Abdominal distention hippocampal peptide and application thereof in preparation of product for improving memory ability and cognitive impairment

Abstract

The invention relates to the field of preparation and application of biological peptides, in particular to a swelling hippocampal peptide and application of the swelling hippocampal peptide in preparation of a product for improving memory ability and cognitive impairment. According to the invention, a green controllable enzymolysis technology is utilized to obtain the protein polypeptide with the main molecular weight of less than 1000 Da and nutritional, health-care and medicinal values, further research is carried out on the basis to obtain an active peptide sequence with high biological activity, and the medicinal value of the active peptide sequence is verified.

Description

Technical Field

[0001] The present invention relates to the preparation and application fields of biological peptides, and in particular to a hippocampal distending peptide and its application in the preparation of products for improving memory ability and improving cognitive impairment. Background Art

[0002] Brain aging is a gradual, long-term process, accompanied by a decline in learning and memory abilities, which may eventually progress to neurodegenerative diseases such as Alzheimer's disease (AD). However, limitations in current diagnostic methods pose significant challenges to Alzheimer's disease intervention. It often takes years, or even decades, from the initial onset of the disease to the onset of overt pathological symptoms such as cognitive impairment. During this time, the pathological progression of AD may quietly accelerate, causing irreversible damage that not only devastatingly impacts patients' quality of life but also places a heavy burden on their families and society. Therefore, improving the aging process, delaying memory loss, and curbing the progression of cognitive impairment have become key concerns for the scientific and medical communities, as well as society at large.

[0003] The bloated seahorse (Hippocampus abdominalis) is a new species introduced to my country in recent years. With the continuous development of the marine aquaculture industry, improved living standards, and the popularization and promotion of the concept of comprehensive health, people's demand for health and wellness is increasing. Exploiting the potential nutritional content of seahorses is conducive to the high-value utilization of seahorses. Currently, the only hippocampal peptides studied for cognitive impairment and Alzheimer's disease are a composite product and a few peptide fragments obtained in the inventor's previous research. This is the case with Chinese patent CN120392956A, which discloses a bloated hippocampal peptide and its use in the preparation of products that enhance memory and improve cognitive impairment. However, this is insufficient research on bloated hippocampal peptides, and further discovery 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 the present invention is that the existing technology has insufficient research on the effect of distended hippocampal active peptide in improving learning and memory ability and improving cognitive impairment, which needs further exploration.

[0005] To solve the above problems, the present invention provides a hippocampal distending peptide and its use in the preparation of products for enhancing memory ability and improving cognitive impairment. By using green controllable enzymatic hydrolysis technology, protein polypeptides with a molecular weight mainly less than 1000 Da and nutritional, health and medicinal value are obtained. On this basis, further research has obtained an active peptide sequence with high biological activity.

[0006] To achieve the above object, the present invention is achieved by the following technical means: a hippocampal distending peptide, the amino acid sequence of which is shown in SEQ ID NO.1: SEQ ID NO.1: DFPLPFS.

[0007] The application of the above-mentioned hippocampal peptide in the preparation of products for improving memory and cognitive impairment. In the early stages of Alzheimer's disease, synaptic damage occurs, leading to a decrease in acetylcholinesterase activity, which precedes neuronal loss and the formation of amyloid protein (Aβ) deposition and is the structural basis for the onset of AD. Excessive accumulation of Aβ in the brain will lead to the massive generation of reactive oxygen species in cells, destroying Ca2+ and Ca2+ in cells. 2+ Abnormal modification of Tau protein triggers a cascade of reactions, leading to neuronal dysfunction and ultimately cognitive impairment. Animal and cell-based 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 that catalyzes the hydrolysis of amyloid precursor protein (APP) to produce β-amyloid (Aβ). Aβ deposition is directly linked to the development of Alzheimer's disease. Further molecular docking revealed that the peptide has a binding energy of -8.0 kcal / mol with BACE1. The peptide can integrate into the active pocket of BACE1 and inhibit its activity by interacting with key amino acid residues through van der Waals forces, carbon-hydrogen bonds, alkyl bonds, and π-alkyl bonds, ultimately improving memory and alleviating cognitive impairment.

