CN1771992A - Brain extract and its prepn and use - Google Patents
Brain extract and its prepn and use Download PDFInfo
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- CN1771992A CN1771992A CNA2005101172947A CN200510117294A CN1771992A CN 1771992 A CN1771992 A CN 1771992A CN A2005101172947 A CNA2005101172947 A CN A2005101172947A CN 200510117294 A CN200510117294 A CN 200510117294A CN 1771992 A CN1771992 A CN 1771992A
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Abstract
The present invention provides one kind of brain extract extracted from brain tissue of mammal, including pig, dog, ox, sheep, deer, horse and rabbit. The brain extract contains polypeptide, free lysine, free glutamic acid and total sugar in the weight ratio of 1-200 to 0.1-100 to 0.1-80 to 0.1-12, and has molecular weight of 5,000-10,000. The brain extract contains active matters capable of regulating and improving cerebral metabolism and may be used in treating cerebral vascular diseases and corresponding cerebral metabolic disturbance sequela.
Description
Technical field
The present invention relates to a kind of brain extract and its production and use, specifically, relate to a kind ofly by the brain extract that contains polypeptide, free amino acid, total sugar that extracts from the mammal cerebral tissue except that the people, and this preparation method of extract and purposes belong to the biological preparation field.
Background technology
Free aminoacid content in the cerebral tissue for the highest, secondly is taurine, aspartic acid besides with glutamic acid.Glutamic acid is main excitatory neurotransmitter in the brain.Brain injury may cause the cerebral tissue ion imbalance, causes the exitotoxicity cascade reaction relevant with other excitatory amino acids with glutamic acid, and causes the neuronal death of initial damage position surrouding brain tissue.Lysine is one of eight kinds of essential amino acids, and it plays an important role in the metabolism of living organism, and the lysine shortage can cause the protein function obstacle, influences growth promoter, causes retardation of growth.Because of generally lacking lysine in the vegetable protein, so nutritionist is referred to as " the first necessary aminoacid ".Lysine pharmaceutically has special purposes, and it is the necessary material of synthetic cranial nerve and sexual cell, nucleoprotein, hemoglobin.
Along with the variation of the natural growth of the population, structure aging and dietary structure, being on the rise of infirmities of age, wherein cerebrovascular mainly causes death one of sick.The animal brain egg has obtained extensive concern in recent years from the specific drug of hydrolysate class medicine as this class disease, and its chemical constituent, pharmacological action, clinical practice, production technology etc. all obtain research in various degree.
The brain egg that has appeared on the market can be divided into tablet, oral liquid and injection from hydrolysate class medicine, main component all is to contain free amino acid and molecular weight 10 through what proteasome degradation produced again after the animal brain defat, the mixture of the small-molecular peptides below 000, but concrete active ingredient has difference, as brain protein hydrolysate injection is not contain protein by what extract in the pig brain tissue, contain 80% free amino acid and 20% small-molecular peptides approximately, with the import cerebrolysin be like product, clinically as the brain metabolism improving medicine get good curative effect.Cerebrolysin Vial can increase the utilization of interior glucose of cerebral tissue and oxygen, thereby increases cerebral tissue antioxidant capacity and physical stress ability, alleviates brain tissue impairment, promotes resuming function of brain cell, has the effect of good brain-strengthening, Fructus Alpiniae Oxyphyllae, reparation brain injury.But except some inconvenience that injection is used, also there are many untoward reaction simultaneously.
Therefore, be necessary to seek that a kind of composition is more reasonable, better effects if, take brain metabolism improving medicine more easily.
Summary of the invention
The object of the present invention is to provide a kind of brain extract, obtain by extracting in the mammal cerebral tissue except that the people.
Another object of the present invention is to provide the preparation method of above-mentioned brain extract.
A further object of the present invention provides the various pharmaceutical dosage forms that contain above-mentioned brain extract.
Last purpose of the present invention provides above-mentioned brain extract at preparation treatment acute and chronic cerebrovascular disease, by raise purposes aspect the edema drugs that causes of sequela such as cerebrovascular disease or the caused brain dysbolismus of cerebral trauma, congenital cerebral dysgenesis, central nervous system infection, inaccessible syndrome, arteriosclerosis, thrombophlebitis, capillary hemorrhage and vascular permeability.
Brain extract of the present invention obtains by extracting from the mammal cerebral tissue except that the people, contains polypeptide, free lysine, free glutamic acid and total sugar, and all components molecular weight is 5000～10000 dalton.
Described mammal except that the people is pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit, considers the preferred pig of above-mentioned mammal except that the people from factors such as production cost, production technology, constant product quality, clinical efficacies.
Above-mentioned brain extract and sulfosalicylic acid reaction, muddiness does not take place in mixing; With biuret reaction, solution should show bluish violet to aubergine; With ninhydrin reaction, should show bluish violet; Show bluish violet or purple with alkaline copper citrate test solution reaction.
Polypeptide in the described brain extract: free lysine: free glutamic acid: the weight part ratio of total sugar is 1～200: 0.1～100: 0.1～80: 0.1～12.
