Acetylcholinesterase inhibitory peptide from trachinotus ovatus as well as preparation method and application of acetylcholinesterase inhibitory peptide

Abstract

The invention discloses a trachinotus ovatus sourced acetylcholin esterase inhibitory peptide as well as a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The acetylcholin esterase inhibitory peptide is any one of polypeptides with amino acid sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 4. According to the invention, trachinotus ovatus is taken as a raw material, heptapeptides DLTDYLM, QGPIGPR, hexapeptide APDPFR and pentapeptide WGDAG with acetylcholin esterase inhibitory activity, antioxidant activity and Ca < 2 + > binding activity are obtained through enzymolysis and immobilized calcium ion affinity chromatography separation, and the action mechanism of chelating with acetylcholin esterase and Keap1 is defined. The polypeptide from trachinotus ovatus can be applied to preparation of acetylcholin esterase inhibitors, antioxidants and calcium supplements, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, in particular to an acetylcholinesterase inhibitory peptide derived from scad ovata, and a preparation method and application thereof. Background Art

[0002] Acetylcholinesterase (AChE) is a key enzyme in the cholinergic system. It hydrolyzes the neurotransmitter acetylcholine (ACh), terminating neural signaling and preventing overexcitation. However, in patients with neurodegenerative diseases such as Alzheimer's disease (AD), ACh deficiency leads to impaired signaling, resulting in memory and cognitive impairment. Therefore, the use of AChE inhibitors to reduce AChE activity can increase ACh levels in the brain and alleviate cognitive impairment. AChE inhibitors have become a mainstay of Alzheimer's disease treatment, with drugs such as donepezil, gramamine, and neostigmine used clinically. However, because small molecule drugs often have significant side effects, the search for naturally occurring, highly effective, and low-toxic active substances has become a new direction in the development of AChE inhibitors. Oxidative stress and excessive calcium influx are also key contributors to AD, making the search for multi-targeted drugs crucial for neuroprotection.

[0003] Currently, substances with AChE inhibitory activity extracted from natural products include alkaloids, flavonoids, polyphenols, and peptides. Food-derived bioactive peptides, due to their low toxicity, high efficacy, ease of absorption, and ability to cross the blood-brain barrier, are ideal raw materials for the development of neuroprotective active foods and pharmaceuticals. Oval pomfret is rich in protein and amino acids, and studies have shown that its enzymatic hydrolysis products have biological activities such as lowering uric acid and blood lipids, making them a high-quality raw material for the preparation of bioactive peptides. Summary of the Invention

[0004] The purpose of the present invention is to provide an acetylcholinesterase inhibitory peptide derived from ovate pomfret, and its preparation method and application, in order to solve the problems existing in the above-mentioned prior art. The polypeptide derived from ovate pomfret provided by the present invention can be used to prepare acetylcholinesterase inhibitors, antioxidants and calcium supplements, and has good application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an acetylcholinesterase inhibitory peptide derived from scad scad. The acetylcholinesterase inhibitory peptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.4.

[0007] The present invention also provides a method for preparing the acetylcholinesterase inhibitory peptide, which is characterized by comprising the following steps:

[0008] The oval pomfret surimi is subjected to enzymatic hydrolysis, and then the enzymatic hydrolysis is carried out through enzyme inactivation, centrifugation, suction filtration, and vacuum freeze-drying to obtain the oval pomfret surimi enzymatic protein powder;

[0009] The oval pomfret enzymatic protein powder is dissolved to prepare an enzymatic hydrolysate, and the enzymatic hydrolysate passes through an immobilized calcium ion affinity chromatography column and is freeze-dried to obtain an oval pomfret enzymatic protein purification component;

[0010] The purified components of the oval pomfret protein hydrolyzed by liquid chromatography-tandem mass spectrometry are separated into peptide segments;

[0011] The peptide segment was docked with acetylcholinesterase and Keap1 through molecular simulation, and the acetylcholinesterase inhibitory peptide was screened and identified.

[0012] Optionally, the enzymatic hydrolysis conditions are: adding papain and reacting at pH 6.0 and 55° C. for 4 hours.

[0013] Optionally, the centrifugal condition is: centrifugation at 10,000 r / min for 20 min.

