CN103656607A - Cerebroprotein hydrolysate in piracetam and cerebroprotein hydrolysate tablets and preparation method of cerebroprotein hydrolysate - Google Patents
Cerebroprotein hydrolysate in piracetam and cerebroprotein hydrolysate tablets and preparation method of cerebroprotein hydrolysate Download PDFInfo
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Abstract
The invention belongs to the field of biological agents, and relates to cerebroprotein hydrolysate in piracetam and cerebroprotein hydrolysate tablets and a preparation method of the cerebroprotein hydrolysate, in particular to cerebroprotein hydrolysate which is extracted from brain tissues of mammals excluding human and contains polypeptide and multiple amino acids including free amino acids and free glutamic acid, as well as a preparation method of the cerebroprotein hydrolysate. The provided method comprises steps such as homogenate, two times of degreasing of acetone, enzymolysis of trypsin and pepsin and the like. The content of amino acids in the cerebroprotein hydrolysate prepared with the method is remarkably higher than that in the prior art.
Description
Technical field
The present invention relates to Cerebrolysin Vial in a kind of piracetam brain protein hydrolysate tablet and preparation method thereof, specifically, relate to a kind of containing polypeptide and comprising free amino acid and the Cerebrolysin Vial of the several amino acids of free glutamic acid of extracting by the mammal cerebral tissue from except people, and the preparation method of this Cerebrolysin Vial, belong to biological preparation field.
Background technology
Cerebral tissue contains rich in protein, people study always the proteolysis in animal brain are become to aminoacid and low molecular peptide for a long time, are used for treating the diseases such as dysfunction that functional disorder, apoplexy, brain memory obstacle and cerebral trauma due to the compensatory deficiency of cerebrovascular cause, cerebral concussion sequela.
Cerebrolysin Vial is exactly a kind of mixture that is rich in several amino acids and micromolecule polypeptide being obtained after protease hydrolysis by animal brain, due to the bioactive peptide that contains the necessary aminoacid of a large amount of human brains and multiple nerve growth, can regulate and improve neuronic metabolism, clinically, Cerebrolysin Vial is mainly used in treating resuming treatment of craniocerebral injury and cerebrovascular (as cerebral infarction and cerebral hemorrhage etc.) sequela, accelerates the functional rehabilitation of brain neuroblastoma system.
Wherein the cerebrolysin clinical practice with the research of Austrian Ebewe pharmaceutical factory must be more extensive.Early than early 1990s, domestic scholars is just studied its technique, quality, pharmacological effect and clinical practice, has delivered a large amount of articles.
The Chinese patent application that is 94104516.1 as application number discloses a kind of preparation method of Cerebrolysin Vial, adopts the steps such as homogenate, defat, air-dry, enzymolysis, ultrafiltration, ion exchange, and in the method, air-dry step can cause the loss of the materials such as polypeptide; Application number is the new preparation method that the Chinese patent application such as 01130523.1,200510064567.6,200710055421.4 disclose Cerebrolysin Vial, in said method, all exist and make a large amount of steps that run off of aminoacid, therefore, said method makes discussion; Application number is 200610065169.0 in preparing the process of Cerebrolysin Vial, has added exogenous amino acid, and this is also the basic condition of the Cerebrolysin Vial medicine of current clinical practice, illustrates that existing method requires further improvement; Application number is the preparation method that 200410022091.5 Chinese patent discloses a kind of pharmaceutics of hydrolysate of brain protein, the method is directly brain tissue homogenate's liquid to be carried out to protease hydrolysis, and obtain Cerebrolysin Vial by further processing, the shortcoming of this technique is exactly, and in brain tissue homogenate's liquid, contains significant quantities of fat, affects the hydrolysis effect of protease, preparation efficiency is low, meanwhile, more lipoid material can be mixed in last Cerebrolysin Vial, affects outward appearance and the stability of final medicine.
Summary of the invention
For the defect of prior art, the invention provides the Cerebrolysin Vial in a kind of piracetam brain protein hydrolysate tablet.In Cerebrolysin Vial provided by the present invention, not only macromolecular substances content is low, and free fatty acid content is low, and free aminoacid content is high, and biological activity is high.
Meanwhile, the preparation method of the Cerebrolysin Vial described in the present invention also provides, its preparation method is simple to operate, is applicable to industrialized great production, and productive rate is high, and Ke Shi enterprise obtains higher economic benefit.
To achieve these goals, the present invention adopts following technical scheme:
A Cerebrolysin Vial in piracetam brain protein hydrolysate tablet, wherein, described Cerebrolysin Vial is adopted with the following method and is prepared:
1) get the mammiferous cerebral tissue except people, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean cerebral tissue;
2) get clean cerebral tissue and carry out homogenate, each homogenate 2～3 minutes, obtains brain tissue homogenate's liquid;
3) to the acetone that adds 2.5～3.5 times of volumes in brain tissue homogenate's liquid, stir 25～35 minutes, standing 3.5～4.5 hours, filter, obtain acetone slag; To the acetone that adds 1.5～2.5 times of volumes in acetone slag, stir 25～35 minutes again, standing 3.5～4.5 hours, filter, filtering residue at 60～70 ℃ dry 3～5 hours, obtains brain egg albumen powder;
4) get trypsin, anhydrous calcium chloride dissolves by cold purified water, is mixed with the trypsin solution that concentration is 0.02%～0.04%g/ml, places activation in 1～2 hour under room temperature; Pepsin dissolves by cold purified water, is mixed with the pepsin solution that concentration is 0.02%～0.04%g/ml;
5) get brain egg albumen powder, add purified water, stir, be heated to 40～45 ℃, add the trypsin having activated, stir, control temperature at enzymolysis between 40～45 ℃ after 3.5～4.5 hours, add again pepsin, stir, control temperature enzymolysis 1.5～2.5 hours between 40～45 ℃, enzymolysis finishes, be heated to 85～95 ℃, be incubated 25～35 minutes, be then cooled to 20～30 ℃, obtain enzymolysis solution;
6) enzymolysis solution of gained is used to centrifugal 15～25 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control pH value to 5.0～7.0 of supernatant liquid;
7) gained supernatant liquid is concentrated, is dried, is pulverized and sieved, obtain Cerebrolysin Vial dry powder.
