Sea cucumber peptide capable of delaying cognitive function decline and application thereof

Abstract

The application discloses a sea cucumber peptide capable of delaying cognitive function decline and application thereof, and the sea cucumber peptide contains at least one of sea cucumber peptides with amino acid sequences of IWNAPHTW, FPKVPGQY or YAPRLISF. The three peptide segments of the application have the functions of delaying cognitive function, improving the anti-inflammatory and antioxidant capacity of a body, improving nerve inflammation and regulating intestinal flora structure. The application excavates the medicinal functions of small molecule active substances (such as sea cucumber peptides) of sea cucumbers, and improves the economic value of sea cucumber products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a sea cucumber peptide that can delay cognitive decline and its applications. Background Technology

[0002] As we age, various physiological functions gradually decline, among which the decline in cognitive and other behavioral functions is one of the most obvious characteristics of aging. Cognitive decline manifests as symptoms such as memory loss, decreased learning ability, and slow reaction. This decline in cognitive function not only affects the quality of life but also increases the risk of developing neurodegenerative diseases.

[0003] Damage to the cholinergic system can impair cognitive processes, leading to cognitive decline. Excessive acetylcholinesterase activity accelerates the degradation of acetylcholine, resulting in decreased acetylcholine levels at synapses, which in turn affects the normal transmission of neurotransmitters and interferes with normal nervous system function. Cholinesterase inhibitors can suppress acetylcholinesterase activity, increasing the availability of acetylcholine at brain synapses, and have been clinically proven to be useful in the prevention or treatment of Alzheimer's disease.

[0004] Sea cucumbers are characterized by high protein and low fat, making them an excellent protein source for preparing bioactive peptides. Numerous studies have demonstrated that the bioactive substances in sea cucumbers possess various physiological activities, including lowering blood pressure, lowering blood lipids, lowering blood sugar, inhibiting angiotensin-converting enzyme, anti-cancer effects, and anti-fatigue properties. However, research on the improvement of age-related cognitive impairment by small-molecule bioactive substances in sea cucumbers (such as sea cucumber peptides) is still scarce. Summary of the Invention

[0005] In order to better prevent and treat cognitive impairment, explore the medicinal functions of sea cucumber products, and improve the economic value of sea cucumber products, this invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a sea cucumber peptide, comprising at least one of the following:

[0007] (1) Sea cucumber peptide with the amino acid sequence IWNAPHTW (SEQ ID NO:1).

[0008] (2) Sea cucumber peptide with the amino acid sequence FPKVPGQY (SEQ ID NO:2).

[0009] (3) Sea cucumber peptide with the amino acid sequence YAPRLISF (SEQ ID NO:3).

[0010] Preferably, the amino acid sequence of the sea cucumber peptide is IWNAPHTW, FPKVPGQY, or YAPRLISF.

[0011] Preferably, the sea cucumber peptide includes (1) and (2) mentioned above.

[0012] Preferably, the sea cucumber peptide includes (1) and (3) mentioned above.

[0013] Preferably, the sea cucumber peptide includes (2) and (3) mentioned above.

[0014] Preferably, the sea cucumber peptides include (1), (2) and (3) mentioned above.

[0015] In a second aspect, the present invention provides a polypeptide mixture comprising the sea cucumber peptide described in the first aspect.

[0016] Thirdly, the present invention provides a composition comprising the sea cucumber peptide described in the first aspect or the polypeptide mixture described in the second aspect.

[0017] Fourthly, the present invention provides a method for preparing the sea cucumber peptide described in the first aspect, the method comprising the following steps:

[0018] (1) Grind the sea cucumber body wall into a paste, add water, adjust the pH, and enzymatically hydrolyze it;

[0019] (2) Inactivate the enzyme in the enzymatic hydrolysis product of step (1), separate by ultrafiltration, freeze-dry the filtrate to obtain freeze-dried powder;

[0020] (3) The freeze-dried powder from step (2) is separated, purified, and identified to obtain the sea cucumber peptide.

[0021] Preferably, the pH in step (1) is 3 to 9, for example: 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.

[0022] Preferably, the enzyme added in step (1) includes one or more of neutral protease, alkaline protease, papain, flavor protease or proteinase K.

[0023] Furthermore, the enzyme activity of each enzyme is 2500–4000 U / g, for example: 2500 U / g, 2800 U / g, 3000 U / g, 3200 U / g, 3500 U / g, 3800 U / g, 4000 U / g.

[0024] Furthermore, the enzymatic hydrolysis time is 2 to 8 hours, for example: 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, and 8 hours.

[0025] Furthermore, the enzymatic hydrolysis temperature is 45–55℃, for example: 45℃, 47℃, 50℃, 53℃, 55℃.

[0026] Preferably, the enzyme inactivation method in step (2) is a water bath.

[0027] Furthermore, the water bath temperature is above 90°C, for example: 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C.

