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

Abstract

The invention discloses a preparation method and application of selenium-rich polypeptide with cognitive function improving activity, and belongs to the technical field of functional food. And the selenium-enriched polypeptide is CSeCGAP, KSeCSeCPK and KHSeCAA (potassium dihydrogen phosphate). The selenium-rich polypeptide is derived from a selenium-rich cardamine violifolia proteolysis product, is safe and nutritional, and has no toxic or side effect. The CSeCGAP, the KSeCSeCPK and the KHSeCAA are combined with the BACE-1 through hydrogen bonds to form a relatively stable structure, so that the generation of A beta is inhibited, and the effect of improving the cognitive function activity is achieved. The invention provides reference for developing a novel food-borne bioactive peptide functional peptide fragment with cognitive function improving activity and a functional product taking selenium-enriched polypeptide as an active ingredient for improving cognitive function.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a selenium-enriched polypeptide capable of improving cognitive function, and belongs to the technical field of functional foods. Background Art

[0002] In recent years, as people's awareness of health has increased, the demand for foods and health products with specific health functions has increased. As an important trace element, selenium plays important physiological roles in humans and animals, such as anti-oxidation, anti-inflammation, and enhancing immunity, and is closely related to the health of the endocrine system, cardiovascular system, muscle function, and central nervous system. In nature, selenium mainly exists in two forms: inorganic selenium and organic selenium. Organic selenium has higher safety and bioavailability than inorganic selenium. Inorganic selenium can be converted into organic selenium by plants. The latter exists in the form of selenoproteins and plays a key role in brain cognitive function. Selenium deficiency is closely related to 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 loss, cognitive decline, and changes in mood and behavior. With the aging of the global population, AD has gradually become one of the major challenges to public health. The pathogenesis of AD is not yet fully understood, but studies have shown that it is related to the abnormal deposition of β-amyloid protein (Aβ) in the brain.

[0004] It is extremely important to develop a method for preparing selenium-rich peptides that can effectively improve cognitive function. Given that appropriate selenium intake has been shown to have a positive effect on enhancing memory and improving attention, especially in delaying cognitive decline in the elderly, it effectively reduces neuroinflammation and protects neuronal structure by exerting powerful antioxidant and anti-inflammatory mechanisms, thereby promoting the optimization of cognitive function. Therefore, it is particularly important and urgent to develop a safe and efficient form of selenium supplement, namely selenium-rich peptides. Summary of the invention

[0005] In order to solve the defects of the prior art, the present invention provides a selenium-rich polypeptide with the activity of improving cognitive function and a preparation method thereof. Selenium is an important antioxidant element. As a core component of various antioxidant enzymes such as glutathione peroxidase (GPx), it can effectively remove reactive oxygen species (ROS) and free radicals in the body. Selenium-rich polypeptides combine the antioxidant properties of polypeptides and the activity of selenium, showing stronger physiological activity, having strong activity of improving cognitive function, and having broad application prospects in the field of functional foods.

[0006] Cardamine violeta is a selenium-rich plant of the genus Cardamine in the cruciferous family. It grows in Enshi, Hubei, China, and its selenium content can be as high as 3000 mg / kg. The plant shows strong selenium tolerance and accumulation ability, and can effectively convert inorganic selenium in the soil into organic selenium. The main form of selenium in Cardamine violeta is selenocystine (SeCys 2 ), it can effectively promote nerve function and regulate metabolism.

[0007] The invention provides a selenium-rich polypeptide having the activity of improving cognitive function. The amino acid sequence of the selenium-rich 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:

[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-rich polypeptide can be used as food, medicine or health care product; it is used to improve cognitive function, significantly reduce Aβ 1-42 Induced PC12 cell damage, improving Alzheimer's disease symptoms, and preventing or treating neurodegenerative diseases.

[0014] In one embodiment of the present invention, the selenium-enriched polypeptide is prepared using Cardamine violaceum as raw material.

