CN107303385B - Preparation method of cerebroprotein hydrolysate solution - Google Patents
Preparation method of cerebroprotein hydrolysate solution Download PDFInfo
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- CN107303385B CN107303385B CN201610235754.4A CN201610235754A CN107303385B CN 107303385 B CN107303385 B CN 107303385B CN 201610235754 A CN201610235754 A CN 201610235754A CN 107303385 B CN107303385 B CN 107303385B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
Abstract
The invention relates to a preparation method of a cerebroprotein hydrolysate solution, which comprises the steps of pretreating animal brain tissues, performing enzymolysis, performing acid-base precipitation, performing ultrafiltration and performing nanofiltration in sequence; the method is characterized in that the nanofiltration step is carried out by adopting a nanofiltration system with the molecular weight cutoff of 150-300 daltons. The method can effectively remove the ineffective components and harmful components in the product, simultaneously reserve the amino acid to the maximum extent and improve the safety of the product.
Description
Technical Field
The invention relates to a preparation method of a cerebroprotein hydrolysate solution, belonging to the field of medicines.
Background
The brain protein hydrolysate is a mixture which is obtained by hydrolyzing animal brain tissue by protease and is rich in various amino acids and small molecular polypeptides, can regulate and improve the metabolism of neurons, and is clinically mainly used for treating craniocerebral injury and sequelae of cerebrovascular diseases (such as cerebral infarction, cerebral hemorrhage and the like) and accelerating the functional recovery of the brain nervous system.
The cerebroprotein hydrolysate contains amino acids and polypeptides with small molecular weight and specific bioactivity, and is used as material for producing cerebroprotein hydrolysate for injection. Amino acids are classified into effective amino acids and impurity amino acids according to their action on the body. The effective amino acids can penetrate blood brain barrier, promote cell DNA synthesis, repair and regeneration, improve adrenal cortex function, and promote whole body metabolism. The impurity amino acid stimulates immune cells of an organism to secrete histamine-like substances, and the histamine-like substances have the effect of reducing blood pressure, so that the substances can cause the blood pressure of a patient to be reduced, and can cause shock in severe cases to threaten life.
Nanofiltration is a membrane separation technology between reverse osmosis and ultrafiltration, the rejection rate of various solutes in a solution is positively correlated with the molar mass of the solutes, and various components in the obtained product are also different by adopting a nanofiltration membrane system with different cut-off molecular weights. The prior art discloses a process for separating and treating brain tissue hydrolysate by adopting a nanofiltration technology, for example, CN 1562339A discloses a preparation method of a cerebroprotein hydrolysate preparation, which comprises the steps of carrying out enzymolysis and ultrafiltration on fresh pig brain, and then carrying out nanofiltration concentration on the fresh pig brain by using a nanofiltration membrane with the molecular weight cutoff of 100-1000; CN 103191403A discloses a preparation method of a cerebroprotein hydrolysate, and particularly discloses that pig brain tissue is subjected to homogenate, degreasing, enzymolysis and ultrafiltration treatment, and then is subjected to nanofiltration concentration by using a nanofiltration machine with the molecular weight cutoff of 90-150 daltons; CN 101019889A discloses that nano-filtration is carried out on cerebroprotein hydrolysate by a nano-filter with the molecular weight of 300-1000. The nanofiltration steps disclosed in the prior art are only aimed at purifying and concentrating the cerebroprotein hydrolysate, or ensuring the qualified refractive index of the preparation, and do not disclose a technical scheme for improving the product safety by the nanofiltration technology.
Disclosure of Invention
The invention aims to provide a preparation method of a cerebroprotein hydrolysate solution, which adopts a 150-300 Dalton nanofiltration system to remove low-molecular ineffective and harmful substances in the cerebroprotein hydrolysate solution, and improves the safety of products.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a brain protein hydrolysate solution comprises the steps of pretreating animal brain tissue, performing enzymolysis, precipitating with acid and alkali, ultrafiltering, and nanofiltering in sequence;
the method is characterized in that the nanofiltration step is carried out by adopting a nanofiltration system with the molecular weight cutoff of 150-300 daltons.