[0028] Furthermore, the water bath time is 10-20 minutes, for example: 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes.

[0029] Preferably, the pore size of the ultrafiltration membrane in step (2) is 0.005-0.05μm, for example: 0.005μm, 0.01μm, 0.02μm, 0.03μm, 0.04μm, 0.05μm.

[0030] Preferably, the freeze-drying temperature in step (2) is -80℃ to 50℃, for example: -80℃, -70℃, -60℃, -50℃.

[0031] Preferably, the separation and purification in step (3) is performed using gel column chromatography and high performance liquid chromatography.

[0032] Preferably, the identification in step (3) is performed using mass spectrometry.

[0033] Fifthly, the present invention provides an acetylcholinesterase inhibitor, wherein the acetylcholinesterase inhibitor comprises the sea cucumber peptide described in the first aspect, the polypeptide mixture described in the second aspect, or the composition described in the third aspect.

[0034] In a sixth aspect, the present invention provides a product for delaying cognitive decline, the product comprising the sea cucumber peptide described in the first aspect, the polypeptide mixture described in the second aspect, or the composition described in the third aspect.

[0035] Preferably, the product is a health food, feed, or medicine.

[0036] Preferably, the product also includes excipients permitted to be added to health foods, feed, or pharmaceuticals.

[0037] In a seventh aspect, the present invention provides the use of the sea cucumber peptide of the first aspect, the polypeptide mixture of the second aspect, or the composition of the third aspect, wherein the use includes at least one of the following;

[0038] (1) Application in the preparation of acetylcholinesterase inhibitors.

[0039] (2) Application in the preparation of products that help improve memory.

[0040] (3) Application in the preparation of products with enhanced anti-inflammatory and antioxidant capabilities.

[0041] (4) Application in the preparation of products that improve neuroinflammation.

[0042] (5) Application in the preparation of products that regulate the structure of intestinal flora.

[0043] The beneficial effects of this invention are:

[0044] This invention isolated three peptides from sea cucumber that inhibit acetylcholinesterase. These three peptides also possess functions such as delaying cognitive decline, enhancing the body's anti-inflammatory and antioxidant capabilities, improving neuroinflammation, and regulating intestinal flora structure. This invention explores the medicinal functions of small-molecule active substances in sea cucumber (such as sea cucumber peptides), thereby increasing the economic value of sea cucumber products. Attached Figure Description

[0045] Figure 1 The diagram shows the docking of IWNAPHTW with acetylcholinesterase.

[0046] Figure 2 The diagram shows the docking of FPKVPGQY with acetylcholinesterase molecules.

[0047] Figure 3 The diagram shows the docking of YAPRLISF with acetylcholinesterase molecules.

[0048] Figure 4 The results show the improvement of cognitive function in aging mice by sea cucumber peptides. A represents the percentage of arm alternation in the Y maze test; B represents the new object recognition index period in the new object recognition test; C represents the time spent in the target quadrant in the water maze test; D represents the number of times the target area was traversed in the water maze test; and E represents the latency of finding the hidden platform in the water maze test.

[0049] Figure 5 The figure shows the effects of sea cucumber peptides on acetylcholine content and acetylcholinesterase expression in the hippocampus of aging mice. A represents the acetylcholine level in the hippocampus; B represents the acetylcholinesterase expression level in the hippocampus.

[0050] Figure 6 The image shown is a pathological diagram of the effect of sea cucumber peptides on the hippocampus of an aging mouse.

[0051] Figure 7 The figure shows the effects of sea cucumber peptides on the anti-inflammatory and antioxidant capacity of aging mice. A is the serum TNF-α level of mice, B is the serum IL-1β level of mice, C is the serum IL-6 level of mice, D is the serum LPS level of mice, E is the serum SOD activity of mice, and F is the serum IL-10 level of mice.

[0052] Figure 8The image shows the effect of sea cucumber peptides on neuroinflammation in the hippocampus of aging mice. A represents the expression of IBA-1, a microglia marker in the hippocampus of mice; B represents the expression of key proteins in the NF-κB pathway in the hippocampus of mice; and C represents the expression of the NLRP3 inflammasome.

[0053] Figure 9 The image shows the effect of sea cucumber peptides on the gut microbiota of aging mice. A represents the α-diversity of gut microbiota, B represents the gut microbiota structure (phylum level), and C represents the gut microbiota structure (family level).

[0054] Figure 10 The figure shows the content of short-chain fatty acids in mouse feces. Detailed Implementation

[0055] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0056] Example 1: Preparation of sea cucumber peptides with the effect of delaying cognitive decline

[0057] (1) Use a high-speed blender to grind 30-80g of sea cucumber body wall into a paste, add 500-1500mL of distilled water, adjust the pH to 3.0-9.0, and add neutral protease, alkaline protease, papain, flavor protease and proteinase K respectively according to the enzyme activity of each enzyme being 3000U / g. The enzymatic hydrolysis temperature is 50℃.