Polypeptide in this brain extract: free lysine: free glutamic acid: the weight part ratio of total sugar more preferably 3～40: 0.3～30: 0.3～20: 0.1～6; The all components molecular weight is preferably 6000～8000 dalton simultaneously;
Polypeptide in this brain extract: free lysine: free glutamic acid: the weight part ratio of total sugar more preferably 15～30: 3～15: 2～6: 1～3; And all components molecular weight is preferably 8000 dalton.
The preparation method of brain extract of the present invention is:
Get the mammal cerebral tissue except that the people, add 0.5-2.5 times of weight water for injection, high-pressure homogenization, freezing under-30 ℃～-40 ℃, freeze real back after 10～40 ℃ of thawings, be heated to 80-95 ℃, kept 15-30 minute, and be cooled to room temperature then naturally, under 0～5 ℃ with 10000～12000 rev/mins speed on High speed refrigerated centrifuge centrifugal 15～30 minutes, separation of supernatant and precipitation then, supernatant is standby;
The proteolytic enzyme that in precipitation, adds 0.1-1.5% precipitation weight, 35～50 ℃ of following enzymolysis 2～8 hours, the enzymolysis pH value is controlled to be 7.5-8.5, add aforementioned supernatant then, regulate pH value to 3.0～4.0, be heated to 80-95 ℃, keep after 15-30 minute, naturally after being cooled to room temperature, under 0～5 ℃ with 3500～4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 20～30 minutes, with supernatant liquid filtering, the filtrate adjust pH is 6.5～7.5 then, is heated to 80～95 ℃, keep after 15～30 minutes, naturally be cooled to room temperature, with 3500～4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 20～30 minutes, the supernatant after centrifugal was freezing at-15 ℃～-20 ℃ under 0～5 ℃, at room temperature melt after freezing in fact, filter, filtrate is the ultrafiltration of 5000-10000 ultrafilter membrane with molecular cut off, and ultrafiltrate is brain extract.
Described high-pressure homogenization preferably uses GRB1500-4S high-pressure homogenization pump (east, Shanghai magnificent high-pressure homogenization factory).
Animal brain should be fresh and healthy.
Described proteolytic enzyme can be pepsin or trypsin; The preferred trypsin that uses; Described trypsin regular size is 1: 250 (a Shanghai uncle bio tech ltd difficult to understand).It is 35-50 ℃ that enzymolysis is fit to temperature, is preferably 37～42 ℃; For keeping temperature stabilization, can take the method for interlayer water bath heat preservation.
It is 7.5-8.5 that enzymolysis is fit to pH value, is preferably 7.8～8.2.
For making production technology of the present invention, constant product quality, the enzyme of use should be as far as possible provided by fixing manufacturer.
Describedly be filtered into paper pulp filtering general in the prior art or the clarification plate filters.
The said process adjust pH uses hydrochloric acid and/or sodium hydroxide.
Above-mentioned ultrafilter membrane is preferably the ultrafilter membrane that molecular cut off is 6000-10000, and more preferably molecular cut off is 8000 ultrafilter membrane.The ultrafilter membrane that uses is the commercially available prod, and its manufacturer should be the production unit of industry approval, and the filter membrane model of use and size can be determined according to concrete volume of production, the ultrafilter size selected for use.
The preparation method of brain extract of the present invention is preferably:
Get the mammal cerebral tissue of fresh and healthy, add 0.8-1.2 times of weight water for injection,, melt down at 10～20 ℃ with the homogenate of high-pressure homogenization pump ,-30 ℃～-40 ℃ quick-freezings; Be heated to 80-85 ℃, kept 15-20 minute, be cooled to behind the room temperature centrifugal on High speed refrigerated centrifuge, under 0～5 ℃ of condition with 10000～12000 rev/mins centrifugal 25～30 minutes, supernatant is standby; Precipitation adds the trypsin that adds 0.4-0.8% by weight, 37～42 ℃ of enzymolysis 3～5 hours, the enzymolysis pH value is controlled to be 7.8-8.2, enzymolysis finishes aforementioned supernatant is added, regulate pH value to 3.3～3.7, be heated to 80-90 ℃, kept 15-20 minute, be cooled to behind the room temperature centrifugal on low speed refrigerated centrifuge, under 0～5 ℃ of condition centrifugal 25～30 minutes with 4000 rev/mins speed.Supernatant liquid filtering, the filtrate adjust pH is 6.8～7.2, be heated to 80～90 ℃, kept 15～20 minutes, be cooled to behind the room temperature centrifugal on low speed refrigerated centrifuge, under 0～5 ℃ of condition centrifugal 25～30 minutes with 4000 rev/mins speed, supernatant-15 ℃～-20 ℃ is freezing, and melt freezing back, handles after filtration again, filtrate is 8000 ultrafilter membrane ultrafiltration with molecular cut off, and ultrafiltrate is brain extract.
Described brain extract can be made acceptable forms on the various pharmaceuticss, as tablet, capsule, granule, oral liquid, small-volume injection, bulk capacity injection or lyophilized injectable powder etc., preferred small-volume injection, bulk capacity injection or lyophilized injectable powder, most preferably oral liquid.
Above-mentioned each dosage form can be carried out according to the conventional method on the pharmaceutics.