As the first preferred version of Cerebrolysin Vial of the present invention, the anhydrous calcium chloride consumption described in step 4) is 12～13wt% of trypsin consumption.
As the second preferred version of Cerebrolysin Vial of the present invention, the tryptic amount that the activation adding in step 5) is good is step 2) 2～3wt% of clean cerebral tissue gross weight; The pepsic amount adding is step 2) 0.5～1wt% of clean cerebral tissue gross weight.
As the third preferred version of Cerebrolysin Vial of the present invention, the pepsin described in step 5) first carries out ultrasonic pretreatment before adding.
As the 4th kind of preferred version of Cerebrolysin Vial of the present invention, described ultrasonic pretreatment for take ultrasonic power as 60～80W, supersonic frequency as 30～35kHz, ultrasonic time carries out ultrasonic pretreatment 8～12min with the intermittent time than the ultrasound wave that is 1:1.
The preparation method of the Cerebrolysin Vial in the piracetam brain protein hydrolysate tablet described in the present invention also further provides, the method comprises the steps:
1) get the mammiferous cerebral tissue except people, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean cerebral tissue;
2) get clean cerebral tissue and carry out homogenate, each homogenate 2～3 minutes, obtains brain tissue homogenate's liquid;
3) to the acetone that adds 2.5～3.5 times of volumes in brain tissue homogenate's liquid, stir 25～35 minutes, standing 3.5～4.5 hours, filter, obtain acetone slag; To the acetone that adds 1.5～2.5 times of volumes in acetone slag, stir 25～35 minutes again, standing 3.5～4.5 hours, filter, filtering residue at 60～70 ℃ dry 3～5 hours, obtains brain egg albumen powder;
4) get trypsin, anhydrous calcium chloride dissolves by cold purified water, is made into the trypsin solution that concentration is 0.02%～0.04%g/ml, places activation in 1～2 hour under room temperature; Pepsin dissolves by cold purified water, is made into the pepsin solution that concentration is 0.02%～0.04%g/ml;
5) get brain egg albumen powder, add purified water, stir, be heated to 40～45 ℃, then add the trypsin having activated, stir, control temperature at enzymolysis between 40～45 ℃ after 3.5～4.5 hours, again add pepsin, stir, control temperature enzymolysis 1.5～2.5 hours between 40～45 ℃, enzymolysis finishes, be heated to 85～95 ℃, be incubated 25～35 minutes, be then cooled to 20～30 ℃, obtain enzymolysis solution;
6) enzymolysis solution of gained is used to centrifugal 15～25 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control pH value to 5.0～7.0 of supernatant liquid;
7) gained supernatant liquid is concentrated, is dried, is pulverized and sieved, obtain Cerebrolysin Vial dry powder.
As the first preferred version of preparation method of the present invention, the anhydrous calcium chloride consumption described in step 4) is 12～13wt% of trypsin consumption.
As the second preferred version of preparation method of the present invention, in step 5), adding the tryptic amount having activated is step 2) 2～3wt% of clean cerebral tissue gross weight; Adding pepsic amount is step 2) 0.5～1wt% of clean cerebral tissue gross weight.
Preferred as the third of preparation method of the present invention, the simmer down to described in step 7) is concentrated into 1/4～1/2 of supernatant liquid original volume at 70～80 ℃; Described is dried as be dried 3～5 hours at 70～80 ℃; Described sieves as crossing 100 mesh sieves.
As the 4th kind of preferred version of preparation method of the present invention, the pepsin described in step 5) first carries out ultrasonic pretreatment before adding; Preferably take ultrasonic power as 60～80W, supersonic frequency as 30～35kHz, ultrasonic time carries out ultrasonic pretreatment 8～12min with the intermittent time than the ultrasound wave that is 1:1.
Below further explanation and description of the technical solution of the present invention are carried out:
The present invention relates to the Cerebrolysin Vial in a kind of piracetam brain protein hydrolysate tablet, in this Cerebrolysin Vial, amino acid whose content is apparently higher than prior art.
The preparation method that the invention still further relates to described Cerebrolysin Vial, the method is: get the mammiferous cerebral tissue except people, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean cerebral tissue; Clean cerebral tissue is carried out to homogenate, and each homogenate 2～3 minutes, adds acetone in brain tissue homogenate's liquid of the backward gained of homogenate, stir 25～35 minutes, standing 3.5～4.5 hours, filter, in filtering residue, add acetone stirring, standing, gained filtering residue is dried, and obtains brain egg albumen powder again.In the present invention, will in backward gained brain tissue homogenate of clean brain tissue homogenate liquid, add at twice acetone defat, make acetone defat more efficient, thorough, thereby can remove more neatly the materials such as fat in cerebral tissue.
Next, preparation trypsin and pepsin solution, wherein stand-by after trypsin activation.Then in the brain egg albumen powder solution that dissolves by purified water and heat, first add trypsin to add again pepsin to carry out enzymolysis.In the present invention, by test example, show first to add trypsin to add again pepsin to carry out enzymolysis and than other modes, add that to carry out the total amino acid content of enzymolysis high.Because in whole enzymolysis process, enzymolysis initial stage concentration of substrate is high, hydrolyzate is few, and enzymolysis speed is fast; Enzymolysis is after a period of time, and trypsin vigor reduces, and at this moment adds a part of pepsin again, has increased the vigor of enzyme, thereby has made hydrolysis rate still keep higher level.
