CN102718857A - Denatured protein powder and brain protein hydrolyzate prepared from same - Google Patents
Denatured protein powder and brain protein hydrolyzate prepared from same Download PDFInfo
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- CN102718857A CN102718857A CN201210235771XA CN201210235771A CN102718857A CN 102718857 A CN102718857 A CN 102718857A CN 201210235771X A CN201210235771X A CN 201210235771XA CN 201210235771 A CN201210235771 A CN 201210235771A CN 102718857 A CN102718857 A CN 102718857A
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Abstract
The invention relates to a denatured protein powder and a brain protein hydrolyzate prepared from the same. The total nitrogen content of the denatured protein powder is more than 120 mg/g; and the denatured protein powder is prepared by adding fresh lysozyme into a pig brain, homogenating, heating, and defatting with acetone. The denatured protein powder, as a raw material, is used for preparing the brain protein hydrolyzate; the contents of amino acids in the prepared brain protein hydrolyzate meet national standards of brain protein hydrolyzate injections; and the content of small molecule peptides with the molecular weight of less than 10000 Da is 25-35% of the brain protein hydrolyzate. The preparation method of the brain protein hydrolyzate mainly comprises the following steps: pre-treating, homogenizing, defatting with the acetone, performing dual enzymatic hydrolysis, centrifugating, refining by column chromatography, and performing ultrafiltration treatment. The invention discloses key technical points for controlling the total nitrogen index of the protein powder and the content of the small molecule peptides for the first time; the obtained brain protein hydrolyzate is a biologically active substance which is really extracted from the pig brain, without adding active components artificially, thus ensuring the efficacy and the safety of the brain protein hydrolyzate; and animal pharmacodynamic experiments show that the brain protein hydrolyzate is more effective than conventional products.
Description
Technical field
The invention belongs to the biochemical drug preparing technical field; The active brain polypeptide series products that relates to a kind of preparation method of active brain polypeptide class material and adopt this method to prepare, the Cerebrolysin Vial of more specifically saying so a kind of metaprotein powder and adopting this protein powder to prepare.
Background technology
Cerebrolysin Vial class medicine is to use one of maximum biochemical drug in recent years.Occupy critical role on Related Research Domain and the market at home and abroad.Cerebrolysin Vial is as healthy, the fresh animal cerebral tissue a kind of active brain polypeptide class material through the multienzyme enzymolysis and extraction; Contain important elements such as necessary amino acid of multiple human brain and kephalin, Yelkin TTS, peptide class NGFF; Can act on nervus centralis in many ways, regulate and improve neuronic metabolism, promote the formation of cynapse; Induce neuronic differentiation, and further neuroprotective cell is avoided the infringement of various ischemics and neurotoxin.It can pass through hemato encephalic barrier, promotes the synthetic of brain internal protein, influences respiratory chain, has the protective capability of anoxia, improves the metabolism of brain self-energy, and other hormone systems of activated adenyl cyclase catalysis provide neurotransmitter, peptide hormone and coenzyme precursors.At present; Mainly adopt Cerebrolysin Vial to process various formulations clinically; Be used for the dull-witted and multiple diseases such as disordered brain function, cerebral dysgenesis, encephalatrophy, neurasthenia, combined external head injuries, central nervous system infection, meningitis, senile dementia, dysthymia disorders, psychosis or cerebrovascular Metabolic disorder of treatment; Simultaneously can effectively strengthen learning memory and physical stress ability, evident in efficacy and safe without toxic side effect.Therefore, further R and D Cerebrolysin Vial class medicine is to curing the sickness to save the patient and to improve patient's life quality significant.
The above significant curative effect of Cerebrolysin Vial; Except 16 seed amino acids that wherein contain, the protein micromolecule polypeptide that obtains of complete hydrolysis not in other the component, especially preparation process; The important pharmacological action of same performance; Numerous research now shows that animal brain proteolysate class medicine can significantly strengthen rat to the anoxybiotic tolerance, and the mixture of the multiple amino acids of homogeneity does not then have this effect; Experiment proof Cerebrolysin Vial is similar with NGFF (NGF) to the neurocyte effect, but different be that NGF is the giant molecule peptide, and Cerebrolysin Vial is a micromolecule polypeptide; By contrast; The latter can pass through hemato encephalic barrier, better brings into play therapeutic action, that is to say; The curative effect of micromolecule polypeptide is that amino acid is irreplaceable, their common guarantee the clinical efficacy of Cerebrolysin Vial.Therefore the concrete moity of Cerebrolysin Vial greatly influences its curative effect, and the quality of control Cerebrolysin Vial is to guarantee the clinical effective basis safe in utilization of this product.
The Cerebrolysin Vial quality controlling means mainly is to measure the content of micromolecule polypeptide (molecular weight is less than 10000Da) and multiple amino acids in the medicine (national standard of its injection is WS
1-(X-018)-200lz).The product of producing had both needed 16 seed amino acid content after the control hydrolysis, also need control the content of incomplete hydrolysate micromolecule polypeptide, arriving rational content and proportioning, thereby guaranteed the curative effect and the security of medicine.Why product has stipulated the content range of 16 seed amino acids; A large amount of clinical trials of carrying out when being based on this medicine of exploitation fully prove that this content and proportioning each other are scientific and reasonable, can play best result of treatment; Simultaneously, clinical use does not have untoward reaction basically.So in case these active constituent contents are not inconsistent in the product, ratio is not inconsistent, all can influence the result of treatment and the security of medicine.
Because the Cerebrolysin Vial product is a multi-component biochemical drug; Its production technique is very complicated; The final composition of the Cerebrolysin Vial of different process production has nothing in common with each other; For example all there are very big influence used proteolytic enzyme kind, enzyme amount and the hydrolysis temperature of pig brain hydrolysis, time etc. to the proteic hydrolysis degree of brain, thereby influence the moity of finished product.Because technical threshold, even the many producers that ratified to produce still can not produce specification product, many producers have to abandon produce.The enterprise that has is because self-technique is immature, and product does not reach the national standard of injection, but orders about from interests; In order to obtain the product that is up to the standards, just directly in product, add corresponding amino acid, allotment content is to up to specification; This artificial effective constituent way of adding itself is violated pharmaceutical production legal provisions (GMP regulation), and more serious consequence is: do not pass through real extraction production stage, the Cerebrolysin Vial effective constituent micromolecule polypeptide that obtains will reduce even lose; Further cause the decline of pharmaceutical effectiveness; Not only do not have therapeutic action, even delay patient and in time treat, cause more serious clinical consequences.For this reason; Country's dispatch on December 15th, 2008: state's food medicine prison is done [2008] No. 734 " about strengthening the notice of brain protein hydrolysate injection supervision and check " files and has been proposed this problem especially; Also stopped simultaneously the production of more than 100 authentication code of domestic all brain protein hydrolysate injections, do not resumed production so far.
