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

Abstract

The invention discloses a sea cucumber peptide capable of delaying cognitive function decline and application of the sea cucumber peptide. The sea cucumber peptide comprises at least one of sea cucumber peptides of which the amino acid sequence is IWNAPHTW, FPKVPGQY or YAPRLISF. The three peptide fragments provided by the invention have the functions of delaying cognition, improving anti-inflammatory and anti-oxidation capacities of a body, improving neuroinflammation and regulating an intestinal flora structure. According to the invention, the medicinal function of sea cucumber micromolecular active substances (such as sea cucumber peptide) is excavated, and the economic value of sea cucumber products is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a sea cucumber peptide capable of delaying cognitive function decline and its application. Background Art

[0002] With the increase of age, various physiological functions gradually degenerate. Among them, the decline of various behavioral functions such as cognition is one of the most obvious characteristics of aging. Cognitive decline is manifested 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 suffering from neurodegenerative diseases.

[0003] Damage to the cholinergic system will affect the cognitive process, thus leading to a decline in cognitive function. Excessive acetylcholinesterase activity will accelerate the degradation of acetylcholine, resulting in a decrease in the level of acetylcholine between synapses, which will affect the normal transmission of neurotransmitters and interfere with the normal function of the nervous system. Cholinesterase inhibitors can inhibit the activity of acetylcholinesterase and increase the availability of acetylcholine at the synapses in the brain, and have been proven clinically to be used for the prevention or treatment of Alzheimer's disease.

[0004] Sea cucumbers have the characteristics of high protein and low fat, and are high-quality protein sources for preparing active peptides. Multiple studies have shown that the active substances in sea cucumbers have various physiological activities such as lowering blood pressure, reducing blood lipid, lowering blood sugar, inhibiting angiotensin-converting enzyme, anti-cancer, and anti-fatigue. However, there are few reports on the study of small molecule active substances (such as sea cucumber peptides) in sea cucumbers to improve senile cognitive dysfunction. Summary of the Invention

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

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

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

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

[0009] (3) A 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 comprises the above (1) and (2).

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

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

[0014] Preferably, the sea cucumber peptide includes the above (1), (2) and (3).

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

[0016] In a third aspect, the present invention provides a composition, which contains the sea cucumber peptide described in the first aspect or the polypeptide mixture described in the second aspect.

[0017] In a fourth aspect, the present invention provides a preparation method of the sea cucumber peptide described in the first aspect, and the preparation method includes the following steps:

[0018] (1) Grind the sea cucumber body wall into minced meat, add water, adjust the pH, and carry out enzymatic hydrolysis;

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

[0020] (3) Separate, purify and identify the freeze-dried powder in step (2) 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 enzymes added for enzymatic hydrolysis in step (1) include one or more of neutral protease, alkaline protease, papain, flavor protease or proteinase K.

[0023] Furthermore, the enzyme activity of each enzyme is 2500 to 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 h, for example: 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h.

[0025] Furthermore, the enzymatic hydrolysis temperature is 45 to 55 °C, for example: 45 °C, 47 °C, 50 °C, 53 °C, 55 °C.

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

[0027] Further, 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] Further, the time of the water bath is 10 - 20 min, for example: 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min.

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

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

[0031] Preferably, the separation and purification in step (3) adopt gel column chromatography and high - performance liquid chromatography separation and purification methods.

[0032] Preferably, the identification in step (3) adopts mass spectrometry detection method.

[0033] In a fifth aspect, the present invention provides an acetylcholinesterase inhibitor, and 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 function decline, and the product 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.

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

[0036] Preferably, the product further comprises excipients allowed to be added in health foods, feeds or medicines.

[0037] In a seventh aspect, the present invention provides the application of 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, and the application includes at least one of the following;

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

[0039] (2) Application in the preparation of a product for assisting in improving memory.

[0040] (3) Application in the preparation of a product for enhancing anti - inflammatory and antioxidant capabilities.

[0041] (4) Use in the preparation of products for improving neuroinflammation.

[0042] (5) Use in the preparation of products for regulating the intestinal flora structure.

