Walnut peptide with neuroprotection function as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioactive peptides, and discloses a walnut peptide with a neuroprotection function and a preparation method and application thereof, and predicts that a batch of small molecular walnut peptides have the neuroprotection function through enzymolysis of natural walnut protein, molecular weight screening and molecular docking. Experiments prove that the walnut peptide disclosed by the invention has a nerve protection function: the cell activity of nerve cells is improved, and the walnut peptide plays a role in preventing neurodegenerative diseases and other nerve-related diseases; the toxic effect of oxidative damage on nerve cells is relieved, nerve cell apoptosis is inhibited, the cell viability of the nerve cells is improved, and the function of treating nerve injury related diseases is achieved; the compound can be used for improving the activity of nerve cells, relieving oxidative damage of the nerve cells, adjusting the protein level of nerve damage related pathways, preparing related drugs for reducing blood pressure or resisting senile dementia or tumors and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptides, and in particular to a walnut peptide with neuroprotective function and a preparation method and application thereof. Background Art

[0002] Walnut (Juglans regia L.) is the most widely distributed nut in the world and is also considered to be the oldest nut in the world. It originated in East Asia, Southeast Europe and North America. Walnut is rich in unsaturated fatty acids, proteins, polyphenols and minerals, and has important biological activities such as antioxidant, anti-tumor, anti-inflammatory, cholesterol-lowering, blood pressure-lowering and reducing the risk of cardiovascular diseases. The fat content of walnut kernel is as high as 52-74%, which can be processed into walnut oil. Walnut oil is rich in polyunsaturated fatty acids and is deeply loved by consumers, and the demand is increasing year by year. The increase in the market demand for walnut oil has led to an increase in walnut residue, a byproduct of walnut oil. Walnut residue contains about 40-45% protein. Walnut protein contains 18 kinds of amino acids, of which the content of 8 essential amino acids meets the needs of the human body, which is close to the standard recommendations formulated by the World Health Organization (WHO), the Food and Agriculture Organization (FAO) and the United Nations University (NUN) in 2007. It is considered to be an important source of plant protein in human food and a plant protein with great development potential.

[0003] Food-derived peptides have many advantages, such as easy absorption, non-toxicity, high specificity and rich biological activity. Some studies have shown that peptides with smaller molecular weights can more easily cross the intestinal barrier and pass through the cell membrane. Bioactive peptides are composed of 2 to 20 amino acids connected by peptide bonds and have received special attention in the fields of nutrition and food science. They are considered to be one of the most promising research areas in the international food industry. Bioactive peptides can have a variety of physiological functions, such as antioxidant, antihypertensive, immune and neuroactive. The main source of bioactive peptides is animal and plant food proteins, which are obtained by enzymatic hydrolysis, fermentation or in vivo or in vitro digestion.

[0004] At present, there are few studies on walnut peptides in the field of neuroprotection, and they are mainly focused on improving learning and memory. There are few studies in the fields of anti-Alzheimer's disease, for example, patent CN202410954437.2 provides a highly active antioxidant walnut-derived peptide for improving learning and memory and its application. Walnut peptide YVPFPLP is used as a template, and the amino acid sequence is replaced to obtain a walnut-derived peptide YVPFPYP with better antioxidant activity and better memory improvement activity. It can protect brain neurons from damage, improve the body's learning and memory, and may help treat neurodegenerative diseases mainly related to oxidative stress. However, its disadvantages are: 1. Walnut-derived peptides with only one amino acid sequence replacement, it can be seen that its preparation method is cumbersome; 2. It mainly acts on oxidative stress-related pathways and synapses, mainly improving learning and memory, and it is impossible to determine its therapeutic effect on neurodegenerative diseases; 3. Although the antioxidant-related content is recorded, it is mainly to improve learning and memory, and it is difficult to infer how much effect it actually plays in neuroprotection.

[0005] Therefore, there is an urgent need to improve the preparation method of walnut peptides and obtain walnut peptides that are related to other neuroprotective pathways, so that they can play a stronger neuroprotective role in addition to improving learning and memory, and play a role in the treatment of neurodegenerative diseases and other neuro-related diseases. Summary of the invention

[0006] In order to solve the above-mentioned existing problems, the present invention discloses a walnut peptide with neuroprotective function and a preparation method and application thereof. By enzymatically hydrolyzing natural walnut protein and screening the molecular weight, a group of small molecule walnut peptides with neuroprotective function are predicted through molecular docking, and experiments verify that the walnut peptide of the present invention has neuroprotective function, and has broad application prospects.

[0007] The neuroprotective function of the present invention has the following meanings: 1. When not damaged, the cell viability of nerve cells is improved, thereby playing a preventive role in neurodegenerative diseases and other nerve-related diseases; 2. When damaged, the toxic effect of the damage on nerve cells is reduced, nerve cell apoptosis is inhibited, and the cell viability of nerve cells is improved, thereby playing a role in treating nerve damage-related diseases; 3. The levels of key proteins in nerve damage-related pathways are regulated, thereby playing a role in preventing or treating neurodegenerative diseases and other nerve-related diseases.

[0008] In one aspect, the present invention provides a walnut peptide having neuroprotective function, including any one or more of the peptides having the amino acid sequences shown in Seq ID NO.1-23.

[0009] The peptide having the amino acid sequence shown in Seq ID NO.1-23 is obtained by enzymatically hydrolyzing natural walnut protein under certain conditions and then screening the molecular weight below 1000Da. Molecular docking simulation shows that the above walnut peptide has good binding with the anti-Alzheimer's target acetylcholinesterase (AChE) and β-amyloid precursor protein cleavage enzyme 1 (BACE1), indicating that it has an anti-Alzheimer's effect; further experiments show that the above walnut peptide has the effect of improving the cell viability of nerve cells. When the nerve cells are treated with hydrogen peroxide, the above walnut peptide improves the cell viability of oxidatively damaged nerve cells, inhibits cell apoptosis, reduces the toxic effect of oxidative damage on nerve cells, downregulates the level of lipid peroxide malondialdehyde (MDA) and increases the levels of antioxidant superoxide dismutase (SOD) and catalase (CAT). Therefore, the above walnut peptide The peptide has the effect of reducing the oxidative damage of nerve cells; combined with walnut peptide to improve cell viability and reduce the oxidative damage of nerve cells, the above-mentioned walnut peptide has a neuroprotective function. The present invention studies the above-mentioned effect, one of the mechanisms of which is to increase the levels of nuclear factor-E2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins in the Nrf2 / HO-1 signaling pathway, and inhibit the level of Kelch-like ECH associated protein 1 (KEAP1) protein to reduce the oxidative damage of H2O2 to human neuroblastoma cells (SH-SY5Y cells); and, compared with natural small molecule walnut peptides, the solubility of enzymatically hydrolyzed walnut peptides with a molecular weight below 1000Da after enzymatic hydrolysis and screening is improved, and the degree of docking with the target molecule is significantly enhanced, which can significantly enhance the improvement of nerve cell viability and reduce the oxidative damage of nerve cells.

[0010] In molecular docking simulation, it is known that the above-mentioned walnut peptide has good binding with the anti-Alzheimer's target AChE or BACE1, which is one of the pathways for exerting neuroprotective effects. Therefore, peptides with any amino acid sequence in Seq ID NO.1-23 have neuroprotective functions; and according to molecular docking simulation, different peptides bind to different sites of the target protein. It can be understood that multiple different peptides can bind to different sites of the target protein at the same time, so the neuroprotective effects between multiple different peptides can be accumulated. At the same time, a mixture of all 23 peptides can be obtained by enzymatic hydrolysis and screening, and all 23 peptides with amino acid sequences in Seq ID NO.1-23 are selected as a composition as a preferred solution.

[0011] Preferably, the walnut peptides having neuroprotective function include any one or more of the peptides having the amino acid sequences shown in Seq ID NO.6, Seq ID NO.13, Seq ID NO.20, Seq ID NO.21, Seq ID NO.16 and Seq ID NO.11.

