Omega3-containing composition and application thereof in promotion of brain and nerve development

Abstract

The invention relates to the field of biological medicine, in particular to a composition containing Omega3 and application of the composition in promotion of brain and nerve development. The invention provides a composition containing Omega3. The composition containing Omega3 comprises a conjugate formed by connecting Omega3 and walnut polypeptide. Wherein the walnut polypeptide is a small molecule active peptide obtained by a walnut kernel enzymolysis method, the amino acid sequence of the walnut polypeptide is as shown in SEQ ID No.1, and the conjugate is obtained by connecting Omega3 and the walnut polypeptide through a click chemistry method. The composition can promote brain and nerve development and has a clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a composition containing Omega3 and its use in promoting brain and nerve development. Background Technology

[0002] Omega-3 polyunsaturated fatty acids (Omega-3 PUFAs) are a class of bioactive substances that the human body cannot synthesize and must obtain from the diet. Their core members include alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Omega-3 PUFAs are essential nutrients for brain and nerve development. For example, DHA is a major structural component of neuronal cell membranes, accounting for 30-40% of the polyunsaturated fatty acids in the cerebral cortex. It can embed itself in the phospholipid bilayer, improving membrane fluidity, ensuring the conformational stability and normal function of neurotransmitter receptors, and accelerating nerve signal transduction. EPA reduces the production of pro-inflammatory factors by competing with metabolic enzymes, promotes the release of anti-inflammatory mediators, and reduces oxidative stress damage to neurons. Furthermore, Omega-3 PUFAs can promote BDNF expression, activate signaling pathways, regulate neuronal survival, differentiation, synapse formation and strengthening, enhance the long-term potentiation effect in the hippocampus, and support learning and memory functions. Walnut polypeptides, small-molecule bioactive peptides prepared by enzymatic hydrolysis of walnut proteins, exert effects on neuroprotection, cognitive enhancement, and nerve damage repair through mechanisms such as antioxidation, promoting neurotrophic function, and regulating synaptic plasticity. They represent a potential substance for nutritional intervention in brain and nerve health. Furthermore, CN119798368 also discloses that walnut polypeptides can cross the blood-brain barrier. Therefore, Omega-3 and walnut polypeptides show promising application prospects in brain and nerve development.

[0003] However, the bioavailability of Omega-3 polyunsaturated fatty acids is affected by factors such as source and dosage form, resulting in limited efficacy of single-drug intervention. Furthermore, walnut peptides are easily degraded in the complex in vivo environment, hindering their effectiveness. Most Omega-3 supplements are simply used in combination with Omega-3, often resulting in a synergistic effect where 1+1<2. Therefore, there is an urgent need for a compound that covalently links Omega-3 to supplements to overcome the technical challenges of single-drug Omega-3 intervention and the susceptibility of walnut peptides to degradation in the body, thereby improving the overall efficacy of the combination. Summary of the Invention

[0004] To address the above problems, the present invention provides a composition comprising a conjugate covalently linked to Omega3 and walnut polypeptide.

[0005] The amino acid sequence of the walnut polypeptide is shown in SEQ ID No. 1, and its specific preparation steps are as follows: 1. Walnut raw material pretreatment: After shelling the walnut kernels, dry them in a 60℃ constant temperature forced-air drying oven for 2-3 hours until the moisture content of the walnut kernels drops to 5%-8%; after drying, pulverize the walnut kernels with a high-speed universal pulverizer and pass them through a 40-mesh sieve to obtain coarse walnut powder; mix the coarse walnut powder with petroleum ether at a material-to-liquid ratio of 1:5 (g:mL), place the mixture in a stoppered conical flask, and defatt it with magnetic stirring in a 30℃ constant temperature water bath for 2 hours, stirring once every 30 minutes to promote oil dissolution; after defatting, filter and collect the filter residue; add petroleum ether to the filter residue again at a material-to-liquid ratio of 1:3, repeat the defatting once, and filter to obtain defatted walnut residue; place the defatted walnut residue in a fume hood, evaporate the petroleum ether at room temperature, pulverize it again and pass it through a 60-mesh sieve to obtain defatted walnut powder, and seal it for later use.

