Chenopodium quinoa willd-derived anti-inflammatory peptide and application thereof

Abstract

The invention discloses an anti-inflammatory peptide from chenopodium quinoa willd and application of the anti-inflammatory peptide, and belongs to the field of polypeptides. The technical problem to be solved by the invention is how to inhibit immune cells from secreting inflammatory mediators. The polypeptide (polypeptide SCAWLLAWSAPK) provided by the invention has an amino acid sequence as shown in SEQ ID NO: 1. The invention also provides a pharmaceutical salt of the polypeptide. The invention further discloses application of the polypeptide or the medicinal salt in preparation of products (such as medicines, reagents or preparations) for inhibiting inflammation. Inflammation inhibition is embodied by inhibiting immune cells from secreting inflammatory mediators.

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 its application. Background Art

[0002] Inflammation is an important defense response of the body to injury, tissue damage or infectious pathogens, and is also an adaptive immune response. Inflammatory cells in the immune system respond to the invasion of foreign substances or the stimulation of inflammatory signals by producing different inflammatory mediators (such as eicosanoids, vasoactive amines, cytokines and chemotactic factors). These mediators interact with different cellular and subcellular components, thereby amplifying the inflammatory response. However, the overproduction of these mediators can lead to tissue damage and loss of immune function. Chronic inflammatory responses are usually closely related to the occurrence of diseases such as type 2 diabetes, obesity, inflammatory bowel disease and neurodegenerative diseases. Therefore, finding effective anti-inflammatory substances and methods is of great significance for the prevention and treatment of related diseases.

[0003] Quinoa is an annual dicotyledonous plant of the genus Chenopodium, which has attracted worldwide attention because of its rich protein, comprehensive and balanced essential amino acids. Quinoa protein beverage is a deep-processed product of quinoa. It is a plant protein beverage prepared from quinoa powder by combining amylase hydrolysis and high-pressure homogenization treatment, and its protein content is higher than 1%. Quinoa protein beverage is suitable for celiac patients because it does not contain gluten. The protein in quinoa protein beverage must be digested by pepsin and pancreatic enzymes in the gastrointestinal tract before it can play a physiological active role in the form of active peptides. However, there is currently no study on screening anti-inflammatory active 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 the secretion of inflammatory mediators by immune cells.

[0005] To solve the above technical problems, the present invention provides an anti-inflammatory peptide derived from quinoa and its application.

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

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

[0008] The medicinal salts include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calciumedetate, 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, glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide,Tannates, hydrochlorides, tartrates, hydroxynaphthoates, teoclates, iodides, tosylates, triethiodides, lactates, valerates, etc. Depending on the use, pharmaceutical salts can be formed by cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, bismuth, etc., or by bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide, etc. These salts can be prepared by standard methods, for example, by the reaction of a free acid with an organic or inorganic base. In the presence of a basic group such as an amino group, acidic salts such as hydrochlorides, hydrobromides, acetates, pamoates, 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 acetates, maleates, pivaloyloxymethyl, etc., and 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 pharmaceutical salt in the preparation of products (such as drugs, reagents or formulations) for inhibiting inflammation.

[0010] The inhibition of inflammation is manifested as the inhibition of the secretion of inflammatory mediators by immune cells.

[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 products (such as drugs, reagents or preparations) for inhibiting inflammation, and the products contain the polypeptide or the medicinal salt.

[0014] The inhibition of inflammation is manifested as inhibiting the secretion of inflammatory mediators by immune cells.

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

[0016] The immune cells are macrophages.

[0017] The products may further include a medicinal carrier.

[0018] The carriers include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, the preferred one is the water-soluble carrier material. Using these materials, various dosage forms can be prepared, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. Among them, the suppository can be a vaginal suppository, or a vaginal ring, or an ointment, cream or gel suitable for vaginal application. It can be an ordinary preparation, a sustained-release preparation, a controlled-release preparation and various microparticle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well-known in the art can be widely used. Examples of the carriers are, for example, 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 paste, dextrin, syrup, honey, glucose solution, acacia mucilage, gelatin mucilage, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dried starch, alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium dodecyl sulfate, etc.; lubricants, such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to prepare the unit dosage form into pills, various carriers well-known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers well-known in the art can be widely used. Examples of the carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into injectable preparations, such as solutions, emulsions, freeze-dried powder injections and suspensions, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc.In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation.