[0058] (2) After enzymatic hydrolysis for 2-8 hours, the solution is quickly placed in a 96℃ water bath for 15 minutes to inactivate the enzyme. The solution is then separated using an ultrafiltration membrane with a pore size of 0.01μm, and the filtrate is collected as a sea cucumber peptide solution.

[0059] (3) Place the sea cucumber peptide solution into a vacuum freeze dryer and freeze dry at -60℃ to obtain sea cucumber peptide freeze-dried powder.

[0060] (4) The sea cucumber peptide freeze-dried powder was purified by gel column chromatography, high performance liquid chromatography, and the peptide sequence was identified by mass spectrometry. Six sea cucumber peptides were obtained, with the sequences being IWNAPHTW (SEQ ID NO:1), FPKVPGQY (SEQ ID NO:2), YAPRLISF (SEQ ID NO:3), FGIDVWEH (SEQ ID NO:4), LDAQKFI (SEQ ID NO:5) and IGDTRFPY (SEQ ID NO:6).

[0061] The specific separation, purification, and identification process is as follows:

[0062] Gel column chromatography: A certain amount of dry powdered Sephadex G-25 was added to 5 times its volume of ultrapure water, swollen in a boiling water bath for 2 hours, cooled, degassed, and packed into a column. The column was equilibrated with 3–5 column volumes of 0.02 mol / L Tris-HCl (pH 7.4). After equilibration, 2 mL (20 mg / mL) of sea cucumber enzymatic hydrolysis ultrafiltration product was added, and ultrapure water was used for elution at a flow rate of 0.6 mL / min. The eluent was collected using an automatic collector, one tube every 5 minutes. The OD value was measured at 220 nm to obtain the distribution curve of the enzymatic hydrolysis ultrafiltration product in each chromatographic fraction, thereby collecting peptide fractions of different molecular weights.

[0063] High-performance liquid chromatography (RP-HPLC) separation and purification: The active components separated by the Sephadex G-25 column were analyzed using RP-HPLC. The analytical column conditions used were: Kromasil C18 (4.6 × 250 mm); sample volume 30 μL; flow rate 6 mL / min; detection wavelength 220 nm; mobile phase: solution A: water containing 0.1% trichloroacetic acid; solution B: acetonitrile containing 0.1% trichloroacetic acid; linear elution program: 0–10 min B 5%; 11–30 min B 5%–48%; 31 min–45 min B 5%; stop at 45 min.

[0064] Mass spectrometry identification: Peptide samples were dissolved in 0.1% formic acid solution and analyzed using an Ultimate 3000 UPLC (Thermo Fisher Scientific) liquid chromatography system. A self-made pre-column and column were used (pre-column: 100 μm inner diameter, packed with 2 cm length of 3 μm C18 packing material; column: 100 μm inner diameter, packed with 30 cm length of 1.9 μm C18 packing material). Liquid chromatography solution A (0.1% (v / v) formic acid aqueous solution) and solution B (0.1% (v / v) formic acid (80% (v / v)) acetonitrile solution) were used. The liquid chromatography gradient increased from 8% solution B to 50%, with an elution flow rate of 300 nL / min and an elution time of 76 min.

[0065] The Eclipse Tribrid Orbitrap (Thermo Fisher Scientific) mass spectrometer was used for data-dependent acquisition (DDA). The spray voltage was 2.2 kV, and the ion transfer tube temperature was 320 °C. Primary spectrum detection was performed using an Orbitrap analyzer with an ion scan range of m / z 50-1500, an Orbitrap resolution of 60,000, and a maximum ion injection time of 40 ms. Secondary spectrum detection was also performed using an Orbitrap analyzer with an ion scan range of m / z 50-1400, a resolution of 15,000, a collision energy of 32%, and a maximum ion injection time of 40 ms. Dynamic exclusion was set to 30 s. Raw files were analyzed using the PD2.4 data analysis platform (Thermo Fisher Scientific) and the Sequest protein proteometry search engine. The corresponding protein sequence was selected as the database. Search parameters were: parent ion mass deviation 20 ppm, daughter ion mass deviation 0.05 Da, and No-Enzyme restriction enzyme mode. The peptide level FDR was 1%. Dynamic modifications were selected from N-terminal acetylation and methionine oxidation of proteins. Peptide lengths were chosen to range from 4 to 30 amino acids.

[0066] Example 2: Experiment on the acetylcholinesterase inhibitory activity of sea cucumber peptides

[0067] In this study, sea cucumber peptide solutions of 10 mg / mL were prepared using peptide powders of IWNAPHTW, FPKVPGQY, and YAPRLISF, respectively, and the inhibition rate of these three sea cucumber peptides on AChE was determined.