[0006] 2. Extraction and dissolution of walnut protein: Prepare 0.2 mol / L phosphate buffer (pH 7.0, for neutral enzymes) / (pH 8.5, for alkaline enzymes); mix defatted walnut powder with the above phosphate buffer at a material-to-liquid ratio of 1:15-1:20 (g:mL), place in an Erlenmeyer flask, and adjust the pH of the system to the optimal pH for the enzyme with 1 mol / L HCl or NaOH; place the Erlenmeyer flask in an ultrasonic cleaner and perform ultrasonic-assisted extraction at room temperature for 30 min (power 200W, frequency 40kHz) to disrupt the spatial structure of walnut protein and promote protein dissolution, thereby obtaining walnut protein extract.

[0007] 3. Enzymatic hydrolysis for preparing walnut polypeptides: Place the walnut protein extract in a constant temperature water bath and preheat to the optimal temperature of the enzyme (45-50℃ for neutral protease, 50-55℃ for alkaline protease), and keep warm for 10 minutes to ensure uniform temperature. Add neutral protease at 2%-4% of the walnut protein mass (enzyme-substrate ratio, E / S), and hydrolyze with magnetic stirring in a constant temperature water bath at 50℃ for 3 hours. During this period, monitor the pH of the system with a pH meter every 30 minutes. If the pH deviates from the optimal value, finely adjust it to the set value with 1mol / L HCl or NaOH. After the neutral protease hydrolysis is completed, raise the system temperature to 55℃, adjust the pH to 8.5, add alkaline protease at 1%-2% of the protein mass, and continue hydrolysis for 2 hours, for a total hydrolysis time of 5 hours. After hydrolysis, place the conical flask in a boiling water bath and heat for 10 minutes to completely inactivate the protease.

[0008] 4. Removal of impurities and crude extraction of enzymatic hydrolysate: After inactivation, the enzymatic hydrolysate is cooled to room temperature, transferred to centrifuge tubes, and placed in a high-speed refrigerated centrifuge. It is centrifuged at 8000 r / min and 4℃ for 20 min to remove insoluble residues. The supernatant is collected, which is the crude enzymatic hydrolysate of walnut polypeptides. The supernatant is filtered through medium-speed qualitative filter paper to further remove tiny suspended impurities, resulting in a clear polypeptide solution.

[0009] 5. Purification of Walnut Peptides: The clarified peptide solution was placed into a pretreated dialysis bag (molecular weight cutoff 3500 Da), the bag was sealed tightly, and dialyzed in a large amount of deionized water at 4°C with magnetic stirring. The deionized water was changed every 4 hours for 24 hours to remove free amino acids, inorganic salts, and small peptide fragments from the system. After dialysis, the purified peptide solution in the dialysis bag was collected. The purified peptide solution was transferred to a beaker and placed in an ice bath. Anhydrous ethanol was slowly added until the ethanol volume fraction of the system reached 70%, while stirring. The mixture was allowed to stand in an ice bath for 12 hours to allow the peptides to precipitate completely. After ethanol precipitation, the mixture was dialyzed at 6000 r / min and 4°C. Centrifuge for 15 min, collect the precipitate, which is the crude walnut polypeptide. Dissolve the crude walnut polypeptide in a small amount of deionized water to obtain a polypeptide aqueous solution, transfer it to a rotary evaporator, concentrate under reduced pressure at 40℃ to remove residual ethanol, and concentrate to 1 / 3-1 / 2 of the original volume to obtain a concentrated walnut polypeptide solution. Pour the concentrated polypeptide solution into a freeze-drying petri dish, spread it evenly (about 0.5-1 cm thick), place it in a freeze dryer, pre-freeze to -40℃ for 2 h, then turn on vacuum freeze-drying and freeze-dry for 24-36 h until the sample is completely dried into a loose white powder. Grind the dried powder into a fine powder using a mortar and pestle, pass it through an 80-mesh sieve to obtain a high-purity active walnut polypeptide product, seal it in a brown reagent bottle, store it in a refrigerator at 4℃ protected from light, and perform amino acid sequence determination.

