An anti-inflammatory peptide derived from quinoa and its application

Abstract

The present invention discloses an anti-inflammatory peptide derived from quinoa and its use, belonging to the field of polypeptides. The technical problem to be solved by the present invention is how to inhibit the secretion of inflammatory mediators by immune cells. The polypeptide provided by the present invention (polypeptide SCAWLLAWSAPK) has an amino acid sequence as shown in SEQ ID NO: 1. The present invention also protects pharmaceutically acceptable salts of the polypeptide. The present invention also protects the use of the polypeptide or pharmaceutical salt in the preparation of products (such as drugs, reagents, or preparations) that inhibit inflammation. The inhibition of inflammation is manifested by inhibiting the secretion of inflammatory mediators by immune cells.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptides, and particularly relates to an anti-inflammatory peptide derived from quinoa and applications thereof. Background Art

[0002] Inflammation is a crucial defensive response to injury, tissue damage, or infectious pathogens, and is also an adaptive immune response. Inflammatory cells in the immune system respond to foreign invasion or inflammatory signaling by producing various inflammatory mediators (such as eicosanoids, vasoactive amines, cytokines, and chemokines). These mediators interact with various cellular and subcellular components, amplifying the inflammatory response. However, excessive production of these mediators can lead to tissue damage and loss of immune function. Chronic inflammatory responses are often closely associated with the development of diseases such as type 2 diabetes, obesity, inflammatory bowel disease, and neurodegenerative disorders. Therefore, the identification of effective anti-inflammatory substances and methods is of great significance for the prevention and treatment of related diseases.

[0003] Quinoa, an annual dicotyledonous plant of the genus Chenopodium, has attracted widespread attention worldwide for its rich protein content and comprehensive, balanced essential amino acid profile. Quinoa protein beverage, a highly processed quinoa product, is made from quinoa flour through a process of enzymatic starch hydrolysis and high-pressure homogenization. Its protein content exceeds 1%. Because it is gluten-free, quinoa protein beverage is suitable for celiac disease patients. The protein in quinoa protein beverage must be digested by pepsin and pancreatic enzymes in the gastrointestinal tract before it can exert its physiologically active effects as active peptides. However, no studies have yet identified anti-inflammatory peptides from processed plant protein beverages. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to inhibit immune cells from secreting inflammatory mediators.

[0005] In order to solve the above technical problems, the present invention provides an anti-inflammatory peptide derived from quinoa and applications thereof.

[0006] The polypeptide provided by the present invention (polypeptide SCAWLLAWSAPK) has an amino acid sequence as shown in SEQ ID NO: 1.

[0007] The present invention also protects pharmaceutically acceptable salts of the polypeptide.

[0008] The pharmaceutically acceptable salts include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate / diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycolylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide,Tannate, hydrochloride, tartrate, hydroxynaphthoate, 8-chlorotheophylline salt (teoclate), iodide, tosylate, triethiodide, lactate, valerate, etc. Depending on the intended use, pharmaceutically acceptable salts can be formed from cations such as sodium, potassium, aluminum, calcium, lithium, manganese, zinc, bismuth, etc., and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts can be prepared by standard methods, for example, by reacting the free acid with an organic or inorganic base. In the presence of a basic group such as an amino group, acidic salts such as hydrochloride, hydrobromide, acetate, pamoate, etc. can be used as dosage forms; in the presence of an acidic group (such as -COOH) or an alcohol group, pharmaceutically acceptable esters such as acetate, maleate, pivaloyloxymethyl, etc., as well as esters known in the literature for improving solubility and hydrolysis, can be used as sustained-release and prodrug formulations.

[0009] The present invention also protects the use of the polypeptide or the pharmaceutically acceptable salt in the preparation of products (such as drugs, reagents or preparations) for inhibiting inflammation.

[0010] The inhibition of inflammation is manifested in the inhibition of immune cells from secreting inflammatory mediators.

[0011] The inflammatory mediators are nitric oxide (NO) and / or interleukin-6 (IL-6) and / or tumor necrosis factor-α (TNF-α).

[0012] The immune cells are macrophages.

