Application of walnut peptide in preparation of cognitive function improver

Abstract

This invention relates to the field of natural active ingredient research technology, specifically to the application of walnut peptides in the preparation of cognitive function improvers. The cognitive function improver is a core functional short peptide derived from walnuts, characterized by high bioactivity, non-toxicity, and favorable pharmacokinetic properties. Its amino acid sequence is AFVHWY. This short peptide is easily synthesized artificially and possesses characteristics such as easy oral administration, easy absorption, strong metabolic stability, and strong blood-brain barrier penetration, providing a novel solution for the prevention and improvement of cognitive impairment.

Description

Technical Field

[0001] This invention relates to the field of natural active ingredient research technology, specifically to the application of walnut peptides in the preparation of cognitive function improvers. Background Technology

[0002] Cognitive function is the core ability of the human central nervous system to receive, process, store, retrieve, and apply information. It encompasses multiple dimensions, including learning, memory, attention, thinking, language comprehension, and executive function, and is fundamental to maintaining normal social, learning, work, and daily living abilities. Cognitive impairment refers to persistent damage to cognitive function caused by various factors, manifesting as a series of symptoms such as decreased learning and memory abilities, poor concentration, slowed thinking, reduced language expression or comprehension, and executive dysfunction. This impairment significantly affects the patient's daily life, learning, or social functions and cannot be alleviated through normal fatigue recovery or emotional regulation. The core characteristic of cognitive impairment is substantial damage to the cognitive domain, distinguishing it from the mild cognitive decline that occurs during normal aging, which usually does not affect normal living abilities and progresses slowly. Cognitive impairment is a broad clinical concept, representing a common pathophysiological manifestation caused by multiple etiologies.

[0003] Cognitive impairment can be classified into several types according to different classification criteria. Currently, the classification methods commonly used in clinical practice and research mainly include the following: 1. Classification by etiology: This can be divided into cognitive impairment related to neurodegenerative diseases (such as cognitive impairment related to Alzheimer's disease, Parkinson's disease, and frontotemporal dementia), cognitive impairment related to cerebrovascular diseases (such as cognitive impairment after ischemic stroke, cognitive impairment after hemorrhagic stroke, and vascular dementia), cognitive impairment related to mental and psychological diseases (such as cognitive impairment associated with anxiety disorders and depression, and cognitive impairment related to schizophrenia), cognitive impairment related to traumatic brain injury (such as post-concussion syndrome and cognitive deficits after severe brain injury), and cognitive impairment related to other etiologies (such as cognitive impairment caused by alcoholic encephalopathy and drug-induced cognitive impairment, cognitive impairment caused by metabolic diseases such as diabetes and thyroid dysfunction, and cognitive impairment caused by infectious diseases such as viral encephalitis). 2. Classification by the severity of cognitive impairment: This can be divided into mild cognitive impairment, moderate cognitive impairment, and severe cognitive impairment. Mild cognitive impairment is the early stage of cognitive dysfunction, with patients experiencing only mild decline in learning and memory, and largely retaining their daily living abilities, although they may encounter difficulties with complex tasks (such as complex learning and fine motor skills). Moderate cognitive impairment involves further cognitive deterioration, manifested as significant memory loss, thought disorder, and varying degrees of dependence on daily living abilities (such as dressing, eating, and traveling). Severe cognitive impairment results in severe cognitive decline, potentially leading to complete memory loss, language loss, and an inability to independently perform basic daily activities, requiring comprehensive care. 3. Classification by the primary cognitive domain affected: Cognitive impairments can be categorized into learning and memory disorders, attention deficits, executive function disorders, language disorders, and visuospatial cognitive disorders. Learning and memory disorders are the most common type, widely present in various cognitive dysfunction diseases.

[0004] Currently, commonly used cognitive function improvement drugs in clinical practice suffer from limited efficacy and significant side effects, failing to meet clinical needs. Meanwhile, in the field of natural products, walnuts, as a traditional food and medicine, have gained widespread public recognition for their "cognitive-boosting" effects. However, current technologies do not yet clearly define the specific active ingredients in walnuts that contribute to their cognitive-boosting effects, and their mechanisms of action lack in-depth research. This significantly limits the efficient development and precise application of walnut resources in the field of cognitive function improvement.

[0005] Therefore, screening and identifying core components with clear cognitive function-improving activities from walnuts, and developing a cognitive function improver that can effectively improve learning and memory impairments, alleviate anxiety-like behaviors, and has high safety, has important clinical value and broad application prospects. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] Based on the aforementioned deficiencies in the prior art, this invention provides an application of walnut peptide in the preparation of cognitive function improvers. This walnut peptide is a core functional short peptide derived from walnuts that is highly bioactive, non-toxic, and has good pharmacokinetic characteristics. This short peptide is very easy to synthesize artificially and has the characteristics of easy oral administration, easy absorption, strong metabolic stability, and strong blood-brain barrier penetration ability, which can provide a new solution for the prevention and improvement of cognitive impairment.

[0008] (II) Technical Solution

[0009] This invention relates to the application of a walnut peptide in the preparation of a cognitive function improver, wherein the amino acid sequence of the walnut peptide is AFVHWY.

[0010] Preferably, the cognitive function improver uses walnut peptide as the sole active ingredient; or the cognitive function improver uses walnut peptide as one of the active ingredients, combined with other active ingredients that have cognitive function improving functions.