Brain extract of the present invention contains the peculiar peptidergic nerve nutrient of a kind of brain; 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.
Described brain extract can pass through blood brain barrier, promotes the synthetic of brain internal protein, influences respiratory chain, protective capability with anti-hypoxia is improved the metabolism of brain self-energy, but other hormone systems of activated adenyl cyclase and catalysis, provide neurotransmitter, peptide hormone and coenzyme precursors; Can be used for senile dementia, cerebral blood supply insufficiency, old mental the degeneration and depression; The Supporting Therapy of parkinson; Cranial nerve function obstacle, schizophrenia; The neurasthenia, nervous headache; Congenital brain development is slow, hypoplasia, mental retardation, dysmnesia etc.; Acute brain-injury and sequela thereof; All types of encephalitis, meningitis acute stage and sequela thereof; Promote neurosurgery (cerebral tumor, craniocerebral trauma, cerebrovascular inflammation etc.) cranial nerve tissue repair; The brain metabolism there is not the auxiliary improvement effect entirely, is used for potein deficiency, patient neurasthenia and yet the case of general protein digestibility malabsorption; Can be used for acute and chronic cerebrovascular disease such as preparation treatment cerebral thrombosis, cerebral embolism, brain spasm, craniocerebral trauma and cerebrovascular disease, incomplete brain blood supply, the caused brain dysbolismus of cerebral infarction and sequela, the edema that inaccessible syndrome, arteriosclerosis, thrombophlebitis, capillary hemorrhage and vascular permeability raise and cause, congenital cerebral dysgenesis, central nervous system infection, alzheimer disease, the peripheral nerve injury disease medicament.
The present invention most preferably specification of dosage form oral liquid is:
10ml/ props up, and it is 1.7～2.3mg that every ml contains polypeptide, and containing free lysine is 0.8～1.2mg, and containing free glutamic acid is 0.4～0.6mg, contains total sugar and is not less than 120 μ g.
Instructions of taking is oral, 3 times on the one, and a 10ml.
The present invention has carried out years of researches on existing technology, beat all discovery, centrifugal being applied to of the low-temperature and high-speed of using in the general biochemical laboratory prepared in the brain extract, can collect more low-molecular-weight active polypeptide and important aminoacid, also macromolecular protease is precipitated simultaneously, avoided degraded active substance; The present invention finds that through test of many times wherein aminoacid than the general technology enrichment, has also been found positive effect based on lysine, glutamic acid simultaneously in pharmacology, clinical trial.
Another beneficial effect of the present invention is to have kept the total sugar in the extract, specifically contain glucose, fructose, reach various polysaccharide, ribose etc., make the present composition have high nutrition on the one hand and have identical material with brain, when using as oral liquid simultaneously, sugar combines with the certain of polypeptide, protected polypeptide class active substance to be subjected to the enzymolysis of enzymes such as gastrointestinal, more active substance is absorbed and used; When pharmaceutical composition of the present invention is used as oral liquid simultaneously, easy to use, help the patient and take for a long time, patient's rehabilitation, mitigate the disease there are very big benefit; Avoided the inconvenience of life-time service injection.
Pharmaceutical composition of the present invention contains the active substance of treatment cerebrovascular disease and the brain dysbolismus sequela that causes thereof, and this active matter mass-energy is regulated and improved the brain metabolism.
Pharmaceutical composition of the present invention contains the active substance of the brain dysbolismus sequela for the treatment of cerebrovascular disease and causing, comprises a large amount of active polypeptide and several amino acids.The contained a large amount of active polypeptide of brain extract, several amino acids etc. can see through blood brain barrier; act on nervus centralis in a variety of forms; adjust and improve the neuron metabolism; and influence its respiratory chain; have activation, improve the activity of neurotransmitter and enzyme in the brain, the neuroprotective cell is avoided the infringement of various ischemias and neurotoxin.Can increase the utilization of cerebral tissue, improve the brain cell anaerobic condition, to the protective effect of anoxybiotic cerebral tissue tool glucose.Can be applicable to prepare the purposes of acute and chronic cerebrovascular disease medicament aspects such as treatment cerebral thrombosis, cerebral embolism, brain spasm, can be used for treating craniocerebral trauma and cerebrovascular disease (incomplete brain blood supply, cerebral infarction) and cause sequela such as brain dysbolismus, and the application in the medicines such as the edema that causes and congenital cerebral dysgenesis, central nervous system infection, alzheimer disease that raise as treatment inaccessible syndrome, arteriosclerosis, thrombophlebitis, capillary hemorrhage and vascular permeability.