[0012] According to the molecular docking model diagram, the above six walnut peptides can tightly bind to the key active centers or key catalytic centers of the anti-Alzheimer's targets acetylcholinesterase (AChE) and β-amyloid precursor protein cleaving enzyme 1 (BACE1) to form a tight complex, thereby significantly inhibiting the activity of AChE and BACE1. Compared with other walnut peptides, they can play a better neuroprotective function. In addition, the above six walnut peptides have a relatively lower molecular weight, which is more conducive to the improvement of absorption and bioavailability; it can be understood that the above six walnut peptides can be used as a walnut peptide composition as a preferred option.

[0013] Preferably, the walnut peptides with neuroprotective function include any one or more of the peptides having amino acid sequences shown in Seq ID NO.1, Seq ID NO.2, Seq ID NO.12, Seq ID NO.14, Seq ID NO.15, Seq ID NO.17, Seq ID NO.19, Seq ID NO.21, Seq ID NO.22 and Seq ID NO.23.

[0014] The molecular docking score of the walnut peptide with the above 10 amino acid sequences with BACE1 is higher than that with AChE, and the difference is ≥30, indicating that the walnut peptide with the above 10 amino acid sequences is more inclined to bind to the target BACE1. It is known that BACE1 is related to neurodegenerative diseases. BACE1 accumulates in axons, causing axonal swelling. Enzymatic walnut peptide is more conducive to binding to BACE1 and inhibiting its activity than natural walnut peptide. Therefore, the walnut peptide with the above 10 amino acid sequences theoretically has a stronger neuroprotective and therapeutic effect on neurodegenerative diseases.

[0015] Further preferably, the walnut peptides having neuroprotective function include any one or more of the peptides having the amino acid sequences shown in Seq ID NO.12, Seq ID NO.14, Seq ID NO.15, Seq ID NO.22 and Seq ID NO.23.

[0016] The molecular docking score of the walnut peptides with the above 5 amino acid sequences with BACE1 is higher than 100, and the molecular docking score with AChE is lower than 100, indicating that it is more conducive to binding with BACE1 and inhibiting its activity. Therefore, it has a stronger effect of protecting nerves or treating neurodegenerative diseases in theory, and is a more preferred group of enzymatic walnut peptides.

[0017] The key to the present invention is that a series of enzymatically hydrolyzed walnut peptides are obtained by enzymatic hydrolysis and molecular weight screening, and their neuroprotective function is predicted and verified. Therefore, the walnut peptides with neuroprotective function can be selectively synthesized according to the amino acid sequence shown in Seq ID NO.1-23; the method of enzymatic hydrolysis of natural walnut protein of the present invention can also be repeated. Due to the difference in walnut raw materials and enzymes, theoretically, walnut peptides with some sequences disclosed by the present invention and other enzymatically hydrolyzed walnut peptides with undisclosed sequences of the present invention can be obtained.

[0018] On the other hand, the present invention provides a method for preparing walnut peptides with neuroprotective function, which comprises: taking a natural walnut protein solution, adding flavor protease for enzymatic hydrolysis, and screening out small molecule walnut peptides of 800Da and below.

[0019] In some embodiments, during the enzymatic hydrolysis process, the concentration of the flavor protease is 2-8%.

[0020] In some embodiments, during the enzymatic hydrolysis process, the concentration of natural walnut protein is 4-10%.

[0021] In some embodiments, during the enzymatic hydrolysis process, the temperature is set at 30-60°C.

[0022] In some embodiments, during the enzymatic hydrolysis process, the pH value is set to 2-8.

[0023] Preferably, during the enzymatic hydrolysis process, the concentration of flavor protease is 8%, the concentration of natural walnut protein is 10%, the temperature is set to 50° C., and the pH value is set to 6.

[0024] After single factor test and orthogonal experiment, the order of factors affecting the yield of walnut peptide in the preparation process of walnut peptide is the concentration of flavor protease, the concentration of natural walnut protein, temperature, pH and time; the optimal enzymatic hydrolysis process parameters are the concentration of flavor protease of 8%, the concentration of natural walnut protein of 10%, the temperature set to 50℃, and the pH value set to 6.

[0025] On the other hand, the present invention provides a use of the walnut peptide with neuroprotective function for preparing an agent for improving the activity of nerve cells.

[0026] Experiments have shown that the walnut peptide with neuroprotective function of the present invention can improve the cell viability of nerve cells regardless of whether they are damaged by oxidation. Improving the cell viability of nerve cells can improve learning and memory, and can also be used to produce brain health products. There are more applications in special scenarios, such as improving the cell viability of motor nerves and promoting the rehabilitation of patients' motor functions.

[0027] On the other hand, the present invention provides a use of the walnut peptide with neuroprotective function for preparing an agent for reducing oxidative damage to nerve cells.

[0028] When nerve cells are damaged by oxidation, the above-mentioned reduction of the oxidative damage to nerve cells has any one or more of the following meanings: improving cell viability, reducing cell apoptosis rate, reducing the toxic effect of oxidative damage on cells, lowering the level of peroxides, and increasing the level of antioxidants.

[0029] The meaning of the above-mentioned oxidative damage can be applied in many specific ways, for example, it can help treat or alleviate neurodegenerative diseases. For example, sequelae of cerebrovascular diseases such as brain trauma, cerebral vascular sclerosis (cerebral hemorrhage, cerebral thrombosis), sequelae of encephalitis and meningitis, demyelinating diseases, etc. can also involve nerve damage. The walnut peptide of the present invention can alleviate the sequelae of the above-mentioned diseases. Since walnut peptide also has the effect of inhibiting cell apoptosis, it can theoretically also have a certain therapeutic effect.

[0030] On the other hand, the present invention provides a use of the walnut peptide with neuroprotective function for preparing an agent for increasing Nrf2 protein level and / or increasing HO-1 protein level and / or inhibiting Keap1 protein level.

[0031] Experiments have shown that one of the mechanisms of the neuroprotective function of the walnut peptide with neuroprotective function of the present invention is: increasing the levels of Nrf2 and HO-1 proteins in the Nrf2 / HO-1 signaling pathway and inhibiting the level of Keap1 protein to reduce the oxidative damage of H2O2 to SH-SY5Y cells; however, the above-mentioned proteins are not limited to the nerve damage pathway in individuals, and other physiological processes and functions related to the above-mentioned proteins can also be regulated by the walnut peptide of the present invention.

[0032] On the other hand, the present invention provides a use of the walnut peptide with neuroprotective function for preparing an agent for lowering blood pressure and / or preventing Alzheimer's disease and / or preventing tumors, wherein the walnut peptide with neuroprotective function is docked with angiotensin converting enzyme, a blood pressure lowering target, and / or AChE, an anti-Alzheimer's disease target, β-amyloid precursor protein cleaving enzyme 1, and / or an anti-tumor target aminopeptidase N molecule.

[0033] The present invention also predicts the molecular docking of enzymatic walnut peptides with angiotensin converting enzyme (ACE) for lowering blood pressure, acetylcholinesterase (AChE) for anti-Alzheimer's disease, beta-amyloid precursor protein cleaving enzyme 1 (BACE1) and aminopeptidase N (APN) for anti-tumor. Based on the results of molecular docking, it can be inferred that the walnut peptide with neuroprotective function of the present invention can be used for lowering blood pressure, anti-Alzheimer's disease and anti-tumor; for example, it is known that the activity of AChE in neuronal cell bodies and proximal axons is enhanced in the early stage of nerve injury, and the walnut peptide of the present invention can bind to it and inhibit its activity, thereby reducing nerve injury, and the effect of reducing nerve injury has been verified by experiments. The walnut peptide of the present invention has a significantly enhanced effect of reducing nerve injury compared with the natural small molecule walnut peptide, which is also due to the higher degree of docking of the walnut peptide of the present invention with the target protein; therefore, the correlation between the molecular docking with the target and the efficacy is also verified. Based on the above mechanism, it can be understood that the efficacy of the walnut peptide of the present invention in reducing blood pressure, anti-Alzheimer's disease and anti-tumor is also significantly higher than that of the natural small molecule walnut peptide.