[0015] In one embodiment of the present invention, the method for preparing the selenium-enriched polypeptide having the activity of improving cognitive function comprises the steps of:

[0016] Using selenium-rich Cardamine violet leaves as raw material, the alkali extraction and acid precipitation method was used to extract the selenium-rich Cardamine violet leaves protein; the selenium-rich polypeptide was obtained by protease complex enzymatic hydrolysis; after ultrafiltration, preparative high performance liquid chromatography separation, cell experiment screening, amino acid sequencing and molecular docking verification, the selenium-rich polypeptide with the activity of improving cognitive function was obtained, and the amino acid sequence was 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 comprises the steps of:

[0018] (1) Selenium-rich Cardamine powder is extracted with water, centrifuged, and the supernatant and precipitate are collected; NaOH is added to the precipitate for extraction, centrifuged, and the supernatant is collected; the supernatants obtained twice are combined, concentrated, pH adjusted, allowed to stand, centrifuged, and freeze-dried to obtain selenium-rich Cardamine protein;

[0019] (2) dissolving the selenium-rich Cardamine protein obtained in step (1) in water to obtain a protein solution, adjusting the pH for the first time, and adding protease I for enzymolysis; adjusting the pH for the second time, adding protease II for enzymolysis, and inactivating the enzyme to terminate the reaction; centrifuging, collecting the supernatant and freeze-drying to obtain the selenium-rich Cardamine polypeptide;

[0020] (3) passing the selenium-rich Cardamine polypeptide obtained in step (2) through a 3 kDa ultrafiltration membrane to obtain a selenium-rich polypeptide component greater than 3 kDa, and lyophilizing the component to prepare a solution;

[0021] (4) purifying the solution obtained in step (3) by high performance liquid chromatography, collecting multiple components according to the peak conditions and time, and screening them for their cognitive function-improving activity to obtain the component with the strongest cognitive function-improving activity;

[0022] (5) Identifying the components obtained in step (4) to obtain peptides, combining them with BACE-1 by molecular docking technology, and screening out peptides with lower binding energy, thereby obtaining the selenium-rich polypeptides CSeCGAP, KSeCSeCPK or KHSeCAA with the activity of improving cognitive function of the present invention.

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

[0024] In one embodiment of the present invention, in step (1), the ratio of selenium-rich Cardamine violacea powder to water 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° C., and the extraction time is 3-4 hours.

[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 condition in step (1) is 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 Cardamine 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, the protease I in step (2) is pepsin, and the added amount is 4.0-5.0% (w / w).

[0033] In one embodiment of the present invention, the protease II in step (2) is trypsin, and the added amount 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 condition in step (2) is 90-100° C. for 10-15 min.

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

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

[0038] In one embodiment of the present invention, the identification in step (5) is specifically as follows: the components screened in step (4) are dissolved in a 0.1% trifluoroacetic acid aqueous solution, desalted by a C18 column, ionized into smaller fragments by nano-liquid chromatography-tandem mass spectrometry, and the amino acid sequence of the original peptide is inferred by measuring the mass of these fragments.

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

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

[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, which contains a therapeutically effective amount of active ingredients and pharmaceutically acceptable pharmaceutical excipients; the active ingredients include any one or more of the above-mentioned selenium-rich polypeptides: Cys-SeCys-Gly-Ala-Pro, Lys-SeCys-SeCys-Pro-Lys or Lys-His-SeCys-Ala-Ala;

[0043] The above selenium-rich peptides 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 auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose, etc.; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch, etc.; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters, etc.; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol, etc.; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate, etc.; in addition, other auxiliary agents such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0046] The present invention also provides a food, medicine, health product or nutritional product, which 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-rich 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 drug 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 auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can 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 injection dosage form includes but is not limited to injection solution and injection powder.

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

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

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

[0056] The present invention also provides the use of at least one or more of the above-mentioned selenium-rich polypeptides in the preparation of food, medicine, health care products or nutritional products; the selenium-rich polypeptides are used to improve cognitive function, significantly reduce Aβ 1-42Induced PC12 cell damage, improving Alzheimer's disease symptoms, and preventing or treating neurodegenerative diseases.

[0057] In one embodiment of the present invention, the amino acid sequence of the selenium-rich 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 drug 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 auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can 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, oral suspensions;

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

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

[0064] In one embodiment of the present invention, the food includes but is not limited to cereal 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 care product further contains acceptable excipients.