[0019] The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracisternal injection or infusion, etc.; administration via body cavities, such as via the rectum, vagina and sublingual; administration via the respiratory tract, such as via the nasal cavity; mucosal administration. The preferred route of administration among the above is injection, and the preferred injection route is subcutaneous injection.

[0020] In the present invention, by combining in vitro digestion, computer-aided screening and molecular docking techniques, novel anti-inflammatory peptides were screened from quinoa protein beverages, which have good anti-inflammatory activities. Description of the Drawings

[0021] Figure 1 Results of cell viability in Example 7.

[0022] Figure 2 Results of NO production in Example 7.

[0023] Figure 3 Results of IL-6 content in Example 7.

[0024] Figure 4 Results of TNF-α content in Example 7.

[0025] Figure 5 Results of cell viability in Example 9.

[0026] Figure 6 Results of NO production in Example 9.

[0027] Figure 7 Results of IL-6 content in Example 9.

[0028] Figure 8 Results of TNF-α content in Example 9. Detailed Description of the Invention

[0029] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0030] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, in the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged. In the accompanying drawings of the specification, English letters represent the degree of significance. Treatments with at least one same letter have no significant difference at the 0.05 level, and treatments without the same letter have a significant difference at the 0.05 level. In the examples, the Kjeldahl method was used to detect the protein concentration. RAW264.7 cells: Beijing Jinyou Technology Co., Ltd. CCK-8 kit: Beijing Wanjing Lizhi Biotechnology Co., Ltd., WJ30025. Nitric oxide detection kit: Shanghai Beyotime 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 detection kit: Shanghai Jianglai Biotechnology Co., Ltd., JL10484. TCA: Trichloroacetic acid, CAS number 76-03-9. TAME: Nα-p-toluenesulfonyl-L-arginine methyl ester hydrochloride, CAS number 1784-03-8.

[0031] Example 1. Preparation of quinoa protein beverage First, the quinoa cereal was ground multiple times to obtain fine quinoa powder.

[0032] Subsequently, the quinoa powder was mixed with purified water at a liquid-to-solid ratio of 1:3, and α-amylase and maltase accounting for 0.5% of the weight of the quinoa powder were added. After mixing evenly, it was enzymatically hydrolyzed in a water bath at 70 °C for 45 min, and then the homogenate was cooled to 55 °C.

[0033] Then, CaCO 3 , Ca 3 (PO 4 ) 2 , edible salt, and edible oil (calculated based on the volume of the final beverage) were added in proportions of 0.16%, 0.14%, 0.1%, and 0.8% respectively. The liquid-to-solid ratio was adjusted to 1:12 with purified water and stirred evenly.

[0034] Then, homogenization treatment was carried out using a high-pressure homogenizer (treated at 30 MPa and 60 MPa for 2 min in sequence) to obtain the quinoa protein beverage.

[0035] The quinoa protein beverage was taken, freeze-dried to obtain a freeze-dried powder, and stored at 4 °C.

[0036] Example 2. Preparation of simulated digestion storage solution Prepare stock solutions of simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) at 1.25-fold concentration according to the INFOGEST in vitro digestion method. The SSF stock solution, SGF stock solution, and SIF stock solution are all composed of salt solutions and water. The various salt solutions added to each 400 mL of the stock solution and their added volumes are shown in Table 1. In Table 1, the volumes of the SSF stock solution, SGF stock solution, and SIF stock solution are all 400 mL, the pH values are all 7, and the concentrations are all 1.25×.

[0037]

[0038] Note: CaCl 2 The aqueous solution is added before use.

[0039] Example 3: Determination of pepsin enzyme activity (spectrophotometry) 1. Prepare a 2% bovine hemoglobin solution 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 the volume to 25 mL.

[0040] 2. Prepare a pepsin dilution

[0041] Take porcine pepsin (commercially available solid preparation), use a buffer solution containing 10 mM Tris and 150 mM NaCl (pH 6.5) as the solvent, and prepare a solution with a pepsin concentration of 1 mg / mL, which is the pepsin enzyme solution.

[0042] Before use, dilute the pepsin enzyme solution with 10 mM HCl solution to make the pepsin concentrations 5, 10, 15, 20, 25, 30, or 35 μg / mL respectively, which are the pepsin dilutions, and store them on ice.

[0043] 3. Prepare enzyme reaction tubes (denoted as Test) and blank tubes (denoted as Blank), add 500 μL of 2% bovine hemoglobin solution to each tube, and incubate in a 37°C water bath for 3 - 4 min.

