CN100522240C - Medicinal composition for treating and improving brain function, its preparation method and application - Google Patents

Abstract

A medicinal composition for preparing the medicine to improve the cerebral function contains proportionally the active polypeptide, amino acid and nucleic acid, which are extracted from the muscle tissue of mammal and ganglioside extracted from the cerebral tissue of mammal. Its preparing process is also disclosed.

Description

Pharmaceutical composition, its preparation method and the application thereof of treatment improving brain function

Technical field

The present invention relates to a kind of pharmaceutical composition, Its Preparation Method And Use for the treatment of improving brain function, specifically, relate to a kind of pharmaceutical composition, its preparation method by the active polypeptide that from the mammal muscular tissue except that the people, extracts, aminoacid, nucleic acid and the ganglioside that from the mammal cerebral tissue except that the people, extracts and the dysfunction that causes at preparation treatment brain diseases aspect medicinal application.

Background technology

Ganglioside is a kind of sialic glycosyl sphingolipid class that contains, and its one or more terminal saccharide are that the nervonic acid of N-acetyl group is a sialic acid.Contain ganglioside up to more than 6% in the ectocinereal film fat, in the conduction of nerve, play an important role.

The clinical medical value of ganglioside has: the functional rehabilitation that is used for the central nervous system structures infringement that a variety of causes causes; Be used for the treatment of cerebrovascular accident wound and post-traumatic central nervous system's sequela; Be used for the treatment of ischemic and hemorrhagic cerebral lesion.

The GM-1 that the gangliosides medicine of present listing has Argentina to produce is the Monostalotetrahexosylgangliside sodium-salt parenteral solution, and it is one-component by gene engineering method production, does not contain active polypeptide, not too is beneficial to absorb and utilization.

Monosialyl tetrahexose base ganglioside (GM1) injection (trade name Sygen) that Italy Fidia company extracts from cow brain tissue is formally sold in China the nineties in 20th century.

Chinese patent application 93112578 discloses a kind of brain health nutriment, its manufacture method and purposes, these health product are the nutritional solution that contains ganglioside, ganglioside content at least every milliliter contain 0.5mg, its manufacture method mainly makes water, chloroform, alcohol extraction, its purposes is to strengthen study, the memory function of human brain, promotes the normal development of human brain and the recovery behind the brain injury.

Chinese patent application 95111569 discloses a kind of production method that ganglioside is master's a glycolipid class material that contains, comprise pulverizing, solvent extraction, filtration and concentrated, it is characterized in that clean animal brain through pulverizing, the ethanol and the Petroleum ether extraction that add 40-100% content, pulverize after perhaps in clean animal brain, adding the ethanol of 40～100% content, add Petroleum ether extraction, the said extracted thing filters, clear liquid concentrates, and the weight ratio of described animal brain, ethanol and petroleum ether is 1:1～20:0.5～8.

Chinese patent application 00133077 discloses a kind of process for preparing high-purity of Monostalotetrahexosylgangliside.

In the organism, micromolecule polypeptide, aminoacid, the various biochemical processes of nucleic acid wide participation, comprise synthetic, the transhipment of material of various materials, the generation and the transmission of information goods and materials, for all vital movements provide energy, especially even more important simultaneously to cerebral tissue.

Method from the extraction ganglioside of domestic and foreign literature report, the total fat of different proportion organic solvent extracting that adopt more, with distributing extracting process to extract ganglioside mixture, reuse ion-exchange chromatography, size-exclusion and silica gel chromatography separate ganglioside then.

Report is arranged, and with tissue homogenate chloroform-methanol solution extracting, centrifugal, supernatant adds the water extraction, evaporate to dryness gets thick fat.Thick liposoluble in methanol, is crossed anion-exchange column, collect ganglioside.

Other has report, will organize to add the extracting of chloroform-methanol solution, and extract concentrates, the rotation evaporate to dryness gets total fat.Total fat is added after diisopropyl ether-n-butyl alcohol dissolution with solvents with sodium chloride solution extraction, centrifugal, and water is acid glycosyl sphingolipid extract.The thick fat of lyophilizing is crossed Sepbadex G-50 column chromatography, collects the lyophilizing of mixed nerve joint glycosides fat component.

At present, the multiple plain injection of cerebral tissue extract class such as brain, the cerebrin injection of living, cerebral tissue injection, heat are hidden brain injection, brain polypeptide injection etc., all for being made by all kinds of cerebral tissue Proteolytic enzyme, contain nervous tissue's active polypeptide, but ganglioside can not detect.

Patent application 01126465 discloses a kind of medicine and health product for the treatment of nervous system disease, contains ganglioside and Folium Ginkgo extract, provides ganglioside as composition component, with the collaborative pharmaceutical composition that plays a role of other component.

The content of the ganglioside in the disclosed brain extract is all lower at present, though ganglioside is also arranged as composition component, with the open report of the collaborative pharmaceutical composition that plays a role of other component, but there are not itself and other body tissues of animal to work in coordination with the relevant report that plays a role yet.

Summary of the invention

The object of the present invention is to provide a kind of pharmaceutical composition of the treatment treatment improving brain function by the active polypeptide that from the mammal muscular tissue except that the people, extracts, aminoacid, nucleic acid and the preparation of the ganglioside that from the mammal cerebral tissue except that the people, extracts.

Another object of the present invention provides this preparation of drug combination method.

Further object of the present invention provides the application of the dysfunction aspect medicine that preparation of pharmaceutical compositions treatment brain diseases causes.

The invention still further relates to the application of pharmaceutical composition on preparation treatment alzheimer disease, cerebrovascular accident wound and post-traumatic central nervous system's sequela, cerebrovascular and ischemic and hemorrhagic cerebral lesion medicine.

The pharmaceutical composition of treatment treatment improving brain function of the present invention, wherein contain animal muscle tissue's extract and cerebral tissue ganglioside extract, contain monosialyl tetrahexose base ganglioside (GM1) in the cerebral tissue ganglioside extract, bifunctional sialyltransferase tetrahexose base ganglioside, multiple gangliosides such as three sialic acid tetrahexose base gangliosides, contain active polypeptide in the muscular tissue extract, aminoacid and nucleic acid, these active component molecular weight are that the ultrafilter membrane of 5000-10000 is controlled when ultrafiltration by molecular cut off, ultrafiltrate is the muscle extracting solution, and the muscle extracting solution contains polypeptide, aminoacid and nucleic acid;

Pharmaceutical composition of the present invention, it is characterized in that containing active polypeptide, aminoacid, nucleic acid that from the mammal muscular tissue except that the people, extracts and the ganglioside that from the mammal cerebral tissue except that the people, extracts, wherein active polypeptide weight is sialic 6.1～100 times, aminoacid weight is sialic 0.1～100 times, total weight nitroxide is sialic 2～50 times, and nucleic acid weight is sialic 0.5～30 times.

Active polypeptide weight is sialic 50～80 times in the described pharmaceutical composition, and aminoacid weight is sialic 1～50 times, and total weight nitroxide is sialic 5～40 times, and nucleic acid weight is sialic 2～20 times.

Active polypeptide weight is sialic 60～70 times in the described pharmaceutical composition, and aminoacid weight is sialic 20～40 times, and total weight nitroxide is sialic 10～20 times, and nucleic acid weight is sialic 3～10 times.

Preferably active polypeptide weight is sialic 64 times in the pharmaceutical composition, and amino acid whose weight is sialic 33 times, and total weight nitroxide is sialic 18.6 times, and nucleic acid weight is sialic 6 times.

Ganglioside content in the mammalian central nervous system is abundant, for example can extract the material that contains ganglioside from fresh cerebral tissue such as pig, Canis familiaris L., cattle, sheep, deer, horse, rabbit, above-mentioned mammiferous muscular tissue also can be extracted active polypeptide, aminoacid and nucleic acid.

Above-mentioned mammal muscular tissue is preferably the rabbit muscle tissue.

Above-mentioned mammal cerebral tissue is preferably cow brain tissue.

In traditional treatment, the Carnis Leporis tissue is used for treating cardiomyopathy more significant curative effect, and considers that from the operability and the effective rate of utilization aspect of production process multiselect extracts active polypeptide, aminoacid and nucleic acid with Carnis Leporis; Extracted amount based on ganglioside considers that the preferred Medulla Bovis seu Bubali of the present invention extracts ganglioside.

Therefore, consider that preferred cow brain tissue and Carnis Leporis tissue are as raw materials for production from factors such as production cost, production technology, constant product quality, clinical efficacies.

Above-mentioned animal muscle tissue can be any part muscle of animal.