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

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

[0068] Beneficial effects:

[0069] (1) The present invention obtains selenium-rich polypeptides with the activity of improving cognitive function, the specific sequences of which are CSeCGAP, KSeCSeCPK and KHSeCAA, which have novel structures and are easy to prepare. They can be obtained by enzymatic hydrolysis of selenium-rich Cardamine serrata, or they can be artificially synthesized, and can significantly reduce Aβ 1-42 The induced PC12 cell damage showed that it had strong activity in improving cognitive function and had broad application prospects in the fields of functional food and medicine.

[0070] (2) The selenium-rich polypeptide of the present invention is derived from selenium-rich Cardamine violaceae, which is a high-selenium-rich plant and was approved as a food raw material in 2021. The present invention provides a scientific basis for the application of selenium-rich Cardamine violaceae in the food nutrition and health industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Figure 2 shows the results of preparative liquid chromatography and activity detection of each component; (A) High performance preparative liquid chromatography; (B) Preparative liquid separation components for Aβ 1-42 Effects on the survival rate of induced damaged PC12 cells;

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

[0073] Figure 3 Visualization diagram of the docking of selenium-rich peptides with BACE-1 molecules; (A) CSeCGAP; (B) KSeCSeCPK; (C) KHSeCAA. DETAILED DESCRIPTION

[0074] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that the examples are intended to better explain the present invention and are not intended to limit the present invention.

[0075] In the following examples, unless otherwise specified, water is used as the solvent.

[0076] In the following examples, the determination was performed according to the following method:

[0077] 1. Determination method of total selenium content

[0078] Take 0.1-0.2g sample in a microwave digestion tube, add 3.5mL concentrated nitric acid, cold digest overnight, add 5.0mL ultrapure water, cover the inner and outer stoppers, and perform microwave digestion. The digestion procedure is shown in Table 1. After the program is completed, the digested solution is diluted to the corresponding volume with ultrapure water and shaken thoroughly. Take about 15mL, pass it through a 0.22μm nylon membrane, and place it in a 15mL centrifuge tube. At the same time, make a reagent blank.

[0079] Total selenium was determined using an inductively coupled plasma mass spectrometer iCAP TQ (instrument conditions see Table 2). 2 Reaction mode, 80 Se isotopes were monitored by the instrument, and the sampling cone was a nickel cone. Using pig liver standard material (selenium content of 1.54±0.29 mg / kg) as the quality control sample for total selenium measurement, a series of standard solutions were prepared, and the selenium content was calculated by external standard method quantitative analysis.

[0080] The calculation formula for selenium content is:

[0081]

[0082] Where X: selenium content of the sample (mg / kg); C: selenium concentration measured after sample digestion and volume adjustment (μg / L); K: dilution factor; V: volume of sample after digestion and volume adjustment (mL); M: amount of digested sample (g).

[0083] Table 1 Microwave digestion procedure

[0084]

[0085] Table 2 iCAP TQ parameters

[0086]

[0087] 2. Determination of Selenium Forms

[0088] HPLC-ICP MS was used to determine the content of organic selenium forms.

[0089] 1) Sample pretreatment:

[0090] Take 100 mg of sample in a 20 mL sealed hydrolysis bottle, add 5% w / w alkaline protease (purchased from DUPONT, enzyme activity ≥580000DU / g), 5% w / w trypsin (purchased from Hefei Bomei Biotechnology Co., Ltd., enzyme activity ≥250N.FU / mg) and 5% w / w proteinase K (purchased from Hefei Bomei Biotechnology Co., Ltd., enzyme activity ≥40U / mg) three kinds of proteases to each hydrolysis bottle in turn, and then add 15 mL 30mmol / L Tris-HCl (pH 8.5) in turn. Stir magnetically at 45°C and 400rpm, react for 4 hours, take the enzymatic solution of the sample, pass it through a 0.22μm water system membrane, and inject it.

[0091] 2) HPLC chromatographic conditions:

[0092] The chromatographic column was a TechMate C18-ST reverse 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. The flow rate was 1.0 mL / min for isocratic elution, the column temperature was 30°C, and the injection volume was 10 μL.