[0043] Advantages of the present invention:

[0044] The present invention has isolated three peptide segments from sea cucumbers that inhibit acetylcholinesterase, and these three peptide segments have the functions of delaying cognitive function, enhancing the anti-inflammatory and antioxidant abilities of the body, improving neuroinflammation, and regulating the intestinal flora structure. The present invention has explored the medicinal functions of small molecule active substances (such as sea cucumber peptides) in sea cucumbers and improved the economic value of sea cucumber products. Description of the drawings

[0045] Figure 1 Shown is the molecular docking diagram of IWNAPHTW and acetylcholinesterase;

[0046] Figure 2 Shown is the molecular docking diagram of FPKVPGQY and acetylcholinesterase;

[0047] Figure 3 Shown is the molecular docking diagram of YAPRLISF and acetylcholinesterase;

[0048] Figure 4 Shown are the results of sea cucumber peptides improving the cognitive function of aging mice. A is the percentage of arm alternation in the Y-maze experiment; B is the new object recognition index period in the novel object recognition experiment, C is the time spent in the target quadrant in the water maze experiment, D is the number of times passing through the target area in the water maze experiment, E is the latency to find the hidden platform in the water maze experiment;

[0049] Figure 5 Shown are the effects of sea cucumber peptides on the acetylcholine content and acetylcholinesterase expression level in the hippocampal tissue of aging mice. A is the acetylcholine level in the hippocampal tissue; B is the expression level of acetylcholinesterase in the hippocampal tissue;

[0050] Figure 6 Shown is the pathological diagram of the hippocampus of aging mice treated with sea cucumber peptides;

[0051] Figure 7 Shown are the effects of sea cucumber peptides on the anti-inflammatory and antioxidant abilities of the body of aging mice. A is the level of TNF-α in the mouse serum, B is the level of IL-1β in the mouse serum, C is the level of IL-6 in the mouse serum, D is the level of LPS in the mouse serum, E is the activity of SOD in the mouse serum, F is the level of IL-10 in the mouse serum;

[0052] Figure 8The figure shows the effects of sea cucumber peptides on neuroinflammation in the hippocampal tissues of aging mice. A shows the expression of the microglial marker IBA-1 in the hippocampal tissues of mice, B shows the expression of key proteins in the NF-κB pathway in the hippocampal tissues of mice, and C shows the expression of NLRP3 inflammasomes.

[0053] Figure 9 The figure shows the effects of sea cucumber peptides on the intestinal flora of aging mice. A shows the α-diversity of the intestinal flora, B shows the intestinal flora structure (at the phylum level), and C shows the intestinal flora structure (at the family level).

[0054] Figure 10 The figure shows the content of short-chain fatty acids in the feces of mice. Specific implementation manners

[0055] The technical solutions of the present invention will be further described below in conjunction with embodiments and the accompanying drawings. The advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention.

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

[0057] (1) 30 - 80 g of sea cucumber body walls were ground into minced meat using a blender, 500 - 1500 mL of distilled water was added, the pH was adjusted to 3.0 - 9.0, and neutral protease, alkaline protease, papain, flavor protease, and proteinase K were added at an enzyme activity of 3000 U / g for each enzyme, and the enzymatic hydrolysis temperature was 50°C.

[0058] (2) After enzymatic hydrolysis for 2 - 8 h, it was quickly placed in a 96°C water bath to inactivate the enzyme for 15 min, and separated using an ultrafiltration membrane with a pore size of 0.01 μm, and the filtrate was collected as the sea cucumber peptide solution.

[0059] (3) The sea cucumber peptide solution was placed in a vacuum freeze dryer and freeze-dried at -60°C to obtain freeze-dried sea cucumber peptides.

[0060] (4) The freeze-dried sea cucumber peptides were separated, purified, and identified for peptide sequences by gel column chromatography, high-performance liquid chromatography, and mass spectrometry detection, and 6 sea cucumber peptides were obtained, with sequences 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) respectively.