The specific embodiment
Embodiment 1
Get the Medulla sus domestica 10Kg of fresh and healthy, add 10L water for injection, use high-pressure homogenization pump (GRB1500-4S, the magnificent high-pressure homogenization in east, Shanghai) homogenate, freezing down at-35 ℃, freeze real back after 15 ℃ of thawings, be heated to 80 ℃, kept 15 minutes, naturally be cooled to room temperature then, under 4 ℃ with 10000 rev/mins speed on High speed refrigerated centrifuge centrifugal 30 minutes, separation of supernatant and precipitation then, supernatant is standby;
Trypsin [the Trypsin that in precipitation, adds 0.5% precipitation weight, 1: 250, Amresco 0458], 37 ℃ of following enzymolysis 4 hours, the enzymolysis pH value is controlled to be 8.0, adds aforementioned supernatant then, regulates pH value to 3.5, be heated to 80 ℃, keep after 15 minutes, be cooled to room temperature naturally after, under 4 ℃ with 4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 25 minutes, then with supernatant liquid filtering, the filtrate adjust pH is 7.0, is heated to 85 ℃, keeps after 15 minutes, naturally be cooled to room temperature, under 4 ℃ with 4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 25 minutes, the supernatant after centrifugal-15 ℃ freezing, freeze and at room temperature melt after real, filter, filtrate is 8000 ultrafilter membrane ultrafiltration with molecular cut off, and ultrafiltrate is brain extract, and its content of peptides is 20mg/ml, free lysine content is 10mg/ml, free glutamic acid content is 5mg/ml, and total sugar content is 1.6mg/ml.
Embodiment 2
Get the Pedis Canitis 10Kg of fresh and healthy, add 10L water for injection, high-pressure homogenization, freezing under-35 ℃, freeze real back after 20 ℃ of thawings, be heated to 85 ℃, kept 15 minutes, and be cooled to room temperature then naturally, under 1 ℃ with 10000 rev/mins speed on High speed refrigerated centrifuge centrifugal 25 minutes, separation of supernatant and precipitation then, supernatant is standby;
Trypsin [the Trypsin that in precipitation, adds 0.3% precipitation weight, 1: 250, Amresco 0458], 40 ℃ of following enzymolysis 4 hours, the enzymolysis pH value is controlled to be 8.0, adds aforementioned supernatant then, regulates pH value to 3.0, be heated to 85 ℃, keep after 20 minutes, be cooled to room temperature naturally after, under 2 ℃ with 3500 rev/mins speed on low speed refrigerated centrifuge centrifugal 30 minutes, then with supernatant liquid filtering, the filtrate adjust pH is 7.0, is heated to 85 ℃, keeps after 30 minutes, naturally be cooled to room temperature, under 2 ℃ with 3500 rev/mins speed on low speed refrigerated centrifuge centrifugal 30 minutes, the supernatant after centrifugal-20 ℃ freezing, freeze and at room temperature melt after real, filter, filtrate is 6000 ultrafilter membrane ultrafiltration with molecular cut off, and ultrafiltrate is brain extract, and its content of peptides is 15mg/ml, free lysine content is 10mg/ml, free glutamic acid content is 2mg/ml, and total sugar content is 3mg/ml.
Embodiment 3
Get the Medulla Bovis seu Bubali 10Kg of fresh and healthy, add 15L water for injection, high-pressure homogenization, freezing under-30 ℃, freeze real back after 30 ℃ of thawings, be heated to 90 ℃, kept 30 minutes, and be cooled to room temperature then naturally, under 2 ℃ with 11000 rev/mins speed on High speed refrigerated centrifuge centrifugal 20 minutes, separation of supernatant and precipitation then, supernatant is standby;
Trypsin [the Trypsin that in precipitation, adds 0.1% precipitation weight, 1: 250, Amresco 0458], 45 ℃ of following enzymolysis 6 hours, the enzymolysis pH value is controlled to be 7.5, adds aforementioned supernatant then, regulates pH value to 3.5, be heated to 90 ℃, keep after 25 minutes, be cooled to room temperature naturally after, under 1 ℃ with 3500 rev/mins speed on low speed refrigerated centrifuge centrifugal 25 minutes, then with supernatant liquid filtering, the filtrate adjust pH is 7.5, is heated to 90 ℃, keeps after 20 minutes, naturally be cooled to room temperature, under 1 ℃ with 3500 rev/mins speed on low speed refrigerated centrifuge centrifugal 25 minutes, the supernatant after centrifugal-20 ℃ freezing, freeze and at room temperature melt after real, filter, filtrate is 10000 ultrafilter membrane ultrafiltration with molecular cut off, and ultrafiltrate is brain extract, and its content of peptides is 3mg/ml, free lysine content is 15mg/ml, free glutamic acid content is 15mg/ml, and total sugar content is 6mg/ml.
Embodiment 4
Get the Medulla caprae seuovis 10Kg of fresh and healthy, add 20L water for injection, high-pressure homogenization, freezing under-35 ℃, freeze real back after 35 ℃ of thawings, be heated to 95 ℃, kept 20 minutes, and be cooled to room temperature then naturally, under 3 ℃ with 12000 rev/mins speed on High speed refrigerated centrifuge centrifugal 15 minutes, separation of supernatant and precipitation then, supernatant is standby;
The pepsin that in precipitation, adds 0.8% precipitation weight, 50 ℃ of following enzymolysis 2 hours, the enzymolysis pH value is controlled to be 8.5, add aforementioned supernatant then, regulate pH value to 4.0, be heated to 95 ℃, keep after 30 minutes, be cooled to room temperature naturally after, under 5 ℃ with 4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 25 minutes, then with supernatant liquid filtering, the filtrate adjust pH is 7.5, is heated to 80 ℃, keeps after 25 minutes, naturally be cooled to room temperature, under 5 ℃ with 4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 25 minutes, the supernatant after centrifugal-15 ℃ freezing, freeze and at room temperature melt after real, filter, filtrate is 5000 ultrafilter membrane ultrafiltration with molecular cut off, and ultrafiltrate is brain extract, and its content of peptides is 40mg/ml, free lysine content is 5mg/ml, free glutamic acid content is 10mg/ml, and total sugar content is 3mg/ml.
