A type of bloating hippocampal peptide and its application in the preparation of products that enhance memory and improve cognitive impairment.

Abstract

This invention relates to the field of preparation and application of bioactive peptides, and more particularly to a bloated hippocampal peptide and its application in the preparation of products that enhance memory and improve cognitive impairment. This invention utilizes green and controllable enzymatic hydrolysis technology to obtain protein polypeptides with molecular weights primarily <1000 Da and possessing nutritional, health-promoting, and medicinal value. Based on this, further research yielded a highly bioactive peptide sequence and verified its medicinal value.

Description

Technical Field

[0001] This invention relates to the field of preparation and application of bioactive peptides, and more particularly to a bloated hippocampal peptide and its application in the preparation of products that enhance memory and improve cognitive impairment. Background Technology

[0002] Brain aging is a gradual process spanning a long period, accompanied by a decline in learning and memory abilities, and may eventually develop into neurodegenerative diseases such as Alzheimer's disease (AD). However, the limitations of current diagnostic methods pose a significant challenge to the intervention of Alzheimer's disease. From the early stages of the disease to the appearance of obvious cognitive impairment and other pathological deterioration symptoms, it often takes several years or even decades. During this period, the pathological progression of AD may quietly accelerate, causing irreversible damage, which not only devastates the patient's quality of life but also places a heavy burden on families and society. Therefore, improving the aging process, delaying memory decline, and curbing the development of cognitive impairment have become a focal point of great concern for the scientific and medical communities and society as a whole.

[0003] Hippocampus abdominalis is a new species introduced to my country in recent years. With the continuous development of the marine aquaculture industry, the improvement of living standards, and the popularization and promotion of the concept of "health and wellness," people's demand for health and wellness is increasing. Exploring its potential nutritional components is beneficial for the high-value utilization of seahorses. Currently, research on hippocampal peptides for cognitive impairment and Alzheimer's disease only includes a complex product and a few peptide segments obtained from the inventor's previous research, namely, a type of abdominalis peptide disclosed in Chinese patent CN120392956A and its application in the preparation of products that enhance memory and improve cognitive impairment. However, this is insufficient for research on abdominalis peptides, and further exploration of more functional peptides is needed to meet the needs of technological development. Summary of the Invention

[0004] The technical problem to be solved by this invention is that the existing technology has insufficient research on the effects of bloated hippocampal active peptides on improving learning and memory abilities and improving cognitive impairment, and further research is needed.

[0005] To address the aforementioned issues, this invention provides a bloated hippocampal peptide and its application in the preparation of products that enhance memory and improve cognitive impairment. Utilizing a green and controllable enzymatic hydrolysis technology, a protein polypeptide with a molecular weight primarily <1000 Da and possessing nutritional, health-promoting, and medicinal value is obtained. Furthermore, a highly bioactive peptide sequence is obtained through further research.

[0006] To achieve the above objectives, the present invention utilizes the following technical means: a bloating hippocampal peptide, the amino acid sequence of which is shown in SEQ ID NO.1:

[0007] SEQ ID NO.1:

[0008] DFPLPFS.

[0009] The above-mentioned expanded hippocampal peptides are used in the preparation of products that enhance memory and improve cognitive impairment. Synaptic damage occurs in the early stages of Alzheimer's disease, leading to a decrease in acetylcholinesterase activity, which precedes neuronal loss and the deposition of amyloid-beta (Aβ), forming the structural basis of AD pathogenesis. Excessive accumulation of Aβ in the brain leads to the excessive generation of intracellular reactive oxygen species, damaging intracellular and extracellular calcium... 2+ Aberrant modification of Tau protein, leading to imbalance, triggers a series of cascade reactions, causing neuronal dysfunction and ultimately cognitive impairment. Animal and cell experiments have shown that hippocampal peptides improve learning and memory by inhibiting the production and accumulation of Aβ in the hippocampus. β-secretase (BACE1) is a key enzyme catalyzing the hydrolysis of amyloid precursor protein (APP) to produce β-amyloid protein (Aβ), and Aβ deposition is directly related to the development of Alzheimer's disease. Further molecular docking results revealed that the peptide binds to BACE1 protein at a rate of -8.0 kcal / mol. The peptide can insert into the active pocket of BACE1, interacting with key amino acid residues to form van der Waals forces, C-H bonds, alkyl groups, and π-alkyl bonds, thereby inhibiting its activity and ultimately improving memory and cognitive impairment.