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

[0062] Gel column chromatography: A certain amount of dry Sephadex G-25 was added to 5 times its volume of ultrapure water, swollen in a boiling water bath for 2 h, cooled, degassed and packed into a column, and equilibrated with 3 - 5 column volumes of 0.02 mol / L Tris-HCl (pH 7.4). After equilibration, 2 mL (20 mg / mL) of the enzymatically hydrolyzed and ultrafiltered product of sea cucumber was added, and eluted with ultrapure water at a flow rate of 0.6 mL / min. The eluate was collected with an automatic collector, 1 tube was collected every 5 min, and the OD value was measured at 220 nm to obtain the distribution curve of the enzymatically hydrolyzed and ultrafiltered product in each chromatographic fraction, thereby collecting polypeptide fractions with different molecular weights;

[0063] High performance liquid chromatography separation and purification: RP-HPLC technology was used to separate and analyze the active components separated by the Sephadex G-25 chromatography column. The analytical column chromatographic conditions used were: Kromasil C18 (4.6×250 mm); sample loading 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: B was 5% from 0 - 10 min; B was 5% - 48% from 11 - 30 min; B was 5% from 31 min - 45 min; stopped at 45 min.

[0064] Mass spectrometry detection and identification: The peptide sample was dissolved with 0.1% formic acid solution, and loaded and separated using an Ultimate 3000 UPLC (Thermo Fisher Scientific) liquid phase system. A self-made pre-column and chromatographic column were used (pre-column: 100 μm inner diameter, packed with 3 μm particle size C18 packing with a length of 2 cm; chromatographic column: 100 μm inner diameter packed with 1.9 μm particle size C18 packing with a length of 30 cm). Liquid phase Solution A (0.1% (V / V) formic acid aqueous solution), Solution B (0.1% V / V) formic acid (80% V / V) acetonitrile solution. The liquid phase gradient increased gradually from 8% Solution B to 50%, the elution flow rate was 300 nL / min, and the elution time was 76 min.

[0065] The Eclipse Tribrid Orbitrap (Thermo Fisher Scientific) mass spectrometer was used for acquisition by data dependent acquisition (DDA). The spray voltage was 2.2 kV, and the temperature of the ion transfer tube was 320 °C. The Orbitrap analyzer was used for the detection of the first-order spectrum. The ion scanning range was m / z 50 - 1500, the Orbitrap resolution was 60,000, and the maximum ion injection time was 40 ms. The Orbitrap analyzer was used for the detection of the second-order spectrum. The ion scanning range was m / z 50 - 1400, the resolution was 15,000, the collision energy was 32%, and the maximum ion injection time was 40 ms. The dynamic exclusion was set for 30 s. The Raw files were analyzed by the PD2.4 data analysis platform (Thermo Fisher Scientific), and the Sequest proteomic analysis search engine was used. The corresponding protein sequence was selected as the database. The database search parameters were as follows: the precursor ion mass deviation was 20 ppm, the product ion mass deviation was 0.05 Da, and the digestion mode was selected as No-Enzyme. The false discovery rate (FDR) at the peptide level was 1%. The dynamic modifications selected were N-terminal acetylation of proteins and methionine oxidation. The peptide length range was selected as 4 - 30 amino acids.

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

[0067] In this study, peptide powders of IWNAPHTW, FPKVPGQY, and YAPRLISF were respectively formulated into 10 mg / mL sea cucumber peptide solutions for experiments, and the inhibition rates of these three sea cucumber peptides against AChE were measured.

[0068] The specific steps for the determination of the enzyme activity inhibition rate were as follows: 30 μL of acetylcholine (7.5 mM), 125 μL of DTNB (3 mM), 40 μL of HEPES (pH 8.0, 50 mM, containing 0.1% bovine serum albumin), and 50 μL of the sample (10 mg / mL) were respectively added to a 96-well plate. After mixing, they were incubated at 37 °C for 15 min. After the incubation, 30 μL of acetylcholinesterase (0.055 U / mL) was added to start the determination. The determination process was carried out in a microplate reader. The determination wavelength was 412 nm, and the determination time was 15 min. The calculation formula for the inhibitory activity of sea cucumber peptides against acetylcholinesterase was:

[0069] Inhibitory activity of acetylcholinesterase (%) = (1 - (A 样品 - A 样品空白 ) / (A 对照 - A 对照空白 )) × 100

[0070] Where, A 样品Absorbance value of the sample group, A 样品空白 Sample group without enzyme, A 对照 Sample group without enzyme and without sample, A 对照空白 Sample group with enzyme and without sample.

[0071] Table 1 Inhibitory rate of acetylcholinesterase activity of sea cucumber peptides

[0072]

[0073] Note: The experimental results are expressed as mean ± SEM. a, b, c in the table represent significant differences, p < 0.05.

