A brain polypeptide material with intelligence and brain health benefits and a preparation method thereof

Abstract

This invention provides a brain polypeptide raw material with brain-boosting and intelligence-enhancing properties and its preparation method. The preparation method includes the following steps: S1, pretreatment; S2, first hydrolysis: adjusting the pH of the pre-hydrolysate to 6.5-7.5, adding neutral protease at 3.5-4.5% of bovine brain protein mass, hydrolyzing at 50-60℃ for 1-3 hours, and obtaining the first hydrolysate after enzyme inactivation; S3, second hydrolysis: adjusting the pH of the first hydrolysate to 6.0-7.0, adding papain at 1.0-3.0% of bovine brain protein mass, hydrolyzing at 45-65℃ for 1.5-2.5 hours, and obtaining the second hydrolysate after enzyme inactivation; S4, post-treatment, to obtain the brain polypeptide raw material powder. This brain polypeptide raw material can significantly scavenge DPPH and ABTS free radicals, inhibit acetylcholinesterase activity, repair PC12 nerve cells, inhibit the expression of the pro-apoptotic gene Bax and promote the expression of the anti-apoptotic gene Bcl-2, thus exerting a brain-boosting and intelligence-enhancing effect and protecting brain nerves.

Description

Technical Field

[0001] This invention relates to a brain polypeptide raw material with brain-boosting and intelligence-enhancing properties and its preparation method. Background Technology

[0002] With the increasing aging of the population, diseases caused by neurotoxic oxidative damage are also on the rise, including brain nerve oxidative damage and neurodegenerative diseases. Brain polypeptide raw materials, extracted from animal brain tissue (such as bovine or porcine brain) through enzymatic hydrolysis, are small molecule active peptides with various brain-boosting functions. ① They promote nerve cell survival and regeneration, activating the secretion of neurotrophic factors (such as BDNF and NGF), promoting neuronal axon growth, and inhibiting apoptosis signaling pathways; ② They improve brain metabolism and energy supply by enhancing mitochondrial function, increasing ATP synthesis efficiency, and alleviating hypoxic-oxidative damage to brain nerve cells.

[0003] Chinese patent CN118726526A discloses a method for preparing brain peptides for preventing dementia. The method involves cold grinding and acid removal, molecular resonance-assisted salting out, molecular resonance-assisted enzymatic hydrolysis, secondary enzymatic hydrolysis, and ultrafiltration drying to obtain the brain peptides for preventing dementia. The extraction rate of the brain peptides is improved by using trypsin and chymotrypsin in synergy. Chinese patent CN120365355A discloses an active peptide for improving memory, a brain peptide enzymatic hydrolysis extract, its preparation method, and its application. This method uses microwave-assisted enzymatic hydrolysis of trypsin and pepsin, and aeration with carbon dioxide gas during microwave treatment to extract small molecule peptides with high activity. One peptide segment, AAVP, was screened to improve sleep and enhance memory.

[0004] The core active peptides in bovine brain peptides with clearly defined brain-boosting functions and their mechanisms of action still lack systematic research. Therefore, developing highly active and specific bovine brain peptide products remains an important research direction. Meanwhile, existing methods for preparing brain-boosting peptides suffer from problems such as complex equipment, low yield of active peptides, and limited functionality. Therefore, developing an efficient, low-cost, and multifunctional brain-boosting peptide preparation process has significant practical implications and application value. Summary of the Invention

[0005] This invention provides a brain polypeptide raw material with brain-boosting and intelligence-enhancing properties and its preparation method, which can effectively solve the above-mentioned problems.

[0006] This invention provides a method for preparing brain polypeptide raw materials that have brain-boosting, cognitive-enhancing, and nerve-protecting properties, comprising the following steps:

[0007] S1, Pretreatment: Remove some of the white fat from the quarantined bovine brain tissue and mince it. Add water at a mass ratio of 1:2 to 1:4 and mix. Heat the mixture at 54 to 56°C for 0.5 to 1.5 hours to obtain a pre-hydrolyzed solution.

[0008] S2, First hydrolysis: Adjust the pH of the pre-hydrolysate to 6.5~7.5, add neutral protease, the amount of which is 3.5~4.5% of the bovine brain protein, and hydrolyze at 50~60℃ for 1~3 hours. After enzyme inactivation, the first hydrolysate is obtained.

[0009] S3, Second hydrolysis: Adjust the pH of the first hydrolysate to 6.0~7.0, add papain, the amount of which is 1.0~3.0% of the bovine brain protein mass, and hydrolyze at 45~65℃ for 1.5~2.5h. After enzyme inactivation, the second hydrolysate is obtained.

[0010] S4, Post-processing: Adjust the pH of the second hydrolysate to 4.0~5.0, and then centrifuge, decolorize and deodorize with activated carbon, filter with diatomaceous earth, ultrafilter, desalinate and concentrate with nanofiltration, and spray dry to obtain brain polypeptide raw material powder; wherein, the molecular weight cutoff of ultrafiltration is <5 kDa, and the mass content of the concentrated brain polypeptide raw material is 10~25%.

[0011] In some embodiments, in step S2, the enzyme activity of the neutral protease is 2800–3600 U / g; in step S3, the enzyme activity of the papain is 4000–12000 U / g.

[0012] In some embodiments, in steps S2 and S3, the enzyme inactivation process involves inactivating the enzyme at 84–86°C for 15–20 minutes.

[0013] In some embodiments, in step S4, the activated carbon decolorization and deodorization and diatomaceous earth filtration steps are as follows: the second hydrolysate is cooled to 50~60℃, activated carbon is added for treatment for 0.5~1.5h, centrifuged, and then passed through a 0.45 μm aqueous membrane and diatomaceous earth; wherein, the amount of activated carbon added is 3.0~5.0% of the bovine brain protein content.

[0014] In some embodiments, the centrifugation conditions are 5000 rpm for 5-10 min.

[0015] This invention provides a brain polypeptide raw material composition that has the functions of improving intelligence, strengthening brain function and protecting brain nerves. The brain polypeptide raw material composition contains active peptide segments with a relative molecular weight of less than 5 kDa, including at least one of the peptide sequences LDAF, DIAF, ALDF, IGEF, FGDI, and LGFD.

[0016] In some embodiments, the brain polypeptide raw material composition that promotes intelligence, strengthens the brain, and protects brain nerves is prepared by the method described above.

[0017] This invention provides a functional product comprising the aforementioned brain polypeptide raw material composition that has the functions of improving intelligence, protecting brain nerves, and enhancing brain function as an active ingredient.

[0018] In some embodiments, the functional product has at least one of the following functions:

[0019] (1) DPPH radical scavenging rate is greater than or equal to 75%;

[0020] (2) ABTS free radical scavenging rate is greater than or equal to 99%;

[0021] (3) Acetylcholinesterase inhibition rate is greater than or equal to 85%;

[0022] (4) Repairs H2O2-induced PC12 neuronal damage, with a cell survival rate of ≥90%.

[0023] This invention provides the application of the aforementioned brain polypeptide raw material composition in the preparation of products for inhibiting acetylcholinesterase activity, scavenging free radicals, or repairing oxidative damage to nerve cells.

[0024] The beneficial effects of this invention are:

[0025] The method for preparing brain polypeptide raw materials of the present invention adopts stepwise enzymatic hydrolysis and conventional separation technology, which has low equipment requirements, simple process and is suitable for large-scale production.

[0026] The brain polypeptide raw material prepared by this invention has a DPPH free radical scavenging rate of over 75% and an ABTS free radical scavenging rate of over 99%, and can significantly inhibit acetylcholinesterase activity with an inhibition rate of over 85%.

[0027] The brain polypeptide raw material obtained by this invention has significant brain-boosting and intelligence-enhancing effects. It can repair H2O2-induced PC12 nerve cell damage, promote the expression of the anti-apoptotic gene Bcl-2, inhibit the expression of the pro-apoptotic gene Bax, and exert brain-boosting and intelligence-enhancing effects through the PI3K / AKT pathway. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 Results of experiments on the neurotoxicity of PC12 with brain peptide raw materials of different processes and concentrations.