[0010] The preparation steps of the coupling compound are as follows: Omega3 alkynylation modification (1) Place 50 mg (0.17 mmol) of Omega-3 in a 25 mL anhydrous round-bottom flask, add 10 mL of anhydrous DCM, stir magnetically until completely dissolved, and purge with nitrogen gas ( )Protect; (2) Add 20 μL (0.20 mmol) of propynyl anhydride (alkynylating agent, 10%-20% excess to ensure complete reaction) to the flask, then slowly add 15 μL (0.11 mmol) of anhydrous TEA at room temperature. Protect the stirring reaction for 4 hours.

[0011] Azide modification of peptides (1) Take 20 mg of walnut polypeptide, dissolve it in 5 mL of 0.1 M PBS buffer (pH 7.4), place it in a 10 mL centrifuge tube, and sonicate to aid dissolution, ensuring no precipitation; (2) Weigh out 5mg (Azide hydration reagent, 5-10 times excess, to avoid cross-linking of the peptide itself), dissolve in 1 mL of anhydrous DMF, slowly add dropwise to the peptide solution, gently shaking continuously during the addition process, and react at room temperature in the dark for 2 hours.

[0012] Click on the chemical reaction to construct Omega3 peptide conjugates (1) Take 20 mg of alkynylated Omega3, dissolve it in 2 mL of anhydrous DMF, transfer it to a 25 mL round-bottom flask, add 15 mg of azido-modified walnut polypeptide, stir magnetically until completely dissolved, and then purge with water. Protect; (2) Take 2mg Dissolve 5 mg of NaAsc in 1 mL of deionized water, and dissolve 5 mg of NaAsc in 1 mL of deionized water. Mix the two solutions and quickly add them to the flask. Then add 3 mg of TBTA (dissolved in 0.5 mL of DMF), and add 0.1 M PBS buffer (pH 7.4) to bring the total volume of the reaction system to 10 mL. Adjust the pH of the system to 7.2-7.5. (3) At room temperature Protect from light and stir the reaction for 6-8 hours.

[0013] Among them, Omega3 is DHA, and the amino acid sequence of the walnut polypeptide is shown in SEQ ID No.1: DDGAFNVPG.

[0014] Another aspect of the present invention is to provide a composition containing Omega3, specifically, the composition comprising the above-described conjugate.

[0015] Another aspect of the present invention is to provide a pharmaceutical composition, specifically, the pharmaceutical composition comprising the above-described conjugate, and the dosage form of the pharmaceutical composition being capsules or tablets. The pharmaceutical composition has the effect of promoting brain and nerve development.

[0016] Another aspect of the present invention is to provide an application in which the above-mentioned conjugate is used to prepare a pharmaceutical composition that promotes brain and nerve development.

[0017] Beneficial effects of this invention: The present invention extracts small molecule active peptides from walnuts, the amino acid sequence of which is shown in SEQ ID No.1. These peptides are covalently linked to DHA via click chemistry, and are carried across the blood-brain barrier by DHA. They work synergistically with DHA to promote brain and nerve development. Attached Figure Description

[0018] Figure 1 : Walnut polypeptide amino acid sequence structure Figure 2 Brain Index Figure 3 NGF protein expression level Figure 4 BDNF protein expression level Detailed Implementation

[0019] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments. Example 1: Preparation of walnut polypeptides

[0020] 1. Walnut raw material pretreatment: After shelling the walnut kernels, place them in a 60℃ constant temperature forced-air drying oven for 2 hours until the moisture content of the walnut kernels drops to 5%; after drying, pulverize the walnut kernels with a high-speed universal pulverizer and pass them through a 40-mesh sieve to obtain coarse walnut powder; mix the coarse walnut powder with petroleum ether at a material-to-liquid ratio of 1:5 (g:mL), place the mixture in a stoppered conical flask, and defatt the mixture in a 30℃ constant temperature water bath with magnetic stirring for 2 hours, stirring once every 30 minutes to promote oil dissolution; after defatting, filter the mixture and collect the filter residue; add petroleum ether to the filter residue again at a material-to-liquid ratio of 1:3, repeat the defatting process once, and filter again to obtain defatted walnut residue; place the defatted walnut residue in a fume hood, evaporate the petroleum ether at room temperature, pulverize it again and pass it through a 60-mesh sieve to obtain defatted walnut powder, and seal it for later use.