Can find the Cerebrolysin Vial relevant report in recent years both at home and abroad has a lot, and at home and abroad the particular content of relevant production technique is less in the relevant report.Existing preparation technology is also very imperfect, can't obtain meeting the product of injection national standard.The brain protein product itself that the technology that many patents are described obtains also all can not reach the national standard of injection, and for example patent 94104516.1; 200910071802.0; 200410073403.5 other patent even the brain protein hydrolysate that directly indicates after extracting also need extra interpolation aminoacid component, just can reach the national standard of injection, for example patent 200710055421.4; 200610065169.0; 200410022091.5 or the like, the characteristics of this several patents all are that pre-treatment, homogenate, degreasing (acetone, ether, propyl carbinol), single enzyme or double-enzyme hydrolysis, centrifugal, ultrafiltration, column chromatography are refining, sterilization obtains; The subject matter of its existence is the Cerebrolysin Vial that obtains according to these technologies and does not meet in the brain protein hydrolysate injection national standard the content of amino acids requirement.
Because the content of micromolecule polypeptide is related with aminoacids content in the Cerebrolysin Vial, under the certain situation of total nitrogen and aminoacids content, the content of small-molecular peptides is also confirmed.Can't confirm the content of activeconstituents micromolecule polypeptide behind the artificial interpolation amino acid especially.Its result can cause this effective constituent of micromolecule polypeptide in the product out of control fully.
Preparation technology sees from Cerebrolysin Vial: existing reported method all has the step of glue homogenate, enzymic hydrolysis; What have also adds chromatography purified process step; But the processing condition that adopt all have nothing in common with each other; Therefore the Cerebrolysin Vial product fraction that causes obtaining has nothing in common with each other, and the product of domestic report does not carry out clearly wherein micromolecule polypeptide content yet.The big pharmaceutical factory of the Austrian Yi Biwei of external like product is the producer that develops this product the earliest, and its product is called Cerebrolysin, but does not have the related process report so far.In sum; From the patent of disclosed Cerebrolysin Vial production technique, can find out; The production technology difficulty of this product is very big, and the key issue of Cerebrolysin Vial existence at present is how to guarantee prepared Cerebrolysin Vial aminoacids content in brain protein hydrolysate injection national Specification scope, and the content of micromolecule polypeptide is high as much as possible under the qualified prerequisite of aminoacids content simultaneously; Guaranteeing security of products and curative effect, even improve its curative effect.
To solve the above problems at present; Key is through metaprotein total nitrogen content (more than the 120mg/g) in the control preparation process; And explore suitable preparation technology and condition; Thereby obtain the high Cerebrolysin Vial of national standard, content of peptides that aminoacids content meets injection, and the pertinent literature of this respect does not appear in the newspapers as yet.The present invention has proposed total nitrogen index and the molecular weight of the control protein powder key technical index less than 10000Da micromolecule polypeptide content for the first time.
Summary of the invention
Main purpose of the present invention is to provide the metaprotein powder and adopts this protein powder to prepare Cerebrolysin Vial.For this reason:
One object of the present invention has been to disclose a kind of metaprotein powder that adopts certain conditions to obtain.
Another object of the present invention has been to disclose a kind of preparation method of metaprotein powder.
A further object of the present invention has been to disclose the purposes of metaprotein powder in preparation Cerebrolysin Vial medicine.
A the present invention also purpose has been to disclose a kind of Cerebrolysin Vial that adopts the preparation of metaprotein powder.
A the present invention also purpose has been to disclose a kind of method that adopts the metaprotein powder to prepare Cerebrolysin Vial.
A the present invention also purpose has been to disclose the purposes of Cerebrolysin Vial in preparation treatment dementia and disordered brain function medicine.
The Cerebrolysin Vial of the present invention's preparation can reach the national standard that aminoacids content meets injection, and micromolecule polypeptide content is up to 25-35%.
Cerebrolysin Vial of the present invention is to obtain with a kind of specific midbody metaprotein powder.The original ground of the present invention is through handle the pig brain with N,O-Diacetylmuramidase under given conditions; Obtained the metaprotein powder that a kind of total nitrogen content is higher than 120mg/g; Use this protein powder as midbody then; Handle under given conditions, meet national injection standard, the Cerebrolysin Vial that micromolecule polypeptide content is high thereby finally obtained each aminoacids content.
One embodiment of the invention relate to the metaprotein powder, and total nitrogen content of this metaprotein powder is more than the 120mg/g; It is through in fresh pig brain, adding N,O-Diacetylmuramidase, through just mix, heating degreasing, acetone degreasing obtain.Its preparation process is following:
(1) preparation just mixes thing: adopt fresh pig brain tissue an amount of, water cleans up the water of dehematizing, and every kg cerebral tissue adds 0.1～1.0g N,O-Diacetylmuramidase and is mixed together stirring colloidal mill or refiner homogenate, through 4～6 homogenate, obtains just mixing thing;
(2) degreasing: will just mix thing, heated and boiled 0.5～1h is cooled to normal temperature, adds 10～20 times of (weight) acetone then, stirs 0.5～1h; Filter, Air drying, obtaining total nitrogen content is the above metaprotein powder of 120mg/g, preserves below-20 ℃.
The present invention further discloses the purposes of metaprotein powder in preparation Cerebrolysin Vial medicine.
The present invention also discloses the Cerebrolysin Vial that adopts the preparation of metaprotein powder simultaneously, and it is characterized in that: each aminoacids content is following in the Cerebrolysin Vial: Aspartic Acid 2.4-3.6mg/ml, Serine 0.21-0.39mg/ml; L-glutamic acid 3.2-4.8mg/ml, glycocoll 1.2-1.8mg/ml, Histidine 1.04-1.56mg/ml; L-arginine 0.3-1.1mg/ml, Threonine 0.21-0.39mg/ml, L-Ala 2.4-3.6mg/ml; Proline(Pro) 1.6-2.4mg/ml, Xie Ansuan 1.6-2.4mg/ml, methionine(Met) 0.35-0.65mg/ml; Methionin 4.8-7.2mg/ml, Isoleucine 1.6-2.4mg/ml, leucine 4.8-7.2mg/ml; Phenylalanine(Phe) 1.6-2.4mg/ml, tryptophane 0.35-0.65mg/ml; Molecular weight accounts for 25～35% of Cerebrolysin Vial less than 10000Da micromolecule polypeptide content.