[0030] Figure 2 The results show the construction of a model of oxidative stress damage to PC12 neurons caused by hydrogen peroxide (H2O2).

[0031] Figure 3The results show the cell viability of PC12 neurons after repairing H2O2 oxidative damage using brain peptide raw materials processed by different methods.

[0032] Figure 4 To investigate the effects of different brain peptide raw materials processed on the SOD enzyme activity of PC12 nerve cells damaged by H2O2 oxidation.

[0033] Figure 5 The effects of different brain peptide raw materials processed on the CAT enzyme activity of PC12 nerve cells damaged by H2O2 oxidation.

[0034] Figure 6 The total antioxidant capacity of brain peptide raw materials processed by different methods against H2O2 oxidative damage to PC12 nerve cells was measured.

[0035] Figure 7 To investigate the effects of different brain peptide raw materials processed on the activity of acetylcholinesterase (AChE) in PC12 neurons damaged by H2O2 oxidation.

[0036] Figure 8 To investigate the effects of different brain peptide raw materials processed on the mRNA gene expression of P13K in PC12 neurons damaged by H2O2 oxidation.

[0037] Figure 9 To investigate the effects of different brain peptide raw materials processed on the mRNA gene expression of AKT in PC12 neurons damaged by H2O2 oxidation.

[0038] Figure 10 The effects of different brain peptide raw materials processed on the mRNA gene expression of Bax in PC12 neurons damaged by H2O2 oxidation.

[0039] Figure 11 The effects of different brain peptide raw materials processed on the mRNA gene expression of Bcl-2 in PC12 neurons damaged by H2O2 oxidation.

[0040] Figures 12 to 29 This is a 2D schematic diagram simulating the interaction between different active peptides (LDAF, DIAF, ALDF, IGEF, FGDI, LGFD) and three receptor proteins, TrkB, TREM2, and AChE, through molecular docking.

[0041] In the 2D schematic diagram of molecular docking interactions, there are overlapping parts of amino acid residues, which are hereby illustrated with numbers. Figure 15 Middle 1: (APSA: 710) and 2: (LEUA: 608); Figure 21 Middle 3: (LGYA: 710) and 4: (LEUA: 560); Figure 24 5 (LEUA: 699) and 6 (ALAA: 586); Figure 277 (LYSA: 643) and 8 (LEUA: 560); Figure 29 In the middle (PROA: 344) and 10 (GLYA: 345); the light blue circle represents the solvent-accessible surface, which refers to the area on the molecular surface that can be contacted by solvent molecules. This helps in analyzing the interaction between molecules and solvents, studying the solubility and stability of molecules, and evaluating the binding sites of ligands and acceptors in molecular docking. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0043] Implementation Column 1

[0044] 1. Pretreatment: Take 500 g of qualified bovine brain tissue (protein content of approximately 12%, or 60 g of protein), remove some visible white fat, and mince it. Add 1500 g of water (feed-to-water ratio 1:3), mix well, and then perform medium-temperature heat treatment at 55℃ for 1.0 hour to obtain pre-hydrolysate.

[0045] 2. First hydrolysis: Adjust the pH of the pre-hydrolysate to 7.0, add 2.4g of neutral protease (accounting for 4.0% of the bovine brain protein mass), and hydrolyze at 55℃ for 2 hours. Then, raise the temperature to 85℃ to inactivate the enzyme for 15 minutes to obtain the first hydrolysate.

[0046] 3. Second hydrolysis: Adjust the pH of the first hydrolysate to 6.5, add 1.2g of papain (accounting for 2.0% of the bovine brain protein content), and hydrolyze at 55℃ for 2 hours. Then, raise the temperature to 85℃ to inactivate the enzyme for 15 minutes to obtain the second hydrolysate.

[0047] 4. Post-treatment: Adjust the pH of the second hydrolysate to 4.5. First, centrifuge at 5000 rpm for 5 min to remove insoluble matter. Take the supernatant, add 2.4 g of activated carbon (4.0% of the bovine brain protein mass), and decolorize and deodorize at 55℃ for 1 hour. Centrifuge again at 5000 rpm for 5 min to remove residue. The supernatant is then filtered sequentially through diatomaceous earth and a 0.45 μm aqueous membrane. The resulting clarified filtrate is ultrafiltered (molecular weight cutoff <5 kDa), then desalted and concentrated by nanofiltration until the mass content of the brain peptide raw material reaches 10-25%. Finally, spray dry the concentrated solution to obtain a powdered brain peptide raw material product.

[0048] Implement column 2

[0049] 1. Pretreatment: Take 500 g of bovine brain tissue (protein content 12%), defatted and minced, add 1000 g of water (material-to-water ratio 1:2), and heat treat at 54℃ for 0.5 hours.

[0050] 2. First hydrolysis: Adjust the pH to 6.5, add 2.1g of neutral protease (accounting for 3.5% of the protein mass), hydrolyze at 50℃ for 1 hour, and then inactivate the enzyme at 84℃ for 15 minutes.

[0051] 3. Second hydrolysis: Adjust the pH to 6.0, add 0.6g of papain (accounting for 1.0% of the protein mass), hydrolyze at 45℃ for 1.5 hours, and then inactivate the enzyme at 84℃ for 15 minutes.

[0052] 4. Post-processing: The subsequent steps are the same as in Example 1.

[0053] Implement column 3

[0054] Pretreatment: Take 500 g of bovine brain tissue (protein content 12%), defatted and minced, add 2000 g of water (material-to-water ratio 1:4), and heat treat at 56℃ for 1.5 hours.

[0055] 2. First hydrolysis: Adjust the pH to 7.5, add 2.7g of neutral protease (accounting for 4.5% of the protein mass), hydrolyze at 60℃ for 3 hours, and then inactivate the enzyme at 86℃ for 20 minutes.

[0056] 3. Second hydrolysis: Adjust the pH to 7.0, add 1.8g of papain (accounting for 3.0% of the protein mass), hydrolyze at 65℃ for 2.5 hours, and then inactivate the enzyme at 86℃ for 20 minutes.

[0057] 4. Post-processing: The subsequent steps are the same as in Example 1.

[0058] Comparative Example

[0059] To demonstrate the key features and superiority of the technical solution of this invention, multiple comparative examples were set up. The main difference lies in the combination of raw materials or enzymatic hydrolysis, while the remaining processes are largely the same as in Example 1:

[0060] Comparative Example A: The enzyme system is the same as in Example 1.

[0061] Comparative Examples 2 and B: Papain in the second step of hydrolysis was replaced with trypsin.

[0062] Comparative Examples 3 and C: The neutral protease in the first step was replaced with a complex lipase, and the papain in the second step was replaced with an alkaline protease.

[0063] Other comparative examples (4-9, DI): different enzymatic hydrolysis combinations were used, including alkaline protease + papain, neutral protease + alkaline protease, alkaline protease + acidic protease, alkaline protease + flavor protease, alkaline protease + trypsin, and pepsin + trypsin.

[0064] Comparative AI: The raw material was changed to pig brain tissue.

[0065] The enzymes used in the above embodiments and comparative examples are as follows:

[0066] Neutral protease: Model 0.8L, enzyme activity 80,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Papain: enzyme activity 400,000 U / g, Nanning Pangbo Bioengineering Co., Ltd.; Trypsin: enzyme activity 200,000 U / g, Nanning Pangbo Bioengineering Co., Ltd.; Complex lipase: enzyme activity 100,000 U / g, Dongheng Huadao Biotechnology Co., Ltd.; Alkaline protease: Model 37071, enzyme activity 400,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Acidic protease: enzyme activity 150,000 U / g, Shandong Longkete Enzyme Preparation Co., Ltd.; Flavor protease: Model 1000L, enzyme activity 100,000 U / g, Novozymes (China) Biotechnology Co., Ltd.; Pepsinase: enzyme activity 300,000 U / g, Shanghai Yuanju Biotechnology Co., Ltd.