[0011] The cognitive function improver also includes pharmaceutically acceptable excipients and / or delivery carriers.

[0012] Preferably, the cognitive function improver is an oral formulation.

[0013] Preferably, the cognitive function improver achieves its effects of alleviating neurodegenerative damage, protecting nerves, and improving cognitive function by increasing the expression of antioxidant proteins related to oxidative stress regulation in the hippocampus, alleviating oxidative stress levels, and inhibiting lipid peroxidation.

[0014] Preferably, the cognitive impairment includes cognitive impairment caused by Alzheimer's-like neurodegenerative diseases.

[0015] Preferably, the cognitive function improver has the function of restoring daily cognitive coordination, short-term memory, recognition memory, and spatial memory.

[0016] Secondly, the present invention also provides a method for screening core functional short peptides derived from walnut peptides, comprising the following steps:

[0017] S1. Preparation of walnut mixed peptides: After dissolving walnut protein, protease was added at 50~60℃ and pH 7.0~8.0 for 2~4h for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the peptide fraction with a molecular weight of less than 10kDa was collected by ultrafiltration fractionation, concentrated and freeze-dried to obtain walnut mixed peptides.

[0018] S2. Peptide sequence identification: The sequence of walnut mixed peptides was identified by using nano-level liquid chromatography-high resolution mass spectrometry combined with a data-independent acquisition mode to obtain peptide sequence information.

[0019] S3. Multi-dimensional screening: The identified peptide sequences are screened for abundance, bioactivity, safety and pharmacokinetic characteristics to obtain core functional short peptides.

[0020] Preferably, the protease in step S1 is trypsin with an enzyme activity ≥2500 U / mg; the ultrafiltration fractionation adopts a tangential flow membrane separation system equipped with a hollow fiber membrane module with a molecular weight cutoff of 10 kDa.

[0021] Preferably, in step S2, the mobile phase A of the nano-level liquid chromatography is an aqueous solution of 2% acetonitrile containing 0.1% formic acid, and the mobile phase B is an aqueous solution of 80% acetonitrile containing 0.1% formic acid, with a total gradient elution time of 6-10 min; the high-resolution mass spectrometry adopts a positive ion detection mode, with an ion source voltage of 1.2-1.8 kV and a mass spectrometry scanning range of 100-1700 m / z.

[0022] Preferably, the method further includes step S4, solid-phase synthesis and purification: using Fmoc-protected amino acids as raw materials, the core functional short peptides screened in step S3 are synthesized by solid-phase synthesis, and high-purity short peptides are obtained after HPLC purification.

[0023] Preferably, the solid-phase synthesis in step S4 uses Fmoc-Phe-Wang resin as the starting support, HBTU as the condensing agent, and NMM as the organic base; the HPLC purification uses a C18 preparative column with a detection wavelength of 220 nm, and obtains functional short peptides with a purity ≥98% through two-step gradient elution.

[0024] Thirdly, the present invention relates to a cognitive function improver, wherein the active ingredient of the cognitive function improver is the core functional short peptide described in the present invention, and also includes pharmaceutically acceptable excipients.

[0025] Furthermore, the dosage form of the cognitive function improver is an oral preparation, including powder, granules, capsules, tablets, or oral liquid.

[0026] (III) Beneficial Effects

[0027] This invention provides an application of walnut peptide in the preparation of cognitive function improvers. This walnut peptide is a core functional short peptide derived from walnuts, characterized by high bioactivity, non-toxicity, and favorable pharmacokinetic properties. This short peptide possesses significant advantages and clear technical effects:

[0028] Firstly, it has a simple structure, is easy to synthesize artificially, and has the characteristics of being easy to take orally, easy to absorb, has strong metabolic stability and strong blood-brain barrier penetration ability, which lays a solid foundation for its clinical translation and practical application.

[0029] Secondly, in vitro experiments have confirmed that, compared with mixed peptides derived from walnuts, the walnut peptide of this invention, as a core functional short peptide, exhibits superior free radical scavenging efficiency and total antioxidant activity, with more outstanding antioxidant performance.

[0030] Third, in vivo experiments (using Alzheimer's disease-like neurodegenerative disease model mice as the research subjects) showed that after oral administration, the short peptide could successfully penetrate the blood-brain barrier and enter the mouse brain. By reducing the content of malondialdehyde (an oxidative stress marker) in brain tissue, it effectively alleviated the level of oxidative stress in the brain, exerted a significant neuroprotective effect, and thus improved oxidative stress-related brain tissue damage.

[0031] Fourth, behavioral experiments have confirmed that this short peptide can significantly improve the nesting test score of model mice, and at the same time, it shows clear improvement effects in core cognitive dimensions such as daily cognitive coordination, short-term memory, spatial memory and recognition memory. It can comprehensively improve the cognitive function of model mice. This invention provides a new solution for the prevention and improvement of cognitive dysfunction. Attached Figure Description

[0032] Figure 1 A schematic diagram of the nesting experiment in a cognitive impairment model mouse model treated with walnut mixed peptides;

[0033] Figure 2 for Figure 1 Nesting score of a cognitive impairment model mouse treated with a mixture of walnut and other peptides;

[0034] Figure 3 A bar chart showing the results of the Y-maze test in mice with cognitive impairment treated with walnut mixed peptides;

[0035] Figure 4 A bar chart showing the improvement in short-term memory in a light-dark box experiment of mice with cognitive impairment treated with walnut mixed peptides.

