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 protein polypeptide which has the main molecular weight of less than 1000 Da and has nutritional, health-care and medicinal values, and four active peptide sequences with high biological activity are obtained by further research on the basis of the protein polypeptide.

Description

Technical Field

[0001] The present invention relates to the field of the preparation and application of bioactive peptides, and particularly to a belly-swollen seahorse peptide and its application in the preparation of products for enhancing memory ability and improving cognitive impairment. Background Art

[0002] Normal aging is accompanied by a mild decline in learning and memory ability, while pathological aging may lead to neurodegenerative diseases such as mild cognitive impairment (MCI) and even Alzheimer's disease (AD). Brain aging plays an important role in cognitive impairment. The hippocampus mainly regulates learning and memory, and the occurrence of cognitive impairment is closely related to the functional changes in the hippocampus. Limited by the diagnostic methods, it takes a long time to identify from the initial stage of Alzheimer's disease to the stage of pathological deterioration such as cognitive impairment in patients, which will cause irreversible and harmful consequences to the pathological progression of AD. Therefore, improving aging accompanied by memory decline and cognitive impairment has become a much-concerned issue, and it is of great significance to develop effective functional foods or nutritional support products to promote brain health.

[0003] The belly-swollen seahorse (Hippocampus abdominalis) is a new variety introduced in China in recent years. With the continuous development of the mariculture industry, the improvement of living standards, and the popularization and promotion of the concept of great health, people's demand for health care is increasing day by day. Exploring its potential nutritional components is beneficial to the high-value utilization of seahorses. At present, the research on cognitive impairment and Alzheimer's disease mainly focuses on walnut peptides (such as Chinese patent CN116705141A, a method for screening Alzheimer's disease prevention peptides from walnut enzymatic hydrolysates based on a CNN-LSTM neural network) and GDF11 protein (such as Chinese patent CN109111517A, a modified growth differentiation factor and its preparation method and application), etc. There is less research on seahorse active peptides at present, and there is no research and application on the belly-swollen seahorse active peptides in improving learning and memory ability and cognitive impairment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is that the current existing technology does not involve the research and application of belly-swollen seahorse active peptides in improving learning and memory ability and cognitive impairment, which is a technical and market gap that needs to be filled.

[0005] To solve the above problems, the present invention provides a belly-swollen seahorse peptide and its application in the preparation of products for enhancing memory ability and improving cognitive impairment. By using a green and controllable enzymatic hydrolysis technology, protein polypeptides with a molecular weight mainly less than 1000 Da and having nutritional and medicinal values are obtained, and on this basis, 4 active peptide sequences with high biological activities are further studied.

[0006] To achieve the above object, the present invention is realized by the following technical means: The application of a puffed belly seahorse peptide in the preparation of products for enhancing memory ability and improving cognitive impairment; the puffed belly seahorse peptide includes polypeptides with amino acid sequences shown in SEQ ID NO.1-4: SEQ ID NO.1: GFDIGF.

[0007] SEQ ID NO.2: FGDGGF.

[0008] SEQ ID NO.3: MPGPMGPMG.

[0009] SEQ ID NO.4: FEGFLPM.

[0010] The preparation method of the above-mentioned puffed belly seahorse peptide powder includes the following steps: Take fresh puffed belly seahorses, wash them and then freeze-dry them, and crush them into powder; add distilled water, add 3% papain and stir evenly, adjust the pH = 6.5 with HCL solution and NaOH solution respectively, and enzymatically hydrolyze for 3 h at 50 °C; After the enzymatic hydrolysate is centrifuged, take the supernatant and filter it through a 0.45 μm microporous filter membrane, and then centrifuge it at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cut-off of 3 kDa. Separate through the ultrafiltration membrane with a molecular weight cut-off of 3 kDa to obtain peptide segments with a molecular weight less than 3 kDa; freeze-dry the peptide solution to obtain puffed belly seahorse peptide powder.

[0011] Further, add distilled water in a ratio of 1:9 of the solid-liquid ratio.

[0012] Further, the concentrations of both the HCL solution and the NaOH solution are 1 mol / L.

[0013] Further, the enzyme activity of the papain is 800,000 U / g.

[0014] The puffed belly seahorse peptide with the amino acid sequences shown in SEQ ID NO.1-4 above.

[0015] Further, the puffed belly seahorse peptide with the amino acid sequences shown in SEQ ID NO.1-4 is artificially synthesized.