Finally, the enzymolysis solution of gained is centrifugal, collect supernatant, to filter, filtrate concentrates, is dried, sieving obtains Cerebrolysin Vial dry powder.By the operations such as further refining and filtration to enzymolysis solution, thereby the impurity in the Cerebrolysin Vial dry powder that makes to obtain and fat wait material to be removed completely, have guaranteed the purity of the Cerebrolysin Vial dry powder that obtains.
As a preferred embodiment of the present invention, the pepsin described in the step 5) of preparation method of the present invention first carries out ultrasonic pretreatment before adding; Most preferably take ultrasonic power as 60～80W, supersonic frequency as 30～35kHz, ultrasonic time carries out ultrasonic pretreatment 8～12min with the intermittent time than the ultrasound wave that is 1:1.
Pepsin solution is carried out before adding to ultrasonic Treatment, can change pepsic conformation, pepsic active center is fully exposed, increase substrate---the chance that brain egg albumen powder is combined with pepsin, accelerated reaction, promote the hydrolysis of brain egg albumen powder, and to greatest extent by the extracts active ingredients in cerebral tissue out.
The preparation method of the Cerebrolysin Vial in piracetam brain protein hydrolysate tablet provided by the present invention is applicable to mammal except people as the cerebral tissue of the animals such as pig, Canis familiaris L., cattle, sheep, deer, horse, rabbit, from factors such as production cost, production technology, constant product quality, clinical efficacies, consider, the preparation method of the Cerebrolysin Vial in piracetam brain protein hydrolysate tablet provided by the present invention is preferably applicable to pig brain tissue.
Compared with prior art, in Cerebrolysin Vial provided by the present invention amino acid whose content apparently higher than prior art.
The specific embodiment
Be below the specific embodiment of the present invention, described embodiment is in order to further describe the present invention, rather than restriction the present invention.
The preparation of the Cerebrolysin Vial in embodiment 1, piracetam brain protein hydrolysate tablet
(1) get the Medulla sus domestica of fresh and healthy, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean Medulla sus domestica;
(2) get clean Medulla sus domestica 10kg, add 10L water for injection, use high-pressure homogenization pump to carry out homogenate, each homogenate 2 minutes, obtains brain tissue homogenate's liquid;
(3) to the acetone that adds 2.5 times of amounts in brain tissue homogenate's liquid, stir 25 minutes, standing 3.5 hours, filter, obtain acetone slag; To the acetone that adds 1.5 times of amounts in acetone slag, stir 25 minutes again, standing 3.5 hours, filter, filtering residue at 60 ℃ dry 3 hours, obtains brain egg albumen powder;
(4) get trypsin, anhydrous calcium chloride (wherein the consumption of anhydrous calcium chloride is the 12wt% of trypsin consumption) dissolves by cold purified water, is made into the trypsin solution that concentration is 0.02%g/ml, places activation in 1 hour under room temperature; Pepsin dissolves by cold purified water, is made into the pepsin solution that concentration is 0.02%g/ml;
(5) get brain egg albumen powder, add purified water, stir, be heated to 40 ℃, by step 2) the 2wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature at enzymolysis between 40 ℃ after 3.5 hours, again by step 2) the 0.5wt% of clean Medulla sus domestica gross weight add pepsin, stir, control temperature enzymolysis 1.5 hours between 40 ℃, enzymolysis finishes, be heated to 85 ℃, be incubated 25 minutes, be then cooled to 20 ℃, obtain enzymolysis solution;
(6) enzymolysis solution of gained is used to centrifugal 15 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control the pH value to 5.0 of supernatant liquid;
(7) gained supernatant liquid is concentrated at 70 ℃ supernatant liquid original volume 1/4, at 70 ℃ dry 3 hours, pulverized 100 mesh sieves, obtain Cerebrolysin Vial dry powder.
The preparation of the Cerebrolysin Vial in embodiment 2, piracetam brain protein hydrolysate tablet
(1) get the Medulla sus domestica of fresh and healthy, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean Medulla sus domestica;
(2) get clean Medulla sus domestica 10kg, add 10L water for injection, use high-pressure homogenization pump to carry out homogenate, each homogenate 2 minutes, obtains brain tissue homogenate's liquid;
(3) to the acetone that adds 3.5 times of amounts in brain tissue homogenate's liquid, stir 35 minutes, standing 4.5 hours, filter, obtain acetone slag; To the acetone that adds 2.5 times of amounts in acetone slag, stir 35 minutes again, standing 4.5 hours, filter, filtering residue at 70 ℃ dry 5 hours, obtains brain egg albumen powder;
(4) get trypsin, anhydrous calcium chloride (wherein the consumption of anhydrous calcium chloride is the 13wt% of trypsin consumption) dissolves by cold purified water, is made into the trypsin solution that concentration is 0.04%g/ml, places activation in 2 hours under room temperature; Pepsin dissolves by cold purified water, is made into the pepsin solution that concentration is 0.04%g/ml;
(5) get brain egg albumen powder, add purified water, stir, be heated to 45 ℃, by step 2) the 3wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature at enzymolysis between 45 ℃ after 4.5 hours, again by step 2) the 1wt% of clean Medulla sus domestica gross weight add pepsin, stir, control temperature enzymolysis 2.5 hours between 45 ℃, enzymolysis finishes, be heated to 95 ℃, be incubated 35 minutes, be then cooled to 30 ℃, obtain enzymolysis solution;
(6) enzymolysis solution of gained is used to centrifugal 25 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control the pH value to 7.0 of supernatant liquid;
(7) gained supernatant liquid is concentrated at 80 ℃ supernatant liquid original volume 1/2, at 80 ℃ dry 5 hours, pulverized 100 mesh sieves, obtain Cerebrolysin Vial dry powder.