[0067] U / g refers to the enzyme activity per gram of enzyme (used to characterize the enzyme's ability to catalyze a specific chemical reaction). This differs in meaning from the enzyme addition amount calculated per gram of brain tissue (%).

[0068] As in Example 1:

[0069] For neutral protease: The enzyme activity of neutral protease is 80,000 U / g; the amount of neutral protease added is 2.4g of brain protein. Based on the protein content of bovine brain (12%), the amount of neutral protease added is 3200 U / g. The specific calculation process is as follows: the enzyme activity of 2.4g of neutral protease is: 80,000 U / g * 2.4g = 192,000 U. Converted to the amount of enzyme added per gram of brain tissue protein, it is: 192,000 U / (500 g * 12%)g = 3200 U / g.

[0070] For papain: Papain has an enzyme activity of 400,000 U / g. Papain is added to 1.2 g of brain protein. Based on the protein content of bovine brain (12%), the amount of papain added is 8,000 U / g. The enzyme activity of 1.2g of enzyme is: 400,000 U / g * 1.2 = 480,000 U. Converted to the amount of enzyme added per g of brain tissue, it is: 480,000 U / (500g * 12%)g = 8,000 U / g.

[0071] In Example 1, the total enzyme addition amount of the two enzymes was 11,200 U / g. The conversion method for the enzyme addition amounts in Examples 2, 3, and each comparative example is the same as that in Example 1. The enzymes with the same names used in the above examples and comparative examples are from the same commercially available company and brand, meaning that the enzyme activity of the enzymes with the same names used in each example and comparative example is consistent.

[0072] Test case

[0073] The performance of the brain polypeptide raw material products prepared in the above embodiments and comparative examples was tested:

[0074] 1. Determination of the DPPH and ABTS free radical scavenging rate of brain polypeptide raw materials

[0075] The brain peptide raw material contains functional groups that can specifically react with DPPH and ABTS free radicals on nerve cells, effectively scavenging DPPH and ABTS free radicals on the surface and inside nerve cells. The brain peptide raw material can rapidly bind to DPPH and ABTS free radicals on nerve cells, reducing them to stable products, lowering their concentration, reducing oxidative damage to nerve cells, maintaining normal nerve cell function, and resisting oxidative stress and neurodegenerative diseases.

[0076] The determination methods for DPPH and ABTS are based on GB / T39100-2020, "Determination of Antioxidants in Brain Polypeptide Raw Materials".

[0077] The specific test results are shown in Table 1 below:

[0078] Table 1. Scavenging rate of DPPH free radicals of brain peptide raw materials processed by different methods

[0079]

[0080] Table 2. Scavenging rate of ABTS free radicals by brain peptide raw materials from different processes

[0081]

[0082] As shown in Table 1, under different conditions in the embodiments, the DPPH removal rate can reach a relatively high level of over 75%, indicating that the embodiments are effective in removing DPPH to a certain extent, and the difference in effect between the embodiments is small. The comparative examples show a large variation in DPPH removal rate. Comparative Example 6 has a removal rate of 53.65%, significantly lower than that of the embodiments, while Comparative Example C has a removal rate of 84.89%, significantly higher than that of the embodiments. Among the comparative examples, except for Comparative Example C, whose removal rate is slightly higher than that of the embodiments, the removal rates of the other comparative examples are all lower than those of the embodiments.

[0083] As shown in Table 2, under different conditions in the embodiments, the ABTS removal rate can reach over 99%, which is a high level, indicating that the embodiments are effective in removing ABTS, and the difference in effect between the embodiments is small. The comparative examples show smaller variations in ABTS removal rate; Comparative Example 5 has a removal rate of 98.80%, slightly lower than that of the embodiments; Comparative Example C has a removal rate of 99.88%, slightly higher than that of the embodiments. The difference in ABTS removal rate between the embodiments and the comparative examples is not significant.

[0084] 2. Determination of acetylcholinesterase activity in brain polypeptide raw materials

[0085] The brain polypeptide raw material contains aromatic and basic amino acid residues, which bind to the active site of acetylcholinesterase through π-π stacking and ionic bonds, blocking the interaction between acetylcholine and the enzyme and inhibiting its hydrolytic activity. This maintains the concentration of acetylcholine in the synaptic cleft, improves the efficiency of nerve signal transmission, helps improve cognitive function, and slows the progression of neurodegenerative diseases. The inhibition rate of acetylcholinesterase was determined using the method described in Wang Wei's "Study on the Effect of Perfume Lotus Extract on Improving Learning and Memory Ability in Alzheimer's Disease".

[0086] Table 3. Inhibition rate of acetylcholinesterase in brain polypeptide raw materials processed by different methods

[0087]

[0088] As shown in Table 3, under different conditions in the examples, the acetylcholinesterase inhibition rate can reach over 85%, which is a relatively high level, indicating that the examples are effective in inhibiting acetylcholinesterase, and the differences in effect between the examples are small. The comparative examples show a larger variation in acetylcholinesterase inhibition rate. Comparative Example B has a rate of 47.88%, lower than the inhibition rate of the examples; Comparative Example 2 has a rate of 92.03%, slightly higher than the inhibition rate of the examples. However, the acetylcholinesterase inhibition rate of the examples is only slightly lower than that of Comparative Examples 2, A, and C, and higher than other comparative examples. This indicates that certain conditions or methods in the comparative examples may produce stronger inhibitory effects under certain circumstances, but there are also cases where the inhibitory effect is low, and the inhibition rate is not as stable as in Example 1.

[0089] In vitro antioxidant capacity and acetylcholinesterase inhibition cannot be used as a single indicator of the performance of brain peptide raw materials. A comprehensive preliminary screening and evaluation are needed before proceeding to nerve cell experiments for comparison and overall evaluation. A comprehensive evaluation was conducted on the DPPH free radical scavenging rate (Table 1), the ABTS free radical scavenging rate (Table 2), and the acetylcholinesterase inhibition rate (Table 3). Examples 1, 2, 3, 4, A, C, G, and h were selected for nerve cell experiments.

[0090] 3. PC12 neurotoxicity test of brain polypeptide raw materials

[0091] PC12 neurons are a cell line derived from pheochromocytoma of the adrenal medulla in rats. They possess neuroendocrine neuronal characteristics and can differentiate into neuron-like neurons under the induction of nerve growth factor (NGF). Their morphology and function are similar to neurons in the central nervous system. Many neurological diseases or cognitive impairments are related to oxidative damage and functional abnormalities of neurons. Brain polypeptide raw materials exert repair and protective effects on highly differentiated PC12 neurons suffering from oxidative damage, suggesting, to some extent, their potential for improving cognitive function and brain health.

[0092] Neurocytotoxicity assays are used to assess the adverse effects of exogenous substances on nerve cell growth, proliferation, metabolism, and nerve cell membrane integrity, thereby providing a preliminary assessment of the substance's safety or biocompatibility. In the CCK-8 reagent, WST-8 is reduced to a water-soluble, orange-yellow formazan product by intracellular dehydrogenases under the action of an electron carrier; the amount produced is directly proportional to the number of viable nerve cells. Absorbance measured at 450 nm reflects nerve cell activity and neurocytotoxicity.

[0093] The effect of brain peptide raw materials on the proliferative activity of PC12 nerve cells was measured using the CCK-8 assay.

[0094] Depend on Figure 1As shown: In the experiments on the toxicity of brain polypeptide raw materials to PC12 neurons with different processes and concentrations, the neuronal survival rates of Examples 1, 2, 3, 4, A, and C at a brain polypeptide raw material concentration of 1.6 mg / mL were all higher than those of the control group (100%), indicating that the brain polypeptide raw material is not toxic to neurons.