[0013] The present invention also protects a product (such as a drug, agent or preparation) for inhibiting inflammation, wherein the product contains the polypeptide or the pharmaceutically acceptable salt.

[0014] The inhibition of inflammation is manifested in the inhibition of immune cells from secreting inflammatory mediators.

[0015] The inflammatory mediators are NO and / or IL-6 and / or TNF-α.

[0016] The immune cells are macrophages.

[0017] The product may further include a pharmaceutically acceptable carrier.

[0018] The carriers include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, and organic acids), poorly soluble carrier materials (such as ethyl cellulose and cholesterol stearate), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose). Water-soluble carrier materials are preferred. These materials can be formulated into a variety of dosage forms, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations, buccal tablets, suppositories, and lyophilized powder injections. Suppositories can be vaginal suppositories, vaginal rings, or ointments, creams, or gels suitable for vaginal application. These formulations can include standard preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. To formulate unit dosage forms into tablets, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucine, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional cosolvents, buffers, pH adjusters, etc. may also be added. In addition, if necessary, colorants, preservatives, fragrances, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation.

[0019] The above dosage forms can be administered by injection, including subcutaneous, intravenous, intramuscular, and intraperitoneal injections, intracisternal injections, or infusions; by cavity administration, such as rectal, vaginal, and sublingual; by respiratory tract administration, such as nasal; or by mucosal administration. The preferred route of administration is injection, and subcutaneous injection is the preferred route of administration.

[0020] The present invention combines in vitro digestion, computer simulation screening and molecular docking technology to screen out new anti-inflammatory peptides from quinoa protein beverage, which have good anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the results of cell activity in Example 7.

[0022] Figure 2 This is the result of NO generation in Example 7.

[0023] Figure 3 This is the result of IL-6 content in Example 7.

[0024] Figure 4 This is the result of TNF-α content in Example 7.

[0025] Figure 5 These are the results of cell activity in Example 9.

[0026] Figure 6 This is the result of NO generation in Example 9.

[0027] Figure 7 This is the result of IL-6 content in Example 9.

[0028] Figure 8 This is the result of TNF-α content in Example 9. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0030] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and were performed according to the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged. In the figures of the specification, English letters indicate the degree of significance of the difference. There was no significant difference at the 0.05 level between treatments with at least one identical letter, and there was a significant difference at the 0.05 level between treatments without the same letter. In the examples, protein concentration was determined by Kjeldahl nitrogen determination. RAW264.7 cells: Beijing Jinyou Technology Co., Ltd. CCK-8 kit: Beijing Wanjing Lizhi Biotechnology Co., Ltd., WJ30025. Nitric oxide detection kit: Shanghai Biyuntian Biotechnology Co., Ltd. S0021M. Mouse interleukin 6 (IL-6) ELISA detection kit: Shanghai Jianglai Biotechnology Co., Ltd., JL20268. Mouse tumor necrosis factor α (TNF-α) ELISA kit: Shanghai Jianglai Biotechnology Co., Ltd., JL10484. TCA: trichloroacetic acid, CAS number 76-03-9. TAME: Nα-p-tosyl-L-arginine methyl ester hydrochloride, CAS number 1784-03-8.

[0031] Example 1: Preparation of quinoa protein beverage

[0032] First, grind the quinoa flakes several times to obtain fine quinoa flour.

[0033] Subsequently, quinoa powder was mixed with purified water at a material-liquid ratio of 1:3, and α-amylase and maltase accounting for 0.5% of the weight of quinoa powder were added. After mixing, the mixture was enzymatically hydrolyzed in a 70°C water bath for 45 minutes, and then the homogenate was cooled to 55°C.

[0034] Then, add CaCO3, Ca3(PO4)2, edible salt and edible oil in the proportions of 0.16%, 0.14%, 0.1% and 0.8% respectively (based on the final beverage volume), adjust the material-liquid ratio to 1:12 with purified water, and stir thoroughly.

[0035] Then, the quinoa protein beverage was obtained by homogenizing the mixture using a high-pressure homogenizer (30 MPa and 60 MPa pressure treatment for 2 min, respectively).

[0036] The quinoa protein beverage was freeze-dried to obtain freeze-dried powder, which was stored at 4°C.