The preparation of the Cerebrolysin Vial in embodiment 3, piracetam brain protein hydrolysate tablet
(1) get the Medulla sus domestica of fresh and healthy, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean Medulla sus domestica;
(2) get clean Medulla sus domestica 10kg, add 10L water for injection, use high-pressure homogenization pump to carry out homogenate, each homogenate 2.5 minutes, obtains brain tissue homogenate's liquid;
(3) to the acetone that adds 3 times of amounts in brain tissue homogenate's liquid, stir 30 minutes, standing 4 hours, filter, obtain acetone slag; To the acetone that adds 2 times of amounts in acetone slag, stir 30 minutes again, standing 4 hours, filter, filtering residue at 65 ℃ dry 4 hours, obtains brain egg albumen powder;
(4) get trypsin, anhydrous calcium chloride (wherein the consumption of anhydrous calcium chloride is the 12.5wt% of trypsin consumption) dissolves by cold purified water, is made into the trypsin solution that concentration is 0.03%g/ml, places activation in 1.5 hours under room temperature; Pepsin dissolves by cold purified water, is made into the pepsin solution that concentration is 0.03%g/ml;
(5) get brain egg albumen powder, add purified water, stir, be heated to 42 ℃, by step 2) the 2.5wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature at enzymolysis between 42 ℃ after 4 hours, again by step 2) the 0.8wt% of clean Medulla sus domestica gross weight add pepsin, stir, control temperature enzymolysis 2 hours between 42 ℃, enzymolysis finishes, be heated to 90 ℃, be incubated 30 minutes, be then cooled to 25 ℃, obtain enzymolysis solution;
(6) enzymolysis solution of gained is used to centrifugal 20 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control the pH value to 6.0 of supernatant liquid;
(7) gained supernatant liquid is concentrated at 75 ℃ supernatant liquid original volume 1/3, at 75 ℃ dry 4 hours, pulverized 100 mesh sieves, obtain Cerebrolysin Vial dry powder.
The preparation of the Cerebrolysin Vial in embodiment 4, piracetam brain protein hydrolysate tablet
(1) get the Medulla sus domestica of fresh and healthy, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean Medulla sus domestica;
(2) get clean Medulla caprae seuovis 10kg, add 10L water for injection, use high-pressure homogenization pump to carry out homogenate, each homogenate 2.8 minutes, obtains brain tissue homogenate's liquid;
(3) to the acetone that adds 2.8 times of amounts in brain tissue homogenate's liquid, stir 28 minutes, standing 3.8 hours, filter, obtain acetone slag; To acetone slag, add again the acetone of 1.8 times of amounts, stir 28 minutes, standing 3.8 hours, filter, filtering residue is dried 3.2 hours at 62 ℃, obtains brain egg albumen powder;
(4) get trypsin, anhydrous calcium chloride (wherein the consumption of anhydrous calcium chloride is the 12.2wt% of trypsin consumption) dissolves by cold purified water, is made into the trypsin solution that concentration is 0.025%g/ml, places activation in 1.2 hours under room temperature; Pepsin dissolves by cold purified water, is made into the pepsin solution that concentration is 0.025%g/ml;
(5) get brain egg albumen powder, add purified water, stir, be heated to 43 ℃, by step 2) the 2.2wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature at enzymolysis between 43 ℃ after 3.8 hours, again by step 2) the 0.6wt% of clean Medulla sus domestica gross weight add pepsin, stir, control temperature enzymolysis 1.8 hours between 43 ℃, enzymolysis finishes, be heated to 86 ℃, be incubated 28 minutes, be then cooled to 22 ℃, obtain enzymolysis solution;
(6) enzymolysis solution of gained is used to centrifugal 22 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control the pH value to 5.5 of supernatant liquid;
(7) gained supernatant liquid is concentrated at 72 ℃ supernatant liquid original volume 1/4, at 72 ℃ dry 3.5 hours, pulverized 100 mesh sieves, obtain Cerebrolysin Vial dry powder.
The preparation of the Cerebrolysin Vial in embodiment 5, piracetam brain protein hydrolysate tablet
(1) get the Medulla sus domestica of fresh and healthy, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean Medulla sus domestica;
(2) get clean Medulla sus domestica 10kg, add 10L water for injection, use high-pressure homogenization pump to carry out homogenate, each homogenate 2.8 minutes, obtains brain tissue homogenate's liquid;
(3) to the acetone that adds 3.2 times of amounts in brain tissue homogenate's liquid, stir 32 minutes, standing 4.2 hours, filter, obtain acetone slag; To the acetone that adds 2.2 times of amounts in acetone slag, stir 32 minutes again, standing 4.2 hours, filter, filtering residue at 68 ℃ dry 4.8 hours, obtains brain egg albumen powder;
(4) get trypsin, anhydrous calcium chloride (wherein the consumption of anhydrous calcium chloride is the 12.8wt% of trypsin consumption) dissolves by cold purified water, is made into the trypsin solution that concentration is 0.03%g/ml, places activation in 1.8 hours under room temperature; Pepsin dissolves by cold purified water, is made into the pepsin solution that concentration is 0.03%g/ml;
(5) get brain egg albumen powder, add purified water, stir, be heated to 44 ℃, by step 2) the 2.8wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature at enzymolysis between 44 ℃ after 4.2 hours, again by step 2) the 0.8wt% of clean Medulla sus domestica gross weight add pepsin, stir, control temperature enzymolysis 2.3 hours between 44 ℃, enzymolysis finishes, be heated to 92 ℃, be incubated 33 minutes, be then cooled to 28 ℃, obtain enzymolysis solution;
(6) enzymolysis solution of gained is used to centrifugal 23 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control the pH value to 6.5 of supernatant liquid;
(7) gained supernatant liquid is concentrated at 78 ℃ supernatant liquid original volume 1/2, at 78 ℃ dry 4.8 hours, pulverized 100 mesh sieves, obtain Cerebrolysin Vial dry powder.