[0016] 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 deposition and is the structural basis for the onset of AD. Aβ is a common pathway for multiple factors that form AD. Excessive accumulation of Aβ in the brain can lead to the massive generation of reactive oxygen species in cells, destroying Ca2+ and Ca2+ molecules inside and outside cells. 2+ Balance, abnormal modification of Tau protein, triggering a series of cascade reactions, causing neuronal dysfunction, and ultimately leading to cognitive dysfunction and neuronal apoptosis. Animal and cell experiments have found that hippocampal peptide improves learning and memory abilities by inhibiting the generation and accumulation of Aβ in the hippocampus of the brain.

[0017] The beneficial effects of the present invention are: (1) The green controlled enzymatic hydrolysis method was used to obtain hippocampal peptide hydrolysates. The SAMP8 naturally aged mouse model, APP / PS1 double transgenic mouse model and AD rat model induced by intraventricular injection of Aβ were used to explore the effects of hippocampal active peptides on improving memory ability and improving cognitive impairment.

[0018] (2) APP protein was used as the target for targeted screening of active peptides in hippocampal peptide hydrolysates. Using mass spectrometry technology and activity prediction methods, four potential hippocampal active peptide sequences with the ability to enhance memory were disclosed: GFDIGF (①), FGDGGF (②), MPGPMGPMG (③), and FEGFLPM (④), providing a theoretical basis for the development of products for enhancing memory and improving cognitive impairment using hippocampal active peptides. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] 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 normal group; * p < 0.05 compared with the model group; ** p < 0.01 compared with the model group.

[0021] Figure 3 Figure 1 shows the Y-maze test for SAMP8 mice. A represents the total number of arm entries, and B represents the spontaneous alternation rate. Note: # compared with the normal group, p < 0.05, ## compared with the normal group, p < 0.01; * compared with the model group, p < 0.05.

[0022] Figure 4Results of the novel object recognition test in SAMP8 mice: A is the exploration time of the object, and B is the recognition index of different objects. Note: ## p < 0.01 compared with the normal group; ** p < 0.01 compared with the model group.

[0023] Figure 5 Results of the novel object recognition test in APP / PS1 double transgenic mice: A is the exploration time of the object, and B is the recognition index of different objects. Note: ## p < 0.01 compared with the normal group; * p < 0.05 compared with the model group.

[0024] Figure 6 Results of the Morris water maze test in AD model rats: A is the latency time of the rats in the place navigation test, B is the number of times the rats crossed the platform in the spatial exploration test, and C is the time the rats stayed in the target quadrant. Note: ## p < 0.01 compared with the normal group; * p < 0.05 compared with the model group, ** p < 0.01 compared with the model group.

[0025] Figure 7 Effects of peptide segments at different concentrations on the survival rate of CHO-APP / PS1 cells.

[0026] Figure 8 Effects of peptide segments on the contents of Aβ40 and Aβ42 in CHO-APP / PS1 cells. Among them, A is the content of intracellular Aβ40, and B is the content of Aβ42. Note: Different letter labels indicate significant differences between groups.

[0027] Figure 9 Effects of peptide segments on the contents of extracellular Aβ40 and Aβ42 in CHO-APP / PS1 cells. Among them, A is the content of extracellular Aβ40, and B is the content of Aβ42. Note: Different letter labels indicate significant differences between groups. Specific implementation manners

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] In addition, all materials used in the embodiments of the present invention are obtained by purchase from the market unless otherwise specified.

[0030] Embodiment 1: A pterothrissus annectens peptide is prepared by the following method: Preparation of Peptide Hydrolysate from Hippocampus abdominalis: Take 100 g of fresh Hippocampus abdominalis, wash it and then freeze-dry it, and crush it into powder. Add distilled water according to the solid-liquid ratio of 1:9 (m / v), add 3% papain and stir evenly. Adjust the pH to 6.5 with HCl solution (1 mol / L) and NaOH solution (1 mol / L) respectively, and enzymatically hydrolyze at 50 °C for 3 h. After the enzymatic hydrolysate is centrifuged (centrifuged at 4 °C and 4000 g / min for 15 min), take the supernatant and filter it through a 0.45 μm microporous membrane, and then centrifuge it at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cut-off of 3 kDa. Separate through the ultrafiltration membrane (molecular weight cut-off 3 kDa) to obtain peptide segments with a molecular weight less than 3 kDa. Freeze-dry the peptide solution to obtain the powder of peptide from Hippocampus abdominalis. Papain (800,000 U / g, G8430-25) was purchased from Solarbio Science & Technology Co., Ltd.