[0008] Furthermore, the hippocampal distending peptide with the amino acid sequence shown in SEQ ID NO.1 is artificially synthesized.

[0009] A method for preparing a powder containing the above-mentioned belly-expanding hippocampal peptide comprises the following steps: Fresh hippocampus distended with diaphragm was washed, freeze-dried, and crushed into powder. Distilled water and 3% papain were 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. After the enzymatic hydrolysate was centrifuged, the supernatant was filtered through a 0.45 μm microporous filter membrane and 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 of less than 3 kDa were separated by ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The peptide solution was freeze-dried to obtain the expanded hippocampal peptide powder.

[0010] Furthermore, distilled water was added at a material-liquid ratio of 1:9.

[0011] Furthermore, the concentrations of the HCL solution and the NaOH solution are both 1 mol / L.

[0012] Furthermore, the enzymatic activity of the papain is 800,000 U / g.

[0013] The beneficial effects of the present invention are: (1) APP protein was used as the target for targeted screening of active peptides from hippocampal peptide hydrolysates. Using mass spectrometry technology and activity prediction methods, a potential hippocampal active peptide sequence DFPLPFS with the ability to enhance memory was disclosed. APP / PS1 (M146L) double-transformed CHO (CHO-APP / PS1) cells were used to verify the activity of the peptide, providing a theoretical basis for the development of products using expanded hippocampal active peptides to enhance memory and improve cognitive impairment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the molecular weight distribution of hippocampal peptide.

[0015] Figure 2 Results of the Morris water maze test in SAMP8 mice: A represents the latency of the mouse in the orientation and heading test, B represents the number of platform crossings in the spatial exploration test, and C represents the time the mouse 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.

[0016] Figure 3 Figure 1 shows the Y-maze test results for SAMP8 mice. A represents the total number of arm entries, and B represents the spontaneous alternation rate. Note: #p < 0.05 compared with the control group, ##p < 0.01 compared with the control group; *p < 0.05 compared with the model group.

[0017] Figure 4 Results of a novel object recognition experiment in SAMP8 mice: A represents the time spent exploring the object, and B represents the index of object recognition. Note: ## p < 0.01 compared with the control group; **p < 0.01 compared with the model group.

[0018] Figure 5 Results of a novel object recognition experiment in APP / PS1 double transgenic mice: A represents the time spent exploring an object, and B represents the index of object recognition. Note: ## p < 0.01 compared with the control group; *p < 0.05 compared with the model group.

[0019] Figure 6 Results of the Morris water maze test in AD model rats: A represents the latency of the rats in the orientation and heading test, B represents the number of platform crossings 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 group; * p < 0.05 compared with the model group; ** p < 0.01 compared with the model group.

[0020] Figure 7The effect of different concentrations of peptides on the survival rate of CHO-APP / PS1 cells.

[0021] Figure 8 The effect of the peptide on the levels of Aβ40 and Aβ42 in CHO-APP / PS1 cells. A represents the level of Aβ40 in the cells, and B represents the level of Aβ42. Note: Different letters indicate significant differences between the groups.

[0022] Figure 9 The effect of the peptide 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 the groups.

[0023] Figure 10 This is the result diagram of the docking of the peptide segment of SEQ ID NO.1 with the BACE1 receptor protein molecule; wherein A is the 3D structure of the docking of the peptide segment and the receptor protein; B is the 2D structure of the docking of the peptide segment and the receptor protein. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] In addition, all materials used in the examples of the present invention, unless otherwise specified, were purchased from the market.