The present invention further discloses the preparation method of Cerebrolysin Vial, it is the step below adopting;
(1) selecting total nitrogen content for use is 120-140mg/g metaprotein powder, with the concentration of purified water dissolving metaprotein powder to total nitrogen 3-30mg/mL, heated and boiled 1 hour;
(2) calculate with total nitrogen, add isodose 3-30mg/mL stomach en-and carry out acidic hydrolysis, 37 ℃～40 ℃ of hydrolysising conditions; Under pH 2.0-3.0 stirring reaction 4-8 hour, hydrolysis was boiled 30 minutes after accomplishing;
(3) the said hydrolyzed product calculates with total nitrogen and continues to divide the trypsinase hydrolysis once more that adds isodose 3-30mg/mL for several times altogether, 45 ℃～50 ℃ of hydrolysising conditions; Stirring reaction is 20～30 hours under pH 7.0-7.5, after the hydrolysis, regulates the pH value to 5.0-6.0, boils 0.5～1h; Centrifugal after the placement room temperature, get supernatant solution, be article in the middle of the Cerebrolysin Vial;
(4) above-mentioned middle article adsorb through the ORGANO Zeo-karb, and adopting pH 5-6, concentration is the sodium-chlor damping fluid desorb of 0.1～0.2mol/L, obtains the Cerebrolysin Vial bullion;
(5) above-mentioned bullion, the membrane ultrafiltration with the 10000Da aperture obtains Cerebrolysin Vial.
Wherein the adding for several times of the described trypsinase branch of step (3) refers to by hydrolysate total nitrogen calculating horizontal and is divided into 3～6 addings.
The ORGANO Zeo-karb that the present invention adopted refers to: model is IRI20B, IRI124,200CT or 252, and preferred model is IRI124 or 200CT.
The adjusting of pH value is accomplished with conventional pH regulator agent in the step of the present invention, and the example comprises without limitation: hydrochloric acid, sodium hydroxide, phosphoric acid, Hydrocerol A and its esters or the like.
The present invention further discloses the purposes of Cerebrolysin Vial in the medicine of preparation treatment dementia and disordered brain function, cerebral dysgenesis, encephalatrophy, neurasthenia, combined external head injuries, central nervous system infection, meningitis, senile dementia, dysthymia disorders, psychosis or cerebrovascular Metabolic disorder.
Key point of the present invention is: at first control total nitrogen content of midbody metaprotein powder, destroy cytolemma through adopting N,O-Diacetylmuramidase, again through skimming treatment, improved the degreasing effect of acetone, thereby improved content of total nitrogen.
The inventor finds in test of many times; When total nitrogen content of the metaprotein powder that is used to prepare Cerebrolysin Vial during less than 120mg/g; Follow-up which kind of hydrolysising condition and the chromatographic separation of no matter passing through; All the time can't let 16 seed amino acids all in above-mentioned acceptability limit, especially Aspartic Acid, L-glutamic acid, L-Ala, Methionin, leucine equal size always have one or more to be lower than the lower limit of above-mentioned content range; Therefore, the present invention discloses first midbody-metaprotein has been controlled this critical step.One of them typical comparative example is:
Conventional method:
(1) preparation just mixes thing: adopt fresh pig brain tissue an amount of, clean, stir, colloidal mill homogenate obtains just mixing thing;
(2) degreasing: will just mix thing heated and boiled 0.5-2h, and be cooled to normal temperature, and add an amount of (weight) acetone then, and stir 0.5-2h; Filter, Air drying does not have the control about total nitrogen content.
Method of the present invention:
(1) preparation just mixes thing: adopt fresh pig brain tissue an amount of, water cleans up the water of dehematizing, and every kg cerebral tissue adds the 0.5g N,O-Diacetylmuramidase and is mixed together stirring, and colloidal mill homogenate through 6 homogenate, obtains just mixing thing;
(2) heating, degreasing: will just mix thing heated and boiled 1h, and be cooled to normal temperature, and add 20 times of (weight) acetone then, and stir 1h; Filter, Air drying, obtaining total nitrogen content is 128mg/g metaprotein powder, preserves below-20 ℃.
More than the protein powder that obtains of two kinds of methods carry out the preparation of follow-up Cerebrolysin Vial respectively according to preparation method of the present invention:
A. with the concentration of purified water dissolving brain protein powder to total nitrogen 15mg/mL, heated and boiled 1 hour; Calculate with total nitrogen, add isodose stomach en-(15mg/mL) and carry out acidic hydrolysis, 37 ℃ of hydrolysising conditions; 2.0 times stirring reactions of pH 4 hours, hydrolysis was boiled 30 minutes after accomplishing;
B. the said hydrolyzed product calculates with total nitrogen and continues to divide the trypsin 15mg/mL that adds isodose for 3 times) hydrolysis once more, 45 ℃ of hydrolysising conditions; 7.0 times stirring reactions of pH 20 hours, after the hydrolysis, regulate pH value to 5.0, boil 1h; Centrifugal after the placement room temperature, get supernatant solution, be article in the middle of the Cerebrolysin Vial;
C. article adsorb through ORGANO Zeo-karb (model 200CT) in the middle of above-mentioned, adopt the sodium-chlor damping fluid desorb of pH value 5.0, concentration 0.2mol/mL, obtain the Cerebrolysin Vial bullion.
D. above-mentioned bullion after the ultrafiltration membrane treatment with the 10000Da aperture, gets Cerebrolysin Vial:
Another relatively more typical instance of the present invention:
With total nitrogen content is that the metaprotein powder of (preferred 120-140mg/g) is a raw material more than the 120mg/g; Prepare in strict accordance with (1) of the present invention-(5) step; Each aminoacids content meets " brain protein hydrolysate injection national standard " in the prepared like this Cerebrolysin Vial, and molecular weight accounts for hydrolysate 25～35% less than the micromolecule polypeptide content of 10000Da.Simultaneously, the hydrolysising condition of metaprotein is even more important, different hydrolysis pH values and time; Can obtain the different product of hydrolysis degree; For example, the pepsin hydrolysis time is too short, can cause other aminoacids contents outside phenylalanine(Phe), tryptophane, the aspartic acid not reach scope; And the trypsin hydrolyzing time is too short, then can cause other aminoacids contents outside l-arginine, Methionin, the Serine to surpass the content range lower limit; On the other hand, the incomplete hydrolysis of protein just can obtain micromolecule polypeptide, in case condition control is unreasonable, hydrolysis degree is excessive, can make protein be hydrolyzed into amino acid completely again, causes micromolecule polypeptide content too low, thereby affects the treatment.