[0044] 4. After completing step 3, take the enzyme reaction tubes, add 100 μL of pepsin dilution to each tube, then incubate in a 37°C water bath for 10 min, and 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, add 1 mL of 5% TCA (w / v) solution to each tube, and then add 100 μL of pepsin dilution to each tube.

[0045] 5. After step 4 is completed, centrifuge all reaction tubes at 6000 g for 30 min, and collect the supernatant separately.

[0046] 6. Take the supernatant obtained in step 5, balance it at room temperature for 5 min, and then measure the absorbance at a wavelength of 280 nm.

[0047] 7. Calculate the pepsin enzyme activity according to formula (1).

[0048]

[0049] Δt: The reaction duration, that is, 10 min; X: The amount of pepsin contained in each mL of the measured enzyme solution, which are 5, 10, 15, 20, 25, or 30 μg respectively; 1000: The dilution factor for converting μg to mg; 0.001: The change in absorbance value (ΔA280) caused by each unit of pepsin.

[0050] Example 4. Determination of trypsin enzyme activity 1. Take porcine pancreatin (commercially available solid preparation), use 1 mM HCl solution as the solvent, and prepare pancreatin solutions with pancreatin contents of 0.25, 0.5, or 1 mg / mL respectively, and place them on ice.

[0051] 2. Prepare enzyme reaction tubes (denoted as Test) and blank tubes (denoted as Blank), add 2.6 mL of working solution and 0.3 mL of substrate solution to each tube, mix well, and then incubate at room temperature for 3 - 4 min.

[0052] Substrate solution: 10 mM TAME solution.

[0053] Working solution: Tris - HCl buffer solution containing 11.5 mM CaCl 2 (pH 8.1, 46 mM).

[0054] 3. After step 2 is completed, add 100 μL of pancreatin solution to each enzyme reaction tube, and add 100 μL of working solution to each blank tube. After rapid mixing, measure the change in absorbance within 10 min at 247 nm (measure once every 10 s).

[0055] 4. Calculate the trypsin activity according to formula (2).

[0056]

[0057] ΔA247: Slope, unit absorbance / min; 1000: Factor for converting mL to μL; 3: Total reaction volume (mL), 3 mL; 540: Molar extinction coefficient of TAME at 247 nm (L / (mol×cm)); X: Corresponding enzyme amount (mg) in 100 μL enzyme solution.

[0058] Example 5. Determination of bile acid content A bile acid assay kit was used (each component for preparing the working solution was provided by the kit).

[0059] Internal standard solution: 80 μM sodium cholate solution.

[0060] Porcine bile solution: Porcine bile extract (commercially available solid preparation) was taken and a 1 mg / mL solution was prepared using ultrapure water as the solvent, which was the porcine bile solution. Sample solution: The porcine bile solution was taken and diluted to 25 times its volume with ultrapure water to obtain the sample solution.

[0061] Working solution 1: Composed of 75 μL Assay Buffer, 8 μL NAD, 4 μL Probe, 1 μL Enzyme A and 1 μL Enzyme B. Working solution 2: Composed of 75 μL Assay Buffer, 8 μL NAD, 4 μL Probe and 1 μL Enzyme B.

[0062] A black microplate was taken. For the internal standard group (denoted as Internal standard), 20 μL of the sample solution, 5 μL of the internal standard solution and 80 μL of working solution 1 were added to each well. For the sample group (denoted as Test), 20 μL of the sample solution, 5 μL of ultrapure water and 80 μL of working solution 1 were added to each well. For the blank group (denoted as Blank), 20 μL of the sample solution, 5 μL of ultrapure water and 80 μL of working solution 2 were added to each well. Then, they were incubated in the dark for 20 min. Then, the fluorescence values of each well (λ ex = 530 nm / λ em = 585 nm) were measured, i.e., the F value. Then, the bile acid content of the porcine bile solution was calculated according to formula (3).

[0063]

[0064] n: Dilution factor for preparing the sample solution with the porcine bile solution, which is 25.

[0065] 20: Added volume of the sample solution (μL), which is 20 μL; 1000: Dilution factor for converting μM to mM.

[0066] Example 6. In vitro simulated digestion The whole in vitro simulated digestion process includes three consecutive stages: oral digestion, gastric digestion, and intestinal digestion.

[0067] Take the freeze-dried powder obtained in Example 1, reconstitute it with ultrapure water to make the protein concentration 60 mg / mL, which is the sample to be digested.