[0026] Example 1: A belly-expanding hippocampal peptide powder is prepared by the following method: Preparation of Hippocampus distended peptide enzymatic hydrolysate: 100 g of fresh Hippocampus distended was washed, freeze-dried, and ground into powder. Distilled water was added to the mixture at a material-liquid ratio of 1:9 (m / v). 3% papain was added and stirred thoroughly. The pH was adjusted to 6.5 with 1 mol / L HCl and 1 mol / L NaOH, respectively. Enzymatic hydrolysis was performed at 50°C for 3 h. The hydrolysate was centrifuged (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 molecular weights less than 3 kDa were separated by ultrafiltration (3 kDa molecular weight cutoff). The peptide solution was freeze-dried to obtain Hippocampus distended peptide powder. Papain (800,000 U / g, G8430-25) was purchased from Solebao Technology Co., Ltd.

[0027] 1. Evaluation of the effects on memory and cognitive impairment in the SAMP8 aging mouse model: Molecular weight determination of distended hippocampal peptide: The molecular weight distribution of distended hippocampal peptide was determined using gel exclusion chromatography. Specific conditions were: 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.

[0028] Gel chromatography was used to detect the molecular weight distribution of papain hydrolyzate of hippocampal peptide. Figure 1 As shown, by comparing with the standard molecular weight curve, it was calculated that the molecular weight of the papain hydrolyzed peptides of the expanded hippocampus peptide was less than 500 Da, accounting for 23.96%, and the short peptides with a molecular weight of 500-1 kDa accounted for the largest proportion of 48.21%, of which 1 kDa-3 kDa short peptides accounted for 27.83% ( Figure 1 ).

[0029] Animal experimentation: 30 SAMP8 mice and 10 SAMR1 mice were purchased from Nanjing Qingzilan Technology Co., Ltd. The animals were housed in a controlled environment with a temperature of 21°C–23°C, a humidity of 45%–55%, and a 12-h light / 12-h dark cycle. Mice had free access to food and water during the experiment. All mice were fed a bar diet (AIN-93G) until four months of age and then a high-fat diet until six months of age. SAMP8 mice were randomly divided into three groups based on body weight: a model group, a low-dose hippocampal peptide group (200 mg / kg body weight), and a high-dose hippocampal peptide group (400 mg / kg body weight). SAMR1 mice served as a control group and received hippocampal peptide supplementation once daily by gavage until nine months of age, at which time behavioral testing began.

[0030] Morris water maze: The pool was 130 cm in diameter, 50 cm high, 30 cm deep, and the water temperature was controlled at 22 ± 1°C. The circular pool was divided into four quadrants using ANY software. The first quadrant was selected, and a circular platform with a diameter of 9 cm was placed 1 cm below the water surface in the center of the quadrant. The camera was placed directly above the circular pool and connected to a computer. During the test, ink was added to the pool to eliminate interference from factors other than landmarks on the mouse's spatial positioning. The entire behavioral testing process included a navigation experiment and a spatial exploration experiment.

[0031] Positioning navigation experiment: During the test, the platform was placed in the first quadrant. Mice were sequentially placed into the pool from each of the four quadrants, facing the pool wall. Each test lasted 60 seconds, and trajectory tracking analysis software automatically recorded the mice's latency and average speed. The order of quadrant entry varied daily, and the experiment lasted for 5 days.

[0032] Space exploration experiment: On the sixth day of the above experiment, the underwater platform was removed, and the time it spent in the original target quadrant and the number of times it entered the quadrant where the original target platform was located within 60 seconds were recorded.

[0033] Y-maze test: Mice were placed in a Y-maze and allowed to explore freely for 8 minutes. The number of times the mouse entered each open arm and the order in which it entered each arm were recorded. A correct exploration was considered to be three consecutive entries into different open arms. The total number of entries into each open arm and the spontaneous alternation rate (%) were recorded (number of correct entries / (number of arm entries - 2) × 100%).

