following steps, (1) stirring carf blood or serum, charging sodium citrate into the plasma, homogenizing and storing by refrigeration, separating and collecting plasma for later use, (2) passing the plasma through pearl form chitosan chromatography column, charging ethanol, stirring and centrifuging the protein precipitate, reclaiming ethanol from the supernatant fluid, charging proteinases, enzymolyzing, (3) charging distilled water into enzymolysis liquid, heating and filtering with chitosan modified activated charcoal column, (4) passing the filter liquor through modified activated charcoal column, passing through microporous filtration film under pressurization, loading, sealing, finally carrying out hot-pressed sterilization.
Description
Technical field
The present invention relates to a kind of injection, relate in particular to a kind of calf blood protein-removed extraction injection and preparation method thereof, belong to drug world.
Background technology
Calf blood protein-removed extraction (solcoseryl) has been applied in countries in the world over 50 years since nineteen fifty-five, plain high pharmaceutical factory was developed successfully by Switzerland.Its injection contains 40～50 milligrams of calf blood protein-removed extractions for every milliliter, and wherein 30% is that aminoacid, saccharide, keto acid, purine, nucleotide and relative molecular weight are 2500～3000 oligopeptides material, and the 70%th, inorganic constituents.Calf blood protein-removed extraction can significantly improve the reticuloendothelial system vigor, improves enzymatic activity, quickens the oxidative phosphorylation reaction of cell and synthesizing of ATP, and active cell is breathed, and enhances metabolism, and accelerates tissue regeneration.Be mainly used in diseases such as treatment senile dementia, acute and chronic cerebrovascular clinically,, some diseases of spinal cord and peripheral blood vessel occlusive disease etc. had good efficacy, nontoxic and better tolerance especially to organic mental disorders such as apoplexy, craniocerebral trauma.
At present, calf blood protein-removed extraction is mainly by calf blood being concentrated, being prepared from after ultrafiltration or the dialysis, and is normal because step is many, and the time is long in the Deproteinization operation of key, and it is more to make active ingredient lose damage.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, and a kind of calf blood protein-removed extraction injection is provided, and this injection preparation technology is simple and direct, active constituent content is high, biological activity is high.
A kind of calf blood protein-removed extraction injection is characterized in that containing solid content 41～60mg in every milliliter of this injection, and 55%～65% weight is inorganic matter in this solid content, and 35%～45% weight is organic active substance.Preferably, the inorganic matter in this injection is for being selected from K
+, Na
+, Cl
-And Ca
2+In a kind of or its mixture, the organic active substance in this injection is for being selected from aminoacid, saccharide, keto acid, purine, nucleotide, a kind of or its mixture in the blood polypeptide.
Calf blood protein-removed extraction injection of the present invention can prepare by following method:
A kind of calf blood protein-removed extraction injection mainly is prepared from by following method:
1) with calf blood or serum by a direction revolve first quick and back slow stir after, take off the gluey agglomerate that twines on the stirring arm, in blood plasma, add sodium citrate and stir evenly the back in 0～4 ℃ of stored refrigerated 1～30 hour, skim the top layer oil film after, tell blood cell, it is standby to collect blood plasma;
2) with resulting blood plasma in the step 1) by pearl chitosan chromatographic column, the blood plasma of collecting behind the post is standby;
3) add ethanol in the blood plasma behind above-mentioned post excessively, stirring, the centrifugal albumen precipitation of removing, supernatant are under 75～85 ℃, and standardize solution reclaims ethanol, adds protease, enzymolysis 10～38h;
4) in enzymolysis solution, add 0.4～0.6 times of (V/V) distilled water, be heated to 75～85 ℃, filter filtrate for later use with the good chitin modified activated-charcoal column of activation in advance while hot;
5) above-mentioned filtrate is concentrated after again by the modified activated carbon post, add and depressed microporous filter membrane, the fill sealing by fusing, pressure sterilizing, promptly.
In the above-mentioned preparation method, wherein in the step 1) calf blood or serum preferably revolved by a direction first quick and back slow with the shaggy stirring arm after the sterilization and stir 15～20min; Add the sodium citrate of 3% weight in the blood plasma in the step 1) after stirring in 0～4 ℃ of stored refrigerated 12～30h, step 2) in blood plasma by 1～3ml/min.cm
2Flow rates is by pearl chitosan chromatographic column; The ethanol that adds 0.5 times of amount (v/v) 95% in the blood plasma in the step 3) after crossing post, the centrifugal albumen precipitation of removing, supernatant reclaims ethanol at 80 ℃ of following standardize solution, preferably is concentrated to the blood plasma identical volume preceding with crossing post and adds protease again, enzymolysis 24h; The weight that adds protease in the step 3) is 1～3% of supernatant, at 45～50 ℃ of following enzymolysis; In enzymolysis solution, add 0.5 times of (V/V) distilled water in the step 4), be heated to 80 ℃ and cross post more while hot; In the step 5) filtrate is concentrated into behind 2/3 volume again by the modified activated carbon post, adds and depressed the 0.2um microporous filter membrane.