A kind of method for preparing described pharmaceutical composition is characterized in that comprising the steps:

(1) preparation of muscle extracting solution:

A) animal any part muscular tissue is added after the water homogenate freezingly, multigelation 1～4 time melts post-heating centrifugal filtration slagging-off, keeps filtrate;

B) filtrate is regulated pH value insulation enzymolysis, regulates pH value and is acid, heats the centrifugal precipitation of removing, and filtering and impurity removing keeps filtrate;

C) filtrate adjusting is neutrality, freezing, melt the centrifugal precipitation of removing in back, supernatant liquid filtering, filtrate is used ultrafilter membrane ultrafiltration 1～2 time, and ultrafiltrate is the muscle extracting solution;

(2) preparation of ganglioside extracting solution:

A) with animal brain's solubilizer homogenate after-filtration, filtering residue extracts with solvent extraction, and extracting solution and filtrate merge the back evaporate to dryness, get dry thing;

B) dry thing is dissolved in the solvent, to the extraction with aqueous solution separatory that wherein adds salt;

C) upper and lower layer of liquid phase of separatory with the mixed aqueous solution washing of at least two kinds of solvents, merges the upper phase part after washing respectively, and concentrating under reduced pressure is used the pre-cooling acetone precipitation then, gets dry thing after the drying;

D) dry thing is dissolved in the mixed aqueous solution of at least two kinds of solvents, chromatographic column separates, and eluent concentrates the back with pre-cooling acetone precipitation, separation, and the precipitation that obtains obtains the ganglioside extracting solution through post processing;

(3) said extracted liquid is mixed, the weight that makes polypeptide in the mixed liquor is 6.1～100 times of sialic acid weight in the ganglioside, making aminoacid weight in the mixed liquor is 0.1～100 times of sialic acid weight in the ganglioside, total weight nitroxide is in the ganglioside sialic 2～50 times, and nucleic acid weight is in the ganglioside sialic 0.5～30 times; Through post processing, get finished product preparation.

Wherein, in the preparation process of muscle extracting solution, add the homogenate of 0.6～2.5 times of weight water, freezing after the homogenate, multigelation 1～4 time.

Enzymolysis adopts trypsin, heat tracing hydrolysis under control pH7.5～8.5 conditions.

Hydrolyzed solution is after the centrifugal filtration of twice adjusting pH value, filtrate is the ultrafilter membrane ultrafiltration 1～2 time of 5000-10000 with molecular cut off, getting content of peptides is 0.5～15mg/ml, amino acid content is 0.5～15mg/ml, total nitrogen content is 0.2～8mg/ml, and nucleic acid content is the muscle extracting solution of 0.05～3mg/ml.

In the preparation process of brain extract, the homogenate solvent for use is an acetone, and (filtrate of merging is used the rotary evaporator evaporate to dryness to filtering residue for 1～20:1～20, the abundant extracting of mixed liquor V/V), gets dry thing with chloroform-methanol.

The solvent that dissolves dry thing is that volume ratio is the halogenated hydrocarbons of 2～30:1～20 and the mixed solvent of alcohol, the preferred KCl aqueous solution of the aqueous solution of salt.

The upper and lower layer liquid phase of separatory is evaporated to 1/8～1/2 of initial volume, then with being lower than 0 ℃ of pre-cooling acetone precipitation respectively with the mixed aqueous solution washing of halogenated hydrocarbons and alcohol.

Dry thing is dissolved in halogenated hydrocarbons and pure mixed aqueous solution, with the mixed aqueous solution gradient elution column chromatography for separation of halogenated hydrocarbons and alcohol.

More particularly, preparation method of the present invention comprises:

(1) preparation of muscle extracting solution:

A) animal any part muscular tissue is added the homogenate of 0.6～2.5 times of weight water, freezing after the homogenate, multigelation 1～4 time melts 60～70 ℃ of post-heating, is incubated 15～30 minutes, the cooling back is centrifugal, centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, keeps supernatant, supernatant liquid filtering gets filtrate;

B) add trypsin insulation hydrolysis in filtrate, trypsinase concentration is 0.03～0.5%, and hydrolysising condition is pH=7.5～8.5, is incubated 35～50 ℃, and temperature retention time is 0.5～5 hour; Behind the enzymolysis, the enzymolysis solution adjust pH is 3.5-4.5, is heated to 70-85 ℃, keeps 15-30 minute, and the cooling back is centrifugal, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate.

C) the filtrate adjust pH is 6.5-7.5, and is freezing, centrifugal after melting, centrifugal condition is 3000～4000 rev/mins, centrifugation time is 15～30 minutes, keeps supernatant, supernatant liquid filtering, filtrate is the ultrafilter membrane ultrafiltration 1～2 time of 5000-10000 with molecular cut off, ultrafiltrate is the muscle extracting solution, and wherein content of peptides is 0.5～15mg/ml, and wherein amino acid content is 0.5～15mg/ml, total nitrogen content is 0.2～8mg/ml, and nucleic acid content is 0.05～3mg/ml.

(2) preparation of ganglioside extracting solution:

A) animal brain is added the acetone homogenate after-filtration of 3～4 times of weight, keep filtering residue and filtrate; Filtering residue with the chloroform-methanol of 3～5 times of weight (1～20:1～20, V/V) the mixed liquor extracting is three times, filters respectively; Above-mentioned filtrate is merged back 37 ℃ of evaporated under reduced pressure in rotary evaporator, get dry thing;

B) dry thing is dissolved in chloroform-methanol (2～30:1～20, V/V) mixed liquor adds the KCl solution of the 0.1mol/L of 1/20～1/80 volume then in mixed liquor, stirred 1 hour under the room temperature, leave standstill separatory after 8～16 hours;

C) behind the separatory, upper phase is with isopyknic chloroform-methanol-water (50～150:5～40:0.5～5, V/V/V) mixed liquor washing, (1～10:30～80:35～85, V/V/V) mixed liquor washing merge the upper phase part after twice washing to lower floor's liquid phase with isopyknic chloroform-methanol-water, be evaporated to 1/8～1/2 of initial volume at 37 ℃, use-20 ℃ of pre-cooling acetone precipitations then, vacuum drying gets dry thing;

D) chloroform-methanol that dry thing is dissolved in-water (30～80:10～50:1～90, V/V/V) mixed liquor, make the solution of 1～5% concentration, add chromatographic column, adjust flow velocity 5mL/ minute, with chloroform-methanol-water (60～90:15～40:1～10, V/V/V) mixed liquor 10～30L eluting, use chloroform-methanol-water (40～80:20～50:5～15 then instead, V/V/V) mixed liquor 8～15L eluting is collected back eluent 6～12L, behind the concentrating under reduced pressure with-20 ℃ of pre-cooling acetone precipitations of 3～5 times of volumes, the supernatant is gone in-20 ℃ of hypsokinesis in static 2 hours, remaining part descended centrifugal 10 minutes at 1000 rev/mins, and the precipitation that obtains is washed 2 times with proper amount of acetone, vacuum drying, with the water-soluble ganglioside extracting solution that gets of dry thing, concentration is that every 1ml contains ganglioside in sialic acid 0.01～0.5mg;

(3) said extracted liquid is mixed, the weight that makes polypeptide in the mixed liquor is 6.1～100 times of sialic acid weight in the ganglioside, making aminoacid weight in the mixed liquor is 0.1～100 times of sialic acid weight in the ganglioside, total weight nitroxide is in the ganglioside sialic 2～50 times, and nucleic acid weight is in the ganglioside sialic 0.5～30 times; Through post processing, get finished product preparation.

More preferred, preparation method of the present invention comprises the steps:

(1) preparation of muscle extracting solution:

A) animal any part muscular tissue is added the homogenate of 1～1.5 times of weight water, freezing after the homogenate, multigelation 2～3 times melts 60～70 ℃ of post-heating, is incubated 20 minutes, the cooling back is centrifugal, centrifugal condition is 4000 rev/mins, and centrifugation time is 20～30 minutes, keeps supernatant, supernatant liquid filtering gets filtrate;

B) add trypsin insulation hydrolysis in filtrate, trypsinase concentration is 0.03～0.5%, and hydrolysising condition is pH=7.8～8.2, is incubated 37～42 ℃, and temperature retention time is 2.5～4 hours; Behind the enzymolysis, the enzymolysis solution adjust pH is 3.8-4.2, is heated to 80-85 ℃, keeps 20 minutes, and the cooling back is centrifugal, and centrifugal condition is 4000 rev/mins, and centrifugation time is 20～30 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate.