[0031] 1. Evaluation of the effects on memory ability and cognitive impairment in the SAMP8 aging mouse model: Determination of the molecular weight of peptide from Hippocampus abdominalis: The molecular weight distribution of SHP was determined by gel exclusion chromatography. The specific conditions were as follows: chromatographic 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; ultraviolet detection wavelength: 214 nm.

[0032] The molecular weight distribution of the papain total enzymatic hydrolysate of peptide from Hippocampus abdominalis was detected by gel chromatography, and the results were as Figure 1 shown. By comparing with the standard molecular weight curve, it was calculated that the proportion of peptide from Hippocampus abdominalis papain total enzymatic hydrolysate with a molecular weight less than 500 Da was 23.96%, and the proportion of short peptides with a molecular weight of 500 - 1 kDa was the largest, which was 48.21%. Among them, the proportion of short peptides with a molecular weight of 1 - 3 kDa was 27.83% ( Figure 1 ).

[0033] Animal experiment: 30 SAMP8 mice and 10 SAMR1 mice were purchased from Nanjing Chinchilla Technology Co., Ltd. and raised in the Biological Resources Center of Ocean University of China. The animal breeding environment was as follows: the temperature was maintained at 21°C - 23°C, the humidity was maintained at 45% - 55%, with a 12 h light / 12 h dark cycle. During the experiment, the mice could freely eat and drink. All mice were fed with rod-shaped feed (AIN-93G type feed) until they were four months old, and then fed with high-fat feed until they were six months old. The SAMP8 mice were randomly divided into 3 groups according to body weight, namely the model group (Model), the low-dose hippocampal peptide group (SHP-L 200 mg / kg BW), and the high-dose hippocampal peptide group (SHP-H 400 mg / kg BW). The SAMR1 mice were used as the control group. Hippocampal peptide was supplemented by gavage once a day until the mice were nine months old, and then behavioral tests were started.

[0034] Morris water maze: The diameter of the pool was 130 cm, the height was 50 cm, the water depth was 30 cm, and the water temperature was controlled at 22 ± 1 °C. The circular pool was equally 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 at the center of the quadrant. The camera device was directly above the circular pool and connected to the computer. During the test, ink was added to the pool to exclude the interference of other factors except markers on the spatial orientation of the mice. The whole process of the behavioral test included a place navigation experiment and a spatial exploration experiment.

[0035] Place navigation experiment: During the test, the platform was placed in the first quadrant. The mice were placed into the pool facing the pool wall from the four quadrants in turn. The time for a single test was 60 s, and the trajectory tracking analysis software automatically recorded the latency and average speed of the mice. The order of the quadrants entered each day changed in turn, and the experiment lasted for 5 d.

[0036] Spatial exploration experiment: On the 6th day of the above experiment, the underwater platform was removed, and the residence time within the original target quadrant and the number of times entering the quadrant where the original target platform was located within 60 s were recorded.

[0037] Y maze test: The mice were placed in the Y maze and allowed to freely explore for 8 min. The number of times the mice entered each open arm for exploration and the order of entering the arms in turn were recorded. One correct exploration should be that the mice entered different open arms in succession 3 times. The total number of times entering each open arm for exploration and the spontaneous alternation rate (%) = number of correct entries / (number of arm entries - 2) × 100% were recorded.

[0038] Novel object recognition test: Three days before training and testing, mice were first acclimated to the chamber. At the start of training, two identical objects, A and B, were placed at the left and right ends of one side wall, and the mice were placed in the arena with their backs to the two objects. Immediately after the mice were placed in the chamber, the video recording device was turned on to record the mice's contact with the two objects, including the exploration time within 2 cm of the objects. After 5 minutes, the mice were immediately returned to their original cages and rested for 24 hours before testing. The next day, object B in the chamber was replaced with object C (different from B), and the mice were still placed with their backs to the two objects, as shown on the right in the following figure. The ANY software was set to record the time the mice approached objects B and C within 5 minutes, and the recognition index was used to reflect the mice's memory. Recognition index = time of contact with object C / time of contact with A + C.