The preparation of the Cerebrolysin Vial in embodiment 6, piracetam brain protein hydrolysate tablet
(1) get the Medulla sus domestica of fresh and healthy, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean Medulla sus domestica;
(2) get clean Medulla sus domestica 10kg, add 10L water for injection, use high-pressure homogenization pump to carry out homogenate, each homogenate 2.8 minutes, obtains brain tissue homogenate's liquid;
(3) to the acetone that adds 3.4 times of amounts in brain tissue homogenate's liquid, stir 33 minutes, standing 4.4 hours, filter, obtain acetone slag; To the acetone that adds 2.2 times of amounts in acetone slag, stir 32 minutes again, standing 4.2 hours, filter, filtering residue at 68 ℃ dry 4.8 hours, obtains brain egg albumen powder;
(4) get trypsin, anhydrous calcium chloride (wherein the consumption of anhydrous calcium chloride is the 12.8wt% of trypsin consumption) dissolves by cold purified water, is made into the trypsin solution that concentration is 0.03%g/ml, places activation in 1.8 hours under room temperature; Pepsin dissolves by cold purified water, is made into the pepsin solution that concentration is 0.03%g/ml;
(5) get brain egg albumen powder, add purified water, stir, be heated to 41 ℃, by step 2) the 2.8wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature at enzymolysis between 41 ℃ after 4.2 hours, again by step 2) the 0.8wt% of clean Medulla sus domestica gross weight add pepsin, stir, control temperature enzymolysis 2.3 hours between 44 ℃, enzymolysis finishes, be heated to 92 ℃, be incubated 33 minutes, be then cooled to 28 ℃, obtain enzymolysis solution;
(6) enzymolysis solution of gained is used to centrifugal 23 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control the pH value to 6.5 of supernatant liquid;
(7) gained supernatant liquid is concentrated at 78 ℃ supernatant liquid original volume 1/2, at 78 ℃ dry 4.8 hours, pulverized 100 mesh sieves, obtain Cerebrolysin Vial dry powder.
The preparation of the Cerebrolysin Vial in embodiment 7, piracetam brain protein hydrolysate tablet
Concrete steps are with embodiment 1, the pepsin of step 5) first carries out ultrasonic pretreatment before adding as different from Example 1, described ultrasonic pretreatment for take ultrasonic power as 60W, supersonic frequency as 30kHz, ultrasonic time carries out ultrasonic pretreatment 8min with the intermittent time than the ultrasound wave that is 1:1.
The preparation of the Cerebrolysin Vial in embodiment 8, piracetam brain protein hydrolysate tablet
Concrete steps are with embodiment 1, the pepsin of step 5) first carries out ultrasonic pretreatment before adding as different from Example 1, described ultrasonic pretreatment for take ultrasonic power as 80W, supersonic frequency as 35kHz, ultrasonic time carries out ultrasonic pretreatment 12min with the intermittent time than the ultrasound wave that is 1:1.
The preparation of the Cerebrolysin Vial in embodiment 9, piracetam brain protein hydrolysate tablet
Concrete steps are with embodiment 1, the pepsin of step 5) first carries out ultrasonic pretreatment before adding as different from Example 1, described ultrasonic pretreatment for take ultrasonic power as 70W, supersonic frequency as 32kHz, ultrasonic time carries out ultrasonic pretreatment 10min with the intermittent time than the ultrasound wave that is 1:1.
The preparation of the Cerebrolysin Vial in embodiment 10, piracetam brain protein hydrolysate tablet
Concrete steps are with embodiment 5, the pepsin of step 5) first carries out ultrasonic pretreatment before adding as different from Example 5, described ultrasonic pretreatment for take ultrasonic power as 75W, supersonic frequency as 33kHz, ultrasonic time carries out ultrasonic pretreatment 9min with the intermittent time than the ultrasound wave that is 1:1.
The preparation of the Cerebrolysin Vial in embodiment 11, piracetam brain protein hydrolysate tablet
The other the same as in Example 1, difference is that cerebral tissue used is Pedis Canitis.
The preparation of the Cerebrolysin Vial in embodiment 12, piracetam brain protein hydrolysate tablet
The other the same as in Example 1, difference is that cerebral tissue used is Medulla Bovis seu Bubali.
The preparation of the Cerebrolysin Vial in embodiment 13, piracetam brain protein hydrolysate tablet
The other the same as in Example 1, difference is that cerebral tissue used is Medulla caprae seuovis.
The preparation of the Cerebrolysin Vial in embodiment 14, piracetam brain protein hydrolysate tablet
The other the same as in Example 1, difference is that cerebral tissue used is horse brain.
The preparation of the Cerebrolysin Vial in embodiment 15, piracetam brain protein hydrolysate tablet
The other the same as in Example 1, difference is that cerebral tissue used is Medulla Leporis seu Oryctolagi.
Test example 1, the impact of degreasing method on brain egg albumen powder quality
Brain egg albumen powder quality good or not is the key of enzyme digestion reaction success or not.If defat is thorough not, the fatty height of brain egg albumen powder, can cause hydrolysis to be difficult to carry out, and hydrolyzation system is muddy suspended substance, brings very large trouble to separating step below, and aminoacid in hydrolyzed solution, low molecular peptide content are also low.Fat is removed totallyer, and proteins react center exposes more fully, is beneficial to the carrying out of enzyme digestion reaction, and hydrolysis terminal is obvious, and solid-liquid is easy to separation.This test example be take the content of fat in brain egg albumen powder as detecting index, has investigated the impact of different degreasing methods on brain egg albumen powder quality.
