Preparation method and application of selenium-rich polypeptide with improved cognitive function activity

Abstract

The application discloses a preparation method and application of selenium-rich polypeptides with improved cognitive function activity, and belongs to the technical field of functional food. The selenium-rich polypeptides are CSeCGAP, KSeCSeCPK and KHSeCAA. The selenium-rich polypeptides are derived from protease hydrolysis products of selenium-rich Pogostemon iseanus, are safe and nutritious, and have no toxic side effects. The CSeCGAP, KSeCSeCPK and KHSeCAA are combined with BACE-1 through hydrogen bonds to form a relatively stable structure, thereby inhibiting the generation of Aβ and playing the effect of improving the cognitive function activity. The application provides a reference for developing new food-derived bioactive peptide functional peptides with improved cognitive function activity and functional products taking the selenium-rich polypeptides as components for improving the cognitive function activity.

Description

Technical Field

[0001] This invention relates to a method for preparing selenium-enriched polypeptides with cognitive-enhancing activity and their applications, belonging to the field of functional food technology. Background Technology

[0002] In recent years, with increasing health awareness, the demand for foods and health products with specific health functions has been growing. Selenium, as an important trace element, plays vital physiological roles in the human and animal bodies, including antioxidation, anti-inflammation, and immune enhancement. It is also closely related to the health of the endocrine system, cardiovascular system, muscle function, and central nervous system. Selenium exists in nature primarily in two forms: inorganic and organic. Organic selenium has higher safety and bioavailability than inorganic selenium. Plants can convert inorganic selenium into organic selenium, which exists in the form of selenoproteins and plays a crucial role in brain cognitive function. Selenium deficiency is closely associated with an increased risk of neurodegenerative diseases such as Alzheimer's disease.

[0003] Alzheimer's disease (AD) is a common neurodegenerative disease characterized by progressive memory decline, cognitive loss, and changes in mood and behavior. With the increasing aging of the global population, AD has gradually become a major public health challenge. The pathogenesis of AD is not fully understood, but research suggests it is related to the abnormal deposition of β-amyloid protein (Aβ) in the brain.

[0004] Developing a method for preparing selenium-enriched peptides that can effectively improve cognitive function is of paramount importance. Given that adequate selenium intake has been proven to have positive effects on enhancing memory and attention, particularly in delaying cognitive decline in the elderly, it effectively reduces neuroinflammation and protects neuronal structure through powerful antioxidant and anti-inflammatory mechanisms, thereby promoting the optimization of cognitive function. Therefore, developing a safe and effective form of selenium supplement—selenium-enriched peptides—is both crucial and urgent. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a selenium-enriched polypeptide with cognitive-enhancing activity and its preparation method. Selenium is an important antioxidant element and, as a core component of various antioxidant enzymes such as glutathione peroxidase (GPx), can effectively scavenge reactive oxygen species (ROS) and free radicals in the body. The selenium-enriched polypeptide combines the antioxidant properties of polypeptides with the activity of selenium, exhibiting stronger physiological activity and significant cognitive-enhancing activity, thus showing broad application prospects in the functional food field.

[0006] Violet-leaved watercress is a selenium-rich plant belonging to the genus *Vigna* in the Brassicaceae family. It grows in Enshi, Hubei Province, China, and its selenium content can reach up to 3000 mg / kg. This plant exhibits extremely strong selenium tolerance and accumulation capacity, effectively converting inorganic selenium in the soil into organic selenium. The main form of selenium in violet-leaved watercress is selenocysteine ​​(SeCys2), which can effectively promote nerve function and regulate metabolism.

[0007] This invention provides a selenium-enriched polypeptide with cognitive function-improving activity, wherein the amino acid sequence of the selenium-enriched polypeptide is Cys-SeCys-Gly-Ala-Pro, Lys-SeCys-SeCys-Pro-Lys, or Lys-His-SeCys-Ala-Ala.

[0008] In one embodiment of the present invention, the amino acid sequence of the polypeptide is as follows:

[0009] Based on SEQ ID NO.1, the sequence after the second cysteine ​​in the sequence is replaced by selenocysteine; or based on SEQ ID NO.2, the sequence after cysteine ​​is replaced by selenocysteine; or based on SEQ ID NO.3, the sequence after cysteine ​​is replaced by selenocysteine.

[0010] SEQ ID NO.1: CCGAP;

[0011] SEQ ID NO.2: KCCPK;

[0012] SEQ ID NO.3: KHCAA.

[0013] In one embodiment of the present invention, the selenium-enriched polypeptide can be used to improve cognitive function and significantly reduce Aβ. 1-42 Induced PC12 cell damage, improved Alzheimer's disease symptoms, and prevention or treatment of neurodegenerative diseases.

[0014] In one embodiment of the present invention, the selenium-enriched polypeptide is prepared from Corydalis yanhusuo as raw material.