Another relatively more typical instance of the present invention:
Tryptic adding mode also has very big influence to the degree of hydrolysis, and the effect that gradation adds is better than disposable adding, and its typical example is following:
A. with the concentration of purified water dissolving brain protein powder to total nitrogen 8mg/mL, heated and boiled 1 hour; Calculate with total nitrogen, add isodose stomach en-(8mg/mL) and carry out acidic hydrolysis, 37 ℃ of hydrolysising conditions; 2.5 times stirring reactions of pH 5 hours, hydrolysis was boiled 30 minutes after accomplishing;
B. the said hydrolyzed product is divided into two duplicate samples, and 1. sample calculates the trypsin 8mg/mL of disposable adding isodose with total nitrogen) hydrolysis once more; 2. sample calculates with total nitrogen and divides the trypsin 8mg/mL that adds isodose for 4 times) hydrolysis once more, subsequent operations is all identical, 40 ℃ of hydrolysising conditions; Stirring reaction is 25 hours under pH7.0, after the hydrolysis, regulates pH value to 5.0, boils 1h; It is centrifugal to put cold back, gets supernatant solution, is article in the middle of the Cerebrolysin Vial;
C. article adsorb through ORGANO Zeo-karb (model 200CT) in the middle of above-mentioned, adopt the sodium-chlor damping fluid desorb of pH value 5.0, obtain the Cerebrolysin Vial bullion.
D. above-mentioned bullion after the ultrafiltration membrane treatment with the 10000Da aperture, gets Cerebrolysin Vial:
Conclusion:
(1) finds through countless tests; The preparation method that we describe has at present taken all factors into consideration various Effect Factors for Sythetic Technology, can control the hydrolysis degree of metaprotein well; Make all aminoacids contents all at acceptability limit, and guarantee to contain higher micromolecule polypeptide.Resulting product meets in the injection national standard aminoacids content scope in 16;
(2) domestic Cerebrolysin Vial product standard is lower, micromolecule polypeptide content is not stipulated; But the product of its list marketing is carried out practical measurement (adopting the mensuration that uses the same method) through us; Micromolecule polypeptide content is all below 20%; As shown in the table, and product of the present invention, micromolecule polypeptide content grinds product considerably beyond state's exogenesis; Reach 25～35%, have better therapeutic.
Be the measured value that Austrian product Cerebrolysin (brain protein hydrolysate injection) 6 is pulled on city's article MV and product of the present invention below:
The quality examination of the Cerebrolysin Vial of the present invention's preparation:
1) measuring method of total nitrogen: mensuration second method according to existing 2010 editions pharmacopeia appendix VIID nitrogen is carried out.
2) multiple amino acids Determination on content method:
The content of amino acids measuring method has a variety of, all is known mature technology, for example available amino end acidity test appearance, AccQ.Tag method or the like, and we have adopted the latter, use following conditions specifically:
1. high performance liquid chromatograph (Tianjin, island LC-2010A) chromatographic column (NOVA-Pak C
185 μ m, 3.9 * 150mm) flow velocity 1.0ml/min, column temperature: 37 ℃
2. derivative reagent box: the special-purpose derivating agent AccQ-Fluor of AccQ.Tag amino acid
TMReagent kit (commercially available);
3. the preparation of moving phase: the preparation of mobile phase A: get sodium acetate trihydrate 19.0g, triethylamine 2.37ml is dissolved in the 1000ml water, with phosphorus acid for adjusting pH value to 4.95.The preparation of Mobile phase B: acetonitrile-water (60: 40).
4. condition determination: gradient
| Time (minute) | A％ | B％ |
| 0.01 | 100 | 0 |
| 17 | 92 | 8 |
| 24 | 76 | 24 |
| 32 | 67 | 33 |
| 34 | 67 | 33 |
5. sample preparation:, get reference substance solution and each 10 μ l sample introduction of need testing solution after deriving respectively, the record color atlas according to the said method of above-mentioned commercially available derivative reagent box.
6. calculate: calculate with external standard method
3) method of calculation of micromolecule polypeptide:
Calculate with formula: peptide content [%]=(N
Total-∑ N
AS)/N
Total* 100
N in the formula
TotalBe total nitrogen content (mg/ml), N
ASNitrogen content (mg/ml) for single total free aminoacids.
Not only staple is identical with imported product for the Cerebrolysin Vial of the present invention preparation, and content is higher than similar imported product with purity, and peptide content wherein can reach 25-35%, far above the level of imported product average 18%.
Be the acute toxicity test of adopting Austrian Cerebrolysin injection liquid, two kinds of Cerebrolysin Vials of the embodiment of the invention 1 product to carry out below:
Summary: acute toxicity test in mice: 100 of mouse (body weight 18-22g), male and female half and half are divided into 10 groups at random; 5 groups of LD that are used for Cerebrolysin Vial (Cerebrolysin) wherein
50Experiment, other 5 groups of LD that are used for embodiment 1 Cerebrolysin Vial
50Experiment.Two receive the reagent thing by the mouse tail vein injection administration, 1 time/day.Adopt the Bliss method to calculate LD
50Result of study shows, the LD of Cerebrolysin Vial (Cerebrolysin)
50And 95% credible 72.3 (69.1～75.3) mL/kg that is limited to, embodiment 1 Cerebrolysin Vial is 74.2 (70.4～78.1) mL/kg; The LD of two preparations
50There was no significant difference.Conclusion: the acute toxicity there was no significant difference of Cerebrolysin Vial (Cerebrolysin) and embodiment 1 Cerebrolysin Vial.
1 test objective
Observe intravenous injection Cerebrolysin Vial (Cerebrolysin), embodiment 1 Cerebrolysin Vial toxic reaction and death condition to mouse, and the two LD relatively
50Difference.
2 test materialss
2.1 trial drug
Brain protein hydrolysate injection (Cerebrolysin): orange-yellow liquid, the big pharmaceutical factory of Austrian Yi Biwei, lot number 92799904.
Cerebrolysin usage: recommend 10～30mL every day, be diluted in the 250mL saline water and instil.Or subcutaneous injection 2mL, intramuscular injection 5mL, the quiet 10mL that pushes away.
Self-control Cerebrolysin Vial stoste (embodiment 1): orange-yellow liquid, lot number 20120306.
2.2 experimental animal
Kunming mouse, body weight 18～22g, male and female half and half; Provide by Institute of Experimental Animals, Chinese Academy of Medical Sciences.
2.3 feeding and management
Feed: provide by the Tianjin animal center.
Animal Lab.: room temperature is controlled at 24 ± 2 ℃ by central air-conditioning, and humidity is 60 ± 15%.
3 experimental techniques and result
3.1 Cerebrolysin Vial (Cerebrolysin) intravenous administration acute toxicity test in mice
50 mouse are divided into 5 groups at random by sex, body weight, and 10 every group, male and female half and half.The tail vein injection administration.Dosage is 90,76.5,65,55.3,47mL/kg, and agent is apart from than being 1: 0.85.Because of the intravenously administrable volume of the every 10g body weight of mouse is 0.2mL, the administration volume that above-mentioned dosage is provided with is excessive, must be with concentrated 10 times of Cerebrolysin Vial (Cerebrolysin) before the experiment.Behind the medicine according to above-mentioned 5 dosage of administration volume intravenous injection of 0.2mL/10g, observe also toxic reaction and the death condition of record animal, observed continuously 14 days, with Bliss method calculating LD
50And 95% fiducial limit, the result sees table 1.
