Compound brain peptide pharmaceutical composition and preparation method thereof

Abstract

According to the compound brain peptide pharmaceutical composition and the preparation method thereof provided by the invention, the compound brain peptide pharmaceutical composition is prepared by the working procedures of increasing freeze-thaw times, controlling freeze-thaw temperature, adding solid matters, adjusting the addition amount and enzymolysis time of trypsin, optimizing process alkaline impure protein and the like; the content of the polypeptide component in the active component rabbit muscle extract in the compound brain peptide pharmaceutical composition is high, uniform and stable, the content of the impurity component histamine is remarkably reduced, and the inherent quality stability of the product is further improved. The optimized and improved preparation method disclosed by the invention has the advantages of simplicity and convenience in operation, stability and reliability, is already used for technological production of the product, and has a good market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide extraction, purification and separation, and particularly relates to a compound brain peptide drug composition and its preparation method. Background Technology

[0002] The compound pharmaceutical composition described in this invention refers to a compound brain peptide ganglioside injection, which is a sterile aqueous solution prepared by mixing extracts from healthy rabbit muscle and pig brain. Its main active ingredients include polypeptides, various gangliosides, and hypoxanthine. Each 1 ml of the solution contains 3.2 mg of polypeptides, 0.24 mg of monosialotetrahexosylganglioside (GM1), and 0.125 mg of hypoxanthine. Its clinical indications are mainly for the treatment of stroke, Alzheimer's disease, traumatic brain injury, spinal cord injury, and traumatic peripheral nerve injury, as well as for the treatment of functional impairment caused by brain diseases.

[0003] A systematic review of existing technical literature on this product reveals that CN1579543A discloses an injectable neurotrophic peptide, comprising: salivary ganglioside GM1 and polypeptide substances. The preparation process of its rabbit muscle extract is as follows: Fresh rabbit muscle is taken, minced, and the minced tissue is mixed with cold water for injection at a weight ratio of 1–3:1, homogenized, and frozen at -40℃ to -10℃, then thawed, repeated 3–8 times. The thawed homogenate is heated to 50℃–100℃, cooled to room temperature, centrifuged, and the supernatant is collected and filtered. The filtrate is filtered through a membrane with a molecular weight of 3000–10000, and the filtrate is collected. Patent document CN101041060A describes a method for preparing a cerebrolysin solution as follows: 100 kg of rabbit brain is melted, meningeal vessels are removed, and impurities are eliminated; the brain homogenate is processed, 10 times the volume of ice-cold acetone is added, stirred, centrifuged, and the precipitate is collected; the precipitate is washed three times with acetone and dried at 80°C to obtain acetone powder; the acetone powder is added to a 2:1 mixture of 10 times the volume of chloroform and methanol, stirred at room temperature, centrifuged, and the supernatant is collected. The mixture is extracted more than three times, the supernatants are combined, extracted, evaporated, and concentrated. The mixture is then subjected to Folch partitioning in a 5% sodium chloride-methanol mixture (1:1, V / V), the upper layer is collected, and the lower layer is partitioned more than three times. The upper layers are combined, evaporated, concentrated to remove methanol, and ultrafiltered. CN113332414A discloses a method for preparing a rabbit muscle extract with the following steps:

[0004] (1) Take rabbit muscle, remove adipose tissue, wash it, add the minced muscle to 10L of water for injection, mix it evenly, homogenize it, place the resulting muscle homogenate at a temperature not lower than -10℃ for 7 days, then place it at 8℃ for 36 hours to thaw. Repeat the freezing and thawing process twice, then heat the resulting rabbit muscle homogenate to 60℃ and keep it warm for 20 minutes. (1) After cooling to room temperature, centrifuge at 4000 rpm for 20 minutes to obtain the first filtrate; (2) Adjust the pH of the first filtrate to 7.5, add 0.2% trypsin, incubate at 37℃ for 2 hours, adjust the pH of the rabbit muscle homogenate to 3.8, heat to 80℃, keep for 20 minutes, cool to room temperature, centrifuge at 4000 rpm for 20 minutes to obtain the second filtrate; (3) Adjust the pH of the second filtrate to 6.8, place it below -20℃, freeze for 6 hours, place the frozen liquid at 10℃, thaw for 4 hours, centrifuge at 4000 rpm for 20 minutes to obtain the third filtrate; (4) Combine the three filtrates, ultrafilter once with a molecular weight cutoff of 8000, concentrate the ultrafiltrate under reduced pressure, concentrate at 60℃ and 0.05 MPa for 2 hours to 900 ml to obtain the final filtrate. CN114272268A discloses a pharmaceutical composition containing sodium monosialotetrahexosylganglioside, the preparation of which includes the following steps: (1) mincing 10 kg of pretreated rabbit muscle, mixing it with water for injection at a weight ratio of 1:2, homogenizing it, freezing the resulting muscle homogenate below -10℃ for 6 days, then thawing it at 9℃ for 40 hours, then heating the thawed rabbit muscle homogenate to 75℃ and keeping it warm for 20 minutes, then cooling it to room temperature and centrifuging it to obtain a primary filtrate; (2) adjusting the pH of the primary filtrate. 2.5, after heating to 37℃, add 0.1% pepsin, keep warm at 37℃ for 2 hours, adjust the pH of the solution to 8, and maintain at 75℃ for 20 minutes. Cool down to 40℃, add 0.1% trypsin, keep warm at 40℃ for 2 hours, adjust the pH to 4, heat to 80℃ for 20 minutes, centrifuge to obtain the secondary filtrate; (3) adjust the pH of the secondary filtrate to 8, freeze and thaw it, centrifuge to obtain the tertiary filtrate; (4) combine the three filtrates, ultrafilter three times with an ultrafiltration membrane with a molecular weight cutoff of 5000-10000, and concentrate the obtained ultrafiltrate under vacuum to obtain the solution; (5) take 4ml of 20-30℃ water for injection, add 0.24g of sodium monosialotetrahexosylganglioside, stir until dissolved, adjust the pH to 7.0-7.5, and prepare a solution with a concentration of 60mg / ml.