[0015] In one embodiment of the present invention, the method for preparing the selenium-enriched polypeptide with cognitive-enhancing activity includes the following steps:

[0016] Using selenium-enriched Corydalis leaf fragments as raw material, the protein of selenium-enriched Corydalis leaf fragments was extracted by alkaline extraction and acid precipitation; selenium-enriched peptides were obtained by enzymatic hydrolysis with protease complex; after ultrafiltration, preparative high performance liquid chromatography separation, cell experiment screening, amino acid sequencing, and molecular docking verification, selenium-enriched peptides with cognitive function improvement activity were obtained, with amino acid sequences of Cys-SeCys-Gly-Ala-Pro, Lys-SeCys-SeCys-Pro-Lys, or Lys-His-SeCys-Ala-Ala.

[0017] In one embodiment of the present invention, the method includes the steps of:

[0018] (1) Take selenium-enriched corydalis powder, add water to extract, centrifuge, and collect the supernatant and precipitate; add NaOH to the precipitate to extract, centrifuge, and collect the supernatant; combine the supernatants obtained twice, concentrate, adjust pH, stand, centrifuge, and freeze dry to obtain selenium-enriched corydalis protein.

[0019] (2) Dissolve the selenium-enriched corydalis protein obtained in step (1) in water to obtain a protein solution. Adjust the pH for the first time and add protease I for enzymatic hydrolysis. Adjust the pH for the second time and add protease II for enzymatic hydrolysis. Inactivate the enzyme to terminate the reaction. Centrifuge, collect the supernatant and freeze dry to obtain selenium-enriched corydalis polypeptide.

[0020] (3) Pass the selenium-enriched corydalis leaf fragments obtained in step (2) through a 3 kDa ultrafiltration membrane to obtain a selenium-enriched polypeptide component with a value greater than 3 kDa, freeze-dry it, and prepare it into a solution.

[0021] (4) The solution obtained in step (3) is purified by high performance liquid chromatography. Multiple components are collected according to the peak conditions and time, and their cognitive function is screened to obtain the component with the strongest cognitive function improvement activity.

[0022] (5) Identify the components obtained in step (4) to obtain peptides. Use molecular docking technology to bind them to BACE-1 respectively. Screen out peptides with lower binding energy to obtain the selenium-rich polypeptides CSeCGAP, KSeCSeCPK or KHSeCAA of the present invention that have the activity of improving cognitive function.

[0023] In one embodiment of the present invention, the total selenium content of the selenium-enriched corydalis leaf powder in step (1) is greater than 1000 mg / kg.

[0024] In one embodiment of the present invention, the ratio of selenium-enriched corydalis leaf powder to water in step (1) is 1:(25~30) (w / v).

[0025] In one embodiment of the present invention, the concentration of NaOH in step (1) is 0.75~0.10 mol / L, and the ratio of precipitate to added NaOH is 1:(25~30) (w / v).

[0026] In one embodiment of the present invention, the temperature for water extraction and NaOH extraction in step (1) is 50~60℃, and the extraction time is 3~4 h.

[0027] In one embodiment of the present invention, the pH is adjusted to 3.0~3.5 in step (1).

[0028] In one embodiment of the present invention, the centrifugation conditions in step (1) are 4000~5000 r / min for 15~20 min.

[0029] In one embodiment of the present invention, the concentration in step (1) is to concentrate to one-quarter of the original volume.

[0030] In one embodiment of the present invention, the ratio of selenium-enriched corydalis leaf broken rice protein to water in step (2) is 1.0~3.0% (w / v).

[0031] In one embodiment of the present invention, in step (2), the pH is adjusted to 2.0~3.0 for the first time and to 7.5~8.0 for the second time.

[0032] In one embodiment of the present invention, in step (2), protease I is pepsin, and the amount added is 4.0~5.0% (w / w).

[0033] In one embodiment of the present invention, in step (2), protease II is trypsin, and the amount added is 4.0~5.0% (w / w).

[0034] In one embodiment of the present invention, the enzymatic hydrolysis conditions in step (2) are 40~50 °C for 2.0~3.0 h.

[0035] In one embodiment of the present invention, the enzyme inactivation conditions in step (2) are 90~100 °C for 10~15 min.

[0036] In one embodiment of the present invention, the solution concentration in step (3) is 2.0~4.0 mg / mL.

[0037] In one embodiment of the present invention, the method of screening for improved cognitive function activity in step (4) is screening using a PC12 cell model; preferably, the method is screening by examining the effect of components on Aβ. 1-42 The protective effect against induced PC12 cell damage was investigated, and the component with the highest cell survival rate was screened.

[0038] In one embodiment of the present invention, the identification in step (5) specifically involves: dissolving the components screened in step (4) in a 0.1% trifluoroacetic acid aqueous solution, desalting them using a C18 column, ionizing them into smaller fragments using nano-liquid chromatography-tandem mass spectrometry, and inferring the amino acid sequence of the original peptide by measuring the mass of these fragments.