[0014] Optionally, the liquid chromatography-tandem mass spectrometry is a liquid chromatography-tandem mass spectrometry equipped with an online nanospray ion source;

[0015] The mobile phase A of the liquid chromatography was ultrapure water containing 0.1% formic acid, and the mobile phase B was acetonitrile containing 0.1% formic acid;

[0016] The tandem mass spectrometry is MS and HCD-MS / MS.

[0017] The present invention also provides the use of the acetylcholinesterase inhibitory peptide in any of the following:

[0018] (1) Application in the preparation of acetylcholinesterase inhibitors;

[0019] (2) Application in the preparation of antioxidants;

[0020] (3) Application in the preparation of calcium supplements.

[0021] The present invention also provides an acetylcholinesterase inhibitor, characterized in that the active ingredient is the acetylcholinesterase inhibitory peptide.

[0022] The present invention also provides an antioxidant, characterized in that the active ingredient is the acetylcholinesterase inhibitory peptide.

[0023] The present invention also provides a calcium supplement, characterized in that the active ingredient is the acetylcholinesterase inhibitory peptide.

[0024] The present invention also provides a neuroprotective oligopeptide oral solution, comprising the following components in parts by weight:

[0025] 40 parts of the acetylcholinesterase inhibitory peptide, 12 parts of acerola cherry powder, 6 parts of vitamin C, 6 parts of citric acid, 10 parts of erythritol, and purified water are added to 100 parts.

[0026] The present invention discloses the following technical effects:

[0027] The present invention uses oval pomfret as raw material, and obtains a protein that can effectively exert acetylcholinesterase inhibitory activity, antioxidant activity and Ca 2+ The active heptapeptides DLTDYLM, QGPIGPR, hexapeptide APDPFR and pentapeptide WGDAG were combined, and their mechanism of action of chelation with acetylcholinesterase and Keap1 was clarified, which provides a new approach for the preparation of multi-target acetylcholinesterase inhibitory peptides and promotes the high-value utilization of oval pomfret.

[0028] This method uses immobilized calcium affinity chromatography to extract peptide components that possess acetylcholinesterase inhibitory, antioxidant, and calcium-binding activities, simplifying the purification process. The acetylcholinesterase inhibitory and antioxidant activities of the extracted peptides are consistent with those obtained by traditional methods. Compared to chemically synthesized acetylcholinesterase inhibitors, bioactive peptides offer the advantages of greater safety, efficacy, and ease of absorption.

[0029] The polypeptides APDPFR, DLTDYLM, QGPIGPR and WGDAG obtained by the present invention can inhibit enzyme activity by chelating the active site of acetylcholinesterase, thereby reducing the degradation of acetylcholine; and release the antioxidant factor Nrf2 by binding to Keap1 (it is well known that in the Keap1-Nrf2 signaling pathway, Nrf2 is a key transcription factor for regulating antioxidant stress. By binding to Keap1, Nrf2 can be released and the antioxidant pathway can be activated), thereby exerting an antioxidant effect; at the same time, it also has a high calcium binding rate. Therefore, the polypeptide derived from the oval pomfret provided by the present invention can be used to prepare acetylcholinesterase inhibitors, antioxidants and calcium supplements, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1This is a schematic diagram of the three-dimensional binding position of the polypeptide APDPFR and acetylcholinesterase;

[0032] Figure 2 Schematic diagram of the two-dimensional interaction between peptide APDPFR and acetylcholinesterase;

[0033] Figure 3 This is a schematic diagram of the three-dimensional binding position of the peptide DLTDYLM and acetylcholinesterase;

[0034] Figure 4 Schematic diagram of the two-dimensional interaction between the peptide DLTDYLM and acetylcholinesterase;

[0035] Figure 5 Schematic diagram of the three-dimensional binding position of the peptide QGPIGPR and acetylcholinesterase;

[0036] Figure 6 Schematic diagram of the two-dimensional interaction between peptide QGPIGPR and acetylcholinesterase;

[0037] Figure 7 Schematic diagram of the three-dimensional binding position of the polypeptide WGDAG and acetylcholinesterase;

[0038] Figure 8 Schematic diagram of the two-dimensional interaction between peptide WGDAG and acetylcholinesterase;

[0039] Figure 9 Schematic diagram of the three-dimensional binding position of peptide APDPFR and Keap1;