[0037] Example 2: Preparation of simulated digestion storage solution

[0038] Simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) stock solutions were prepared at 1.25x concentrations according to the INFOGEST in vitro simulated digestion method. The SSF, SGF, and SIF stock solutions all consisted of saline and water. The saline solutions added per 400 mL of stock solution and their volumes are shown in Table 1. In Table 1, the volume of the SSF, SGF, and SIF stock solutions was 400 mL, the pH was 7, and the concentration was 1.25x.

[0039]

[0040] Note: CaCl2 aqueous solution was added before use.

[0041] Example 3. Pepsin activity determination (spectrophotometry)

[0042] 1. Prepare 2% bovine hemoglobin solution

[0043] Dissolve 0.5 g of bovine hemoglobin in 20 mL of ultrapure water, adjust the pH to 2 with 300 mM HCl solution, and make up to 25 mL.

[0044] 2. Prepare pepsin dilution solution.

[0045] Porcine pepsin (commercially available solid preparation) was taken and a buffer solution (pH 6.5) containing 10 mM Tris and 150 mM NaCl was used as a solvent to prepare a pepsin solution with a concentration of 1 mg / mL, which was the pepsin enzyme solution.

[0046] Before use, dilute the pepsin solution with 10 mM HCl solution to a pepsin concentration of 5, 10, 15, 20, 25, 30, or 35 μg / mL, respectively. This is the pepsin dilution solution and store on ice.

[0047] 3. Prepare enzyme reaction tubes (indicated by Test) and blank tubes (indicated by Blank). Add 500 μL of 2% bovine hemoglobin solution to each tube and incubate in a 37°C water bath for 3-4 minutes.

[0048] 4. After completing step 3, take the enzyme reaction tubes and add 100 μL of pepsin diluent to each tube. Then, incubate at 37°C in a water bath for 10 min. Then, add 1 mL of 5% TCA (w / v) solution to each tube to terminate the reaction. After completing step 3, take the blank tubes and add 1 mL of 5% TCA (w / v) solution to each tube. Then, add 100 μL of pepsin diluent to each tube.

[0049] 5. After completing step 4, centrifuge all reaction tubes at 6000 g for 30 min and collect the supernatant.

[0050] 6. Take the supernatant obtained in step 5, equilibrate it at room temperature for 5 minutes, and then measure the absorbance at a wavelength of 280 nm.

[0051] 7. Calculate the pepsin activity according to formula (1).

[0052]

[0053] Δt: reaction duration, i.e. 10 min;

[0054] X: the amount of pepsin contained in each mL of the assay enzyme solution, which is 5, 10, 15, 20, 25, or 30 μg;

[0055] 1000: dilution factor to convert μg to mg;

[0056] 0.001: Absorbance change (ΔA280) caused by one unit of pepsin.

[0057] Example 4, trypsin activity assay

[0058] 1. Prepare porcine pancreatic enzyme solutions (commercially available solid preparation) with 1 mM HCl solution as solvent, with the contents of 0.25, 0.5, or 1 mg / mL, respectively, and place on ice.

[0059] 2. Prepare enzyme reaction tubes (indicated by Test) and blank tubes (indicated by Blank). Add 2.6 mL of working solution and 0.3 mL of substrate solution to each tube, mix well, and incubate at room temperature for 3-4 minutes.

[0060] Substrate solution: 10 mM TAME solution.

[0061] Working solution: Tris-HCl buffer (pH 8.1, 46 mM) containing 11.5 mM CaCl2.

[0062] 3. After completing step 2, add 100 μL of trypsin solution to each enzyme reaction tube and 100 μL of working solution to each blank tube. After rapid mixing, measure the absorbance change at 247 nm within 10 minutes (measure every 10 seconds).

[0063] 4. Calculate trypsin activity according to formula (2).

[0064]

[0065] ΔA247: slope, unit absorbance / min;

[0066] 1000: Factor for converting mL to μL;

[0067] 3: total reaction volume (mL), 3 mL;

[0068] 540: molar extinction coefficient of TAME at 247 nm (L / (mol×cm));

[0069] X: The amount of enzyme in 100 μL of enzyme solution (mg).