[0039] In the place navigation experiment stage, as the number of training days increased, the time for the 4 groups of mice to find the platform all became shorter (as shown in A - C below), but there was no significant difference between groups (P > 0.05). In the spatial exploration experiment stage, compared with the SAMR1 group of mice, the residence time of the SAMP8 group and the SHP group of mice in the target quadrant both decreased, and the number of times the P8 mice crossed the platform decreased significantly (P < 0.05). Compared with the SAMP8 group of mice, the residence time of the SHP - H group of mice in the target quadrant was significantly prolonged (P < 0.05), and the number of crossings increased significantly, as shown in Figure 2 Figure C below. The results showed that compared with the SAMR1 group, the memory ability of the SAMP8 group of mice decreased. The memory ability of the SAMP8 + SHP - H group of mice was improved compared with that of the SAMP8 group of mice. Figure 2

[0040] Figure 3 As shown in A - B below, the results of the Y - maze experiment showed that compared with the control group, the number of entries into the arms and the spontaneous alternation rate of the model group of mice decreased significantly (P < 0.05); compared with the model group, the number of entries into the arms and the spontaneous alternation rate of the SHP - L group showed an increasing trend, but there was no statistical significance (P > 0.05). The spontaneous alternation rate of the SHP - H group increased significantly (P < 0.05), and there was no significant difference in the total number of entries into the arms and the spontaneous alternation rate among the hippocampal peptide groups.

[0041] The novel object recognition test experiment is a learning and memory test method established based on the principle that animals have an innate tendency to explore novel objects. Compared with the water maze experiment, it has less stress and can more closely simulate human learning and memory behavior. In addition, by changing the shape, size, etc. of the objects, this method can be applied to the detection of long - term or short - term memory formation in animals. The results are as Figure 4As shown in A-B of the figure, compared with the control group, the exploration time and discrimination index of the mice in the model group were significantly decreased (P < 0.05). Compared with the model group, the exploration time and discrimination index of the mice in the SHP-H group were increased (P < 0.05). Although SHP-L intervention increased the exploration time of new things, the effect on the discrimination index was not significant, and there was no significant difference compared with the model group (P > 0.05). The above results indicate that SHP-H effectively improves the cognitive memory impairment of aging mice.

[0042] 2. Evaluation of memory ability and cognitive impairment in the APP / PS1 transgenic mouse model: 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 AD. APP / PS1 double transgenic mice produce more Aβ compared with normal mice. 1-40 and Aβ 1-42 , which is an ideal transgenic animal model for AD.

[0043] Animal grouping and intervention: 30 six-month-old male APP / PS1 double transgenic mice and 10 non-transgenic C57BL / 6 mice of the same strain background (used as controls) were purchased from Beijing Huafukang Biotechnology Co., Ltd. All mice were housed in the resource center of Ocean University of China, and the housing conditions were the same as those in Example 1. After 1 week of adaptive feeding, the mice were grouped. The 30 APP / PS1 double transgenic mice were randomly divided into a model group (Model), 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 weights. The C57BL / 6 mice were used as the control group (Normal). Hippocampal peptide was supplemented by gavage once a day. After continuous gavage for 60 days, behavioral tests were started.

[0044] The Morris water maze test was the same as 1.

[0045] The novel object recognition test was the same as 1.

[0046] As shown in Table 1, there was no statistical significance in the swimming speed of the mice in each group (P > 0.05). Compared with the Normal group, the escape latency time and the total average swimming distance of the mice in the Model group were significantly increased (P < 0.01), and the number of times of crossing the platform was significantly decreased (P < 0.01). Compared with the Model group, the escape latency time of the mice in the SHP-L group and the SHP-H group was significantly decreased (P < 0.01), and the number of times of crossing the platform in the SHP-H group was significantly increased (P < 0.05).

[0047] Table 1 Effects of hippocampal peptides on behavioral indices of mice in each group: Group Total average swimming distance (m) Average swimming speed (mm / s) Escape latency (s) Number of platform crossings (times) Control group: Normal 3.60 ± 0.34 141.26±10.07 13.62±3.61 4.6±1.25 Model group: Model <![CDATA[1.69 ± 0.32 ## > 136.55±14.21 <![CDATA[58.63±2.40 ## > <![CDATA[0.8±0.45 ## > Low-dose hippocampal peptide group: SHP-L 200 mg / kg BW 1.82 ± 0.51 137.48±12.06 <![CDATA[40.36±7.62 * > 2.1±0.99 High-dose hippocampal peptide group: SHP-H 400 mg / kg BW <![CDATA[3.01 ± 0.38 ** > 138.92±14.61 <![CDATA[31.36±7.07 ** > <![CDATA[2.9±0.76 * > Note: ## p < 0.01 compared with the normal group; * p < 0.05 compared with the model group, ** p < 0.01 compared with the model group.