[0068] Oral digestion stage: First, preheat the SSF stock solution in a 37°C water bath; then, add 4 mL of the preheated SSF stock solution to 5 mL of the sample to be digested, make up to 10 mL with ultrapure water, and then incubate in a 37°C water bath for 2 min (with magnetic stirring during the process) to obtain the digestion product of simulated oral digestion. Since the quinoa protein beverage has been enzymatically hydrolyzed with two enzymes during the preparation process, its carbohydrate content has been significantly reduced, and the quinoa protein purity of the quinoa protein beverage is higher than 90%. Therefore, salivary amylase was not added in this stage.

[0069] Gastric digestion stage: Prepare a 40000 U / mL solution of porcine pepsin with ultrapure water (the method for measuring enzyme activity is shown in Example 3), which is the porcine pepsin solution; take 10 mL of the digestion product of simulated oral digestion, add 8 mL of the preheated SGF stock solution in a 37°C water bath, then adjust the pH of the system to 3.0 with 1 M HCl solution, then add 1 mL of the preheated porcine pepsin solution in a 37°C water bath, and then add ultrapure water to make the total volume of the system 20 mL; finally, incubate the whole system in a 37°C water bath for 2 h (with magnetic stirring during the process) to obtain the digestion product of simulated gastric digestion.

[0070] Intestinal digestion stage: Prepare an 800 U / mL solution of porcine pancreatin with SIF stock solution (the method for measuring enzyme activity is shown in Example 4), which is the porcine pancreatin solution; prepare a solution containing 160 mM bile acid of porcine bile extract with SIF stock solution (the method for measuring bile acid content is shown in Example 5), which is the porcine bile solution; take 20 mL of the digestion product of simulated gastric digestion, add 8.5 mL of the preheated SIF stock solution in a 37°C water bath, then adjust the pH of the system to 7.0 with 1 M HCl solution or 1 M NaOH solution, then add 2.5 mL of the preheated porcine bile solution in a 37°C water bath and 5 mL of the preheated porcine pancreatin solution in a 37°C water bath, and then add ultrapure water to make the total volume of the system 40 mL; finally, incubate the whole system in a 37°C water bath for 2 h (with magnetic stirring during the process) to obtain the product of in vitro simulated digestion.

[0071] The product of in vitro simulated digestion is freeze-dried to obtain a powdery substance, named the digestion product, and stored at -20°C.

[0072] Replace the sample to be digested with 5 mL of ultrapure water, and successively carry out the above oral digestion stage, gastric digestion stage and intestinal digestion stage, and then carry out freeze-drying to obtain a dry powder substance, named digestion control, and store it at -20 °C.

[0073] Example 7. Effects of in vitro simulated digestion products on cells Complete medium: DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0074] Incubator conditions: 37 °C, 5% CO 2 .

[0075] I. Determination of cell viability The test articles are respectively: the digestion products or digestion controls prepared in Example 6.

[0076] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash them with PBS buffer, and then resuspend them with complete medium to obtain a cell suspension of 4×10 5 cells / mL.

[0077] 2. Take a 96-well cell culture plate, add 200 μL of the cell suspension prepared in step 1 to each test well and negative control well, add 200 μL of complete medium to each blank control well, and then place the cell culture plate in the incubator and culture for 24 h. Three replicates are set for each of the blank control well and the negative control well.

[0078] 3. After completing step 2, take the cell culture plate, aspirate the supernatant, add 200 μL of complete medium containing the test article to each test well (different polypeptide contents are set for each test article, which are 0.625, 1.25, 2.5 or 5 mg / mL respectively, and three replicates are set for each polypeptide content of each test article; the polypeptide concentration is measured by the Ortho-Phthalaldehyde method), add 200 μL of complete medium to each negative control well, and add 200 μL of complete medium to each blank control well, and then place the cell culture plate in the incubator and culture for 24 h.

[0079] 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 the incubator and culture for 1 h.

[0080] 5. After completing step 4, measure the absorbance of each well at 450 nm, and calculate the cell viability according to formula (4).

[0081]

[0082] A s : Absorbance of the test well; A b : Absorbance of the blank control well; Ac : Absorbance of the negative control well.

[0083] The results are shown in Figure 1 . The results showed that the cell viability of RAW264.7 cells treated with the digestion product at a polypeptide concentration of 2.5 mg / mL was 96.46%. In the general field, it is considered that a cell viability higher than 85% is judged as non-toxic.

[0084] II. Determination of NO production, IL-6 content and TNF-α content The test articles were respectively: the digestion product prepared in Example 6 or the digestion control.

[0085] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash them with PBS buffer, and then resuspend them with complete medium to obtain a cell suspension of 4×10 5 cells / mL.