[0074] From the inhibitory rate results in Table 1, it can be seen that the three sea cucumber peptides all have inhibitory effects on AChE activity. Among them, FPKVPGQY and YAPRLISF have the highest inhibitory rate on AChE activity, indicating that sea cucumber peptides have the potential to delay the decline of cognitive function.

[0075] Example 3 Molecular docking simulation of the binding of sea cucumber peptides to acetylcholinesterase

[0076] Through LibDock molecular docking screening, sea cucumber peptides that can dock with AChE can be selected. The higher the LibdockScore, the higher the activity of the ligand-receptor binding, and the easier it is for the ligand and receptor to interact.

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

[0078] Table 2 LibdockScore of the docking of sea cucumber peptides with acetylcholinesterase

[0079]

[0080]

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

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

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

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

[0085] Example 4 Effect of Sea Cucumber Peptide on Cognitive Dysfunction

[0086] Sixty 8-week-old SPF-grade male C57BL / 6J mice (20±2 g) were selected and raised according to the SPF-grade animal feeding standards. The animal experiment was reviewed and approved by the Animal Experiment Committee of China Agricultural University. The mice were acclimated for 1 week in an environment with a temperature of 20±2 °C, a relative humidity of 55±5%, and a 12-hour light-dark cycle, with free access to water and food. The mice were randomly divided into 6 groups, namely the normal control group (NC group) (n = 10), the D-galactose-induced rapid aging model group (D-gal group) (n = 10), the IWNAPHTW sea cucumber peptide group (IWNAPHTW group) (n = 10), the FPKVPGQY sea cucumber peptide group (FPKVPGQY group) (n = 10), the YAPRLISF sea cucumber peptide group (YAPRLISF group) (n = 10), and the donepezil positive drug group (Don group) (n = 10). The body weight and food intake of the mice were recorded weekly during the feeding period.

[0087] The IWNAPHTW group was intragastrically administered IWNAPHTW sea cucumber peptide powder at a dose of 400 mg / kg BW, the FPKVPGQY group was intragastrically administered FPKVPGQY sea cucumber peptide powder at a dose of 400 mg / kg BW, the YAPRLISF group was intragastrically administered YAPRLISF sea cucumber peptide powder at a dose of 400 mg / kg BW, the Don group was intragastrically administered donepezil at a dose of 1 mg / kg BW, and the NC group and the D-gal group were both intragastrically administered sterile normal saline at a dose of 0.1 mL / 10 g BW.

[0088] At 4 h after intragastric administration, mice in the D-gal group, IWNAPHTW group, FPKVPGQY group, YAPRLISF group, and Don group were intraperitoneally injected with D-gal at a dose of 150 mg / kg BW; mice in the NC group were intraperitoneally injected with sterile saline at a dose of 0.1 mL / 10 g BW. Intragastric administration and intraperitoneal injection were performed daily for a total of 9 weeks.

[0089] The body weight, diet, and health status of all mice were checked every day. After 9 weeks of intervention, the Y-maze test, novel object recognition test, and Morris water maze test were performed to evaluate the learning and memory abilities of the mice.

[0090] As age increases, various physiological functions gradually degenerate. Among them, the decline of various behavioral functions such as cognition is one of the most obvious characteristics of aging, and the decline in learning and memory ability is a relatively common manifestation of the decline in cognitive function.

[0091] As Figure 4 shown in

[0092] As Figure 4 shown in

[0093] As Figure 4As shown in Figure C, the time that the mice in the D-gal group stayed in the target quadrant was 11.53 ± 1.62 s, which was significantly lower than that of the NC group. There were no significant differences in the time that the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group stayed in the target quadrant compared with the NC group and the Don group. Compared with the mice in the D-gal group, the time that the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group stayed in the target quadrant increased significantly, by 6.32 ± 1.17 s, 10.69 ± 1.95 s, and 9.30 ± 1.66 s respectively.

[0094] As Figure 4 shown in Figure D, the number of times that the mice in the D-gal group passed through the target area was 1.87 ± 0.58 times, which was significantly lower than that of the NC group. There were no significant differences in the number of times that the mice in the FPKVPGQY group and YAPRLISF group passed through the target area compared with the NC group and the Don group. Compared with the D-gal group, the number of times that the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group passed through the target area increased significantly, by 1.38 ± 0.45 times, 3.375 ± 0.49 times, and 2.00 ± 0.55 times respectively; there was no significant difference in the number of times that the mice in the FPKVPGQY group and YAPRLISF group passed through the target area, and the number of times that the mice in the FPKVPGQY group passed through the target area was significantly higher than that of the IWNAPHTW group.