[0040] Figure 10 Schematic diagram of the two-dimensional interaction between peptide APDPFR and Keap1;

[0041] Figure 11 Schematic diagram of the three-dimensional binding position of the peptide DLTDYLM and Keap1;

[0042] Figure 12 Schematic diagram of the two-dimensional interaction between peptide DLTDYLM and Keap1;

[0043] Figure 13 Schematic diagram of the three-dimensional binding position of peptide QGPIGPR and Keap1;

[0044] Figure 14 Schematic diagram of the two-dimensional interaction between peptide QGPIGPR and Keap1;

[0045] Figure 15 Schematic diagram of the three-dimensional binding position of peptide WGDAG and Keap1;

[0046] Figure 16Schematic diagram of the two-dimensional interaction between peptide WGDAG and Keap1;

[0047] Figure 17 This is the chromatogram of immobilized calcium ion affinity chromatography. DETAILED DESCRIPTION

[0048] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0049] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0050] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0051] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0052] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0053] Example 1 Preparation of Acetylcholinesterase Inhibitory Peptide from Pomfret

[0054] (1) The oval pomfret meat was taken and made into fish paste using a meat grinder. The mixture was dissolved in deionized water at a ratio of 1:3 (m:V), and 0.3% papain was added. The mixture was reacted at a constant temperature of pH 6.0 and 55°C for 4 h. After the reaction, the enzyme was inactivated in a boiling water bath for 15 min, and the mixture was centrifuged at 10,000 rpm for 20 min. The supernatant was collected, filtered, and vacuum freeze-dried to obtain the oval pomfret protein powder.

[0055] (2) The enzymatic hydrolyzed protein powder of the oval pomfret was dissolved in a buffer solution to prepare an enzymatic hydrolyzate, and an immobilized calcium ion affinity chromatography column was prepared with a 0.2 mol / L calcium chloride solution. The enzymatic hydrolyzate was passed through the immobilized calcium ion affinity chromatography column, with a sample load of 10 mg / time and a chelation time of 90 min. The unchelated components were washed away with a buffer solution having a pH value of 7.4, and the chelated peptides were eluted with a NaCl solution having a pH value of 6.0 and a concentration of 0.5 mol / L as an eluent at an elution rate of 4 mL / min. The eluted sample solution was dialyzed (100 Da) for 24 h and then freeze-dried to obtain the purified component of the oval pomfret enzymatic hydrolyzed protein ( Figure 17 ).

[0056] (3) The purified components of the enzymatic hydrolyzed protein of the oval pomfret were analyzed by liquid chromatography-tandem mass spectrometry equipped with an online nanospray ion source. A total of 5 μL of sample was loaded and the sample was separated with a gradient of 60 min. The column flow rate was controlled at 300 nL / min, the column temperature was 40°C, the electrospray voltage was 2 kV, and the gradient started from 4% B phase (mobile phase A: ultrapure water containing 0.1% (volume fraction) formic acid, mobile phase B: acetonitrile containing 0.1% (volume fraction) formic acid) and increased to 50% in 53 minutes and 40 seconds with a nonlinear gradient, and increased to 95% in 40 seconds, and maintained for 5 minutes and 40 seconds. Mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 100–1500; resolution: 120,000; normalized AGC target: 200%; maximum injection time: 100 ms; (2) HCD-MS / MS: resolution: 50,000; normalized AGC target: 200%; maximum injection time: 86 ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30 s. Tandem mass spectra were analyzed using PEAKS Studio version 10.6, and the final components were selected based on the Uniprot-Trachinotus ovatus database.

[0057] (4) Molecular docking screening: The obtained peptides were simulated and docked using the molecular docking software Autodock Vina, and four peptides that chelated most tightly with acetylcholinesterase were screened out. The sequences of these four peptides were APDPFR (SEQ ID NO.1), DLTDYLM (SEQ ID NO.2), QGPIGPR (SEQ ID NO.3) and WGDAG (SEQ ID NO.4), and they were visualized using Pymol and Discovery Studio software.