[0086] 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 h.

[0087] 3. After completing step 2, take the cell culture plate, aspirate the supernatant. Add 200 μL of complete medium to each well of the negative control well and the positive control well, and add 200 μL of complete medium containing the test article to each well of the sample wells (each test article has different polypeptide contents, which are 0.625, 1.25 or 2.5 mg / mL respectively, and each polypeptide content of each test article has three replicates), and then place the cell culture plate in an incubator for 2 h. Each of the negative control well and the positive control well has three replicates.

[0088] 4. After completing step 3, take the cell culture plate, add 10 μL of complete medium containing 20 μg / mL LPS to each well of the positive control well and the sample wells, and add 10 μL of complete medium to each well of the negative control well, and then place the cell culture plate in an incubator for 24 h.

[0089] 5. After completing step 4, take the cell culture plate, collect the supernatant of each well, and detect the NO production, IL-6 content and TNF-α content in the supernatant.

[0090] The results of NO production 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 well is represented by CK, and the positive control well is represented by LPS.

[0091] Example 8. Discovery of anti-inflammatory active peptides I. Mass spectrometry identification of polypeptide sequences The digested product prepared in Example 6 was identified and analyzed by a system combining nano liquid chromatography (Nano LC1000, Thermo Fisher Scientific) with an electrostatic field orbitrap high-resolution mass spectrometer (Orbitrap Q Exactive, Thermo Fisher Scientific, San Jose, CA) (Shevchenko et al., 2006). The analytical column was a C18 reversed-phase chromatography column (75 μm × 15 cm, packed with 3 μm ReproSil-Pur C18-AQ), and the loading column was a C18 column (150 μm × 3 cm, packed with 5 μm ReproSil-Pur C18-AQ). Mobile phase A was an aqueous solution of 0.5% formic acid, and mobile phase B was an acetonitrile solution of 0.5% formic acid. The gradient was optimized based on a nanoflow rate of 300 nL / min to ensure effective separation of the target polypeptides. Mass spectrometry acquisition was performed in data-dependent scanning mode. The data was analyzed using Proteome Discoverer (version 2.1, Thermo Fisher Scientific). The MS2 spectra were searched against the uniprotkb_Chenopodium_quinoa.fasta database using the SEQUEST search engine with the following parameters: trypsin digestion (allowing 2 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. The search results were filtered using the Percolator algorithm ( q value <1%, FDR ≤ 1%).

[0092] II. Computer simulation to screen for potential anti-inflammatory active peptides First, the ToxinPred tool (http: / / webs.iiitd.edu.in / raghava / toxinpred / ) was used to predict the potential toxicity of all polypeptides. After excluding the toxic polypeptides, the PeptideRanker tool (http: / / distilldeep.ucd.ie / PeptideRanker / ) was further used to screen for potential bioactive peptides with scores higher than or equal to 0.5. Finally, the PreAIP tool (http: / / kurata14.bio.kyutech.ac.jp / PreAIP / index.php) was used to predict the scores of these potential bioactive peptides for possible anti-inflammatory activity, thereby screening for potential anti-inflammatory active peptides.

[0093] III. Molecular docking First, download the tertiary structure of the receptor TLR4 / MD2 / LPS (PDBID: 3FXI) complex from the Protein Data Bank (https: / / www.rcsb.org / ), and observe it using PyMOL (version 2.6.0a0) software. Based on the screened potential anti-inflammatory active peptide sequences, predict their three-dimensional structures 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 in the TLR4 / MD2 complex were removed, and Autodock Vina 1.5.6 software was used to dock it with the potential anti-inflammatory active peptides. The docking conditions were as follows: for the TLR4 / MD2 complex, the grid spacing was set to 1 Å; the box grid sizes were 33, 40.5, and 35.25 Å (x, y, z) respectively; the box center coordinates were 25.692, -5.342, and 14.883 (x, y, z) respectively. The docking results were visually analyzed using Discovery Studio 2021 Client software.

[0094] The active peptide discovered based on the above steps was named polypeptide SCAWLLAWSAPK. The connection between polypeptide SCAWLLAWSAPK and the TLR4 / MD2 complex was mainly through hydrophobic interactions, hydrogen bonds, and electrostatic interactions, etc.

[0095] The amino acid sequence of polypeptide SCAWLLAWSAPK (SEQ ID NO: 1): SCAWLLAWSAPK.