C) the filtrate adjust pH is 6.8-7.2, and is freezing, centrifugal after melting, centrifugal condition is 4000 rev/mins, centrifugation time is 20～30 minutes, keeps supernatant, supernatant liquid filtering, filtrate is the ultrafilter membrane ultrafiltration 1～2 time of 8000-10000 with molecular cut off, ultrafiltrate is the muscle extracting solution, and wherein content of peptides is 0.5～15mg/ml, and wherein amino acid content is 0.5～15mg/ml, total nitrogen content is 0.2～8mg/ml, and nucleic acid content is 0.05～3mg/ml.

(2) preparation of ganglioside extracting solution:

A) animal brain is added the acetone homogenate after-filtration of 3～4 times of weight, keep filtering residue and filtrate; Filtering residue with the chloroform-methanol of 3～5 times of weight (1:1, V/V) the mixed liquor extracting is three times, filters respectively; Above-mentioned filtrate is merged back 37 ℃ of evaporated under reduced pressure in rotary evaporator, get dry thing;

B) dry thing is dissolved in chloroform-methanol (2:1, V/V) mixed liquor adds the KCl solution of the 0.1mol/L of 1/80 volume then in mixed liquor, stirred 1 hour under the room temperature, leave standstill separatory after 8～16 hours;

C) behind the separatory, upper phase is with isopyknic chloroform-methanol-water (86:14:1, V/V/V) mixed liquor washing, (3:48:47, V/V/V) mixed liquor washing merge the upper phase part after twice washing to lower floor's liquid phase with isopyknic chloroform-methanol-water, be evaporated to 1/4 of initial volume at 37 ℃, use-20 ℃ of pre-cooling acetone precipitations then, vacuum drying gets dry thing;

D) chloroform-methanol that dry thing is dissolved in-water (65:25:4, V/V/V) mixed liquor, make the solution of 2.5% concentration, add chromatographic column, adjust flow velocity 5mL/ minute, with chloroform-methanol-water (65:25:4, V/V/V) mixed liquor 20L eluting, use chloroform-methanol-water (60:35:8 then instead, V/V/V) mixed liquor 12L eluting is collected back eluent 10L, behind the concentrating under reduced pressure with-20 ℃ of pre-cooling acetone precipitations of 4 times of volumes, the supernatant is gone in-20 ℃ of hypsokinesis in static 2 hours, remaining part descended centrifugal 10 minutes at 1000 rev/mins, and the precipitation that obtains is washed 2 times with proper amount of acetone, vacuum drying, with the water-soluble ganglioside extracting solution that gets of dry thing, concentration is that every 1ml contains ganglioside in sialic acid 0.01～0.5mg;

(3) said extracted liquid is mixed, the weight that makes polypeptide in the mixed liquor is 6.1～100 times of sialic acid weight in the ganglioside, making aminoacid weight in the mixed liquor is 0.1～100 times of sialic acid weight in the ganglioside, total weight nitroxide is in the ganglioside sialic 2～50 times, and nucleic acid weight is in the ganglioside sialic 0.5～30 times; Through post processing, get finished product preparation.

The B of above-mentioned steps (1)) in, the trypsin of use is conventional commercially available prod, in order to make production technology of the present invention, constant product quality, should be as far as possible provided by fixing manufacturer; The enzymolysis pH value is preferably 7.8～8.2, and hydrolysis temperature is preferably 37～42 ℃; For keeping temperature stabilization, can take the method for interlayer water bath heat preservation.After finishing, insulation should regulate pH3.8～4.2 back reheat.

The C of above-mentioned steps (1)) in, 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 B of above-mentioned steps (2)) in, chloroform-methyl alcohol mixed liquor addition is as the criterion to dissolve dry thing fully.

The addition of acetone is as the criterion to no longer include to precipitate to separate out in the above-mentioned steps (2).

The D of above-mentioned steps (2)) in, used chromatographic column prepares by following process: silica gel particle is crossed screening 60～80 purpose single-sizes, drying is 1 hour under 110 ℃, make its activation, use then 5 times of column volumes chloroform-methanol (5～20:0.5～3, V/V) mixed liquor is as carrier, with silica gel furnishing pasty state, make the chromatographic column of the 80 * 10cm that packs into behind its homodisperse, promptly.Wherein preferred chloroform-methanol mixing ratio is 9:1 (V/V).

The pressure of reduction vaporization of the present invention is controlled at about 0.04～0.08MPa.

The dosage form of finished product preparation is an acceptable forms on the pharmaceutics in the above-mentioned steps (3), such as tablet, capsule, oral liquid, small-volume injection, infusion solutions or freeze-dried powder etc., be preferably small-volume injection, infusion solutions or freeze-dried powder, most preferably be small-volume injection.Therefore, the described post processing of described step (3) can be to carry out according to the conventional method of acceptable forms on the preparation pharmaceutics.

Preparation method of the present invention adopts the silicagel column single step purification to get mixed nerve joint glycosides fat, use the pre-cooling acetone precipitation behind elder generation's concentrating under reduced pressure, be dissolved in distilled water behind the vacuum drying, standby as the ganglioside extracting solution, make the extract effective ingredient yield that makes higher, component is more reasonable, has also saved the operating time simultaneously, has reduced consumption of raw and auxiliary materials.Ganglioside has the function of external information in perception, the transfer sell, participates in the processes such as identification, adhesion, growth, differentiation and the transmission of cell information of cell.As the receptor of some neurotransmitter, hormone, virus and interferon, have the differentiation, regeneration, the reparation that participate in nervous tissue, with the conduction of neural impulse, intercellular recognition reaction.Can quicken the Regeneration and Repair of the nervous tissue of damage, promote the innervation functional rehabilitation, lower the release of excitatory amino acid, thereby alleviate cytotoxicity and angioedema, be the good medicine of cerebrovascular accident treatment, be used for the treatment of alzheimer disease and other nervous system disease is obtained good efficacy.

Adopt freeze thawing in conjunction with enzyme solution in the preparation method of the present invention, extract polypeptide, aminoacid, nucleic acid quickly and easily from muscular tissue, this method has the yield height, and the operating time is short, the characteristics that cost is low.

Contain active polypeptide, aminoacid, nucleic acid and multiple ganglioside in the pharmaceutical composition of the present invention, contain active polypeptide, aminoacid, nucleic acid and multiple ganglioside simultaneously in the pharmaceutical composition of the present invention and be beneficial to absorption and utilization, can promote the metabolism of cerebral tissue, participate in the neuronic growth of cerebral tissue, differentiation and regenerative process, improve cerebral blood circulation and brain metabolic function, can be applicable to prepare the dysfunction medicine aspect that the treatment brain diseases causes, can be used for diseases such as treatment dementia, cerebral trauma, cerebral edema; Pharmaceutical composition of the present invention is as the application in preparation treatment alzheimer disease, cerebrovascular accident wound and the medicines such as post-traumatic central nervous system's sequela, cerebrovascular and ischemic and hemorrhagic cerebral lesion.

Research worker of the present invention is tested discovery in a large number, when active polypeptide weight in this pharmaceutical composition is in the ganglioside sialic 64 times, aminoacid weight is 33 times of sialic acides in the ganglioside, total weight nitroxide is in the ganglioside sialic 18.6 times, nucleic acid weight is in the ganglioside sialic 6 times the time, synergism is best between component, is more conducive to absorption by human body and utilization.

The conventional amount used of preferred pharmaceutical compositions injection of the present invention is:

Specification 2ml:

(6.4mg polypeptide): 100 μ g (sialic acid): 3.3mg (aminoacid): 1.86mg (total nitrogen): 0.6mg (nucleic acid);

5ml：

(16.0mg polypeptide): 250 μ g (sialic acid): 8.25mg (aminoacid): 4.65mg (total nitrogen): 1.5mg (nucleic acid);

10ml：

(32.0mg polypeptide): 500 μ g (sialic acid): 16.5mg (aminoacid): 9.3mg (total nitrogen): 3.0mg (nucleic acid);

Intramuscular injection: 2～4ml/ time, 2 times/day or follow the doctor's advice;

Intravenous drip: 10-20ml/ time, add 300ml 0.9% sodium chloride injection or 5% glucose injection, slowly instil (per minute 2ml), 1 time/day, two weeks were a course of treatment.

Pharmaceutical composition of the present invention contains the active substance of the special cerebral tissue of nutritious when injected organism tissue, adopts the synergism of ganglioside and polypeptide, aminoacid and nucleic acid, has reached best clinical effectiveness.

Medicine composition injection of the present invention contains the particularly active substance of cerebral tissue of nutritious when injected organism tissue, comprise materials such as active polypeptide, aminoacid, nucleic acid, ganglioside, can promote the metabolism of cerebral tissue, participate in the neuronic growth of cerebral tissue, differentiation and regenerative process, improve cerebral blood circulation and brain metabolic function, can be used for treating the dysfunction that brain diseases causes; Be used for the treatment of diseases such as dementia, cerebral trauma, cerebral edema; The purposes of pharmaceutical composition of the present invention on preparation treatment alzheimer disease, cerebrovascular accident wound and post-traumatic central nervous system's sequela, cerebrovascular and ischemic and hemorrhagic cerebral lesion medicine.