Table 1 Cerebrolysin Vial (Cerebrolysin) intravenous administration acute toxicity test in mice
Startling, trembling appears in Cerebrolysin Vial (Cerebrolysin) mouse immediately, and muscular tension increases, urinary incontinence, and is prostrate motionless, until death.Strengthen sx with dosage.Mostly after administration in 30 minutes, dead mouse is dissected immediately in death, and visual inspection is not found obviously unusual, survival mice after 1 day outward appearance, activity all recover normally, 1 week, 2 all body weight all increase.
3.2 embodiment 1 Cerebrolysin Vial intravenous administration acute toxicity test in mice
50 mouse are divided into 5 groups at random by sex, body weight, and 10 every group, male and female half and half.The tail vein injection administration.Dosage is 110,93.5,79.5,67.6,57.4,48.8mL/kg, and agent is apart from than being 1: 0.85.Because of the intravenously administrable volume of the every 10g body weight of mouse is 0.2mL, the administration volume that above-mentioned dosage is provided with is excessive, must be with concentrated 10 times of embodiment 1 Cerebrolysin Vial before the experiment.Behind the medicine according to above-mentioned 5 dosage of administration volume intravenous injection of 0.2mL/10g, observe also toxic reaction and the death condition of record animal, observed continuously 14 days, with Bliss method calculating LD
50And 95% fiducial limit, the result sees table 2.
Table 2 embodiment 1 Cerebrolysin Vial drug administration by injection acute toxicity test in mice
After the embodiment 1 Cerebrolysin Vial intravenous injection, mouse occurs accelerated breathing immediately, runs, startles, urinary incontinence, and is prostrate few moving, until death.Strengthen sx with dosage.Mostly after administration in 30 minutes, dead mouse is dissected immediately in death, and visual inspection is not found obviously unusual, survival mice after 1 day outward appearance, activity all recover normally, 1 week, 2 all body weight all increase.
Brief summary: result of study shows, the LD of Cerebrolysin Vial (Cerebrolysin)
50And 95% credible 72.3 (69.1～75.3) mL/kg that is limited to, embodiment 1 Cerebrolysin Vial is 74.2 (70.4～78.1) mL/kg; The LD of two preparations
50There was no significant difference.Conclusion: the acute toxicity there was no significant difference of Cerebrolysin Vial (Cerebrolysin) and embodiment 1 Cerebrolysin Vial.
Above determination data shows with the toxicological test result: not only staple is identical with imported product for the Cerebrolysin Vial of the present invention's preparation; All meet national injection standard; And total nitrogen is higher than similar imported product with micromolecule polypeptide; Wherein molecular weight can reach 25-35% less than the small-molecular peptides content of 10000Da, is higher than the level of imported product average 18%; And security and imported product are suitable.The increase of small-molecular peptides does not increase its toxicity.
Whether the raising for micromolecule polypeptide content has better therapeutic really; We have carried out the further animal test of pesticide effectiveness; The unexpected discovery compared the Austrian product of import, and the increase of small-molecular peptides has significantly improved the curative effect of Cerebrolysin Vial treatment dementia and disordered brain function aspect in the product of the present invention; Concrete TP and result such as embodiment 5 are said, and the result proves: Cerebrolysin Vial drug effect of the present invention obviously is better than existing Cerebrolysin Vial product.
The positively effect that the Cerebrolysin Vial of the present invention's preparation is compared with prior art had is:
(1) obtains meeting the Cerebrolysin Vial of injection national standard, guarantee drug quality.
(2) each aminoacids content is qualified, and molecular weight is high less than the micromolecule polypeptide content of 10000Da, compares with existing Cerebrolysin Vial product, and toxicity did not increase when drug effect significantly improved.。
(3) the real biologically active substance that from the pig brain, extracts, inartificial interpolation effective constituent guarantees that drug safety is effective.
| 35 | 0 | 100 |
| 37 | 0 | 100 |
| 38 | 100 | 0 |
| 45 | 100 | 0 |
Embodiment
Below in conjunction with embodiment the present invention is described; The scheme of embodiment described here; Do not limit the present invention; One of skill in the art can make improvements and change according to spirit of the present invention, and described these improvement and variation all should be regarded as within the scope of the invention, and scope of the present invention and essence are limited claim.Used reagent all has commercially available.Under the situation of not mentioning temperature, all carry out at ambient temperature.
Embodiment 1
The preparation of metaprotein powder:
The first step brain tissue homogenate
Fresh pig brain tissue is an amount of, and water cleans up the water of dehematizing.Add the 0.5g/kg N,O-Diacetylmuramidase and be mixed together stirring, colloidal mill homogenate.Obtain just mixing thing through 6 homogenate.
The second step protein denaturation
With above-mentioned cerebral tissue heated and boiled 1h, be cooled to normal temperature.To the acetone that wherein adds 20 times of amounts (weight), stir 1h, filter, Air drying gets the metaprotein powder, contains total nitrogen 135mg/g.Preserve the brain protein powder below-20 ℃.
The preparation of Cerebrolysin Vial:
The 3rd step enzymolysis
(1) get above-mentioned protein powder, according to total nitrogen, with the concentration of purified water soluble protein powder to 3mg/mL total nitrogen, heated and boiled 1 hour.
(2) add isodose 3mg/ml (calculating with total nitrogen) stomach en-, carry out acidic hydrolysis, hydrolysising condition is specially: 37 ℃; With 10% phosphoric acid adjust pH to 2.0, stirring reaction under pH2.0.4 hours, hydrolysis was boiled 30 minutes after accomplishing.
(3) hydrolysate continues to add the trypsin hydrolyzing of isodose (calculating with total nitrogen) 3mg/ml, and hydrolysising condition is specially: 45 ℃, with 10% sodium hydroxide adjust pH to 7.0, stirring reaction is 22 hours under pH7.0.The adding method of pancreatin is to be divided into 5 parts, divides 5 addings.After the hydrolysis, below the 10% phosphoric acid adjust pH to 6.0, boil 0.5h.
(4) hydrolysate is centrifugal, removes dregs.Get supernatant solution, be article in the middle of the Cerebrolysin Vial.
The 4th one-step refining
Zeo-karb (ORGANO PK 200CT is available from ORGANO): with 10% phosphoric acid the pH value of article in the middle of the hydrolysate of above-mentioned (4) is transferred to 6.0, adsorb the 0.12mol/L sodium-chlor damping fluid desorb of employing pH6.0 through Zeo-karb.Obtain the Cerebrolysin Vial bullion.