Embodiment 5
Get the horse brain 10Kg of fresh and healthy, add 25L water for injection, high-pressure homogenization, freezing under-40 ℃, freeze real back after 40 ℃ of thawings, be heated to 80 ℃, kept 25 minutes, and be cooled to room temperature then naturally, under 4 ℃ with 12000 rev/mins speed on High speed refrigerated centrifuge centrifugal 25 minutes, separation of supernatant and precipitation then, supernatant is standby;
The pepsin that in precipitation, adds 1.5% precipitation weight, 40 ℃ of following enzymolysis 4 hours, the enzymolysis pH value is controlled to be 8.0, add aforementioned supernatant then, regulate pH value to 3.5, be heated to 85 ℃, keep after 25 minutes, be cooled to room temperature naturally after, under 4 ℃ with 4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 20 minutes, then with supernatant liquid filtering, the filtrate adjust pH is 6.5, is heated to 85 ℃, keeps after 15 minutes, naturally be cooled to room temperature, under 4 ℃ with 4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 20 minutes, the supernatant after centrifugal-15 ℃ freezing, freeze and at room temperature melt after real, filter, filtrate is 8000 ultrafilter membrane ultrafiltration with molecular cut off, and ultrafiltrate is brain extract, and its content of peptides is 65mg/ml, free lysine content is 20mg/ml, free glutamic acid content is 4mg/ml, and total sugar content is 5mg/ml.
Embodiment 6
Get the Medulla Leporis seu Oryctolagi 10Kg of fresh and healthy, add 15L water for injection, high-pressure homogenization, freezing under-30 ℃, freeze real back after 15 ℃ of thawings, be heated to 85 ℃, kept 25 minutes, and be cooled to room temperature then naturally, under 5 ℃ with 11000 rev/mins speed on High speed refrigerated centrifuge centrifugal 30 minutes, separation of supernatant and precipitation then, supernatant is standby;
The pepsin that in precipitation, adds 1.0% precipitation weight, 45 ℃ of following enzymolysis 5 hours, the enzymolysis pH value is controlled to be 8.5, add aforementioned supernatant then, regulate pH value to 3.0, be heated to 80 ℃, keep after 20 minutes, be cooled to room temperature naturally after, under 0 ℃ with 4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 30 minutes, then with supernatant liquid filtering, the filtrate adjust pH is 7.0, is heated to 90 ℃, keeps after 30 minutes, naturally be cooled to room temperature, under 0 ℃ with 4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 30 minutes, the supernatant after centrifugal-20 ℃ freezing, freeze and at room temperature melt after real, filter, filtrate is 6000 ultrafilter membrane ultrafiltration with molecular cut off, and ultrafiltrate is brain extract, and its content of peptides is 25mg/ml, free lysine content is 13mg/ml, free glutamic acid content is 11mg/ml, and total sugar content is 8mg/ml.
Embodiment 7～12
Get embodiment 1～6 brain extract respectively, handle by known method, make granule, dry back tabletting gets tablet.
Embodiment 13～18
With reference to embodiment 1～6, add suitable flavoring agent (as sucrose, citric acid etc.), carry out the degerming fill, or by known method, the oral liquid of canned, sterilization, packing, each constituent content is as follows in the oral liquid:
| Polypeptide should be mg/mL | Free lysine mg/mL | Free glutamic acid mg/mL | Total sugar μ g/mL | |
| Embodiment 13 | 1.7 | 1.2 | 0.4 | 125 |
| Embodiment 14 | 2.3 | 0.8 | 0.6 | 132 |
| Embodiment 15 | 2.1 | 0.9 | 0.5 | 137 |
| Embodiment 16 | 2.2 | 1.1 | 0.4 | 151 |
| Embodiment 17 | 1.8 | 0.8 | 0.6 | 144 |
| Embodiment 18 | 2.0 | 1.0 | 0.5 | 160 |
Embodiment 19～24
With reference to embodiment 1～6, add suitable excipient (as Dextran 40, mannitol etc.), carry out the degerming fill, by known method, the freeze-dried powder product is made in lyophilization.
Embodiment 25～30
With reference to embodiment 1～6, brain extract is put in the dilute preparing tank; In dense preparing tank, drop into the sodium chloride of troxerutin and 0.85% final preparation cumulative volume, add 0.4 ‰ active carbons and boil decarbonization filtering, after the cooling, with medical filtration to dilute preparing tank, add water for injection to the dosing amount in dilute preparing tank, transferring PH is 6.8～7.0, filters.Embedding is in infusion bottle, and 100 ℃ of flowing steam sterilizations 45 minutes promptly get infusion products.
Experimental example 1
This experimental example is the detection of the oral agents inspection item of embodiment 18 oral liquid appearance characters, pH value, protein and other regulation.