[0034] Preferably, the walnut peptides having neuroprotective function include any one or more of the peptides having the amino acid sequences shown in Seq ID NO.6, Seq ID NO.13, Seq ID NO.20, Seq ID NO.21, Seq ID NO.16 and Seq ID NO.11.

[0035] Preferably, the walnut peptides with neuroprotective function include any one or more of the peptides having amino acid sequences shown in Seq ID NO.1, Seq ID NO.2, Seq ID NO.12, Seq ID NO.14, Seq ID NO.15, Seq ID NO.17, Seq ID NO.19, Seq ID NO.21, Seq ID NO.22 and Seq ID NO.23.

[0036] Further preferably, the walnut peptides having neuroprotective function include any one or more of the peptides having the amino acid sequences shown in Seq ID NO.12, Seq ID NO.14, Seq ID NO.15, Seq ID NO.22 and Seq ID NO.23.

[0037] For the above uses, the term "reagent" includes any product that can exert the neuroprotective function of the walnut peptide of the present invention, such as food preparations, cosmetic or skin care preparations, and pharmaceutical preparations.

[0038] In summary, the present invention includes the following beneficial technical effects:

[0039] 1. The present invention provides a walnut peptide with neuroprotective function, which improves the cell viability of nerve cells when not damaged, thereby preventing neurodegenerative diseases and other nerve-related diseases; when damaged, it reduces the toxic effect of the damage on nerve cells, inhibits nerve cell apoptosis, and improves the cell viability of nerve cells, thereby treating nerve damage-related diseases; regulates the levels of key proteins in nerve damage-related pathways, thereby preventing or treating neurodegenerative diseases and other nerve-related diseases;

[0040] 2. The present invention provides the specific amino acid sequence of the walnut peptide with neuroprotective function, which can be selectively synthesized according to the amino acid sequence shown in Seq ID NO.1-23; the method of the present invention for enzymatic hydrolysis of natural walnut protein can also be repeated to theoretically obtain walnut peptides with a sequence disclosed in part of the present invention;

[0041] 3. The present invention found through molecular docking mode that some walnut peptides can produce tighter binding with the key active center or catalytic center of AChE or BACE1 to form a tight complex, thereby significantly inhibiting the activity of AChE and BACE1, and selected six walnut peptides, TDELL, VPQNF, LPEDV, VALEP, TPSLV and VALVT, as the preferred scheme;

[0042] 4. The present invention analyzes the molecular docking prediction results between enzymatic walnut peptides and targets, and finds that 10 enzymatic walnut peptides have a significant tendency to bind to BACE1, from which 5 walnut peptides with higher binding tendency are selected, which can be used for early intervention or treatment of neurodegenerative diseases;

[0043] 5. The present invention predicts and verifies the neuroprotective function of walnut peptides having the amino acid sequence shown in Seq ID NO.1-23. The walnut peptides of the present invention can improve the cell viability of oxidatively damaged neurons, reduce the apoptosis ratio of neurons under oxidative damage, reduce the oxidative damage of neurons under oxidative stress, and alleviate the toxic effects of oxidative damage on neurons. After the intervention of the walnut peptides of the present invention, the level of lipid peroxide MDA is significantly downregulated and the levels of antioxidants SOD and CAT are significantly increased. From the perspective of mechanism, the present invention finds that enzymatic walnut peptides can reduce the oxidative damage of H2O2 to SH-SY5Y cells by increasing the levels of Nrf2 and HO-1 proteins in the Nrf2 / HO-1 signaling pathway and inhibiting the level of Keap1 protein.

[0044] 6. The present invention also proposes the specific application of the above-mentioned walnut peptide with neuroprotective function, which can be used to improve the vitality of nerve cells, reduce the oxidative damage of nerve cells, regulate the protein level of nerve damage-related pathways, and prepare blood pressure-lowering or anti-Alzheimer's or anti-tumor related drugs, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Effect of enzymatic hydrolysis pH (2, 4, 6, 8) on peptide yield;

[0046] Figure 2 :Effect of substrate mass concentration (1:10, 1:15, 1:20, 1:25) on peptide yield;

[0047] Figure 3 : Effect of enzyme addition amount (2%, 4%, 6%, 8%) on peptide yield;

[0048] Figure 4 : Effect of enzymatic hydrolysis temperature (30℃, 40℃, 50℃, 60℃) on peptide yield;

[0049] Figure 5 : Effect of enzymatic hydrolysis time (4h, 5h, 6h, 7h) on peptide yield;

[0050] Figure 6 : Mass spectrum of walnut protein sample without enzymatic hydrolysis;

[0051] Figure 7 : Mass spectrum of walnut protein sample treated with enzymatic hydrolysis;

[0052] Figure 8 : Molecular docking diagram of enzymatically hydrolyzed walnut peptide TDELL and anti-Alzheimer's target AChE;

[0053] Fig. 9 : Molecular docking diagram of enzymatically hydrolyzed walnut peptide TDELL and anti-Alzheimer's target BACE1;

[0054] Fig.10 : Molecular docking diagram of other enzymatically hydrolyzed walnut peptides and anti-Alzheimer's target AChE;

[0055] Fig.11 : Molecular docking diagram of other enzymatically hydrolyzed walnut peptides and the Alzheimer's disease target BACE1;

[0056] Fig.12 : Cell viability test results after walnut peptide treatment for 12 hours;

[0057] Fig.13 :The results of cell viability test when H2O2 series concentrations acted on SH-SY5Y cells for 2h;

[0058] Fig.14 :The results of cell viability detection when SH-SY5Y cells were pre-protected by walnut peptide for 12h with H2O2;

[0059] Fig.15 : The results of LDH release rate detection in the cell supernatant after walnut peptide treatment;

[0060] Fig.16 :Flow cytometry results of distinguishing necrotic cells, early apoptotic cells and late apoptotic cells by Annexin-V / PI double staining;

[0061] Fig.17 :Flow cytometry detection of mitochondrial membrane potential of SH-SY5Y cells;

[0062] Fig.18 : Detection results of SOD levels in cells;

[0063] Fig.19 :Detection results of MDA levels in cells;

[0064] Fig. 20 : CAT level detection results in cells;

[0065] Fig.21 :WB images of nerve injury-related protein expression;

[0066] Fig. 22 : Quantitative detection results of the expression of nerve damage-related protein Nrf2 in cells;

[0067] Fig.23 : Quantitative detection results of the expression of nerve injury-related protein HO-1 in cells;

[0068] Fig.24 : Quantitative detection results of the expression of nerve injury-related protein Keap1 in cells;

[0069] Wherein, WAP-1 represents natural walnut peptide, WAP-2 represents walnut peptides that have been enzymatically hydrolyzed and screened for a molecular weight of less than 1000Da according to the present invention, and when there is no special distinction between 1 and 2, WAP also refers to walnut peptides that have been enzymatically hydrolyzed and screened for a molecular weight of less than 1000Da according to the present invention; in the flow cytometry result diagram, G2-3 represents normal cells, G2-4 represents early apoptotic cells, G2-2 represents late apoptotic cells, G2-1 represents dead cells, 0 represents a blank group, 1 represents a model group, and 2, 3, and 4 represent walnut peptide treatment groups with concentrations of 1, 1.5, and 2 mg / L, respectively;

[0070] SPSS26.0 and GraphPad Prism 8.0 were used for data analysis and drawing, and Image-PRO was used for grayscale analysis. One-way ANOVA was used for comparison among multiple groups, and the LSD method was used to verify the significance of pairwise comparisons. In the letters abcd, if the two groups are marked with the same letter, it means p>0.05, and the difference is not significant. If the two groups are marked with different letters, it means P<0.05, which is statistically significant. Compared with the model group, "#" indicates P<0.05, which is statistically significant; compared with the control group, "*" indicates P<0.05, which is statistically significant. DETAILED DESCRIPTION

[0071] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. The materials, reagents, etc. used in the following examples are reagents and materials available from commercial sources unless otherwise specified.