[0095] Comparative Examples G and H showed higher neuronal survival rates at a brain peptide raw material concentration of 1.2 mg / mL compared to the control group (100%), while their neuronal survival rates at a concentration of 1.6 mg / mL were lower than the control group (100%). However, both comparison examples were still greater than 80%, indicating that Comparative Examples G and H exhibited slight toxicity to neurons at a concentration of 1.6 mg / mL. Comparative Examples A, C, and G showed the highest neuronal survival rate at 1.2 mg / mL, and their neuronal survival rates remained high in other examples and comparative examples. Therefore, a 1.2 mg / mL concentration of brain peptide raw material will be used in subsequent experiments.

[0096] 4. Model of oxidative stress damage to PC12 nerve cells by hydrogen peroxide (H2O2)

[0097] H2O2, as a reactive oxygen species (ROS), can induce the production of hydroxyl radicals in nerve cells, resulting in oxidative stress. This damages nerve cell membrane lipids, proteins, and DNA, leading to nerve cell apoptosis or necrosis. It is often used to construct models of oxidative damage in nerve cells to simulate the oxidative stress pathological process of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0098] This experiment used the CCK-8 method to determine the effect of H2O2 on the oxidative damage activity of PC12 nerve cells during modeling. Figure 2 As shown, our laboratory needs to subject nerve cells to severe oxidative damage to investigate the antioxidant, repair, and acetylcholinesterase inhibition effects of brain polypeptide raw materials on nerve cells. At a H2O2 solution concentration of 300 mM, the survival rate of PC12 nerve cells was less than 50%, indicating that significant oxidative damage to nerve cells was induced without causing a large number of nerve cell deaths. This demonstrates that PC12 nerve cells at 300 mM exhibit a good effect on oxidative stress and oxidative damage, indicating successful model establishment.

[0099] 4. The repair effect of brain polypeptide raw materials on PC12 nerve cells damaged by H2O2 oxidation.

[0100] Brain peptides can inhibit neuronal apoptosis by regulating apoptosis proteins such as Bax and Bcl-2, thus maintaining neuronal survival; promote neuronal differentiation by activating signaling pathways such as PI3K / AKT, inducing neurite growth and synaptic protein synthesis; and enhance the activity of antioxidant enzymes, scavenging free radicals and protecting mitochondrial function. The repair effect of brain peptides on PC12 neurons is the result of a multi-mechanism synergy, achieving neuronal survival maintenance and functional reconstruction through a combined effect of "anti-apoptosis-promoting differentiation-antioxidation".

[0101] Experimental methods: The effect of brain polypeptide raw materials on the repair of PC12 nerve cells damaged by H2O2 oxidation was measured by the CCK-8 method.

[0102] Depend on Figure 3 As shown, in the repair of PC12 neurons damaged by H2O2 oxidation by different brain peptide raw materials, the survival rate of PC12 neurons in the model group was less than 80%, indicating that H2O2 solution can promote apoptosis of PC12 neurons and has weak resistance to H2O2. Among them, the neuronal survival rate of Example 1 reached 102%, and the survival rate of PC12 neurons in other comparative examples was also above 90%, indicating that brain peptide raw materials can inhibit neuronal apoptosis and protect PC12 neurons, with Example 1 showing the best protective effect.

[0103] 5. Effects of brain polypeptide raw materials on the antioxidant activity of PC12 nerve cells damaged by H2O2 oxidation.

[0104] The effects of brain peptide raw materials on the catalase (CAT) activity of PC12 neurons damaged by H2O2 oxidation were determined using a SOD enzyme, CAT enzyme, and total antioxidant activity kit. The assays were performed according to the instructions for the SOD enzyme, CAT enzyme, and total antioxidant activity kits.

[0105] Depend on Figure 4 As shown: In the effect of different brain peptide raw materials on SOD enzyme activity of PC12 neurons damaged by H2O2 oxidation, the model group and the control group were compared. The SOD enzyme activity of PC12 neurons in the model group was significantly higher than that in the control group. This indicates that when PC12 neurons are damaged by H2O2 oxidation, the SOD enzyme activity will significantly increase, and it can specifically remove superoxide anions (O2). - It disproportionates into H2O2 and O2, laying the foundation for subsequent antioxidant reactions.

[0106] Comparison of Example 1 and Model Group: The SOD enzyme activity of PC12 nerve cells in Example 1 was significantly lower than that in the control group and the model group, indicating that its brain polypeptide raw material had the strongest antioxidant capacity.

[0107] Comparison with the model group: The SOD enzyme activity of comparative examples 2, 3, 4, A, C, G and h was significantly lower than that of the model group, and slightly lower or slightly higher than that of the control group. Among them, the SOD enzyme activity of comparative example A was close to that of Example 1.

[0108] Overall, this indicates that the brain peptide raw material has strong antioxidant capacity, can undergo redox reactions with H2O2, reduce the attack intensity of H2O2 on PC12 nerve cells, reduce SOD enzyme activity, and decrease the interaction between SOD enzyme and O2. - A redox reaction occurs, leading to excessive consumption of SOD enzymes. The control group showed slightly lower or slightly higher activity than the model group, indicating that the control group also possesses strong antioxidant capabilities, can protect nerve cells, and reduce SOD enzyme activity.

[0109] Depend on Figure 5 As shown: In the effect of different brain peptide raw materials on the CAT enzyme activity of PC12 neurons damaged by H2O2 oxidation, the model group and the control group were compared. The CAT enzyme activity of PC12 neurons in the model group was significantly higher than that in the control group. This indicates that when PC12 neurons are damaged by H2O2 oxidation, the CAT enzyme activity will increase significantly and can catalyze the decomposition of H2O2 into water and oxygen.

[0110] Comparison of Example 1 and Model Group: The CAT enzyme activity of PC12 nerve cells in Example 1 was significantly lower than that in the control group and the model group.

[0111] Comparison with the model group: The CAT enzyme activity of all comparative groups was significantly lower than that of the model group, and slightly lower or slightly higher than that of the control group. The CAT enzyme activity of all comparative groups was higher than that of Example 1. Lower CAT enzyme activity is better, indicating stronger antioxidant properties of the peptide. Lower CAT enzyme activity also reduces CAT enzyme consumption. The CAT enzyme activity of Example 1 was the lowest, exhibiting the strongest antioxidant properties.

[0112] Overall, this demonstrates that the brain peptide raw material possesses strong antioxidant capacity. By scavenging reactive oxygen species (ROS), it mitigates the attack of H2O2 on PC12 neurons, protects the structure of CAT enzymes within PC12 neurons, and maintains their catalytic activity. The CAT enzyme activity in Example 1 was significantly lower than that in the model group and the control group, indicating that the brain peptides of this example have strong antioxidant capacity, effectively scavenging ROS and protecting PC12 neurons. The CAT enzyme activities in the comparative examples were all significantly lower than those in the model group and close to those in the control group, indicating that the antioxidant capacity of the comparative brain peptides was slightly weaker than that of the examples, but they still possess the ability to protect neurons and reduce CAT enzyme activity.

[0113] Depend on Figure 6As shown: In the effect of different brain peptide raw materials on the total antioxidant capacity of PC12 nerve cells damaged by H2O2 oxidation, the model group and the control group were compared. The antioxidant capacity of PC12 nerve cells in the model group was significantly lower than that in the control group, indicating that the antioxidant capacity of PC12 nerve cells will be significantly reduced when they are damaged by H2O2 oxidation.

[0114] Comparison of Example 1 and Model Group: The antioxidant capacity of PC12 nerve cells in Example 1 was significantly higher than that in the model group and slightly higher than that in the control group.

[0115] Comparison of comparative examples and model groups: The antioxidant activity of the comparative examples was significantly higher than that of the model group, and slightly higher or slightly lower than that of the control group. Among them, the antioxidant capacity of comparative example 3 was close to that of example 1, indicating that there are certain differences in the antioxidant capacity of brain peptide raw materials processed by different methods.