[0036] Figure 5 A bar chart showing the mood improvement results in a light-dark chamber for a cognitive impairment model mouse after intervention with walnut mixed peptides;

[0037] Figure 6 A bar chart showing the results of proteomics experiments in the hippocampus of mice after treatment with walnut mixed peptides;

[0038] Figure 7 The changes in MDA content in the brain tissue of mice after treatment with walnut mixed peptides;

[0039] Figure 8 The results of in vitro ABTS free radical scavenging experiments on different molecular weight peptides in walnut mixed peptides;

[0040] Figure 9The screening results for core functional short peptides from walnut mixed peptides;

[0041] Figure 10 A comparative study on the in vitro antioxidant activity of the core functional short peptide AF and the walnut mixed peptide Mix was conducted to screen them.

[0042] Figure 11 A comparison of the MDA content ratios in the brain tissue of mice with an Alzheimer's disease-like neurodegenerative disease model induced by SCo (scopolamine) intervention, with the core functional short peptides AF, FV, and HA respectively as the interventions.

[0043] Figure 12 Nesting test scores were obtained by intervening with SCo-induced Alzheimer's disease-like neurodegenerative disease model mice using the core functional short peptides AF, FV, and HA, respectively.

[0044] Figure 13 The study compared the latency time improved by the intervention of the core functional short peptides AF, walnut peptide FV, and walnut peptide HA in SCo-induced Alzheimer's disease-like neurodegenerative disease model mice in light-dark box training.

[0045] Figure 14 The results of the Y-maze experiment were obtained by intervening in SCo-induced Alzheimer's disease-like neurodegenerative disease model mice with the core functional short peptides AF, FV, and HA, respectively.

[0046] Figure 15 The core functional short peptides AF, walnut peptide FV, and walnut peptide HA were used to intervene in the novel object recognition index (DI) of SCo-induced Alzheimer's disease-like neurodegenerative disease model mice. Detailed Implementation

[0047] To better explain and facilitate understanding of the present invention, a detailed description of the invention is provided below with reference to the accompanying drawings and specific embodiments. Data in each experiment are expressed as "mean ± standard deviation (Mean ± SD)". Statistical analysis was performed using SPSS 26.0 software. One-way ANOVA was used for comparisons between groups, and P < 0.05 indicated statistical significance.

[0048] Example 1

[0049] The walnut peptide disclosed in this invention is a core functional short peptide derived from walnuts. First, walnut mixed peptides are prepared using a combined enzymatic hydrolysis-membrane separation technology. Then, polypeptide fractions with potential antioxidant and cognitive protective activities are screened through experiments, laying the foundation for subsequent screening of core functional short peptides.

[0050] This embodiment details the preparation method of walnut mixed peptides and the initial screening for antioxidant and cognitive protective activities through experiments, including:

[0051] 1.1 Experimental Objective:

[0052] Walnut mixed peptides were prepared using a combined enzymatic hydrolysis-membrane separation technique, and then peptide fractions with potential antioxidant and cognitive protective activities were screened through experiments.

[0053] 1.2 Experimental Materials and Instruments:

[0054] Walnut protein (purity ≥90%); trypsin (enzyme activity ≥2500 U / mg); AKTA Flux 6 tangential flow membrane separation system (GE Healthcare Systems Trading & Development Co., Ltd.), equipped with a 10 kDa molecular weight cutoff hollow fiber membrane module; rotary evaporator; freeze dryer; ABTS free radical scavenging kit (Nanjing Jiancheng Bioengineering Institute); 8-week-old healthy male C57BL / 6J mice (SPF grade, Jicui Pharmaceutical Experimental Animal Co., Ltd.); scopolamine (purity ≥98%).

[0055] 1.3 Experimental Methods:

[0056] (1) Preparation of walnut mixed peptides: Walnut protein was dissolved and placed in a buffer system at 55℃ and pH 7.5. Trypsin was added to initiate the enzymatic hydrolysis reaction, which lasted for 3 hours. The mixed solution after enzymatic hydrolysis was immediately subjected to ultrafiltration fractionation using an AKTA Flux 6 tangential flow membrane separation system, and peptide fractions with a molecular weight less than 10 kDa were collected. The obtained filtrate was concentrated by rotary evaporation and then freeze-dried to obtain walnut mixed peptide powder samples, which were then sealed and refrigerated for later use.

[0057] (2) Initial screening of in vivo cognitive protective activity through the construction of animal models of cognitive impairment and behavioral experiments:

[0058] ① The method for constructing a learning and memory-based cognitive impairment model induced by scopolamine:

[0059] Eight-week-old healthy male C57BL / 6J mice (SPF grade) were purchased from Jicui Pharmaceutical Experimental Animal Co., Ltd. All animals were housed in a specific pathogen-free (SPF) environment with a laboratory temperature maintained at 22±2℃ and relative humidity at 50±10%, using a 12h light / 12h dark cycle. Mice had free access to standard experimental feed and water. After one week of acclimatization, the mice were randomly divided into four groups (n=10): normal control group 1 (N1), model group 1 (M1), high-dose walnut mixed peptide intervention group (H, 600mg / kg·BW), and low-dose walnut mixed peptide intervention group (L, 300mg / kg·BW). First, the model was constructed: except for the normal control group 1, all other groups were drug treatment groups. Mice in each drug treatment group were intraperitoneally injected daily with scopolamine (1.0 mg / kg BW) to induce a learning and memory-type cognitive impairment model for 8 consecutive days. After successful modeling (verified by preliminary experiments that cognitive function was significantly reduced), each drug treatment group was then administered the predetermined dose by gavage for 21 consecutive days, while the normal control group and model group 1 were given an equal volume of physiological saline. During the drug treatment period, the cognitive function of mice was assessed using the nesting test, Y-maze test, and light-dark box test to verify the restorative effect of walnut mixed peptides on damaged cognitive function.