Further, the nanofiltration process is that a nanofiltration system with the molecular weight cutoff of 150-300 daltons is adopted for filtration, the return liquid is collected to be nanofiltration liquid, and the nanofiltration is finished when the volume of the nanofiltration liquid is 1/5-1/2.
Further, water for injection can be added into the nanofiltration liquid to reach the volume before nanofiltration, the nanofiltration system with the volume of 150-300 daltons is used for filtering again, the reflux liquid is collected, the nanofiltration is finished when the reflux liquid is 1/5-1/2, and the process is repeated for 1-4 times.
Further, the animal brain tissue is fresh or frozen pig brain which is preserved for no more than 3 months.
Further, the pretreatment comprises the steps of removing structures attached to the brain tissue and homogenizing the brain tissue to obtain a brain tissue homogenate. Furthermore, injection water with the weight 1-3 times of that of the brain tissue is added in the homogenization process.
Further, the enzymolysis process comprises a process of sequentially carrying out enzymolysis by using pepsin and pancreatin to obtain an enzymolysis solution. Furthermore, the adding amount of the pepsin and the pancreatin is 0.2 to 1 percent of the brain tissue homogenate, the enzymolysis temperature is 40 to 70 ℃, and the enzymolysis time is 2 to 8 hours; further, the enzymolysis temperature is 50-60 ℃, and the enzymolysis time is 3-6 hours; furthermore, the enzymolysis temperature is 55 ℃, and the enzymolysis time is 4 hours.
Further, the acid-base precipitation process comprises the steps of adjusting the pH of the enzymolysis liquid to 1-2 by using an acidic solution, standing for 8-12 hours, and taking a supernatant; and regulating the pH of the supernatant to 8-10 by using an alkaline solution, standing for 1-2 hours, and taking the supernatant.
Further, the ultrafiltration process comprises the steps of sequentially carrying out ultrafiltration by using ultrafiltration devices with the molecular weight cutoff of 100000-150000 daltons and 10000-50000 daltons, and taking the permeate as ultrafiltrate; further, the ultrafiltration process is sequentially carried out by ultrafiltration devices with molecular weight cut-off of 100000 daltons and 10000 daltons.
The method can effectively remove the ineffective components and harmful components in the product, simultaneously reserve the amino acid to the maximum extent and improve the safety of the product.
Detailed Description
Example 1
Pretreatment: taking the pig brain of a fresh healthy pig, and removing attached bones, fat, connective tissues and the like; homogenizing pig brain and 2 times of injection water for 2 times to obtain homogenate;
enzymolysis: adding pepsin into the homogenate according to 0.5% of the weight of the corresponding pig brain, adjusting the pH value of the homogenate to 3.5 by hydrochloric acid, and hydrolyzing at 55 ℃ for 4 h; then adjusting pH to 8.5 with sodium hydroxide solution, adding 0.02mol/L CaCL into the homogenate at a ratio of 0.5% of pig brain weight2Hydrolyzing the solution-activated pancreatin at 55 deg.C for 4 h; heating and boiling pancreatin hydrolysate for 60 min;
acid-base precipitation: regulating the pH value of the pancreatic enzyme hydrolysate to 1.5 by using hydrochloric acid, standing for 8 hours, and separating supernatant; regulating the pH of the supernatant to 9.0 with sodium hydroxide solution, standing for 2h, and separating the supernatant;
and (3) ultrafiltration: ultrafiltering the supernatant with ultrafiltration membrane with cut-off molecular weight of 100000 Dalton, collecting the filtrate, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 10000 Dalton, and collecting the filtrate.