[0039] The present invention also provides an expression vector or recombinant microorganism, wherein the expression vector or recombinant microorganism contains at least one of the above-mentioned selenium-enriched polypeptides.

[0040] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0041] In one embodiment of the present invention, the recombinant microorganism is a bacterium or a fungus.

[0042] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprising any one or more of the above-mentioned selenium-enriched polypeptides: Cys-SeCys-Gly-Ala-Pro, Lys-SeCys-SeCys-Pro-Lys, or Lys-His-SeCys-Ala-Ala;

[0043] The above-mentioned selenium-enriched polypeptides can significantly inhibit Aβ 1-42 Induced PC12 cell damage.

[0044] In one embodiment of the present invention, the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field;

[0045] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose, etc.; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch, etc.; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides, etc.; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol, etc.; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate, etc.; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0046] The present invention also provides a food, medicine, health product or nutritional product, wherein the food, medicine, health product or nutritional product contains an effective dose of at least one of the above-mentioned selenium-enriched polypeptides.

[0047] In one embodiment of the present invention, the amino acid sequence of the selenium-enriched polypeptide is Cys-SeCys-Gly-Ala-Pro, Lys-SeCys-SeCys-Pro-Lys, or Lys-His-SeCys-Ala-Ala.

[0048] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0049] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0050] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0051] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0052] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0053] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0054] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods;

[0055] In one embodiment of the present invention, the health product also contains acceptable excipients.

[0056] The present invention also provides the use of at least one or more of the above-mentioned selenium-enriched polypeptides in the preparation of food, pharmaceuticals, health products or nutritional products.

[0057] In one embodiment of the present invention, the amino acid sequence of the selenium-enriched polypeptide is Cys-SeCys-Gly-Ala-Pro, Lys-SeCys-SeCys-Pro-Lys, or Lys-His-SeCys-Ala-Ala.

[0058] In one embodiment of the present invention, the pharmaceutical product further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0059] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0060] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form.

[0061] In one embodiment of the present invention, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0062] In one embodiment of the present invention, the injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

[0063] In one embodiment of the present invention, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0064] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods.

[0065] In one embodiment of the present invention, the health product also contains acceptable excipients.

[0066] The present invention also provides a method for preparing the above-mentioned food, medicine, health product or nutritional product, the method comprising mixing at least one of the above-mentioned selenium-enriched polypeptides with at least one acceptable excipient.

[0067] In one embodiment of the present invention, the amino acid sequence of the selenium-enriched polypeptide is Cys-SeCys-Gly-Ala-Pro, Lys-SeCys-SeCys-Pro-Lys, or Lys-His-SeCys-Ala-Ala.

[0068] Beneficial effects:

[0069] (1) This invention yields selenium-enriched polypeptides with cognitive-enhancing activity, specifically the sequences CSeCGAP, KSeCSeCPK, and KHSeCAA. These polypeptides have novel structures and are easy to prepare. They can be obtained through enzymatic hydrolysis of selenium-enriched corydalis leaves or synthesized artificially. They can significantly reduce Aβ. 1-42 The induced damage to PC12 cells indicates that it has strong cognitive function-improving activity and has broad application prospects in the fields of functional foods and medicines.

[0070] (2) The selenium-enriched polypeptide of the present invention is derived from selenium-enriched Viola yezoensis, a plant rich in selenium that was approved as a food ingredient in 2021. The present invention provides a scientific basis for the application of selenium-enriched Viola yezoensis in the food nutrition and health industry. Attached Figure Description

[0071] Figure 1 To prepare liquid chromatography and activity detection results of each component; (A) High-performance liquid chromatography (HPLC) chromatogram; (B) Preparation of liquid chromatography to separate components against Aβ 1-42 The effect of induced damage on the survival rate of PC12 cells;

[0072] Figure 2 Mass spectra of selenium-enriched peptides; (A) CSeCGAP; (B) KSeCSeCPK; (C) KHSeCAA;

[0073] Figure 3 Visualization of the docking of selenium-enriched peptides with BACE-1 molecules; (A) CSeCGAP; (B) KSeCSeCPK; (C) KHSeCAA. Detailed Implementation

[0074] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0075] In the following examples, unless otherwise specified, the solutions mentioned use water as the solvent.

[0076] In the following embodiments, the measurements were performed according to the following methods:

[0077] 1. Method for determining total selenium content

[0078] Take 0.1–0.2 g of sample into a microwave digestion tube, add 3.5 mL of concentrated nitric acid, and allow to digest overnight. Then add 5.0 mL of ultrapure water, seal the tube with both inner and outer stoppers, and proceed with microwave digestion as shown in Table 1. After the program is complete, dilute the digested solution to the appropriate volume with ultrapure water and mix thoroughly. Take approximately 15 mL of the solution, pass it through a 0.22 μm nylon membrane, and place it in a 15 mL centrifuge tube. Perform a reagent blank simultaneously.