[0060] ② Behavioral experimental methods:

[0061] The Light / Dark Box Test (LDBT) assesses anxiety-like behavior and learning / memory abilities in mice using the Passive Avoidance Test. Before testing, mice are placed in darkness for 30 minutes to acclimatize. They are then placed in a bright room and allowed to actively enter an adjacent dark room during exploration. Upon first entering the dark room, a mild electric shock is administered to create aversive memory. The mice remain still after the shock until they move again; this time interval is recorded as the initial training latency. The mice are then placed in the bright room again, and the training is repeated, with a mild electric shock administered immediately upon entering the dark room. The training lasts for 1 hour before testing. The mice are then placed in the bright room again, and the test latency is recorded. Mice with impaired learning / memory function typically show insufficient or no significant increase in latency after training.

[0062] The Y-Maze Test: The Y-Maze consists of three equal-arm channels with an angle of 120°, used to assess the spatial working memory of mice. Mice are placed in the center of the Y-Maze and allowed to explore freely for 5 minutes. The order and number of times they enter each arm are recorded. Entering three different arms consecutively (e.g., ABC, BCA, etc.) is defined as one spontaneous alternation. The percentage of spontaneous alternations is calculated as: (Number of actual alternations / (Total number of entries - 2)) × 100%. A lower alternation rate indicates impaired memory.

[0063] Nesting Test: Used to assess the daily activity levels, motivation, and social behavior of mice. Before the test, each mouse is placed individually in a clean cage and provided with the same weight (approximately 3g) of cotton balls. After 24 hours, nesting performance is observed and scored: 0 points indicates no touching, 1 point indicates partial tearing, 2-3 points indicate significant accumulation, and 4-5 points indicate the formation of a complete nest. Decreased nesting ability indicates impaired cognition and motivation.

[0064] (3) Detection of oxidative stress indicators in hippocampal tissue:

[0065] ① After the behavioral experiments, mice were euthanized and brain tissue was collected. A 10% (w / v) brain tissue homogenate was prepared by adding pre-cooled physiological saline at a specific mass ratio. The homogenate was centrifuged at 4°C and 10,000 rpm for 10 minutes, and the supernatant was collected. The malondialdehyde (MDA) content in the brain tissue homogenate was determined using a commercially available kit (Nanjing Jiancheng Bioengineering Institute). The MDA content was expressed as nmol / mg protein and was used to reflect the level of oxidative stress in the brain tissue.

[0066] ② Detection of superoxide dismutase (SOD) and related antioxidant proteins in mouse hippocampus: Mouse hippocampal tissue was collected and pre-chilled RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added at a mass-to-volume ratio of 1:10. The mixture was homogenized on ice and centrifuged at 12,000 r / min for 15 min at 4°C. The supernatant (i.e., total protein extract) was collected. Detection procedure: Following the instructions of the ELISA kit for the corresponding antioxidant protein, the standard and sample were sequentially added to the wells of an ELISA plate coated with specific antibodies and incubated at 37°C for 30 min. After washing, enzyme-labeled secondary antibody was added and incubated at 37°C for 30 min. After washing again, substrate solution was added for color development. After terminating the reaction, the absorbance (OD value) was measured using an ELISA reader at the corresponding wavelength (e.g., 450 nm). Result calculation: A standard curve was plotted based on the OD values ​​of the standards. The absolute content (ng / mg protein) of the target antioxidant protein was calculated by substituting the OD values ​​of the samples.

[0067] (4) In vitro antioxidant activity detection: The antioxidant activity of walnut mixed peptides of different molecular weights was verified by ABTS free radical scavenging assay. Walnut mixed peptide sample groups with different molecular weights (0-3kDa, 3-10kDa, <10kDa, >10kDa, WPH walnut peptide) were set up. The scavenging rate of ABTS+ free radicals of the samples was detected according to the kit instructions to evaluate their antioxidant potential.

[0068] 1.4 Experimental Results:

[0069] (1) Improved cognitive function in vivo:

[0070] Walnut protein was first hydrolyzed using trypsin to obtain walnut mixed peptides. These peptides were then fractionated by ultrafiltration, and the fractions with a molecular weight less than 10 kDa were collected. Animal experiments were then conducted to evaluate their neuroprotective effects. Behavioral results showed that the walnut mixed peptide group performed superiorly in several cognitive-related experiments.

[0071] Specifically, such as Figure 1 , Figure 2 As shown, the nesting experiment results of cognitive impairment model mice treated with walnut mixed peptides demonstrate the effect of walnut mixed peptides on improving the daily executive function and living functions of the model mice.