[0093] The ICP MS conditions are shown in Table 2. A nickel cone with a high sensitivity gasket was used. The measurement mode was TQ-O 2 mode, collecting isotope positions 80 Se, dwell time is 0.05s.

[0094] The external standard method was used to calculate the content of methylselenocysteine ​​(MeSeCys), selenomethionine (SeMet), and selenocysteine ​​(SeCys 2 ), tetravalent selenium (Se(IV)), and hexavalent selenium (Se(VI)).

[0095] 3. Screening method for improving cognitive function activity

[0096] Construction of Aβ 1-42 The rat adrenal pheochromocytoma (PC12) cell injury model was induced to study the effects of different samples on cell survival. During the cell culture process, a control group, a model group, and a sample group were set up and treated according to the following methods:

[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 in an atmosphere of 5% CO. 2Culture the cells in a 37°C cell culture incubator. Replace the culture medium every 2-3 days and perform cell subculture at a ratio of 1:3. When the cells cover more than 80% of the bottom of the culture flask, digest them with 0.25% trypsin and set aside.

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

[0100] First, prepare Aβ monomers: take 1 mg Aβ 1-42 The powder was dissolved in 1 mL HFIP, incubated at room temperature for 1 h, and then sonicated for 10 min to completely dissolve it. The powder was divided into 10 1.5 mL centrifuge tubes, evaporated overnight in a fume hood until HFIP was completely removed, and placed in a -20 °C refrigerator for later use.

[0101] Next, the Aβ oligomer solution was prepared: 10 μL DMSO was added to the 1.5 mL centrifuge tube from the previous step to dissolve it again, and then it was diluted with 433 μL PBS. The tube was incubated at room temperature for 20 min, and the supernatant was aspirated after centrifugation at 14000 g for 15 min. The supernatant was incubated at 37 °C for 24 h to obtain 50 μmol / L Aβ. 1-42 The oligomer solution was finally diluted to a concentration of 10 μmol / L with cell culture medium to obtain 10 μmol / L Aβ 1-42 Solution.

[0102] (3) Cell experiments:

[0103] The cells were cultured in a constant temperature and humidity incubator at 37°C, 25% relative humidity, and 5% carbon dioxide concentration:

[0104] Control group: 5×10 3 Cells were inoculated at a density of 100 μL / mL in a 96-well plate, with a volume of 100 μL per well. After the cells adhered to the wall (24 h), the medium was replaced, and the culture was continued for 12 h, and then the medium was replaced again for another 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 at a wavelength of 570 nm was measured by an ELISA reader.

[0105] Model group: The cultured PC12 cells were cultured at 5×10 3 The cells were inoculated at a density of 100 cells / mL in a 96-well plate, with a volume of 100 μL per well. After the cells adhered to the wall (24 h), the culture medium was replaced, and after culturing for 12 h, the culture medium was removed, and 100 μL of 10 μmol / L Aβ was added to each well. 1-42Solution, culture for 24 hours. Add 50 μL MTT to each well, incubate for 4 hours and carefully remove it, add 150 μL DMSO, and measure the absorbance of the cells at a wavelength of 570 nm using an enzyme reader.

[0106] Sample group: The cultured PC12 cells were cultured at 5×10 3 The cells were inoculated at a density of 100 cells / mL in a 96-well plate, with a volume of 100 μL per well. After the cells adhered (24 h), the culture medium was removed, and the sample was dissolved in the cell culture medium to prepare 1 mg / mL, and added to the 96-well plate, with a volume of 100 μL per well. After 12 h of culture, the sample solution was removed, and 100 μL of 10 μmol / L Aβ was added to each well. 1-42 Solution, incubate for 24 hours. Add 50 μL MTT to each well, incubate for 4 hours and carefully remove it, add 150 μL DMSO, and measure the absorbance of the cells at a wavelength of 570 nm using an enzyme reader.