[0010] Furthermore, the bloated hippocampal peptide with the amino acid sequence shown in SEQ ID NO.1 is artificially synthesized.

[0011] A method for preparing a powder containing the above-mentioned expanded hippocampal peptide includes the following steps:

[0012] Fresh bloated seahorses were taken, washed, freeze-dried, and pulverized into powder. Distilled water was added, and 3% papain was added and stirred evenly. The pH was adjusted to 6.5 with HCl solution and NaOH solution, respectively, and enzymatic hydrolysis was carried out at 50°C for 3 h.

[0013] After centrifugation, the supernatant of the enzymatic hydrolysate was filtered through a 0.45 μm microporous membrane and then centrifuged at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The peptides with a molecular weight cutoff of 3 kDa were obtained by separation through the ultrafiltration membrane. The peptide solution was freeze-dried to obtain expanded hippocampal peptide powder.

[0014] Furthermore, distilled water is added at a material-to-liquid ratio of 1:9.

[0015] Furthermore, the concentrations of both the HCl solution and the NaOH solution are 1 mol / L.

[0016] Furthermore, the papain has an enzyme activity of 800,000 U / g.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) Active peptides in hippocampal peptide hydrolysates were screened using APP protein as the target. Using mass spectrometry and activity prediction methods, a potential hippocampal active peptide sequence DFPLPFS with memory-enhancing ability was disclosed. The activity of the peptide was verified using APP / PS1 (M146L) double-transformed CHO (CHO-APP / PS1) cells, providing a theoretical basis for the development of products that enhance memory and improve cognitive impairment by expanding hippocampal active peptides. Attached Figure Description

[0019] Figure 1 This is the molecular weight distribution of the expanded hippocampal peptide.

[0020] Figure 2 These are the results of the Morris water maze experiment in SAMP8 mice: A represents the latency of mice in the orientation test; B represents the number of times mice crossed platforms in the spatial exploration test; and C represents the time mice spent in the target quadrant. Note: ## p < 0.01 compared with the control group; * p < 0.05 compared with the model group; ** p < 0.01 compared with the model group.

[0021] Figure 3 In the Y-maze experiment, A represents the total number of arm advances and B represents the spontaneous alternation rate in SAMP8 mice. Note: # Compared with the control group, p < 0.05; ## Compared with the control group, p < 0.01; * Compared with the model group, p < 0.05.

[0022] Figure 4 These are the results of the novel object recognition experiment in SAMP8 mice: A represents the time spent exploring the object, and B represents the recognition index of different objects. Note: ## p < 0.01 compared with the control group; ** p < 0.01 compared with the model group.

[0023] Figure 5 These are the results of the novel object recognition experiment in APP / PS1 double transgenic mice: A represents the time spent exploring the object, and B represents the recognition index of different objects. Note: ## p < 0.01 compared with the control group; * p < 0.05 compared with the model group.

[0024] Figure 6These are the results of the Morris water maze test in AD model rats: A represents the latency time of the rats in the orientation test; B represents the number of times the rats crossed the platform in the spatial exploration test; and C represents the time the rats spent in the target quadrant. Note: ## p < 0.01 compared with the sham-operated group; * p < 0.05 compared with the model group; ** p < 0.01 compared with the model group.

[0025] Figure 7 The effect of different concentrations of peptides on the survival rate of CHO-APP / PS1 cells.

[0026] Figure 8 This study investigated the effect of peptides on the levels of Aβ40 and Aβ42 in CHO-APP / PS1 cells. A represents the intracellular Aβ40 level, and B represents the Aβ42 level. Note: Different letters indicate significant differences between groups.

[0027] Figure 9 This study investigated the effect of peptides on the extracellular Aβ40 and Aβ42 levels in CHO-APP / PS1 cells. A represents the extracellular Aβ40 level, and B represents the Aβ42 level. Note: Different letters indicate significant differences between groups.