[0068] The specific steps for determining the enzyme activity inhibition rate are as follows: Add 30 μL of acetylcholine (7.5 mM), 125 μL of LDTNB (3 mM), 40 μL of HEPES (pH 8.0, 50 mM, containing 0.1% bovine serum albumin), and 50 μL of sample (10 mg / mL) to a 96-well plate, mix well, and incubate at 37°C for 15 min. After incubation, add 30 μL of acetylcholinesterase (0.055 U / mL) to begin the assay. The assay was performed using a microplate reader. The measurement wavelength was 412 nm, and the measurement time was 15 min. The formula for calculating the inhibitory activity of sea cucumber peptide against acetylcholinesterase is:

[0069] Acetylcholinesterase inhibitory activity (%) = (1-(A) 样品 -A 样品空白 ) / (A 对照 -A 对照空白 ))×100

[0070] Among them, A 样品A represents the absorbance of the sample group. 样品空白 For the sample group without enzymes, A 对照 For the group without enzymes and without samples, A 对照空白 The sample group was not added because of enzyme addition.

[0071] Table 1. Inhibition rate of acetylcholinesterase activity by sea cucumber peptides

[0072]

[0073] Note: Experimental results are expressed as mean ± SEM. In the table, a, b, and c represent significant differences (p < 0.05).

[0074] As shown in Table 1, all three sea cucumber peptides inhibited AChE activity, with FPKVPGQY and YAPRLISF exhibiting the highest inhibition rates, indicating that sea cucumber peptides have the potential to delay cognitive decline.

[0075] Example 3: Molecular docking simulates the binding of sea cucumber peptides to acetylcholinesterase.

[0076] Sea cucumber peptides that can dock with AChE are screened using LibDock molecular docking. The higher the LibdockScore, the higher the activity of ligand-receptor binding, and the easier it is for the ligand and receptor to interact.

[0077] IWNAPHTW, FPKVPGQY, YAPRLISF, FGIDVWEH (SEQ ID NO:4), LDAQKFI (SEQ ID NO:5), and IGDTRFPY (SEQ ID NO:6) were docked with acetylcholinesterase, respectively.

[0078] Table 2. Libidock Score for the docking of sea cucumber peptides with acetylcholinesterase

[0079]

[0080]

[0081] As shown in Table 2, the LibdockScore of the three peptides IWNAPHTW, FPKVPGQY, and YAPRLISF when docked with AChE was significantly higher than that of the other three peptides, indicating that IWNAPHTW, FPKVPGQY, and YAPRLISF have a stronger inhibitory effect on AChE activity.

[0082] like Figure 1As shown, the peptide IWNAPHTW is linked to GLU358, PHE346, and ARG296 of AChE via conventional hydrogen bonds, and to GLY342, GLY345, and TYR341 of AChE via carbon-hydrogen bonds.

[0083] like Figure 2 As shown, the peptide FPKVPGQY is connected to HIS284, ASN283, GLN279, VAL73, TYR72, and SER293 of AChE via conventional hydrogen bonds, and to PHE295 of AChE via carbon-hydrogen bonds.

[0084] like Figure 3 As shown, the peptide YAPRLISF is linked to SER293, TRP286, and LEU289 of AChE via conventional hydrogen bonds, and to GLY342 and GLU292 of AChE via carbon-hydrogen bonds.

[0085] Example 4: Effects of sea cucumber peptides on cognitive impairment

[0086] Sixty 8-week-old SPF-grade male C57BL / 6J mice (20±2g) were selected and fed according to SPF-grade animal feeding standards. The animal experiments were reviewed and approved by the Animal Experiment Committee of China Agricultural University. Mice were acclimatized for one week in an environment with a temperature of 20±2℃, relative humidity of 55±5%, and alternating light and dark cycles of 12 hours, with free access to water and food. The mice were randomly divided into 6 groups: a normal control group (NC group) (n=10), an Igal-induced rapid aging model group (D-gal group) (n=10), an IWNAPHTW sea cucumber peptide group (IWNAPHTW group) (n=10), an FPKVPGQY sea cucumber peptide group (FPKVPGQY group) (n=10), a YAPRLISF sea cucumber peptide group (YAPRLISF group) (n=10), and a Donepezil positive drug group (Don group) (n=10). Body weight and food intake were recorded weekly during the feeding period.

[0087] The IWNAPHTW group was administered IWNAPHTW sea cucumber peptide powder by gavage at a dose of 400 mg / kg BW; the FPKVPGQY group was administered FPKVPGQY sea cucumber peptide powder by gavage at a dose of 400 mg / kg BW; the YAPRLISF group was administered YAPRLISF sea cucumber peptide powder by gavage at a dose of 400 mg / kg BW; the Don group was administered Donepezil by gavage at a dose of 1 mg / kg BW; and the NC and D-gal groups were administered sterile saline by gavage at a dose of 0.1 mL / 10 g BW.

