Anti-inflammatory peptide slsallqlir derived from quinoa and application thereof

Abstract

The application discloses an anti-inflammatory peptide SLSALLQLIR derived from quinoa and application thereof. The application provides a polypeptide, also known as polypeptide SLSALLQLIR, and the amino acid sequence of the polypeptide is SEQ ID NO:1. The application also protects a medicinal salt of the polypeptide. The application also protects application of the polypeptide or the medicinal salt in preparation of a product (such as a medicine, a reagent or a preparation) for inhibiting inflammation. The inhibition of inflammation is embodied as inhibition of secretion of inflammatory mediators by immune cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptides, and relates to an anti-inflammatory peptide SLSALLQLIR derived from quinoa and application thereof. BACKGROUND

[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, excessive production of these mediators can lead to tissue damage and loss of immune function. Chronic inflammatory response is often closely related to the occurrence of diseases such as type 2 diabetes, obesity, inflammatory bowel disease and neurodegenerative disease. Therefore, it is of great significance to find effective anti-inflammatory substances and methods for the prevention and treatment of related diseases.

[0003] Quinoa is an annual dicotyledonous plant of the Chenopodium genus, which has attracted worldwide attention due to its high protein content and balanced essential amino acids. Quinoa protein beverage is a deep-processed product of quinoa, which is prepared by combining starch enzymolysis and high-pressure homogenization treatment with quinoa powder as raw material. The protein content of the quinoa protein beverage is higher than 1%. Quinoa protein beverage is suitable for celiac patients to eat 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 exert physiological activity in the form of active peptides. However, there is no research on screening anti-inflammatory active peptides from processed plant protein beverages. SUMMARY

[0004] The technical problem to be solved by the present application is how to inhibit the secretion of inflammatory mediators by immune cells.

[0005] To solve the above technical problem, the present application provides a polypeptide (polypeptide SLSALLQLIR), whose amino acid sequence is SEQ ID NO: 1.

[0006] The present application also protects the pharmaceutical salts of the polypeptide.

[0007] The pharmaceutical 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, glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide,tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, triethiodide, lactate, valerate, and the like. Depending on the use, pharmaceutical salts can be formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, and zinc, bismuth, 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 from the free acid by standard methods, for example by reaction of the free acid with the organic or inorganic base. In the presence of an alkaline group such as an amino group, acidic salts such as hydrochloride, hydrobromide, acetate, pamoate, and the like can be used as dosage forms; in the presence of an acidic group (such as -COOH) or an alcoholic group, pharmaceutically acceptable esters such as acetate, maleate, pivaloyloxymethyl, and the like, and esters known in the literature for improving solubility and hydrolysis can be used as sustained release and prodrug formulations.

[0008] The present application also protects the use of the polypeptide or the pharmaceutical salt in the preparation of a product (such as a drug, reagent, or preparation) for inhibiting inflammation.

[0009] The inhibition of inflammation is manifested as the inhibition of the secretion of inflammatory mediators by immune cells.

[0010] The inflammatory mediator is nitric oxide (NO) and / or interleukin-6 (IL-6) and / or tumor necrosis factor-alpha (TNF-alpha).

[0011] The immune cell is a macrophage.

[0012] The present application also protects a product (such as a medicament, reagent or preparation) for inhibiting inflammation, the product comprising the polypeptide or the pharmaceutical salt.

[0013] The inhibition of inflammation is manifested by inhibition of secretion of inflammatory mediators by immune cells.

[0014] The inflammatory mediator is NO and / or IL-6 and / or TNF-alpha.

[0015] The immune cell is a macrophage.

[0016] The product can also include a pharmaceutical carrier.

[0017] The carrier, including but not limited to water-soluble carrier material (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), insoluble carrier material (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier material (such as cellulose acetate phthalate and carboxymethyl cellulose, etc.). Among them, the water-soluble carrier material is preferred. Using these materials can be made into a variety of dosage forms, including but not limited to tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. Among them, the suppository can be a vaginal suppository, also can be a vaginal ring, also can be a cream, cream or gel suitable for vaginal application. It can be a general preparation, sustained-release preparation, controlled-release preparation and various microparticle drug delivery systems. In order to make the unit administration dosage form into tablets, a variety of carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders such as water, glycerol, polyethylene glycol, ethanol, propyl alcohol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, alginic acid, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glycerol tri-stearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salt, sodium dodecyl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further coated, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit administration dosage form into pills, a variety of carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to make the unit administration dosage form into suppositories, a variety of carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to make the unit administration dosage form into injection 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, polyoxyethylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc.In addition, for the preparation of isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation, and in addition, a conventional solubilizing agent, a buffer, a pH adjustor, etc. can be added. In addition, if necessary, a coloring agent, a preservative, a flavoring agent, a sweetener or other materials can be added to the pharmaceutical preparation.