[0021] 2. Extraction and dissolution of walnut protein: Prepare 0.2 mol / L phosphate buffer (pH 7.0, for neutral enzymes) / (pH 8.5, for alkaline enzymes); mix defatted walnut powder with the above phosphate buffer at a material-to-liquid ratio of 1:15 (g:mL), place in an Erlenmeyer flask, and adjust the pH of the system to the optimal pH of the enzyme with 1 mol / L HCl or NaOH; place the Erlenmeyer flask in an ultrasonic cleaner and perform ultrasonic-assisted extraction at room temperature for 30 min (power 200W, frequency 40kHz) to disrupt the spatial structure of walnut protein and promote protein dissolution, thereby obtaining walnut protein extract.

[0022] 3. Enzymatic hydrolysis for preparing walnut polypeptides: Place the walnut protein extract in a constant temperature water bath and preheat to the optimal temperature of the enzyme (45-50℃ for neutral protease, 50-55℃ for alkaline protease), and keep warm for 10 minutes to ensure uniform temperature. Add neutral protease at 2% of the walnut protein mass (enzyme-substrate ratio, E / S), and hydrolyze with magnetic stirring in a constant temperature water bath at 50℃ for 3 hours. During this period, monitor the pH of the system with a pH meter every 30 minutes. If the pH deviates from the optimal value, finely adjust it to the set value with 1mol / L HCl or NaOH. After the neutral protease hydrolysis is completed, raise the system temperature to 55℃, adjust the pH to 8.5, add alkaline protease at 1% of the protein mass, and continue hydrolysis for 2 hours, for a total hydrolysis time of 5 hours. After hydrolysis, place the conical flask in a boiling water bath and heat for 10 minutes to completely inactivate the protease.

[0023] 4. Removal of impurities and crude extraction of enzymatic hydrolysate: After inactivation, the enzymatic hydrolysate is cooled to room temperature, transferred to centrifuge tubes, and centrifuged in a high-speed refrigerated centrifuge at 8000 r / min and 4℃ for 20 min to remove insoluble residues. The supernatant is collected, which is the crude enzymatic hydrolysate of walnut polypeptides. The supernatant is then filtered through medium-speed qualitative filter paper to further remove tiny suspended impurities, resulting in a clear polypeptide solution.

[0024] 5. Purification of Walnut Peptides: The clarified peptide solution was placed into a pretreated dialysis bag (molecular weight cutoff 3500 Da), the bag was sealed tightly, and dialyzed in a large amount of deionized water at 4°C with magnetic stirring. The deionized water was changed every 4 hours for 24 hours to remove free amino acids, inorganic salts, and small peptide fragments from the system. After dialysis, the purified peptide solution in the dialysis bag was collected. The purified peptide solution was transferred to a beaker and placed in an ice bath. Anhydrous ethanol was slowly added until the ethanol volume fraction of the system reached 70%, while stirring. The mixture was allowed to stand in an ice bath for 12 hours to allow the peptides to precipitate completely. After ethanol precipitation, the mixture was dialyzed at 6000 r / min and 4°C. Centrifuge for 15 min, collect the precipitate, which is the crude walnut polypeptide. Dissolve the crude walnut polypeptide in a small amount of deionized water to obtain a polypeptide aqueous solution, transfer it to a rotary evaporator, concentrate under reduced pressure at 40℃ to remove residual ethanol, concentrate to 1 / 2 of the original volume to obtain a concentrated walnut polypeptide solution. Pour the concentrated polypeptide solution into a freeze-drying petri dish, spread it evenly, place it in a freeze dryer, pre-freeze to -40℃ for 2 h, then turn on vacuum freeze-drying for 36 h until the sample is completely dried into a loose white powder. Grind the dried powder into a fine powder in a mortar and pestle, pass it through an 80-mesh sieve to obtain a high-purity active walnut polypeptide product, seal it in a brown reagent bottle, store it in a refrigerator at 4℃ protected from light, and perform amino acid sequence determination. The amino acid sequence of the walnut polypeptide is shown in SEQ ID No. 1: DDGAFNVPG ( Figure 1 ). Example 2: Preparation of Couplings