Example 2
Pretreatment: taking the pig brain of a fresh healthy pig, and removing attached bones, fat, connective tissues and the like; homogenizing pig brain and injection water 3 times of the weight of pig brain for 2 times to obtain homogenate;
enzymolysis: adding pepsin into the homogenate according to 0.2% of the weight of the corresponding pig brain, adjusting the pH value of the homogenate to 3.5 by hydrochloric acid, and hydrolyzing at 50 ℃ for 6 h; then adjusting pH to 8.5 with sodium hydroxide solution, adding 0.02mol/L CaCL into the homogenate at a ratio of 0.2% of pig brain weight2Hydrolyzing with solution-activated trypsin at 50 deg.C for 6 hr; heating and boiling pancreatin hydrolysate for 60 min;
acid-base precipitation: regulating the pH value of the pancreatic enzyme hydrolysate to 1.0 by using hydrochloric acid, standing for 3 hours, and separating supernatant; regulating the pH of the supernatant to 10.0 with sodium hydroxide solution, standing for 3h, and separating the supernatant;
and (3) ultrafiltration: ultrafiltering the supernatant with ultrafiltration membrane with cut-off molecular weight of 150000D, collecting the filtrate, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 50000D, and collecting the filtrate.
Example 3
Pretreatment: taking the pig brain of a fresh healthy pig, and removing attached bones, fat, connective tissues and the like; homogenizing medulla sus domestica and water for injection for 2 times to obtain homogenate;
enzymolysis: adding pepsin into the homogenate according to the weight of 1 percent of the homogenate corresponding to the weight of the pig brain, adjusting the pH value of the homogenate to 3 by hydrochloric acid, and hydrolyzing for 4 hours at 60 ℃; then adjusting pH to 9 with sodium hydroxide solution, adding 0.02mol/L CaCL into the homogenate according to 1% of pig brain weight2Hydrolyzing with solution-activated trypsin at 60 deg.C for 4 hr; heating and boiling pancreatin hydrolysate for 60 min;
acid-base precipitation: regulating the pH value of the pancreatic enzyme hydrolysate to 2.0 by using hydrochloric acid, standing for 1.5h, and separating supernatant; regulating the pH of the supernatant to 8.0 with sodium hydroxide solution, standing for 1.5h, and separating the supernatant;
and (3) ultrafiltration: ultrafiltering the supernatant with ultrafiltration membrane with cut-off molecular weight of 120000D, collecting the filtrate, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 30000D, and collecting the filtrate as ultrafiltrate.
Example 4
And (4) nanofiltration: nanofiltration of the ultrafiltrate prepared in any of the embodiments 1-3 by a nanofiltration membrane separation system with the molecular weight cutoff of 150 daltons, discarding the permeate, and collecting the reflux to the volume 1/2 before nanofiltration; adding water for injection into the reflux liquid to a volume before nanofiltration, performing nanofiltration again by using a separation membrane system with the molecular weight cutoff of 150 daltons, discarding the permeation liquid, collecting the reflux liquid to a volume before nanofiltration of 1/4, and finishing nanofiltration; filtering with 0.22um filter membrane to obtain cerebroprotein hydrolysate.
Example 5
And (4) nanofiltration: nanofiltration of the ultrafiltrate prepared in any of the embodiments 1-3 by a nanofiltration membrane separation system with the molecular weight cutoff of 200 daltons, discarding the permeate, and collecting the reflux to the volume 1/2 before nanofiltration; adding water for injection into the reflux liquid to a volume before nanofiltration, performing nanofiltration again by using a separation membrane system with the molecular weight cutoff of 200 daltons, discarding the permeation liquid, and collecting the reflux liquid to a volume before nanofiltration of 1/2; adding water for injection into the reflux liquid to the volume before nanofiltration, performing nanofiltration for the third time by using a separation membrane system with the molecular weight cutoff of 200 daltons, and finishing the nanofiltration when the reflux liquid reaches 1/4 of the original volume; filtering with 0.22um filter membrane to obtain cerebroprotein hydrolysate.