[0005] The existing technology has several shortcomings: the preparation process of rabbit muscle extract involves complex steps and parameters, numerous processing steps, and a large amount of waste residue (solid matter). This not only generates hazardous substances, affecting the product and the environment, but also requires additional costs for disposal. Furthermore, the use of organic solvents such as acetone, chloroform, and methanol poses significant quality risks during production. Specifically, the thawing temperature in the above preparation process is not clearly defined, which cannot effectively guarantee the intrinsic quality of the extract's active pharmaceutical ingredients. This is especially true for traditional Chinese medicine injections containing rabbit muscle extract, where strict quality control is required in large-scale production and clinical practice to ensure the efficacy and safety of the medication for patients. Therefore, this project, "Conducting Research on Optimization and Improvement of the Preparation Process of Rabbit Muscle Extract," aiming to obtain an invention solution with simplified process, high content of effective substances, low impurity content, and stable and uniform quality, is particularly urgent. Summary of the Invention

[0006] This invention provides a compound brain peptide pharmaceutical composition and its preparation method. The active ingredient, rabbit muscle extract, in this compound brain peptide pharmaceutical composition exhibits high, uniform, and stable polypeptide content. Furthermore, by recycling solids, the content of components that cause side effects is significantly reduced / avoided, further enhancing the intrinsic quality stability of the product. The optimized preparation method of this invention is simple to operate, stable, and reliable, and has already been used in the industrial production of this product, demonstrating promising market application prospects.

[0007] One of the objectives of this invention is to improve the content of active peptides in rabbit muscle extract and enhance batch-to-batch consistency. This invention addresses the issues of fluctuations in active ingredient control and insufficient extraction efficiency caused by various steps in the original process by increasing the number of freeze-thaw cycles, controlling freeze-thaw temperatures, adding solids, adjusting the amount of trypsin added and the enzymatic hydrolysis time, and optimizing the process to remove alkaline impurities. Ultimately, this results in a higher content of active substances in rabbit muscle extract and a high degree of consistency in quality both within and between batches.

[0008] The second objective of this invention is to precisely locate histamine, a key impurity component affecting the abnormality of hypotensive substances, through repeated freeze-thaw tests and by controlling the freeze-thaw temperature. Furthermore, by optimizing the production process, the metabolic rate of histamine-producing bacteria is slowed down, effectively controlling histamine production. This ensures that the histamine limit (≤0.04 μg / ml) of the key impurity component affecting the quality and safety of rabbit muscle extract is met, further guaranteeing the intrinsic quality of the rabbit muscle extract.

[0009] The closest prior art solution to this invention (CN1824292A) is as follows:

[0010] Preparation of rabbit muscle extract:

[0011] Take 10 kg of fresh rabbit muscle, add 10 L of water to homogenize, freeze the homogenate, thaw it, heat it to 75-80℃ and keep it warm for 20 minutes, cool it down and centrifuge it at 4000 rpm for 20 minutes. Retain the supernatant and filter it to obtain the filtrate. Add trypsin to the filtrate and incubate it at 0.2% concentration for hydrolysis at pH 7.8-8.2 for 2 hours at 37-42℃. After enzymatic hydrolysis, adjust the pH of the hydrolysate to 3.8-4.2, heat it to 80-85℃ and keep it warm for 20 minutes. After cooling it down, centrifuge it at 4000 rpm for 20 minutes. Retain the supernatant and filter it to obtain the filtrate.

[0012] Adjust the pH of the filtrate to 6.8-7.2, heat to 80-85℃ and maintain for 20 minutes. After cooling, centrifuge at 4000 rpm for 20 minutes. Retain the supernatant and filter it. Use an 8000-molecular-weight cutoff ultrafiltration membrane to ultrafilter the filtrate. The ultrafiltrate is the muscle extract. Concentrate the filtrate under reduced pressure to 800 ml.

[0013] Based on long-term production practice and research, the preparation process of the above-mentioned existing technology has been found to have the following technical defects, as detailed below.

[0014] ① Existing technology only describes "freezing after homogenization". "One freezing" alone cannot meet the process requirements. Repeated freeze-thaw cycles are beneficial for cell disruption and release of effective substances.

[0015] ② The existing technology describes "retaining the supernatant, filtering the supernatant, and then enzymatically hydrolyzing the filtrate". At this time, a large amount of protein exists in the centrifuge residue, which is wasteful and will cause environmental pollution and increase the additional cost of disposal.

[0016] ③ The existing enzymatic hydrolysis technology, which involves "adding 0.2% trypsin and hydrolyzing for 2 hours", cannot guarantee the hydrolysis effect and has problems such as unstable hydrolysis speed, and cannot guarantee that the protein is fully hydrolyzed.

[0017] ④ The existing technology involves heating to 80-85℃ after enzymatic hydrolysis, but the heating temperature is insufficient to ensure that the trypsin used for hydrolysis is completely inactivated.

[0018] ⑤ In addition to the initial centrifugation before enzymatic hydrolysis, the existing technology has a "two-stage centrifugation process". The purpose of these two centrifugation processes is only to remove impurities, and the operation of large-capacity centrifugation is cumbersome.

[0019] ⑥ Existing technology adjusts the pH of the filtrate to 6.8-7.2 and heats it to 80-85℃. However, the target pH and temperature are not conducive to the removal of alkaline impurities and improve the purity of the product.

[0020] ⑦ The existing technology of "direct ultrafiltration after centrifugation and concentration under reduced pressure to obtain the extract" is too simple. It does not make use of the further removal of impurities in the extract, and the target pH value is different from the pH value of human blood, which does not help to improve the patient's medication safety and clinical compliance.

[0021] ⑧. The existing technology does not specify the thawing temperature after freezing, which cannot effectively guarantee the quality of the extract. The testing items for the depressant substances in the extract directly depend on the thawing temperature and effect. Once the temperature exceeds 25℃, it is very easy to cause a large amount of histamine to be produced, which will directly affect the product quality.

[0022] The specific solutions of this invention patent application include the following:

[0023] A compound brain peptide pharmaceutical composition, comprising the following active ingredients: polypeptides, gangliosides, and hypoxanthine, characterized in that the preparation method of the rabbit muscle extract includes the following steps:

[0024] (1) Processing of rabbit muscle

[0025] Take frozen white rabbit carcasses, thaw them in a water bath, remove the muscles required for production, mince the rabbit muscle tissue with a meat grinder, add water in a 1:1 ratio to form a homogenate, freeze the homogenate, and freeze and thaw repeatedly, with the thawing temperature below 25℃.

