CN1228082C - Method for preparing pharmaceutics of hydrolysate of brain protein - Google Patents
Method for preparing pharmaceutics of hydrolysate of brain protein Download PDFInfo
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Images
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a method for preparing a brain protein hydrolysate preparation, which is characterized in that purified water with volume of 1 to 2 times of the volume of fresh pig brain or pig brain stored in frozen mode is added to the fresh pig brain or the pig brain stored in a freezing mode to be homogenized, heated to 85 DEG C and cooled down; a pH value is adjusted to be 1.5 to 2.0, and pepsin is used for enzymolysis for 3 to 12 hours; after a temperature is adjusted to be 42 DEG C, a pH value is adjusted to be 7.7 to 8.0 by using sodium hydroxide; pancreatin multienzyme is used for enzymolysis for 2 to 4 hours at a temperature of 38 to 42 DEG C, and a pH value is adjusted to be 2.5 to 3.0; still standing for freezing is carried out at a temperature of-20 to-10DEGC, filtration is carried out at a room temperature, and a pH value is adjusted to be neutral by adding sodium oxide; clear liquid is taken and is ultrafiltered by using an ultrafiltration membrane with interception molecular weight of 10, 000; penetration liquid is collected and processed through nano-filtration and concentration to obtain concentrated liquid which is sterilized by using steam of 121 DEG C for 30 minutes to obtain degermed solution named as a brain protein hydrolysate highly finished product; prepared L-tryptophan, prepared L-tyrosine, added various amino acids, a prepared peptide map and the solution are prepared into concentrated preparation liquid which is diluted and filled to be prepared into a finished product; the finished product is sterilized by using steam to be prepared into a sterile preparation. The present invention has the advantages of production period shortening, production increase, simple and convenient operation, production procedure reduction and medicine contamination avoidance.
Description
Technical field
The present invention relates to the preparation method of medicine, specifically the preparation method of Cerebrolysin Vial preparation.
Background technology
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 multiple human brain essential amino acids and cephalin, lecithin, peptide class nerve growth factor; can act on nervus centralis in many ways; regulate and improve neuronic metabolism; promote the formation of synapse; induce neuronic differentiation, and further neuroprotective cell is avoided the infringement of various ischemias and neurotoxin.It can pass through blood brain barrier, promotes the synthetic of brain internal protein, influences respiratory chain, has the protective capability of anti-hypoxia, improves the metabolism of brain self-energy.Other hormone systems of activated adenyl cyclase catalysis.Neurotransmitter, peptide hormone and coenzyme precursors are provided.
Brain protein hydrolysate injection be applicable to craniocerebral trauma and cerebrovascular disease (incomplete brain blood supply, cerebral infarction) sequela with hypomnesis and the attention doing well,improving of concentrating obstacle; Be applicable to the prevention and the treatment of disordered brain functioies such as cognition that cranial nerve such as nervous vascular Headache, oligophrenia, cerebral arteriosclerosis, alzheimer disease, brain injury sequela, cerebrovascular, vascular conditions cause, memory, motion, sensation, balance, no obvious toxic-side effects.Parkinson, fatigue syndrome, sub-health state had certain effect.