[0072] In the figure, N1 represents the normal control group 1, M1 represents the model group 1, H represents the high-dose intervention group of walnut mixed peptides (600 mg / kg BW), and L represents the low-dose intervention group of walnut mixed peptides (300 mg / kg BW). In the nesting experiment, compared with the normal control group 1 (N1), the nesting score of the model group 1 (M1) was significantly lower, indicating that the mice in the model group 1 (M1) had the worst executive and daily living abilities. The nesting score of the high-dose intervention group H of walnut mixed peptides showed significant recovery, followed by the low-dose intervention group L. This reflects that walnut mixed peptides with a molecular weight less than 10 kDa can improve the executive and daily living abilities of mice and alleviate daily behavioral impairment caused by cognitive impairment.

[0073] like Figure 3 The results of the Y-maze test in a cognitive impairment model mouse after intervention with walnut mixed peptides were presented to demonstrate the restorative effect of walnut mixed peptides on spatial working memory in the model mice. Compared with the normal control group 1 (N1), the spontaneous alternation ratio in model group 1 (M1) was significantly reduced, while the spontaneous alternation ratio in the low-dose walnut mixed peptide intervention group L and the high-dose walnut mixed peptide intervention group H was increased. Furthermore, the spontaneous alternation ratio in the high-dose walnut mixed peptide intervention group H recovered to a level close to that of the normal control group, i.e., group N1. This indicates that walnut mixed peptides can promote the recovery of spatial working memory.

[0074] like Figure 4 , Figure 5The bar charts shown above illustrate the improvement in short-term memory and mood in a light-and-dark chamber experiment of mice with cognitive impairment treated with walnut mixed peptides. These bar charts represent the effects of walnut mixed peptides on improving short-term memory and regulating emotional stability in the model mice, respectively. The light-and-dark chamber experiment showed that short-term memory and emotional stability were decreased in model group 1 (M1), but intervention with walnut mixed peptides improved both short-term memory and emotional stability (increased latency and reduced number of shocks). These results suggest that walnut mixed peptides have a positive impact on multidimensional cognitive function and possess potential for protection against neurodegenerative disorders.

[0075] In summary, this study used a scopolamine-induced cognitive impairment model to evaluate the neuroprotective effect of walnut mixed peptides. The results showed that, compared to the model group M1, mice treated with walnut mixed peptides exhibited significant behavioral improvements, with markedly enhanced learning and memory abilities (P<0.05). This preliminary in vivo experiment demonstrates that walnut mixed peptides can effectively alleviate scopolamine-induced cognitive impairment, suggesting a potential neuroprotective effect.

[0076] (2) Regulation of oxidative stress level

[0077] like Figure 6 The bar chart shown presents the results of proteomics experiments in the hippocampus of mice after intervention with walnut mixed peptides. It illustrates the changes in protein expression in the hippocampus of mice after intervention with walnut mixed peptides, providing a basis for subsequent correlation analysis of core functional short peptides.

[0078] After treatment with walnut mixed peptides, the expression of SOD3, an antioxidant protein related to oxidative stress regulation, was significantly increased in the hippocampus of mice, suggesting that walnut mixed peptides may alleviate oxidative damage and promote cognitive function recovery by enhancing the antioxidant defense system.

[0079] like Figure 7 The figure shows the changes in MDA content in the brain tissue of mice after treatment with walnut mixed peptides, which is used to present the regulatory effect of walnut mixed peptides on the level of oxidative stress in the brain tissue of model mice (MDA is a marker of oxidative stress). Compared with the normal control group 1 (N1), the MDA content in the brain tissue of model group 1 (M1) was significantly increased, indicating that model group 1 had obvious oxidative stress damage. The MDA content in the brain tissue of mice in groups L and H after walnut mixed peptide intervention was significantly decreased (P<0.05), indicating that the walnut mixed peptides can effectively inhibit lipid peroxidation, alleviate the level of oxidative stress in the brain, and exert a neuroprotective effect.

[0080] (3) In vitro antioxidant activity

[0081] Figure 8This study presents the results of in vitro ABTS free radical scavenging experiments on different molecular weight peptides in walnut mixed peptides. The aim is to compare the differences in antioxidant activity among different molecular weight fractions of walnut mixed peptides and to verify the antioxidant potential of small molecule peptides (<10 kDa). Walnut mixed peptides were divided into 0-3 kDa, 3-10 kDa, <10 kDa, >10 kDa, and unseparated initial walnut peptide powder (WPH) for ABTS free radical scavenging experiments. The results are as follows: Figure 8 As shown, walnut peptides with a molecular weight of less than 10 kDa have good free radical scavenging ability, and the scavenging efficiency increases in a dose-dependent manner as the molecular weight of the peptide decreases, indicating that this fraction has significant in vitro antioxidant potential.

[0082] Example 2

[0083] Although walnut mixed peptides have good antioxidant and cognitive protective effects as a whole, their composition is complex, their mechanisms are unclear, and their artificial synthesis is difficult. In this embodiment, a multi-index prediction method was used to systematically screen the components of walnut mixed peptides <10kDa, and core functional short peptides with neuroprotective potential were selected. The experiment is as follows:

[0084] 2.1 Experimental Objective:

[0085] By combining proteomics analysis with multidimensional activity prediction, core functional short peptides with high bioactivity, non-toxicity and good pharmacokinetic characteristics were screened and identified from the walnut mixed peptides prepared in Example 1.