[0026] (2) After melting, heat to 70-85℃ and keep warm for 15-25 minutes. After cooling, centrifuge at 4000 rpm for 20 minutes. Retain the supernatant and add the centrifuged solids to the supernatant. Add trypsin to the supernatant and incubate for hydrolysis. The trypsin concentration is 0.2-0.4%, and the pH is 7.6-8.2. Incubate at 35-40℃ for 2.5-3.5 hours. Adjust the pH of the hydrolysate to 3.8-4.2, heat to 85-95℃, and incubate for 20 minutes. Remove the residue from the solution, clarify, and filter to obtain the filtrate.

[0027] (3) Take the filtrate from step (2), adjust the pH value to 8.3-8.7, heat to 85-95℃, keep warm for 15-25 minutes, cool to room temperature, clarify and filter the solution to obtain the filtered solution.

[0028] (4) In step (3), the pH value of the drug solution is adjusted, heated, kept warm, and cooled to room temperature. The drug solution is filtered and frozen in a -20℃ cold storage. After the filtrate is thawed, it is ultrafiltered twice using an ultrafiltration membrane. The ultrafiltrate is rabbit muscle extract.

[0029] Preferably, the preparation method includes the following steps: in step (1) the number of freeze-thaw cycles in the treatment of rabbit muscle is 2 to 4.

[0030] Preferably, the preparation method includes the following steps: in step (1) the freeze-thaw cycle in the treatment of rabbit muscle is 3 times.

[0031] Preferably, the preparation method includes the following steps: in step (1) of the rabbit muscle treatment, the freeze-thaw time is 36 to 60 hours each time, and the freeze-thaw temperature is 16 to 22°C.

[0032] Preferably, the preparation method includes the following steps: in step (1) the treatment of rabbit muscle, the freeze-thaw time is 52 hours and the freeze-thaw temperature is 18°C.

[0033] Preferably, the method for preparing the rabbit muscle extract includes the following steps: the amount of solids added after centrifugation in step (2) is 2.1%-2.6%.

[0034] Preferably, the preparation method includes the following steps: in step (2), the trypsin concentration is 0.3% and the enzymatic hydrolysis time is 3 hours.

[0035] Preferably, the preparation method includes the following steps: in step (3), the pH value is adjusted to 8.4-8.5, heated to 90°C, and kept warm for 20 minutes.

[0036] Preferably, the preparation method includes the following steps: in step (4), the pH value is adjusted to 7.0-7.4, heated to 85-95°C, and kept warm for 20 minutes.

[0037] Preferably, the preparation method includes the following steps: the thawing temperature of the filtrate in step (4) is below 25°C, and the ultrafiltration membrane has a molecular weight cutoff of 8000.

[0038] Preferably, the thawing temperature of the filtrate in step (4) is 12-15°C.

[0039] Based on rabbit muscle technology, this invention was discovered by chance during extensive scientific research. The key steps and parameters of the process in this invention, compared with the original preparation process (patented technology solution CN1824292A), significantly improved the effective components of polypeptides and hypoxanthine in rabbit muscle extract, while ensuring the significant removal of impurity histamine components, thereby more effectively ensuring the stability of the product's intrinsic quality.

[0040] The testing of antihypertensive substances in the rabbit muscle extract of this invention directly depends on the thawing temperature and effect. Once the temperature exceeds 25°C, it is very easy to cause a large amount of histamine to be produced, which will directly affect the product quality.

[0041] 1. Detailed explanation of the increase in effective ingredients before and after optimization

[0042] The original preparation process yielded a final peptide content of 5 mg / ml and a hypoxanthine content of 0.2 mg / ml, which does not meet national quality standards. Therefore, we need to optimize the entire process to increase the content of the two active ingredients. However, the two active ingredients have different metabolic degradation pathways, requiring individual analysis and optimization.

[0043] 1.1 Optimization to increase xanthine content – ​​Adjusting the freeze-thaw cycle of the homogenate to three times.

[0044] The pathway by which rabbit muscle metabolizes hypoxanthine after slaughter is illustrated in the flowchart attached to the instruction manual. Figure 1 Key steps:

[0045] ① Starting point (slaughter and ATP depletion): After a rabbit is slaughtered, blood circulation stops, and muscle cells become hypoxic. Mitochondrial aerobic respiration ceases, preventing the synthesis of ATP (adenosine triphosphate), while the cell's own metabolism continues to consume ATP, causing ATP levels to drop rapidly;

[0046] ② Degradation pathway of ATP: ATP is degraded along a specific pathway: ATP → ADP (adenosine diphosphate) → AMP (adenosine monophosphate);

[0047] ③ IMP (Inosine Monophosphate) Formation: This is a crucial step. In living organisms, AMP can be regenerated into ATP. However, under post-mortem conditions, AMP deaminase is activated, removing an amino group from AMP and directly converting it into IMP. IMP is one of the main sources of umami flavor in meat.

[0048] ④ Formation of hypoxanthine: IMP, under the action of enzymes such as acid phosphatase in muscle, loses its ribose-phosphate (i.e., loses ribose) to form hypoxanthine. This process occurs gradually over a period of time after slaughter.

[0049] As can be seen from the flowchart above, hypoxanthine synthesis is mainly a metabolic process in rabbit muscle. However, without the interference of the manufacturing process, the amount metabolized spontaneously is negligible. Therefore, a freeze-thaw cycle is added to the production process. The purpose is to cause ice crystals of varying sizes to form inside and outside the cells of the rabbit muscle during freezing. These sharp ice crystals pierce delicate organelle structures such as cell membranes, mitochondrial membranes, and lysosomal membranes. After thawing, these damaged organelles release their internal enzymes (including AMP deaminase, acid phosphatase, and cathepsins), which are normally separated in intact cells. At the same time, substrates (such as ATP, AMP, and IMP) that were originally separated by membrane structures are also released in large quantities. This large-scale, unimpeded contact between "enzymes" and "substrates" causes a sharp increase in the rate of all biochemical reactions in the ATP degradation chain.

[0050] Theoretically, rabbit muscle extract can increase the metabolic rate of hypoxanthine during freeze-thaw cycles. However, the original process only involved one freeze-thaw cycle, which was insufficient to ensure the effective release of all cell structures during the limited freezing and thawing. Therefore, the improved process added two more freeze-thaw cycles to ensure the effective destruction of all cell structures and stabilize batch-to-batch content.