[0034] Novel Object Recognition Test: Three days before training and testing, mice were acclimated to the cage. At the beginning of training, two identical objects, A and B, were placed at the left and right ends of one wall, with the mice placed inside facing the objects. Immediately after the mice were placed inside the cage, a video camera was activated to record their interactions with the two objects, including the time spent exploring within a 2 cm radius. After 5 minutes, the mice were immediately returned to their original cages and allowed to rest for 24 hours before testing. The following day, object B in the cage was replaced with object C (different from object B), with the mice still facing away from the two objects, as shown in the figure below (right). ANY software was configured to record the time the mice spent approaching objects B and C over a 5-minute period. The recognition index was used to assess the mice's memory performance. The recognition index was calculated as the time spent approaching object C divided by the time spent approaching object A + C.

[0035] In the navigation experiment, as the number of training days increased, the time it took for the four groups of mice to find the platform became shorter (e.g. Figure 2 In the spatial exploration experiment, compared with the control group, the time that the model group and the hippocampal peptide group mice stayed in the target quadrant decreased, and the number of times the model group mice crossed the platform was significantly reduced (P < 0.05). Compared with the model group mice, the time that the high-dose hippocampal peptide group mice stayed in the target quadrant was significantly prolonged (P < 0.05), and the number of times they crossed the platform was significantly increased, as shown in Figure 5. Figure 2 Middle C. The results showed that compared with the control group, the memory ability of the mice in the model group was reduced, while high-dose hippocampal peptide significantly improved the memory ability of mice.

[0036] like Figure 3 As shown in Figures AB, the results of the Y-maze test showed that compared with the control group, the number of arm entries and 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 spontaneous alternation rate in the low-dose hippocampal peptide group showed an increasing trend, but without statistical significance (P > 0.05), while the spontaneous alternation rate in the high-dose hippocampal peptide group was significantly increased (P < 0.05).

[0037] The novel object recognition test is a learning and memory test method established by using the principle that animals have an innate tendency to explore new objects. Compared with the water maze test, it is less stressful and can more closely simulate human learning and memory behavior. In addition, by changing the shape and size of the object, this method can be applied to the detection of long-term or short-term memory formation in animals. Figure 4As shown in Figures AB, compared with the control group, the model group mice had significantly reduced object exploration time and recognition index (P < 0.05). Compared with the model group, the high-dose hippocampal peptide group had increased object exploration time and recognition index (P < 0.05). Although low-dose hippocampal peptide intervention increased exploration time for novel objects, the recognition index did not show a significant effect, with 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 aging mice.

[0038] 2. Evaluation of the effects on memory and cognitive impairment in the APP / PS1 transgenic mouse model: Amyloid precursor protein APP gene, presenilin-1 (PS-1) gene, presenilin-2 (PS-2) gene and apolipoprotein E (ApoE) gene have been confirmed to be closely related to the onset of AD. APP / PS1 double transgenic mice produce more Aβ than normal mice. 1-40 and Aβ 1-42 , is an ideal transgenic animal model of AD.

[0039] Animal Grouping and Intervention: Thirty six-month-old APP / PS1 double-transgenic male mice and ten syngeneic non-transgenic C57BL / 6 mice (used as controls) were purchased from Beijing Huafukang Biotechnology Co., Ltd. and housed under the same conditions as in Example 1. After one week of adaptive feeding, the mice were grouped. Thirty APP / PS1 double-transgenic mice were randomly divided according to body weight into a model group (Model), a low-dose hippocampal peptide group (SHP-L 200 mg / kg body weight), and a high-dose hippocampal peptide group (SHP-H 400 mg / kg body weight). C57BL / 6 mice served as the control group (Normal) and were supplemented with hippocampal peptide by gavage once daily for 60 days before behavioral testing began.

[0040] Morris water maze test was the same as 1.

[0041] The new object recognition test is the same as 1.