[0058] The structures of acetylcholinesterase (PDB ID: 4M0E) and Keap1 (PDB ID: 2FLU) were obtained from the RCSB protein library. Prior to docking, all water molecules and cocrystallized ligands in acetylcholinesterase were removed using Pymol, and hydrogen atoms were added. The secondary structures of the heptapeptides DLTDYLM and QGPIGPR, the hexapeptide APDPFR, and the pentapeptide WGDAG were mapped and energy minimized. In AutoDock Vina software, the GridBox was set, and the X, Y, and Z coordinates, as well as the grid center, were adjusted. The box size was set to encompass the entire protein molecule. The "-Docker Energy" value was used as a screening metric; a smaller value indicates a greater likelihood of binding.

[0059] Molecular docking screening of the peptide sequences identified by liquid chromatography-tandem mass spectrometry yielded four peptides with the highest activity: APDPFR, DLTDYLM, QGPIGPR, and WGDAG. Their molecular weights and docking results are shown in Table 1. Docking results showed that the minimum binding energies of the four peptides with acetylcholinesterase and Keap1 were all less than -7 kcal / mol, indicating that the ligands and receptors bind spontaneously. Furthermore, it is generally considered that a binding free energy of ≤-5.0 kcal / mol indicates that the binding of a ligand to the target protein is meaningful.

[0060] Table 1 Molecular weight and molecular docking results of acetylcholinesterase inhibitory peptides from scad ovata

[0061]

[0062] Figure 1 、 Figure 3 、 Figure 5 and Figure 7 The binding positions of peptides APDPFR, DLTDYLM, QGPIGPR and WGDAG after docking with acetylcholinesterase are shown respectively, and all of them bind to the active site of acetylcholinesterase. Figure 2 、 Figure 4 and Figure 6 and Figure 8It can be seen that the four peptides form hydrogen bonds and hydrophobic forces with the active site of acetylcholinesterase. Among them, APDPFR interacts with the amino acid residues Trp286, Gln291, Ser293 and Tyr341 of AChE through hydrogen bonds, forms Π-alkyl forces with Leu76, and forms Π-Π stacking forces with Trp286 and Tyr341. DLTDYLM forms hydrogen bond interactions with the amino acid residues Trp286, Ser293, His287, and Thr75 of AChE, forms Π-Π stacking with the Trp286 residue, and forms alkyl forces with the Tyr72 and Val365 residues. QGPIGPR forms hydrogen bonds with Pro368, Gln369, Asn233, His405, Gln413, Asn533, Pro235, Arg247, Thr238, and Trp236, undergoes electrostatic attraction with Glu313, and forms alkyl bonds with His405, Trp532, Leu536, Leu540, and Val370. WGDAG forms hydrogen bonds with Pro235, Phe297, Trp236, Arg247, and Glu313 in AChE. The residues binding to acetylcholinesterase in each peptide have some similarity.

[0063] Figure 9 、 Figure 11 、 Figure 13 and Figure 15 The binding positions of peptides APDPFR, DLTDYLM, QGPIGPR and WGDAG after docking with Keap1 are shown respectively. Figure 10 、 Figure 12 and Figure 14 and Figure 16It can be seen that APDPFR forms conventional hydrogen bonds with amino acid residues Val418, Val463, Val465, Val467, Val512, Val561 and Ile416 of Keap1, and forms alkyl and π-alkyl forces with amino acid residues Ala607, Cys368, Val369 and Val420; DLTDYLM forms conventional hydrogen bonds with amino acid residues Val369, Val561, Val608, Arg326 and Arg470 of Keap1, and forms alkyl forces with amino acid residues Leu515, Val514 and Ala466; QGPIGPR forms conventional hydrogen bonds with amino acid residues Val369, Val561, Val608, Arg326 and Arg470 of Keap1, and forms alkyl forces with amino acid residues Leu515, Val514 and Ala466; The amino acid residues Gly371, Gly372, Gly367, Val608, Val467, Val606, Val463, Ile559, and Ile416 of the Keap1 molecule form conventional hydrogen bonds, and form alkyl forces with the amino acid residues Val514, Val561, Ala607, Cys368, and Cys513; WGDAG forms conventional hydrogen bonds with the amino acid residues Val512, Val561, Val465, Val463, Val604, Ile559, and Leu365 of the Keap1 molecule, and forms π-alkyl forces with the amino acid residues Arg415 and Ala556.