[0070] Example 5, Bile Acid Content Determination

[0071] A bile acid assay kit was used (the components for preparing the working solution were provided by the kit).

[0072] Internal standard solution: 80 µM sodium cholate solution.

[0073] Pig bile solution: Prepare a 1 mg / mL solution of pig bile extract (commercially available solid preparation) using ultrapure water as the solvent. Sample solution: Prepare the sample solution by diluting the pig bile solution to 25 times its volume with ultrapure water.

[0074] Working Solution 1: consists of 75 µL Assay Buffer, 8 µL NAD, 4 µL Probe, 1 µL Enzyme A, and 1 µL Enzyme B. Working Solution 2: consists of 75 µL Assay Buffer, 8 µL NAD, 4 µL Probe, and 1 µL Enzyme B.

[0075] Take a black ELISA plate, add 20 μL sample solution, 5 μL internal standard solution and 80 μL working solution 1 to each well of the internal standard group (represented by Internal standard), add 20 μL sample solution, 5 μL ultrapure water and 80 μL working solution 1 to each well of the sample group (represented by Test), and add 20 μL sample solution, 5 μL ultrapure water and 80 μL working solution 2 to each well of the blank group (represented by Blank), and then incubate in the dark for 20 minutes. Then, detect the fluorescence value (λ ex = 530 nm / λ em = 585 nm), that is, the F value, and then the bile acid content of the pig bile solution was calculated according to formula (3).

[0076]

[0077] n: The dilution factor of the sample solution prepared with pig bile solution is 25.

[0078] 20: added volume of sample solution (µL), which is 20 μL;

[0079] 1000: Dilution factor to convert μM to mM.

[0080] Example 6: In vitro simulated digestion

[0081] The entire in vitro simulated digestion process includes three consecutive stages: oral digestion, gastric digestion and intestinal digestion.

[0082] The lyophilized powder obtained in Example 1 was reconstituted with ultrapure water to a protein concentration of 60 mg / mL, which was the sample to be digested.

[0083] Oral digestion: First, preheat the SSF stock solution in a 37°C water bath. Then, add 4 mL of preheated SSF stock solution to 5 mL of the sample to be digested, make up to 10 mL with ultrapure water, and incubate in a 37°C water bath for 2 minutes (with magnetic stirring during the process) to obtain a digestion product that simulates oral digestion. Because the quinoa protein beverage has already been enzymatically hydrolyzed with two enzymes during preparation, its carbohydrate content has been significantly reduced. Furthermore, the quinoa protein purity in the quinoa protein beverage is greater than 90%, so salivary amylase was not added during this stage.

[0084] Gastric digestion stage: porcine pepsin was prepared into a 40,000 U / mL solution using ultrapure water (see Example 3 for the enzyme activity determination method), which is the porcine pepsin solution. 10 mL of the digestion product simulating oral digestion was added to 8 mL of SGF stock solution preheated in a 37°C water bath. The pH of the system was then adjusted to 3.0 with 1 M HCl solution. 1 mL of preheated porcine pepsin solution preheated in a 37°C water bath was then added. Ultrapure water was then added to a total volume of 20 mL. Finally, the entire system was incubated in a 37°C water bath for 2 h (with magnetic stirring during the process) to obtain the digestion product simulating gastric digestion.

[0085] Intestinal digestion stage: porcine pancreatic enzyme was prepared into an 800 U / mL solution using SIF storage solution (for the enzyme activity determination method, see Example 4), which is the porcine pancreatic enzyme solution; porcine bile extract was prepared into a solution containing 160 mM bile acid using SIF storage solution (for the bile acid content determination method, see Example 5), which is the porcine bile solution; 20 mL of the digestion product of simulated gastric digestion was added to 8.5 mL of SIF storage solution preheated at 37°C, and the pH of the system was adjusted to 7.0 using 1 M HCl solution or 1 M NaOH solution. 2.5 mL of porcine bile solution preheated at 37°C and 5 mL of porcine pancreatic enzyme solution preheated at 37°C were then added, and ultrapure water was added to a total volume of 40 mL; finally, the entire system was incubated in a 37°C water bath for 2 h (with magnetic stirring during the process) to obtain the product of in vitro simulated digestion.