Each sample makes in accordance with the following methods:
Sample 1: the brain egg albumen powder making according to the step of the embodiment of the present invention 1 (1)-(3);
Sample 2: get the Medulla sus domestica of fresh and healthy, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean Medulla sus domestica; Get clean Medulla sus domestica 10Kg, add 10L water for injection, use high-pressure homogenization pump to carry out homogenate, each homogenate 2 minutes, obtains brain tissue homogenate's liquid; To adding in brain tissue homogenate's liquid 3 times of amount acetone to soak, stir, place 20 hours in-5 ℃, filter, then soak with 3 times of amount acetone, 3 times repeatedly, fling to acetone, in 60 ℃ dry 5 hours, pulverizing, obtains brain egg albumen powder.
Analytical method: adopt Soxhlet extraction process to measure the content of fat in each sample.The results are shown in Table shown in 1.
The content of fat in the brain egg albumen powder that table 1, different degreasing method obtain
| Sample | Fat content (%) |
| Sample 1 | 1.6 |
| Sample 2 | 3.8 |
From the above results, can find out, adopt the content of fat in the brain egg albumen powder that degreasing method of the present invention obtains lower, show that fat removes totallyer.
Test example 2, the impact of different enzyme hydrolysis on amino acid content
This test example has been investigated the impact of different enzyme hydrolysiss on amino acid content in gained enzymolysis solution.
Sample number into spectrum is as follows:
Sample 1: according to the enzymolysis solution that the step of the embodiment of the present invention 1 (1)-(5) make;
Sample 2: other steps with embodiment 1 (1)-(4), step (5) is: get brain egg albumen powder, add purified water, stir, be heated to 40 ℃, by step 2) the 2.5wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature enzymolysis 5 hours between 40 ℃, enzymolysis finishes, be heated to 85 ℃, be incubated 25 minutes, be then cooled to 20 ℃, obtain enzymolysis solution; (hereinafter to be referred as trypsin, once adding)
Sample 3: other steps with embodiment 1 (1)-(4), step (5) is: get brain egg albumen powder, add purified water, stir, be heated to 40 ℃, by step 2) the 2wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature at enzymolysis between 40 ℃ after 3.5 hours, again by step 2) the 0.5wt% of clean Medulla sus domestica gross weight add trypsin, stir, control temperature enzymolysis 1.5 hours between 40 ℃, enzymolysis finishes, be heated to 85 ℃, be incubated 25 minutes, then be cooled to 20 ℃, obtain enzymolysis solution, (hereinafter to be referred as trypsin, dividing 2 times adds)
Sample 4: other steps with embodiment 1 (1)-(4), step (5) is: get brain egg albumen powder, add purified water, stir, be heated to 40 ℃, by step 2) the 2wt% of clean Medulla sus domestica gross weight add pepsin, stir, control temperature at enzymolysis between 40 ℃ after 3.5 hours, again by step 2) the 0.5wt% of clean Medulla sus domestica gross weight add the trypsin having activated, stir, control temperature enzymolysis 1.5 hours between 40 ℃, enzymolysis finishes, be heated to 85 ℃, be incubated 25 minutes, then be cooled to 20 ℃, obtain enzymolysis solution, (hereinafter to be referred as first adding pepsin, adding again trypsin)
Aminoacid quantitative assay: get each sample 10ml, with 0.1mol/L hydrochloric acid solution or 0.1mol/L sodium hydroxide solution, regulate pH value to 7.0, then add the formalin 10ml that regulates in advance pH value to 9.0, stir evenly, the pH value that is titrated to solution with sodium hydroxide volumetric solution (0.1mol/L) is 9.0, by adding the amount (ml) of the sodium hydroxide volumetric solution (0.1mol/L) consuming after formalin to calculate.The sodium hydroxide volumetric solution (0.1mol/L) of every 1ml is equivalent to the aminoacid of 1.401mg.
Amino acid whose content (mg/ml) in table 2, each enzymolysis solution
| Sample | Amino acid content (mg/ml) |
| Sample 1 | 53.7 |
| Sample 2 | 29.4 |
| Sample 3 | 33.1 |
| Sample 4 | 40.2 |
From above-mentioned result of the test, can find out, in hydrolytic process, trypsin is added at twice higher than once adding the total amino acid content being hydrolyzed, and than trypsin, add at twice the aminoacid of hydrolysis high with the total amino acid content that trypsin and two kinds of enzymes of pepsin are combined hydrolysis, but in the process by trypsin stomach function regulating enzyme linked Heshui solution, first adding trypsin, to add the aminoacid of pepsin hydrolysis higher with the aminoacid of trypsin hydrolyzing again than first adding pepsin again again.Visible, adopt of the present inventionly first to add trypsin to add the amino acid content of pepsin hydrolysis the highest again.
Test example 3, the impact of ultrasonic pretreatment on amino acid content in enzymolysis solution
This test example is to investigate the impact of ultrasonic pretreatment on amino acid content in enzymolysis solution.