[0025] Omega3 alkynylation modification (1) Place 50 mg (0.17 mmol) of Omega-3 in a 25 mL anhydrous round-bottom flask, add 10 mL of anhydrous DCM, stir magnetically until completely dissolved, and then purge with nitrogen gas ( )Protect; (2) Add 20 μL (0.20 mmol) propynyl anhydride (alkynylating agent, 10%-20% excess to ensure complete reaction) to the flask, then slowly add 15 μL (0.11 mmol) anhydrous TEA at room temperature. Protect the stirring reaction for 4 hours.

[0026] Azide modification of peptides (1) Take 20 mg of walnut polypeptide, dissolve it in 5 mL of 0.1 M PBS buffer (pH 7.4), place it in a 10 mL centrifuge tube, and sonicate to aid dissolution, ensuring no precipitation; (2) Weigh out 5mg (Azide hydration reagent, 5-10 times excess, to avoid cross-linking of the peptide itself), dissolve in 1 mL of anhydrous DMF, slowly add dropwise to the peptide solution, gently shaking continuously during the addition process, and react at room temperature in the dark for 2 hours.

[0027] Click on the chemical reaction to construct Omega3 peptide conjugates (1) Take 20 mg of alkynylated Omega3, dissolve it in 2 mL of anhydrous DMF, transfer it to a 25 mL round-bottom flask, add 15 mg of azido-modified walnut polypeptide, stir magnetically until completely dissolved, and then purge with... Protect; (2) Take 2mg Dissolve 5 mg of NaAsc in 1 mL of deionized water, and dissolve 5 mg of NaAsc in 1 mL of deionized water. Mix the two solutions and quickly add them to the flask. Then add 3 mg of TBTA (dissolved in 0.5 mL of DMF), and add 0.1 M PBS buffer (pH 7.4) to bring the total volume of the reaction system to 10 mL. Adjust the pH of the system to 7.5. (3) At room temperature Protect from light and stir the reaction for 8 hours. Example of results:

[0028] Forty 3-week-old mice were randomly divided into four groups of 10 mice each: group A (saline), group B (DHA), group C (walnut polypeptide), and group D (DHA-walnut polypeptide). There were no significant differences in initial body weight and health status among the groups.

[0029] After 7 days of feeding, mice with abnormal health conditions were removed, ensuring 6 mice per group. Mice were then administered gavage according to the above groupings: Group A received saline, Group B received DHA, Group C received walnut peptides, and Group D received DHA-walnut peptides. The concentrations of DHA, walnut peptides, and DHA-walnut peptides were all 200 mg / kg·d, with a gavage volume of 0.2 ml / 10 g. Gavage was performed every morning for 21 consecutive days.

[0030] The day after gavage, five mice from each group were randomly selected and fasted for 3 hours before being weighed. They were then euthanized by cervical dislocation, and their brain tissue was quickly dissected, rinsed with saline, and dried with filter paper. The brains were then weighed, and the brain index was calculated (brain index = brain weight / mouse body weight × 100%).

[0031] The results showed that compared with the saline group, the DHA group, walnut peptide group, and DHA-walnut peptide group had higher brain indices, with the DHA-walnut peptide group having the highest brain indices. This indicates that coupling DHA and walnut peptides can effectively increase brain weight and promote brain development. Figure 2 ).

[0032] Proteins were extracted from the brain tissue using a standard protein extraction kit and Western blot was performed to detect the expression levels of NGF and BDNF proteins.