The specific embodiment

Following embodiment further describes the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.

Embodiment 1

Step (1):

Get fresh rabbit muscle 10kg, add the homogenate of 10L water, freezing after the homogenate, multigelation 2 times melts 60 ℃ of post-heating, is incubated 20 minutes, and the cooling back is centrifugal, and centrifugal condition is 4000 rev/mins, and centrifugation time is 20 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate; Add trypsin insulation hydrolysis in filtrate, trypsinase concentration is 0.2%, and hydrolysising condition is pH=7.8, is incubated 37 ℃, and temperature retention time is 2 hours; Behind the enzymolysis, the enzymolysis solution adjust pH is 3.8, is heated to 80 ℃, keeps 20 minutes, and the cooling back is centrifugal, and centrifugal condition is 4000 rev/mins, and centrifugation time is 20 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate.

The filtrate adjust pH is 6.8, and is freezing, and melting the centrifugal centrifugal condition in back is 4000 rev/mins, and centrifugation time is 20 minutes, keep supernatant, supernatant liquid filtering, filtrate is 8000 ultrafilter membrane ultrafiltration 1 time with molecular cut off, ultrafiltrate is the muscle extracting solution, and filtrate decompression is concentrated into 800ml

Wherein content of peptides is 5mg/ml, and wherein amino acid content is 2.6mg/ml; Total nitrogen content is 1.45ml, and nucleic acid content is 0.46mg/ml.

Step (2):

Get the 40L acetone homogenate of the fresh Medulla Bovis seu Bubali of 10kg, the homogenate filter paper filtering gets filtering residue 2500g, filtering residue filters the back merging filtrate respectively with 10L chloroform-methanol (1:1) mixed liquor extracting three times, uses rotary evaporator, 37 ℃ of evaporated under reduced pressure, get dry thing 1.25g.(2:1 V/V) dissolves dry thing, adds the 0.1mol/L KCl solution of 250ml with 20L chloroform-methyl alcohol mixed liquor.Stirring at room 1 hour is transferred to and is left standstill 8 hours in the separatory funnel.Behind the separatory, on use isopyknic chloroform-methanol-water mutually (86:14:1 V/V/V) wash 1 time; (3:48:47 V/V/V) washes 1 time to use isopyknic chloroform-methanol-water mutually down; Merge 2 times and upward be evaporated to about 5L ,-20 ℃ of pre-cooling acetone precipitations, vacuum drying in 37 ℃.

Chromatographic column prepares by following process: silica gel particle is crossed screening 80 purpose single-sizes, drying is 1 hour under 110 ℃, make its activation, use the chloroform-methanol (9:1 of 5 times of column volumes then, V/V) mixed liquor is as carrier, with silica gel furnishing pasty state, make the chromatographic column of the 80 * 10cm that packs into behind its homodisperse, get final product.

Aforementioned vacuum drying thing 1.2g is dissolved in chloroform-methanol-water (65:25:4, V/V/V) 50ml adds chromatographic column, adjust flow velocity 5ml/ minute, with chloroform-methanol-water (65:25:4, V/V/V) 20L eluant solution, use chloroform-methanol-water (60:35:8) 12L eluting then instead, collect back eluent 10L, use-20 ℃ of pre-cooling acetone precipitations of 40L behind the concentrating under reduced pressure,-20 ℃ static 2 hours, the supernatant of inclining, centrifugal 10 minutes (1000rpm), precipitation is washed 2 times with proper amount of acetone, and the precipitation vacuum drying spends the night.

The dry thing of 1.2g is dissolved in 1200ml water for injection, and standby as the ganglioside extracting solution, concentration is that every 1ml contains ganglioside in sialic acid 0.1mg/ml.

Step (3):

Rabbit muscle extracting solution 1280ml and Medulla sus domestica ganglioside extracting solution 100ml are mixed, add the injection water, contain 6.4mg polypeptide, 100 μ g sialic acides, 3.33mg aminoacid, 1.86mg total nitrogen, 0.6mg nucleic acid in wherein every ml soln to 2L; Fill becomes 2ml/ to prop up after the aseptic filtration, autoclaving, and the packing warehouse-in obtains injection with small volume.

Embodiment 2

Step (1):

Get fresh deer muscle muscle 10kg, add the homogenate of 16L water, freezing after the homogenate, 70 ℃ of post-heating are melted in freeze thawing 1 time, are incubated 25 minutes, and the cooling back is centrifugal, and centrifugal condition is 3500 rev/mins, and centrifugation time is 30 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate; Add trypsin insulation hydrolysis in filtrate, trypsinase concentration is 0.8%, and hydrolysising condition is pH=8.2, is incubated 45 ℃, and temperature retention time is 3 hours; Behind the enzymolysis, the enzymolysis solution adjust pH is 4.1, is heated to 80 ℃, keeps 15 minutes, and the cooling back is centrifugal, and centrifugal condition is 4000 rev/mins, and centrifugation time is 30 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate.

The filtrate adjust pH is 7.1, and is freezing, centrifugal after melting, centrifugal condition is 4000 rev/mins, and centrifugation time is 30 minutes, keeps supernatant, supernatant liquid filtering, filtrate is 10000 ultrafilter membrane ultrafiltration 1 time with molecular cut off, the reuse molecular cut off is 8000 ultrafilter membrane ultrafiltration 1 time, ultrafiltrate is the muscle extracting solution, filtrate decompression is concentrated into 800ml, and wherein content of peptides is 8mg/ml, and wherein amino acid content is 3.5mg/ml, total nitrogen content is 2.4mg/ml, and nucleic acid content is 0.9mg/ml.

Step (2):

Get the 35L acetone homogenate of the fresh Medulla Bovis seu Bubali of 10kg, the homogenate filter paper filtering gets filtering residue 2500g, filtering residue filters the back merging filtrate respectively with 7.5L chloroform-methanol (5:1) mixed liquor extracting three times, uses rotary evaporator, 37 ℃ of evaporated under reduced pressure, get dry thing 1.25g.(10:1 V/V) dissolves dry thing, adds the 0.1mol/L KCl solution of 1000ml with 20L chloroform-methyl alcohol mixed liquor.Stirring at room 1 hour is transferred to and is left standstill 10 hours in the separatory funnel.Behind the separatory, on use isopyknic chloroform-methanol-water mutually (50:10:0.5 V/V/V) wash 1 time; (1:30:35 V/V/V) washes 1 time to use isopyknic chloroform-methanol-water mutually down; Merge 2 times and upward be evaporated to about 2.5L ,-20 ℃ of pre-cooling acetone precipitations, vacuum drying in 37 ℃.

Chromatographic column prepares by following process: silica gel particle is crossed screening 70 purpose single-sizes, drying is 1 hour under 110 ℃, make its activation, use the chloroform-methanol (20:3 of 5 times of column volumes then, V/V) mixed liquor is as carrier, with silica gel furnishing pasty state, make the chromatographic column of the 80 * 10cm that packs into behind its homodisperse, get final product.

Aforementioned vacuum drying thing 1.2g is dissolved in chloroform-methanol-water (30:50:40, V/V/V) 120ml adds chromatographic column, adjust flow velocity 5ml/ minute, with chloroform-methanol-water (90:15:6, V/V/V) 30L eluant solution, use chloroform-methanol-water (80:20:5) 8L eluting then instead, collect back eluent 12L, use-20 ℃ of pre-cooling acetone precipitations of 35L behind the concentrating under reduced pressure,-20 ℃ static 2 hours, the supernatant of inclining, centrifugal 10 minutes (1000rpm), precipitation is washed 2 times with proper amount of acetone, and the precipitation vacuum drying spends the night.

The dry thing of 1.16g is dissolved in 800ml water for injection, and standby as the ganglioside extracting solution, concentration is that every 1ml contains ganglioside in sialic acid 1.45mg/ml.

Step (3):

Deer muscle extracting solution 800ml and ox cranial nerve joint glycosides fat extracting solution 200ml are mixed, add the water injection water, contain 6.4mg polypeptide, 290 μ g sialic acides, 2.8mg aminoacid, 1.92mg total nitrogen, 0.72mg nucleic acid in wherein every ml soln to 2L; Fill becomes 2ml/ to prop up after the aseptic filtration, autoclaving, and the packing warehouse-in obtains injection with small volume.