Extracting method of the present invention is owing to adopt the technology of absorption, and there is the protease of adding to carry out this effective protein degradation step of enzymolysis, can effectively remove multiple protein contained in the calf blood rapidly, increase so the content of its active ingredient is also corresponding in the injection of the present invention, biological activity is higher relatively.
Further describe beneficial effect of the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit the scope of the invention.
The specific embodiment
[embodiment 1]
Gather totally 10 liters in the venous blood of 6 months calves, put in the wide-mouth Plastic Drum of sterilization back, stir 5min soon with shaggy stirring paddle clockwise direction is first, stir 10min then slowly, mention stirring arm, the clean blood plasma of drop removes gluing agglomerate on the stirring paddle; Residue blood plasma adds 30 gram sodium citrate and stirs evenly, and after 4 ℃ of cold preservations were spent the night, skimmed the top layer oil film, the centrifuge tube of packing into was told blood cell with the centrifugal 15min of 3000r/min speed, leached upper plasma with double gauze and got 9.5 liters approximately; By pearl chitosan chromatographic column (blood volume: the chitosan amount is 10: 1), flow speed control is at 3ml/min.cm with blood plasma
2, colourless with 500ml distillation washing post to effluent, get 10 liters of chromatographic solutions; The ethanol that adds 5 liter 95% in the blood plasma under stirring behind chromatography stirs 15min, and the centrifuge tube of packing into the centrifugal 15min of 5000r/min speed, is removed albumen precipitation; Supernatant reclaims 5 liters of ethanol down at 80 ℃, surplus 10 liters of distillate; Distillate is transferred in the hydrolytic decomposition pot, when being cooled to 45 ℃, adds 25 gram papains, stirs evenly insulation hydrolysis 24h; In hydrolyzed solution, add 5 liters of distilled water dilutings, be heated to 80 ℃, act while hot prior to the chitin modified activated carbon powder post filtration of 120 ℃ of activation 4h.(liquid: charcoal is 10: 1), filtrate is concentrated into 10 liters, once more by the modified activated carbon post; With spray pump filtrate was pressed the 0.2um microporous filter membrane, fill, 2ml/ props up, sealing by fusing, 115 ℃ of pressure sterilizing 30min preserve in 4 ℃ of refrigerators.
[embodiment 2]
Gather 15 liters in 6 monthly age calf venous blood, put in the wide mouthed bottle after the sterilization, stir 5min soon with shaggy stirring paddle clockwise direction, slowly stir 10min then, mention the clean blood plasma of stirring paddle drop, remove the gluing agglomerate on the stirring paddle, adding 45 gram sodium citrate in the residue blood plasma stirs, spend the night in 4 ℃ of cold preservations, after skimming the oil film on top layer morning next day, the centrifuge tube of packing into leaches upper plasma with double-deck husky cloth and gets 14.2 liters (holding back blood cell) approximately with the centrifugal 15min of 3000r/min speed, blood plasma is crossed pearl chitosan layer folding post (plasma volume: chitosan is 10: 1), and flow speed control is at 2ml/min.cm
2, adding the 800ml distilled water, wash post from the top colourless to effluent, 15 liters of layers folding liquid.Stir the ethanol that behind this chromatography, adds 75 liter 95% down in the blood plasma, stir 15min, the centrifuge tube of packing into, with 5000r/min speed from 15min, remove albumen precipitation, supernatant reclaims 7.5 liters of ethanol down, surplus 15 liters of distillate in 85 ℃, this distillate is transferred in the hydrolytic decomposition pot, when waiting to be cooled to 40 ℃, add trypsin 45 grams, stir evenly, keep 40 ℃ of hydrolysis 24h, 7.5 liters of distilled water of adding are heated to 85 ℃ while hot with the good chitin modified activated carbon powder filtration of activation in advance in hydrolyzed solution.(liquid: charcoal is 10: 1).Filtrate vacuum concentration to 15 liter filters by the modified activated carbon post once more, and with the spray pump 0.2um microporous filter membrane that pressurizeed, fill 2ml/ props up, and sealing by fusing is preserved in 4 ℃ of refrigerators behind 115 ℃ of pressure sterilizing 30min.