This product is colourless or light yellow clear liquid.
PH value: should be 6.0～8.0 (two VIH of Chinese Pharmacopoeia version in 2000).
Protein: get this product 2ml, add 20% sulfosalicylic acid solution 1ml, muddiness or precipitation must not take place in mixing.
Other: should meet every regulation relevant under the oral solution item (two appendix IO of Chinese Pharmacopoeia version in 2000).
Experimental example 2
This experimental example is the qualitative determination of key component in the embodiment 18 oral agents liquid.
Get 1 of this product, add water 20ml dissolving, as need testing solution.
(1) get need testing solution 5ml, add ninhydrin solution number droplet, heating, solution should show bluish violet.
(2) get need testing solution 5ml, hydro-oxidation sodium solution (6 → 10) 2ml adds alkaline copper citrate test solution 0.2ml, mixing, and room temperature was placed 15 minutes, and solution should show bluish violet or purple.
More than three kinds of experiments be the qualitative reactions of the contained component of pharmaceutical composition of the present invention, illustrate and contain definite component in the drug regimen of the present invention.
Experimental example 3
This experimental example is embodiment 18 oral liquid polypeptide quantitative assay.
It is an amount of to get this product, adds water and makes the solution that every 1ml contains polypeptide 0.15mg, as need testing solution.Precision is measured need testing solution 1.0ml, measures according to forint phenol algoscopy (attached), obtains content of peptides from regression equation.
Reagent alkaline copper solution is got sodium hydroxide 10g, and sodium carbonate 50g adds water 400ml and makes dissolving, as first liquid; Get Soluble tartar. 0.5g, add water 50ml and make its dissolving, other gets copper sulfate 0.25g, adds water 30ml and makes its dissolving, two liquid is mixed, as second liquid.
Before facing usefulness, merge first, second two liquid, and add water to 500ml.
The bovine serum albumin reference substance is got in the preparation of maneuver reference substance solution, adds water and makes the solution that contains 0.3mg among every 1ml.
Preparing of need testing solution according to the method preparation of regulation down of each kind item.
The preparation precision of standard curve is measured reference substance solution 0.0ml, 0.1ml, 0.3ml, 0.5ml, 0.7ml, 0.9ml, put respectively in the tool plug test tube, respectively add water to 1.0ml, add alkaline copper test solution 1.0ml more respectively, shake up, each adds forint phenol test solution (getting the stock solution 1 → 16 in the forint test solution) 4.0ml, mixing immediately, put in 55 ℃ of water-baths accurate response 5 minutes, put psychrolusia 10 minutes, and measured trap at the wavelength place of 650nm according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 B); Manage as blank with No. 0.Calculate regression equation with reference substance solution concentration and respective absorption degree.
The algoscopy precision is measured need testing solution 1.0ml, the method under the preparation of sighting target directrix curve, and " from adding the alkaline copper test solution " measured in accordance with the law, from the content of regression equation calculation polypeptide, and multiply by extension rate, promptly.
Experimental example 4
This experimental example is the quantitative assay of total sugar in embodiment 18 oral liquids.
The preparation of reference substance solution: take by weighing the about 50mg of the glucose that is dried to constant weight, put in the 50ml measuring bottle, the adding distil water dissolving also is settled to scale, mixing.Get this solution 5.0ml, add water and be settled to 50ml, mixing, promptly.
The preparation of need testing solution: get this product 10ml, add water and be settled to 50ml, as need testing solution.
The preparation of standard curve: precision is measured reference substance solution 0.0,0.2,0.4,0.6,0.8,1.0ml puts respectively in the tool plug test tube, respectively add entry to 1.0ml, each adds 5% phenol solution 1.0ml, shake up, add 5ml sulphuric acid, put in 37 ℃ of water-baths and accurately heated 10 minutes, be cooled to room temperature rapidly, with No. 0 pipe is blank, according to spectrophotography (two appendix IVA of Chinese Pharmacopoeia version in 2000), measure trap at the wavelength place of 487nm, be abscissa with concentration, trap is that the vertical coordinate drawing standard curve line linearity of going forward side by side returns, and its correlation coefficient should be greater than 0.99.
Algoscopy: precision is measured need testing solution 1ml, and the preparation method of sighting target directrix curve from " respectively adding 5% phenol solution 1.0ml ", is measured in accordance with the law.From regression equation, obtain total sugar content.
Experimental example 5
This experimental example has illustrated the effect of embodiment 18 oral liquid treatment craniocerebral trauma sequela.
1, physical data is originally organized 38 examples, 38～70 years old age; The course of disease 1 month to 3 years; Cranium contusion and laceration of brain 20 examples, cerebral concussion 10 examples, intracranial hematoma postoperative 8 examples wherein have stupor medical history 22 examples.Sequela mainly show as the headache that all has in various degree, giddy, dizziness, insomnia, dreaming often and waking easily, recent amnesia, hypomnesis, mental disorder and hemiplegia, aphasia, indivedual cases still have paroxysmal to twitch.