[0088] Four hours after gavage, mice in the D-gal, IWNAPHTW, FPKVPGQY, YAPRLISF, and Don groups were intraperitoneally injected with D-gal at a dose of 150 mg / kg body weight (BW); mice in the NC group were intraperitoneally injected with sterile saline at a dose of 0.1 mL / 10 g BW. Gavage and intraperitoneal injections were administered daily for a total of 9 weeks.

[0089] The weight, diet, and health status of all mice were checked daily. After 9 weeks of intervention, the mice underwent the Y-maze test, the new object recognition test, and the water maze test to evaluate their learning and memory abilities.

[0090] As we age, various physiological functions gradually decline, among which the decline in cognitive and other behavioral functions is one of the most obvious characteristics of aging. A decline in learning and memory abilities is a common manifestation of cognitive decline.

[0091] like Figure 4 As shown in Figure A, the percentage of Y-maze alternation in the D-gal group mice was 47.88±2.74%, significantly lower than that in the NC group. The percentages of Y-maze alternation in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups showed no significant difference compared to the NC and Don groups. Compared to the D-gal group, the percentages of Y-maze alternation in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were significantly increased, by 18.40±2.89%, 20.16±1.57%, and 20.00±2.79%, respectively.

[0092] like Figure 4 As shown in Figure B, the novel object recognition index of mice in the D-gal group was 45.71±3.43%, significantly lower than that in the NC group. The novel object recognition indices of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups showed no significant difference compared to the NC and Don groups. Compared to the D-gal group, the novel object recognition indices of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were significantly increased, by 6.73±1.43%, 15.62±0.84%, and 12.93±1.26%, respectively.

[0093] like Figure 4As shown in Figure C, the time spent in the target quadrant by mice in the D-gal group was 11.53 ± 1.62 s, significantly shorter than that in the NC group. The time spent in the target quadrant by mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups was not significantly different from that in the NC and Don groups. Compared with the D-gal group, the time spent in the target quadrant by mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups was significantly increased, by 6.32 ± 1.17 s, 10.69 ± 1.95 s, and 9.30 ± 1.66 s, respectively.

[0094] like Figure 4 As shown in Figure D, the number of times mice in the D-gal group traversed the target area was 1.87±0.58, significantly lower than that in the NC group. The number of times mice in the FPKVPGQY and YAPRLISF groups traversed the target area was not significantly different from that in the NC and Don groups. Compared with the D-gal group, the number of times mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups traversed the target area significantly increased, by 1.38±0.45, 3.375±0.49, and 2.00±0.55 times, respectively. Among these, there was no significant difference in the number of times mice in the FPKVPGQY and YAPRLISF groups traversed the target area, but the number of times mice in the FPKVPGQY group traversed the target area was significantly higher than that in the IWNAPHTW group.

[0095] like Figure 4 As shown in Figure E, starting from day 2 of the water maze experiment, the escape latency of mice in the D-gal group was significantly higher than that in the NC group. On day 5 of the water maze experiment, the escape latency of mice in the D-gal group was 34.63±9.80s, significantly higher than that in the NC group. The escape latency of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups did not differ significantly from that in the NC and Don groups. Compared with the D-gal group, the escape latency of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups was significantly reduced, decreasing by 24.06±4.45s, 24.73±3.85s, and 26.73±2.61s, respectively. This indicates that with increasing training time, mice gradually remember the location of the platforms, and sea cucumber peptides can significantly reduce the latency of aging mice in finding hidden platforms.

[0096] Figure 4 The results showed that the learning and memory abilities of aging mice were significantly reduced, and that supplementing with sea cucumber peptides could delay the decline in the learning and memory abilities of aging mice.

[0097] like Figure 5As shown in Figure A, the ACh content in the hippocampus of mice in the D-gal group was 39.26±0.68 pg / mg prot, significantly lower than that in the NC group. Compared with the D-gal group, the ACh levels in the hippocampus of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were significantly increased, by 2.30±0.42 pg / mg prot, 5.56±0.75 pg / mg prot, and 7.12±0.53 pg / mg prot, respectively. The ACh level in the hippocampus of mice in the YAPRLISF group was not significantly different from that in the NC group, nor was it significantly different from that in the Don group.

[0098] like Figure 5 As shown in Figure B, compared with the NC group, the expression level of AChE in the hippocampus of mice in the D-gal group was significantly increased. Compared with the D-gal group, the AChE levels in the hippocampus of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were significantly decreased, with relative protein expression reduced to 94%, 78%, and 57% of that in the D-gal group, respectively. The differences between the FPKVPGQY and YAPRLISF groups and the D-gal group were significant. The AChE expression level in the YAPRLISF group was significantly lower than that in the NC group.

[0099] Figure 5 The results showed that supplementing with sea cucumber peptides could inhibit the expression of AChE in the seahorse, thereby inhibiting the degradation of ACh.