[0107] The cell viability was calculated according to the following formula:

[0108]

[0109] In the formula, the experimental group contains cell culture medium and samples (ie, model group or sample group); the control group contains only cell culture medium (ie, 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), which is 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 Improving Cognitive Function

[0112] 1. Preparation of selenium-enriched Cardamine polypeptide

[0113] Take 100g of dried selenium-rich Cardamine powder, add deionized water at a solid-liquid ratio of 1:30 (w / v), extract at 50°C for 4h, centrifuge at 4000r / min for 15min, and collect the supernatant. Then add 0.1mol / L NaOH at 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 rotary evaporator. Adjust the pH to 3.0 with 1mol / L HCl, let stand at 4°C for 24h, centrifuge at 4000r / min for 15min, and obtain selenium-rich Cardamine protein after the precipitate is dried.

[0114] The obtained selenium-rich Cardamine violifolia protein was dissolved in ultrapure water at a ratio of 1% (w / v) to obtain a protein solution. The protein solution was adjusted to pH 2.0 with 1 mol / L HCl, and after adding pepsin (5% w / w) (purchased from Sigma-Aldrich, enzyme activity ≥ 250 U / mg), it was enzymatically hydrolyzed at 45 °C for 2 h. Subsequently, the pH was adjusted to 7.5 with 1 mol / L NaOH, and trypsin (5% w / w) (purchased from Sigma-Aldrich, enzyme activity 8×USP) was added, and enzymatic hydrolysis was continued at 45 °C for 2 h. The reaction was terminated by heat inactivation in a boiling water bath at 100 °C for 10 min, and centrifuged at 4000 r / min for 10 min at 4 °C. The supernatant was collected and freeze-dried to obtain selenium-rich Cardamine violifolia polypeptide.

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

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

[0117] The selenium-rich Cardamine violifolia polypeptide obtained in the above step 1 was separated through ultrafiltration membranes with a molecular weight cut-off of 1 kDa and 3 kDa respectively, to obtain three ultrafiltration fractions: 1# (MW < 1 kDa), 2# (1 kDa < MW < 3 kDa), and 3# (MW > 3 kDa) (where MW is the molecular weight). Each fraction was freeze-dried and formulated into a 2.0 mg / mL solution with water.

[0118] The three obtained ultrafiltration fractions were determined according to the above-mentioned "3. Screening method for improving cognitive function activity". After detection, the cell survival rate of fraction 3# was 62.34%, the cell survival rate of fraction 1# was 45.16%, and the cell survival rate of fraction 2# was 51.67%. The cell survival rate of fraction 3# was significantly higher than the other two fractions.

[0119] The three obtained ultrafiltration fractions were determined according to the above-mentioned "1. Determination method of total selenium content" and "2. Determination method of selenium species". The selenium content and selenocysteine content of fraction 3# were the highest, being 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-rich polypeptide

[0121] The 3# component obtained in step 2 above was further purified by preparative high performance liquid chromatography, and the chromatographic column used was a Waters XBridge Prep C18 column (250×19 mm, 5 μm). The mobile phase was 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] According to the peak conditions, 10 components were collected according to time ( Figure 1 A), freeze-dried, labeled as SP1 to SP10, and prepared into 1 mg / mL aqueous solution. According to the above "3. Screening method for improving cognitive function activity", the effects of each component on Aβ 1-42 induced PC12 cell injury.

[0123] The results showed that these ten components have an impact on Aβ 1-42 The survival rates of PC12 cells induced by the SP3 group were affected to varying degrees, among which SP3 showed the highest cell survival rate (73.01%), which was significantly different from that of the model group (40.12%) (P<0.0001) ( Figure 1 B).

[0124] The total selenium content of each component was determined according to the above-mentioned "1. Determination of total selenium content". The SP3 group contained the highest selenium content of 4556.753 mg / kg, indicating that selenium may be present in Aβ 1-42 It plays a key role in the protection of PC12 cells from induced injury.

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

[0126] The SP3 component obtained in step 3 above was identified by nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). The polypeptide of the SP3 component was dissolved in 0.1% trifluoroacetic acid aqueous solution to prepare a 0.5 mg / mL solution, 1 mL was taken for desalting using a C18 column, and after passing through a 0.22 μm microporous filter membrane, it was tested on the machine.