[0042] The results are shown in Table 1. There was no statistically significant difference in swimming speed between the groups (P > 0.05). Compared with the control group, the escape latency and average total swimming distance of the mice in the model group were significantly increased (P < 0.01), and the number of platform crossings was significantly reduced (P < 0.01). Compared with the model group, the escape latency of the mice in the low-dose and high-dose hippocampal peptide groups was significantly reduced (P < 0.01), and the number of platform crossings in the high-dose hippocampal peptide group was significantly increased (P < 0.05).

[0043] Table 1 Effects of hippocampal peptide on behavioral indicators of mice in each group: ; Note: ## compared with the control group, p < 0.01; * compared with the model group, p < 0.05; ** compared with the model group, p < 0.01.

[0044] like Figure 5 As shown in Figures AB, there was no statistically significant difference in the total exploration time of objects A and B among the mice in each group (P > 0.05). Excluding the influence of the motor ability of the mice in each group on the experimental results, compared with the control group, the exploration time and recognition index of the mice in the model group were significantly reduced (P < 0.01); compared with the model group, the exploration time and recognition index of the mice in the high-dose hippocampal peptide group were significantly increased (P < 0.05).

[0045] 3. Evaluation of the effects on memory and cognitive impairment in an AD rat model induced by intraventricular injection of Aβ: Intraventricular injection of Aβ can induce oxidative stress, glial cell activation, increased tau protein toxicity, and neuronal apoptosis in the brain. A rat model of AD was established by intracerebroventricular injection of Aβ1-42 to validate the efficacy of hippocampal bioactive peptides in improving learning, memory, and cognitive impairment in Aβ-induced AD rats. Preparation of aggregated Aβ1-42: Aβ1-42 was dissolved in sterile saline to a 1 mg / mL stock solution. This stock solution was incubated at 37°C for 5 days to obtain soluble oligomeric Aβ1-42.

[0046] Establishment of an AD animal model: Forty male Sprague-Dawley rats (weighing 280-300 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and acclimated for one week with free access to food and water. Surgery was performed under sterile conditions, and the rats were anesthetized before their heads were placed in a stereotaxic apparatus. The left lateral ventricle was selected as the injection target according to Paxinos's "Rat Stereotaxic Atlas." The skull was drilled 1.0 mm posterior to the bregma and 1.4 mm lateral to the midline. Aβ1-42 was injected 4 mm below the dura mater using a microinfusion pump (5 μL / 5 min). The Aβ1-42 dose 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 minutes, then slowly removed. The wound was disinfected with penicillin and sutured.

[0047] Seven days after modeling, rats injected with Aβ intraventricularly were randomly divided into three groups according to body weight, namely model group, low-dose hippocampal peptide group (200 mg / kg BW) and high-dose hippocampal peptide group (400 mg / kg BW).

[0048] Morris water maze test was the same as 1.

[0049] The results are as follows Figure 6As shown in Figures AC, on day 1, there was no significant difference in the latency to find the platform between the experimental groups compared with the model group. However, on day 3 of the learning period, hippocampal peptide significantly reduced the learning latency of rats (P < 0.05), with the time to find the platform reduced by 27% compared with the model group to 40.6 s and 37.3 s, respectively.

[0050] In the spatial exploration experiment, the platform was removed, and the number of times the rats crossed the original platform and the time they spent exploring the target quadrant were recorded over a 60-second period 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 and 4.2 times, respectively) and the time spent in the target quadrant (25.31 and 36.62 seconds, respectively) (P < 0.05). After supplementation with hippocampal peptide, the number of platform crossings and the time spent exploring the platform quadrant were significantly increased, indicating that hippocampal peptide ameliorates Aβ-induced cognitive deficits in rats.

[0051] Example 2: Based on the evaluation results of the effect of hippocampal peptides on the three models in Example 1, affinity chromatography was used to enrich potential active peptides from the hippocampal peptide mixture in Example 1 using immobilized APP and peptide fingerprint identification was performed.