[0100] Pathological staining of hippocampal tissue, such as Figure 6 As shown, the NC group mice had a greater number of hippocampal neurons, which were arranged neatly and densely. At the black arrows, the neuronal cell structures were clear, stained normally, and the nuclei were intact. In contrast, the D-gal group had a reduced number of hippocampal neurons, which were loosely arranged. At the red arrows, the neuronal cell bodies were shrunken, irregular in shape, and showed signs of deep staining. Compared to the D-gal group, the IWNAPHTW, FPKVPGQY, and YAPRLISF groups showed a significantly increased number of hippocampal neurons, which were more orderly arranged, had regular morphology, and showed less deep staining.

[0101] Figure 6 The results showed that supplementing with sea cucumber peptides could effectively reduce the severity of hippocampal neuronal lesions.

[0102] Example 5: Verification of the anti-inflammatory and antioxidant capabilities of sea cucumber peptides

[0103] The levels of inflammatory factors in the serum of mice in Example 4 were detected, and the results are shown in [the table below]. Figure 7 .

[0104] like Figure 7As shown in A, B, C, and D, the serum levels of TNF-α, IL-1β, IL-6, and LPS in the D-gal group were significantly higher than those in the NC group. Compared with the D-gal group, the serum levels of TNF-α, IL-1β, IL-6, and LPS in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were significantly decreased. Specifically, TNF-α decreased by 14.07%, 30.48%, and 27.30%, respectively; IL-1β decreased by 6.12%, 21.92%, and 17.94%, respectively; IL-6 decreased by 9.79%, 21.32%, and 20.81%, respectively; and LPS decreased by 24.27%, 51.56%, and 44.03%, respectively. The serum levels of TNF-α, IL-1β, and IL-6 in the FPKVPGQY and YAPRLISF groups were not significantly different from those in the NC group or the Don group.

[0105] like Figure 7 As shown in Figure E, the serum SOD activity in the D-gal group was 74.17±1.18 U / mL, significantly lower than that in the NC group. Compared with the D-gal group, the serum SOD activity in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups was significantly increased, by 5.875±1.47 U / mL, 14.59±1.61 U / mL, and 19.24±4.01 U / mL, respectively. The serum SOD activity in the FPKVPGQY group was not significantly different from that in the NC and Don groups, and the serum SOD activity in the YAPRLISF group was not significantly different from that in the Don group.

[0106] like Figure 7 As shown in Figure F, the serum IL-10 level in the D-gal group was 14.02±1.74 pg / mL, significantly lower than that in the NC group. Compared with the D-gal group, the serum IL-10 levels in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were significantly increased, by 6.83±0.89 pg / mL, 17.17±2.02 pg / mL, and 15.39±2.94 pg / mL, respectively. The serum IL-10 levels in the FPKVPGQY and YAPRLISF groups were not significantly different from those in the NC and Don groups.

[0107] Figure 7 The results showed that aging mice had an inflammatory response and decreased antioxidant capacity. Supplementing with sea cucumber peptides could effectively improve the inflammatory state of aging mice and enhance their anti-inflammatory and antioxidant capabilities.

[0108] Example 6: Effects of sea cucumber peptides on neuroinflammation

[0109] Sixty 8-week-old SPF-grade male C57BL / 6J mice (20±2g) were selected and fed according to SPF-grade animal feeding standards. The animal experiments were reviewed and approved by the Animal Experiment Committee of China Agricultural University. Mice were acclimatized for one week in an environment with a temperature of 20±2℃, relative humidity of 55±5%, and alternating light and dark cycles of 12 hours, with free access to water and food. The mice were randomly divided into 6 groups: a normal control group (NC group) (n=10), an Igal-induced rapid aging model group (D-gal group) (n=10), an IWNAPHTW sea cucumber peptide group (IWNAPHTW group) (n=10), an FPKVPGQY sea cucumber peptide group (FPKVPGQY group) (n=10), a YAPRLISF sea cucumber peptide group (YAPRLISF group) (n=10), and a Donepezil positive drug group (Don group) (n=10). Body weight and food intake were recorded weekly during the feeding period.

[0110] The IWNAPHTW group was administered IWNAPHTW sea cucumber peptide powder by gavage at a dose of 400 mg / kg BW; the FPKVPGQY group was administered FPKVPGQY sea cucumber peptide powder by gavage at a dose of 400 mg / kg BW; the YAPRLISF group was administered YAPRLISF sea cucumber peptide powder by gavage at a dose of 400 mg / kg BW; the Don group was administered Donepezil by gavage at a dose of 1 mg / kg BW; and the NC and D-gal groups were administered sterile saline by gavage at a dose of 0.1 mL / 10 g BW.

[0111] Four hours after gavage, mice in the D-gal, IWNAPHTW, FPKVPGQY, YAPRLISF, and Don groups were intraperitoneally injected with D-gal at a dose of 150 mg / kg body weight (BW); mice in the NC group were intraperitoneally injected with sterile saline at a dose of 0.1 mL / 10 g BW. Gavage and intraperitoneal injections were administered daily for a total of 9 weeks.