[0095] As Figure 4 shown in Figure E, starting from the second day of the water maze experiment, the escape latency of the mice in the D-gal group was significantly higher than that of the NC group. On the fifth day of the water maze experiment, the escape latency of the mice in the D-gal group was 34.63 ± 9.80 s, which was significantly higher than that of the NC group. There were no significant differences in the escape latency of the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group compared with the NC group and the Don group. Compared with the mice in the D-gal group, the escape latency of the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group decreased significantly, by 24.06 ± 4.45 s, 24.73 ± 3.85 s, and 26.73 ± 2.61 s respectively. This indicates that as the training time increases, the mice can gradually remember the position of the platform, and sea cucumber peptides can significantly reduce the latency of aging mice to find the hidden platform.

[0096] Figure 4 The results show that the learning and memory ability of aging mice decreases significantly, and supplementing with sea cucumber peptides can delay the decline of the learning and memory ability of aging mice.

[0097] As Figure 5As shown in , in the hippocampal tissues of the mice in the D-gal group, the content of ACh was 39.26 ± 0.68 pg / mg prot, which was significantly lower than that in the NC group. Compared with the D-gal group, the ACh levels in the hippocampal tissues of the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group 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. There was no significant difference in the ACh level in the hippocampal tissues of the mice in the YAPRLISF group from that in the NC group, nor was there a significant difference from the Don group.

[0098] As Figure 5 shown in , compared with the NC group, the expression level of AChE in the hippocampal tissues of the mice in the D-gal group was significantly increased. Compared with the D-gal group, the AChE levels in the hippocampal tissues of the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly decreased, and the relative protein expressions were decreased to 94%, 78%, and 57% of the D-gal group respectively. There were significant differences between the FPKVPGQY group and YAPRLISF group and the D-gal group. The AChE expression level in the hippocampal tissues of the mice 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 hippocampus, and thus inhibit the degradation of ACh.

[0100] The pathological staining of the hippocampal tissues was as Figure 6 shown. In the NC group, there were more hippocampal neurons, which were arranged neatly and closely; at the black arrow, the neuronal cell structure was clear, the staining was normal, and the cell nucleus was intact. While in the D-gal group, the number of hippocampal neurons decreased, and the neurons were arranged loosely; at the red arrow, the cell bodies of the neurons shrank, the morphology was irregular, and there was deep staining. Compared with the D-gal group, the number of hippocampal neurons in the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group increased significantly, the arrangement was more orderly, the morphology was regular, and the deep staining decreased.

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

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

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

[0104] As Figure 7As shown in A, B, C, and D, the levels of TNF-α, IL-1β, IL-6, and LPS in the serum of mice in the D-gal group were significantly higher than those in the NC group. Compared with the D-gal group, the levels of TNF-α, IL-1β, IL-6, and LPS in the serum of mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly decreased. Among them, TNF-α was decreased by 14.07%, 30.48%, and 27.30% respectively; IL-1β was decreased by 6.12%, 21.92%, and 17.94% respectively; IL-6 was decreased by 9.79%, 21.32%, and 20.81% respectively; LPS was decreased by 24.27%, 51.56%, and 44.03% respectively. There were no significant differences in the levels of TNF-α, IL-1β, and IL-6 in the serum of mice in the FPKVPGQY group and YAPRLISF group compared with the NC group and the Don group.

[0105] As Figure 7 shown in E, the SOD activity in the serum of mice in the D-gal group was 74.17 ± 1.18 U / mL, which was significantly lower than that in the NC group. Compared with the D-gal group, the SOD activities in the serum of mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly increased, by 5.875 ± 1.47 U / mL, 14.59 ± 1.61 U / mL, and 19.24 ± 4.01 U / mL respectively. There were no significant differences in the SOD activity in the serum of mice in the FPKVPGQY group compared with the NC group and the Don group, and there was no significant difference in the SOD activity in the serum of mice in the YAPRLISF group compared with the Don group.