[0072] Example 1: Preparation method and process optimization of walnut peptide

[0073] Materials and reagents: Walnut meal (provided by Shanxi Matan Shunkang Company), flavor protease (Shanghai McLean Company), petroleum ether (analytical grade), and other reagents were all of analytical grade.

[0074] Instruments and equipment: centrifuge (Eppendorf, 5430R); water purifier (Thermo Scientific, GenPure); ultrasonic extractor; water bath.

[0075] Preparation of walnut peptide: Weigh 50g of walnut meal into a conical flask, add about twice the volume of petroleum ether, and shake on a 110r / min shaker for 48h to remove the oil in the walnut meal. Separate the walnut meal by vacuum filtration, place it in an oven at 50℃ and dry it to constant weight, and pass it through a 60-mesh sieve to obtain the walnut meal after secondary fat removal. Dissolve the walnut meal in water according to the ratio of 1:25 of solid-liquid ratio, adjust the pH to 9, and ultrasonically treat it at 55℃ and 550w for 1h. After the ultrasound, cool it to room temperature, centrifuge it at 4000r / min for 20min, and take the supernatant to adjust it to acidity (pH=5.5). Centrifuge it again at 4000r / min for 20min, and wash it twice at 40℃ with distilled water twice the mass of the precipitate to neutrality. Put the precipitate in a freeze dryer and vacuum freeze-dry it at -55℃ for 48h to obtain walnut protein.

[0076] Process optimization-single factor experiment:

[0077] 1) Enzymatic hydrolysis pH value: Under the conditions of material ratio 1:10, enzymatic hydrolysis time 6h, enzymatic hydrolysis temperature 50℃, and enzyme addition amount 8%, the effect of enzymatic hydrolysis pH value (2, 4, 6, 8) on peptide yield was investigated. The results are as follows: Figure 1 As shown in the figure, when the pH value of enzymatic hydrolysis is 6-8, the yield of walnut peptide is higher. This is because the pH value of the solution is one of the main parameters that determine the catalytic activity of the protease. On the one hand, the pH value will change the dissociation state of the test substrate and affect its binding with the protease. On the other hand, the over-acidic and over-alkaline test environment can also change the spatial conformation of the protease and lead to the inactivation of the protease. Therefore, the pH value of the enzymatic hydrolysis is determined to be around 8.

[0078] 2) Substrate mass concentration (material-liquid ratio): Under the conditions of enzymatic hydrolysis time 6h, enzymatic hydrolysis temperature 50℃, enzymatic hydrolysis pH=6, and enzyme addition amount 8%, the effect of substrate mass concentration (1:10, 1:15, 1:20, 1:25) on peptide yield was investigated. The results are as follows: Figure 2 As shown in the figure, when the material-liquid ratio is between 1:15 and 1:10, the yield of walnut peptide is higher; this is because as the substrate mass concentration increases, the enzymatic hydrolysis rate accelerates, but when the substrate mass concentration is too high, on the one hand, a large amount of substrate accumulates on the enzyme molecules, generating inactive intermediates, and the enzyme molecules cannot be released; on the other hand, the high mass concentration substrate reduces the effective concentration of water and the molecular diffusivity, thereby affecting the yield of walnut peptide, so the material-liquid ratio is determined to be 1:15;

[0079] 3) Enzyme dosage (enzyme concentration): Under the conditions of material ratio 1:10, enzymatic hydrolysis time 6h, enzymatic hydrolysis pH=6, and enzymatic hydrolysis temperature 50℃, the effect of enzyme dosage (2%, 4%, 6%, 8%) on peptide yield was investigated. The results are as follows: Figure 3 As shown in the figure, when the enzyme dosage is 6-8%, the walnut peptide yield is higher. With the increase of enzyme dosage, the walnut peptide yield shows a rising trend, so the enzyme dosage is about 8%;

[0080] 4) Enzymatic hydrolysis temperature: Under the conditions of material ratio 1:10, enzyme addition amount 8%, enzymatic hydrolysis pH = 6, and enzymatic hydrolysis time 6h, the effect of enzymatic hydrolysis temperature (30℃, 40℃, 50℃, 60℃) on peptide yield was investigated. The results are as follows Figure 4 As shown in the figure, when the enzymatic hydrolysis temperature is 40-50℃, the walnut peptide yield is higher. Increasing the temperature within a certain range can increase the walnut peptide yield through the catalytic action of the enzyme, but too high a temperature will lead to enzyme inactivation, so the enzymatic hydrolysis temperature is determined to be around 50℃;

[0081] 5) Enzymatic hydrolysis time: Under the conditions of material ratio 1:10, enzyme addition amount 8%, enzymatic hydrolysis pH = 6, and enzymatic hydrolysis temperature 50°C, the effect of enzymatic hydrolysis time (4h, 5h, 6h, 7h) on peptide yield was investigated. The results are as follows: Figure 5As shown in the figure, when the enzymatic hydrolysis time is 6 to 8, the yield of walnut peptide is higher. As time goes by, the peptide yield increases, and after more than 5 hours, it tends to be flat or even decreases, so the enzymatic hydrolysis time is determined to be about 5 hours.

[0082] Orthogonal experiment: On the basis of single factor experiment, with enzymatic hydrolysis time (A), pH (B), material-liquid ratio (C), enzyme concentration (D) as experimental factors and walnut peptide yield as index, orthogonal design was adopted to explore the influence of different factors on the preparation process of walnut peptide. The orthogonal factor level table is shown in Table 1, the orthogonal test results and analysis are shown in Table 2, and the orthogonal test variance analysis is shown in Table 3.

[0083] Table 1: Orthogonal factor level table

[0084]

[0085] Table 2: Orthogonal test results and analysis

[0086]

[0087] Table 3: Orthogonal test variance analysis

[0088]

[0089] It can be seen that the order of factors affecting the yield of walnut peptide in the preparation process of walnut peptide is D>C>B>A, and the enzyme concentration is a significant influencing factor (p<0.05). The optimal preparation process of walnut peptide is A4B3C1D4, that is, the optimal process conditions for preparing walnut peptide are: pH 6, enzyme addition 8%, material-liquid ratio 1:10, and enzymatic hydrolysis time 5h.

[0090] Example 2: Structural Identification of Walnut Peptide

[0091] Materials and reagents: walnut meal (provided by Shanxi Matan Shunkang Company), flavor protease (Shanghai McLean Company), petroleum ether (analytical grade), formic acid, acetonitrile, trifluoroacetic acid, trimercaptopropanesulfonic acid, aminomethyldicarboxylic acid (all mass spectrometry grade).

[0092] Instruments and equipment: centrifuge (Eppendorf, 5430R); water purifier (Thermo Scientific, GenPure); mass spectrometer (Thermo Scientific, Q-Exactive HF-X); chromatography system (Easy-nLC1200, ThermoScientific); chromatographic column (Trap column (Reverse-phase), 100μm*20mm (5um, C18)); chromatographic analysis column (Thermo scientific EASY column (Reverse-phase), 75μm*150mm (3um, C18))

[0093] Structural identification of walnut peptides: Walnut peptides were divided into two groups (natural walnut peptides without enzymatic hydrolysis and walnut peptides after enzymatic hydrolysis) for structural identification.

[0094] Natural walnut peptide: Take an appropriate amount of sample and add an appropriate amount of UA buffer (8M Urea, 150mM Tris-HCl, pH 8.0), ultrasonically break in an ice bath, incubate at 37°C for 1h, centrifuge at 20000g for 5min, and take the supernatant to obtain a peptide solution sample. The sample was transferred to a 10KD ultrafiltration centrifuge tube, centrifuged at 12000g for 15min, added 200μL 0.1% TFA and centrifuged at 12000g for 15min, repeated twice, collected the filtrate, desalted using C18 StageTip, and vacuum dried. After drying, the peptide was re-dissolved with 0.1% FA, and the peptide concentration was determined for LC-MS analysis.