[0112] Systemic inflammation, oxidative stress, impaired neuronal generation capacity, changes in the expression of neurotrophic factors, and alterations in the synthesis and breakdown of neurotransmitters are all possible factors contributing to age-induced cognitive impairment. Several genes involved in immune and inflammatory responses increase in expression with age.

[0113] IBA-1 is a marker of microglia activation. Excessive activation of microglia can further induce neuroinflammation in the brain, exacerbate oxidative stress, and induce cellular senescence.

[0114] like Figure 8As shown in Figure A, the expression level of IBA-1 in the hippocampus of mice in the D-gal group was significantly higher than that in the NC group. Compared with the D-gal group, the expression level of IBA-1 in the hippocampus of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups was significantly reduced, with relative protein expression decreasing to 78%, 72%, and 49% of that in the D-gal group, respectively. The expression level of IBA-1 in the YAPRLISF group was significantly lower than that in the NC and Don groups.

[0115] NF-κB is a transcription factor that participates in regulating the transcription of various inflammation-related genes. When stimulated, NF-κB enters the cell nucleus and initiates the transcription of inflammation-related genes.

[0116] like Figure 8 As shown in Figure B, compared with the NC group, the expression levels of p-IKK / IKK, p-IκBα / IκBα, and p-p65 / p65 in the hippocampus of mice in the D-gal group were increased. Compared with the D-gal group, the expression levels of p-IKK / IKK, p-IκBα / IκBα, and p-p65 / p65 in the hippocampus of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were all significantly decreased. The expression level of p-IκBα / IκBα in the hippocampus of mice in the YAPRLISF group was not significantly different from that in the NC group, and the expression level of p-p65 / p65 in the hippocampus of mice in the YAPRLISF group was not significantly different from that in the Don group, but significantly lower than that in the NC group.

[0117] like Figure 8 As shown in Figure C, compared with the NC group, the expression levels of NLRP3, ASC, and caspase-1 in the hippocampus of mice in the D-gal group were significantly increased. Compared with the D-gal group, the expression levels of NLRP3, ASC, and caspase-1 in the hippocampus of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were significantly decreased. The expression level of NLRP3 in the hippocampus of mice in the YAPRLISF group was not significantly different from that in the NC group or the Don group; the expression levels of ASC and caspase-1 were significantly lower than those in the NC and Don groups.

[0118] Figure 8 The results indicate that the hippocampus of aging mice is in an inflammatory state, and that supplementing with sea cucumber peptides can inhibit microglia activation and inhibit the activation of the NF-κB / NLRP3 pathway to suppress neuroinflammation.

[0119] Example 7: Effects of sea cucumber peptides on gut microbiota structure

[0120] The gut microbiota structure of older adults differs from that of younger adults, and its richness decreases with age. Gut microbiota dysbiosis may disrupt the blood-brain barrier (BBB) ​​through the gut-brain axis via LPS and other pro-inflammatory factors, leading to cognitive decline.

[0121] Alpha diversity refers to the biodiversity within each sample. The Chao index represents richness, the Shannon index represents diversity, and the Pielou_e index represents evenness.

[0122] Akkermansia is a mucus-degrading anaerobic bacterium belonging to the family Verrucomicrobiaceae. It colonizes the intestinal mucosa and enhances barrier function by producing short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. These SCFAs repair blood-brain barrier (BBB) ​​damage, maintain the integrity of the gut microbiome and neurovascular system, and help prevent neurological diseases. A strong correlation exists between SCFA levels and cognitive function.

[0123] Lachnospiraceae is a potential probiotic and one of the butyrate-producing bacteria. It belongs to the family Lachnospiraceae in the phylum Firmicutes. It can improve the intestinal barrier function of aged rats, and its abundance is negatively correlated with the level of inflammation. It can also improve cognitive impairment caused by sleep deprivation and has neuroprotective effects.

[0124] The bacterial flora sequence and short-chain lipid content in mouse feces from Example 4 were analyzed using 16SRNA sequencing technology. The results are shown in [Figure 4]. Figure 9 and Figure 10 .

[0125] like Figure 9 As shown in Figure A, the Chao, Shannon, and Pielou_e indices of mice in the D-gal group were significantly lower than those in the NC group. Compared with the D-gal group, the Chao index of mice in the FPKVPGQY and YAPRLISF groups was significantly increased; the Shannon and Pielou_e indices of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups were significantly increased. The Chao, Shannon, and Pielou_e indices of mice in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups showed no significant differences compared to the NC group or the Don group. This indicates that sea cucumber peptides can improve the richness, diversity, and evenness of the gut microbiota in aging mice.