Above-mentioned bullion after the ultrafiltration membrane treatment with the 10000Da aperture, gets Cerebrolysin Vial.
The aminoacids content of measuring is following, and micromolecule polypeptide content (molecular weight is less than 10000Da) accounts for 34.69% of hydrolysate
Embodiment 2
The preparation of metaprotein powder:
The first step brain tissue homogenate
Fresh pig brain tissue is an amount of, and water cleans up the water of dehematizing.Add the 0.2g/kg N,O-Diacetylmuramidase and be mixed together stirring, all pulp grinder homogenate.Obtain just mixing thing through 4 homogenate.
The second step protein denaturation
With above-mentioned cerebral tissue heated and boiled 1h.Be cooled to normal temperature.To the acetone that wherein adds 10 times of amounts (weight).Stir 1h.Filter.Air drying.Get the metaprotein powder, contain total nitrogen 123mg/g.Preserve the brain protein powder below-20 ℃.
The preparation of Cerebrolysin Vial:
The 3rd step enzymolysis
(1) according to the total nitrogen of protein powder, with the concentration of purified water soluble protein powder to 3mg/mL total nitrogen, heated and boiled 1 hour.
(2) add isodose 3mg/ml (calculating) gastric enzyme and carry out acidic hydrolysis with total nitrogen.37 ℃ of hydrolysising conditions; Stirring reaction under the pH2.5,6 hours.With 10% phosphoric acid adjust pH to 2.0, stirring reaction under pH2.0.Hydrolysis was boiled 30 minutes after accomplishing.
(3) hydrolysate continues to add the pancreatin hydrolysis of isodose 3mg/ml (calculating with total nitrogen).Stirring reaction under 45 ℃ of hydrolysising conditions, the pH7.5.With 10% sodium hydroxide adjust pH to 7.0, stirring reaction is 22 hours under pH7.0.The adding method of pancreatin is to be divided into 5 parts, divides 5 addings.After the hydrolysis, 10% phosphoric acid adjust pH to 6.0 boils 0.5h.
(4) hydrolysate is centrifugal, removes dregs.Get supernatant solution, be article in the middle of the Cerebrolysin Vial.
The 4th one-step refining
Zeo-karb (ORGANO PK 200CT; Available from ORGANO): with 10% phosphoric acid the pH value of article in the middle of the hydrolysate of above-mentioned (4) is transferred to 6.0; Adsorb through Zeo-karb, adopt the 0.12mol/L sodium-chlor damping fluid desorb of pH6.0, obtain the Cerebrolysin Vial bullion.
Above-mentioned bullion after the ultrafiltration membrane treatment with the 10000Da aperture, gets Cerebrolysin Vial, and the aminoacids content of mensuration is following, and micromolecule polypeptide content (molecular weight is less than 10000Da) accounts for 29.7% of hydrolysate:
Embodiment 3
The preparation of metaprotein powder:
The first step brain tissue homogenate
Fresh pig brain tissue is an amount of, and water cleans up the water of dehematizing.Add the 0.8g/kg N,O-Diacetylmuramidase and be mixed together stirring, colloidal mill homogenate.Obtain just mixing thing through 5 homogenate.
The second step protein denaturation
With above-mentioned cerebral tissue heated and boiled 1h.Be cooled to normal temperature.To the acetone that wherein adds 20 times of amounts (weight), stir 1h, filter, Air drying gets the metaprotein powder, contains total nitrogen 135mg/g.Preserve the brain protein powder below-20 ℃.
The preparation of Cerebrolysin Vial:
The 3rd step enzymolysis
(1) get above-mentioned protein powder, according to total nitrogen, with the concentration of purified water soluble protein powder to 3mg/mL total nitrogen, heated and boiled 1 hour.
(2) add isodose 3g/ml (calculating with total nitrogen) stomach en-, carry out acidic hydrolysis, hydrolysising condition is specially: 37 ℃; With 10% phosphoric acid adjust pH to 2.0, stirring reaction is 4 hours under pH2.0, and hydrolysis was boiled 30 minutes after accomplishing.
(3) hydrolysate continues to add the trypsin hydrolyzing of isodose (calculating with total nitrogen) 3mg/ml, and hydrolysising condition is specially: 45 ℃, with 10% sodium hydroxide adjust pH to 7.0, stirring reaction is 22 hours under pH7.0.The adding method of pancreatin is to be divided into 5 parts, divides 5 addings.After the hydrolysis, below 10% phosphoric acid adjust pH to 6.0, boil 0.5h.
(4) hydrolysate is centrifugal, removes dregs.Get supernatant solution, be article in the middle of the Cerebrolysin Vial.
The 4th one-step refining
Zeo-karb (ORGANO 200CT; Available from ORGANO): with 10% phosphoric acid the pH value of article in the middle of the hydrolysate of above-mentioned (4) is transferred to 6.0; Adsorb through Zeo-karb, adopt the 0.12mol/L sodium-chlor damping fluid desorb of pH6.0, obtain the Cerebrolysin Vial bullion.
Above-mentioned bullion after the ultrafiltration membrane treatment with the 10000Da aperture, gets Cerebrolysin Vial.
The aminoacids content of measuring is following, and micromolecule polypeptide content (molecular weight is less than 10000Da) accounts for 26.1% of hydrolysate:
Embodiment 4
(1) preparation just mixes thing: adopt fresh pig brain tissue an amount of, water cleans up the water of dehematizing, and every kg cerebral tissue adds the 0.2g N,O-Diacetylmuramidase and is mixed together stirring, colloidal mill or refiner homogenate, through 4 homogenate, obtains just mixing thing;
(2) heating, degreasing: will just mix thing, heated and boiled 0.5h is cooled to normal temperature, adds 20 times of (weight) acetone then, stirs 1h, filter, and Air drying, obtaining total nitrogen content is the metaprotein powder of 135mg/g, preserves below-20 ℃.
Adopt the method for the Cerebrolysin Vial of metaprotein powder preparation:
(1) selecting total nitrogen content for use is 135mg/g metaprotein powder, with the concentration of purified water dissolving metaprotein powder to total nitrogen 20mg/mL, heated and boiled 1 hour;
(2) calculate with total nitrogen, add isodose 20mg/mL stomach en-and carry out acidic hydrolysis, 37 ℃ of hydrolysising conditions; Under pH2.0-3.0 stirring reaction 4-8 hour, hydrolysis was boiled 30 minutes after accomplishing;
(3) the said hydrolyzed product calculates the trypsinase hydrolysis once more that continues to add for 3 times isodose 20mg/mL, 50 ℃ of hydrolysising conditions with total nitrogen; Stirring reaction is 30 hours under pH7.5, after the hydrolysis, regulates pH value to 5.0, boils 1h; Centrifugal after the placement room temperature, get supernatant solution, be article in the middle of the Cerebrolysin Vial;
(4) above-mentioned middle article adsorb through the ORGANO Zeo-karb, and adopting pH5-6, concentration is the sodium-chlor damping fluid desorb of 0.1-0.2mol/L, obtains the Cerebrolysin Vial bullion;
(5) above-mentioned bullion, the membrane ultrafiltration (model 200CT) with the 10000Da aperture obtains Cerebrolysin Vial.