[0106] As Figure 7 shown in F, the IL-10 level in the serum of mice in the D-gal group was 14.02 ± 1.74 pg / mL, which was significantly lower than that in the NC group. Compared with the D-gal group, the IL-10 levels in the serum of mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly increased, by 6.83 ± 0.89 pg / mL, 17.17 ± 2.02 pg / mL, and 15.39 ± 2.94 pg / mL respectively. There were no significant differences in the IL-10 levels in the serum of mice in the FPKVPGQY group and YAPRLISF group compared with the NC group and the Don group.

[0107] Figure 7 The results indicate that there is an inflammatory response in the body of aging mice, and the antioxidant capacity decreases. Supplementing with sea cucumber peptides can effectively improve the inflammatory state of the body of aging mice and enhance their anti-inflammatory and antioxidant capacities.

[0108] Example 6: Effect of Sea Cucumber Peptides on Neuroinflammation

[0109] Sixty 8-week-old SPF male C57BL / 6J mice (20±2 g) were selected and raised according to the SPF animal feeding standards. The animal experiment was reviewed and approved by the Animal Experiment Committee of China Agricultural University. The mice were acclimated for 1 week in an environment with a temperature of 20±2 °C, a relative humidity of 55±5%, and a 12-h light-dark cycle, with free access to water and food. The mice were randomly divided into 6 groups: normal control group (NC group) (n = 10), D-galactose-induced rapid aging model group (D-gal group) (n = 10), IWNAPHTW sea cucumber peptide group (IWNAPHTW group) (n = 10), FPKVPGQY sea cucumber peptide group (FPKVPGQY group) (n = 10), YAPRLISF sea cucumber peptide group (YAPRLISF group) (n = 10), and donepezil positive drug group (Don group) (n = 10). The body weight and food intake of the mice were recorded weekly during the feeding period.

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

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

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

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

[0114] Such as Figure 8As shown in , the expression level of IBA-1 in the hippocampal tissue of mice in the D-gal group was significantly higher than that in the NC group. Compared with the D-gal group, the expression levels of IBA-1 in the hippocampal tissues of mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly decreased, and the relative protein expressions were decreased to 78%, 72%, and 49% of the D-gal group, respectively. The expression level of IBA-1 in the YAPRLISF group was significantly lower than that in the NC group and the Don group.

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

[0116] As Figure 8 shown in , compared with the NC group, the expression levels of p-IKK / IKK, p-IκBα / IκBα, and p-p65 / p65 in the hippocampal tissue 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 hippocampal tissues of mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly decreased. There was no significant difference in the expression of p-IκBα / IκBα in the hippocampal tissue of mice in the YAPRLISF group compared with the NC group, and there was no significant difference in the expression of p-p65 / p65 in the hippocampal tissue of mice in the YAPRLISF group compared with the Don group, but it was significantly lower than that in the NC group.

[0117] As Figure 8 shown in , compared with the NC group, the expression levels of NLRP3, ASC, and caspase-1 in the hippocampal tissue 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 hippocampal tissues of mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly decreased. There was no significant difference in the expression level of NLRP3 in the hippocampal tissue of mice in the YAPRLISF group compared with the NC group and also no significant difference compared with the Don group; the expression levels of ASC and caspase-1 were significantly lower than those in the NC group and the Don group.

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

[0119] Example 7 Effect of Sea Cucumber Peptides on the Structure of Intestinal Flora

[0120] There are differences in the gut microbiota structure between the elderly and the young, and with the increase of age, the gut microbiota richness will decrease. Gut microbiota dysregulation may disrupt the blood-brain barrier (BBB) through LPS and other pro-inflammatory factors via the gut-brain axis, leading to cognitive decline.

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

[0122] The genus Akkermansia is a mucus-degrading anaerobic bacterium belonging to the family Verrucomicrobiaceae of the phylum Verrucomicrobia. It colonizes the intestinal mucosa, has the function of enhancing the barrier function, can produce short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate, repair blood-brain barrier (BBB) damage, maintain the integrity of the gut microbiota and neurovascular system, and prevent nervous system diseases. There is a strong correlation between SCFA levels and cognitive function.

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

[0124] The bacterial flora sequences and short-chain fat content in the feces of the mice in Example 4 were detected and analyzed using 16sRNA sequencing technology, and the results are shown in Figure 9 and Figure 10 .