[0064] Example 2 Detection of Acetylcholinesterase Inhibition Rate of Polypeptides

[0065] The acetylcholinesterase inhibition rates of the above-mentioned oval pomfret enzymatic protein powder, oval pomfret enzymatic protein purified fraction, APDPFR, DLTDYLM, QGPIGPR and WGDAG were detected as follows:

[0066] To a 96-well plate, 30 μL of ATCI (7.5 mM), 125 μL of DTNB (3 mM), and 50 μL of sample solutions of varying concentrations were added sequentially. After preincubation at 37°C for 15 minutes, 30 μL of acetylcholinesterase (0.055 U / mL) was added and the assay was performed continuously at 412 nm for 15 minutes. A blank control was replaced with PBS (pH 8.0, 50 mM). The acetylcholinesterase inhibitory activity was calculated as follows:

[0067] Acetylcholinesterase inhibitory activity (%) = [1-(A sample-A sample blank) / (A control-A control blank)] × 100;

[0068] Among them, A sample is the absorbance value of the sample group, A sample blank is the absorbance value of the sample group without enzyme, A control is the absorbance value of the enzyme-added but no sample group, and A control blank is the absorbance value of the enzyme-added but no sample group.

[0069] The acetylcholinesterase inhibitory activities of 10 mg / mL oval pomfret enzymatic protein powder and its purified fraction were 18.02±0.78% and 25.17±0.65%, respectively. The four peptides APDPFR, DLTDYLM, QGPIGPR and WGDAG obtained through identification and screening had good acetylcholinesterase inhibitory activity, and their acetylcholinesterase inhibition rates IC 50 The results of the value detection are shown in Table 2. As shown in Table 2, the acetylcholinesterase inhibitory activity of the four peptides is ranked as DLTDYLM>APDPFR>QGPIGPR>WGDAG.

[0070] Table 2 Acetylcholinesterase half inhibition rate IC of peptides 50

[0071]

[0072] Example 3 Detection of ABTS free radical scavenging rate of polypeptide

[0073] The ABTS clearance rates of the above-mentioned oval pomfret enzymatic protein powder, oval pomfret enzymatic protein purified fraction, APDPFR, DLTDYLM, QGPIGPR and WGDAG were detected as follows:

[0074] The ABTS free radical scavenging ability of each sample was determined according to the instructions of the total antioxidant capacity (T-AOC) kit. Each sample was prepared into a 1 mg / mL sample solution with deionized water. Reagent 1 and Reagent 2 were mixed at a ratio of 1:1. After reacting in the dark for 12 hours, the working solution was diluted 40 times. 10 μL of sample solution and 190 μL of working solution were added to a 96-well plate in sequence, mixed, and allowed to stand in the dark for 6 minutes at room temperature. The absorbance value A was measured at 414 nm. Deionized water was used instead of the working solution in the control tube, and deionized water was used instead of the sample solution in the blank tube. The formula for calculating the ABTS free radical scavenging ability is as follows:

[0075] ABTS free radical scavenging rate (%) = [A blank - (A sample - A control) / A blank] × 100;

[0076] Among them, A sample is the absorbance value of the sample group, A blank is the absorbance value of the sample group without enzyme, and A control is the absorbance value of the sample group with enzyme but no sample.

[0077] The ABTS free radical scavenging rate of 1 mg / mL oval pomfret enzymatic protein powder was 52.54±0.89%, and the ABTS free radical scavenging rate of the protein purification fraction was 62.78±0.47%. The inhibition rate of peptides APDPFR, DLTDYLM, QGPIGPR and WGDAG was further improved. According to the molecular weight of each peptide, IC 50 The mass concentration unit of the value is converted into the molar concentration unit, and the IC shown in Table 3 is obtained.50 As a result, the ABTS scavenging activity was ranked as WGDAG>DLTDYLM>APDPFR>QGPIGPR.

[0078] Table 3 ABTS scavenging activity IC of peptides 50

[0079]

[0080] Example 4 Detection of the Ca of Polypeptides 2+ Binding activity

[0081] Detection of the Ca content of the above-mentioned oval pomfret enzymatic protein powder, oval pomfret enzymatic protein purification fraction, APDPFR, DLTDYLM, QGPIGPR and WGDAG 2+ The inhibition rate was determined as follows:

[0082] Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the concentration of the samples and Ca 2+ To determine the binding capacity of the sample, mix the sample with the metal salt in a mass ratio of 3:1 to prepare a solution of a certain concentration. Adjust the pH to 5.0 and place the sample in a 37°C water bath, maintaining constant temperature and oscillation for 90 minutes to allow for full binding. The binding solution is then removed and dialyzed for 48 hours, with the water changed every 4 hours. The total volume of the solution expanded after dialysis is recorded. After microwave digestion, the volume is adjusted to 50 mL, and the metal ion content in the solution is determined by ICP-MS. The binding rate formula is as follows:

[0083]

[0084] Among them, R T is the metal ion binding rate (%); m is the total mass of added metal (mg); c is the mass concentration of metal ions after dialysis (μg / mL); V is the total expansion volume (mL); and n represents the dilution multiple.