[0095] Walnut hydrolysate: Take an appropriate amount of sample and add an appropriate amount of SDT lysis solution (4% SDS, 100mM Tris-HCl), ultrasonically break in an ice bath, boil in 100℃ water bath for 5min, centrifuge at 20000g for 5min, take the supernatant, obtain a protein solution sample, add an appropriate amount of TCEP / CAA mixture to the sample, and boil in 100℃ water bath for 5min to reduce the protein. Add an appropriate amount of methanol, chloroform and water to the sample, mix well, centrifuge at 4℃9000g for 15min to precipitate the protein; remove the supernatant, add an appropriate amount of methanol to mix well, centrifuge at 4℃20000g for 5min, remove the supernatant, repeat twice to wash the protein, and air dry. Add an appropriate amount of 50mM NH4HCO3 buffer to re-dissolve the protein, add an appropriate amount of HCl to adjust the pH value to 6, add 8% enzyme, the material-liquid ratio is 1:10, and the enzymatic hydrolysis is carried out at 50℃ for 5h; the peptides after enzymatic hydrolysis are desalted using C18 StageTip and vacuum dried. After drying, the peptides were re-dissolved in 0.1% FA and the peptide concentration was determined for LC-MS analysis.

[0096] An appropriate amount of peptides was taken from each sample and chromatographically separated using the nanoliter flow rate Easy nLC 1200 chromatography system (ThermoScientific). Buffer: Liquid A is a 0.1% formic acid aqueous solution, and liquid B is a mixed solution of 0.1% formic acid, acetonitrile and water (of which acetonitrile is 80%). The chromatographic column was balanced with 100% liquid A. The sample was injected into the Trap Column (100μm*20mm, 5μm, C18, Dr.Maisch GmbH) and then passed through the chromatographic analysis column (75μm*150mm, 3μm, C18, Dr.MaischGmbH) for gradient separation at a flow rate of 300nl / min. The liquid phase separation gradient was as follows: 0 min to 2 min, liquid B linear gradient from 2% to 5%; 2 min to 44 min, liquid B linear gradient from 5% to 28%; 44 min to 51 min, liquid B linear gradient from 28% to 40%; 51 min to 53 min, liquid B linear gradient from 40% to 100%; 53 min to 60 min, liquid B maintained at 100%. After peptide separation, DDA (data dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). The analysis time was 60 min, detection mode: positive ion, parent ion scanning range: 350-1800 m / z, primary mass spectrometry resolution: 60,000@m / z 200, AGC target: 3e6, primary Maximum IT: 50 ms. The peptide secondary mass spectrometry analysis was collected according to the following method: after each full scan, the secondary mass spectra of 20 parent ions with the highest intensity were triggered (MS2 scan), secondary mass spectrometry resolution: 15,000@m / z 200, AGC target: 1e5, secondary Maximum IT: 50ms, MS2 Activation Type: HCD, Isolation window: 1.6m / z, Normalized collision energy: 28. The mass spectrometry database search software used was MaxQuant 2.0.1.0.

[0097] The mass spectrum of the walnut protein sample without enzymatic hydrolysis is as follows Figure 6 As shown, Shanghai Baipu Biotechnology Co., Ltd. was commissioned to conduct mass spectrometry analysis and library search qualitative analysis, and 127 natural walnut peptides were identified from 33 proteins. The number of amino acids in these peptides ranged from 5 to 30, and the molecular weight distribution ranged from 423.1754 to 3381.515 Da.

[0098] The mass spectrum of the walnut protein sample treated with enzymatic hydrolysis is shown in Figure 7As shown, Shanghai Baipu Biotechnology Co., Ltd. was commissioned to conduct mass spectrometry analysis and library search qualitative analysis, and 1,348 natural walnut peptides were identified, which came from 64 proteins. The number of amino acids in these peptides ranged from 5 to 30, and the molecular weight distribution ranged from 501.3163 to 3634.611 Da.

[0099] Example 3: Prediction of toxicity, solubility and neuroprotective function of walnut peptides

[0100] Small molecule peptides are more conducive to human absorption. By predicting the toxicity and solubility of peptides at an early stage, problems can be identified in advance to avoid further development of inappropriate drugs, thereby saving time and resources. Therefore, small molecule walnut peptides of 1000Da and below were screened out from the identification results of Example 2 for further analysis, and Shanghai Baipu Biotechnology Co., Ltd. was commissioned to separate small molecule walnut peptides with a molecular weight of 1000Da and below after enzymatic hydrolysis.

[0101] Prediction of toxicity and solubility: Toxicity and solubility prediction of walnut peptides can help in safety assessment, bioavailability optimization, drug delivery system design, and save time and resources. The online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to predict the toxicity of the selected peptides based on their physicochemical properties. Subsequently, the solubility of these peptides was evaluated using the peptide property calculator (http: / / www.innovagen.com / ).

[0102] Prediction of neuroprotective function of walnut peptides: The X-ray crystal structures of angiotensin converting enzyme (ACE), an antihypertensive target, AChE, β-site amyloid precursor protein cleavage enzyme 1 (BACE1), and an anti-tumor target, aminopeptidase N (APN) were obtained through the RCSB PDB database. After removing water molecules and adding hydrogen atoms to the receptor target using Discovery Studio (DS) 2019R2 (Dassault Systemes Biovia, San Diego, CA, USA) software, the active center was defined in the "Receptor Ligand Interactions" module. The information of the active center (x, y, z) and docking radius (r) is shown in Table 4. The structure of the peptide was drawn using the "Macromolecules" module of the DS software, and the energy of the peptide structure was optimized using the "Minimize Ligands" and "Prepare Ligands" tools under the CHARMm force field. The receptor and peptide were molecularly docked using the CDOCKER tool in the Receptor-Ligand Interactions module, and the natural walnut active peptides with potential anti-hypertensive, anti-Alzheimer's and anti-tumor effects were screened according to the -CDOCKER_Energy value. For the application in anti-Alzheimer's disease, the enzymatically hydrolyzed walnut peptides were screened for peptides with relevant effects.

[0103] Table 4: Active center (x, y, z) and docking radius (r) information of receptor targets

[0104]

[0105] The structure, properties and target binding ability of natural walnut peptides are shown in Table 5, and the structure, properties and target binding ability of enzymatic walnut peptides are shown in Table 6.

[0106] Table 5: Structure, properties and target binding ability of natural walnut peptides

[0107]

[0108] Table 6: Structure, properties and target binding ability of enzymatic walnut peptides

[0109]

[0110] The smallest molecular weight peptide GGGSF identified in natural walnut peptides can dock with the antihypertensive target ACE and the anti-tumor target APN to varying degrees, but its solubility is poor. In terms of docking with AChE and BACE1 targets, the VEDEL-AChE complex is stabilized by 5 conventional hydrogen bond interactions, 2 carbon-hydrogen bond interactions, 1 alkyl interaction, 2 π-alkyl interactions, 2 π-cation interactions, and 1 π-anion interaction. The VEDEL-BACE1 complex binds to the active catalytic center Asp32 of BACE1 and forms a salt bridge, but unfavorable negative-negative interactions also occur at the same time. However, Asp228 is also the active catalytic center of BACE1, and VEDEL also forms a salt bridge with it, which makes VEDEL-BACE1 show a tighter binding overall. In terms of docking with APN targets, the active site of APN is composed of three pockets: S1 (Glu355, His388), S1' (Glu411, His392, Tyr477) and S2'. Existing studies have clarified that the amino acid residues involved in the binding mode of APN inhibitors or substrates include Glu355, which plays a key role in stabilizing the transition state, His388 and Glu389, which cooperate with water molecules, His392 and Glu411, which interact with zinc ions, and Tyr477, which forms hydrogen bonds with inhibitors. Although the VEDEL peptide forms a carbon-hydrogen bond with Tyr477, it also has an unfavorable negative-negative interaction with Glu411. In summary, the small molecule peptides in natural walnut peptides have adverse effects on the binding of targets such as AChE and BACE1, and have poor solubility.