[embodiment 3]
Gather 20 liters in 6 monthly age calf venous blood, put in the wide mouthed bottle after the sterilization, stir 5min with shaggy stirring paddle soon by direction, slowly stir l0min then, mention the clean blood plasma of stirring paddle control, discard the gluing group on the oar, add 60 gram sodium citrates in the bottle in the blood plasma and stir evenly the back more than 4 ℃ of cold preservation 12h, after removing the top layer oil film then, the centrifuge tube of packing into is with the centrifugal 15min of 3000r/min speed, filter with double gauze, hold back blood cell and discard, supernatant blood plasma must be about 19 liters, and this blood plasma is adsorbed foreign protein (plasma volume: chitosan is 10: 1) flow speed control at 1ml/min.cm by pearl chitosan chromatographic column
2It is colourless to effluent to get 1 liter of distillation washing post, gets 20 liters of chromatographic solutions.Add 10 liter 95% ethanol in the blood plasma behind chromatography while stirring again, after continuing to stir 15min, the centrifuge tube of packing into is with the centrifugal 15min of 5000r/min speed, filtering bottom precipitation, supernatant reclaims down ethanol to 10 liter at 75 ℃, and surplus distillate is 20 liters, is transferred in the hydrolytic decomposition pot, when being cooled to 40 ℃, add compound protease 60 grams, stir evenly, be incubated 40 ℃ of hydrolysis 24h, in hydrolyzed solution, add 10 liters, distilled water is heated to 80 ℃ of 0.5h, uses chitin modified activated carbon powder post (prior to 120 ℃ of activation 4h) to filter (liquid: charcoal is 10: 1) while hot, filtrate vacuum concentration to 20 liter, after a modified activated carbon post, descended 0.2um microporous filter membrane, fill with the spray pump pressurization at last, 2ml/ props up, sealing by fusing, 115 ℃ of pressure sterilizing 30min store in 4 ℃ of refrigerators.
[test example] injection active constituent content of the present invention detects
One, test material:
1, for test agent: the injection that the embodiment of the invention 1 is prepared.
2, control sample: commercially available calf blood protein-removed extraction injection.
Two, detection method:
1, detecting instrument
Ten thousand/balance, amino-acid analyzer, weigh bang, vacuum pump, electric oven, crucible, alcohol burner.
2, detection method
Total solids content: precision measure sample 2ml to weighing bang at 53 ± 3 ℃ of following vacuum dryings, calculate to constant weight (2000 editions 2 appendix VIII L of Chinese Pharmacopoeia).
Free chloro acid: get the acid of this product and standard chloro in right amount, analyze with chloro acid analysis instrument.
Inorganic salt: dry total solid matters residue on ignition method, ashing is weighed later on.Small organic molecule: the amount of total solid matters deducts the inorganic salt amount, obtains small organic molecule.
Three, testing result
Each active constituent content testing result of sample sees Table 1.
Table 1 injection active constituent content of the present invention testing result
| Detect liquid measure | The total solid dry weight | ??Na +、K +、Ca +、CC -Deng inorganic ions | Nucleotide, aminoacid micromolecule polypeptide etc. | |
| For test agent | ????2ml | ??82.1mg | ??59.2％ | ??40.8％ |
| Control sample | ????2ml | ??80mg | ??69％ | ??31％ |
The testing result explanation, the prepared injection of the present invention contains the higher effective composition, compares with control sample, and its content of effective obviously will be higher than control sample.
[test example] calf blood (protein removed) extracting solution biological activity test of the present invention
Calf blood (protein removed) extracting solution of the present invention is the Cellular respiration activator, can promote picked-up and the utilization of cell to oxygen.Breathe inspection by the WARBURG trace and press instrument to measure it promotes Cellular respiration in the Cavia porcellus hepatocyte vigor, calculate its biological activity by the oxygen consumption recruitment.Measure the biological activity of calf blood (protein removed) injection liquid samples, compare, find the QO of calf blood (protein removed) extracting solution of the present invention with commercially available calf blood (protein removed) extracting solution sample
2(μ lO
2/ mgh) all being higher than 6.40, the corresponding numerical value of reference substance all is lower than 5.20.
Claims (11)
1, a kind of calf blood protein-removed extraction injection is characterized in that containing solid content 41～60mg in every milliliter of this injection, and wherein 55%～65% weight is inorganic matter in this solid content, and 35%～45% weight is organic active substance.
2, according to the calf blood protein-removed extraction injection of claim 1, wherein said inorganic matter is selected from K
+, Na
+, Cl
-And Ca
2+In a kind of or its mixture.
3, according to the calf blood protein-removed extraction injection of claim 1, wherein organic active substance is selected from a kind of or its mixture in aminoacid, saccharide, keto acid, purine, nucleotide, the blood polypeptide.