[0086] The products of the simulated in vitro digestion were freeze-dried to obtain dry powdery substances, named digestion products, and stored at -20°C.

[0087] 5 mL of ultrapure water was used to replace the sample to be digested, and the above-mentioned oral digestion stage, gastric digestion stage, and intestinal digestion stage were carried out in sequence. Then, the sample was freeze-dried to obtain a dry powder substance, which was named digestion control and stored at -20°C.

[0088] Example 7: Effects of in vitro simulated digestion products on cells

[0089] Complete culture medium: DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0090] Incubator conditions: 37°C, 5% CO2.

[0091] 1. Determination of Cell Viability

[0092] The test samples were: digestion products prepared in Example 6 or digestion controls.

[0093] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash with PBS buffer, and then resuspend in complete culture medium to obtain 4×105 cells / mL of cell suspension.

[0094] 2. Add 200 μL of the cell suspension prepared in step 1 to each well of a 96-well cell culture plate, including the test wells and negative control wells. Add 200 μL of complete culture medium to each well of the blank control wells. Then, place the plate in an incubator and culture for 24 hours. Set up three replicates for each blank control well and negative control well.

[0095] 3. After completing step 2, take the cell culture plate, discard the supernatant, add 200 μL of complete medium containing the test sample to each well of the test well (each test sample is set with a different polypeptide content, namely 0.625, 1.25, 2.5 or 5 mg / mL, and three replicate wells are set for each polypeptide content of each test sample; the polypeptide concentration is measured using the Ortho-Phthal aldehyde method), add 200 μL of complete medium to each well of the negative control well, and add 200 μL of complete medium to each well of the blank control well. Then, place the cell culture plate in an incubator and culture for 24 hours.

[0096] 4. After completing step 3, take the cell culture plate, add 10 μL of CCK solution to each well, and then place the cell culture plate in an incubator for 1 hour.

[0097] 5. After completing step 4, measure the absorbance of each well at 450 nm and calculate the cell activity according to formula (4).

[0098]

[0099] A s : absorbance of test well; A b : absorbance of blank control well; A c : Absorbance of negative control well.

[0100] See the results Figure 1 The results showed that the cell viability of RAW264.7 cells treated with the digestion product at a concentration of 2.5 mg / mL peptide was 96.46%. Generally speaking, a cell viability above 85% is considered non-toxic in the field.

[0101] II. Determination of NO Production, IL-6 Content, and TNF-α Content

[0102] The test samples were: digestion products prepared in Example 6 or digestion controls.

[0103] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash with PBS buffer, and then resuspend in complete culture medium to obtain 4×10 5 cells / mL of cell suspension.

[0104] 2. Take a 96-well cell culture plate, add 200 μL of the cell suspension prepared in step 1 to each well, and then place the cell culture plate in an incubator for 24 hours.

[0105] 3. After completing step 2, remove the cell culture plate, discard the supernatant, add 200 μL of complete medium to each negative and positive control well, and add 200 μL of complete medium containing the test article to each sample well (each test article has a different peptide concentration, 0.625, 1.25, or 2.5 mg / mL, with three replicates per well for each peptide concentration). Then, place the cell culture plate in an incubator and incubate for 2 hours. Set up three replicates for each negative and positive control well.

[0106] 4. After completing step 3, take the cell culture plate, add 10 μL of complete culture medium containing 20 μg / mL LPS to each of the positive control wells and sample wells, and add 10 μL of complete culture medium to each of the negative control wells, and then place the cell culture plate in an incubator and culture for 24 hours.

[0107] 5. After completing step 4, take the cell culture plate, collect the supernatant from each well, and detect the NO production, IL-6 content, and TNF-α content in the supernatant.

[0108] The results of NO generation are shown in Figure 2 The results of IL-6 content are shown in Figure 3 The results of TNF-α content are shown in Figure 4 . Figures 2 to 4 In the negative control wells, CK was used, and the positive control wells were used, LPS was used.