[0096] Example 9, Synthesis and Functional Verification of Active Peptide Complete medium: DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0097] Incubator conditions: 37 °C, 5% CO 2 。

[0098] I. Synthesis of Active Peptide Synthetic polypeptide SCAWLLAWSAPK (i.e., the polypeptide shown in SEQ ID NO: 1).

[0099] II. Determination of Cell Viability Test sample: Polypeptide SCAWLLAWSAPK prepared in step 1.

[0100] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash them with PBS buffer, and then resuspend them with complete medium to obtain 4×10 5Cell suspension at [X] cells / mL.

[0101] 2. Take a 96-well cell culture plate. Add 200 μL of the cell suspension prepared in step 1 to each test well and negative control well, and add 200 μL of complete medium to each blank control well. Then place the cell culture plate in an incubator and culture for 24 h. Set three replicate wells for both the blank control well and the negative control well.

[0102] 3. After completing step 2, take the cell culture plate, aspirate the supernatant. Add 200 μL of complete medium containing the test article to each test well (set different test article contents, namely 0.1, 0.5, 1, or 2 mg / mL, and set three replicate wells for each content), add 200 μL of complete medium to each negative control well, and add 200 μL of complete medium to each blank control well. Then place the cell culture plate in an incubator and culture for 24 h.

[0103] 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 and culture for 1 h.

[0104] 5. After completing step 4, measure the absorbance of each well at 450 nm, and calculate the cell viability according to formula (4).

[0105] The results are shown in Figure 5 (CK corresponds to the negative control well). The results show that the cell viability of RAW264.7 cells treated with the polypeptide SCAWLLAWSAPK at a concentration of 1 mg / mL is 91.74%. In the general field, it is considered that a cell viability higher than 85% is judged as non-toxic.

[0106] III. Determination of NO production, IL-6 content, and TNF-α content Test article: The polypeptide SCAWLLAWSAPK prepared in step one.

[0107] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash them with PBS buffer, and then resuspend them with complete medium to obtain a cell suspension at [X] cells / mL. 5 Cell suspension at [X] cells / mL.

[0108] 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 and culture for 24 h.

[0109] 3. After completing Step 2, take the cell culture plate, aspirate and discard the supernatant. Add 200 μL of complete medium to each well of the negative control wells and positive control wells, and add 200 μL of complete medium containing the test article to each well of the sample wells (set different test article contents, namely 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, or 1 mg / mL, and set three replicates for each content), then place the cell culture plate in an incubator and culture for 2 h. Set three replicates for each of the negative control wells and positive control wells.

[0110] 4. After completing Step 3, take the cell culture plate, add 10 μL of complete medium containing 20 μg / mL LPS to each well of the positive control wells and sample wells, and add 10 μL of complete medium to each well of the negative control wells, then place the cell culture plate in an incubator and culture for 24 h.

[0111] 5. After completing Step 4, take the cell culture plate, collect the supernatant of each well, and detect the NO production amount, IL-6 content, and TNF-α content in the supernatant.

[0112] The results of the NO production amount are shown in Figure 6 . The results of the IL-6 content are shown in Figure 7 . The results of the TNF-α content are shown in Figure 8 . Figures 6 to 8 Among them, the negative control wells are represented by CK, and the positive control wells are represented by LPS. When the concentration of the polypeptide SCAWLLAWSAPK is 1 mg / mL, the inhibition rates of the production amounts of the three inflammatory mediators (NO, IL-6, TNF-α) are 69.15 ± 3.48%, 94 ± 0.65%, and 86.32 ± 1.36% respectively. Inhibition rate = ((content of positive control well - content of sample well) / (content of positive control well - content of negative control well)) × 100%.

[0113] The above has detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application. Some basic features can be applied according to the scope of the following appended 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 a product for inhibiting inflammation.

4. The use according to claim 3, characterized in that: The inhibition of inflammation is manifested in the inhibition of the secretion of inflammatory mediators by immune cells.

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

6. The use according to claim 4 or 5, characterized in that: The immune cells are macrophages.

7. A product for inhibiting inflammation, characterized in that: The product contains the polypeptide according to claim 1 or the pharmaceutically acceptable salt according to claim 2.

8. The product according to claim 7, characterized in that: The inhibition of inflammation is manifested in the inhibition of the secretion of inflammatory mediators by immune cells.

9. The product according to claim 8, characterized in that: The inflammatory mediators are nitric oxide and / or interleukin-6 and / or tumor necrosis factor-α.

10. The product according to claim 8 or 9, characterized in that: The immune cells are macrophages.

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