[0033] The results showed that the DHA group, walnut peptide group, and DHA-walnut peptide group had higher NGF and BDNF protein expression levels compared with the saline group, with the DHA-walnut peptide group showing the highest expression level. This indicates that coupling DHA and walnut peptide can effectively increase the expression levels of NGF and BDNF proteins and promote brain and nerve development. Figure 3 and Figure 4 ).

[0034] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A composition, characterized in that, The composition comprises a conjugate covalently linked to Omega3 and walnut polypeptide.

2. The composition according to claim 1, characterized in that, The conjugate is obtained by linking Omega3 and walnut polypeptide via click chemistry, wherein the Omega3 is DHA and the amino acid sequence of the walnut polypeptide is shown in SEQ ID No. 1: DDGAFNVPG.

3. The composition according to claim 1, characterized in that, The preparation method of the walnut polypeptide is as follows: (1) Pretreatment of walnut raw materials: After shelling the walnut kernels, they are placed in a 60℃ constant temperature forced air drying oven for 2-3 hours until the moisture content of the walnut kernels drops to 5%-8%; the dried walnut kernels are crushed by a high-speed universal pulverizer and passed through a 40-mesh sieve to obtain coarse walnut powder; the coarse walnut powder is mixed with petroleum ether at a material-to-liquid ratio of 1:5 (g:mL), placed in a stoppered conical flask, and degreased by magnetic stirring in a 30℃ constant temperature water bath for 2 hours, stirring once every 30 minutes to promote the dissolution of oil; after degreasing, the mixture is filtered and the filter residue is collected; the filter residue is added with petroleum ether again at a material-to-liquid ratio of 1:3, and the degreasing is repeated once, and the degreased walnut residue is obtained by filtration; the degreased walnut residue is placed in a fume hood, the petroleum ether is evaporated at room temperature, and it is crushed again and passed through a 60-mesh sieve to obtain degreased walnut powder, which is then sealed for later use; (2) Extraction and dissolution of walnut protein: Prepare 0.2 mol / L phosphate buffer (pH 7.0, for neutral enzymes) / (pH 8.5, for alkaline enzymes); mix defatted walnut powder with the above phosphate buffer at a material-to-liquid ratio of 1:15-1:20 (g:mL), place in an Erlenmeyer flask, and adjust the pH of the system to the optimal pH of the enzyme with 1 mol / L HCl or NaOH; place the Erlenmeyer flask in an ultrasonic cleaner and perform ultrasonic-assisted extraction at room temperature for 30 min (power 200W, frequency 40kHz) to destroy the spatial structure of walnut protein and promote protein dissolution, thereby obtaining walnut protein extract; (3) Enzymatic hydrolysis to prepare walnut polypeptides: Place the walnut protein extract in a constant temperature water bath and preheat it to the optimal temperature of the enzyme (45-50℃ for neutral protease and 50-55℃ for alkaline protease). Keep it warm for 10 min to make the system temperature uniform. Add neutral protease at 2%-4% of the walnut protein mass (enzyme-substrate ratio, E / S). Stir the mixture magnetically in a constant temperature water bath at 50℃ for 3 h. During this period, monitor the pH of the system with a pH meter every 30 min. If the pH deviates from the optimal value, finely adjust it to the set value with 1 mol / L HCl or NaOH. After the neutral protease hydrolysis is completed, raise the system temperature to 55℃, adjust the pH to 8.5, add alkaline protease at 1%-2% of the protein mass, and continue the hydrolysis for 2 h. The total hydrolysis time is 5 h. After the hydrolysis is completed, place the conical flask in a boiling water bath and heat for 10 min to completely inactivate the protease. (4) Removal of impurities and crude extraction of enzymatic hydrolysate: After the enzymatic hydrolysate is deactivated, it is cooled to room temperature, transferred to a centrifuge tube, placed in a high-speed refrigerated centrifuge, centrifuged at 8000 r / min and 4℃ for 20 min to remove insoluble residues and collect the supernatant, which is the crude enzymatic hydrolysate of walnut polypeptide; the supernatant is filtered with medium-speed qualitative filter paper to further remove tiny suspended impurities and obtain a clear polypeptide solution; (5) Purification of walnut polypeptides: The clarified polypeptide solution was placed into a pretreated dialysis bag (molecular weight cutoff 3500 Da), the bag was sealed tightly, and dialyzed in a large amount of deionized water at 4°C with magnetic stirring. The deionized water was changed every 4 hours and dialyzed continuously for 24 hours to remove free amino acids, inorganic salts, and small molecule peptides from the system. After dialysis, the purified polypeptide solution in the dialysis bag was collected. The purified polypeptide solution was transferred to a beaker and placed in an ice bath. Anhydrous ethanol was slowly added until the volume fraction of ethanol in the system reached 70%, while stirring. The mixture was allowed to stand in an ice bath for 12 hours to allow the polypeptides to precipitate fully. After ethanol precipitation, the mixture was dialyzed at 6000 r / min and 4°C. Centrifuge for 15 min, collect the precipitate, which is the crude walnut polypeptide. Dissolve the crude walnut polypeptide in a small amount of deionized water to obtain a polypeptide aqueous solution, transfer it to a rotary evaporator, concentrate under reduced pressure at 40℃ to remove residual ethanol, and concentrate to 1 / 3-1 / 2 of the original volume to obtain a concentrated walnut polypeptide solution. Pour the concentrated polypeptide solution into a freeze-drying petri dish, spread it evenly (about 0.5-1 cm thick), place it in a freeze dryer, pre-freeze to -40℃ for 2 h, then turn on vacuum freeze-drying and freeze-dry for 24-36 h until the sample is completely dried into a loose white powder. Grind the dried powder into a fine powder using a mortar and pestle, pass it through an 80-mesh sieve to obtain a high-purity active walnut polypeptide product, seal it in a brown reagent bottle, store it in a refrigerator at 4℃ protected from light, and perform amino acid sequence determination.