[0116] Overall, this indicates that the brain peptide raw material has a strong antioxidant capacity. When the ROS level in nerve cells increases, it can maintain the antioxidant capacity of nerve cells by timely neutralizing excess free radicals (ROS). The antioxidant activity of Comparative Example 4 was significantly higher than that of the model group and slightly lower than that of the control group, indicating that the antioxidant capacity of the comparative example brain peptide raw material was slightly weaker, but it could still neutralize excess free radicals (ROS) and maintain the antioxidant capacity of nerve cells.

[0117] 6. Effects of brain polypeptide raw materials on acetylcholinesterase (AChE) activity in PC12 neurons damaged by H2O2 oxidation.

[0118] Acetylcholinesterase (AChE) is a key enzyme in the central nervous system that catalyzes the hydrolysis of acetylcholine. Abnormal AChE activity is closely associated with neurodegenerative diseases such as Alzheimer's disease (AD). H2O2 treatment can mimic β-amyloid (Aβ)-induced oxidative stress. Oxidative damage disrupts neuronal structure and accelerates the hydrolysis of neurotransmitters. Increased AChE activity is positively correlated with neuronal apoptosis. Oxidative stress disrupts the dynamic homeostasis of AChE, whose activity is regulated by post-translational modifications such as phosphorylation and ubiquitination. H2O2 may inhibit the degradative enzyme activity of AChE or promote its phosphorylation, leading to the accumulation and upregulation of AChE activity within neuronal cells.

[0119] The effect of brain peptide raw materials on the acetylcholinesterase (AChE) activity of PC12 neurons damaged by H2O2 oxidation was determined using an acetylcholinesterase (AChE) activity assay kit. Logarithmic-phase PC12 neurons were collected and cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at a cell density of 1×10⁻⁶ cells / mL. 4Neurons were seeded at a density of 1 / mL in 96-well plates and incubated at 37°C for 24 hours in a CO2 incubator to allow for cell adhesion. The culture medium was aspirated, and culture medium containing 1.2 mg / mL of the brain polypeptide raw material solution from the examples and comparative studies was added to create the experimental group; fresh culture medium was used as the control group; and culture medium without neurons was used as the blank group. All plates were incubated at 37°C for 24 hours. After incubation, 6-well plates were removed, and except for the control group, 1 mL of 300 mM H2O2 solution was added to each well. The plates were then incubated for 20 minutes in a constant temperature incubator, and the acetylcholinesterase (AChE) activity assay was performed according to the instructions of the kit.

[0120] Depend on Figure 7 As shown: In the effect of different brain peptide raw materials on the acetylcholinesterase (AChE) activity of PC12 neurons damaged by H2O2 oxidation, the model group and the control group were compared. The AChE activity of PC12 neurons in the model group was significantly higher than that in the control group. This indicates that H2O2 oxidation damage to PC12 neurons disrupts the dynamic balance of AChE activity, leading to disordered acetylcholine metabolism and abnormally increased AChE activity. Neurons may accelerate the hydrolysis of acetylcholine by upregulating AChE activity to avoid its excessive accumulation and excitotoxicity.

[0121] Comparison of Example 1 with the model group: The AChE activity of Example 1 was significantly lower than that of the model group and slightly lower than that of the control group, with the AChE activity of Example 1 being 90.28%.

[0122] Comparison between comparative examples and the model group: The AChE activity of the comparative examples was significantly lower than that of the model group. The AChE activity of comparative examples 4, A, and G was slightly higher than that of the control group; the AChE activity of comparative examples 2, 3, C, and h was slightly lower than that of the control group; the AChE activity of examples 1, 2, and 3 was relatively close. This indicates that there are certain differences in the antioxidant capacity of brain peptide raw materials produced by different processes. The lower the AChE activity value, the better. Examples 1, 2, and 3 are relatively close, all showing good reduction of acetylcholinesterase activity.

[0123] Overall, this indicates that the brain peptide raw material has a strong antioxidant capacity, can undergo redox reactions with H2O2, reduce the attack intensity of H2O2 on PC12 nerve cells, inhibit the abnormal increase of AChE activity, avoid excessive hydrolysis of acetylcholine, and prevent the dynamic balance of AChE activity from being disrupted, which could lead to abnormal motor regulation, synaptic dysfunction, and the risk of central excitotoxicity.

[0124] The AChE activity of PC12 neurons in the model group was significantly higher than that in the control group, indicating that H2O2 oxidative damage to PC12 neurons disrupts the dynamic balance of AChE activity, leading to disordered acetylcholine metabolism and abnormally elevated AChE activity. Neurons may accelerate the hydrolysis of acetylcholine by upregulating AChE activity to prevent excessive accumulation and excitotoxicity. Similar compensatory mechanisms have been observed in models of neurodegenerative diseases such as Alzheimer's disease (AD), where increased AChE activity under oxidative stress may exacerbate the decline in cholinergic system function.

[0125] The AChE activity of the examples and comparative examples was significantly lower than that of the model group. The AChE activity of Examples 1, 2, 3, C, and h was slightly lower than that of the control group. The AChE activity of Example 1 was 90.28%, indicating that the brain polypeptide raw material has a strong antioxidant capacity and can undergo redox reaction with H2O2, reducing the attack intensity of H2O2 on PC12 nerve cells, inhibiting the abnormal increase of AChE activity, avoiding excessive hydrolysis of acetylcholine, and preventing the disruption of the dynamic balance of AChE activity, which could lead to abnormal motor regulation, synaptic dysfunction, and the risk of central excitotoxicity.

[0126] 7. The effect of brain polypeptide raw materials on the expression of related genes after H2O2 oxidative damage to PC12 neurons.

[0127] The phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) pathways are core pathways for nerve cell survival. PI3K catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), which activates AKT phosphorylation. Phosphorylated AKT (p-AKT) can inhibit the activity of pro-apoptotic proteins (such as Bax) while activating the expression of anti-apoptotic proteins (such as Bcl-2), thereby inhibiting nerve cell apoptosis.

[0128] Bax is a pro-apoptotic gene that increases mitochondrial membrane permeability, releases neuronal cytochrome C, and activates the caspase cascade. Bcl-2 is an anti-apoptotic gene that maintains neuronal cell survival by inhibiting mitochondrial oxidative damage and caspase activation. Significance of expression balance: The Bax / Bcl-2 protein ratio is a key indicator determining the sensitivity of neuronal cells to apoptosis; an increased ratio promotes apoptosis, while a decreased ratio inhibits it.

[0129] The expression of genes related to H2O2 oxidative damage in PC12 neurons was measured using qPCR with brain peptide raw materials. Primer information for these neurons is shown in Table 4.

[0130] Table 4. Primers for PC12-related neural cell genes

[0131]

[0132] Depend on Figure 8 As shown: In the effect of different brain polypeptide raw materials on the expression of PI3K mRNA in PC12 nerve cells damaged by H2O2 oxidation, the model group and the control group showed that the expression of PI3K gene in the model group (H2O2 oxidation damage) was significantly lower than that in the control group, indicating that H2O2 oxidation damage inhibited the expression of PI3K gene.

[0133] Comparison of Example 1 with the model group: The expression of PI3K gene after intervention with brain polypeptide raw materials in Example 1 was significantly higher than that in the model group, indicating that brain polypeptide raw materials can reverse H2O2 oxidative damage to a certain extent and promote the upregulation of PI3K gene expression.

[0134] Comparison between the comparative and model groups: The PI3K expression of the comparative group G was higher than that of Example 1, while some comparative groups were lower or close to it. The effects of different brain peptide raw materials on PI3K expression varied, but the brain peptide raw material of Example 1 showed a promoting effect on PI3K gene expression relative to the model group.

[0135] Overall, brain peptide raw materials can significantly upregulate the mRNA expression of the PI3K gene in PC12 neurons damaged by H2O2 oxidation. This may be achieved by activating PI3K and catalyzing the generation of PIP3, which then acts as a second messenger to activate AKT to the cell membrane, initiate anti-apoptotic signals, and exert the potential to improve intelligence and brain function.