[0079] Total selenium was determined using an iCAP TQ inductively coupled plasma mass spectrometer (instrument conditions are shown in Table 2), employing the TQ-O2 reaction mode. 80 Se isotopes were monitored by the instrument, and the sampling cone was a nickel cone. A pig liver standard (selenium content 1.54 ± 0.29 mg / kg) was used as the quality control sample for total selenium determination. A series of standard solutions were prepared, and the selenium content was calculated using the external standard method.

[0080] The formula for calculating selenium content is:

[0081]

[0082] in X Selenium content of the sample (mg / kg); C Selenium concentration (μg / L) measured after sample digestion and volume adjustment; K： Dilution factor; V : The volume (mL) of the sample after digestion and final volume adjustment; M : Amount of sample digested (g).

[0083] Table 1 Microwave Digestion Procedure

[0084]

[0085] Table 2 iCAP TQ Parameters

[0086]

[0087] 2. Methods for determining selenium speciation

[0088] The content of organic selenium forms was determined by HPLC-ICP MS.

[0089] 1) Sample pretreatment:

[0090] Take 100 mg of sample into a 20 mL sealed hydrolysis flask. Add 5% w / w alkaline protease (purchased from DUPONT, enzyme activity ≥580000 DU / g), 5% w / w trypsin (purchased from Hefei Bomei Biotechnology Co., Ltd., enzyme activity ≥250 NFU / mg), and 5% w / w proteinase K (purchased from Hefei Bomei Biotechnology Co., Ltd., enzyme activity ≥40 U / mg) sequentially to each flask. Then add 15 mL of 30 mmol / L Tris-HCl (pH 8.5). Stir magnetically at 45 ℃ and 400 rpm for 4 h. After reaction, collect the enzymatic hydrolysate, filter it through a 0.22 μm aqueous membrane, and inject it into the sample.

[0091] 2) HPLC chromatographic conditions:

[0092] The chromatographic column was a TechMate C18-ST reversed-phase column (4.6 mm × 250 mm, 5 μm); the mobile phase was a 30 mmol / L diammonium hydrogen phosphate solution containing 0.5 mmol / L tetrabutylammonium hydroxide and 3.0% (v / v) methanol, and the pH of the mobile phase was adjusted to 6.0 with 10% formic acid. Isocratic elution was performed at a flow rate of 1.0 mL / min, the column temperature was 30 ℃, and the injection volume was 10 μL.

[0093] The ICP-MS conditions are shown in Table 2. A nickel cone with a high-sensitivity pad was used. The measurement mode was TQ-O2 mode, and the isotope sites were collected as follows: 80 Se, the dwell time is 0.05 s.

[0094] The contents of five selenium forms in the sample, namely methylselenocysteine ​​(MeSeCys), selenomethionine (SeMet), selenocysteine ​​(SeCys2), tetravalent selenium (Se(IV)), and hexavalent selenium (Se(VI)), were calculated using the external standard method.

[0095] 3. Screening methods for improving cognitive function

[0096] Construct Aβ 1-42 A rat model of induced pheochromocytoma (PC12) cell damage was established to investigate the effects of different samples on cell viability. Control, model, and sample groups were set up during cell culture and treated as follows:

[0097] (1) Culture of PC12 cells:

[0098] PC12 cells were cultured in DMEM high-glucose medium containing 5% fetal bovine serum and 1% penicillin-streptomycin, and then in a cell culture incubator containing 5% CO2 at 37 °C. The medium was changed every 2-3 days, and cells were passaged at a ratio of 1:3. When the cells covered more than 80% of the bottom of the culture flask, they were digested with 0.25% trypsin and ready for use.

[0099] (2) 10 μmol / L Aβ 1-42 Solution preparation:

[0100] First, the preparation of Aβ monomer: Take 1 mg of Aβ 1-42 Dissolve the powder in 1 mL of HFIP, incubate at room temperature for 1 h, sonicate for 10 min to completely dissolve, dispense into 10 1.5 mL centrifuge tubes, and evaporate overnight in a fume hood until all HFIP is removed. Store at -20°C for later use.

[0101] Next, the Aβ oligomer solution was prepared: Take the 1.5 mL centrifuge tube from the previous step, add 10 µL DMSO to redissolve the oligomer, dilute with 433 µL PBS, incubate at room temperature for 20 min, centrifuge at 14000 g for 15 min, collect the supernatant, and incubate the supernatant at 37 ℃ for 24 hours to obtain 50 μmol / L Aβ. 1-42 The oligomer solution was then diluted with cell culture medium to a concentration of 10 μmol / L to obtain 10 μmol / L Aβ. 1-42 Solution.

[0102] (3) Cell experiments:

[0103] Cells were cultured in a constant temperature and humidity incubator at 37 ℃, 25% RH, and 5% CO2.