Example 6
And (4) nanofiltration: nano-filtering the ultrafiltrate prepared in any one of the embodiments 1-3 by a nano-filtration membrane separation system with the molecular weight cutoff of 300 daltons, discarding the permeation liquid, and finishing the nano-filtration when the reflux liquid reaches 1/4 of the original volume; filtering the nano filtrate with 0.22um filter membrane to obtain cerebroprotein hydrolysate solution.
Comparative example 1
And (4) nanofiltration: nano-filtering the ultrafiltrate prepared in the embodiment 1 by using a nano-filtration membrane separation system with the molecular weight cutoff of 400, discarding the permeation liquid, and finishing the nano-filtration when the reflux liquid reaches 1/4 of the original volume; filtering with 0.22um filter membrane to obtain cerebroprotein hydrolysate.
Comparative example 2
And (4) nanofiltration: nano-filtering the ultrafiltrate prepared in the embodiment 1 by using a nano-filtration membrane separation system with the molecular weight cut-off of 100, discarding a permeate, and finishing the nano-filtration when the reflux reaches 1/4 of the original volume; filtering with 0.22um filter membrane to obtain cerebroprotein hydrolysate.
Comparative example 3
The ultrafiltrate prepared in example 1 is collected and concentrated under vacuum degree of-0.08 mpa + -0.005 mpa and temperature of 60 deg.C to 75 deg.C to 1/4 of the original volume, and the concentrated solution is filtered through 0.22um filter membrane to obtain the cerebroprotein hydrolysate.
Examination of production method
1 Experimental drugs
Samples 1 to 3: the brain protein hydrolysates prepared in examples 4-6, respectively, and the ultrafiltrate used was the ultrafiltrate prepared in example 1;
samples 4 to 6: the brain protein hydrolysates prepared in comparative examples 1-3, respectively.
2 Experimental content and methods
2.1 measurement of physical and chemical Components
2.1.1 Total Nitrogen content
2.1.1.1 detection method
Instrument and appliance
Graduated pipette (0.5ml, 2ml, 3ml), KDY-9810 Kjeldahl nitrogen determination apparatus, KXL-1010 type temperature control digestion furnace, acid burette (25ml), electronic balance (AL204-IC or BS224S)
Test solution and reagent
Potassium sulfate (analytically pure), sulfuric acid (analytically pure), copper sulfate (analytically pure), sulfuric acid titration solution (0.1mol/L)
30% copper sulfate solution: weighing 30g of copper sulfate, placing the copper sulfate in a 100ml measuring flask, adding water to dissolve and dilute the copper sulfate into 100ml, and shaking up the copper sulfate to obtain the copper sulfate.
Inspection method and result determination
The total nitrogen content is 0.3ml, and is determined according to the standard operation procedure for measuring nitrogen in cerebroprotein hydrolysate, and 2 parts are measured in parallel and corrected by blank test.
And (3) calculating:
in the formula V1The volume (ml) of sulfuric acid titration solution (0.005mol/L) was consumed for the test sample
V0Is emptyVolume (ml) of white spent sulfuric acid titration solution (0.005mol/L)
F is a correction factor of sulfuric acid titration solution (0.005mol/L)
T is the titer of the sample corresponding to the sulfuric acid titration solution (0.005mol/L)
C is total nitrogen amount (mg/ml) of the sample
2.1.1.2 sample testing
Samples 1-6 were taken respectively and the total nitrogen content was determined as above.