Embodiment 3

Step (1):

Get fresh rabbit muscle 10kg, add the homogenate of 25L water, freezing after the homogenate, 65 ℃ of post-heating are melted in freeze thawing 3 times, are incubated 25 minutes, and the cooling back is centrifugal, and centrifugal condition is 3800 rev/mins, and centrifugation time is 30 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate; Add trypsin insulation hydrolysis in filtrate, trypsinase concentration is 0.2%, and hydrolysising condition is pH8.4, is incubated 42 ℃, and temperature retention time is 1.5 hours; Behind the enzymolysis, the enzymolysis solution adjust pH is 4.0, is heated to 85 ℃, keeps 20 minutes, and the cooling back is centrifugal, and centrifugal condition is 4000 rev/mins, and centrifugation time is 20 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate.

The filtrate adjust pH is 7.4, is heated to 85 ℃, keeps 20 minutes, the cooling back is centrifugal, and centrifugal condition is 4000 rev/mins, and centrifugation time is 20 minutes, keep supernatant, supernatant liquid filtering, filtrate is 6000 ultrafilter membrane ultrafiltration with molecular cut off, ultrafiltrate is the muscle extracting solution, filtrate decompression is concentrated into 800ml, and wherein content of peptides is 4mg/ml, and amino acid content is 2.8mg/ml, total nitrogen content is 1.2mg/ml, and nucleic acid content is 0.6mg/ml.

Step (2):

Get the 30L acetone homogenate of the fresh horse brain of 10kg, the homogenate filter paper filtering gets filtering residue 2500g, filtering residue filters the back merging filtrate respectively with 12.5L chloroform-methanol (20:1) mixed liquor extracting three times, uses rotary evaporator, 37 ℃ of evaporated under reduced pressure, get dry thing 1.25g.(30:1 V/V) dissolves dry thing, adds the 0.1mol/L KCl solution of 500ml with 20L chloroform-methyl alcohol mixed liquor.Stirring at room 1 hour is transferred to and is left standstill 16 hours in the separatory funnel.Behind the separatory, on use isopyknic chloroform-methanol-water mutually (120:35:5 V/V/V) wash 1 time; (10:68:77 V/V/V) washes 1 time to use isopyknic chloroform-methanol-water mutually down; Merge 2 times and upward be evaporated to about 10L ,-20 ℃ of pre-cooling acetone precipitations, vacuum drying in 37 ℃.

Chromatographic column prepares by following process: silica gel particle is crossed screening 80 purpose single-sizes, drying is 1 hour under 110 ℃, make its activation, use the chloroform-methanol (15:2 of 5 times of column volumes then, V/V) mixed liquor is as carrier, with silica gel furnishing pasty state, make the chromatographic column of the 80 * 10cm that packs into behind its homodisperse, get final product.

Aforementioned vacuum drying thing 1.2g is dissolved in chloroform-methanol-water (80:10:20, V/V/V) 24ml adds chromatographic column, adjust flow velocity 5ml/ minute, with chloroform-methanol-water (75:15:10, V/V/V) 20L eluant solution, use chloroform-methanol-water (40:50:15) 15L eluting then instead, collect back eluent 8L, use-20 ℃ of pre-cooling acetone precipitations of 40L behind the concentrating under reduced pressure,-20 ℃ static 2 hours, the supernatant of inclining, centrifugal 10 minutes (1000rpm), precipitation is washed 2 times with proper amount of acetone, and the precipitation vacuum drying spends the night.

The dry thing of 1.25g is dissolved in 500ml water for injection, and standby as the ganglioside extracting solution, concentration is that every 1ml contains ganglioside in sialic acid 2.5mg/ml.

Step (3):

Rabbit muscle extracting solution 800ml and horse brain ganglioside extracting solution 200ml are mixed, add the water injection water, contain 3.2mg polypeptide, 500 μ g sialic acides, 2.24mg aminoacid, 0.96mg total nitrogen, 0.48mg nucleic acid in wherein every ml soln to 2L; Fill becomes 2ml/ to prop up after the aseptic filtration, autoclaving, and the packing warehouse-in obtains injection with small volume.

Embodiment 4

Step (1):

Get fresh deer muscle 10kg, add the homogenate of 25L water, standby with reference to embodiment 1 step (1) preparation deer muscle extracting solution, ultrafiltrate is the muscle extracting solution, filtrate decompression is concentrated into 800ml, and wherein content of peptides is 5.4mg/ml, and wherein amino acid content is 2.7mg/ml, total nitrogen content is 1.5mg/ml, and nucleic acid content is 0.6mg/ml.

Step (2):

Get the fresh horse brain of 10kg with the homogenate of 45L acetone, with reference to embodiment 1 step, preparation ganglioside extracting solution is standby, and concentration is that every 1ml contains 1 and contains ganglioside in sialic acid 10mg/ml.

Step (3):

According to the operation of embodiment 3, obtain containing the solution of each component; Make injection.

Embodiment 5

Step (1):

With reference to embodiment 1 step (1), wherein content of peptides is 5mg/ml, and wherein amino acid content is 2.6mg/ml, and total nitrogen content is 1.45ml, and nucleic acid content is 0.46mg/ml.

Step (2):

Get the fresh Pedis Canitis of 10kg with the homogenate of 40L acetone, with reference to embodiment 1 step, preparation ganglioside extracting solution is standby, and concentration is that every 1ml contains 1 and contains ganglioside in sialic acid 100 μ g/ml.

Step (3):

Muscle extracting solution 800ml and Pedis Canitis ganglioside extracting solution 800ml mix, and add the injection water to 2L, contain 4.0mg polypeptide, 80 μ g sialic acides, 2.08mg aminoacid, 1.16mg total nitrogen, 0.37mg nucleic acid in wherein every ml soln; Fill becomes 2ml/ to prop up after the aseptic filtration, autoclaving, and the packing warehouse-in obtains injection with small volume.

Embodiment 6

Step (1):

With embodiment 1 step (1).

Step (2):

With embodiment 1 step (2).Get the fresh Medulla sus domestica of 10kg with the homogenate of 35L acetone, with reference to embodiment 1 step, preparation Medulla sus domestica ganglioside extracting solution is standby, and concentration is that every 1ml contains sialic acid 400 μ g sialic acides.

Operation according to embodiment 5 makes injection.

Embodiment 7

Step (1):

With embodiment 1 step (1).

Step (2):

Get the fresh Medulla caprae seuovis of 10kg with the homogenate of 35L acetone, with reference to embodiment 1 step, preparation Medulla caprae seuovis ganglioside extracting solution is standby, and concentration is that every 1ml contains sialic acid 400 μ g.

Step (3):

Rabbit muscle extracting solution 1500ml and Medulla caprae seuovis ganglioside extracting solution 250ml are mixed, add the injection water, contain 7.5mg polypeptide, 100 μ g sialic acides, 3.9mg aminoacid, 2.2mg total nitrogen, 0.69mg nucleic acid in wherein every ml soln to 2L; Fill becomes 2ml/ to prop up after the aseptic aseptic filtration, autoclaving, and the packing warehouse-in obtains injection with small volume.

Embodiment 7～11

With reference to step (1) and (2) among the embodiment 1～9, step (3) by well known to a person skilled in the art method, is made granule after muscle extracting solution and cranial ganglia glycosides fat extracting solution are mixed, and the compacting of dry back promptly gets the tablet of pharmaceutical composition in flakes.

Embodiment 12～21

With reference to step (1) and (2) among the embodiment 1～9, after step (3) is mixed muscle extracting solution and cranial ganglia glycosides fat extracting solution, add suitable excipient (as Dextran 40, mannitol etc.), after carrying out the degerming fill, by well known to a person skilled in the art method, make the freeze-dried powder product through lyophilization.

Embodiment 22～28

With reference to step (1) and (2) among the embodiment 1～9, step (3) adds an amount of water for injection and puts in the dilute preparing tank after muscle extracting solution and cranial ganglia glycosides fat extracting solution are mixed; Add sodium chloride by 0.9% of final preparation cumulative volume in dense preparing tank, add 0.4 ‰ activated carbon and boil decarbonization filtering, after the cooling, medical filtration to dilute preparing tank, is added water for injection to the dosing amount in dilute preparing tank, transferring PH is 7.2～7.5, filters.Embedding is in infusion bottle, and 100 ℃ of flowing steam sterilizations 45 minutes promptly get infusion products.

Experimental example 1

The medicine composition injection that the embodiment of the invention 1 is obtained is colourless or little glistening yellow prescribed liquid.

Two appendix VIH measure according to Chinese Pharmacopoeia version in 2000, and medicine composition injection pH value of the present invention is 6.0-8.0, meets the injection standard.