4. The composition according to claim 2, characterized in that, The preparation method of the aforementioned coupling compound is as follows: a. Omega3 alkynylation modification (1) Take 50 mg (0.17 mmol) of Omega-3 and place it in a 25 mL anhydrous round-bottom flask. Add 10 mL of anhydrous DCM and stir magnetically until completely dissolved. Then, purge with nitrogen gas. Protect; (2) Add 20 μL (0.20 mmol) propynyl anhydride (alkynylating agent, 10%-20% excess to ensure complete reaction) to the flask, then slowly add 15 μL (0.11 mmol) anhydrous TEA at room temperature. Protect the stirring reaction for 4 hours; b. Azide modification of peptides (1) Take 20 mg of walnut polypeptide, dissolve it in 5 mL of 0.1 M PBS buffer (pH 7.4), place it in a 10 mL centrifuge tube, and sonicate to aid dissolution, ensuring no precipitation; (2) Weigh out 5mg (Azide hydration reagent, 5-10 times excess, to avoid cross-linking of the peptide itself), dissolve in 1 mL of anhydrous DMF, slowly add dropwise to the peptide solution, gently shaking continuously during the addition process, and react at room temperature in the dark for 2 hours; c. Click on chemical reactions to construct Omega3 peptide conjugates (1) Take 20 mg of alkynylated Omega3, dissolve it in 2 mL of anhydrous DMF, transfer it to a 25 mL round-bottom flask, add 15 mg of azido-modified walnut polypeptide, stir magnetically until completely dissolved, and then purge with... Protect; (2) Take 2mg Dissolve 5 mg of NaAsc in 1 mL of deionized water, and dissolve 5 mg of NaAsc in 1 mL of deionized water. Mix the two solutions and quickly add them to the flask. Then add 3 mg of TBTA (dissolved in 0.5 mL of DMF), and add 0.1 M PBS buffer (pH 7.4) to bring the total volume of the reaction system to 10 mL. Adjust the pH of the system to 7.2-7.

5. (3) At room temperature Protect from light and stir the reaction for 6-8 hours.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the conjugate according to any one of claims 1-4, and the dosage form of the pharmaceutical composition is a capsule or a tablet.

6. Use of the composition according to any one of claims 1-4 in the preparation of a medicament for promoting brain and nerve development.

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