One of preparation method of existing Cerebrolysin Vial preparation is: fresh Medulla sus domestica is removed the mucosa on surface and bloodstain etc., and homogenate adds the acetone precipitation of 5 times of volumes, and adding the washing of equivalent ether after the precipitate and separate again soaked 1 hour, refilters, vacuum drying is made the brain powder; The brain powder with water dissolution after, use pepsin, pancreatin double-enzyme hydrolysis successively, hydrolyzed solution centrifuging and taking supernatant, ebuillition of heated and rapidly cooling get hydrolyzed solution through plate-and-frame filtration, ultrafiltration and concentrating under reduced pressure then; The hydrolyzed solution branch is pulled on Sephadex G-25 gel chromatography column chromatography, liquid is collected at active peak made elaboration liquid through concentrating under reduced pressure, 0.45 μ m microporous filter; According to required amount of liquid medicine, drug standard (numbering WS1-XG-25-2000) by the promulgation on the 28th of National Drug Administration's December in 2000 calculates the aminoacid consumption, add corresponding amino acid ligand and make concentrated wiring liquid, rarely then join, the fill finished product, become sterile preparation through steam sterilization.Its shortcoming is: (1), equipment investment are big, and the production cycle is long, and production efficiency is low; With the chromatography method effective ingredient of purifying, each process need 30～48 hours, the production cycle is long; Because the production cycle is long,, require to carry out Sephadex G-25 gel filtration chromatography, and the necessary import of Sephadex G-25 gel chromatography material costs an arm and a leg in 2～8 ℃ of freezer workshops for ensuring drug quality; Produce 1,000,000 (10ml) brain protein hydrolysate injections per year and calculate, above-mentioned input just reaches more than 2,000,000 yuans; Use concentrating under reduced pressure and centrifugal step, production efficiency is low, is unfavorable for the control to drug quality.(2), use a large amount of organic chemistry solvents, the boiling point of acetone and ether is extremely low, it is inflammable, explosive, deleterious hazardous chemical, ether also is used as anesthetis, uses a large amount of acetone and ether earlier the brain albumen precipitation to be come out to make the brain powder, and this step is abnormally dangerous, produce in must be between hoolivan, operate loaded down with trivial details, contaminated environment, harm production operation personnel's is healthy.
Tool, Chinese invention patent discloses a kind of " process for preparing injection liquid of brain protein hydrolyzate ", its content is: fresh Medulla sus domestica is removed surperficial mucosa and bloodstain etc., add the water homogenate of 2 times of volumes, filter with double gauze, filtrate is used earlier pepsin hydrolysis, transfer pH to 1.5-1.6 with hydrochloric acid, bath temperature 40-45 ℃, the time is 6-8 hour, the reuse sodium hydroxide transfers pH to 7.6-7.7, add pancreatin (pancreatin is dissolved in earlier in the calcium chloride solution of 0.02N in 4 ℃ of activation 30 minutes down) hydrolysis, 40-45 ℃ of water-bath hydrolysis 4-6 hour obtains hydrolyzed solution, add hydrochloric acid adjust pH 2.5-3.0, left standstill 10-30 hour under-20～-10 ℃; At ambient temperature, filter with double gauze, the water that adds 1/2 volume again, hydro-oxidation sodium adjust pH is to neutral, with the aperture is the filtering with microporous membrane clarification of 0.5-1.0 μ m, get clear liquor, be 70,000 and 10,000 ultrafilter membrane ultrafiltration with the molecular weight that dams successively, the ultrafiltrate reuse that the obtains molecular weight that dams is that 1000 ultrafilter membrane concentrates, the concentrated solution that obtains is heated to 100 ℃ of boilings 15 minutes, be cooled to below 25 ℃ then rapidly, be 70,000 and 10,000 ultrafilter membrane more successively through the microporous filter membrane in 0.5-1 μ m aperture and the molecular weight that dams, the degerming solution that the ultrafiltrate that obtains obtained through 121 ℃ of steam sterilizations in 30 minutes again is called the proteoclastic mixed polypeptide of brain, Freamine highly finished product: according to required amount of liquid medicine, by drug standard (the numbering WS of National Drug Administration's December in 2000 promulgation on the 28th
1-XG-25-2000) calculate the aminoacid consumption that needs interpolation, be mixed with concentrated wiring liquid with above-mentioned solution, rare then equipped pin finished product medicinal liquid became sterile preparation in 30 minutes through 121 ℃ of steam sterilizations.Its shortcoming is: it is low that (1) uses ultrafilter membrane to carry out the ultrafiltration and concentration response rate: though this technology has adopted hyperfiltration technique, but what use is that the molecular weight that dams is that 1000 ultrafilter membrane concentrates, make effective active composition brain polypeptide losing in various degree, just in permeate, run off, greatly reduce yield.(2) repeatedly be 70,000 and 10,000 ultrafilter membrane, obtain ultrafiltrate, make operation more loaded down with trivial details through the microporous filter membrane in 0.5-1 μ m aperture and the molecular weight that dams.
Summary of the invention
The object of the invention provides a kind of organic chemistry solvent that do not use, and extracts the preparation method that Cerebrolysin Vial is produced preparation with the method substituted chemistry reagent sedimentation method of physics, can reach the separating effect of existing technology.