Get the medicine composition injection 1ml that the embodiment of the invention 1 is obtained, add 20% sulfosalicylic acid solution 1ml, do not have muddy the generation, illustrate that injection protein inspection of the present invention is up to specification.

Experimental example 2

Get the medicine composition injection 1ml that the embodiment of the invention 2 is obtained, add 3 of ninhydrin solutions, heating is purple, and (characteristic reaction CO-NH-) illustrates that medicine composition injection of the present invention contains polypeptide for peptide bond.

Get this product 2ml, add 40% sodium hydroxide solution 0.5ml, shake up, add 1% copper-bath 1ml again, solution shows bluish violet.

Experimental example 3

This experimental example is the toxicity test parameter of the pharmaceutical composition injection with small volume that obtained of the embodiment of the invention 3:

The √ bacterial endotoxin is measured: two appendix XIE check according to Chinese Pharmacopoeia version in 2000, contain the amount of bacterial endotoxin less than 4EU among the every 1ml of medicine composition injection of the present invention.

The √ undue toxicity measures: checks according to two appendix XIC of Chinese Pharmacopoeia version in 2000 intravenous injection, and up to specification.

The √ toxicity test is measured: LD50 is 298 ± 79mg/Kg, and maximum tolerated dose surpasses 250 times of adult's consumption.

The √ long term toxicity is measured: successive administration was observed blood, urine index in three months, and pathologic finding there is no unusually.

√ sensitivity test: get 6 of body weight 250-350g healthy guinea pigs, every other day lumbar injection medicine composition injection 0.5ml of the present invention, continuous 3 times, fortnight after injection for the third time, from intravenous injection this product 1.0ml, inject in back 15 minutes and observe animal, all do not occur continuous dry cough, continuously fore paw grab nose, obviously alarm that hair, extremity feel like jelly, couch, dyspnea, spasm, collapse and the phenomena of mortality.Observed 3 days continuously, animal is strong deposits.

Conclusion: medicine composition injection of the present invention meets the injection requirement, does not have any toxic action, uses human body safety.

Experimental example 4

This experimental example is that the pharmaceutical composition injection with small volume high molecular weight material that the embodiment of the invention 4 is obtained is measured:

Measure according to high performance liquid chromatography among two appendix VD of Chinese Pharmacopoeia version in 2000.

Chromatographic condition and system suitability test: the usefulness gel chromatographic columns (TSK GEL2000SWx17.8mm*300mm, 5um); Mobile phase is trifluoracetic acid-acetonitrile-water (0.05:10:90); The detection wavelength is 214nm.Number of theoretical plate calculates by insulin spikes should be lower than 3000.

The preparation of reference substance solution: it is an amount of to get insulin (molecular weight 5800), makes the solution that contains 1mg among every 1ml with mobile phase, in contrast product solution.

Algoscopy: get reference substance solution and each 20ul of medicine composition injection of the present invention, inject chromatograph of liquid respectively, the record chromatogram less than the peak area of insulin spikes retention time, calculates by area normalization method, must not surpass 5.0%.

This medicine composition injection according to an expert view, increases the high molecular weight material inspection by the intravenous drip administration.According to the measurement result of three batch samples, high molecular weight material is defined as " less than the peak area of insulin spikes retention time, calculate by area normalization method, be no more than 5.0% ".

Experimental example 5

It is an amount of to get the pharmaceutical composition that the embodiment of the invention 1 obtained, and adds water and makes the solution that contains the polypeptide of 0.15mg among every 1ml approximately, and as need testing solution, precision is measured 1.0ml, measures according to forint phenol algoscopy and obtains content of peptides.

Folin-phenol algoscopy:

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 6

The sialic acid content of the pharmaceutical composition injection with small volume that the embodiment of the invention 1 is obtained (containing ganglioside in sialic acid) adopts the resorcinol algoscopy to measure.

The resorcinol algoscopy:

Reagent:

1, resorcinol stock solution: get resorcinol 2g, add water 100ml dissolving, as stock solution (can 4 ℃ preserve the several months).

2, copper-bath (0.1mol/L): get copper sulfate 2.5g, add water 100ml dissolving, promptly.

3, resorcinol solution: get resorcinol stock solution 10ml, add copper-bath 0.25ml, add hydrochloric acid 80ml, add water to 100ml.

Maneuver:

(1) preparation of reference solution: it is an amount of to get sialic acid reference reagent, adds 0.9% sodium chloride solution and makes the solution that contains 50 μ g among every 1ml.

(2) preparation of standard curve: precision is measured reference solution 0.0,0.5,1.0,1.5,2.0ml, puts respectively in the tool plug test tube, respectively adds water to 2.0ml, add resorcinol solution 2.0ml more respectively, shake up, put in the water-bath reaction 15 minutes, take out and put in the cold water 10 minutes.Add n-butyl acetate-n-butyl alcohol (85:15) 5ml respectively, placed 15 minutes after the violent jolting.Get upper strata liquid and measure trap in 585nm wavelength place; Manage as blank with 0 simultaneously.Calculate regression equation with the trap that records with corresponding concentration, its correlation coefficient should be greater than 0.995.

(4) algoscopy: precision is measured this product 2.0ml, and the method under the preparation of sighting target directrix curve from " the accurate resorcinol solution that adds ", is measured in accordance with the law, tries to achieve the concentration of need testing solution from regression equation, and multiply by extension rate, is sialic acid content.

Experimental example 7

The amino acid content of the pharmaceutical composition that the embodiment of the invention 1 is obtained is measured.

Get this product and standard amino acid is an amount of, carry out amino acid analysis, calculate the free amino acid total amount with amino-acid analyzer or HPLC.

Experimental example 8

The total nitrogen content of the pharmaceutical composition that the embodiment of the invention 1 is obtained is measured.

Get this product, measure (two appendix VIID of Chinese Pharmacopoeia version in 2000) in accordance with the law.

Experimental example 9

The nucleic acid content of the pharmaceutical composition that the embodiment of the invention 1 is obtained is measured.

Get this product 2.0ml, thin up is to 25ml, as need testing solution.Measure absorption value down according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A) fourth 260nm wavelength, press 200 calculate nucleic acid content.Comparative example 1

The explanation of this comparative example is in the senile acute apoplexy of treatment, and the present invention most preferably pharmaceutical composition injection with small volume (every ml contains 3.2mg polypeptide, 50 μ g sialic acides, 1.65mg free amino acid, 0.93mg total nitrogen, 0.30mg nucleic acid) curative effect is better than brain protein hydrolysate injection.

1, object: the standard that all person's cases are all formulated by Chinese Medical Association the 2nd cerebrovascular academic conference, through detailed medical history-taking, neurologic check and head CT and (or) MR checks and makes a definite diagnosis.All patients are first morbidity, by improvement Scandinavia, Edinburg (improvement SSS scoring), disease degree be in, severe, all do not give thromboembolism treatment, because of economic problems do not finish treatment course of treatment do not participate in estimate.38 examples are organized in treatment, male 25 examples, women 13 examples, 74 years old mean age (64-84 year), cerebral infarction 22 examples wherein, cerebral hemorrhage 11 examples, subarachnoid hemorrhage 5 examples; Selecting in the past patient's 40 examples with the brain protein hydrolysate injection treatment at random was matched group, male 25 examples, women 15 examples, 73 years old mean age (60-86 year), cerebral infarction 22 examples wherein, cerebral hemorrhage 14 examples, subarachnoid hemorrhage 4 examples.Two groups of basic datas have comparability.

2, Therapeutic Method:

The treatment group: 10-20ml of medicine composition injection of the present invention adds in the 250ml sodium chloride injection or in 5% glucose injection, slowly instils every day 1 time, keeps 4-6 week.

Matched group: brain protein hydrolysate injection, one time 10-20ml adds in the 250ml sodium chloride injection or in 5% glucose injection, slowly instils every day 1 time, keeps 4-6 week.

Give corresponding symptomatic treatment to two groups of patients with diabetes and hyperpietic.All check hepatic and renal function, routine blood test and electrocardiogram before and after the treatment of treatment group.

3, efficacy evaluation: according to improvement SSS point system, patient's nervous function damage is estimated when being admitted to hospital and when leaving hospital.

Be almost recovered: the SSS scoring reduces more than 90%;

Marked improvement: SSS scoring reduces 46-89%;

Progressive: the SSS scoring reduces 18-45%;

No change: SSS scoring changes below 18%;

Worsen: the SSS scoring increases greater than 18%.

4, result:

Before and after the treatment of treatment group, hepatic and renal function, routine blood test and electrocardiogram do not have significant change.