[0051] Table 1 Comparison of hypoxanthine content before and after increasing the number of freeze-thaw cycles

[0052] Before process optimization After process optimization (increasing the number of freeze-thaw cycles) Hypoxanthine content without increasing freeze-thaw cycles Increasing the number of freeze-thaw cycles increases the hypoxanthine content. Hypoxanthine: 0.23 mg / ml Hypoxanthine: 0.42 mg / ml

[0053] 1.2 Optimization to increase peptide content—adding solid substrate before enzymatic hydrolysis

[0054] Rabbit muscle extract is the main raw material of compound brain peptide ganglioside extract, whose active ingredients are polypeptides and hypoxanthine. The national standard stipulates that the polypeptide content in compound brain peptide ganglioside extract should be 2.72-3.68 mg / ml (3.2±15%), and the hypoxanthine content should be 0.20-0.28 mg / ml (0.24±16%). Specifically, each 1 ml of rabbit muscle extract should contain no less than 6.4 mg of polypeptide and no less than 0.25 mg of hypoxanthine. From the above content standards, it can be seen that there is a certain ratio between polypeptides and hypoxanthine, with the optimal ratio being 25.6. After optimizing the freeze-thaw process, we obtained a higher inosine content, reaching 0.42 mg / ml. However, the ratio of peptides to inosines was unbalanced at this point, making it difficult to meet the quality standards of the compound brain peptide ganglioside extract. Therefore, we started with the centrifugation process before enzymatic hydrolysis. After centrifugation, we added a portion of the solids (centrifugation residue) to the enzymatic hydrolysis solution to increase the substrate and improve the peptide content.

[0055] 1.3 Optimization to increase peptide content – ​​Adjusting the amount of trypsin added and the enzymatic hydrolysis time

[0056] The core value of trypsin in rabbit muscle extraction lies in its "precise disassembly of tissue structure and gentle release of target components." Rabbit muscle is mainly composed of myofibrillar proteins (such as myosin and actin), sarcoplasmic proteins (enzymes involved in metabolism), and connective tissue proteins (such as collagen). Trypsin scans all these muscle proteins, searching for lysine (Lys) and arginine (Arg) residues. The enzyme cleaves the peptide chain after the C-terminus of each Lys and Arg residue. This means that a complete protein molecule is "cut" into many segments. After a period of reaction, the originally complex and large-molecule muscle protein network is completely destroyed, generating a mixture of peptides of varying sizes. For details on the process of adding trypsin to rabbit muscle extract to break down it into peptides, please refer to the instruction manual. Figure 2 .

[0057] From the above mechanism of action, it can be seen that trypsin is the main substance determining the polypeptide content in rabbit muscle extract. Therefore, the substrate, the amount of enzyme added, and the hydrolysis time are all important factors determining whether trypsin can efficiently catalyze the target compound. In the original process, the enzyme addition was 0.2% and the hydrolysis time was 2 hours. However, after the previous optimization of the polypeptide, solids (the solids being mainly denatured muscle fibrous protein, along with small amounts of connective tissue collagen, elastin, residual fat particles, and minerals) were added before hydrolysis. With the increased substrate and unchanged enzyme amount, it was difficult to obtain the target content. Therefore, the enzyme addition was adjusted to 0.3%, and the hydrolysis time was correspondingly extended to 3 hours to facilitate sufficient hydrolysis reaction with increased substrate.

[0058] Table 2 Comparison of peptide content in rabbit muscle extracts after addition of solids and adjustment of enzymatic hydrolysis.

[0059]

[0060] The present invention has surprisingly discovered that, based on the adjustment of the enzyme dosage to 0.3%, the addition of solids not only increases the polypeptide content in the product, but also stabilizes the enzyme activity, making the enzymatic hydrolysis reaction more controllable and thus avoiding the generation of impurities.

[0061] To further demonstrate the inventiveness of the preparation process of this invention, the technical improvements of this invention compared to the closest prior art are summarized as follows:

[0062] Innovation 1: The homogenate is subjected to three freeze-thaw cycles, which can maximize the dissolution and release of effective substances in cells and increase and stabilize the content of hypoxanthine.

[0063] Innovation Point Two: The thawing temperature for each step is clearly defined to be below 25℃, ensuring that the thawing process of the extract avoids the temperature most likely to cause the formation of histidine decarboxylase, reducing the production of histamine substances, and ensuring the stability of the extract's hypotensive substances.

[0064] Innovation Point 3: The "supernatant filtration" process is eliminated. A certain amount of the solids after centrifugation is weighed and added to the supernatant to increase the substrate for enzymatic hydrolysis and ensure the stability of the extract content.

[0065] Innovation Point 4: The concentration of added trypsin is adjusted to 0.3%, and the enzymatic hydrolysis is carried out for 3 hours. Sufficient enzyme quantity and hydrolysis time can ensure complete hydrolysis of proteins.

[0066] Innovation Point 5: The heating temperature of the enzymatically hydrolyzed drug solution is increased from 80-85℃ to 85-95℃. Increasing the temperature can fully ensure that the trypsin used for enzymatic hydrolysis is completely inactivated, avoiding the impact of residual enzymes on subsequent production processes.

[0067] Innovation point six: The original technology has been optimized by replacing the "two centrifugation processes" (in addition to the initial enzymatic hydrolysis) with a "deep filtration process", which greatly shortens the process time, improves work efficiency, and the final result is the same as centrifugation.

[0068] Table 3 compares the time required to optimize the two centrifugation processes into a deep filtration process.

[0069]

[0070] Innovation point seven: The original technology required adjusting the pH of the filtrate to 6.8-7.2 and heating it to 80-85℃. Instead, the pH of the filtrate is adjusted to 8.3-8.7 and heated to 85-95℃. The higher pH and temperature are more conducive to the removal of alkaline impurities and can improve the purity of the product.

[0071] Table 4 Comparison of changes in alkaline contaminant levels during optimized process

[0072]

[0073] Innovation Point 8: The original technology, which involved "direct ultrafiltration after centrifugation followed by concentration under reduced pressure to obtain the extract," has been optimized to "adjusting the pH of the drug solution to 7.0–7.4, heating to 85–95°C, holding at that temperature for 20 minutes, cooling to room temperature, filtering the solution, and freezing it in a -20°C cold storage. After thawing the filtrate below 25°C, it is then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 8000." By further adjusting the pH value to match that of human blood, the safety and comfort of patients taking the medication can be improved.

[0074] Innovation Point Nine: Select frozen white rabbits of the same species and with the same number of days of feeding. After passing inspection, remove the rabbit meat required for production and store it in a cold storage at -20℃. The source of raw materials is stable, which can provide a guarantee for subsequent production.