[0028] Figure 10 This is a diagram showing the docking results of the peptide segment of SEQ ID NO.1 with the BACE1 receptor protein molecule; where A is the 3D structure of the docking between the peptide segment and the receptor protein; and B is the 2D structure of the docking between the peptide segment and the receptor protein. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0031] Example 1:

[0032] A type of bloating hippocampal peptide powder is prepared by the following method:

[0033] Preparation of enzymatic hydrolysate of expanded hippocampus peptides: 100 g of fresh expanded hippocampus was washed, freeze-dried, and pulverized into powder. Distilled water was added at a material-to-liquid ratio of 1:9 (m / v), and 3% papain was added and stirred evenly. The pH was adjusted to 6.5 with HCl solution (1 mol / L) and NaOH solution (1 mol / L), respectively, and enzymatic hydrolysis was carried out at 50 °C for 3 h. After centrifugation (4 °C, 4000 g / min for 15 min), the supernatant was filtered through a 0.45 μm microporous membrane and then centrifuged at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. Peptides with a molecular weight cutoff of less than 3 kDa were obtained by separation through the ultrafiltration membrane. The peptide solution was freeze-dried to obtain expanded hippocampus peptide powder. Papain (800,000 U / g, G8430-25) was purchased from Solarbio Science & Technology Co., Ltd.

[0034] 1. Evaluation of the effects on memory and cognitive impairment in the SAMP8 aging mouse model:

[0035] Molecular weight determination of expanded hippocampal peptide: The molecular weight distribution of expanded hippocampal peptide was determined by gel size exclusion chromatography. Specific conditions were as follows: column: Superdex peptide PE10 / 300GL; sample concentration: 2 mg / mL; injection volume: 100 μL; mobile phase: 25% acetonitrile (0.1% formic acid); flow rate: 0.35 mL / min; column temperature: room temperature; UV detection wavelength: 214 nm.

[0036] The molecular weight distribution of the total enzymatic hydrolysate of papain from expanded hippocampus was determined by gel electrophoresis, and the results are as follows: Figure 1 As shown, by comparing with the standard molecular weight curve, it was calculated that 23.96% of the peptides hydrolyzed by papain in the expanded hippocampus peptide were less than 500 Da, and the largest proportion (48.21%) were short peptides with a molecular weight of 500-1 kDa, of which 27.83% were short peptides with a molecular weight of 1 kDa-3 kDa. Figure 1 ).

[0037] Animal experiments: Thirty SAMP8 mice and ten SAMR1 mice were purchased from Nanjing Qingzilan Technology Co., Ltd. The animal housing environment was maintained at 21°C–23°C and 45%–55% humidity, with a 12-hour light / 12-hour dark cycle. Mice had free access to food and water during the experiment. All mice were fed bar-shaped feed (AIN-93G type feed) until four months of age, then a high-fat diet until six months of age. SAMP8 mice were randomly divided into three groups according to body weight: a model group, a low-dose hippocampal peptide group (200 mg / kg BW), and a high-dose hippocampal peptide group (400 mg / kg BW). SAMR1 mice served as the control group and were supplemented with hippocampal peptide via gavage once daily until nine months of age, at which point behavioral tests began.

[0038] Morris Water Maze: The pool was 130 cm in diameter, 50 cm high, and 30 cm deep, with the water temperature controlled at 22 ± 1°C. Using ANY software, the circular pool was divided into four quadrants. The first quadrant was selected, and a circular platform with a diameter of 9 cm was placed 1 cm below the water surface at the center of the quadrant. A camera was positioned directly above the pool and connected to the computer. To eliminate interference from markers on the mice's spatial orientation during the test, ink was added to the pool. The entire behavioral testing process included a navigational orientation experiment and a spatial exploration experiment.

[0039] Orientation and navigation experiment: During the test, the platform was placed in the first quadrant. Mice were placed into the pool sequentially from each of the four quadrants, facing the pool wall. Each test lasted 60 seconds, and trajectory tracking and analysis software automatically recorded the mice's latency and average speed. The order of entry into the water changed each day, and the experiment lasted for 5 days.

[0040] Space exploration experiment: On day 6 of the above experiment, the underwater platform was removed, and the time spent in the original target quadrant within 60 seconds and the number of times it entered the quadrant where the original target platform was located were recorded.