The preparation method of brain protein hydrolysate injection of the present invention is: remove surperficial peplos and blood vessel etc. with fresh (or frozen) Medulla sus domestica, add the purified water homogenate of 1～2 times of volume, water proof is steam heated to 80～85 ℃, 15 minutes, during fast cooling to 40～45 ℃, transfer pH to 1.5～2.0 with hydrochloric acid, with pepsin enzymolysis 3～12 hours (or precipitate and separate is got supernatant), homoiothermic to 40～42 ℃, transfer pH to 7.7～8.0 with sodium hydroxid, with pancreatin multienzyme (comprising trypsin, pancreatic lipase and pancreatic amylase), add 0.04mol/L CaCl before using
2Activation pancreatin multienzyme 0.5～1 hour, under 38～42 ℃ of conditions, enzymolysis 2～4 hours, and then left standstill freezing 12～48 hours with under 2.5～3.0 ,-20～-10 ℃ of the hydrochloric acid adjust pHs; At ambient temperature, use the multilamellar filtered through gauze, hydro-oxidation sodium adjust pH is the microporous inorganic membrane filtration clarification of 0.2～1.0 μ m to neutral with the aperture, gets clear liquor, it with the molecular weight that dams 10,000 ultrafilter membrane ultrafiltration, collect permeate, the reuse molecular weight that dams is that 100～1000 NF membrane is carried out nanofiltration and concentrated, and abandons permeate, the concentrated solution that obtains was through 121 ℃ of steam sterilizations 30 minutes, and this degerming solution is the Cerebrolysin Vial highly finished product; Allotment L-tryptophan, L-tyrosine, according to required amount of liquid medicine, drug standard (numbering WS1-XG-25-2000) by the promulgation on the 28th of National Drug Administration's December in 2000 calculates each seed amino acid consumption that need add, allotment peptide figure, be mixed with concentrated wiring liquid with above-mentioned solution, rarely then join, fill becomes semi-finished product, again through 115～121 ℃ of steam sterilizations 30 minutes, make sterile preparation, reach national drug (WS
1-XG-25-2000) quality standard.
Cerebrolysin Vial preparation of the present invention is injection and lyophilized injectable powder.
The preparation method of lyophilized injectable powder and the method for injection are basic identical, just after being mixed with concentrated wiring liquid, add excipient (for example mannitol, dextran etc.), with microporous filter membrane aseptic filtration, packing, the cryogenic vacuum lyophilization finished product in 0.2 μ m aperture, make aseptic freeze-dried injectable powder.
The present invention compared with prior art has the following advantages:
1, given up the chemical precipitation step, the fresh animal cerebral tissue is directly through the multienzyme enzymolysis, and reuse clarification mode is removed water-insoluble, purify, concentrate through clarification, ultrafiltration, nanofiltration, can remove foreigh protein removing, make impurity peak area, reach the separating effect of existing technology less than 5%; Allocate L-tryptophan, L-tyrosine again, drug standard (numbering WS1-XG-25-2000) by the promulgation on the 28th of National Drug Administration's December in 2000 calculates each seed amino acid consumption that need add, allotment peptide figure, be mixed with concentrated wiring liquid with above-mentioned solution, rarely then join, fill becomes semi-finished product, make sterile preparation, with high performance liquid chromatography (HPLC), with the gel chromatographic columns analysis, its chromatogram (peptide figure) should with the chromatogram basically identical of contrast solution, and in its solution chromatogram with the contrast solution chromatogram in inconsistent peak area must not cross 5.0%.This technology has safety, efficient, processing speed fast, the equipment less investment, advantage such as pollution-free, easy to operate.
2, adopt the multienzyme enzymatic isolation method, purify, concentrate Cerebrolysin Vial with clarification technique, hyperfiltration technique and nanofiltration, advantage be shorten greatly the production cycle, output improves, production environment requires to reduce, easy to operation.Simultaneously, reduce production link, shorten the production cycle just mean reduced medicine may contaminated chance, just can reduce the probability that medicine goes bad in process of production.
3, reduced production cost, production can be adopted closed production, adopts physical method, does not have leakage, pollution-free, GMP compatible.