[0127] The detection system was Orbitrap Exploris 480 coupled with Easy-nLC 1200. 3 μL of sample was loaded, and the analytical column was PepMap C18 (75 μm×50 cm). The separation gradient was 70 min with the mobile phase B (0.1% formic acid in acetonitrile) increasing from 5% to 35%. The chromatographic flow rate was 300 nL / min, the column temperature was 60°C, 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; the lowest scan range was fixed at m / z = 110; the maximum ion introduction time for MS / MS was 50 ms; AGC control was set to 2e5; the parent ion selection window was set to 1.6 Da; MS / MS acquisition was performed for ions with charge numbers of 1, 2, 3, and 4, with a dynamic exclusion time of 40 s.

[0128] A total of 8 seleno-peptides with a 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 in Figure 2. Figure 2 .

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

[0130] In order to further screen selenium-rich peptides with the activity of improving cognitive function, the eight selenium-containing peptides identified in Example 1 were respectively bound to BACE-1 using molecular docking technology, and active peptides were screened according to the strength of the binding site and binding energy.

[0131] BACE-1 is the rate-limiting enzyme that cleaves amyloid precursor protein to produce Aβ peptides, which subsequently aggregate to form amyloid plaques, the main pathological feature of AD.

[0132] The crystal structure of BACE-1 was obtained from the PDB database (PDB ID: 1TQF) with a resolution of At the same time, ChemBio3D software was used to draw the three-dimensional structure of the seleno-peptide. The structures of BACE-1 and the seleno-peptide were imported into Autodock vina1.1.2 software, and after dehydration and hydrogenation steps, molecular docking simulation was started ( Figure 3 ).

[0133] The results showed that three of the eight peptide sequences had low binding energy with 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, KHSeCAA) bind to BACE-1 through hydrogen bonds, and the binding sites are: Arg128, Pro129, Asp131; Lys350, Glu380; Glu380, ASP381; the binding energies are -5.96, -4.43 and -4.23 kcal / mol, respectively, forming a relatively stable structure.

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

[0136] Example 3: Cell experiment verification of selenium-rich polypeptide improving cognitive function activity

[0137] CSeCGAP, KSeCSeCPK and KHSeCAA were artificially synthesized by solid phase synthesis and prepared into 1 mg / mL aqueous solution. According to the above “3. Screening method for improving cognitive function activity”, the effects of three selenium-rich peptides on Aβ 1-42 induced PC12 cell injury.

[0138] The specific steps are as follows:

[0139] Construction of Aβ 1-42 The rat adrenal pheochromocytoma (PC12) cell injury model was induced to study the effects of different samples on cell survival. During the cell culture process, a control group, a model group, and a sample group (i.e., KSeCSeCPK group, CSeCGAP group, and KHSeCAA group) were set up and treated according to the following methods:

[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 cultured in a 37°C cell culture incubator containing 5% CO2. The medium was replaced every 2-3 days, and the 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 set aside.

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

[0143] First, prepare Aβ monomers: take 1 mg Aβ 1-42 The powder was dissolved in 1 mL HFIP, incubated at room temperature for 1 h, and then sonicated for 10 min to completely dissolve it. The powder was divided into 10 1.5 mL centrifuge tubes, evaporated overnight in a fume hood until HFIP was completely removed, and placed in a -20 °C refrigerator for later use.

[0144] Next, the Aβ oligomer solution was prepared: 10 μL DMSO was added to the 1.5 mL centrifuge tube from the previous step to dissolve it again, and then it was diluted with 433 μL PBS. The tube was incubated at room temperature for 20 min, and the supernatant was aspirated after centrifugation at 14000 g for 15 min. The supernatant was incubated at 37 °C for 24 h to obtain 50 μmol / L Aβ. 1-42 The oligomer solution was finally diluted to a concentration of 10 μmol / L with cell culture medium to obtain 10 μmol / L Aβ 1-42 Solution.

[0145] (3) Cell experiments:

[0146] The cells were cultured in a constant temperature and humidity incubator at 37°C, 25% relative humidity, and 5% carbon dioxide concentration:

[0147] Control group: 5×10 3 The cells were inoculated in a 96-well plate at a density of 100 μL cells / mL, with a volume of 100 μL per well. After the cells adhered to the wall (24 h), the culture medium was replaced, and the culture was continued for 12 h, and then the culture medium was replaced again and cultured for 24 h.