[0086] 2.2 Experimental Materials and Instruments:

[0087] Walnut mixed peptide powder prepared in Example 1; Vanquish Neo nano-level liquid chromatography system (Thermo Fisher Scientific, USA); uPAC High Throughput analytical column (75 μm × 5.5 cm, Thermo, USA); Orbitrap Astral high-resolution mass spectrometer (Thermo Fisher Scientific, USA); PeptideRanker, AIPpred, AntiInflam, AntioxPred, AOPs, ToxinPred, AllergenFP, SwissADME, ADMETlab2.0 and other activity prediction platforms.

[0088] 2.3 Experimental Methods:

[0089] (1) Peptide sequence identification: After quantification, the walnut mixed peptide powder was dissolved in mass spectrometry loading buffer to form an equal volume of sample, and mass spectrometry analysis was performed using data-independent acquisition (DIA) mode. Peptide separation was performed using a Vanquish Neo nano-level liquid chromatography system. Mobile phase A was an aqueous solution of 2% acetonitrile containing 0.1% formic acid, and mobile phase B was an aqueous solution of 80% acetonitrile containing 0.1% formic acid. The total gradient elution time was 8 min. The liquid chromatography system was coupled with an Orbitrap Astral high-resolution mass spectrometer, using positive ion detection mode, ion source voltage of 1.5 kV, and mass spectrometry scanning range of 100–1700 m / z. Data acquisition and control were performed using Thermo Xcalibur 4.7 software to obtain accurate sequence information of the peptides.

[0090] (2) In silico screening of core functional short peptides: The identified peptide sequences were input into a multi-model activity prediction platform for comprehensive screening. The screening criteria included: ① peptide abundance > 0.1%; ② PeptideRanker bioactivity score > 0.45; ③ free radical scavenging ability prediction value > 0.4; ④ predicted by ToxinPred and AllergenFP to be non-toxic and non-sensitizing; ⑤ predicted by SwissADME and ADMETlab2.0 to have good oral absorption, metabolic stability and blood-brain barrier penetration ability.

[0091] 2.4 Experimental Results

[0092] More than 200 mixed peptide sequences of walnut were identified using LC-MS / MS technology. After screening using the aforementioned multi-dimensional screening criteria, a core functional short peptide with high bioactivity, non-toxicity, and excellent pharmacokinetic characteristics was finally obtained. Its amino acid sequence is Ala–Phe–Val–His–Trp–Tyr (hexapeptide, named AFVHWY, abbreviated as AF, sequence SEQ ID NO. 1). This peptide is rich in key antioxidant residues such as Trp, Tyr, and His, providing a structural basis for its subsequent antioxidant and neuroprotective functions. Figure 9 As shown, this core functional short peptide ranks among the top in all scores: it has advantages in abundance, predicted activity, and anti-inflammatory potential. It also performs well in free radical scavenging prediction, Peptide Ranker score, anti-inflammatory activity, and sequence stability, and has good reactive active sites and functional potential, especially ranking high in antioxidant-related indicators.

[0093] Compared with walnut mixed peptides or long-chain peptides, the core functional short peptide AF is easier to synthesize, more stable, and more rapidly absorbed, and has higher cell penetration and lower immunogenicity, making it suitable for subsequent in vitro and in vivo functional validation and drug development.

[0094] Example 3

[0095] This example describes the solid-phase synthesis and purification of the core functional short peptide AFVHWY. The experiments included:

[0096] 3.1 Experimental Objective:

[0097] High-purity core functional short peptide AFVHWY was prepared using solid-phase synthesis to meet the needs of subsequent in vivo and in vitro functional verification.

[0098] 3.2 Experimental Materials and Instruments:

[0099] Fmoc-Phe-Wang resin; Fmoc protected amino acids (Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-His-OH, Fmoc-Trp-OH, Fmoc-Tyr-OH); HBTU, NMM, DIEA, DCM, DMF, hexahydropyridine, TFA, anisole, phenol, EDT; Kaiser reagents (phenol / ethanol solution, redistilled pyridine, ninhydrin / anhydrous ethanol solution); high performance liquid chromatograph (equipped with a C18 (10Å) preparative column, 10cm × 25cm); vacuum desiccator; centrifuge.

[0100] 3.3 Experimental Methods:

[0101] (1) Solid-phase synthesis: Using Fmoc-Phe-Wang resin as the starting support, the resin was swollen with DCM for 30 min, and then deprotected by adding 20% ​​hexahydropyridine / DMF solution. The resin was washed three times with DMF. Fmoc protected amino acids and HBTU were added at 3 times the molar amount of resin, and NMM was added as an organic base at 6 times the molar amount. The condensation was carried out under nitrogen agitation for 30 min. The degree of condensation was detected by Kaiser reagent. If the color was yellow, the deprotection, washing and condensation steps were repeated. Tyr, Trp, His, Val, Phe and Ala amino acids were coupled in sequence until the complete sequence was synthesized.

[0102] (2) Peptide cleavage and precipitation: After synthesis, the resin was washed three times with methanol and DCM alternately and dried under vacuum for 12 hours. The cleavage solution (87.5% TFA + 5% benzyl sulfide + 5% phenol + 2.5% EDT + 0.5% H2O) was added to cleave the peptide from the resin. After precipitating with ether, the peptide was centrifuged at 4000 rpm and washed five times to collect the precipitate.