[0111] Most of the walnut peptides after enzymatic hydrolysis have good solubility. The CDOCKER energy values ​​of 7 enzymatic hydrolyzed walnut peptides in the process of binding with the Alzheimer's disease targets AChE and BACE1 are all less than -100 (the absolute value of the CDOCKER energy value is the Docking score). Among them, the TDELL peptide with the smallest molecular weight is 589.29591Da; The molecular docking diagram of the enzymatic hydrolyzed walnut peptide TDELL and the anti-Alzheimer's disease target AChE is shown in Figure 8 As shown in Figure 1, the active center of AChE includes the peripheral anionic site (Tyr70, Asp72, Tyr121, Trp279, Tyr334) and the catalytic site (Trp84, Gly118, Gly119, Ala201, Tyr130, His440, Ser200, Glu327, Tyr330, Phe331), among which the aromatic residues Trp279 and Trp84 play a key role; the molecular docking diagram of the enzymatic walnut peptide TDELL and the anti-Alzheimer's target BACE1 is shown in Figure 1. Fig. 9As shown in the figure, in the process of binding with BACE1, TDELL binds to the active catalytic center Asp32 and Asp228 of BACE1 through attractive charge and hydrogen bond interactions to form a salt bridge. In addition, Thr232, Gln12, and Gly230 residues on BACE1 also form carbon-hydrogen bonds with TDELL. The above factors enable TDELL to form a tight complex with BACE1.

[0112] From the perspective of molecular docking prediction scores, on the one hand, the highest prediction score of natural walnut peptides with target AChE is only 107.545, and the highest prediction score with target BACE1 is only 116.526; among the enzymatically hydrolyzed walnut peptides, the highest prediction score with target AChE is 119.695, and there are 6 walnut peptides with scores higher than 107.545, and the highest prediction score with target BACE1 is 154.582, and there are 14 walnut peptides with scores higher than 116.526. Whether it is the tightness of molecular docking or the number of peptides with tight docking, enzymatic walnut peptides are superior to natural walnut peptides. Therefore, enzymatic walnut peptides theoretically have a stronger effect in reducing nerve damage.

[0113] On the other hand, the predicted scores of most natural walnut peptides for docking with the two targets are similar. For example, the predicted score difference between GGGSF and AChE and BACE1 is only 6.2. It can be seen that GGGSF does not differentiate between forming complexes with a certain target protein when acting on nerve cells, which is not conducive to the neuroprotective effect of walnut peptides. Among the enzymatic walnut peptides, a large number of walnut peptides such as SGGYDED have a predicted score difference of greater than or equal to 30 with AChE and BACE1, which is reflected in a stronger tendency to form tight complexes with BACE1. In practical applications, they can produce tighter complexes with BACE1. It is known that BACE1 is related to neurodegenerative diseases. BACE1 accumulates in axons, causing axonal swelling. Enzymatic walnut peptides are more conducive to binding to BACE1 and inhibiting its activity than natural walnut peptides. Therefore, enzymatic walnut peptides can theoretically achieve the effect of early intervention or treatment of neurodegenerative diseases.

[0114] Based on the above, it can be seen that NDNAKIN, EQGEGLL, SGGYDED, TDELLAE, VALEPSN, YTPHW, VALEP, LPEEVLA, and ALPEEVL (corresponding to Seq ID NO. 1, 2, 12, 14, 15, 17, 19, 21, 22, and 23, respectively) are more preferred because their molecular docking scores with BACE1 are at least 30 points higher than their molecular docking scores with AChE; among them, the molecular docking scores of SGGYDED, TDELLAE, VALEPSN, LPEEVLA, and ALPEEVL with BACE1 (corresponding to Seq ID NO. 12, 14, 15, 22, and 23, respectively) are higher than 100, and the molecular docking scores with AChE are lower than 100, indicating that they are more conducive to binding to BACE1 and inhibiting its activity. Therefore, theoretically, they have stronger effects in protecting nerves or treating neurodegenerative diseases, and are a more preferred group of enzymatic walnut peptides.

[0115] Except for TDELL, the solubility of most walnut peptides obtained by enzymatic hydrolysis was significantly improved, and the docking with AChE and BACE1 targets was tighter; among them, VPQNF, LPEDV, VALEP, TPSLV and VALVT were more tightly bound to the tight complex formed with the anti-Alzheimer's targets AChE and BACE1, and the binding sites were all the key active centers or catalytic centers of the target proteins. Therefore, the present invention provides a molecular docking diagram thereof, which is shown in the molecular docking diagram with AChE. Fig.10 As shown in the figure, the molecular docking diagram with BACE1 is shown in Fig.11 As shown, Fig.10 and Fig.11 In the figure, from left to right and from top to bottom, they are VPQNF, LPEDV, VALEP, TPSLV and VALVT. Based on the molecular docking results, TDELL, VPQNF, LPEDV, VALEP, TPSLV and VALVT (corresponding to Seq ID NO. 6, 13, 20, 21, 16, 11, respectively) are more preferred among all the walnut peptides of the present invention. It can be understood that the combination of the above six walnut peptides can be used as a preferred walnut peptide composition.

[0116] Example 4: Walnut peptide improves cell viability of nerve cells

[0117] Reagents and instruments: SH-SY5Y human neuroblastoma cells (Starfish Biotechnology), SH-SY5Y complete medium; DMEM medium, SH-SY5Y special medium; phosphate buffered saline (PBS1×); serum-free cell freezing solution; BCA kit, MDA detection kit, total SOD activity detection kit, LDH release detection kit, mitochondrial membrane potential detection kit (JC-1), RIPA lysis buffer; DMSO, CCK-8 cell activity detection kit, Keap1 (Solebo Technology Co., Ltd.); AnnexinV-FITC Apoptosis Detection Kit (BD); PVDF membrane 0.45μm, ECL (Millipore); developing and fixing reagent (Servicebio); Caspase3, β-ACTIN (CST); Nrf2, HO-1 (Proteintech); Bcl-2, Bax (ABCAM); HRP-labeled goat anti-rabbit (Jackson). Cell culture incubator (Herocell 180) from Shanghai Rundu Biotechnology Co., Ltd.; low-temperature refrigerated centrifuge from Sigma, Germany; flow cytometer CytoFLEX; microplate reader (spectra MAX190) from Molecular Devices; electrophoresis instrument; gel imaging system (FluorChen HD2); inverted optical microscope Olympus BX53; vertical electrophoresis instrument and transfer electrophoresis instrument from Tanon; scanner from EPSON.

[0118] SH-SY5Y cells were cultured in a 37°C saturated humidity, 5% CO2 incubator. New culture medium was replaced every 48 hours, and subculture was performed the next day. Cells in the logarithmic growth phase were used for subsequent experiments.

[0119] Cells in the logarithmic growth phase were cultured at 1.5×10 4 The cells were inoculated in a 96-well plate at a density of 100 μL per well, and 3 replicate wells were set for each group. The walnut peptides were the natural walnut peptide (denoted as WAP-1) in Example 1 and the enzymatic walnut peptide (denoted as WAP-2) obtained from Example 1-3 after enzymatic hydrolysis with a sieve molecular weight of less than 1000 Da. The walnut peptide intervention group was added with DMEM medium containing walnut peptide concentrations (0.5, 1.0, 1.5, 2 mg / mL), and the blank group was added with an equal amount of DMEM medium. At the end of the culture, 10 μL of CCK-8 working solution was added to each well, incubated for a suitable time, and the absorbance (optical density, OD) value of each well was measured by adjusting the microplate reader to 450 nm.

[0120] After the cells were treated with walnut peptide for 12 h, the cell viability was detected. Fig.12As shown. Whether it is enzymatic peptide or natural peptide, compared with the blank control group, the cell viability of the cells treated with walnut peptide group showed a certain upward trend, but the cell viability of the enzymatic peptide treatment group was significantly higher than that of the natural peptide treatment group, indicating that enzymatic peptide has a significant effect on improving the cell viability of nerve cells; and the cell viability of the enzymatic peptide treatment group was the largest at a concentration of 1.5 mg / ml, and its cell viability was significantly different from that of the control group (P<0.05).