2, Therapeutic Method treatment group: the embodiment of the invention 15 oral liquid 10ml, every day 3 times, 14 days is a course of treatment 3, observation of curative effect
3, criterion of therapeutical effect recovery from illness: symptom disappears substantially, recovers operate as normal.Produce effects: symptom is most of to disappear, the time slight headache, giddy, dizziness, the more preceding descender of memory are arranged, but can adhere to work.Invalid: through treating 4 courses of treatment, the state of an illness does not have obvious improver.
Recovery from illness 32 examples in therapeutic outcome 38 examples, produce effects 3 examples, invalid 5 examples, total effective rate is 92.1%.
This evidence brain extract oral liquid treatment of the present invention craniocerebral trauma sequela has better curative effect.
Comparative example
1, case is selected: this organizes 50 examples, male 30 examples, women 20 examples, 40～78 years old age, average 56 years old; Cerebral thrombosis 28 examples, cerebral hemorrhage 16 examples (following 9 examples of amount of bleeding 25ml, hematoma clearance postoperative 7 examples), cerebral infarction 6 examples; Acute stages 12 example, convalescent periods 21 example, sequela stage 17 examples; The elder of the course of disease is 8 years, and the patient is made head CT and MRI checks, cerebral malacia or brain atrophy is in various degree all arranged, and make mensuration such as hematuria stool routine, liver function, blood fat, whole blood contrast viscosity, Fibrinogen, erythrocyte sedimentation rate; Be divided into treatment group, matched group.
2, medication and observational technique: treatment group: the 5%GS250ml+ embodiment of the invention 18 oral liquid 10ml, iv gtt, qd, 14 days is a course of treatment; Matched group: 5%GS250ml+ piracetam 8g+ citicoline pin 0175g, iv gtt, qd, 14 days is a course of treatment.Carry out the simple intelligent test before and after the medication, dysnoesia, aphasis degree and hemiplegia degree detect and assessment with the simple and easy intelligence scale in international standard Chang Gu river.
3, clinical efficacy evaluation: produce effects: intelligence is recovered normal or aphasis improves the I level, and muscular strength improves the I level on former basis; Effectively: intelligence, language improve the I level on former basis, and muscular strength improves the I level; Invalid: intelligence, language and muscular strength no change.
4, statistical procedures: treatment group and matched group neurological deficits score relatively adopt the t check, the capable X 2 test of clinical efficacy.Treatment group and matched group clinical symptoms are improved effective percentage relatively, see Table 1.The result: treatment group produce effects 47 examples, effective 28 examples, 4 examples are invalid, total effective rate reaches 94%, and wherein muscular strength improves above person's 10 examples of I level after the 23 routine mental retardation person medications, feels limb adynamia after the 3 routine medications, the no significant change of 1 example, 22 illustrative phrases speech obstacle person, 18 examples improve more than the I level
Table 1 liang group curative effect relatively
| Group | Produce effects | Effectively | Invalid | Total effective rate (%) |
| Treatment group (n=25) | 18 | 5 | 2 | 92 |
| Matched group (n=25) | 11 | 8 | 6 | 76 |
Cerebrovascular disorders causes cerebral hypoxia ischemia, causes brain cell necrosis, and neural cell injury makes it lose function, along with the development of the state of an illness, cerebral malacia, brain atrophy occur.Traditional treatment all with anticoagulant, thrombolytic, blood fat reducing, blood vessel dilating, microcirculation improvement, improve brain blood supply, reduce brain cell death as far as possible, improve clinical symptoms, but neural powerless to dead brain cell or loss of function, and brain extract main component of the present invention is a brain organ specificity polypeptide, and contain free amino acid and total sugar, can pass through blood brain barrier, participate in the metabolism of maincenter and peripheral nervous tissue directly, make the nucleic acid metabolism of damaged nerve tissue and carbohydrate metabolism recover normal.It can promote neuronic growth of effect and differentiation such as sympathetic, sensation.The sophisticated effector neuron of nutrition, keep its survival and function, the brain atrophy patient that causes of our clinical observation cerebrovascular disorders has obtained satisfactory effect at this point, it to some sequela stage patient intelligence recover, aphasis improves and limb functional recover plays good effect.
Claims (9)
1, a kind of brain extract obtains by extracting in the mammal cerebral tissue except that the people, it is characterized in that described brain extract contains polypeptide, free lysine, free glutamic acid and total sugar, and all components molecular weight is 5000～10000 dalton.
2, brain extract according to claim 1 is characterized in that, described mammal except that the people is pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit, preferred pig.
3, brain extract according to claim 2 is characterized in that, polypeptide in the described brain extract: free lysine: free glutamic acid: the weight part ratio of total sugar is 1～200: 0.1～100: 0.1～80: 0.1～12; More preferably 3～40: 0.3～30: 0.3～20: 0.1～6; The all components molecular weight is preferably 6000～8000 dalton simultaneously; More preferably 15～30: 3～15: 2～6: 1～3; And all components molecular weight is preferably 8000 dalton.