[0041] Y-maze test: Place mice in a Y-maze and allow them to explore freely for 8 minutes. Record the number of times the mouse enters each open arm and the order in which it enters the arms. One correct exploration should be three consecutive attempts by the mouse to enter different open arms. Record the total number of times the mouse enters each open arm and the spontaneous alternation rate (%) = (Number of correct entries / (Number of arm entries - 2)) × 100%.

[0042] New Object Recognition Test: Three days before training and testing, mice were allowed to acclimatize to the enclosure. At the start of training, two identical objects, A and B, were placed at opposite ends of one side wall, with the mice placed in the enclosure facing away from the objects. Immediately after placement, a recording device was activated to record the mice's interactions with the objects, including the time spent exploring within 2 cm of the objects. After 5 minutes, the mice were immediately returned to their original cages and allowed to rest for 24 hours before the next test. The following day, object B was replaced with object C (different from B), and the mice were again placed facing away from the objects, as shown in the right image below. The ANY software was used to record the time the mice spent approaching objects B and C within 5 minutes. A recognition index was used to reflect the mice's memory. Recognition Index = Time spent contacting object C / Time spent contacting A + C.

[0043] During the positioning and navigation experiment, as the number of training days increased, the time it took for all four groups of mice to find the platform decreased (e.g., Figure 2 (As shown in AC). During the space exploration experiment, compared with the control group, the time spent in the target quadrant was decreased in both the model group and the hippocampal peptide group, and the number of platform crossings was significantly reduced in the model group (P < 0.05). However, compared with the model group, the time spent in the target quadrant was significantly prolonged in the high-dose hippocampal peptide group (P < 0.05), and the number of platform crossings was significantly increased (see AC). Figure 2 The results showed that, compared with the control group, the memory ability of mice in the model group was reduced, while high doses of hippocampal peptides significantly improved the memory ability of mice.

[0044] like Figure 3 As shown in Figure AB, the results of the Y-maze experiment showed that, compared with the control group, the number of arm entries and the spontaneous alternation rate of mice in the model group were significantly reduced (P < 0.05); compared with the model group, the number of arm entries and the spontaneous alternation rate in the low-dose hippocampal peptide group showed an increasing trend, but there was no statistical significance (P > 0.05), while the spontaneous alternation rate in the high-dose hippocampal peptide group was significantly increased (P < 0.05).

[0045] The novel object recognition test is a learning and memory testing method based on the innate tendency of animals to explore new objects. Compared to the water maze test, it is less stressful and can more closely simulate human learning and memory behavior. Furthermore, by changing the shape and size of the object, this method can be applied to detect the formation of long-term or short-term memory in animals. Results are as follows... Figure 4As shown in Figure AB, compared with the control group, the exploration time and recognition index of objects in the model group mice were significantly reduced (P < 0.05). Compared with the model group, the exploration time and recognition index of objects in the high-dose hippocampal peptide group mice were both increased (P < 0.05). Although low-dose hippocampal peptide intervention improved the exploration time of new objects, the effect on the recognition index was not significant, and there was no significant difference compared with the model group (P > 0.05). These results indicate that high-dose hippocampal peptide effectively improves cognitive memory impairment in aged mice.

[0046] 2. Evaluation of the effects on memory and cognitive impairment in the APP / PS1 transgenic mouse model:

[0047] The amyloid precursor APP gene, presenilin-1 (PS-1) gene, presenilin-2 (PS-2) gene, and apolipoprotein E (ApoE) gene have all been confirmed to be closely related to the pathogenesis of Alzheimer's disease (AD). APP / PS1 double transgenic mice produce more Aβ compared to normal mice. 1-40 and Aβ 1-42 It is an ideal transgenic animal model for AD.

[0048] Animal grouping and intervention: Thirty 6-month-old male APP / PS1 double transgenic mice and ten syngeneic non-transgenic C57BL / 6 mice (used as controls) were purchased from Beijing Huafukang Biotechnology Co., Ltd., and were housed under the same conditions as in Example 1. After one week of acclimatization, the mice were grouped. The 30 APP / PS1 double transgenic mice were randomly divided into a model group, a low-dose hippocampal peptide group (SHP-L 200 mg / kg BW), and a high-dose hippocampal peptide group (SHP-H 400 mg / kg BW) according to their body weight. The C57BL / 6 mice served as the control group and were supplemented with hippocampal peptide by gavage once a day for 60 consecutive days before behavioral testing began.