[0052] Prepare an APP protein affinity chromatography column: Wash the CNBr-activated 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 in a 1:1 volume ratio and incubate with rotation at room temperature for 2 hours (or overnight at 4°C). Remove unbound protein solution and wash three times with coupling buffer. Add blocking buffer (containing 1 M ethanolamine, pH 8.0) and incubate at room temperature for 1 hour. Wash sequentially with 3 column volumes of coupling buffer and binding buffer. Store at 4°C (containing 0.02% NaN3). Determine the protein concentration before and after coupling using the BCA assay to calculate the coupling efficiency.

[0053] Enrichment of hippocampal active peptides: The hippocampal peptide hydrolysate was exchanged into binding buffer (pH 7.4) using a PD-10 desalting column. Insoluble matter was removed by centrifugation at 12,000 rpm for 10 minutes, and 1 mM PMSF (final concentration) was added. The column was kept on ice to prevent peptide degradation. The pretreated hippocampal peptide hydrolysate was loaded onto an APP protein-immobilized chromatography column at a flow rate of 0.5-1 mL / min. The loading was repeated 2-3 times to improve binding efficiency. Unbound impurities were removed by washing with 10 column volumes of binding buffer (pH 7.4). Subsequently, the column was eluted with 5 column volumes of 0.1 M glycine-HCl (pH 2.5). The collected solution was immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 8.5) to prevent peptide denaturation. The column was then regenerated with 5 column volumes of binding buffer and stored at 4°C in a buffer containing 0.02% NaN₃. The eluate was concentrated using a 3 kDa ultrafiltration centrifuge tube and replaced with PBS or pure water.

[0054] The peptide sequences obtained from de novo sequencing were searched against the NCBI database for hippocampal protein peptide sequences. The results indicated that the peptide sequences were not publicly available. The search parameters were as follows: potential dynamic modification: oxidation (M, P, K); enzyme specificity: nonspecific; fragment mass tolerance: 0.02 Da; precursor mass tolerance: 10 ppm; and a false discovery rate (FDR) of ≤ 1% for peptide identification. Peptide Ranker was used to predict the likelihood of biological activity for the identified peptides.

[0055] By predicting the activity of Peptide Ranker (Table 2), this example discloses a hippocampal active peptide sequence with memory-enhancing ability.

[0056] Molecular docking screening for active peptides: Using BACE1 as the key target protein, peptides were subjected to homology modeling and molecular docking with the primary binding active site of the BACE1 protein. Peptide sequence homology modeling: 3D modeling was performed using Chemdraw 18.1 software to input the peptide sequence and generate the peptide structure. Energy minimization of the peptide structure was performed using the CHARMm program in Discover Studio 2019.

[0057] Receptor preprocessing: The X-ray crystal structure of human BACE1 protein (PDB ID: 4D89) was downloaded from the PDB database. Using Discover Studio 2019 software, we removed the existing ligand, performed chemical bond correction, and added charges. We then identified the active pocket and covered the pocket site. We then performed molecular docking of the receptor and peptide using the CDOCKER program. Based on the highest CDOCKER Energy score, we analyzed binding free energy, hydrogen bonds, hydrophobic groups, and charged groups to calculate the docking score.

[0058] Table 2 Sequence identification and activity prediction of active peptides from Hippocampus distended ; a From PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0059] The predicted bioactive peptide from Hippocampus distendedus was synthesized by Shanghai Bioengineering Co., Ltd. using the Fmoc-peptide solid-phase synthesis method. HPLC and MS sequence analysis revealed a purity of greater than 98.89% for the DFPLPFS peptide. To further validate the effect of the activity of the selected peptides on Aβ production, this example utilized CHO-APP / PS1 cells. CHO-APP / PS1 double-transfected cells, carrying the APP / PS1 plasmid, secrete more Aβ1-40 and Aβ1-42 than normal neurons and can be considered a transgenic cell model for AD. The cells are stored in the Human Health Laboratory, College of Food Science and Engineering, Ocean University of China.