2.1.2 Total amino acid content
2.1.2.1 detection method
Instrument and tool
High performance liquid chromatograph (LC-10ATVP, LC-15C), DH-101 type electric heating constant temperature blast drying box
Electronic balance (BP211D), measuring flask (50ml), measuring cylinder (1000ml), beaker, derivative tube
25 mul glass microsyringe, vortex mixer, micro-pipetting gun, drying suction head
Reagent, solution, or solution
Amino acid standard sample reference, AccQ. Tag mobile phase A concentrate (sealed in a refrigerator at 4 ℃), AccQ. Fluor Rcaging Kit { derivatization reagent: AccQ. flow reagent powder (2A), AccQ. flow diluent (2B), AccQ. flow borate buffer (buffer) }, high purity water prepared on the same day, acetonitrile (chromatographic grade),
Preparation of amino acid standards for derivatization: opening 1 bottle of 17 amino acid standard samples, placing the standard samples in a 10ml volumetric flask, diluting the standard samples to the scale with high-purity water, and uniformly mixing the standard samples for later use.
Preparation of derivatizing agent 2A: before opening the 2A bottle, flicking lightly to ensure that all the AccQ. flow reagent powder falls on the bottom of the bottle; using a 1ml micropipette to suck 1ml of AccQ. flow diluent from the 2B bottle and discharging the diluent so as to clean the micropipette head; sucking 1ml of AccQ-flow diluent, putting the AccQ-flow diluent into a 2A bottle filled with AccQ-flow derivative powder, and sealing the bottle by a cover; shake until dissolved.
Inspection method and result determination
Chromatographic condition setting parameters
A chromatographic column: waters AccQ. Tag C-18150 mm. times.3.9 mm
Column temperature: 37 ℃, detection wavelength: flow rate at 248 nm: 1.0ml/min sample size: 10 μ l
Mobile phase:
solution A: according to the following steps: 10(V/V) AccQ. Tag A concentrate (WAT052890) is diluted with water, filtered, poured into a clean solvent bottle, and ultrasonically degassed in an ultrasonic cleaner for 1 minute for use. (sealing and storing in a refrigerator at 4 deg.C)
And B, liquid B: taking acetonitrile and water 60: pouring 40(V/V) of the mixture into a beaker, stirring and mixing the mixture evenly, filtering the mixture, pouring the mixture into a clean solvent bottle, and placing the solvent bottle into an ultrasonic cleaner for ultrasonic degassing for 1 minute for later use. (Ready-to-use)
Gradient tables and single run times were set as in table 1:
TABLE 1 elution procedure
Chromatographic system pre-balancing
After the pump head pipelines are respectively filled with A, B mobile phases (namely, pump exhaust operation is carried out), the system is firstly flushed for 15min by the liquid B at the flow rate of 1ml/min, then the system is balanced for 60min by the liquid A, parameters are set according to the AccQ & Tag chromatographic conditions, and blank gradient balance is carried out.
Derivatization of amino acid standards
Using a clean pipette tip to remove 10ul of the diluted standard sample, injecting the diluted standard sample into the bottom of a clean derivatization tube, replacing the bottom of the clean micropipette tip, adding 70ul AccQ. flow borate buffer (buffer) into the derivatization tube, and carrying out vortex mixing; another clean micropipette tip was replaced to aspirate 20ul of the AccQ flor derivative (2A) that had been dispensed, added to the derivative tube in a vortexed state, vortexed for 10 seconds, and allowed to stand at room temperature for 1 minute; sealing the opening of the derivative tube with a sealing film, and heating in an oven at 55 ℃ for 10 minutes; and after heating, taking out the sample to be used for sample injection.
Derivatization of test solutions
Precisely measuring 1ml of the product, placing the product in a 25ml measuring flask, adding water to dissolve the product, and diluting the product to a scale to obtain a test solution. The method of derivatizing the test solution is the same as that of derivatizing the amino acid standard. And calculating the content of the amino acid by peak area according to an external standard method.
Wherein C is the percentage content of amino acid in the test sample
Molecular weight molecular weights of various amino acids
AFor supplying toPeak areas for various amino acids
ATo pairPeak area for the corresponding amino acid
n dilution factor (ml) of test article
2.1.2.2 sample testing
Samples 1-6 were taken, and the amino acid content in the samples was determined as above.