[0049] Morris water maze test is the same as 1.

[0050] The new object recognition test is the same as 1.

[0051] The results are shown in Table 1. There was no statistically significant difference in swimming speed among the groups (P > 0.05). Compared with the control group, the escape latency and average total swimming distance of the model group mice were significantly increased (P < 0.01), while the number of platform crossings was significantly decreased (P < 0.01). Compared with the model group, the escape latency of mice in the low-dose and high-dose hippocampal peptide groups was significantly decreased (P < 0.01), while the number of platform crossings in the high-dose hippocampal peptide group was significantly increased (P < 0.05).

[0052] Table 1. Effects of hippocampal peptides on behavioral indicators in mice of each group:

[0053] ;

[0054] Note: ## p < 0.01 compared with the control group; * p < 0.05 compared with the model group; ** p < 0.01 compared with the model group.

[0055] like Figure 5 As shown in Figures AB, there was no statistically significant difference in the total exploration time of mice in each group for objects A and B (P > 0.05). After excluding the influence of the mice's motor ability on the experimental results, compared with the control group, the exploration time and recognition index of mice in the model group were significantly reduced (P < 0.01); compared with the model group, the exploration time and recognition index of mice in the high-dose hippocampal peptide group were significantly increased (P < 0.05).

[0056] 3. Evaluation of the effects of intraventricular injection of Aβ on memory and cognitive impairment in an AD rat model:

[0057] Intraventricular injection of Aβ can induce oxidative stress in the brain, activate glial cells, increase the toxicity of Tau protein, and induce neuronal apoptosis. An AD model rat was established by intraventricular injection of Aβ1-42 to verify the ameliorative effect of hippocampal active peptides on learning, memory, and cognitive impairment in AD rats after Aβ production. Preparation of aggregated Aβ1-42: Aβ1-42 was dissolved in sterile physiological saline to prepare a 1 mg / mL stock solution, which was incubated at 37°C for 5 days to obtain soluble oligomeric Aβ1-42.

[0058] Establishment of the AD animal model: Forty male SD rats (weighing 280-300 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. They were acclimatized for one week with free access to food and water. Surgery was performed under sterile conditions. The rats were anesthetized before placing their heads in a stereotaxic brain localization device. Referring to Paxinos's "Stereotaxic Map of the Rat Brain," the left lateral ventricle was selected as the injection target area. Using the anterior fontanelle as the origin, 1.0 mm posteriorly and 1.4 mm lateral to the midline, the skull was opened with a cranial drill. Aβ1-42 was injected 4 mm below the dura mater using a micro-infusion pump (5 μL / 5 min). The dosage of Aβ1-42 was selected based on previous laboratory studies. Rats in the sham-operated group received an equal volume of sterile saline, and all other procedures were the same. The needle was left in place for 5 min, then slowly withdrawn. The wound was disinfected with penicillin and sutured.

[0059] Seven days after modeling, rats injected with Aβ in the ventricle were randomly divided into three groups according to their body weight: the model group, the low-dose hippocampal peptide group (200 mg / kg BW), and the high-dose hippocampal peptide group (400 mg / kg BW).

[0060] Morris water maze test is the same as 1.

[0061] The results are as follows Figure 6 As shown in the AC diagram, on day 1, there was no significant difference in the latency to find the platform among the experimental groups compared to the model group. However, on day 3 of the learning period, hippocampal peptides significantly reduced the learning latency of rats (P < 0.05), with the time to find the platform reduced by 27% compared to the model group, to 40.6 s and 37.3 s, respectively.

[0062] In the spatial exploration experiment, the platform was removed, and the number of times rats crossed the original platform and the exploration time in the target quadrant were recorded within 60 seconds to assess memory impairment. Compared with the sham-operated group, the model group showed a significant decrease in the number of platform crossings (1.5 times and 4.2 times, respectively) and the time spent in the target quadrant (25.31 s and 36.62 s, respectively) (P < 0.05). After supplementation with hippocampal peptides, the number of platform crossings and the exploration time in the platform quadrant were significantly increased, indicating that hippocampal peptides improve Aβ-induced cognitive deficits in rats.