4, a kind of method for preparing the calf blood protein-removed extraction injection of claim 1 may further comprise the steps:
1) with calf blood or serum by a direction revolve first quick and back slow stir after, gluey agglomerate is removed, add sodium citrate in the blood plasma after stirring, stir evenly the back in 0～4 ℃ of stored refrigerated 1～30h, skim the top layer oil film after, tell blood cell, it is standby to collect blood plasma;
2) with resulting blood plasma in the step 1) by pearl chitosan chromatographic column, the blood plasma of collecting behind the post is standby;
3) add ethanol in the blood plasma behind above-mentioned post excessively, stirring, the centrifugal albumen precipitation of removing, supernatant reclaims ethanol at 75～85 ℃ of following standardize solution, adds protease, enzymolysis 10～38h;
4) in enzymolysis solution, add 0.4～0.6 times of (V/V) distilled water, be heated to 75～85 ℃, filter filtrate for later use with the good chitin modified activated-charcoal column of activation in advance while hot;
5) above-mentioned filtrate is concentrated after again by the modified activated carbon post, add and depressed microporous filter membrane, the fill sealing by fusing, pressure sterilizing, promptly.
5, according to the described preparation method of claim 1, it is characterized in that: in the step 1) calf blood or serum revolved by a direction first quick and back slow with shaggy stirring arm and stir 15～20min.
6, according to the described preparation method of claim 1, it is characterized in that: add the sodium citrate of 3% weight in the blood plasma in the step 1) after stirring, stir evenly the back in 0～4 ℃ of stored refrigerated 12～30h.
7, according to the described preparation method of claim 1, it is characterized in that: step 2) in blood plasma by 1～3ml/min.cm
2Flow rates is by pearl chitosan chromatographic column.
8, according to the described preparation method of claim 1, it is characterized in that: the ethanol that adds 0.5 times of amount (v/v) 95% in the blood plasma in the step 3) after crossing post, the centrifugal albumen precipitation of removing, supernatant reclaims ethanol at 80 ℃ of following standardize solution, keep the blood plasma identical volume preceding with crossing post, add protease, enzymolysis 24h.
9, according to the described preparation method of claim 1, it is characterized in that: the weight that adds protease in the step 3) is 1～3% of supernatant, at 45～50 ℃ of following enzymolysis.
10, according to the described preparation method of claim 1, it is characterized in that: in enzymolysis solution, add 0.5 times of (V/V) distilled water in the step 4), be heated to 80 ℃ and cross post more while hot.
11, according to the described preparation method of claim 1, it is characterized in that: in the step 5) filtrate is concentrated into behind 2/3 volume again by the modified activated carbon post, adds and depressed the 0.2um microporous filter membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100769147A CN100396291C (en) | 2005-06-09 | 2005-06-09 | Injection of calf blood de-protein extract and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100769147A CN100396291C (en) | 2005-06-09 | 2005-06-09 | Injection of calf blood de-protein extract and preparation method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1698653A true CN1698653A (en) | 2005-11-23 |
| CN100396291C CN100396291C (en) | 2008-06-25 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100374123C (en) * | 2006-05-15 | 2008-03-12 | 锦州奥鸿药业有限责任公司 | Method for extracting and purifying calf serum protein-removing extract |
| CN101230090B (en) * | 2007-01-24 | 2011-11-02 | 中国生化制药工业协会 | Blood peptide X and derivative thereof as well as preparation method and use thereof |
| CN104940237A (en) * | 2015-07-15 | 2015-09-30 | 符健 | Crocodile blood protein removing extracting object and extracting method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062135C (en) * | 1996-11-27 | 2001-02-21 | 锦州医学院科技开发部 | Method for extracting deproteinized calf blood extract |
| CN100352450C (en) * | 2004-01-12 | 2007-12-05 | 江勇 | Albumen removed extraction of calf blood for injection and its preparing process |
| CN1286471C (en) * | 2004-07-08 | 2006-11-29 | 兆科药业（合肥）有限公司 | Calf blood deproteinizing extract, its eye ointment preparation and its preparing method |
-
2005
- 2005-06-09 CN CNB2005100769147A patent/CN100396291C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100374123C (en) * | 2006-05-15 | 2008-03-12 | 锦州奥鸿药业有限责任公司 | Method for extracting and purifying calf serum protein-removing extract |
| CN101230090B (en) * | 2007-01-24 | 2011-11-02 | 中国生化制药工业协会 | Blood peptide X and derivative thereof as well as preparation method and use thereof |
| CN104940237A (en) * | 2015-07-15 | 2015-09-30 | 符健 | Crocodile blood protein removing extracting object and extracting method and application thereof |
| CN104940237B (en) * | 2015-07-15 | 2019-12-27 | 符健 | Crocodile blood deproteinized extract and extraction method and application thereof |
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| CN100396291C (en) | 2008-06-25 |
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Assignee: Hubei Renmin Pharmaceutical Co.,Ltd. Assignor: Zhao Hongmei Contract fulfillment period: 2009.7.1 to 2011.12.31 contract change Contract record no.: 2009990001246 Denomination of invention: Injection of calf blood de-protein extract and preparation method thereof Granted publication date: 20080625 License type: General permission Record date: 20091111 |
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