2.2 evaluation of safety
In the clinical application process of the brain protein hydrolysate for injection, adverse reactions occur in a few cases, wherein the adverse reactions are common allergic reactions, the symptoms comprise chill, fever, rapid blood pressure reduction and the like, and severe cases can cause shock. The legal standards of the product only contain abnormal toxicity for safety evaluation, and no evaluation related to anaphylaxis is included. The process of the invention refers to a blood pressure reducing substance inspection method in appendix XI G of second part of Chinese pharmacopoeia 2010 edition, compares the degree of blood pressure reduction of anesthetized cats caused by histamine comparison products (S) and test products (T), judges whether the limit of the blood pressure reducing substance contained in the test products meets the regulation, compares the experimental results of the blood pressure reducing substances of samples prepared by different processes, and confirms the advancement of the process of the invention.
2.2.1 test methods
Preparation of control solution A proper amount of histamine phosphate control is precisely weighed, based on histamine, water is added to dissolve the histamine phosphate control into a solution containing 1.0mg per 1ml, and the solution is subpackaged in a proper container and stored at 4-8 ℃.
Preparation of reference substance diluent 0.1ml of histamine phosphate standard substance stock solution is precisely measured, placed in a 200ml volumetric flask, added with normal saline to scale, and shaken up to obtain 0.5 mug/ml.
Preparation of test solution
Filtering samples 1-5 with 0.22um filter membrane respectively, subpackaging in sterilized container under aseptic condition, sealing, and storing at 4-8 deg.C.
The inspection method comprises taking cat with qualified health and weight of more than 2kg, making female sterile, anesthetizing with appropriate amount of anesthetic (sodium pentobarbital 40mg/2ml/kg), fixing on thermal insulation operating table, and separating trachea. A cannula is inserted to allow smooth breathing if necessary, or artificial breathing may be performed. An arterial cannula connected to a manometer is inserted into one carotid artery and filled with a suitable anticoagulant solution to record blood pressure, or other suitable instruments may be used to record blood pressure. A venous cannula is inserted into one femoral vein for injecting liquid medicine, and the dosage is as follows: the dosage is 10mg/kg calculated according to the total nitrogen content of the sample. Care was taken in the experiment to maintain the animal body temperature. After all operations are finished, the manometer is adjusted to the height equivalent to the animal blood pressure, the artery clamp is opened, and after the blood pressure is stable, the medicine injection can be carried out.
3 results of the experiment
3.1 results of measurement of physical and chemical Components
The results are shown in Table 2.
TABLE 2 physicochemical component contents
| Sample (I) | Total nitrogen (mg/ml) | Total amino acids (mg/ml) |
| 1 | 9.33 | 14.61 |
| 2 | 9.20 | 14.26 |
| 3 | 8.92 | 13.52 |
| 4 | 5.32 | 7.40 |
| 5 | 9.60 | 15.10 |
| 6 | 10.10 | 15.89 |
The above experimental results show that the cerebroprotein hydrolysate (i.e. sample 5) treated by the reduced pressure concentration process has the highest total nitrogen and amino acid contents; the total nitrogen and amino acid content of the cerebroprotein hydrolysate treated by the nanofiltration process is lower than that of the sample 5, and the total nitrogen and amino acid content of the cerebroprotein hydrolysate is reduced along with the increase of the permeable molecular weight range of the nanofiltration membrane.
3.2 evaluation results of safety
The results are shown in Table 3.
TABLE 3 evaluation results of safety
The above experimental results show that, after the administration of the samples 4 and 5, the blood pressure reduction amplitude of the tested cat is larger than the blood pressure reduction value caused by the administration of the reference substance, and the experimental results do not meet the regulations. The preparation processes of the two samples are shown to be incapable of removing the substances for reducing the pressure to an acceptable level, and the safety of the product is poor. After the samples 1-4 are administrated, the blood pressure reduction amplitude of the tested cat is 0.5 times smaller than that of the control substance after the administration, and the experimental result meets the regulation, so that the content of substances for reducing blood pressure can be remarkably reduced in the product subjected to separation by adopting a 150-400 Dalton nanofiltration technology, and the product has high safety.