[0060] Cell culture: CHO-APP / PS1 cells in the logarithmic growth phase were obtained and digested with trypsin to prepare a 5×10 4 100 μL / well of a 96-well culture plate containing 100 μg / mL cell suspension was inoculated and cultured at 37°C in 5% CO2. After 24 hours, the cells were incubated with different concentrations of peptide samples from the examples (DFPLPFS concentrations of 0, 25, 50, and 100 μg / mL, respectively) at 200 μL per well, with four replicates per group. After 48 hours, cell viability was determined by MTT assay.

[0061] Effects of hippocampal active peptides on Aβ40 and Aβ42 in supernatant and cells: CHO-APP / PS1 double-transfected cells in logarithmic growth phase were obtained and digested with trypsin to prepare a 2.5×10 5After 1 mL / well of a 12-well culture plate containing 100 μg / mL of cell suspension, the cells were seeded at 1 mL / well and cultured at 37°C in 5% CO2. After cell attachment, they were incubated with a 100 μg / mL DFPLPFS peptide solution (n = 3). After 48 hours, the culture medium was removed, and the cells were recovered with pre-chilled PBS. After sonication, the cells were lysed and centrifuged, and the supernatant was collected. ELISA kits were used to measure Aβ40 and Aβ42 levels, respectively. Protein content in each cell group was also measured, and the results were expressed as pg / mg protein.

[0062] Effects of different concentrations of 0, 25, 50, 75, and 100 μg / mL of peptides on the survival rate of CHO-APP / PS1 cells Figure 7 In the range of 0-75 μg / mL, each peptide had no significant effect on cell viability and showed no damage to CHO-APP / PS1 cells. Therefore, a dose of 50 μg / mL was selected for subsequent experimental studies.

[0063] Effects on intracellular Aβ40 and Aβ42 levels Figure 8 As shown in Figures 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 of the DFPLPFS peptide for 48 h significantly reduced the intracellular Aβ40 and Aβ42 levels.

[0064] Effects of peptides on extracellular Aβ40 and Aβ42 concentrations Figure 9 As shown in Figures AB, compared with the model group, the DFPLPFS peptide can significantly reduce the concentrations of Aβ40 and Aβ42 secreted into the extracellular space. After the action of the DFPLPFS peptide, the concentrations of extracellular Aβ40 and Aβ42 decreased by 49% and 31%, respectively, and the measurement results were statistically significant.

[0065] In order to further explore whether the inhibition of Aβ production by the above peptides is related to its key enzyme BACE1, molecular docking experiments were performed to achieve the interaction between the above new peptides and receptor proteins. The above peptides were homologously modeled and molecularly docked with BACE1 protein. The docking results are as follows Figure 10 The docking pattern between the peptide and β-secretase 1 (Figure AB) represents the optimal simulated docking position. The binding energy of the DFPLPFS peptide to the BACE1 protein is -8.0 kcal / mol. The peptide's structure is capable of integrating into the active pocket of BACE1. By interacting with key amino acid residues, forming conventional hydrogen bonds and hydrophobic interactions, it inhibits BACE1 enzyme activity and thereby reduces Aβ production. These results demonstrate that the DFPLPFS peptide is a promising BACE1 inhibitor with the potential to be developed as an active ingredient for enhancing memory and improving cognitive impairment.

[0066] Finally, it should be noted that while the above embodiments describe specific implementations of the present invention, they are not intended to limit the present invention. Those skilled in the art will understand that these are merely illustrative and that the scope of the present invention is defined by the appended claims. All modifications and equivalent substitutions are intended to be within the scope of the present invention.

Claims

1. A hippocampal peptide for abdominal distension, characterized by: The amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the hippocampal distending peptide according to claim 1 in the preparation of a product for improving memory ability and improving cognitive impairment.

3. The hippocampal peptide for abdominal distension according to claim 1, wherein: The belly-expanding hippocampal peptide is artificially synthesized.

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