Description of drawings
Fig. 1 is a standard substance peptide figure collection of illustrative plates of the present invention.
Fig. 2 is the peptide figure collection of illustrative plates of sample of the present invention.
The specific embodiment
Embodiment 1:
1, gets 100 kilograms of fresh Medulla sus domestica, remove surperficial peplos and blood vessel etc., homogenate limit, limit adds the purified water (being designated hereinafter simply as water) of 100L, and water proof is steam heated to 85 ℃, 15 minutes, during fast cooling to 40 ℃, transfer pH to 1.5 with 6mol/L hydrochloric acid, add 3Kg pepsin enzymolysis after 3 hours, transfer pH to 8.0 with the 12mol/L sodium hydroxid, add 2Kg pancreatin multienzyme (trypsin, pancreatic lipase and pancreatic amylase), add 0.04mol/L CaCl before using
2 Activation pancreatin multienzyme 1 hour, under 42 ℃ of conditions, enzymolysis 2 hours, and then with 6mol/L hydrochloric acid adjust pH to 3.0 left standstill freezing 12 hours under-20 ℃; At ambient temperature, use the multilamellar filtered through gauze, hydro-oxidation sodium adjust pH is the filtering with microporous membrane clarification of 1.0 μ m to neutral with the aperture, gets clear liquor, it with the molecular weight that dams 10,000 ultrafilter membrane ultrafiltration, collect permeate, the reuse molecular weight that dams is that 400 NF membrane is carried out nanofiltration and concentrated, and abandons permeate, the concentrated solution that obtains was through 121 ℃ of steam sterilizations 30 minutes, and this degerming solution is the Cerebrolysin Vial highly finished product; Allotment L-tryptophan, L-tyrosine, according to required amount of liquid medicine, drug standard (numbering WS1-XG-25-2000) by the promulgation on the 28th of National Drug Administration's December in 2000 calculates each seed amino acid consumption that need add, allotment peptide figure, with high performance liquid chromatography (HPLC), with the gel chromatographic columns analysis, its chromatogram (peptide figure sees Fig. 1) should with the chromatogram (see figure 2) basically identical of contrast solution, and in its solution chromatogram with the contrast solution chromatogram in inconsistent peak area must not cross 5.0%, be mixed with concentrated wiring liquid with above-mentioned solution, rare then joining, fill becomes semi-finished product, through 115 ℃ of steam sterilizations 30 minutes, makes aseptic parenteral solution again.
Embodiment 2,
Get 100 kilograms of fresh Medulla sus domestica, remove surperficial peplos and blood vessel etc., homogenate limit, limit adds the purified water (being designated hereinafter simply as water) of 200L, and water proof is steam heated to 80 ℃, 15 minutes, during fast cooling to 45 ℃, transfer pH to 2.0 with 6mol/L hydrochloric acid, add 3Kg pepsin enzymolysis after 12 hours, transfer pH to 7.7 with the 12mol/L sodium hydroxid, add 2Kg pancreatin multienzyme, add 0.04mol/L CaCl before using
2Activation pancreatin multienzyme 0.5 hour, under 38 ℃ of conditions, enzymolysis 4 hours, and then with 6mol/L hydrochloric acid adjust pH to 2.0 left standstill freezing 48 hours under-10 ℃; At ambient temperature, use the multilamellar filtered through gauze, hydro-oxidation sodium adjust pH is the filtering with microporous membrane clarification of 0.2 μ m to neutral with the aperture, gets clear liquor, it with the molecular weight that dams 10,000 ultrafilter membrane ultrafiltration, collect permeate, the reuse molecular weight that dams is that 400 NF membrane is carried out nanofiltration and concentrated, and abandons permeate, the concentrated solution that obtains obtains degerming solution and is called the Cerebrolysin Vial highly finished product through 121 ℃ of steam sterilizations 30 minutes; Allotment L-tryptophan, L-tyrosine, according to required amount of liquid medicine, drug standard (numbering WS1-XG-25-2000) by the promulgation on the 28th of National Drug Administration's December in 2000 calculates each seed amino acid consumption that need add, allotment peptide figure, see Fig. 1, be mixed with concentrated wiring liquid, add excipient dextran 40 with above-mentioned solution, with filtering with microporous membrane, packing, the cryogenic vacuum lyophilization finished product in 0.2 μ m aperture, make aseptic freeze-dried injectable powder.