By combining the above experimental results, although the total nitrogen and amino acid contents in samples 5 and 6 are high, the reduced pressure test is not satisfactory, and the corresponding ineffective components and harmful components in the product are also high, which does not meet the requirement for the safety of the product. The pressure reduction test of the samples 1-4 meets the requirement, which shows that the corresponding process can effectively remove harmful ingredients in the product, and the safety meets the requirement.
The total nitrogen and amino acid content in sample 4 was only 52.7% and 46.6% of sample 6, the total nitrogen content in sample 1, sample 2 and sample 3 was 92.3, 90% and 85% of sample 6, respectively, and the amino acid content was 91.9%, 89.7% and 85.1% of sample 6, respectively. The solution treated by the 400 dalton nanofiltration membrane system has the loss of about 50 percent of effective components, and more amino acids need to be added when preparing the cerebroprotein hydrolysate for injection. The safety and the economy are considered, the processes of the samples 1, 2 and 3 are more reasonable, namely the 150-dalton 300-dalton nanofiltration technology is selected to have the best effect.
Claims (9)
1. A preparation method of a brain protein hydrolysate solution comprises the steps of pretreating animal brain tissue, performing enzymolysis, precipitating with acid and alkali, ultrafiltering, and nanofiltering in sequence;
the method is characterized in that the nanofiltration step is carried out by adopting a nanofiltration system with the molecular weight cutoff of 200-300 daltons; the ultrafiltration process is that ultrafiltration is carried out by using ultrafiltration devices with the molecular weight cutoff of 100000-150000 daltons and 10000-50000 daltons in sequence, and permeate is taken as ultrafiltrate.
2. The preparation method of claim 1, wherein the nanofiltration process comprises the steps of filtering by using a nanofiltration system with the molecular weight cutoff of 200-300 daltons, collecting the reflux liquid as nanofiltration liquid, and finishing nanofiltration when the nanofiltration liquid is in an original volume of 1/5-1/2.
3. The preparation method of claim 2, wherein the nanofiltration solution is added with water for injection to a volume before nanofiltration, and then filtered again by a nanofiltration system with 200 to 300 daltons, and the reflux is collected, and the nanofiltration is terminated when the reflux is 1/5 to 1/2, and the process is repeated for 1 to 4 times.
4. The method of claim 1, wherein the pre-treatment comprises the steps of removing structures attached to the brain tissue and homogenizing the brain tissue to obtain a brain tissue homogenate.
5. The method according to claim 1, wherein the enzymatic hydrolysis process comprises a process of sequentially subjecting to enzymatic hydrolysis with pepsin and pancreatin to obtain an enzymatic hydrolysate.
6. The preparation method according to claim 4, wherein the pepsin and pancreatin are added in an amount of 0.2-1% of the brain tissue homogenate, the enzymolysis temperature is 40-70 ℃, and the enzymolysis time is 2-8 hours.
7. The preparation method according to claim 5, wherein the enzymolysis temperature is 50-60 ℃ and the enzymolysis time is 3-8 hours.
8. The preparation method according to claim 1, wherein the acid-base precipitation process comprises the steps of firstly adjusting the pH of the enzymatic hydrolysate to 1-2 by using an acidic solution; and adjusting the pH value to 8-10 by using an alkaline solution.
9. A cerebroprotein hydrolysate prepared by the method according to any one of claims 1 to 8.
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| CN101940596A (en) * | 2009-07-09 | 2011-01-12 | 张树信 | New method for preparing cerebroprotein hydrolysate |
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| CN101940596A (en) * | 2009-07-09 | 2011-01-12 | 张树信 | New method for preparing cerebroprotein hydrolysate |
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