The aminoacids content of measuring is following, and micromolecule polypeptide content (molecular weight is less than 10000Da) accounts for 31.5% of hydrolysate:
Embodiment 5
The present invention further discloses the purposes of Cerebrolysin Vial in preparation treatment dementia and disordered brain function, cerebral dysgenesis, encephalatrophy, neurasthenia, combined external head injuries, central nervous system infection, meningitis, senile dementia, dysthymia disorders, psychosis and cerebrovascular Metabolic disorder medicine.Be the pharmacodynamic experiment of Cerebrolysin Vial (embodiment 3) aspect preparation treatment dementia and disordered brain function that the present invention prepares below:
Cerebrolysin Vial is for the influence of dementia rats ability of learning and memory
1, material
1.1 animal: the SD male rat, body weight 250～300g is provided by Institute of Experimental Animals, Chinese Academy of Medical Sciences.
1.2 the Cerebrolysin Vial of reagent medicine: trial drug: embodiment 3 preparations.Positive control drug is selected brain protein hydrolysate injection (Cerebrolysin, Ebewe Pharma Ges M. B. H. Nfg KG, lot number: 93373504) for use.A β 25-35 adopts the SPSS dissolving to be mixed with the solution of 10mmol/L available from Sigma company, places incubator to wear out 3 days for 37 ℃, is stored in-20 ℃ of refrigerators and preserves subsequent use.
1.3 laboratory apparatus: SN-3N stereotaxic apparatus (Japanese Narishige), DMS-2Morris water maze system.
2, method
2.1 make up the AD rat model
After rat flexibility is fed 3d, with 3% vetanarcol intraperitoneal injection of anesthesia (40mg/kg body weight).Anesthetized rat is fixed on the stereotaxic apparatus, the head unhairing, skin is cut in tincture of iodine sterilization back, with reference to bag new people's " rat brain stereotaxic atlas ", selects the right side tricorn to be the injection target area.The 1.7mm place is opened on 1.0mm, center line side backward in bregma; Open skull with the dental burr brill; Expose endocranium, again with microsyringe from brain Surface Vertical inserting needle 3.8mm, the A β 25-35 body lotion 5 μ l of 10mmol/L are slowly injected (injection length is no less than 5min); Slowly remove pin behind let the acupuncture needle remain at a certain point the 2min, sew up a wound; Blank group injection equivalent SPSS.The conventional abdominal injection dibenzylethylenediamine dipenicillin G of each treated animal postoperative is anti-infective, treats that the clear-headed back treatment of animal group promptly begins the tail vein injection administration.
2.2 medication
At random rat is divided into four groups: blank group, model group, embodiment 3 Cerebrolysin Vial groups and Cerebrolysin positive drug control group, 6 every group.Dosage is 20 times of adult's clinical dosage, i.e. 40mg/kg/d; Volumetric injection is 0.3ml/100g (calculating exact value according to total amount and drug concentration), every day 2 times.Blank and model group are then injected with the equal-volume SPSS.Successive administration 15d after the rat modeling carries out water maze laboratory next day; Duration of test is administration still.
2.3 study of behaviour experimental technique
Water maze is made up of round pool (black inwall), black platform and register system 3 parts.Pool diameter 120cm, high 60cm, depth of water 40cm dyes black with ink with Chi Shui, and in 2cm under water, water temperature does not remain on 23 ℃-24 ℃ to platform.The labyrinth places laboratory central authorities, and the position of indoor each object keeps immobilizing.The water maze test comprises orientation navigation test (place navigation) and the primary space exploratory experiment (spatial probe test) of continuous 5d.
The orientation navigation test:
The test proxima luce (prox. luc) is put into pond (not putting into platform) free swimming 2min with rat, makes it be familiar with the testing circumstance environment.Official test lasts 5d, divides 2 time periods of upper and lower noon every day, training of every period 4 times, respectively from east, south, west, north 4 somes entry.During entry, rat is towards pool wall, and it is escape latency that the record rat is found the platform required time; In 2min, do not find platform like rat, then by the experimenter it is guided into platform and stops 10s, be 120s its latent period.Camera system writes down time (escape latency) and the swimming approach (process) that rat seeks platform automatically, and computer writes down above-mentioned data automatically; Setting 120s is the longest escape latency, stops record behind the 120s automatically.
The space exploration test:
6d removes platform, lets rat from the mid point entry free swimming 120s of original platform offside quadrant tub wall, writes down its swimming track, and software analysis calculates animal in used time of all quadrants and all quadrants course length.Calculate animal and belong to the per-cent that the quadrant swimming time accounts for escape latency, account for the per-cent of detection range in the course length of original platform quadrant at original platform.
2.4 the statistical method data adopt the SPSS10.0 statistical analysis software with mean ± standard deviation
expression.Orientation navigation experiment and space exploration experiment knot state adopt one-way analysis of variance (One way ANOVA), relatively adopt the t check between group in twos.
3, result
Table 1, medicine are to the influence
of escape latency and detection range in the AD rat orientation navigation experiment
| Group | Escape latency (s) | Detection range (cm) |
| The blank group | 13.25±4.85 1) | 264.12±68.26 1) |
| Model group | 57.18±7.58 | 778.36±122.12 |
| Embodiment 3 Cerebrolysin Vials | 22.86±5.38 1) | 312.40±75.72 1) |
| Austria's brain protein hydrolysate injection | 38.44±7.93 1)2) | 504.72±88.94 1)2) |
Annotate: compare with model group
1)Compare with embodiment 3 Cerebrolysin Vial groups P＜0.05
2)P＜0.05
Table 2, medicine are tested the influence
of Central Plains quadrant search time and distance to the space exploration of AD rat
| Group | Per-cent search time (%) | Detection range per-cent (%) |
| The blank group | 45.12±5.13 1) | 47.12±4.33 1) |
| Model group | 16.26±4.37 | 15.96±5.17 |
| Embodiment 3 Cerebrolysin Vials | 37.02±4.23 1) | 35.02±5.43 1) |
| Austria's brain protein hydrolysate injection | 28.33±5.72 1)2) | 26.26±4.12 1)2) |
Annotate: compare with model group
1)Compare with embodiment 3 Cerebrolysin Vial groups P＜0.0
2)P＜0.05
The result shows: rat orientation navigation and space exploration experimental result show; Embodiment 3 Cerebrolysin Vials and Austrian brain protein hydrolysate injection all can significantly improve space learning, the memory capability of AD rat model; Promptly shorten escape latency and detection range, and significantly increase space exploration time and the detection range of rat at the original platform quadrant.In addition, embodiment 3 Cerebrolysin Vials significantly are superior to Austrian brain protein hydrolysate injection to the improvement effect of the study of AD rat, memory capability.