[0136] Depend on Figure 9 As shown: In the effect of different brain peptide raw materials on the expression of AKT mRNA in PC12 nerve cells damaged by H2O2 oxidation, the model group and the control group showed that the expression of AKT gene in the model group (H2O2 oxidation damage) was significantly higher than that in the control group, indicating that H2O2 oxidation damage promoted AKT gene expression and inhibited oxidative stress-induced nerve cell apoptosis.

[0137] Comparison of Example 1 with the model group: In Example 1, the expression of AKT gene after intervention with brain polypeptide raw materials was significantly lower than that in the model group, indicating that the brain polypeptide raw materials undergo redox reaction with H2O2, inhibiting the apoptosis of PC12 neurons induced by H2O2 oxidative stress, resulting in a slight upregulation of AKT gene expression promoted by brain polypeptide raw materials.

[0138] Comparison between comparative examples and the model group: The AKT expression of comparative examples 4 and 4 was higher than that of example 1, while some comparative examples were lower than or close to that of the model group. The effects of different brain peptide raw materials on AKT expression varied, but the brain peptide raw material of example 1 showed an inhibitory effect on AKT gene expression relative to the model group.

[0139] Overall, when H2O2 does not excessively damage PC12 neurons, PC12 neurons themselves upregulate AKT gene expression, enhance AKT phosphorylation, strengthen downstream anti-apoptotic signals, and inhibit oxidative stress-induced neuronal apoptosis. Brain peptide raw materials possess strong antioxidant activity and can bind to free radicals, inhibiting H2O2 oxidative stress-induced PC12 neuronal apoptosis, leading to a slight upregulation of AKT gene expression promoted by brain peptide raw materials. This may exert its potential for improving cognitive function and brain health through antioxidant activity and the promotion of AKT gene expression.

[0140] Depend on Figure 10 As shown: In the effect of different brain peptide raw materials on the mRNA gene expression of Bax in PC12 nerve cells damaged by H2O2 oxidation, the model group and the control group showed that the Bax gene expression in the model group (H2O2 oxidation damage) was significantly higher than that in the control group, indicating that H2O2 oxidation damage promoted Bax gene expression.

[0141] Comparison of Example 1 with the model group: In Example 1, the expression of Bax gene after intervention with brain polypeptide raw materials was significantly lower than that in the model group and the control group, indicating that brain polypeptide raw materials can reverse H2O2 oxidative damage to a certain extent and promote the downregulation of Bax gene expression.

[0142] Comparison of comparative examples with the model group: The Bax gene expression of comparative examples C and G was higher than that of Example 1 and close to that of the model group; comparative examples 2, 3, 4, A and h were close to the control and significantly lower than that of the model group; indicating that the brain polypeptide raw materials of different processes have different effects on Bax expression, but the brain polypeptide raw material of Example 1 showed an inhibitory effect on Bax gene expression compared with the model group.

[0143] Overall, upon H2O2 stimulation, Bax translocates from the cytoplasm to the mitochondria, oligomerizes to form transmembrane channels, leading to mitochondrial membrane rupture and cytochrome C release. Overexpression exacerbates mitochondrial damage and promotes apoptosis in PC12 neurons under H2O2-induced damage. Brain peptides can downregulate Bax gene mRNA expression in H2O2-oxidatively damaged PC12 neurons, thereby exerting their potential for cognitive enhancement by inhibiting the Bax pathway.

[0144] Depend on Figure 11 As shown: In the effect of different brain polypeptide raw materials on the mRNA gene expression of Bcl-2 in PC12 nerve cells damaged by H2O2 oxidation, the model group and the control group showed that the Bcl-2 gene expression in the model group (H2O2 oxidation damage) was significantly lower than that in the control group, indicating that H2O2 oxidation damage inhibited the expression of Bcl-2 gene.

[0145] Comparison of Example 1 with the model group: In Example 1, the expression of Bcl-2 gene was significantly higher than that in the model group after intervention with brain polypeptide raw materials, indicating that brain polypeptide raw materials can reverse H2O2 oxidative damage to a certain extent and promote the upregulation of Bcl-2 gene expression.

[0146] Comparison of comparative examples with the model group: Comparative examples 4 and A were lower than or close to the control group, while the remaining comparative examples were higher than the control group and the model group. The effects of brain peptide raw materials from different processes on Bcl-2 expression were different, but the brain peptide raw material in Example 1 showed a promoting effect on the upregulation of Bcl-2 gene expression relative to the model group.

[0147] Overall, brain peptide raw materials can significantly upregulate the mRNA expression of the Bcl-2 gene in PC12 neurons damaged by H2O2 oxidation, and may exert the potential to improve intelligence and brain function by activating the Bcl-2 pathway.

[0148] In summary, the regulation of the PI3K / AKT and Bax / Bcl-2 pathways by brain peptide raw materials provides new targets for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. For example, enhancing the activity of the PI3K / AKT pathway can protect dopaminergic neurons from oxidative stress damage, while reducing the Bax / Bcl-2 ratio can delay the process of neuronal apoptosis. Neuronal cell experimental data indicate that brain peptide raw materials have a clear potential for improving cognitive function and brain health.

[0149] 8. Molecular docking experiment

[0150] Chromatographic conditions, mass spectrometry conditions, and data analysis were based on the report "Screening of URAT1-regulated active ingredients in Tibetan medicine *Short-tube Rabbit Ear Grass* based on target cell capture and molecular docking". Mass spectrometry identification was commissioned to Beijing Baitaipaike Biotechnology Co., Ltd., which provided the "Peptide Sequence Analysis Project Report Based on LC-MS / MS".

[0151] The raw files acquired by mass spectrometry contained over 3000 peptides ranging from 2 to 21. Due to the prevalence of studies on dipeptides, they were excluded from this screening. First, peptides with peak areas (abundance) of 3 or more were selected based on their relative abundance (>0.5%), totaling 42 peptides. Activity scoring was performed on the data from (http: / / distilldeep.ucd.ie / PeptideRanker / ), selecting 6 peptides with a score >0.60 and containing 3 or more peptides. Further searching was performed on the BIOPEP (http: / / bis.zju.edu.cn / biopepdbr / index.pHp) database, revealing 6 previously unreported peptides. Toxicity analysis of these 6 peptides was conducted, and a peptide toxicity prediction website (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.pHp) predicted them to be non-toxic. After searching the Byonic database, the peptide list is shown below. The table below shows the six peptide sequences obtained in Example 1, sorted by peak area (abundance) after screening based on peptide sequence, number of amino acids, activity score, peak area (abundance), relative molecular mass, and toxicity test results. See Table 5 for details.

[0152] Table 5. Brain polypeptide raw material sequences and information scores

[0153]

[0154] The three-dimensional structures of the above four peptide sequences with TrkB receptor (ID: 4AT3), TREM2 (ID: 5UD7), and AChE (ID: 4EY7) were downloaded from the PDB database (http: / / www.rcsb.org / ) using molecular docking software. The docking binding energies of the six peptide sequences with different receptors are shown in Table 6.

[0155] Table 6. MoE (Measure of Efficacy) Analysis of Molecular Docking of Brain Peptide Raw Materials

[0156]

[0157] CDOCKERENERGY is an energy value calculated during CDOCKER docking. It primarily reflects the overall binding energy between the ligand and acceptor during docking. This energy comprehensively considers various interactions between the ligand and acceptor, including van der Waals forces and electrostatic interactions. Physically speaking, a lower CDOCKERENERGY value indicates a tighter and more stable binding between the ligand and acceptor.