[0104] Control group: Cultured PC12 cells were inoculated at 5 × 10⁻⁶ cells per cell line. 3 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), the culture medium was changed, and the cells were cultured for another 12 h, followed by another 24 h. 50 µL of MTT was added to each well, and after incubation for 4 h, the MTT was carefully removed, and 150 µL of DMSO was added. The absorbance of the cells was measured at 570 nm using a microplate reader.

[0105] Model group: Cultured PC12 cells were sputtered at 5 × 10⁻⁶. 3Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), the culture medium was changed, and the cells were cultured for another 12 h. The culture medium was then removed, and 100 μL of 10 μmol / L Aβ solution was added to each well. 1-42 The solution was incubated for 24 h. 50 µL of MTT was added to each well, and after incubation for 4 h, it was carefully removed, and 150 µL of DMSO was added. The absorbance of the cells was measured at 570 nm using a microplate reader.

[0106] Sample group: Cultured PC12 cells were subjected to a 5×10⁻⁶ incubation period. 3 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), the culture medium was aspirated, and the sample was dissolved in cell culture medium to a concentration of 1 mg / mL, which was then added to each well of the 96-well plate at a concentration of 100 μL. After culturing for 12 h, the sample solution was aspirated, and 100 μL of 10 μmol / L Aβ solution was added to each well. 1-42 The solution was incubated for 24 h. 50 µL of MTT was added to each well, and after incubation for 4 h, it was carefully removed, and 150 µL of DMSO was added. The absorbance of the cells was measured at 570 nm using a microplate reader.

[0107] Cell viability is calculated using the following formula:

[0108]

[0109] In the formula, the experimental group contains cell culture medium and sample (i.e., model group or sample group); the control group contains only cell culture medium (i.e., control group); and the blank group contains only 250 μL PBS buffer.

[0110] Among them, Aβ 1-42 It is a form of β-amyloid protein (Aβ), a toxic polypeptide. Studies have shown that the pathogenesis of AD is related to the abnormal deposition of Aβ in the brain.

[0111] Example 1: Preparation of selenium-enriched polypeptides with cognitive-enhancing activity

[0112] 1. Preparation of selenium-enriched Corydalis leaf and rice bran polypeptide

[0113] Take 100 g of dry powder of selenium-enriched Cardamine violifolia, add deionized water according to the solid-liquid ratio of 1:30 (w / v), extract at 50 °C for 4 h, centrifuge at 4000 r / min for 15 min, and collect the supernatant. Subsequently, add 0.1 mol / L NaOH with a solid-liquid ratio of 1:25 (w / v) to the precipitate, extract and centrifuge again under the same conditions, and collect the supernatant. Combine the supernatants obtained twice and concentrate to one-fourth of the original volume by a rotary evaporator. Adjust the pH to 3.0 with 1 mol / L HCl, stand at 4 °C for 24 h, centrifuge at 4000 r / min for 15 min, and dry the precipitate to obtain selenium-enriched Cardamine violifolia protein.

[0114] Dissolve the obtained selenium-enriched Cardamine violifolia protein in ultrapure water at a ratio of 1% (w / v) to obtain a protein solution. Adjust the pH of the protein solution to 2.0 with 1 mol / L HCl, add pepsin (5% w / w) (purchased from Sigma-Aldrich, enzyme activity ≥250 U / mg), and enzymatically hydrolyze at 45 °C for 2 h. Subsequently, adjust the pH to 7.5 with 1 mol / L NaOH, add trypsin (5% w / w) (purchased from Sigma-Aldrich, enzyme activity 8×USP), and continue enzymatic hydrolysis at 45 °C for 2 h. Terminate the reaction by boiling water bath at 100 °C for 10 min, centrifuge at 4000 r / min at 4 °C for 10 min, collect the supernatant and freeze-dry to obtain selenium-enriched Cardamine violifolia polypeptide.

[0115] Determine the obtained selenium-enriched Cardamine violifolia polypeptide according to the above "1. Determination method of total selenium content" and "2. Determination method of selenium speciation". The total selenium content of the obtained selenium-enriched polypeptide is 2106.85 mg / kg, of which the proportion of organic selenium in the total selenium is 80.7%, and the content of selenocysteine is 1691.34 mg / kg, accounting for 97.5% of the total organic selenium content.

[0116] 2. Ultrafiltration separation and screening of selenium-enriched polypeptide

[0117] Separate the selenium-enriched Cardamine violifolia polypeptide obtained in step 1 above through ultrafiltration membranes with molecular weight cut-offs of 1 kDa and 3 kDa, and obtain three ultrafiltration components: 1# (MW < 1 kDa), 2# (1 kDa < MW < 3 kDa), and 3# (MW > 3 kDa) (where MW is the molecular weight). Freeze-dry each component and prepare a 2.0 mg / mL solution with water.

[0118] The three ultrafiltration components were measured according to the above-mentioned "3. Screening method for improving cognitive function". The cell survival rate of component 3 was 62.34%, that of component 1 was 45.16%, and that of component 2 was 51.67%. The cell survival rate of component 3 was significantly higher than that of the other two components.