[0125] As Figure 9 shown in A, the Chao index, Shannon index, and Pielou_e index of the 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 the mice in the FPKVPGQY group and the YAPRLISF group was significantly increased; the Shannon index and Pielou_e index of the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group were significantly increased. The Chao index, Shannon index, and Pielou_e index of the mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group had no significant difference from those in the NC group and also had no significant difference from those in the Don group. This shows that sea cucumber peptides can increase the richness, diversity, and evenness of the gut microbiota in aging mice.

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

[0127] As Figure 9 As shown in Figure C, the dominant bacteria in the mouse intestine are Muribaculaceae and Lachnospiraceae. Compared with the NC group, there was no significant change in Lactobacillaceae in the D-gal group of mice, the abundance of Muribaculaceae was significantly increased, the abundance of Lachnospiraceae was significantly decreased, the abundance of Prevotellaceae was significantly increased, and the abundance of Verrucomicrobiaceae was significantly decreased. Compared with the D-gal group, the abundances of Muribaculaceae in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group of mice were significantly decreased by 13.74%, 12.02%, and 14.79%, respectively; the abundances of Lachnospiraceae were significantly increased by 3.17%, 0.20%, and 3.99%, respectively; the abundances of Prevotellaceae were significantly decreased by 2.51%, 1.19%, and 2.40%, respectively; the abundances of Verrucomicrobiaceae were significantly increased by 3.74%, 2.54%, and 0.97%, respectively. This indicates that supplementing with sea cucumber peptides can improve the disorder of the intestinal flora in aging mice.

[0128] As 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 decreased, and the contents of propionic acid, isovaleric acid, and valeric acid showed no significant difference compared with the NC group. Compared with the D-gal group, the acetic acid content in the feces of mice in the IWNAPHTW group, FPKVPGQY group, and YAPRLISF group was significantly increased, by 1.04-fold, 1.11-fold, and 1.12-fold respectively; the propionic acid content was significantly increased, by 1.20-fold, 1.35-fold, and 1.23-fold respectively; the isobutyric acid content was significantly increased, by 1.91-fold, 3.63-fold, and 1.82-fold respectively; the butyric acid and valeric acid contents in the feces of mice in the FPKVPGQY group were significantly increased, by 2.23-fold and 2.33-fold respectively; the isovaleric acid content in the feces of mice in the FPKVPGQY group and YAPRLISF group was significantly increased, by 2.83-fold and 2.20-fold respectively. This indicates that supplementing with sea cucumber peptides can increase the content of short-chain fatty acids in the feces of aging mice.

[0129] The abundances of Akkermansia and Lachnospiraceae in the feces of aging mice intervened with sea cucumber peptides were significantly higher than those of the aging mice in the model group ( Figure 9 ). Sea cucumber peptides can prevent the decrease in the contents of acetic acid, isobutyric acid, butyric acid, and valeric acid in the feces of aging mice, and increase the contents of propionic acid and isobutyric acid in the feces of aging mice ( Figure 10 ). This indicates that sea cucumber peptides can prevent intestinal flora dysregulation in aging mice at the phylum, family, and genus levels, thereby promoting short-chain fatty acid metabolism, increasing the content of short-chain fatty acids in the feces, and having a neuroprotective effect.

[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 solutions 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 described above include at least one of the following: (1) A sea cucumber peptide with the amino acid sequence IWNAPHTW; (2) A sea cucumber peptide with the amino acid sequence FPKVPGQY; (3) A sea cucumber peptide with the amino acid sequence YAPRLISF.

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

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

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

5. A product for delaying cognitive function decline, characterized in that, The product contains the sea cucumber peptide described in claim 1, the polypeptide mixture described in claim 2, or the composition described in 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, wherein, The product also includes excipients allowed to be added in health foods, feeds, or medicines.

8. Use of the sea cucumber peptide according to claim 1, the polypeptide mixture according to claim 2, or the composition according to claim 3, characterized in that, The application includes at least one of the following; (1) Application in the preparation of an acetylcholinesterase inhibitor; (2) Application in the preparation of a product for assisting in improving memory; (3) Application in the preparation of a product for enhancing anti-inflammatory and antioxidant capabilities; (4) Application in the preparation of a product for improving neuroinflammation; (5) Application in the preparation of a product for regulating the intestinal flora structure.

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