[0158] Figure 12As shown, there are interactions between LDAF and TrkB receptor protein mainly consisting of van der Waals forces, π-cations, salt bridges, alkyl groups, conventional hydrogen bonds, π-alkyl groups, and C-H bonds. Figure 13 As shown, there is an interaction between LDAF and the TREM2 receptor protein, mainly consisting of van der Waals forces, conventional hydrogen bonds, salt bridges, C-H bonds, and attractive charges. Figure 14 As shown, there are van der Waals forces, C-H bonds, salt bridges, conventional hydrogen bonds, and alkyl-based interactions between LDAF and the AChE receptor protein. Figure 15 As shown, there are interactions between DIAF and TrkB receptor protein mainly consisting of van der Waals forces, C-H bonds, salt bridges, alkyl groups, attractive charges, π-alkyl groups, and conventional hydrogen bonds. Figure 16 As shown, there are interactions between DIAF and the TREM2 receptor protein, mainly consisting of van der Waals forces, C-H bonds, salt bridges, attractive charges, alkyl groups, and conventional hydrogen bonds. Figure 17 As shown, there is an interaction between DIAF and the AChE receptor protein, mainly consisting of van der Waals forces, C-H bonds, and conventional hydrogen bonds. Figure 18 As shown, there are interactions between LDAF and TrkB receptor protein mainly consisting of van der Waals forces, salt bridges, π-anions, attractive charges, alkyl groups, conventional hydrogen bonds, π-alkyl groups, and C-H bonds. Figure 19 As shown, there is an interaction between LDAF and the TREM2 receptor protein, mainly consisting of van der Waals forces, conventional hydrogen bonds, salt bridges, C-H bonds, and attractive charges. Figure 20 As shown, there are van der Waals forces, C-H bonds, conventional hydrogen bonds, and alkyl-based interactions between LDAF and the AChE receptor protein. Figure 21 As shown, there are interactions between IGEF and the TrkB receptor protein, mainly involving van der Waals forces, π-cations, attractive charges, alkyl groups, conventional hydrogen bonds, π-alkyl groups, and C-H bonds. Figure 22 As shown, there is a predominantly van der Waals force, salt bridge, conventional hydrogen bond, alkyl group and C-H bond interaction between IGEF and the TREM2 receptor protein. Figure 23 As shown, there are van der Waals forces, C-H bonds, attractive charges, conventional hydrogen bonds, and alkyl-based interactions between IGEF and the AChE receptor protein. Figure 24 As shown, there are van der Waals forces, C-H bonds, attractive charges, π-πT-shape, conventional hydrogen bonds and alkyl-based interactions between FGDI and the TrkB receptor protein; Figure 25 As shown, there are van der Waals forces, conventional hydrogen bonds, salt bridges, C-H bonds, attractive charges, and alkyl-based interactions between FGDI and the TREM2 receptor protein. Figure 26 As shown, FGDI interacts with the AChE receptor protein primarily through van der Waals forces, C-H bonds, attractive charges, π-anions, and conventional hydrogen bonds. Figure 27As shown, there are interactions between LGFD and TrkB receptor protein, mainly consisting of van der Waals forces, salt bridges, π-cations, conventional hydrogen bonds, alkyl groups, C-H bonds, and π-alkyl groups. Figure 28 As shown, there are van der Waals forces, conventional hydrogen bonds, salt bridges, C-H bonds, attractive charges, and alkyl-based interactions between LGFD and the TREM2 receptor protein. Figure 29 As shown, LGFD interacts with the AChE receptor protein primarily through van der Waals forces, C-H bonds, conventional hydrogen bonds, and amide-π stacking.

[0159] The TrkB receptor (PDBID: 4AT3), a member of the neurotrophic factor receptor family, is an important subtype of tyrosine kinase receptor (RTK). It is mainly expressed on the surface of neurons and neural progenitor cells in the central nervous system. It can specifically bind to brain-derived neurotrophic factor (BDNF) and is a key molecule for regulating neural plasticity and synaptic regeneration.

[0160] TrkB activates downstream pathways by mimicking neurotrophic factors through brain peptides. When BDNF binds to the extracellular domain of TrkB, it induces receptor dimerization and triggers autophosphorylation of the intracellular kinase domain, thereby activating multiple signaling pathways, including PI3K / AKT. TrkB activation significantly promotes neuronal survival through mechanisms including: 1) upregulating the expression of the anti-apoptotic protein Bcl-2; 2) enhancing mitochondrial biosynthesis; and 3) inhibiting caspase cascade reactions. Simultaneously, TrkB signaling can directly regulate synaptic plasticity by increasing postsynaptic AMPA receptor aggregation and promoting dendritic spine morphological remodeling, thus improving neural network connectivity efficiency.

[0161] The TREM2 receptor (PDBID: 5UD7) belongs to the immunoglobulin superfamily and is highly expressed in microglia. As a neuroinflammatory regulatory hub, it recognizes pathologically relevant molecular patterns such as lipoproteins and β-amyloid (Aβ). Its activation depends on coupling with its adaptor protein DAP12: when the ligand binds to TREM2, DAP12 recruits Syk kinase to initiate downstream signaling, inducing microglia functional reprogramming. TREM2's cognitive and brain-boosting effects are manifested in: 1) enhancing microglia's phagocytic clearance of Aβ; 2) inhibiting excessive inflammatory cytokine release; and 3) promoting myelin repair through the Wnt / β-catenin pathway. In Alzheimer's disease, TREM2 dysfunction leads to microglia metabolic disorders and accelerates neurodegeneration.

[0162] Acetylcholinesterase (AChE) (PDBID: 4EY7), a core hydrolase of the cholinergic system, terminates neural signal transmission by rapidly degrading acetylcholine (ACh) in the synaptic cleft. Its active site contains a substrate channel composed of a catalytic triplet and aromatic amino acids. AChE inhibitors (such as donepezil) can prolong the duration of ACh's action and improve cognitive function by competitively binding to the active site. Recent studies have also discovered that AChE has non-catalytic functions: its C-terminal peptide can promote β-amyloid protein aggregation, forming a positive feedback loop that exacerbates neurotoxicity.

[0163] Synergistic mechanism: Cross-dialogue between TrkB and TREM2: The BDNF-TrkB pathway can upregulate TREM2 expression, and TREM2 activation enhances the neuronal response to BDNF through DAP12 signaling, forming a positive cycle of brain health and intelligence.

[0164] AChE regulatory network: AChE inhibitors can indirectly activate TrkB signaling, while inhibiting AChE-mediated Aβ toxicity, and synergistically delay neurodegeneration with the clearance function of TREM2.

[0165] Targeted Therapy Potential: The brain peptide raw materials designed in this application (including sequences such as LDAF and DIAF) can simultaneously bind to the TrkB ligand-binding domain, the TREM2 immune recognition region, and the peripheral anion site of AChE, achieving the following through multi-target synergy: 1) enhancing BDNF / TrkB signal transduction efficiency; 2) improving microglia phagocytosis efficiency; and 3) selectively inhibiting AChE non-catalytic toxicity. The resulting "neural repair-immune regulation-metabolic balance" triple regulatory network provides a novel intervention strategy for neurodegenerative diseases.

[0166] Because in the brain tissue active brain polypeptide raw materials prepared in the embodiments of this application, the six peptides LDAF, DIAF, ALDF, IGEF, FGDI, and LGFD successfully docked with the three receptors TrkB receptor, TREM2 receptor, and acetylcholinesterase AChE, and there is a certain binding energy.

[0167] Summary of Experimental Results: Through in vitro antioxidant and nerve cell experiments, a brain polypeptide raw material with optimal brain-boosting activity was screened for use in amino acid sequence and neuroprotective activity studies. The brain-boosting effects of different enzymatic hydrolysis compositions were evaluated using in vitro antioxidant, in vitro acetylcholinesterase, PC12 nerve cell proliferation activity, repair of PC12 nerve cells damaged by hydrogen peroxide, CAT enzyme activity, SOD enzyme activity, total antioxidant capacity, acetylcholinesterase activity in PC12 nerve cells damaged by hydrogen peroxide, PI3K, AKT, Bax, and Bcl-2. The brain polypeptide raw material prepared according to the embodiments of this application was selected as having the best comprehensive performance and optimal brain-boosting activity.