Claims (12)
1. metaprotein powder, the total nitrogen content that it is characterized in that this metaprotein powder is more than the 120mg/g; It is through in fresh pig brain, adding N,O-Diacetylmuramidase, obtaining through homogenate, heating degreasing, acetone degreasing.
2. the said metaprotein powder of claim 1, its total nitrogen content is 120-140mg/g.
3. the said metaprotein powder of claim 1, it prepares according to following steps:
(1) preparation just mixes thing: adopt fresh pig brain tissue an amount of, water cleans up the water of dehematizing, and every kg cerebral tissue adds 0.1～1.0g N,O-Diacetylmuramidase and is mixed together stirring, colloidal mill or refiner homogenate, through 4～6 homogenate, obtains just mixing thing;
(2) degreasing: will just mix thing, heated and boiled 0.5～1h is cooled to normal temperature, adds 10～20 times of (weight) acetone then, stirs 0.5～1h; Filter, Air drying, obtaining total nitrogen content is the above metaprotein powder of 120mg/g.
4. method for preparing the metaprotein powder is characterized in that being undertaken by following step:
(1) preparation just mixes thing: adopt fresh pig brain tissue an amount of, water cleans up the water of dehematizing, and every kg cerebral tissue adds 0.1～1.0g N,O-Diacetylmuramidase and is mixed together stirring, colloidal mill or refiner homogenate, through 4～6 homogenate, obtains just mixing thing;
(2) heating degreasing: will just mix thing, heated and boiled 0.5～1h is cooled to normal temperature, adds 10～20 times of (weight) acetone then, stirs 0.5～1h; Filter, Air drying, obtaining total nitrogen content is the above metaprotein powder of 120mg/g.
5. each said metaprotein powder of claim 1-3 is in the purposes of preparation in the Cerebrolysin Vial.
6. Cerebrolysin Vial that adopts each said metaprotein powder preparation of claim 1-3, it is characterized in that: each aminoacids content is following in the Cerebrolysin Vial: Aspartic Acid 2.4-3.6mg/ml, Serine 0.21-0.39mg/ml; L-glutamic acid 3.2-4.8mg/ml, glycocoll 1.2-1.8mg/ml, Histidine 1.04-1.56mg/ml; L-arginine 0.3-1.1mg/ml, Threonine 0.21-0.39mg/ml, L-Ala 2.4-3.6mg/ml; Proline(Pro) 1.6-2.4mg/ml, Xie Ansuan 1.6-2.4mg/ml, methionine(Met) 0.35-0.65mg/ml; Methionin 4.8-7.2mg/ml, Isoleucine 1.6-2.4mg/ml, leucine 4.8-7.2mg/ml; Phenylalanine(Phe) 1.6-2.4mg/ml, tryptophane 0.35-0.65mg/ml; Molecular weight accounts for 25～35% of Cerebrolysin Vial less than the micromolecule polypeptide of 10000Da.
7. method for preparing the said Cerebrolysin Vial of claim 6 is characterized in that carrying out as follows:
(1) selects each said metaprotein powder of claim 1-3 for use, with the concentration of purified water dissolving metaprotein powder to total nitrogen 3-30mg/mL, heated and boiled 1 hour;
(2) calculate with total nitrogen, add isodose 3-30mg/mL stomach en-and carry out acidic hydrolysis, 37 ℃～40 ℃ of hydrolysising conditions; Under pH 2.0-3.0 stirring reaction 4-8 hour, hydrolysis was boiled 30 minutes after accomplishing;
(3) the said hydrolyzed product calculates with total nitrogen and continues to divide the trypsinase hydrolysis once more that adds isodose 3-30mg/mL for several times altogether, 45 ℃～50 ℃ of hydrolysising conditions; Stirring reaction is 20～30 hours under pH value 7.0-7.5, after the hydrolysis, regulates the pH value to 5.0-6.0, boils 0.5～1h; Centrifugal after the placement room temperature, get supernatant solution, be article in the middle of the Cerebrolysin Vial;
(4) above-mentioned middle article adsorb through the ORGANO Zeo-karb, and adopting pH5-6, concentration is the sodium-chlor damping fluid desorb of 0.1～0.2mol/L, obtains the Cerebrolysin Vial bullion;
(5) above-mentioned bullion, the membrane ultrafiltration with the following aperture of 10000Da obtains Cerebrolysin Vial.
8. the described method of claim 7, wherein the described trypsinase of step (3) divides to add for several times and refers to the trypsinase amount of calculating by the hydrolysate total nitrogen and be equally divided into 3～6 addings.
9. the described method of claim 7, wherein ORGANO Zeo-karb model is IRI20B, IRI124,200CT or 252.
10. the described method of claim 9, wherein ORGANO Zeo-karb model is IRI124 or 200CT.
11. the described method of claim 7, wherein the described film of step (5) is the 10000Da aperture.
12. the purposes of Cerebrolysin Vial in the medicine of preparation treatment dementia and disordered brain function, cerebral dysgenesis, encephalatrophy, neurasthenia, combined external head injuries, central nervous system infection, meningitis, senile dementia, dysthymia disorders, psychosis or cerebrovascular Metabolic disorder of each said method preparation of Cerebrolysin Vial described in the claim 6 or claim 7-11.
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| CN103980343A (en) * | 2014-05-27 | 2014-08-13 | 河北智同生物制药有限公司 | Multi-functional extracting, filtering and drying machine for albumen powder and applications thereof |
| CN104096214A (en) * | 2014-06-10 | 2014-10-15 | 广州一品红制药有限公司 | Cerebroprotein hydrolysate freeze-dried powder injection and preparation method thereof |
| CN104587444A (en) * | 2014-12-24 | 2015-05-06 | 内蒙古天奇生物科技有限公司 | Preparation method of high-protein-content cerebroprotein hydrolysate |
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| CN105331665A (en) * | 2015-12-09 | 2016-02-17 | 梁尚文 | Method for preparing brain polypeptide and brain small-molecule peptide by means of pig brain protein through enzymolysis |
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| CN114208938A (en) * | 2021-12-08 | 2022-03-22 | 天津泰创生物科技有限公司 | Cerebroprotein hydrolysate, preparation method thereof and composition containing the same |
| CN114208938B (en) * | 2021-12-08 | 2023-10-24 | 天津泰创生物科技有限公司 | Brain protein hydrolysate, preparation method thereof and composition containing brain protein hydrolysate |
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