[0063] Example 2:

[0064] Based on the effect evaluation results of hippocampal peptides on three models in Example 1, potential active peptides were enriched from the hippocampal peptide mixture in Example 1 using affinity chromatography and immobilized APP, and then identified by peptide fingerprinting.

[0065] Preparation of APP protein affinity chromatography column: Wash the CNBr activation medium with 10 column volumes of 1 mM HCl to remove the protective agent; immediately equilibrate the column with coupling buffer (pH=8.3). Dissolve the purified APP protein in coupling buffer (final concentration 2-5 mg / mL), mix the protein solution with the activation medium at a 1:1 volume ratio, and incubate at room temperature for 2 h (or overnight at 4°C). Remove the unbound protein solution and wash 3 times with coupling buffer. Add blocking buffer (containing 1 M ethanolamine, pH=8.0) and incubate at room temperature for 1 h. Wash sequentially with 3 column volumes of coupling buffer and binding buffer, and store at 4°C (containing 0.02% NaN3). Determine the protein concentration in the solution before and after coupling using the BCA method to calculate the coupling efficiency.

[0066] Hippocampal active peptide enrichment: Hippocampal peptide hydrolysate samples were desalted using a PD-10 column, and the hydrolysate was replaced with binding buffer (pH 7.4). The samples were centrifuged at 12,000 rpm for 10 min to remove insoluble matter. 1 mM PMSF (final concentration) was added, and the process was performed on ice to prevent peptide degradation. The pretreated hippocampal peptide hydrolysate was loaded onto an APP protein immobilization column at a flow rate of 0.5–1 mL / min, and this loading was repeated 2–3 times to improve binding efficiency. The column was washed with 10 column volumes of binding buffer (pH 7.4) to remove unbound impurities. Subsequently, elution was performed with 5 column volumes of 0.1 M glycine-HCl (pH=2.5), and the collected solution was immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 8.5) (to avoid peptide denaturation). The column was regenerated with 5 column volumes of binding buffer and stored at 4°C in a buffer containing 0.02% NaN3. Concentrate the elution buffer using a 3 kDa ultrafiltration centrifuge tube and replace it with PBS or pure water.

[0067] The peptide sequences obtained from de novo sequencing were searched for hippocampal protein peptide sequences in the NCBI database. The results showed that the peptide sequences were not publicly available. The search parameters were set as follows: potential dynamic modification: oxidation (M, P, K); enzyme specificity: non-specific; fragment mass tolerance: 0.02 Da; precursor mass tolerance: 10 ppm; false discovery rate (FDR) of peptide identification results ≤ 1%. Peptide Ranker was used to predict the bioactivity of the identified peptide sequences.

[0068] By performing activity prediction on Peptide Ranker (Table 2), this example discloses a bioactive peptide sequence from the bloated hippocampus that enhances memory.

[0069] Molecular docking screening of active peptides: Using BACE1 as the key target protein, peptides were homology-modeled and molecularly docked with the major binding active sites of the BACE1 protein. Peptide sequence homology modeling: The peptide sequence was input into Chemdraw3D 18.1 software for 3D modeling to generate the peptide structure. Energy minimization of the peptide structure was performed using the CHARMm program in Discover Studio 2019 molecular simulation software.

[0070] Receptor pretreatment: The X-ray crystal structure of human BACE1 protein (PDBID: 4D89) was downloaded from the PDB database. Existing ligands were removed using Discover Studio 2019 software, and chemical bonds were corrected and charges were added. Active pockets were further identified and covered. Molecular docking of the receptor and peptide was performed using the CDOCKER program. The docking score was calculated based on the highest CDOCKER Energy score, combined with indicators such as free energy, hydrogen bonds, hydrophobic groups, and charged groups.

[0071] Table 2. Sequence identification and activity prediction of active peptides from distended hippocampus:

[0072] ;

[0073] a Source: PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0074] The predicted bioactive peptides from the bloated hippocampus were biosynthesized by Shanghai Sangon Biotech Co., Ltd. using the Fmoc-peptide solid-phase synthesis method. HPLC and MS sequence analysis showed that the purity of the DFPLPFS peptide was greater than 98.89%. To further verify the effect of the screened peptide activity on Aβ production, this example used CHO-APP / PS1 cells. CHO-APP / PS1 double-transgenic cells, carrying the APP / PS1 plasmid, secrete more Aβ1-40 and Aβ1-42 compared to normal nerve cells and can be considered a transgenic cell model for AD. The cells were preserved in the Human Health Laboratory of the College of Food Science and Engineering, Ocean University of China.