[0121] Example 5: Protective effect of walnut peptide on oxidatively damaged nerve cells

[0122] H2O2-induced cellular oxidative damage reactions are easy to occur, and H2O2 is easily obtained and has relatively stable and controllable properties. It is the most commonly used in vitro evaluation model for studying cellular oxidative stress damage.

[0123] 1. Model validation of H2O2-induced cellular oxidative damage

[0124] When H2O2 concentration series were applied to SH-SY5Y cells for 2 h, the results were as follows: Fig.13 As shown in the figure, H2O2 can inhibit the proliferation of SH-SY5Y cells in a dose-dependent manner. When the H2O2 concentration is 200μmol / L, the cell damage is small, and the cell survival rate is about 80%, which does not meet the modeling requirements. When the concentration is greater than 600μmol / L, the inhibitory effect is too strong. At 600μmol / L, the cell survival rate is about 55%, which is relatively moderate, and the difference is statistically significant (P<0.05). Therefore, the final H2O2 modeling condition is 600μmol / L for 2h.

[0125] 2. Enzymatic walnut peptide has the effect of significantly improving the cell viability of oxidatively damaged neurons

[0126] Cells in the logarithmic growth phase were cultured at 1.5×10 4 The cells were inoculated in a 96-well plate at a density of 100 μL per well, and 3 replicates were set for each group. A blank group, a H2O2 model group (600 μmol / L), and a walnut peptide (enzymatically hydrolyzed walnut peptide obtained by enzymatic hydrolysis in Example 1-3 with a sieve molecular weight of less than 1000 Da) + H2O2 (600 μmol / L) intervention group (0.5, 1.0, 1.5, 2 mg / mL) were set up. After 24 hours of culture, 10 μL of CCK-8 solution was added to each well, incubated for a suitable time, and the OD value of each well was measured at 450 nm using a microplate reader.

[0127] When H2O2 was applied to SH-SY5Y cells pre-protected by walnut peptide for 12 h, the cell viability was detected. Fig.14As shown. Compared with the blank control group, the cell viability of the model group was significantly lower than that of the control group (P < 0.05), while the walnut peptide group could significantly improve the cell viability of the oxidatively damaged cell model at three concentrations of 1, 1.5 and 2 μmol / L (P < 0.005). Overall, the protective effect of enzymatic walnut peptide was significantly stronger than that of natural walnut peptide, indicating that enzymatic walnut peptide has the effect of significantly improving the cell viability of oxidatively damaged neurons.

[0128] 3. Enzymatic walnut peptide can alleviate the cytotoxic effect caused by H2O2

[0129] After treating the cells for 12 hours, the cell supernatant treated with enzymatic walnut peptide was taken and the LDH release rate was detected using a kit. The specific method was carried out according to the instructions of the kit. The results are as follows: Fig.15 As shown. Compared with the blank control group, the LDH content of cells in the H2O2 treatment group increased significantly (P < 0.05). H2O2 induced a large amount of LDH release, resulting in cytotoxicity, indicating that cells died. After the intervention of enzymatic walnut peptide, compared with the model group, the LDH content of each enzymatic walnut peptide intervention group decreased significantly (P < 0.05), indicating that enzymatic walnut peptide can alleviate the cytotoxic effect caused by H2O2.

[0130] 4. Enzymatic walnut peptide intervention can inhibit cell apoptosis caused by H2O2

[0131] After treating the cells for 12 hours, stain according to the instructions of the Annexin V-FITC Apoptosis Detection Kit, incubate at room temperature for 15 minutes in the dark, and detect cell apoptosis using a CytoFLEX flow cytometer within 30 minutes after the incubation to calculate the apoptosis rate. Annexin-V / PI double staining can be used to distinguish necrotic cells, early apoptotic cells, and late apoptotic cells. Fig.16 In the result graph shown, G2-3 is normal cells, G2-4 is early apoptotic cells, G2-2 is late apoptotic cells, G2-1 is dead cells, 0 is blank control, 1 is model group, 2, 3, 4 are 1.0, 1.5, 2 mg / mL concentration enzymatic walnut peptide treatment groups, the operation in the experiment will also cause mechanical cell death, which is shown in the G2-1 quadrant of the figure. According to the flow cytometry results, compared with the blank group, the model group had a significant increase in apoptotic cells due to H2O2 treatment. After the intervention of enzymatic walnut peptide, the number of apoptotic cells decreased significantly compared with the model group.

[0132] 5. Enzymatic walnut peptide group can significantly increase mitochondrial membrane potential and reduce oxidative damage in SH-SY5Y cells

[0133] After 12 hours of treatment, the cells were stained according to the instructions of the Mitochondrial Membrane Potential Detection Kit (JC-1), incubated at room temperature for 15 minutes in the dark, and the mitochondrial membrane potential of the cells was detected by CytoFLEX flow cytometer within 30 minutes after the incubation. The decrease of mitochondrial membrane potential (MMP) is an early indicator of mitochondrial membrane damage and cell apoptosis. The imbalance of mitochondrial membrane potential indicates the occurrence of cell apoptosis. The results are shown in Fig.17 As shown in the results, 600 μmol / L H2O2 can significantly reduce the mitochondrial membrane potential, indicating that H2O2 induces a decrease in the potential difference between the inside and outside of the mitochondrial membrane and destroys the integrity of the mitochondrial membrane. Compared with the control group, the difference is statistically significant. The enzymatic walnut peptide group can significantly increase the mitochondrial membrane potential and reduce the oxidative damage of SH-SY5Y cells. Compared with the model group, the difference is statistically significant.

[0134] 6. Enzymatic walnut peptide can reduce the level of peroxide MDA and increase the level of antioxidants SOD and CAT

[0135] After treating the cells for 12 hours, centrifuge at 1000r / min for 5 minutes, collect the cell pellet, use pre-cooled RIPA protein lysis buffer to lyse the cells, place at -4℃ for 30 minutes, and oscillate for 30 seconds every 5 minutes. After the lysis is completed, centrifuge at 12000r / min, 4℃, 5 minutes, take the supernatant, calculate the total cell protein according to the BCA kit, and then detect the absorbance value with a microplate reader according to the operating steps of the SOD, MDA, and CAT instructions. MDA is a lipid peroxide, which reflects the degree of oxidation in the cell to a certain extent, and the antioxidant enzymes SOD, CAT, etc. are important factors in anti-oxidation, which maintain the oxidation-antioxidation balance in the cell by restricting oxidative damage. Fig.18 , Fig.19 and Fig. 20 The results of the SOD, MDA and CAT level tests showed that after H2O2 induction, the MDA content in the model group cells was significantly higher than that in the blank control group (P < 0.05), while the SOD and CAT levels were significantly decreased (P < 0.05). After the intervention of enzymatic walnut peptide, the lipid peroxide MDA level was significantly downregulated and the antioxidant SOD and CAT levels were increased. This suggests that enzymatic walnut peptide can alleviate the oxidative damage caused by hydrogen peroxide.