Sample number into spectrum:
Sample 1: according to the enzymolysis solution that the step of the embodiment of the present invention 1 (1)-(5) make;
Sample 2: according to the enzymolysis solution that the step of the embodiment of the present invention 7 (1)-(5) make;
Sample 3: specifically, with step (1)-(5) of embodiment 1, difference is in step (5), before adding trypsin, to substrate---brain acetone powder solution carries out ultrasonic pretreatment, and described ultrasonic pretreatment is with embodiment 7;
Sample 4: specifically, with step (1)-(5) of embodiment 1, difference is in step (5), before adding trypsin, the trypsin activating is carried out to ultrasonic pretreatment, and described ultrasonic pretreatment is with embodiment 7;
Sample 5: specifically with step (1)-(5) of embodiment 1, difference is in step (5), after adding trypsin to stir, brain acetone powder and tryptic reaction solution are carried out to ultrasonic pretreatment, described ultrasonic pretreatment is with embodiment 7.
Amino acid whose content (mg/ml) in table 3, each enzymolysis solution
| Sample | Amino acid content (mg/ml) |
| Sample 1 | 53.7 |
| Sample 2 | 61.2 |
| Sample 3 | 19.7 |
| Sample 4 | 38.1 |
| Sample 5 | 20.2 |
From the result of upper table, can find out, when using same ultrasound wave to carry out different processing modes, the result of its acquisition is completely different.When ultrasonic pretreatment substrate---after brain acetone powder solution, in gained enzymolysis solution, amino acid whose content is very low.May be due to ultrasonic pretreatment substrate---after brain acetone powder solution, because making the structure of brain acetone powder solution, ul-trasonic irradiation changes, make to reduce with tryptic binding site, thereby enzyme reaction is slowed down rapidly, until the no longer reaction after adding trypsin of the protein in brain acetone powder solution.
After ultrasonic pretreatment trypsin, compared with ultrasonic pretreatment substrate, to compare, in gained enzymolysis solution, amino acid whose content increases, and may to the increase of tryptic activity, have facilitation due to ultrasound wave.
After ultrasonic pretreatment brain acetone powder and tryptic reaction solution, although ultrasound wave has facilitation to the increase of tryptic activity, but because making the structure of brain acetone powder solution, ul-trasonic irradiation changes, make to reduce with tryptic binding site, thereby enzyme reaction is slowed down, and in gained enzymolysis solution, amino acid whose content is not significantly improved.
And after ultrasonic pretreatment pepsin, amino acid whose content improves significantly in gained enzymolysis solution, may to the increase of pepsin activity, there is obvious facilitation due to ultrasound wave.
By above-mentioned test, the present invention is ultrasonic pretreatment pepsin most preferably.
Main amino acid whose content in test example 4, Cerebrolysin Vial
This test example has been investigated main amino acid whose content in the Cerebrolysin Vial that the inventive method and art methods make.
Sample number into spectrum is as follows:
Sample 1-10: the Cerebrolysin Vial dry powder making according to the method for embodiment of the present invention 1-10 respectively;
Contrast 1: the brain extract making according to the method for the embodiment of CN200510117294.7 1;
Contrast 2: according to " preparation of brain egg albumen powder and the research of enzyme solution thereof " [Yu Libi, Shi Yan, etc. the preparation of brain egg albumen powder and the research of enzyme solution [J] thereof, aminoacid and living resources, the Cerebrolysin Vial that method 2000,22(3): 18-21] makes;
Contrast 3: the Cerebrolysin Vial making according to the method for the embodiment of CN201310113215.X one;
To above-mentioned sample, adopt the quick amino-acid analyzer of the 835-50 of Hitachi type to analyze, several main amino acid whose content results see the following form 4.
Main amino acid whose content (mg/ml) in table 4, each Cerebrolysin Vial
| Sample | Glu | Ala | Arg | Lys | Val | Phe | Ile | Leu |
| Sample 1 | 6.32 | 3.59 | 3.18 | 12.37 | 3.89 | 4.12 | 4.03 | 6.89 |
| Sample 2 | 6.29 | 3.55 | 3.12 | 12.39 | 3.88 | 4.07 | 4.01 | 6.85 |
| Sample 3 | 6.31 | 3.51 | 3.13 | 12.41 | 3.87 | 4.06 | 4.05 | 6.83 |
| Sample 4 | 6.30 | 3.54 | 3.12 | 12.38 | 3.86 | 4.11 | 4.06 | 6.87 |
| Sample 5 | 6.28 | 3.53 | 3.13 | 12.39 | 3.87 | 4.12 | 4.07 | 6.86 |
| Sample 6 | 6.30 | 3.52 | 3.12 | 12.40 | 3.86 | 4.11 | 4.08 | 6.89 |
| Sample 7 | 7.81 | 5.51 | 5.79 | 13.26 | 4.13 | 5.31 | 5.39 | 7.98 |
| Sample 8 | 7.80 | 5.52 | 5.79 | 13.25 | 4.12 | 5.30 | 5.38 | 7.99 |
| Sample 9 | 7.83 | 5.53 | 5.80 | 13.24 | 4.14 | 5.29 | 5.37 | 7.97 |
| Sample 10 | 7.82 | 5.52 | 5.81 | 13.23 | 4.15 | 5.28 | 5.39 | 7.98 |
| Contrast 1 | 5.01 | 1.78 | 2.01 | 10.03 | 1.21 | 1.32 | 1.47 | 3.02 |
| Contrast 2 | 2.84 | 2.33 | 2.26 | 3.65 | 2.48 | 2.44 | 2.10 | 4.71 |
| Contrast 3 | 2.86 | 2.41 | 2.31 | 3.61 | 2.41 | 2.18 | 2.01 | 4.63 |
From the result of upper table, can find out, adopt in the Cerebrolysin Vial that method of the present invention makes main amino acid whose content apparently higher than prior art, and in method of the present invention, adopt main amino acid whose content in the Cerebrolysin Vial of the gained that is hydrolyzed after ultrasonic pretreatment pepsin again apparently higher than not adopting ultrasonic pretreatment.