[0103] (3) Purification and purity detection: The precipitate was dissolved by ultrasonication with deionized water and acetonitrile at a volume ratio of 3:1 and filtered through a 0.45 μm microporous membrane; purification was performed by HPLC with a detection wavelength of 220 nm. Mobile phase A was 0.1% trifluoroacetic acid / acetonitrile and mobile phase B was 0.1% trifluoroacetic acid / water. The first step purification gradient was 25%-35% (0 min A pump 25%, 100 min A pump 35%, 120 min A pump 70%). The target peptide peak was collected and the molecular weight was confirmed by mass spectrometry. The second step purification gradient was 26%–36% (other conditions were the same as the first step). The qualified fractions were combined, concentrated by rotary evaporation, and then freeze-dried to obtain the high-purity core functional short peptide AFVHWY.

[0104] 3.4 Experimental Results:

[0105] After two-step HPLC purification, the core functional short peptide AFVHWY was obtained with a purity ≥98%, and its molecular weight was consistent with the theoretical value, meeting the requirements for subsequent in vivo and in vitro functional verification experiments. For comparison, walnut peptides HA (sequence HAGUGVM, SEQ ID NO.2) and FV (FVHPSLI, SEQ ID NO.3) were also synthesized using the same method. Both HA and AF (AFVHWY, SEQ ID NO.1) were isolated from walnut mixed peptides, with walnut peptide FV exhibiting the highest abundance. Subsequent experiments compared the antioxidant activity and neuroprotective activity of the core functional short peptide AF with other walnut peptides FV and HA.

[0106] Example 4

[0107] This embodiment verifies the effect of the high-purity core functional short peptide AFVHWY prepared in Example 3 on improving cognitive impairment, including:

[0108] 4.1 Experimental Objective:

[0109] Using scopolamine-induced Alzheimer's disease-like neurodegenerative disease model mice as the research subjects, this study verified the in vivo antioxidant activity and cognitive function improvement effect of the core functional short peptide AFVHWY.

[0110] 4.2 Experimental Materials and Instruments:

[0111] Example 3: High-purity core functional short peptides AF, walnut peptide HA, and walnut peptide FV synthesized; 8-week-old healthy male C57BL / 6J mice (SPF grade); scopolamine; malondialdehyde (MDA) detection kit; light and dark chamber experimental setup, Y-maze experimental setup, new object recognition experimental setup; centrifuge; enzyme-linked immunosorbent assay (ELISA) reader.

[0112] 4.3 Experimental Methods:

[0113] (1) Animal grouping and treatment: Eight-week-old healthy male C57BL / 6J mice (SPF grade) were purchased from Jicui Pharmaceutical Experimental Animal Co., Ltd. All animals were housed in a specific pathogen-free (SPF) environment with a laboratory temperature maintained at 22±2℃ and a relative humidity of 50±10%, using a 12h light / 12h dark cycle. Mice had free access to standard experimental feed and water. After one week of acclimatization, the mice were randomly divided into 5 groups (n=10): normal control group 2 (N2), model group 2 (M2), AF intervention group (AF orally administered, 100mg / kg·BW), HA intervention group (HA orally administered, 100mg / kg·BW), and FV intervention group (FV orally administered, 100mg / kg·BW).

[0114] First, an Alzheimer's disease-like neurodegenerative disease model was established: Except for the normal control group 2 (N2), the other groups of mice were drug-treated. Each drug-treated group received daily intraperitoneal injections of scopolamine SCo (1.5 mg / kg) to induce the Alzheimer's disease-like neurodegenerative disease model for 8 consecutive days. After successful modeling (verified by preliminary experiments showing a significant decline in cognitive function), each drug-treated group was administered the prescribed dose by gavage for 28 consecutive days (4 weeks). The normal control group 2 (N2) and model group 2 (M2) received an equal volume of physiological saline. SCo is a classic muscarinic receptor antagonist that blocks the central cholinergic signaling pathway, leading to learning and memory impairment, and is widely used to simulate mild to moderate cognitive deficits. Cognitive impairment was induced in mice by intraperitoneal injection of SCo (1.5 mg / kg), and the intervention effect of AF was evaluated through behavioral experiments.

[0115] (2) In vitro total antioxidant capacity detection: The total antioxidant capacity (T-AOC) of the core functional short peptides AF, walnut peptide HA and walnut peptide FV was determined by chemical colorimetric method to evaluate the difference in antioxidant activity between the two.

[0116] (3) Behavioral experiments: Behavioral tests were conducted during the drug administration period to assess the recovery of cognitive function in mice. The specific methods are as follows:

[0117] ① Light and dark chamber experiment: Before the test, mice were adapted to the dark chamber for 30 minutes. After being placed in the light chamber, the training latency was recorded when they first entered the dark chamber. The test latency was recorded 1 hour later.

[0118] ②Y-maze experiment: Mice were placed in the center of the maze and allowed to explore freely for 5 minutes. The order and number of times mice entered each arm were recorded, and the percentage of spontaneous alternation was calculated.

[0119] ③ Nest building experiment: Mice were raised alone and given 3g cotton balls. After 24 hours, the nest building score was assessed according to the 0-5 standard.

[0120] For detailed methods of experiments ①-③ above, please refer to Example 1.