[0109] Example 8: Discovery of anti-inflammatory peptides

[0110] 1. Mass spectrometry identification of peptide sequences

[0111] The digested product prepared in Example 6 was identified and analyzed by nano-liquid chromatography (Nano LC1000, Thermo Fisher Scientific) coupled to an Orbitrap Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific, San Jose, CA) (Shevchenko et al., 2006). The analytical column was a C18 reversed-phase column (75 μm × 15 cm, 3 μm ReproSil-Pur C18-AQ packing), and the loading column was a C18 column (150 μm × 3 cm, 5 μm ReproSil-Pur C18-AQ packing). Mobile phase A consisted of 0.5% formic acid in water, and mobile phase B consisted of 0.5% formic acid in acetonitrile. A gradient was optimized at a nanoflow rate of 300 nL / min to ensure efficient separation of the target peptides. Mass spectra were acquired in data-dependent scan mode. Data were analyzed using Proteome Discoverer (version 2.1, ThermoFisher Scientific). MS2 spectra were retrieved from the uniprotkb_Chenopodium_quinoa.fasta database using the SEQUEST search engine. Parameters were set as follows: trypsin digestion (allowing two missed cleavage sites), precursor ion mass error <10 ppm, fragment ion mass error <20 ppm; fixed modification was cysteine ​​alkylation, and variable modification was methionine oxidation. Search results were filtered using the Percolator algorithm ( q value <1%, FDR ≤ 1%).

[0112] 2. Computer simulation screening of potential anti-inflammatory peptides

[0113] First, the potential toxicity of all peptides was predicted using the ToxinPred tool (http: / / webs.iiitd.edu.in / raghava / toxinpred / ). After eliminating toxic peptides, the PeptideRanker tool (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to screen for potential bioactive peptides with a score of 0.5 or higher. Finally, the PreAIP tool (http: / / kurata14.bio.kyutech.ac.jp / PreAIP / index.php) was used to predict the potential anti-inflammatory activity of these potential bioactive peptides, thereby identifying potential anti-inflammatory peptides.

[0114] 3. Molecular Docking

[0115] First, the tertiary structure of the TLR4 / MD2 / LPS complex (PDBID: 3FXI) was downloaded from Protein Databank (https: / / www.rcsb.org / ) and visualized using PyMOL (version 2.6.0a0). Based on the sequences of the selected potential anti-inflammatory peptides, their three-dimensional structures were predicted using PEP-FOLD 4 (https: / / mobyle2.rpbs.univ-paris-diderot.fr / cgi-bin / portal.py#forms::PEP-FOLD4). For molecular docking analysis, water and redundant ligands were removed from the TLR4 / MD2 complex, and docking analysis with the potential anti-inflammatory peptides was performed using Autodock Vina 1.5.6 software. Docking conditions were as follows: for the TLR4 / MD2 complex, the grid spacing was set to 1 Å; the box grid dimensions were 33, 40.5, and 35.25 Å (x, y, z), respectively; and the box center coordinates were 25.692, -5.342, and 14.883 (x, y, z), respectively. The docking results were visualized and analyzed using Discovery Studio 2021 Client software.

[0116] The active peptide discovered based on the above steps was named SCAWLLAWSAPK. The peptide SCAWLLAWSAPK is connected to the TLR4 / MD2 complex mainly through hydrophobic interactions, hydrogen bonds and electrostatic interactions.

[0117] Amino acid sequence of polypeptide SCAWLLAWSAPK (SEQ ID NO: 1): SCAWLLAWSAPK.

[0118] Example 9: Synthesis and functional verification of active peptides

[0119] Complete culture medium: DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0120] Incubator conditions: 37°C, 5% CO2.

[0121] 1. Synthesis of active peptides

[0122] Artificially synthesized polypeptide SCAWLLAWSAPK (i.e., the polypeptide shown in SEQ ID NO: 1).

[0123] 2. Determination of Cell Viability

[0124] Test sample: peptide SCAWLLAWSAPK prepared in step 1.

[0125] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash with PBS buffer, and then resuspend in complete culture medium to obtain 4×10 5 cells / mL of cell suspension.

[0126] 2. Add 200 μL of the cell suspension prepared in step 1 to each well of a 96-well cell culture plate, including the test wells and negative control wells. Add 200 μL of complete culture medium to each well of the blank control wells. Then, place the plate in an incubator and culture for 24 hours. Set up three replicates for each blank control well and negative control well.