[0075] In summary, the technical solution of this invention can greatly improve the effective components of polypeptides and hypoxanthine in rabbit muscle extract, while ensuring the removal of histamine impurities. It can also be used in the production process of compound brain peptide injection and for internal control component detection, thereby more effectively ensuring the intrinsic quality of the product and further improving its uniformity and stability.

[0076] The beneficial effects of the technical solution of this invention are as follows:

[0077] (1) Through extensive experimentation and trial, the optimal preparation process parameters were finally found: by optimizing the production process of the extract, the yield of the extract was improved and the intrinsic quality of the product was stabilized. The specific advantages are as follows:

[0078] ① The content of peptides and hypoxanthine in the extract is stabilized to achieve a stable ratio. The quality standard of this product specifies that the content of peptides is not less than 6.4 mg / ml and the content of hypoxanthine is not less than 0.25 mg / ml, with a ratio of 25.6:1. By optimizing the process, the protein concentration during the extraction process can be stabilized, so that the content of peptides and hypoxanthine in the final extracted product reaches the standard ratio, ensuring the usability of the extract.

[0079] ② Improved extract yield: Through repeated freeze-thaw cycles and optimization of process temperature, added solids, pH value, enzyme amount, and enzymatic hydrolysis time, the present invention maximizes the dissolution of effective components from rabbit muscle. Subsequent purification processes further improve the yield of effective components in the extract.

[0080] The specific technical effects are as follows: Following the original preparation process (CN1824292A), the average peptide content was 5.8 mg / ml, and the average hypoxanthine content was 0.23 mg / ml. After optimizing and improving the preparation method in Example 1, the average peptide content was 10.8 mg / ml, and the average hypoxanthine content was 0.42 mg / ml. Therefore, the optimized preparation process significantly improved the content of the extracts (peptides and hypoxanthine).

[0081] ③ To improve the stability of the intrinsic quality of rabbit muscle extract, this invention selects frozen white-feathered rabbits that meet quality standards as stable raw materials, eliminating batch-to-batch differences at the source. Furthermore, by optimizing each stage of the process, the differences in control at each stage caused by the original simplified process are eliminated, ultimately achieving the goal of ensuring batch-to-batch and batch-to-batch consistency of the extract.

[0082] The specific technical effects are as follows: According to the original preparation process (CN1824292A), the peptide content fluctuated within a range of 5.0–7.1 mg / ml, and the hypoxanthine content fluctuated within a range of 0.20–0.26 mg / ml. After process optimization and improvement of the preparation method in Example 1, the peptide content fluctuated within a range of 10.3–11.2 mg / ml, and the hypoxanthine content fluctuated within a range of 0.40–0.43 mg / ml. It can be seen that, compared with the original preparation process, the improved preparation process has higher consistency in the content of each substance between batches, making the quality of the rabbit muscle extract of the present invention more uniform and stable.

[0083] ④ Ensure that the antihypertensive substances in rabbit muscle extract are qualified. The production of antihypertensive substances is mainly related to histamine. When certain bacteria grow, they produce a metabolite of histidine decarboxylase. This histidine decarboxylase reacts with free histidine to produce histamine, causing the antihypertensive substances to be unqualified.

[0084] This invention reduces the reaction of histamine-related impurities by optimizing process parameters such as the number of freeze-thaw cycles and freeze-thaw temperatures. Through adjustments and optimizations of different process parameters and procedures, the probability of the rabbit muscle extract failing the pressure test is significantly reduced. Extensive experimental research has shown that, especially when the histamine content is ≤0.04 μg / ml, the pass rate can reach 100%. Furthermore, the improved preparation process in Example 1 of this invention controls the histamine content to ≤0.01 μg / ml, achieving a 100% pass rate, thus effectively ensuring the intrinsic quality of the extract.

[0085] ⑤ Compared with the original production process, the present invention further optimizes the two centrifugation processes into deep filtration, reducing the time of this process from 120 minutes to 60 minutes, thereby further improving production efficiency. Attached Figure Description

[0086] Figure 1 The pathway by which rabbit muscle metabolizes hypoxanthine after slaughter;

[0087] Figure 2 The diagram shows the process of rabbit muscle extraction being broken down into polypeptides after the addition of trypsin. Detailed Implementation

[0088] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0089] Example 1

[0090] Take 10kg of frozen white rabbit, thaw in a water bath, remove the muscle required for production, mince the rabbit muscle tissue with a meat grinder, add water in a 1:1 ratio to homogenize, freeze after homogenization, freeze and thaw 3 times, freeze and thaw for 48 hours, freeze and thaw at 18℃.

[0091] (2) Take the freeze-thawed liquid from step (1), thaw it, heat it to 70°C, keep it at that temperature for 25 minutes, cool it down, and centrifuge it at 4000 rpm for 20 minutes. Keep the supernatant, and add the solids after centrifugation to the supernatant. Add trypsin to the supernatant and incubate it for hydrolysis. The trypsin concentration is 0.2% and the pH is 7.6. Incubate the hydrolysis at 35°C for 2.5 hours. Adjust the pH of the hydrolysate to 4.2, heat it to 95°C, and keep it at that temperature for 20 minutes. Remove the residue from the solution, clarify it, and filter it to obtain the filtrate.

[0092] (3) Take the filtrate from step (2), adjust the pH to 8.5, heat to 90°C, keep warm for 20 minutes, cool to room temperature, clarify and filter the solution to obtain the filtered solution.

[0093] (4) In step (3), the pH of the drug solution is adjusted to 7.2, heated to 90°C, kept warm for 20 minutes, cooled to room temperature, filtered, and stored in a -20°C cold storage. After the filtrate is thawed at 14°C, it is subjected to two ultrafiltrations with a molecular weight cutoff of 8000 using an ultrafiltration membrane. The ultrafiltrate is rabbit muscle extract.

[0094] Example 2

[0095] Take 10kg of frozen white rabbit, thaw in a water bath, remove the muscle required for production, mince the rabbit muscle tissue with a meat grinder, add water in a 1:1 ratio to form a homogenate, freeze after homogenization, freeze and thaw twice, freeze and thaw twice, freeze and thaw for 36 hours, freeze and thaw at 20℃.

[0096] (2) Take the freeze-thawed liquid from step (1), thaw it, heat it to 85°C, keep it at that temperature for 15 minutes, cool it down, and then centrifuge it at 4200 rpm for 20 minutes. Keep the supernatant and add the solids from the centrifugation to the supernatant. Add trypsin to the supernatant and incubate it for hydrolysis. The trypsin concentration is 0.2% and the pH is 7.6. Incubate the hydrolysis at 35°C for 3.5 hours. Adjust the pH of the hydrolysate to 3.8, heat it to 85°C, and keep it at that temperature for 20 minutes. Remove the residue from the solution, clarify it, and filter it to obtain the filtrate.