[0126] like Figure 9As shown in B, at the phylum level, the dominant bacteria in the mouse gut are Bacteroidetes and Firmicutes. Compared with the NC group, the abundance of Bacteroidetes and Proteobacteria was significantly increased, while the abundance of Firmicutes and Verrucomicrobia was significantly decreased in the D-gal group. Compared with the D-gal group, the abundance of Bacteroidetes in the IWNAPHTW, FPKVPGQY, and YAPRLISF groups was significantly reduced, by 19.96%, 15.40%, and 17.70%, respectively; the abundance of Proteobacteria was significantly reduced, by 2.19%, 2.67%, and 2.79%, respectively; the abundance of Firmicutes was significantly increased, by 18.88%, 15.76%, and 17.42%, respectively; and the abundance of Verrucomicrobia was significantly increased, by 3.74%, 2.54%, and 0.81%, respectively.

[0127] like Figure 9 As shown in Figure C, the dominant bacteria in the mouse gut were S24-7 family (Muribaculaceae) and Lachnospiraceae family. Compared with the NC group, the D-gal group mice showed no significant change in the abundance of Lactobacillaceae, but a significantly increased abundance of S24-7 family (Muribaculaceae), a significantly decreased abundance of Lachnospiraceae, a significantly increased abundance of Prevotellaceae, and a significantly decreased abundance of Verrucomicrobiaceae. Compared with the D-gal group, the abundance of S24-7 family (Muribaculaceae) was significantly decreased in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group, decreasing by 13.74%, 12.02%, and 14.79%, respectively; the abundance of Lachnospiraceae was significantly increased, increasing by 3.17%, 0.20%, and 3.99%, respectively; the abundance of Prevotellaceae was significantly decreased, decreasing by 2.51%, 1.19%, and 2.40%, respectively; and the abundance of Verrucomicrobiaceae was significantly increased, increasing by 3.74%, 2.54%, and 0.97%, respectively. This indicates that supplementation with sea cucumber peptides can improve intestinal flora imbalance in aging mice.

[0128] like Figure 10As shown, compared with the NC group, the contents of acetic acid, isobutyric acid, and butyric acid in the feces of mice in the D-gal group were significantly lower, while the contents of propionic acid, isovaleric acid, and valerate were not significantly different from those in the NC group. Compared with the D-gal group, the contents of acetic acid in the feces of mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly higher, increasing by 1.04 times, 1.11 times, and 1.12 times, respectively; the contents of propionic acid were significantly higher, increasing by 1.20 times, 1.35 times, and 1.23 times, respectively; and the contents of isobutyric acid were significantly higher, increasing by 1.91 times, 3.63 times, and 1.82 times, respectively. The contents of butyric acid and valerate in the feces of mice in the FPKVPGQY group were significantly higher, increasing by 2.23 times and 2.33 times, respectively. The contents of isovaleric acid in the feces of mice in the FPKVPGQY group and YAPRLISF group were significantly higher, increasing by 2.83 times and 2.20 times, respectively. The study indicates that supplementing with sea cucumber peptides can increase the content of short-chain lipids in the feces of aging mice.

[0129] The abundance of Akkermansia and Lachnospiraceae bacteria in aging mice treated with sea cucumber peptides was significantly higher than that in the model group of aging mice. Figure 9 Sea cucumber peptides can prevent the decrease in the levels of acetic acid, isobutyric acid, butyric acid, and valeric acid in the feces of aging mice, and increase the levels of propionic acid and isobutyric acid in the feces of aging mice. Figure 10 This indicates that sea cucumber peptides can prevent intestinal flora imbalance in aging mice at three levels (phylum, family, and genus), thereby promoting short-chain fatty acid metabolism, increasing the content of short-chain fatty acids in feces, and exhibiting neuroprotective effects.

[0130] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A sea cucumber peptide, characterized in that, The sea cucumber peptides mentioned above contain at least one of the following: (1) Sea cucumber peptide with the amino acid sequence IWNAPHTW; (2) Sea cucumber peptide with the amino acid sequence FPKVPGQY; (3) Sea cucumber peptide with the amino acid sequence YAPRLISF.

2. A polypeptide mixture, characterized in that, The polypeptide mixture contains the sea cucumber peptide of claim 1.

3. A composition, characterized in that, The composition comprises the sea cucumber peptide of claim 1 or the polypeptide mixture of claim 2.

4. An acetylcholinesterase inhibitor, characterized in that, The acetylcholinesterase inhibitor comprises the sea cucumber peptide of claim 1, the polypeptide mixture of claim 2, or the composition of claim 3.

5. A product for delaying cognitive decline, characterized in that, The product comprises the sea cucumber peptide of claim 1, the polypeptide mixture of claim 2, or the composition of claim 3.

6. The product according to claim 5, characterized in that, The product is a health food, feed, or medicine.

7. The product according to claim 5, characterized in that, The products also include excipients that are permitted to be added to health foods, feed, or pharmaceuticals.

8. The use of the sea cucumber peptide of claim 1, the polypeptide mixture of claim 2, or the composition of claim 3 in the preparation of a product that helps improve memory dysfunction.

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