[0127] 3. After completing step 2, take the cell culture plate, discard the supernatant, add 200 μL of complete medium containing the test article to each well of the test well (set different test article concentrations, respectively, 0.1, 0.5, 1 or 2 mg / mL, and set three replicates for each concentration), add 200 μL of complete medium to each well of the negative control well, and add 200 μL of complete medium to each well of the blank control well. Then place the cell culture plate in an incubator and culture for 24 hours.

[0128] 4. After completing step 3, take the cell culture plate, add 10 μL of CCK solution to each well, and then place the cell culture plate in an incubator for 1 hour.

[0129] 5. After completing step 4, measure the absorbance of each well at 450 nm and calculate the cell activity according to formula (4).

[0130] See the results Figure 5 (CK corresponds to the negative control well.) The results showed that the cell viability of RAW264.7 cells treated with the peptide SCAWLLAWSAPK at a concentration of 1 mg / mL was 91.74%. Generally, a cell viability above 85% is considered non-toxic in the field.

[0131] 3. Determination of NO Production, IL-6 Content, and TNF-α Content

[0132] Test sample: peptide SCAWLLAWSAPK prepared in step 1.

[0133] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash with PBS buffer, and then resuspend in complete culture medium to obtain 4×10 5 cells / mL of cell suspension.

[0134] 2. Take a 96-well cell culture plate, add 200 μL of the cell suspension prepared in step 1 to each well, and then place the cell culture plate in an incubator for 24 hours.

[0135] 3. After completing step 2, remove the cell culture plate, discard the supernatant, add 200 μL of complete medium to each of the negative and positive control wells, and add 200 μL of complete medium containing the test article to each of the sample wells (set different test article concentrations: 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, or 1 mg / mL, with three replicates for each concentration). Then, place the cell culture plate in an incubator and incubate for 2 hours. Set up three replicates for each of the negative and positive control wells.

[0136] 4. After completing step 3, take the cell culture plate, add 10 μL of complete culture medium containing 20 μg / mL LPS to each of the positive control wells and sample wells, and add 10 μL of complete culture medium to each of the negative control wells, and then place the cell culture plate in an incubator and culture for 24 hours.

[0137] 5. After completing step 4, take the cell culture plate, collect the supernatant from each well, and detect the NO production, IL-6 content, and TNF-α content in the supernatant.

[0138] The results of NO generation are shown in Figure 6 The results of IL-6 content are shown in Figure 7 The results of TNF-α content are shown in Figure 8 . Figures 6 to 8 In the negative control wells, CK is used, and the positive control wells, LPS, are used. At a concentration of 1 mg / mL, the SCAWLLAWSAPK peptide inhibited the production of three inflammatory mediators (NO, IL-6, and TNF-α) by 69.15 ± 3.48%, 94 ± 0.65%, and 86.32 ± 1.36%, respectively. Inhibition rate = ((positive control well content - sample well content) / (positive control well content - negative control well content)) × 100%.

[0139] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

1.

2. A pharmaceutically acceptable salt of the polypeptide according to claim 1.

3. Use of the polypeptide according to claim 1 or the pharmaceutically acceptable salt according to claim 2 in the preparation of an agent for inhibiting inflammation; the inhibition of inflammation is the inhibition of the secretion of inflammatory mediators by immune cells in vitro.

4. The use according to claim 3, characterized in that: The inflammatory mediators are nitric oxide and / or interleukin-6 and / or tumor necrosis factor-α.

5. The use according to claim 3, characterized in that: The immune cells are macrophages.

6. An agent for inhibiting inflammation, characterized in that: The reagent contains the polypeptide according to claim 1 or the pharmaceutically acceptable salt according to claim 2; and the inflammation inhibition is the in vitro inhibition of the secretion of inflammatory mediators by immune cells.

7. The reagent according to claim 6, wherein: The inflammatory mediators are nitric oxide and / or interleukin-6 and / or tumor necrosis factor-α.

8. The reagent according to claim 6, wherein: The immune cells are macrophages.

Images