[0097] (3) Take the filtrate from step (2), adjust the pH to 8.3, heat to 95°C, keep warm for 15 minutes, cool to room temperature, clarify and filter the solution to obtain the filtered solution.

[0098] (4) In step (3), the pH of the drug solution is adjusted to 7.2, heated to 90°C, kept warm for 20 minutes, cooled to room temperature, filtered, and stored in a -20°C cold storage. After the filtrate is thawed, it is subjected to two ultrafiltrations with a molecular weight cutoff of 8000 using an ultrafiltration membrane. The ultrafiltrate is rabbit muscle extract.

[0099] Example 3

[0100] Take 10kg of frozen white rabbit, thaw in a water bath, remove the muscle required for production, mince the rabbit muscle tissue with a meat grinder, add water in a 1:1 ratio to homogenize, freeze after homogenization, freeze and thaw 3 times, freeze and thaw for 48 hours, freeze and thaw at 18℃.

[0101] (2) Take the freeze-thawed liquid from step (1), thaw it, heat it to 80°C, keep it at that temperature for 20 minutes, cool it down, and then centrifuge it at 4000 rpm for 20 minutes. Keep the supernatant and add the solids from centrifugation to the supernatant. Add trypsin to the supernatant and incubate it for hydrolysis. The trypsin concentration is 0.3% and the pH is 8.0. Incubate the hydrolysis at 38°C for 3 hours. Adjust the pH of the hydrolysate to 4.0, heat it to 90°C, and keep it at that temperature for 20 minutes. Remove the residue from the solution, clarify it, and filter it to obtain the filtrate.

[0102] (3) Take the filtrate from step (2), adjust the pH to 8.5, heat to 90°C, keep warm for 20 minutes, cool to room temperature, clarify and filter the solution to obtain the filtered solution.

[0103] (4) In step (3), the pH of the drug solution is adjusted to 7.2, heated to 90°C, kept warm for 20 minutes, cooled to room temperature, filtered, and stored in a -20°C cold storage. After the filtrate is thawed at 12°C, it is subjected to two ultrafiltrations with a molecular weight cutoff of 8000 using an ultrafiltration membrane. The ultrafiltrate is rabbit muscle extract.

[0104] Example 4

[0105] Take 10kg of frozen white rabbit, thaw in a water bath, remove the muscle required for production, mince the rabbit muscle tissue with a meat grinder, add water in a 1:1 ratio to form a homogenate, freeze after homogenization, freeze and thaw 4 times, freeze and thaw for 60 hours, freeze and thaw at 22℃.

[0106] (2) Take the freeze-thawed liquid from step (1), thaw it, heat it to 72°C, keep it at that temperature for 18 minutes, cool it down, and then centrifuge it at 4000 rpm for 20 minutes. Keep the supernatant and add the solids from the centrifugation to the supernatant. Add trypsin to the supernatant and incubate it for hydrolysis. The trypsin concentration is 0.3% and the pH is 8.0. Incubate the hydrolysis at 38°C for 3 hours. Adjust the pH of the hydrolysate to 4.0, heat it to 90°C, and keep it at that temperature for 20 minutes. Remove the residue from the solution, clarify it, and filter it to obtain the filtrate.

[0107] (3) Take the filtrate from step (2), adjust the pH to 8.7, heat to 85°C, keep warm for 25 minutes, cool to room temperature, clarify and filter the solution to obtain the filtered solution.

[0108] (4) In step (3), the pH of the drug solution is adjusted to 7.2, heated to 90°C, kept warm for 20 minutes, cooled to room temperature, filtered, and stored in a -20°C cold storage. After the filtrate is thawed at 14°C, it is subjected to two ultrafiltrations with a molecular weight cutoff of 8000 using an ultrafiltration membrane. The ultrafiltrate is rabbit muscle extract.

[0109] Example 5

[0110] Take 10kg of frozen white rabbit, thaw in a water bath, remove the muscle required for production, mince the rabbit muscle tissue with a meat grinder, add water in a 1:1 ratio to homogenize, freeze after homogenization, freeze and thaw 4 times, freeze and thaw for 48 hours, freeze and thaw at 19℃.

[0111] (2) Take the freeze-thawed liquid from step (1), thaw it, heat it to 75°C, keep it at that temperature for 20 minutes, cool it down, and then centrifuge it at 4000 rpm for 20 minutes. Keep the supernatant and add the solids from the centrifugation to the supernatant. Add trypsin to the supernatant and incubate it for hydrolysis. The trypsin concentration is 0.3% and the pH is 7.8. Incubate the hydrolysis at 36°C for 3.5 hours. Adjust the pH of the hydrolysate to 4.1, heat it to 88°C, and keep it at that temperature for 20 minutes. Remove the residue from the solution, clarify it, and filter it to obtain the filtrate.

[0112] (3) Take the filtrate from step (2), adjust the pH to 8.2, heat to 92°C, keep warm for 22 minutes, cool to room temperature, clarify and filter the solution to obtain the filtered solution;

[0113] (4) In step (3), the pH of the drug solution is adjusted to 7.0, heated to 85°C, kept warm for 20 minutes, cooled to room temperature, filtered, and stored in a -20°C cold storage. After the filtrate is thawed at 12°C, it is subjected to two ultrafiltrations with a molecular weight cutoff of 8000 using an ultrafiltration membrane. The ultrafiltrate is rabbit muscle extract.

[0114] Example 6

[0115] Take 10kg of frozen white rabbit, thaw in a water bath, remove the muscle required for production, mince the rabbit muscle tissue with a meat grinder, add water in a 1:1 ratio to form a homogenate, freeze after homogenization, freeze and thaw twice, freeze and thaw twice, freeze and thaw twice, freeze and thaw twice, freeze and thaw twice, freeze and thaw at 18℃.

[0116] (2) Take the freeze-thawed liquid from step (1), thaw it, heat it to 80°C, keep it at that temperature for 20 minutes, cool it down, and then centrifuge it at 3900 rpm for 20 minutes. Keep the supernatant and add the solids from centrifugation to the supernatant. Add trypsin to the supernatant and incubate it for hydrolysis. The trypsin concentration is 0.2% and the pH is 8.1. Incubate the hydrolysis at 37°C for 3 hours. Adjust the pH of the hydrolysate to 3.9, heat it to 90°C, and keep it at that temperature for 20 minutes. Remove the residue from the solution, clarify it, and filter it to obtain the filtrate.