4, a kind of preparation method of brain extract, it is characterized in that, described method is: get the mammal cerebral tissue except that the people, add 0.5-2.5 times of weight water for injection, high-pressure homogenization, freezing down at-30 ℃～-40 ℃, freeze real back after 10～40 ℃ of thawings, be heated to 80-95 ℃, kept 15-30 minute, and be cooled to room temperature then naturally, under 0～5 ℃ with 10000～12000 rev/mins speed on High speed refrigerated centrifuge centrifugal 15～30 minutes, separation of supernatant and precipitation then, supernatant is standby; The proteolytic enzyme that in precipitation, adds 0.1-1.5% precipitation weight, 35～50 ℃ of following enzymolysis 2～8 hours, the enzymolysis pH value is controlled to be 7.5-8.5, add aforementioned supernatant then, regulate pH value to 3.0～4.0, be heated to 80-95 ℃, keep after 15-30 minute, naturally after being cooled to room temperature, under 0～5 ℃ with 3500～4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 20～30 minutes, with supernatant liquid filtering, the filtrate adjust pH is 6.5～7.5 then, is heated to 80～95 ℃, keep after 15～30 minutes, naturally be cooled to room temperature, with 3500～4000 rev/mins speed on low speed refrigerated centrifuge centrifugal 20～30 minutes, the supernatant after centrifugal was freezing at-15 ℃～-20 ℃ under 0～5 ℃, at room temperature melt after freezing in fact, filter, filtrate is the ultrafiltration of 5000-10000 ultrafilter membrane with molecular cut off, and ultrafiltrate is brain extract.
5, preparation method according to claim 4 is characterized in that, described proteolytic enzyme is pepsin or trypsin; The preferred trypsin that uses; Described trypsin regular size is 1: 250; It is 35-50 ℃ that enzymolysis is fit to temperature, is preferably 37～42 ℃; For keeping temperature stabilization, can take the method for interlayer water bath heat preservation; It is 7.5-8.5 that enzymolysis is fit to pH value, is preferably 7.8～8.2.
6, preparation method according to claim 4 is characterized in that, describedly is filtered into paper pulp filtering general in the prior art or the clarification plate filters; The said process adjust pH uses hydrochloric acid and/or sodium hydroxide; Above-mentioned ultrafilter membrane is preferably the ultrafilter membrane that molecular cut off is 6000-10000, and more preferably molecular cut off is 8000 ultrafilter membrane.
7, preparation method according to claim 4 is characterized in that, described mammal except that the people is pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit, preferred pig.
8, the various drug forms that contain the described brain extract of claim 1, described dosage form is an acceptable forms on the various pharmaceuticss, as tablet, capsule, granule, oral liquid, small-volume injection, bulk capacity injection or lyophilized injectable powder etc., preferred small-volume injection, bulk capacity injection or lyophilized injectable powder, most preferably oral liquid.
9, the described brain extract of claim 1 is at preparation treatment acute and chronic cerebrovascular disease, by raise purposes aspect the edema drugs that causes of sequela such as cerebrovascular disease or the caused brain dysbolismus of cerebral trauma, congenital cerebral dysgenesis, central nervous system infection, inaccessible syndrome, arteriosclerosis, thrombophlebitis, capillary hemorrhage and vascular permeability.
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| CNA2005101172947A CN1771992A (en) | 2005-11-02 | 2005-11-02 | Brain extract and its prepn and use |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188447A (en) * | 2010-03-16 | 2011-09-21 | 博安兄弟制药（中国）有限公司 | Preparation method for cerebroprotein hydrolysate in piracetam cerebroprotein hydrolysate tablets |
| CN101343317B (en) * | 2007-07-09 | 2012-01-04 | 锡林郭勒盟鑫泰生物制品有限责任公司 | Brain peptide, preparation and uses thereof |
| EP3095451A1 (en) | 2015-05-22 | 2016-11-23 | Bioiberica, S.A. | Process for preparing an animal brain extract |
| CN113908174A (en) * | 2021-10-29 | 2022-01-11 | 北京四环制药有限公司 | Efficient and safe preparation method and application of cerebroprotein hydrolysate |
| WO2024164927A1 (en) * | 2023-02-09 | 2024-08-15 | Alis Pharma Ltd. | Use of fetal sheep brain extracts for managing neurodegenerative diseases |
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2005
- 2005-11-02 CN CNA2005101172947A patent/CN1771992A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343317B (en) * | 2007-07-09 | 2012-01-04 | 锡林郭勒盟鑫泰生物制品有限责任公司 | Brain peptide, preparation and uses thereof |
| CN102188447A (en) * | 2010-03-16 | 2011-09-21 | 博安兄弟制药（中国）有限公司 | Preparation method for cerebroprotein hydrolysate in piracetam cerebroprotein hydrolysate tablets |
| EP3095451A1 (en) | 2015-05-22 | 2016-11-23 | Bioiberica, S.A. | Process for preparing an animal brain extract |
| WO2016188684A1 (en) | 2015-05-22 | 2016-12-01 | Bioiberica, S.A. | Process for preparing an animal brain extract |
| CN113908174A (en) * | 2021-10-29 | 2022-01-11 | 北京四环制药有限公司 | Efficient and safe preparation method and application of 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 |
| WO2024164927A1 (en) * | 2023-02-09 | 2024-08-15 | Alis Pharma Ltd. | Use of fetal sheep brain extracts for managing neurodegenerative diseases |
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