[0048] As Figure 5 shown in A - B, there was no statistical significance in the total exploration time of the old 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 Normal group, the exploration time recognition index and exploration frequency recognition index of the mice in the Model group were significantly decreased (P < 0.01); compared with the Model group, the exploration time recognition index and exploration frequency recognition index of the mice in the SHP - H group were significantly increased (P < 0.05), and the effect increased in a dose - dependent manner.

[0049] 3. Evaluation of the effects on memory ability and cognitive impairment in an AD rat model induced by intracerebroventricular injection of Aβ: Intracerebroventricular injection of Aβ can trigger intracerebral oxidative stress, activation of glial cells, increase the toxicity of Tau protein, and induce neuronal apoptosis. An AD model rat was established by intracerebroventricular injection of Aβ1 - 42 to verify the improvement effect of hippocampal active peptide on learning, memory, and cognitive impairment in AD rats after Aβ generation. Preparation of aggregated Aβ1 - 42: Aβ1 - 42 was dissolved in sterile physiological saline to a stock solution of 1 mg / mL, and this stock solution was incubated at 37 °C for 5 d to obtain soluble oligomeric Aβ1 - 42.

[0050] Establishment of an AD animal model: Forty male SD rats (body weight 280 - 300 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. and were individually housed in an SPF environment and adaptively fed for one week under the conditions of 12 h light - dark alternation, temperature maintained at 23 ± 1 °C, and relative humidity 55 ± 5%. During this period, they had free access to food and water. The surgery was performed under sterile conditions. The rats were anesthetized before placing their heads in the stereotaxic apparatus. Referring to Paxinos' "Stereotaxic Atlas of the Rat Brain", the left lateral ventricle was selected as the injection target area. With the bregma as the origin, 1.0 mm posterior and 1.4 mm lateral to the midline, a cranial drill was used to drill through the skull, and Aβ1 - 42 was injected into the area 4 mm below the dura mater (5 μl / 5 min) using a micro - infusion pump. The dose of Aβ1 - 42 was selected based on previous laboratory research. Rats in the sham - operation group were infused with an equal volume of sterile physiological saline, and other operations were the same. After leaving the needle in place for 5 min, the needle was slowly withdrawn, and the wound was sutured after disinfection with penicillin.

[0051] Seven days after model establishment, the rats with Aβ ventricular injection were randomly divided into 3 groups according to body weight, namely the model group (Model), the low-dose hippocampal peptide group (SHP-L 200 mg / kg BW), and the high-dose hippocampal peptide group (SHP-H 400 mg / kg BW).

[0052] The Morris water maze test was the same as in 1.

[0053] The results were as Figure 6 shown in A - C in the figure. On the 1st day, compared with the model group, there was no significant difference in the latency of finding the platform in each experimental group. However, on the 3rd day of the learning period, the latency of rats in the SHP-H group was significantly shortened (P < 0.05), and the time to find the platform was reduced by 27% compared with the model group, which were 40.6 s and 37.3 s respectively. On the last training day, compared with the rats supplemented with SHP, the Aβ1-42 rats in the model group still needed more time to find the platform.

[0054] In the spatial exploration experiment, the platform was removed, and the number of times each group of rats crossed the original platform and the exploration time in the target quadrant within 60 s were recorded to evaluate the memory impairment of the rats. Compared with the sham operation group, the number of times the model group rats crossed the platform (1.5 times and 4.2 times respectively) and the time in the target quadrant (25.31 s and 36.62 s) both decreased significantly (P < 0.05). In addition, compared with the Aβ1-42 rats, after supplementing SHP, the number of times the rats in the group crossed the platform and the exploration time in the quadrant where the platform was located both showed significant improvement, indicating that the intervention of SHP can improve the cognitive defects of Aβ1-42 injected rats.

[0055] Example 2: Based on the evaluation results of the effects of SHP in Example 1 on three models, an affinity chromatography method was used to enrich potential active peptides from the hippocampal peptide mixture in Example 1 using immobilized amyloid precursor protein (APP) and perform peptide fingerprint identification.