[0085] As shown in Table 4, the Ca content of the purified protein fraction from the oval pomfret enzymatic hydrolysis was 2+ The binding rate was greatly improved, and the calcium binding rate of the peptides DLTDYLM, QGPIGPR and WGDAG identified from them was further improved, showing good binding to Ca 2+ ability.

[0086] Table 4 Ca of each sample 2+ Binding rate

[0087]

[0088] Example 5 Neuroprotective Oligopeptide Oral Solution

[0089] According to the above experimental results, the polypeptide identified by the present invention can be used to prepare a neuroprotective oligopeptide oral liquid, which includes, by weight, 40 parts of acetylcholinesterase inhibitory peptide (DLTDYLM), 12 parts of acerola cherry powder, 6 parts of vitamin C, 6 parts of citric acid, 10 parts of erythritol, and purified water to 100 parts.

[0090] According to the above raw material formula, the raw materials are mixed evenly to obtain the neuroprotective oligopeptide oral solution.

[0091] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An acetylcholinesterase inhibitory peptide derived from scad scad, characterized in that: The acetylcholinesterase inhibitory peptide is a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.

4.

2. The method for preparing the acetylcholinesterase inhibitory peptide according to claim 1, wherein: The following steps are involved: The oval pomfret surimi is subjected to enzymatic hydrolysis, and then the enzymatic hydrolysis is carried out through enzyme inactivation, centrifugation, suction filtration, and vacuum freeze-drying to obtain the oval pomfret surimi enzymatic protein powder; The oval pomfret enzymatic protein powder is dissolved to prepare an enzymatic hydrolysate, and the enzymatic hydrolysate passes through an immobilized calcium ion affinity chromatography column and is freeze-dried to obtain an oval pomfret enzymatic protein purification component; The purified components of the oval pomfret protein hydrolyzed by liquid chromatography-tandem mass spectrometry are separated into peptide segments; The peptide segment was docked with acetylcholinesterase and Keap1 through molecular simulation, and the acetylcholinesterase inhibitory peptide was screened and identified.

3. The preparation method according to claim 2, wherein The enzymatic hydrolysis conditions are: adding papain and reacting at pH 6.0 and 55° C. for 4 hours.

4. The preparation method according to claim 2, wherein The centrifugal condition is: 10000 r / min for 20 min.

5. The preparation method according to claim 2, wherein The liquid chromatography-tandem mass spectrometry is a liquid chromatography-tandem mass spectrometry equipped with an online nanospray ion source; The mobile phase A of the liquid chromatography was ultrapure water containing 0.1% formic acid, and the mobile phase B was acetonitrile containing 0.1% formic acid; The tandem mass spectrometry is MS and HCD-MS / MS.

6. Use of the acetylcholinesterase inhibitory peptide according to claim 1 in any of the following: (1) Application in the preparation of acetylcholinesterase inhibitors; (2) Application in the preparation of antioxidants; (3) Application in the preparation of calcium supplements.

7. An acetylcholinesterase inhibitor, characterized in that The active ingredient is the acetylcholinesterase inhibitory peptide according to claim 1.

8. An antioxidant, characterized in that The active ingredient is the acetylcholinesterase inhibitory peptide according to claim 1.

9. A calcium supplement, characterized in that The active ingredient is the acetylcholinesterase inhibitory peptide according to claim 1.

10. A neuroprotective oligopeptide oral solution, characterized in that: Calculated by weight, it includes the following components: 40 parts of the acetylcholinesterase inhibitory peptide according to claim 1, 12 parts of acerola cherry powder, 6 parts of vitamin C, 6 parts of citric acid, 10 parts of erythritol, and purified water to 100 parts.

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