Claims (2)
1. the preparation method of a Cerebrolysin Vial preparation, it is characterized in that: peplos and the blood vessel of removing the surface with fresh or frozen Medulla sus domestica, add the purified water homogenate of 1～2 times of volume, water proof is steam heated to 80～85 ℃, 15 minutes, during fast cooling to 40～45 ℃, transfer pH to 1.5～2.0 with hydrochloric acid, get supernatant with pepsin enzymolysis 3～12 hours or precipitate and separate, behind homoiothermic to 40～42 ℃, transfer pH to 7.7～8.0 with sodium hydroxid, with the pancreatin multienzyme under 38～42 ℃ of conditions, enzymolysis 2～4 hours, and then left standstill freezing 12～48 hours with under 2.5～3.0 ,-20～-10 ℃ of the hydrochloric acid adjust pHs; At ambient temperature, filter, hydro-oxidation sodium adjust pH is the microporous inorganic membrane filtration clarification of 0.2～1.0 μ m to neutral with the aperture, gets clear liquor, it with the molecular weight that dams 10,000 ultrafilter membrane ultrafiltration, collect permeate, the reuse molecular weight that dams is that 100～1000 NF membrane is carried out nanofiltration and concentrated, and abandons permeate, the concentrated solution that obtains was through 121 ℃ of steam sterilizations 30 minutes, and this degerming solution is the Cerebrolysin Vial highly finished product; Allotment L-tryptophan, L-tyrosine according to required amount of liquid medicine, calculate each the seed amino acid consumption that needs interpolation, allotment peptide figure is mixed with concentrated wiring liquid with above-mentioned solution, rarely then joins, fill becomes semi-finished product, through 115～121 ℃ of steam sterilizations 30 minutes, make sterile preparation again.
2, the preparation method of Cerebrolysin Vial preparation according to claim 1 is characterized in that described preparation is injection and lyophilized injectable powder.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100431599C (en) * | 2006-03-23 | 2008-11-12 | 夏中宁 | Brain protein hydrolysate and production process of its freeze dried preparation |
| CN104225572A (en) * | 2014-08-19 | 2014-12-24 | 北华大学 | Cerebroprotein hydrolysate and preparation method thereof |
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| CN101940596B (en) * | 2009-07-09 | 2014-07-16 | 张树信 | New method for preparing cerebroprotein hydrolysate |
| CN102093440B (en) * | 2010-12-28 | 2013-04-03 | 山东新时代药业有限公司 | Coproduction process of pig brain protein hydrolysate and monosialoganglioside |
| CN102166200A (en) * | 2011-04-12 | 2011-08-31 | 罗诚 | Freeze-drying composition containing cerebroprotein hydrolysates and preparation method of freeze-drying composition |
| CN102302768A (en) * | 2011-09-04 | 2012-01-04 | 潘首德 | Method for preparing brain protein hydrolysate oral liquid |
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| CN104096215B (en) * | 2014-07-07 | 2015-10-28 | 湖南利诺生物药业有限公司 | Pure natural Cerebrolysin Vial method for preparing raw material |
| CN104587444B (en) * | 2014-12-24 | 2018-04-03 | 内蒙古天奇生物科技有限公司 | A kind of preparation method of high protein content Cerebrolysin Vial |
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| CN112063677A (en) * | 2020-09-30 | 2020-12-11 | 江苏恒新药业有限公司 | Preparation process of oral cerebroprotein hydrolysate |
| CN113908174B (en) * | 2021-10-29 | 2022-06-24 | 北京四环制药有限公司 | Efficient and safe preparation method and application of cerebroprotein hydrolysate |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100431599C (en) * | 2006-03-23 | 2008-11-12 | 夏中宁 | Brain protein hydrolysate and production process of its freeze dried preparation |
| CN104225572A (en) * | 2014-08-19 | 2014-12-24 | 北华大学 | Cerebroprotein hydrolysate and preparation method thereof |
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