[0119] The three ultrafiltration fractions were analyzed according to the methods described in "1. Determination of total selenium content" and "2. Determination of selenium speciation" above. Fraction #3 had the highest selenium content and selenocysteine ​​content, at 2194.44 mg / kg and 1332.67 mg / kg, respectively. Therefore, fraction #3 was selected for further purification.

[0120] 3. Purification and screening of selenium-enriched peptides

[0121] The fraction 3# obtained in step 2 above was further purified by preparative high-performance liquid chromatography (HPLC) using a Waters XBridge Prep C18 column (250 × 19 mm, 5 μm). The mobile phase consisted of solvent A (acetonitrile containing 0.05% trifluoroacetic acid) and solvent B (ultrapure water containing 0.05% trifluoroacetic acid), with a flow rate of 10 mL / min; the detection wavelength was 220 nm. The gradient elution conditions were: 0–30 min, 5%–20% solvent A; 30–50 min, 20%–50% solvent A; 50–53 min, 50%–50% solvent A; 53–55 min, 50%–5% solvent A; 55–60 min, 5%–5% solvent A.

[0122] Based on the peak elution, 10 components were collected according to time. Figure 1 A) Lyophilized, labeled SP1~SP10 respectively, and prepared as 1 mg / mL aqueous solution. Based on "3. Screening Methods for Cognitive Function Improvement Activities" above, the effects of each component on Aβ were studied. 1-42 Protective effect against induced PC12 cell damage.

[0123] The results showed that these ten components had an effect on Aβ. 1-42 The induced PC12 cell survival rate was affected to varying degrees, with SP3 showing the highest cell survival rate (73.01%), which was significantly different from the model group (40.12%). P <0.0001)( Figure 1 B).

[0124] The total selenium content of components SP1 to SP10 obtained above was determined according to "1. Determination of Total Selenium Content" above. Group SP3 had the highest selenium content, at 4556.753 mg / kg, indicating that selenium may be present in Aβ. 1-42It plays a key role in protecting against induced PC12 cell damage.

[0125] 4. Mass spectrometry identification of selenium-enriched peptides

[0126] The SP3 fraction obtained in step 3 above was identified by nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). The polypeptide of the SP3 fraction was dissolved in 0.1% trifluoroacetic acid aqueous solution to prepare a 0.5 mg / mL solution. 1 mL of the solution was desalted using a C18 column, filtered through a 0.22 µm microporous membrane, and then analyzed by the instrument.

[0127] The detection system consisted of an Orbitrap Exploris 480 coupled with an Easy-nLC 1200. 3 μL of sample was loaded, and the analytical column was a PepMap C18 (75 μm × 50 cm). The separation gradient was achieved by increasing mobile phase B (0.1% formic acid in acetonitrile) from 5% to 35% over 70 min. The chromatographic flow rate was 300 nL / min, the column temperature was 60 ℃, and the ion source spray voltage was 2.3 kV. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z) 150-1600; resolution set to 120,000; maximum ion introduction time 50 ms; automatic gain control (AGC) set to 5e5; (2) HCD-MS / MS (Top20): scan resolution set to 17500; minimum scan range fixed at m / z=110; maximum ion introduction time during MS / MS is 50 ms; AGC control set to 2e5; precursor ion selection window set to 1.6 Da; for ions with charge numbers of 1, 2, 3, and 4, MS / MS acquisition was performed, with a dynamic exclusion time of 40s.

[0128] Eight selenium-containing peptides with an confidence level (ALC) greater than 80% were identified by NanoLC-MS / MS: KSeCSeCPK, VQRSeMDQ, VESeCLKK, KSSeCSK, VERSeCK, CSeCGAP, STTSeMY, and KHSeCAA (SeC represents selenocysteine, and SeM represents selenomethionine). The mass spectra of CSeCGAP, KSeCSeCPK, and KHSeCAA are shown below. Figure 2 .

[0129] Example 2: Molecular docking simulates the binding of selenium-enriched peptides to BACE-1

[0130] To further screen selenium-enriched peptides with cognitive-enhancing activity, molecular docking technology was used to bind eight selenium-containing peptides identified in Example 1 to BACE-1, and active peptides were screened based on the binding site and binding energy.

[0131] BACE-1 is the rate-limiting enzyme that cleaves amyloid precursor proteins to produce Aβ peptides, which then aggregate to form amyloid plaques, a major pathological feature of Alzheimer's disease (AD).

[0132] The crystal structure of BACE-1 (PDB ID: 1TQF) with a resolution of 1.8 Å was obtained from the PDB database. Simultaneously, the 3D structure of the selenium-containing peptide was plotted using ChemBio3D software. The structures of BACE-1 and the selenium-containing peptide were imported into Autodock vina 1.1.2 software. After dehydration and hydrogenation steps, molecular docking simulations were initiated. Figure 3 ).