Claims (10)
1. the Cerebrolysin Vial in piracetam brain protein hydrolysate tablet, is characterized in that, described Cerebrolysin Vial is adopted with the following method and prepared:
1) get the mammiferous cerebral tissue except people, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean cerebral tissue;
2) get clean cerebral tissue and carry out homogenate, each homogenate 2～3 minutes, obtains brain tissue homogenate's liquid;
3) to the acetone that adds 2.5～3.5 times of volumes in brain tissue homogenate's liquid, stir 25～35 minutes, standing 3.5～4.5 hours, filter, obtain acetone slag; To the acetone that adds 1.5～2.5 times of volumes in acetone slag, stir 25～35 minutes again, standing 3.5～4.5 hours, filter, filtering residue at 60～70 ℃ dry 3～5 hours, obtains brain egg albumen powder;
4) get trypsin, anhydrous calcium chloride dissolves by cold purified water, is mixed with the trypsin solution that concentration is 0.02%～0.04%g/ml, places activation in 1～2 hour under room temperature; Pepsin dissolves by cold purified water, is mixed with the pepsin solution that concentration is 0.02%～0.04%g/ml;
5) get brain egg albumen powder, add purified water, stir, be heated to 40～45 ℃, add the trypsin having activated, stir, control temperature at enzymolysis between 40～45 ℃ after 3.5～4.5 hours, add again pepsin, stir, control temperature enzymolysis 1.5～2.5 hours between 40～45 ℃, enzymolysis finishes, be heated to 85～95 ℃, be incubated 25～35 minutes, be then cooled to 20～30 ℃, obtain enzymolysis solution;
6) enzymolysis solution of gained is used to centrifugal 15～25 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control pH value to 5.0～7.0 of supernatant liquid;
7) gained supernatant liquid is concentrated, is dried, is pulverized and sieved, obtain Cerebrolysin Vial dry powder.
2. Cerebrolysin Vial according to claim 1, is characterized in that, the consumption of the anhydrous calcium chloride described in step 4) is 12～13wt% of trypsin consumption.
3. Cerebrolysin Vial according to claim 1, is characterized in that, the good tryptic amount of activation adding in step 5) is step 2) 2～3wt% of clean cerebral tissue gross weight; The pepsic amount adding is step 2) 0.5～1wt% of clean cerebral tissue gross weight.
4. according to the Cerebrolysin Vial described in claim 1～3 any one, it is characterized in that, the pepsin described in step 5) first carries out ultrasonic pretreatment before adding.
5. Cerebrolysin Vial according to claim 4, it is characterized in that, described ultrasonic pretreatment for take ultrasonic power as 60～80W, supersonic frequency as 30～35kHz, ultrasonic time carries out ultrasonic pretreatment 8～12min with the intermittent time than the ultrasound wave that is 1:1.
6. a preparation method for the Cerebrolysin Vial in piracetam brain protein hydrolysate tablet claimed in claim 1, is characterized in that, described preparation method comprises the steps:
1) get the mammiferous cerebral tissue except people, natural negative catalysis, to without ice slag, is rejected impurity, obtains clean cerebral tissue;
2) get clean cerebral tissue and carry out homogenate, each homogenate 2～3 minutes, obtains brain tissue homogenate's liquid;
3) to the acetone that adds 2.5～3.5 times of volumes in brain tissue homogenate's liquid, stir 25～35 minutes, standing 3.5～4.5 hours, filter, obtain acetone slag; To the acetone that adds 1.5～2.5 times of volumes in acetone slag, stir 25～35 minutes again, standing 3.5～4.5 hours, filter, filtering residue at 60～70 ℃ dry 3～5 hours, obtains brain egg albumen powder;
4) get trypsin, anhydrous calcium chloride dissolves by cold purified water, is made into the trypsin solution that concentration is 0.02%～0.04%g/ml, places activation in 1～2 hour under room temperature; Pepsin dissolves by cold purified water, is made into the pepsin solution that concentration is 0.02%～0.04%g/ml;
5) get brain egg albumen powder, add purified water, stir, be heated to 40～45 ℃, add the trypsin having activated, stir, control temperature at enzymolysis between 40～45 ℃ after 3.5～4.5 hours, add again pepsin, stir, control temperature enzymolysis 1.5～2.5 hours between 40～45 ℃, enzymolysis finishes, be heated to 85～95 ℃, be incubated 25～35 minutes, be then cooled to 20～30 ℃, obtain enzymolysis solution;
6) enzymolysis solution of gained is used to centrifugal 15～25 minutes of centrifuge, collect supernatant, filter and obtain supernatant liquid, control pH value to 5.0～7.0 of supernatant liquid;
7) gained supernatant liquid is concentrated, is dried, is pulverized and sieved, obtain Cerebrolysin Vial dry powder.
7. preparation method according to claim 6, is characterized in that, the consumption of the anhydrous calcium chloride described in step 4) is 12～13wt% of trypsin consumption.
8. preparation method according to claim 6, is characterized in that, in step 5), adding the tryptic amount having activated is step 2) 2～3wt% of clean cerebral tissue gross weight; Adding pepsic amount is step 2) 0.5～1wt% of clean cerebral tissue gross weight.
9. preparation method according to claim 6, is characterized in that, the simmer down to described in step 7) is concentrated into 1/4～1/2 of supernatant liquid original volume at 70～80 ℃; Described is dried as be dried 3～5 hours at 70～80 ℃; Described sieves as crossing 100 mesh sieves.
10. according to the preparation method described in claim 6～9 any one, it is characterized in that, the pepsin described in step 5) first carries out ultrasonic pretreatment before adding; Preferably take ultrasonic power as 60～80W, supersonic frequency as 30～35kHz, ultrasonic time carries out ultrasonic pretreatment 8～12min with the intermittent time than the ultrasound wave that is 1:1.
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