[0056] Preparation of the App protein affinity chromatography column: Rinse the CNBr-activated medium with 10 column volumes of 1 mM HCl to remove the protective agent; immediately equilibrate the chromatography column with coupling buffer (pH = 8.3). Dissolve the purified App protein in the coupling buffer (final concentration 2 - 5 mg / mL), mix the protein solution with the activated medium at a volume ratio of 1:1, and incubate with rotation at room temperature for 2 h (or overnight at 4℃). Remove the unbound protein solution and wash 3 times with the coupling buffer. Add the blocking buffer (containing 1 M ethanolamine, pH = 8.0) and incubate at room temperature for 1 h. Wash successively with 3 column volumes of the coupling buffer and the binding buffer, and store at 4℃ (containing 0.02% NaN3). Determine the protein concentration in the solution before and after coupling by the BCA method and calculate the coupling efficiency.

[0057] Enrichment of seahorse active peptides: The seahorse peptide hydrolysate sample was desalted using a PD-10 desalting column, and the seahorse peptide hydrolysate was replaced with binding buffer (pH 7.4). It was centrifuged at 12,000 rpm for 10 min to remove insoluble matter, and 1 mM PMSF (final concentration) was added. The operation was carried out on ice to prevent peptide degradation. The pretreated seahorse peptide hydrolysate was loaded onto the App protein immobilized chromatography column at a flow rate of 0.5 - 1 mL / min, and the loading was repeated 2 - 3 times to improve the binding efficiency. It was rinsed with 10 column volumes of binding buffer (pH 7.4) to remove unbound impurities. Subsequently, it was eluted with 5 column volumes of 0.1 M glycine-HCl (pH = 2.5), and the collected eluate 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. The eluate was concentrated using a 3 kDa ultrafiltration centrifugal tube and replaced with PBS or pure water.

[0058] The peptide sequences obtained by de novo sequencing were searched for seahorse 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 polypeptide identification results ≤ 1%. Peptide Ranker was used to predict the possibility that the identified peptide sequences have biological activity.

[0059] Through activity prediction of Peptide Ranker (Table 2), this example discloses 5 seahorse abdominal distension active peptide sequences with the ability to enhance memory.

[0060] Table 2 Identification and activity prediction of seahorse abdominal distension active peptide sequences: Sequence Mass-to-charge ratio of nucleoplasmic ratio m / z Charge Source protein Source protein sequence <![CDATA[Peptide activity score a > GFDIGF 655.31 1 PREDICTED: collagen alpha-1(I) chain-like isoform X1 XP_019740420.1 0.95 FGDGGF 681.37 1 E3 ubiquitin-protein ligase RNF185 isoform X1 XP_051906873.1 0.94 MPGPMGPMG 840.40 1 collagen alpha-1(I) chain-like isoform X1 XP_051926668.1 0.93 FEGFLPM 699.38 1 myosin light chain 1, skeletal muscle isoform XP_051937666.1 0.92 a From PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0061] The above-predicted bioactive peptides of *Hippocampus abdominalis* were biosynthesized separately by Shanghai Sangon Biotech Co., Ltd. through Fmoc-solid phase peptide synthesis. Through HPLC and MS sequence analysis, the purity of the four peptide segments GFDIGF, FGDGGF, MPGPMGPMG, and FEGFLPM was greater than 98.89%. In order to further verify the effect of the activities of the above-screened peptide segments on Aβ production, in this example, APP / PS1 (M146L) double-transfected CHO (CHO-APP / PS1) cells were used for verification. APP / PS1 (M146L) double-transfected cells, because they carry the APP / PS1 plasmid, can secrete more Aβ1-40 and Aβ1-42 compared with normal nerve cells, and can be regarded as a transgenic cell model of AD. The cells are stored in the Human Health Laboratory of the College of Food Science and Engineering, Ocean University of China.

[0062] Cell culture: CHO-APP / PS1 cells in the logarithmic growth phase were taken, digested with trypsin, and made into a cell suspension with a concentration of 5×10 4 cells / mL. 100 μL / well was inoculated into a 96-well culture plate and cultured under the conditions of 37 °C and 5% CO2. After 24 h, peptide samples of different concentrations in the examples were incubated, namely GFDIGF, FGDGGF, MPGPMGPMG, and FEGFLPM at concentrations of 0, 25, 50, and 100 μg / mL, 200 μL per well, with 4 replicates in each group. After 48 h, the cell viability was detected by the MTT method.