[0168] The brain polypeptide raw material prepared in this application has the characteristics of high antioxidant capacity, significant in vitro acetylcholinesterase inhibition, promotion of PC12 nerve cell proliferation, significant repair of PC12 nerve cells damaged by hydrogen peroxide, significant enhancement of total antioxidant capacity, significant inhibition of acetylcholinesterase in PC12 nerve cells damaged by hydrogen peroxide, promotion of expression of PI3K, AKT and Bcl-2 related survival genes, and inhibition of expression of pro-apoptotic gene Bax. The brain polypeptide raw material has strong antioxidant activity, and by binding with hydrogen peroxide, it can scavenge free radicals and reduce excessive oxidative stress of SOD and CAT enzymes.

[0169] In this application embodiment, six amino acid peptides with brain-boosting activities—LDAF, DIAF, ALDF, IGEF, FGDI, and LGFD—were screened from all identified peptides using mass spectrometry, virtual screening, and molecular docking. These peptides exert their brain-boosting effects by promoting the repair of PC12 neurons, clearing Aβ aggregates, inhibiting acetylcholinesterase, promoting the expression of survival genes, and inhibiting the expression of pro-apoptotic genes.

[0170] In summary, the method for preparing brain polypeptide raw materials provided in this application has at least the following mechanisms of action and technical effects:

[0171] (1) This application found that brain tissue, after being crushed, homogenized, pretreated with medium-temperature heat treatment, and then subjected to a specific enzymatic hydrolysis combination (first enzymatic hydrolysis with neutral protease and second enzymatic hydrolysis with papain), produces a brain polypeptide raw material powder with a high level of brain-boosting and intelligence-enhancing effects.

[0172] (2) The proposed solution uses a specific combination of neutral protease and papain for enzymatic hydrolysis to achieve the desired effect; the effect of the proposed solution can not be achieved by hydrolyzing the solution with a combination of two enzymes or any other combination of proteases.

[0173] For the combination of neutral protease and papain:

[0174] Neutral proteases exhibit stable activity within a mild pH range of 6–8, making them well-suited for processing in neutral environments. They also possess some adaptability to temperature variations, maintaining good catalytic efficiency within the 30–50°C range. Most neutral proteases are metalloproteinases, functioning by hydrolyzing peptide bonds within proteins, and are suitable for conventional industrial applications such as food processing and detergents.

[0175] Papain has a wide pH range, maintaining certain activity between pH 5 and 8, and performing particularly well in acidic to neutral environments. It is also relatively tolerant of high temperatures, maintaining its catalytic activity at around 60°C, and exhibits some resistance to organic solvents, making it suitable for specialized industrial applications in food, pharmaceuticals, and cosmetics.

[0176] (3) The method of this application uses intact bovine brain tissue as raw material and adopts a combination of enzymatic hydrolysis and ultrafiltration. First, the nerve cell structure is destroyed by grinding and homogenizing. Then, the brain polypeptide raw material is released by enzymatic hydrolysis with a specific combination of enzymes. Finally, the target molecular weight brain polypeptide raw material is separated by ultrafiltration. The desired brain polypeptide raw material can be obtained by simply combining mixing, enzymatic hydrolysis and filtration. It combines the advantages of multiple technologies and has the advantages of low equipment requirements, simple and easy operation, and high extraction efficiency, which is convenient for large-scale industrial production.

[0177] In summary, this application method obtains brain polypeptide raw material powder with brain-boosting, brain-protecting, and intelligence-enhancing properties by extracting, separating, and identifying active brain polypeptide raw materials in brain tissue.

[0178] Furthermore, this application utilizes intact bovine brain tissue to obtain brain polypeptide raw materials through enzymatic hydrolysis. These brain polypeptide raw materials possess high levels of antioxidant capacity, significant in vitro acetylcholinesterase inhibition, promotion of PC12 neuronal proliferation, significant repair of PC12 neuronal damage caused by hydrogen peroxide, significant enhancement of total antioxidant capacity, significant inhibition of acetylcholinesterase in PC12 neuronal cells damaged by hydrogen peroxide, promotion of the expression of PI3K, AKT, and Bcl-2-related survival genes, and inhibition of the expression of the pro-apoptotic gene Bax. The brain polypeptide raw materials exhibit strong antioxidant activity, scavenging free radicals and reducing excessive oxidative stress from SOD and CAT enzymes by binding to hydrogen peroxide. The amino acid sequences of the brain polypeptide raw materials were identified using LC-MS / MS analysis. Using activity prediction tools and molecular docking, amino acid sequences of brain polypeptide raw materials with brain-boosting and neuroprotective effects were screened. These sequences were then artificially synthesized to verify their brain-boosting activities and further elucidate their mechanism of action. This provides theoretical support for the predictive screening and structure-activity relationship studies of brain polypeptide raw materials for brain-boosting and neurological benefits. Among these methods, mass spectrometry analysis of the prepared brain peptide raw materials was used to determine their amino acid linkage sequence, molecular docking was used to determine their active sites, and synthesis was used to verify whether they had the corresponding activity. This provides a scientific theoretical basis for the production and application of brain peptide raw materials and has important theoretical value and practical significance for the research of brain peptide raw materials.

[0179] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing a brain polypeptide raw material with brain-boosting, intelligence-enhancing, and nerve-protecting properties, characterized in that, Includes the following steps: S1, Pretreatment: Remove some of the white fat from the quarantined bovine brain tissue, mince it, add water at a mass ratio of 1:2 to 1:4, mix, and heat at 54 to 56°C for 0.5 to 1.5 hours to obtain a pre-hydrolyzed solution; S2, First hydrolysis: Adjust the pH of the pre-hydrolysate to 6.5-7.5, add neutral protease at a concentration of 3.5-4.5% of the bovine brain protein mass, and hydrolyze at 50-60℃ for 1-3 hours. After enzyme inactivation, the first hydrolysate is obtained. The neutral protease has an enzyme activity of 2800-3600 U / g. S3, Second hydrolysis: Adjust the pH of the first hydrolysate to 6.0~7.0, add papain, the amount added is 1.0~3.0% of the bovine brain protein mass, and hydrolyze at 45~65℃ for 1.5~2.5h. After enzyme inactivation, the second hydrolysate is obtained; the papain has an enzyme activity of 4000~12000 U / g. S4, Post-processing: Adjust the pH of the second hydrolysate to 4.0~5.0, centrifuge, decolorize and deodorize with activated carbon, filter with diatomaceous earth, ultrafilter, desalinate and concentrate with nanofiltration, and spray dry to obtain brain polypeptide raw material powder; wherein, the molecular weight cutoff of ultrafiltration is <5 kDa, and the mass content of the concentrated brain polypeptide raw material is 10~25%.

2. The method for preparing the brain polypeptide raw material with brain-boosting, intelligence-enhancing, and nerve-protecting properties according to claim 1, characterized in that, In steps S2 and S3, the enzyme inactivation process involves inactivating the enzyme at 84–86°C for 15–20 minutes.

3. The method for preparing brain polypeptide raw materials with brain-boosting, intelligence-enhancing, and nerve-protecting properties according to claim 1, characterized in that, In step S4, the activated carbon decolorization and deodorization and diatomaceous earth filtration steps are as follows: the second hydrolysate is cooled to 50-60°C, activated carbon is added and treated for 0.5-1.5 hours, and after centrifugation, it is filtered through diatomaceous earth and a 0.45 μm aqueous membrane.

4. The method for preparing brain polypeptide raw materials with brain-boosting, intelligence-enhancing, and nerve-protecting properties according to claim 3, characterized in that, The amount of activated carbon added is 3.0-5.0% of the bovine brain protein content.

5. The method for preparing the brain polypeptide raw material with brain-boosting, intelligence-enhancing, and nerve-protecting properties according to claim 3, characterized in that, The centrifugation conditions are 5000 rpm for 5-10 min.

6. A brain polypeptide raw material composition with brain-boosting, brain-protecting, and intelligence-enhancing properties, characterized in that, Prepared by the method according to any one of claims 1 to 5.

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