[0133] The results showed that three of the eight peptide sequences had low binding energies to BACE-1 and all contained selenocysteine, namely Cys-SeCys-Gly-Ala-Pro (CSeCGAP), Lys-SeCys-SeCys-Pro-Lys (KSeCSeCPK), and Lys-His-SeCys-Ala-Ala (KHSeCAA).

[0134] These three peptides (CSeCGAP, KSeCSeCPK, and KHSeCAA) bind to BACE-1 via hydrogen bonds at the following sites: Arg128, Pro129, Asp131; Lys350, Glu380; and Glu380, ASP381, respectively. The binding energies are -5.96, -4.43, and -4.23 kcal / mol, respectively, forming a relatively stable structure.

[0135] Specifically, CSeCGAP, KSeCSeCPK, and KHSeCAA peptides have a good inhibitory effect on BACE-1, thereby reducing the production of Aβ and improving cognitive function.

[0136] Example 3: Cellular experimental verification of the cognitive function-enhancing activity of selenium-enriched peptides

[0137] CSeCGAP, KSeCSeCPK, and KHSeCAA were artificially synthesized using a solid-phase synthesis method and prepared as 1 mg / mL aqueous solutions. Based on the above-mentioned "3. Screening methods for improving cognitive function," the effects of these three selenium-enriched peptides on Aβ were investigated. 1-42 Protective effect against induced PC12 cell damage.

[0138] The specific steps are as follows:

[0139] Construct Aβ 1-42A rat model of induced pheochromocytoma (PC12) cell damage was established to investigate the effects of different samples on cell viability. During cell culture, control, model, and sample groups (KSeCSeCPK, CSeCGAP, and KHSeCAA groups) were established and treated as follows:

[0140] (1) Culture of PC12 cells:

[0141] PC12 cells were cultured in DMEM high-glucose medium containing 5% fetal bovine serum and 1% penicillin-streptomycin, and then in a cell culture incubator containing 5% CO2 at 37 °C. The medium was changed every 2-3 days, and cells were passaged at a ratio of 1:3. When the cells covered more than 80% of the bottom of the culture flask, they were digested with 0.25% trypsin and ready for use.

[0142] (2) 10 μmol / L Aβ 1-42 Solution preparation:

[0143] First, the preparation of Aβ monomer: Take 1 mg of Aβ 1-42 Dissolve the powder in 1 mL of HFIP, incubate at room temperature for 1 h, sonicate for 10 min to completely dissolve, dispense into 10 1.5 mL centrifuge tubes, and evaporate overnight in a fume hood until all HFIP is removed. Store at -20°C for later use.

[0144] Next, the Aβ oligomer solution was prepared: Take the 1.5 mL centrifuge tube from the previous step, add 10 µL DMSO to redissolve the oligomer, dilute with 433 µL PBS, incubate at room temperature for 20 min, centrifuge at 14000 g for 15 min, collect the supernatant, and incubate the supernatant at 37℃ for 24 hours to obtain 50 μmol / L Aβ. 1-42 The oligomer solution was then diluted with cell culture medium to a concentration of 10 μmol / L to obtain 10 μmol / L Aβ. 1-42 Solution.

[0145] (3) Cell experiments:

[0146] Cells were cultured in a constant temperature and humidity incubator at 37 ℃, 25% RH, and 5% CO2.

[0147] Control group: Cultured PC12 cells were inoculated at 5 × 10⁻⁶ cells per cell line. 3 Cells were seeded at a density of 100 μL / mL in 96-well plates. After the cells adhered (24 h), the culture medium was changed, and the cells were cultured for another 12 h. Then, the culture medium was changed again, and the cells were cultured for another 24 h.

[0148] Model group: Cultured PC12 cells were sputtered at 5 × 10⁻⁶. 3 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), the culture medium was changed, and the cells were cultured for another 12 h. The culture medium was then removed, and 100 μL of 10 μmol / L Aβ solution was added to each well. 1-42 Incubate in solution for 24 hours.

[0149] KSeCSeCPK group: Cultured PC12 cells were inoculated at 5×10⁻⁶ 3 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), the culture medium was aspirated, and the sample was dissolved in cell culture medium to prepare a 1 mg / mL KSeCSeCPK solution, which was then added to each well of the 96-well plate at a volume of 100 μL. After culturing for 12 h, the sample solution was aspirated, and 100 μL of 10 μmol / L Aβ solution was added to each well. 1-42 Incubate in solution for 24 hours.

[0150] CSeCGAP group: Cultured PC12 cells were inoculated at 5 × 10⁶ 3 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), the culture medium was aspirated, and the sample was dissolved in cell culture medium to prepare a 1 mg / mL CSeCGAP solution, which was then added to each well of the 96-well plate at a volume of 100 μL. After culturing for 12 h, the sample solution was aspirated, and 100 μL of 10 μmol / L Aβ was added to each well. 1-42 Incubate in solution for 24 hours.