[0063] Effects of hippocampal bioactive peptide segments on Aβ40 and Aβ42 in the supernatant and cells: CHO-APP / PS1 double-transfected cells in the logarithmic growth phase were taken, digested with trypsin, and made into a cell suspension with a concentration of 2.5×10 5 cells / mL, and then inoculated into a 12-well culture plate at 1 mL / well and cultured under the conditions of 37 °C and 5% CO2. After the cells adhered, 100 μg / mL of GFDIGF, FGDGGF, MPGPMGPMG, and FEGFLPM peptide solutions (n = 3) were incubated. After 48 h, the culture medium was recovered, the cells were recovered with pre-cooled PBS, lysed with an ultrasonic cell disruptor, and centrifuged to obtain the supernatant. An ELISA kit was used to measure the levels of Aβ40 and Aβ42 respectively, and the protein content of each group of cells was measured at the same time. The results were expressed as pg / mg prot.

[0064] The effects of peptide segments with different concentrations of 0, 25, 50, 75, and 100 μg / mL on the viability of APP / PS1 cells are as Figure 7 shown. In the range of 0-75 μg / mL, each peptide segment had no obvious 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.

[0065] The effects on the intracellular contents of Aβ40 and Aβ42 are as Figure 8 shown in A - B of [Figure]. The intracellular Aβ40 content in the model group was 568 pg / mg prot, and the Aβ42 content was 422.6 pg / mg prot. Incubation with the four peptide segments for 48 h could significantly reduce the intracellular levels of Aβ40 and Aβ42. The improvement effects of the GFDIGF and FGDGGF peptide segments were significantly better than those of MPGPMGPMG and FEGFLPM.

[0066] The effects of the peptide segments on the extracellular concentrations of Aβ40 and Aβ42 are as Figure 9 shown in A - B of [Figure]. Compared with the model group, the four peptide segments could all significantly reduce the extracellular concentrations of Aβ40 and Aβ42. After the action of the five test substances, the reduction amplitudes of extracellular Aβ40 were 45%, 34%, 52%, 33% and 39% respectively, and there was statistical significance among the test substances. The inhibition degrees of extracellular Aβ42 concentration were 34%, 52%, 39%, 27% and 54% respectively. The improvement effects of the GFDIGF and FGDGGF peptide segments were significantly better than those of MPGPMGPMG and FEGFLPM.

[0067] Finally, it should be noted that although the above embodiments describe the specific implementation manners of the present invention, they do not limit the present invention; those skilled in the art should understand that these are only examples. The protection scope of the present invention is defined by the appended claims. Any modifications or equivalent replacements shall be included within the protection scope of the present invention.

Claims

1. A ventral hippocampal peptide and its application in the preparation of products for enhancing memory ability and improving cognitive impairment, characterized in that: The belly-swelling seahorse peptide comprises polypeptides with the amino acid sequences shown in SEQ ID NO.1-4.

2. The preparation method of the seahorse abdominal peptide powder according to claim 1, characterized in that It includes the following steps: Take fresh belly-swelling seahorses, wash them, freeze-dry them, and crush them into powder; add distilled water, add 3% papain and stir evenly, adjust the pH = 6.5 with HCL solution and NaOH solution respectively, and enzymatically hydrolyze at 50 °C for 3 h; After centrifuging the enzymatic hydrolysate, take the supernatant, filter it through a 0.45 μm microporous membrane, and then centrifuge it at 4000 g / min for 20 min using an ultrafiltration tube with a molecular weight cut-off of 3 kDa. Separate through the ultrafiltration membrane with a molecular weight cut-off of 3 kDa to obtain peptide segments with a molecular weight less than 3 kDa; freeze-dry the peptide solution to obtain belly-swelling seahorse peptide powder.

3. The preparation method according to claim 2, characterized in that: Add distilled water at a ratio of 1:9 of the material-liquid ratio.

4. The preparation method according to claim 2, wherein: The concentrations of the HCL solution and the NaOH solution are both 1 mol / L.

5. The preparation method according to claim 2, characterized in that: The enzyme activity of the papain is 800,000 U / g.

6. The belly-swelling seahorse peptide powder prepared by any of the methods according to claims 2-5.

7. The ventral-bloating seahorse peptide according to claim 1, characterized in that: The belly-swelling seahorse peptide with the amino acid sequences shown in SEQ ID NO.1-4.

8. The puffed belly seahorse peptide according to claim 7, wherein: The belly-swelling seahorse peptide is artificially synthesized.

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