[0117] (3) Take the filtrate from step (2), adjust the pH to 8.5, heat to 90°C, keep warm for 20 minutes, cool to room temperature, clarify and filter the solution to obtain the filtered solution.

[0118] (4) In step (3), the pH of the drug solution is adjusted to 7.0, heated to 95°C, kept warm for 20 minutes, cooled to room temperature, filtered, and stored in a -20°C cold storage. The thawing temperature of the filtrate is 15°C. Ultrafiltration is performed twice with a molecular weight cutoff of 8000 using an ultrafiltration membrane. The ultrafiltrate is rabbit muscle extract.

[0119] Description of the overall quality improvement effect of the rabbit muscle extract of this invention

[0120] This invention optimizes the process steps and parameters throughout the entire production process by increasing the number of freeze-thaw cycles, controlling the freeze-thaw temperature, adding solids, adjusting the amount of trypsin added and the enzymatic hydrolysis time, and optimizing the process to remove alkaline impurities. The specific experimental exploration and research process is as follows.

[0121] Table 5 Comparison of Optimization Parameters for Rabbit Muscle Extract Process

[0122]

[0123]

[0124] The results of the determination of the effective components of the rabbit muscle extract, peptides and hypoxanthine, prepared according to the closest prior art (CN1824292A) are shown in Table 6.

[0125] Table 6: List of increased rabbit muscle extract content before and after improvement:

[0126]

[0127] As shown in Table 6 above, the average peptide content obtained according to the original preparation process (CN1824292A) was 5.8 mg / ml, and the average hypoxanthine content was 0.23 mg / ml. After process optimization and improvement of the preparation method in Example 1, the average peptide content was 10.8 mg / ml, and the average hypoxanthine content was 0.42 mg / ml. Therefore, the optimized preparation process significantly improved the content of extracts (peptides and hypoxanthine).

[0128] According to the original preparation process (CN1824292A), the peptide content fluctuated within the range of 5.0–7.1 mg / ml, and the hypoxanthine content fluctuated within the range of 0.20–0.26 mg / ml. After process optimization and improvement of the preparation method in Example 1, the peptide content fluctuated within the range of 10.3–11.2 mg / ml, and the hypoxanthine content fluctuated within the range of 0.40–0.43 mg / ml. It can be seen that, compared with the original preparation process, the improved preparation process has higher consistency in the content of each substance between batches, making the quality of the rabbit muscle extract of the present invention more uniform and stable.

[0129] 2. Explanation of the reduction in toxic side effects (histamine) before and after optimization

[0130] The detection of antihypertensive substances is a crucial test item in rabbit muscle extract. Abnormalities in this test can lead to the side effect of decreased blood pressure during use of Compound Brain Peptide Ganglioside Injection, preventing product release. Analysis of this item revealed that histamine is the primary substance causing the failure to meet the antihypertensive substance standard.

[0131] The following is a detailed explanation of the relationship between histamine and the failure of antihypertensive substances to meet standards:

[0132] Rabbit muscle tissue is rich in protein, which is hydrolyzed to produce L-histidine. Intracellular histidine decarboxylase recognizes L-histidine and initiates a decarboxylation reaction, catalyzing the removal of a carboxyl group (-COOH) from the L-histidine molecule to form histamine. However, histamine is a typical source of antihypertensive substances; its mechanism of action involves the strong dilation of small blood vessels (especially arterioles and precapillary sphincters), leading to a decrease in peripheral vascular resistance and an increase in vascular permeability, thereby lowering blood pressure. This process is primarily mediated by the co-mediation of H1 and H2 receptors.

[0133] 1. The process of H1 receptor-mediated vasodilation (endothelial cell dependent):

[0134] Histamine binds to H1 receptors on endothelial cells, activating intracellular phospholipase C (PLC), which promotes the hydrolysis of phosphoinositol to generate inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates the release of Ca2+ from intracellular calcium stores in endothelial cells, activating endothelial nitric oxide synthase (eNOS), which catalyzes the conversion of L-arginine into nitric oxide (NO). NO diffuses to adjacent vascular smooth muscle cells, activating guanylate cyclase (GC), which increases intracellular cGMP levels, inhibits sarcoplasmic reticulum Ca2+ release and promotes Ca2+ efflux, leading to smooth muscle relaxation and vasodilation.

[0135] 2. The process of H2 receptor-mediated vasodilation (acting directly on smooth muscle):

[0136] Histamine binds to H2 receptors in smooth muscle cells, activating adenylate cyclase (AC), which increases intracellular cAMP levels. cAMP inhibits the activity of smooth muscle contraction-related proteins (such as myosin light chain kinase), directly leading to smooth muscle relaxation and vasodilation.

[0137] As can be seen from the above description of the relationship between histamine and the failure of pressure-reducing substances, histidine decarboxylase is the key substance that causes histamine production. In the whole process, histamine mainly comes from microbial metabolism. Therefore, reducing the generation of histamine by controlling the reproduction of microorganisms is the only way to reduce the failure rate of pressure-reducing substances in products.

[0138] (1) Adjust the defrosting temperature

[0139] After pretreatment, rabbit muscle contains Staphylococcus bacteria (such as Staphylococcus epidermidis) and Morganella morganii, which thrive at temperatures between 30°C and 37°C, growing particularly rapidly near 32.2°C. During growth, these bacteria produce histidine decarboxylase metabolites. This histidine decarboxylase reacts with free histidine to produce histamine. The original preparation process specified a thawing temperature of 35°C, which falls within the optimal growth range for Staphylococcus bacteria (such as Staphylococcus epidermidis) and Morganella morganii, frequently resulting in substandard levels of the hypotensive substance in the final extract.

[0140] Through optimization, we adjusted the thawing temperature of the five freeze-thaw cycles of the extract to below 25°C, avoiding the suitable production temperature of Staphylococcus bacteria (such as Staphylococcus epidermidis) and Morganella morganii, thus reducing the formation of histamine.

[0141] During animal experiments, the histamine content, which caused the antihypertensive substance to fail the test, was 1.6 μg / ml. Histamine levels can be quickly determined using an Agilent liquid chromatograph (Agilent HC18 column), allowing for process analysis and adjustments.