Two groups of curative effects see Table 6:

Table 6: Comparison of therapeutic

Group	The example number	Be almost recovered	Marked improvement	Progressive	No change	Worsen	Obvious effective rate	Effective percentage
									The treatment group	38	6(15.8)	20(52.6)	10(26.3)	2(5.3)	0	26(68.4) *	36(94.7) **
Matched group	40	3(7.5)	10(25.0)	15(39.5)	9(22.5)	3(7.5)	13(30.0)	28(70.0)

Annotate: treatment group and matched group compare, p＜0.01.

More than explanation is in the senile acute apoplexy of treatment, and medicine composition injection curative effect of the present invention is better than brain protein hydrolysate injection.

Comparative example 2

This comparative example explanation the present invention most preferably pharmaceutical composition injection with small volume (every ml contains 3.2mg polypeptide, 50 μ g sialic acides, 1.65mg free amino acid, 0.93mg total nitrogen, the 0.30mg nucleic acid) curative effect that is used for the treatment of the cerebrovascular accident paralysis is better than sarcosine peptide glycoside injection liquid.

Matched group: sarcosine peptide glycoside injection liquid.Treatment group: injection of the present invention.

Two groups of curative effects see Table 7:

Table 7: Comparison of therapeutic

Group	The example number	Be almost recovered	Show number	Progressive	No change	Worsen	Obvious effective rate	Effective percentage
									The treatment group	50	8(16.0)	24(48.0)	15(28.0、)	3(7.5)	0	32(64.0) *	47(94.0)
Matched group	50	4(8.0)	16(32.0)	16(32.0)	14(28.0)	0	20(40.0)	36(72.0)

Annotate: treatment group and matched group compare, p＜0.01.

Claims (14)

1. medicine for improving brain function compositions, it is characterized in that described pharmaceutical composition contains active polypeptide, aminoacid, nucleic acid that extracts and the ganglioside that extracts from the mammal muscular tissue except that the people from the mammal cerebral tissue except that the people, be contrast wherein with the sialic acid in the ganglioside in the pharmaceutical composition, active polypeptide weight is sialic 6.1～100 times, aminoacid weight is sialic 0.1～100 times, total weight nitroxide is sialic 2～50 times, and nucleic acid weight is sialic 0.5～30 times; Described medicine for improving brain function prepares according to following steps:

(1) preparation of muscle extracting solution:

A) animal any part muscular tissue is added after the water homogenate freezingly, multigelation 1～4 time melts post-heating centrifugal filtration slagging-off, keeps filtrate;

B) filtrate is regulated pH value insulation trypsin digestion, regulates pH value and is acid, heats the centrifugal precipitation of removing, and filtering and impurity removing keeps filtrate;

C) filtrate adjusting is neutrality, freezing, melt the centrifugal precipitation of removing in back, supernatant liquid filtering, filtrate is used ultrafilter membrane ultrafiltration 1～2 time, and ultrafiltrate is the muscle extracting solution;

(2) preparation of ganglioside extracting solution:

A) with animal brain's solubilizer homogenate after-filtration, filtering residue extracts with solvent extraction, and extracting solution and filtrate merge the back evaporate to dryness, get dry thing;

B) dry thing is dissolved in the solvent, to the extraction with aqueous solution separatory that wherein adds salt;

C) upper and lower layer of liquid phase of separatory with the mixed aqueous solution washing of at least two kinds of solvents, merges the upper phase part after washing respectively, and concentrating under reduced pressure is used the pre-cooling acetone precipitation then, gets dry thing after the drying;

D) dry thing is dissolved in the mixed aqueous solution of at least two kinds of solvents, chromatographic column separates, and eluent concentrates the back with pre-cooling acetone precipitation, separation, and the precipitation that obtains obtains the ganglioside extracting solution through post processing;

(3) said extracted liquid is mixed, the weight that makes polypeptide in the mixed liquor is 6.1～100 times of sialic acid weight in the ganglioside, aminoacid weight is 0.1～100 times of sialic acid weight, total weight nitroxide is sialic 2～50 times, nucleic acid weight is sialic 0.5～30 times, through post processing, get finished product preparation.

2. medicine for improving brain function compositions according to claim 1 is characterized in that described medicine for improving brain function prepares according to following steps:

(1) preparation of muscle extracting solution:

A) animal any part muscular tissue is added the homogenate of 0.6～2.5 times of weight water, freezing after the homogenate, multigelation 1～4 time melts 60～70 ℃ of post-heating, is incubated 15～30 minutes, the cooling back is centrifugal, centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, keeps supernatant, supernatant liquid filtering gets filtrate;

B) add trypsin insulation hydrolysis in filtrate, trypsinase concentration is 0.03～0.5%, and hydrolysising condition is pH=7.5～8.5, is incubated 35～50 ℃, and temperature retention time is 0.5～5 hour; Behind the enzymolysis, the enzymolysis solution adjust pH is 3.5-4.5, is heated to 70-85 ℃, keeps 15-30 minute, and the cooling back is centrifugal, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate;

C) the filtrate adjust pH is 6.5-7.5, and is freezing, centrifugal after melting, centrifugal condition is 3000～4000 rev/mins, centrifugation time is 15～30 minutes, keeps supernatant, supernatant liquid filtering, filtrate is the ultrafilter membrane ultrafiltration 1～2 time of 5000-10000 with molecular cut off, ultrafiltrate is the muscle extracting solution, and wherein content of peptides is 0.5～15mg/ml, and wherein amino acid content is 0.5～15mg/ml, total nitrogen content is 0.2～8mg/ml, and nucleic acid content is 0.05～3mg/ml;

(2) preparation of ganglioside extracting solution:

A) animal brain is added the acetone homogenate after-filtration of 3～4 times of weight, keep filtering residue and filtrate; Filtering residue filters respectively with the chloroform of 3～5 times of weight-methyl alcohol mixed liquor extracting three times; Above-mentioned filtrate is merged back 37 ℃ of evaporated under reduced pressure in rotary evaporator, get dry thing; Wherein chloroform-methanol volume ratio is 1～20:1～20;

B) dry thing is dissolved in chloroform-methyl alcohol mixed liquor, adds the KCl solution of the 0.1mol/L of 1/20～1/80 volume then in mixed liquor, stirs 1 hour under the room temperature, leaves standstill separatory after 8～16 hours; Wherein chloroform-methanol volume ratio is 2～30:1～20;

C) behind the separatory, upper phase is washed with isopyknic chloroform-methanol-water mixed liquid, and wherein chloroform-methanol-water volume ratio is 50～150:5～40:0.5～5; Lower floor's liquid phase is washed with isopyknic chloroform-methanol-water mixed liquid, and wherein chloroform-methanol-water volume ratio is 1～10:30～80:35～85; Merge the upper phase part after twice washing, be evaporated to 1/8～1/2 of initial volume, use-20 ℃ of pre-cooling acetone precipitations then at 37 ℃, vacuum drying, dry thing;

D) dry thing is dissolved in chloroform-methanol-water mixed liquid, makes the solution of 1～5% concentration, wherein chloroform-methanol-water volume ratio is 30～80:10～50:1～90; Add chromatographic column, adjust flow velocity 5ml/ minute, with chloroform-methanol-water mixed liquid 10～30L eluting, wherein chloroform-methanol-water volume ratio is 60～90:15～40:1～10; Use chloroform-methanol-water mixed liquid 8～15L eluting then instead, wherein chloroform-methanol-water volume ratio is 40～80:20～50:5～15; Collect back eluent 6～12L, behind the concentrating under reduced pressure with-15～-22 ℃ of pre-cooling acetone precipitations of 3～5 times of volumes, the supernatant is gone in-15～-22 ℃ of hypsokinesis in static about 2 hours, remaining part descended centrifugal 8～12 minutes at 800～1200 rev/mins, the precipitation that obtains is washed 1～3 time with proper amount of acetone, vacuum drying, with the water-soluble ganglioside extracting solution that gets of dry thing, concentration is that every 1ml contains ganglioside in sialic acid 0.01～0.5mg;

(3) said extracted liquid is mixed, the weight that makes polypeptide in the mixed liquor is 6.1～100 times of sialic acid weight in the ganglioside, aminoacid weight is 0.1～100 times of sialic acid weight, and total weight nitroxide is sialic 2～50 times, and nucleic acid weight is sialic 0.5～30 times.

3. medicine for improving brain function compositions according to claim 1 and 2, the D that it is characterized in that step in the described method (2)) chromatographic column used in prepares by following process: silica gel particle is crossed screening 60～80 purpose single-sizes, drying is about 1 hour under 100 ℃～120 ℃, make its activation, the chloroform-methanol mixed liquor of using 4～6 times of column volumes then is as carrier, with silica gel furnishing pasty state, make the chromatographic column of packing into behind its homodisperse, promptly; Wherein the chloroform-methanol volumetric mixture ratio is 5～20:0.5～3.