[0136] VII. Enzymatic walnut peptides regulate the expression levels of nerve injury-related proteins Nrf2, HO-1 and Keap1

[0137] After treating the cells for 12 hours, centrifuge at 1000r / min for 5 minutes, collect the cell pellet, use pre-cooled RIPA protein lysis buffer to lyse the cells, place at -4℃ for 30 minutes, and oscillate for 30 seconds every 5 minutes. After the lysis is completed, 12000r / min, 4℃, 5 minutes. Take the supernatant and perform protein quantification. According to the instructions for use of the BCA protein quantification kit, determine the protein concentration. Draw a standard curve and calculate the protein concentration. Configure 8-20% concentration separation gel and 5% concentration stacking gel. When the protein is in the stacking gel, the voltage is 75V. When the bromophenol blue band enters the interface between the stacking gel and the separation gel, the pre-stained protein marker begins to separate multiple bands. Increase the voltage to 120V and continue electrophoresis. When the electrophoresis is about 0.5cm from the bottom of the bromophenol blue gel, the electrophoresis can be terminated and the membrane transfer can be prepared. Use a gel spatula to cut the separation gel from the edge of the concentrated gel to 0.5cm below the bromophenol blue, and place it on the filter paper on the negative side of the transfer clip. There should be no bubbles above or below. Slowly and evenly cover the PVDF membrane on the gel from one side. Gently cover the filter paper and sponge on the positive side of the transfer clip on the PVDF membrane. Then clamp the transfer clip and put it into the transfer core of the electrophoresis tank with transfer solution, confirming that the gel is at the negative pole and the PVDF membrane is at the positive pole. After the transfer is completed, prepare for the immune reaction. Add 5% BSA prepared with 0.5% TBST to the antibody incubation tank with PVDF membrane, shake slowly on the decolorization shaker at room temperature, and block for 1h. Dilute the primary antibody with 5% BSA dissolved in TBST according to the antibody instructions, and add the diluted primary antibody correctly to the corresponding incubation tank. Wrap the incubation tank with the antibody with plastic wrap, put it in a foam box, put the foam box on the decolorization shaker, shake slowly, and incubate at 4℃ for 16h. Take out the incubation tank, recover the primary antibody correctly, and store it in a -20 or -80℃ refrigerator for later use. Rinse the PVDF membrane quickly with TBST 3 times, then quickly wash it three times with TBST at room temperature on a decolorizing shaker for 5 minutes each time, and discard the TBST to prepare for the incubation of the secondary antibody. Dilute the secondary antibody with TBST according to the instructions, add it to the incubation tank correctly, and incubate it at room temperature for 30 minutes. Rinse the PVDF membrane incubated with the secondary antibody quickly with TBST 3 times, then decolorize it with TBST at room temperature, and quickly wash it three times on a shaker for 5 minutes each time. Turn on the CLINX ChemiScope6100 chemiluminescence imaging system. Mix the two reagents ECLA and ECLB in equal volumes in a centrifuge tube. Use clean toilet paper or filter paper to absorb the TBST of the PVDF membrane to be detected, place it on the work platform of the luminescence imaging system, and evenly add the mixed ECL on the PVDF membrane. Gently place the workbench in the darkroom of the luminescence imaging system, adjust the lens until the preview image is clear, select the appropriate exposure time, and start exposure using time superposition.

[0138] Fig.21The WB diagram of related protein expression. Fig. 22 , Fig.23 and Fig.24 These are the quantitative analysis results of the expression of nerve injury-related proteins Nrf2, HO-1, and Keap1. Nuclear factor erythropoietin-related factor-2 (Nrf2) is an important antioxidant transcription factor acting in the body's oxidative stress regulation center. Activated Nrf2 will bind to the antioxidant response element (ARE) and be transferred to the cell nucleus to upregulate the expression of heme oxygenase 1 (HO-1) to achieve the purpose of reducing oxidative stress. Normally, Keap1 binds to Nrf2 in the cytoplasm as a dimer and acts as an endogenous inhibitor of Nrf2 to maintain intracellular Nrf2 homeostasis. Compared with the blank control group, the levels of Nrf2 and HO-1 proteins in the H2O2 treatment group decreased significantly (P < 0.05), and the level of Keap1 protein, which has a negative regulatory function on the Nrf2 / HO-1 signaling pathway, increased significantly (P < 0.05). The enzymatic walnut peptide intervention group can significantly increase the levels of Nrf2 and HO-1 proteins (P < 0.05); and significantly downregulate the level of Keap1 protein (P < 0.05). The above results show that enzymatic walnut peptide can reduce the oxidative damage of H2O2 to SH-SY5Y cells by increasing the levels of Nrf2 and HO-1 proteins in the Nrf2 / HO-1 signaling pathway and inhibiting the level of Keap1 protein.

[0139] In summary, enzymatic walnut peptides can improve the cell viability of oxidatively damaged neurons, reduce the apoptosis ratio of neurons under oxidative damage, reduce the oxidative damage of neurons under oxidative stress, and alleviate the toxic effects of oxidative damage on neurons. After the intervention of enzymatic walnut peptides, the level of lipid peroxide MDA was significantly downregulated and the levels of antioxidants SOD and CAT were significantly increased. Therefore, enzymatic walnut peptides have the effect of reducing nerve damage, and their effect of reducing nerve damage is stronger than that of natural walnut peptides, which also verifies the conclusion obtained in molecular docking. From a mechanistic perspective, in addition to the close binding with the target proteins AChE and BACE1, the present invention also found that enzymatic walnut peptides can reduce the oxidative damage of H2O2 to SH-SY5Y cells by increasing the levels of Nrf2 and HO-1 proteins in the Nrf2 / HO-1 signaling pathway and inhibiting the level of Keap1 protein.

[0140] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto, and any equivalent modifications or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention.

[0141] Sequence Listing

[0142] Seq ID NO.1 NDNAKIN Seq ID NO.2 EQGEGLL Seq ID NO.3 EEVLATA Seq ID NO.4 CERQF Seq ID NO.5 NIADAQR Seq ID NO.6 TDELL Seq ID NO.7 VDTETAR Seq ID NO.8 DTPVVTV Seq ID NO.9 YPTSVLA Seq ID NO.10 PEDEIR Seq ID NO.11 VALVT Seq ID NO.12 SGGYDED Seq ID NO.13 VPQ Seq ID NO.14 TDELLAE Seq ID NO.15 VALEPSN Seq ID NO.16 TPSLV Seq ID NO.17 YTPHW Seq ID NO.18 DANPNTF Seq ID NO.19 YV Seq ID NO.20 LPEDV Seq ID NO.21 VALEP Seq ID NO.22 LPEEVLA Seq ID NO.23 ALPEEVL

Claims

1. A walnut peptide with neuroprotective function, characterized in that: It includes any one or more of the peptides having the amino acid sequences shown in Seq ID NO.1-23.

2. A method for preparing walnut peptide with neuroprotective function, characterized in that: The preparation method of the walnut peptide with neuroprotective function comprises: taking a natural walnut protein solution, adding flavor protease for enzymolysis, and screening out small molecule walnut peptides of 800Da and below.

3. The method for preparing the walnut peptide with neuroprotective function according to claim 2, characterized in that: During the enzymatic hydrolysis process, the concentration of flavor protease is 2-8%.

4. The method for preparing the walnut peptide with neuroprotective function according to claim 2, characterized in that: During the enzymatic hydrolysis process, the concentration of natural walnut protein is 4-10%.

5. The method for preparing the walnut peptide with neuroprotective function according to claim 2, characterized in that: During the enzymolysis process, the temperature is set at 30-60°C.

6. The method for preparing the walnut peptide with neuroprotective function according to claim 2, characterized in that: During the enzymatic hydrolysis process, the pH value is set to 2-8.

7. Use of the walnut peptide with neuroprotective function as claimed in claim 1 or prepared according to any one of claims 2 to 6 for preparing an agent for improving the activity of nerve cells.

8. Use of the walnut peptide with neuroprotective function as claimed in claim 1 or prepared according to any one of claims 2 to 6 for preparing an agent for reducing oxidative damage to nerve cells.

9. Use of the walnut peptide with neuroprotective function as claimed in claim 1 or prepared according to any one of claims 2 to 6 for preparing an agent for increasing Nrf2 protein level and / or increasing HO-1 protein level and / or inhibiting Keap1 protein level.

10. Use of the walnut peptide with neuroprotective function as claimed in claim 1 or prepared according to any one of claims 2 to 6 for preparing an agent for lowering blood pressure and / or preventing Alzheimer's disease and / or preventing tumors, characterized in that: The walnut peptide with neuroprotective function is docked with angiotensin converting enzyme, a target for lowering blood pressure, and / or AChE, a target for resisting Alzheimer's disease, beta-amyloid precursor protein cleaving enzyme 1, and / or aminopeptidase N, a target for resisting tumors.

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