[0142] Table 7 shows the comparative experimental results of the reduction of impurity components (histamine content) before and after the optimization of the rabbit muscle extract process.

[0143]

[0144] As shown in Table 7 above, when the thawing temperature is controlled below 25℃, the histamine content shows a significant decreasing trend. After adjusting and optimizing different process parameters, the histamine content in rabbit muscle extract is significantly reduced.

[0145] Table 8. Statistical table of the probability of non-compliance of antihypertensive substances in rabbit muscle extract before and after process optimization.

[0146]

[0147] As can be seen from Table 8, after adjusting and optimizing different process parameters and procedures, the failure rate of the rabbit muscle extract gradually decreased significantly from 20.50%, 20.45%, 12.50%, 9.18%, 1.34%, and 0.98% in the pressure reduction test results of Comparative Examples 1 to 6.

[0148] Extensive experimental research revealed that, especially when the histamine content is ≤0.04 μg / ml, the pass rate can reach 100%. Furthermore, the improved preparation process in Example 1 of this invention controls the histamine content to ≤0.01 μg / ml, achieving a pass rate of 100%, demonstrating that the preparation process of this invention has achieved a high level of control.

[0149] Compared with existing technologies, the preparation process of this invention has the following advantages:

[0150] 1. Overcoming the core pain points of extract quality compliance: Through process optimization, the protein concentration during the extraction process is stabilized, ensuring that the content of peptides and hypoxanthine in the final product meets the standard ratio requirements. At the same time, it ensures that the core components of the extract meet national standards, fully guaranteeing its application feasibility.

[0151] 2. Significantly Improved Extract Yield: Through repeated freeze-thaw experiments and systematic optimization of key parameters such as process temperature, pH value, enzyme dosage, and enzymatic hydrolysis time, the effective components in rabbit muscle were maximized for dissolution. Subsequent purification processes further enhanced the extract yield. As shown in the content statistics table, taking peptides as an example, the original process had an average content of 5.8 mg / ml, which was increased to 10.8 mg / ml after optimization, representing an 86.28% increase in yield.

[0152] 3. Enhance the stability control of extracts: Select frozen white rabbits that meet quality standards at the source to eliminate batch-to-batch differences in raw materials; and then optimize the entire process to solve the fluctuation problems in the control of each process caused by the original simplified process, so as to achieve a high degree of consistency in the quality of extracts within and between batches.

[0153] 4. To ensure the quality of the antihypertensive substances in rabbit muscle extract, we comprehensively analyzed the entire production process, accurately identified the key factor affecting the abnormality of antihypertensive substances—histamine, and optimized the production process to slow down the metabolic rate of bacteria related to histamine production, effectively controlling histamine production, achieving stable antihypertensive substance indicators, and ensuring the intrinsic quality stability of rabbit muscle extract.

[0154] Finally, it should be noted that this invention is not limited to the specific embodiments listed above. These specific embodiments are merely illustrative and normative, and not restrictive. Any changes, equivalent substitutions, adjustments, or partial improvements made by those skilled in the art, based on the guidance or instruction of this patent application, within the scope of this invention, shall fall within the protection scope of this invention.

Claims

1. A compound brain peptide pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition is prepared by mixing rabbit muscle extract and pig brain extract, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: (1) Processing of rabbit muscle Take frozen white rabbit carcasses, thaw them in a water bath, remove the muscle required for production, mince the rabbit muscle tissue with a meat grinder, add water in a 1:1 ratio to form a homogenate, freeze the homogenate, and freeze and thaw repeatedly, with the thawing temperature below 25℃. (2) Take the freeze-thawed liquid from step (1), thaw it, heat it to 70-85℃, keep it at that temperature for 15-25 minutes, cool it down, and centrifuge it at 4000 rpm for 20 minutes. Keep the supernatant, and add the solids after centrifugation to the supernatant. Add trypsin to the supernatant and incubate it for hydrolysis. The trypsin concentration is 0.2-0.4%, the pH is 7.6-8.2, and the hydrolysis is carried out at 35-40℃ for 2.5-3.5 hours. Adjust the pH of the hydrolysate to 3.8-4.2, heat it to 85-95℃, keep it at that temperature for 20 minutes, remove the residue, clarify and filter the solution to obtain the filtrate. (3) Take the filtrate from step (2), adjust the pH value to 8.3-8.7, heat to 85-95℃, keep warm for 15-25 minutes, cool to room temperature, clarify and filter the solution to obtain the filtered solution. (4) In step (3), the pH value of the drug solution is adjusted, heated, kept warm, and cooled to room temperature. The drug solution is filtered and frozen in a -20℃ cold storage. After the filtrate is thawed, it is ultrafiltered twice using an ultrafiltration membrane. The ultrafiltrate is rabbit muscle extract.

2. The compound brain peptide pharmaceutical composition according to claim 1, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: in step (1), the number of freeze-thaw cycles in the treatment of the rabbit muscle is 2 to 4.

3. The compound brain peptide pharmaceutical composition as described in claim 1, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: In step (1), the freeze-thaw time for each step of the rabbit muscle treatment is 36 to 60 hours, and the freeze-thaw temperature is 16 to 22°C.

4. The compound brain peptide pharmaceutical composition according to claim 1, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: In step (1), the freeze-thaw time for each step of the rabbit muscle treatment is 52 hours, and the freeze-thaw temperature is 18°C.

5. The compound brain peptide pharmaceutical composition as described in claim 1, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: the amount of solids added after centrifugation in step (2) is 2.1%-2.6%.

6. The compound brain peptide pharmaceutical composition according to claim 1, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: in step (2), the concentration of trypsin is 0.3% and the enzymatic hydrolysis time is 3 hours.

7. The compound brain peptide pharmaceutical composition according to claim 1, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: in step (3), the pH value is adjusted to 8.4-8.5, heated to 90°C, and kept warm for 20 minutes.

8. The compound brain peptide pharmaceutical composition according to claim 1, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: in step (4), the pH value is adjusted to 7.0-7.4, heated to 85-95℃, and kept warm for 20 minutes.

9. The compound brain peptide pharmaceutical composition according to claim 1, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: in step (4), the thawing temperature of the filtrate is below 25°C, and the ultrafiltration membrane has a molecular weight cutoff of 8000.

10. The compound brain peptide pharmaceutical composition according to claim 9, characterized in that, The method for preparing the rabbit muscle extract includes the following steps: the thawing temperature of the filtrate in step (4) is 12-15℃.

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