[0121] ④ Novel Object Recognition Experiment: Used to evaluate the recognition and memory abilities of mice. The experiment is divided into an adaptation period, a learning period, and a testing period. During the adaptation period, mice are allowed to freely explore an empty box for 5 minutes. During the learning period, two objects of the same shape are placed in the box, and the mice explore freely for 5 minutes. After 1 hour, the testing period begins, where one of the old objects is replaced with a new object of a different shape, and the mice explore again for 5 minutes. The exploration time for the new and old objects is recorded. Recognition Index (DI) = (New object exploration time / (New object exploration time + Old object exploration time) × 100%. A higher DI value indicates better memory ability.

[0122] The new object recognition experiment is divided into an adaptation period (exploration of an empty box for 5 minutes), a learning period (exploration of two identical objects for 5 minutes), and a testing period (replacing one object with a new object and exploring for 5 minutes), and the recognition index (DI) is calculated.

[0123] (4) Detection of MDA content in brain tissue: After the behavioral experiment, a 10% homogenate was prepared from mouse brain tissue, centrifuged at 4℃ and 10000r / min for 10min, and the supernatant was collected. The MDA content was determined according to the instructions of the MDA detection kit.

[0124] 4.4 Experimental Results:

[0125] (1) In vitro antioxidant activity:

[0126] like Figure 10 As shown, at the same mass concentration, the total antioxidant capacity of the core functional short peptide AF was significantly higher than that of walnut peptide HA, walnut peptide FV and walnut mixed peptide Mix (<10kDa), indicating that the core functional short peptide AF has better free radical scavenging efficiency and confirms that it has more outstanding antioxidant potential.

[0127] (2) Effect on alleviating oxidative stress in the body:

[0128] like Figure 11 As shown, the comparison of MDA content in the brain tissue of mice with SCo (scopolamine)-induced Alzheimer's disease-like neurodegenerative disease is illustrated. This comparison was used to evaluate the alleviating effects of these three peptides on oxidative stress levels in the brain tissue of the model mice. Compared to model group 2 (M2), the MDA content in the brain tissue of the AF intervention group was significantly reduced (P<0.05), reaching the lowest value among all experimental groups and approaching the level of the normal control group 2 (N2). This indicates that the core functional peptide AF can effectively inhibit lipid peroxidation in brain tissue, alleviate oxidative stress damage, and exert a neuroprotective effect.

[0129] (3) Improvement in cognitive function:

[0130] Figure 12 The results of the nesting test in mice with SCo-induced Alzheimer's disease-like neurodegenerative disease were shown, using the core functional short peptide AF, walnut peptide FV, and walnut peptide HA to compare their effects on improving daily cognitive coordination in the model mice. Compared with model group 2 (M2), the nesting score of mice in the AF intervention group was significantly higher, and also higher than that in the FV intervention group and the HA intervention group.

[0131] Figure 13 The study compared the latency time of light-dark box training in mice with SCo-induced Alzheimer's disease-like neurodegenerative disease model by intervention with the core functional short peptides AF, FV, and HA. This was used to compare the effects of AF and FV / HA on improving short-term memory in model mice. Compared with model group 2 (M2), the latency time of the light-dark box test in the AF intervention group was significantly prolonged, and the latency time improved by training was greater than that in the FV intervention group and the HA intervention group.

[0132] Figure 14 The results of the Y-maze test in mice with SCo-induced Alzheimer's disease-like neurodegenerative disease were compared to those of the core functional short peptides AF, FV, and HA, respectively, to assess their effects on restoring spatial memory in the model mice. Compared with model group 2 (M2), the proportion of spontaneous alternation in the Y-maze test was improved in the AF intervention group, FV intervention group, and HA intervention group, with the improvement in the AF intervention group being greater than that in the FV intervention group and HA intervention group.

[0133] Figure 15 The novel object recognition index (DI) of SCo-induced Alzheimer's disease-like neurodegenerative disease model mice was evaluated using core functional peptides AF, FV, and HA. The effects of these peptides on improving cognitive and memory abilities in the model mice were compared. Compared with model group 2 (M2) and the FV intervention group, the AF intervention group showed a significant increase in the novel object recognition index (DI) (P<0.05). These results indicate that among various walnut peptides, core functional peptide AF has a superior effect on improving cognitive function in Alzheimer's disease-like neurodegenerative disease model mice compared to the FV and HA intervention groups. This demonstrates that core functional peptide AFVHWY can significantly improve daily cognitive coordination, short-term memory, spatial memory, and cognitive and memory abilities in model mice, and has a significant ameliorative effect on scopolamine-induced cognitive impairment.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a walnut peptide in the preparation of a cognitive function improver, characterized in that: The cognitive function improver improves cognitive impairment, the amino acid sequence of the walnut peptide is AFVHWY, and the cognitive impairment is cognitive impairment caused by Alzheimer's disease-like neurodegenerative disease.

2. The application of the walnut peptide according to claim 1 in the preparation of cognitive function improvers, characterized in that: The cognitive function improver uses the walnut peptide as the sole active ingredient; or the cognitive function improver uses the walnut peptide as one of its active ingredients.

3. The application of the walnut peptide according to claim 2 in the preparation of cognitive function improvers, characterized in that: The cognitive function improver also includes pharmaceutically acceptable excipients and / or delivery carriers.

4. The application of the walnut peptide according to claim 2 or 3 in the preparation of cognitive function improvers, characterized in that: The cognitive function improver is an oral formulation.

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