[0075] Cell culture: Logarithmic growth phase CHO-APP / PS1 cells were digested with trypsin to prepare a concentration of 5×10⁻⁶ cells / mL. 4 Cell suspension at 100 μL / well was seeded into 96-well plates and cultured at 37°C and 5% CO2. After 24 h, different concentrations of peptide samples from the examples were incubated, with DFPLPFS concentrations of 0, 25, 50, and 100 μg / mL, 200 μL per well, and 4 replicates per group. Cell viability was assessed by MTT assay after 48 h.

[0076] Effects of hippocampal active peptides on Aβ40 and Aβ42 in supernatant and cells: Log-phase CHO-APP / PS1 double-transformed cells were digested with trypsin to prepare a concentration of 2.5 × 10⁻⁶. 5Cells were seeded at 1 mL / well in a 12-well culture plate after being suspended in a 1 / mL cell suspension and cultured at 37°C and 5% CO2. After cell adhesion, the cells were incubated with 100 μg / mL DFPLPFS peptide solution (n=3). After 48 h, the culture medium was recovered, and the cells were recovered with pre-cooled PBS. After lysing with sonicated cell disruptors, the supernatant was collected by centrifugation. The levels of Aβ40 and Aβ42 were measured using an ELISA kit, and the protein content of each group of cells was also measured. The results were expressed as pg / mg prot.

[0077] The effects of different concentrations of peptides (0, 25, 50, 75, 100 μg / mL) on the survival rate of CHO-APP / PS1 cells are as follows: Figure 7 As shown, within the range of 0-75 μg / mL, each peptide had no significant effect on cell viability and did not show any damaging effect on CHO-APP / PS1 cells. A dose of 50 μg / mL was selected for subsequent experimental studies.

[0078] Effects on intracellular Aβ40 and Aβ42 levels, such as Figure 8 As shown in Figure AB, the intracellular Aβ40 content in the model group was 538 pg / mg prot, and the Aβ42 content was 372.7 pg / mg prot. Incubation with DFPLPFS peptide for 48 h significantly reduced the intracellular Aβ40 and Aβ42 levels.

[0079] Effects of peptides on extracellular Aβ40 and Aβ42 concentrations, such as Figure 9 As shown in Figure AB, compared with the model group, the DFPLPFS peptide significantly reduced the concentrations of Aβ40 and Aβ42 secreted into the extracellular space. After the action of the DFPLPFS peptide, the concentrations of Aβ40 and Aβ42 in the extracellular space decreased by 49% and 31%, respectively, and the results were statistically significant.

[0080] To further investigate whether the inhibition of Aβ production by the aforementioned peptides is related to their key enzyme BACE1, molecular docking experiments were conducted to achieve the interaction between the novel peptides and the receptor protein. Homology modeling was performed on the aforementioned peptides, and molecular docking was performed with the BACE1 protein. The docking results are shown below. Figure 10 As shown in Figure AB, the docking mode between the peptide and β-secretase 1 represents the optimal posture after simulation. The binding energy of the DFPLPFS peptide to BACE1 protein is -8.0 kcal / mol. The peptide structure can insert into the active pocket of BACE1, inhibiting BACE1 enzyme activity through interactions with key amino acid residues, forming conventional hydrogen bonds and hydrophobic interactions, thereby reducing Aβ production. These results indicate that the DFPLPFS peptide is a good BACE1 inhibitor and has the potential to be developed into an active ingredient for improving memory and alleviating cognitive impairment.

[0081] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the invention. Those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. All modifications or equivalent substitutions should be included within the scope of protection of the present invention.

Claims

1. A type of bloating hippocampal peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the expanded hippocampal peptide according to claim 1 in the preparation of products that enhance memory and improve cognitive impairment.

3. The bloating hippocampal peptide as described in claim 1, characterized in that: The bloated hippocampal peptide is artificially synthesized.

Images