4. pharmaceutical composition according to claim 1, it is characterized in that active polypeptide weight is sialic 50～80 times in the described pharmaceutical composition, aminoacid weight is sialic 1～50 times, and total weight nitroxide is sialic 5～40 times, and nucleic acid weight is sialic 2～20 times.

5. pharmaceutical composition according to claim 4, it is characterized in that active polypeptide weight is sialic 60～70 times in the described pharmaceutical composition, aminoacid weight is sialic 20～40 times, and total weight nitroxide is sialic 10～20 times, and nucleic acid weight is sialic 3～10 times.

6. according to claim 1,2,4 or 5 any described pharmaceutical compositions, it is characterized in that described muscular tissue is any part muscular tissue of pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit; Described cerebral tissue is the cerebral tissue of pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit.

7. pharmaceutical composition according to claim 3 is characterized in that described muscular tissue is any part muscular tissue of pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit; Described cerebral tissue is the cerebral tissue of pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit.

8. a method for preparing the described pharmaceutical composition of claim 1 is characterized in that described method comprises the steps:

(1) preparation of muscle extracting solution:

A) animal any part muscular tissue is added after the water homogenate freezingly, multigelation 1～4 time melts post-heating centrifugal filtration slagging-off, keeps filtrate;

B) filtrate is regulated pH value insulation trypsin digestion, regulates pH value and is acid, heats the centrifugal precipitation of removing, and filtering and impurity removing keeps filtrate;

C) filtrate adjusting is neutrality, freezing, melt the centrifugal precipitation of removing in back, supernatant liquid filtering, filtrate is used ultrafilter membrane ultrafiltration 1～2 time, and ultrafiltrate is the muscle extracting solution;

(2) preparation of ganglioside extracting solution:

A) with animal brain's solubilizer homogenate after-filtration, filtering residue extracts with solvent extraction, and extracting solution and filtrate merge the back evaporate to dryness, get dry thing;

B) dry thing is dissolved in the solvent, to the extraction with aqueous solution separatory that wherein adds salt;

C) upper and lower layer of liquid phase of separatory with the mixed aqueous solution washing of at least two kinds of solvents, merges the upper phase part after washing respectively, and concentrating under reduced pressure is used the pre-cooling acetone precipitation then, gets dry thing after the drying;

D) dry thing is dissolved in the mixed aqueous solution of at least two kinds of solvents, chromatographic column separates, and eluent concentrates the back with pre-cooling acetone precipitation, separation, and the precipitation that obtains obtains the ganglioside extracting solution through post processing;

(3) said extracted liquid is mixed, the weight that makes polypeptide in the mixed liquor is 6.1～100 times of sialic acid weight in the ganglioside, aminoacid weight is 0.1～100 times of sialic acid weight, total weight nitroxide is sialic 2～50 times, nucleic acid weight is sialic 0.5～30 times, through post processing, get finished product preparation.

9. preparation method according to claim 8 is characterized in that described method comprises the steps:

(1) preparation of muscle extracting solution:

A) animal any part muscular tissue is added the homogenate of 0.6～2.5 times of weight water, freezing after the homogenate, multigelation 1～4 time melts 60～70 ℃ of post-heating, is incubated 15～30 minutes, the cooling back is centrifugal, centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, keeps supernatant, supernatant liquid filtering gets filtrate;

B) add trypsin insulation hydrolysis in filtrate, trypsinase concentration is 0.03～0.5%, and hydrolysising condition is pH=7.5～8.5, is incubated 35～50 ℃, and temperature retention time is 0.5～5 hour; Behind the enzymolysis, the enzymolysis solution adjust pH is 3.5-4.5, is heated to 70-85 ℃, keeps 15-30 minute, and the cooling back is centrifugal, and centrifugal condition is 3000～4000 rev/mins, and centrifugation time is 15～30 minutes, keeps supernatant, and supernatant liquid filtering gets filtrate;

C) the filtrate adjust pH is 6.5-7.5, and is freezing, centrifugal after melting, centrifugal condition is 3000～4000 rev/mins, centrifugation time is 15～30 minutes, keeps supernatant, supernatant liquid filtering, filtrate is the ultrafilter membrane ultrafiltration 1～2 time of 5000-10000 with molecular cut off, ultrafiltrate is the muscle extracting solution, and wherein content of peptides is 0.5～15mg/ml, and wherein amino acid content is 0.5～15mg/ml, total nitrogen content is 0.2～8mg/ml, and nucleic acid content is 0.05～3mg/ml;

(2) preparation of ganglioside extracting solution:

A) animal brain is added the acetone homogenate after-filtration of 3～4 times of weight, keep filtering residue and filtrate; Filtering residue filters respectively with the chloroform of 3～5 times of weight-methyl alcohol mixed liquor extracting three times; Above-mentioned filtrate is merged back 37 ℃ of evaporated under reduced pressure in rotary evaporator, get dry thing; Wherein chloroform-methanol volume ratio is 1～20:1～20;

B) dry thing is dissolved in chloroform-methyl alcohol mixed liquor, adds the KCl solution of the 0.1mol/L of 1/20～1/80 volume then in mixed liquor, stirs 1 hour under the room temperature, leaves standstill separatory after 8～16 hours; Wherein chloroform-methanol volume ratio is 2～30:1～20;

C) behind the separatory, upper phase is washed with isopyknic chloroform-methanol-water mixed liquid, and wherein chloroform-methanol-water volume ratio is 50～150:5～40:0.5～5; Lower floor's liquid phase is washed with isopyknic chloroform-methanol-water mixed liquid, and wherein chloroform-methanol-water volume ratio is 1～10:30～80:35～85; Merge the upper phase part after twice washing, be evaporated to 1/8～1/2 of initial volume, use-20 ℃ of pre-cooling acetone precipitations then at 37 ℃, vacuum drying, dry thing;

D) dry thing is dissolved in chloroform-methanol-water mixed liquid, makes the solution of 1～5% concentration, wherein chloroform-methanol-water volume ratio is 30～80:10～50:1～90; Add chromatographic column, adjust flow velocity 5ml/ minute, with chloroform-methanol-water mixed liquid 10～30L eluting, wherein chloroform-methanol-water volume ratio is 60～90:15～40:1～10; Use chloroform-methanol-water mixed liquid 8～15L eluting then instead, wherein chloroform-methanol-water volume ratio is 40～80:20～50:5～15; Collect back eluent 6～12L, behind the concentrating under reduced pressure with-15～-22 ℃ of pre-cooling acetone precipitations of 3～5 times of volumes, the supernatant is gone in-15～-22 ℃ of hypsokinesis in static about 2 hours, remaining part descended centrifugal 8～12 minutes at 800～1200 rev/mins, the precipitation that obtains is washed 1～3 time with proper amount of acetone, vacuum drying, with the water-soluble ganglioside extracting solution that gets of dry thing, concentration is that every 1ml contains ganglioside in sialic acid 0.01～0.5mg;

(3) said extracted liquid is mixed, the weight that makes polypeptide in the mixed liquor is 6.1～100 times of sialic acid weight in the ganglioside, aminoacid weight is 0.1～100 times of sialic acid weight, and total weight nitroxide is sialic 2～50 times, and nucleic acid weight is sialic 0.5～30 times.

10. according to Claim 8 or 9 described preparation methoies, the D that it is characterized in that step in the described method (2)) chromatographic column used in prepares by following process: silica gel particle is crossed screening 60～80 purpose single-sizes, drying is about 1 hour under 100 ℃～120 ℃, make its activation, the chloroform-methanol mixed liquor of using 4～6 times of column volumes then is as carrier, with silica gel furnishing pasty state, make the chromatographic column of packing into behind its homodisperse, promptly; Wherein the chloroform-methanol volumetric mixture ratio is 5～20:0.5～3.

11. according to Claim 8 or 9 described preparation methoies, it is characterized in that step in the described method (3) gained pharmaceutical composition through post processing, finished product preparation, the dosage form of finished product preparation is an acceptable forms on the pharmaceutics.

12. preparation method according to claim 11 is characterized in that acceptable forms is on the described pharmaceutics: tablet, capsule, oral liquid, small-volume injection, infusion solutions or freeze-dried powder.

13. according to Claim 8 or 9 described preparation methoies, it is characterized in that described muscular tissue is any part muscular tissue of pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit; Described cerebral tissue is the cerebral tissue of pig, Canis familiaris L., cattle, sheep, deer, horse or rabbit.

14. the purposes of any described pharmaceutical composition of claim 1～5 aspect preparation treatment apoplexy and cerebrovascular accident paralysis medicine.