[0148] Model group: The cultured PC12 cells were cultured at 5×10 3 The cells were inoculated at a density of 100 cells / mL in a 96-well plate, with a volume of 100 μL per well. After the cells adhered to the wall (24 h), the culture medium was replaced, and after culturing for 12 h, the culture medium was removed, and 100 μL of 10 μmol / L Aβ was added to each well. 1-42 Solution, cultured for 24h.

[0149] KSeCSeCPK group: The cultured PC12 cells were plated at 5×10 3 The cells were inoculated at a density of 100 cells / mL in a 96-well plate, with a volume of 100 μL per well. After the cells adhered (24 h), the culture medium was removed, the sample was dissolved in the cell culture medium and prepared into a 1 mg / mL KSeCSeCPK solution, which was added to the 96-well plate, with a volume of 100 μL per well. After culturing for 12 h, the sample solution was removed, and 100 μL of 10 μmol / L Aβ was added to each well.1-42 Solution, cultured for 24h.

[0150] CSeCGAP group: The cultured PC12 cells were cultured at 5×10 3 The cells were inoculated at a density of 100 cells / mL in a 96-well plate, with a volume of 100 μL per well. After the cells adhered (24 h), the culture medium was removed, and the sample was dissolved in the cell culture medium and prepared into a 1 mg / mL CSeCGAP solution, which was added to the 96-well plate, with a volume of 100 μL per well. After culturing for 12 h, the sample solution was removed, and 100 μL of 10 μmol / L Aβ was added to each well. 1-42 Solution, cultured for 24h.

[0151] KHSeCAA group: The cultured PC12 cells were plated at 5×10 3 The cells were inoculated at a density of 100 cells / mL in a 96-well plate, with a volume of 100 μL per well. After the cells adhered (24 h), the culture medium was removed, the sample was dissolved in the cell culture medium and prepared into a 1 mg / mL KHSeCAA solution, which was added to the 96-well plate, with a volume of 100 μL per well. After culturing for 12 h, the sample solution was removed, and 100 μL of 10 μmol / L Aβ was added to each well. 1-42 Solution, cultured for 24h.

[0152] 50 μL of MTT was added to each well of the above groups, carefully removed after incubation for 4 h, and 150 μL of DMSO was added, and the absorbance (OD) of the cells at a wavelength of 570 nm was measured by an enzyme-labeled instrument.

[0153] The cell viability was calculated according to the following formula:

[0154]

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

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

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

[0158]

[0159] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of 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 as food, medicine or health product; it is used to improve cognitive function, significantly reduce Aβ 1-42 Induced PC12 cell damage, improving Alzheimer's disease symptoms, and preventing or treating neurodegenerative diseases.

3. An expression vector or a recombinant microorganism, characterized in that: The expression vector or recombinant microorganism comprises at least one of the polypeptides according to claim 1; Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector; Preferably, the recombinant microorganism is a bacterium or a fungus.

4. A food, medicine, health product or nutritional product, characterized in that: The food, medicine, health product or nutritional product comprises an effective dose of at least one of the polypeptides according to claim 1.

5. The food, medicine, health product or nutritional product according to claim 4, characterized in that: The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition; Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

6. The food, medicine, health product or nutritional product according to claim 4, characterized in that: The food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods; The health care product also contains acceptable auxiliary materials.

7. Use of one or more of the polypeptides of claim 1 in the preparation of food, medicine, health care product or nutritional product; for improving cognitive function, significantly reducing Aβ 1-42 Induced PC12 cell damage, improving Alzheimer's disease symptoms, and preventing or treating neurodegenerative diseases.

8. The use according to claim 7, characterized in that: The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition; Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

9. The use according to claim 7, characterized in that: The food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods; The health care product also contains acceptable auxiliary materials.

10. A method for preparing the food, medicine, health product or nutritional product according to any one of claims 4 to 6, characterized in that: The method comprises mixing at least one of the polypeptides described in claim 1 with at least one acceptable excipient.

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