[0151] KHSeCAA group: Cultured PC12 cells were inoculated at 5×10⁻⁶ 3 Cells were seeded at a density of 100 μL / mL in 96-well plates. After cell attachment (24 h), the culture medium was aspirated, and the sample was dissolved in cell culture medium to prepare a 1 mg / mL KHSeCAA solution, which was then added to each 96-well plate at a volume of 100 μL. After culturing for 12 h, the sample solution was aspirated, and 100 μL of 10 μmol / L Aβ solution was added to each well. 1-42 Incubate in solution for 24 hours.

[0152] Add 50 µL of MTT to each well of each group, incubate for 4 h, carefully remove the incubator, and add 150 µL of LDMSO. Measure the absorbance (OD) of the cells at a wavelength of 570 nm using a microplate reader.

[0153] Cell viability is calculated using the following formula:

[0154]

[0155] Here, the experimental group contains cell culture medium and samples (i.e., model group or sample group); the control group contains only cell culture medium (i.e., control group); and the blank group contains only 250 μL PBS buffer.

[0156] The results showed that CSeCGAP, KSeCSeCPK, and KHSeCAA had an effect on Aβ. 1-42 The induced PC12 cells showed significant protective effects, with cell survival rates increasing to 76.54%, 72.26%, and 78.06% respectively compared to the model group (40.12%) (Table 3).

[0157] Table 3. Peptide effect on Aβ 1-42 Effect of induced PC12 cell survival

[0158]

[0159] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is Cys-SeCys-Gly-Ala-Pro, Lys-SeCys-SeCys-Pro-Lys, or Lys-His-SeCys-Ala-Ala.

2. The polypeptide according to claim 1, characterized in that, It is used to significantly alleviate Aβ 1-42 Induced PC12 cell damage may improve Alzheimer's disease symptoms.

3. An expression carrier, characterized in that, The expression vector encodes at least one of the polypeptides of claim 1.

4. The expression vector according to claim 3, characterized in that, The expression vector is selected from DNA vectors, RNA vectors, or viral vectors.

5. The expression vector according to claim 4, characterized in that, The DNA vector is a plasmid, transposon vector, or CRISPR / Cas9 vector.

6. A recombinant microorganism, characterized in that, The recombinant microorganism comprises at least one of the polypeptides of claim 1.

7. The microorganism according to claim 6, characterized in that, The recombined microorganisms are bacteria or fungi.

8. A food, medicine, health product, or nutritional product, characterized in that, The food, medicine, health product, or nutritional product contains at least one of the polypeptides of claim 1 in an effective dose.

9. The food, medicine, health product, or nutritional product according to claim 8, characterized in that, The medicine also contains pharmaceutically acceptable excipients.

10. The food, medicine, health product, or nutritional product according to claim 9, characterized in that, The pharmaceutical excipients include one or more of the following: binders including cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents including starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers including starch and sucrose; humectants including glycerol; disintegrants including sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers including quaternary ammonium compounds; surfactants including polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants including titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants including hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials including acrylic resins, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients including flavoring agents and sweeteners may also be added to the composition.

11. The food, medicine, health product, or nutritional product according to claim 8, characterized in that, The dosage forms of the drug include oral dosage forms, injectable dosage forms, and inhaled dosage forms; The oral dosage forms include tablets, capsules, granules, oral liquids, and oral suspensions; The injectable dosage forms include injection solutions and injection powders for injection; The inhalation formulations include aerosols and powder inhalers.

12. The food, medicine, health product, or nutritional product according to claim 8, characterized in that, The food products include grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food products also include special dietary foods. The health products also contain acceptable excipients.

13. The use of one or more of the polypeptides of claim 1 in the preparation of food, pharmaceuticals, health products or nutritional products; wherein the pharmaceuticals are used to improve symptoms of Alzheimer's disease.

14. The application according to claim 13, characterized in that, The medicine also contains pharmaceutically acceptable excipients.

15. The application according to claim 14, characterized in that, The pharmaceutical excipients include one or more of the following: binders including cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents including starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers including starch and sucrose; humectants including glycerol; disintegrants including sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers including quaternary ammonium compounds; surfactants including polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants including titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants including hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials including acrylic resins, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients including flavoring agents and sweeteners may also be added to the composition.

16. The application according to claim 13, characterized in that, The dosage forms of the drug include oral dosage forms, injectable dosage forms, and inhaled dosage forms; The oral dosage forms include tablets, capsules, granules, oral liquids, and oral suspensions; The injectable dosage forms include injection solutions and injection powders for injection; The inhalation formulations include aerosols and powder inhalers.

17. The application according to claim 13, characterized in that, The food products include grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food products also include special dietary foods. The health products also contain acceptable excipients.

18. A method for preparing the food, medicine, health product, or nutritional product according to any one of claims 8 to 12, characterized in that, The method includes mixing at least one of the polypeptides of claim 1 with at least one acceptable excipient.

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