[0018] The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intraperitoneal injection, intracisternal injection or infusion, etc.; cavity administration, such as rectal, vaginal and sublingual; respiratory tract administration, such as nasal cavity; mucosal administration. The above administration route is preferably injection administration, and the preferred injection route is subcutaneous injection.

[0019] The present application also provides a pharmaceutical compound, wherein the active ingredient of the pharmaceutical compound is the polypeptide or the pharmaceutical salt.

[0020] The pharmaceutical compound is used for inhibiting inflammation.

[0021] The inhibition of inflammation is embodied in the inhibition of immune cells from secreting inflammatory mediators.

[0022] The inflammatory mediators are NO and / or IL-6 and / or TNF-α.

[0023] The immune cells are macrophages.

[0024] The present application also provides a method for inhibiting inflammation of an animal, comprising the step of administering the polypeptide or the pharmaceutical salt to the recipient animal.

[0025] The inhibition of inflammation is embodied in the inhibition of immune cells from secreting inflammatory mediators.

[0026] The inflammatory mediators are NO and / or IL-6 and / or TNF-α.

[0027] The immune cells are macrophages.

[0028] The animal is a mammal or a human.

[0029] As an example, the animal is a mouse.

[0030] In the present application, a new anti-inflammatory peptide is screened from quinoa protein beverage by combining in vitro digestion, computer simulation screening and molecular docking technology, and has good anti-inflammatory activity. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Results of cell activity in Example 7.

[0032] Figure 2 Results of NO production amount in Example 7.

[0033] Figure 3Results for IL-6 content in Example 7.

[0034] Figure 4 Results for TNF-α content in Example 7.

[0035] Figure 5 Results for cell viability in Example 9.

[0036] Figure 6 Results for NO production in Example 9.

[0037] Figure 7 Results for IL-6 content in Example 9.

[0038] Figure 8 Results for TNF-α content in Example 9. DETAILED DESCRIPTION

[0039] The application will be further described in conjunction with the preferred embodiments thereof, given solely by way of illustration of the present application and not by way of limitation thereof. The following examples provided serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation of the present application.

[0040] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. In the following examples, the materials, reagents, etc. used are commercially available, unless otherwise specified. In the following examples, the quantitative tests are performed in triplicate, and the results are averaged, unless otherwise specified. In the drawings of the specification, English letters represent the degree of significant difference, and treatments with at least one identical letter are not significantly different at the 0.05 level, and treatments without identical letters are significantly different at the 0.05 level. In the examples, the Kjeldahl method is 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 Biyun Tian 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.

[0041] Example 1, Preparation of Quinoa Protein Beverage

[0042] Firstly, the quinoa flakes were ground several times to obtain fine quinoa powder.

[0043] Subsequently, the quinoa powder was mixed with purified water at a solid-liquid ratio of 1:3, and 0.5% of α-amylase and maltase by weight of the quinoa powder were added. After mixing, the enzyme hydrolysis was carried out at 70°C for 45 min in a water bath, and then the homogenate was cooled to 55°C.

[0044] Then, CaCO3, Ca3(PO4)2, edible salt, and edible oil were added in the proportions of 0.16%, 0.14%, 0.1%, and 0.8%, respectively, based on the final beverage volume. The solid-liquid ratio was adjusted to 1:12 with purified water, and the mixture was thoroughly stirred and mixed.

[0045] Then, the high-pressure homogenizer was used for homogenization treatment (30 MPa and 60 MPa pressure treatment for 2 min, respectively), and the quinoa protein beverage was obtained.

[0046] The quinoa protein beverage was taken and freeze-dried to obtain a freeze-dried powder, which was stored at 4°C.

[0047] Example 2, preparation of simulated digestion storage solution

[0048] According to the INFOGEST in vitro simulated digestion method, 1.25-fold concentrated simulated salivary fluid (SSF) storage solution, simulated gastric fluid (SGF) storage solution, and simulated intestinal fluid (SIF) storage solution were prepared. The SSF storage solution, SGF storage solution, and SIF storage solution were all composed of salt solutions and water. The various salt solutions and their added volumes for each 400 mL of storage solution are shown in Table 1. In Table 1, the volume of the SSF storage solution, SGF storage solution, and SIF storage solution is 400 mL, the pH value is 7, and the concentration is 1.25×.

[0049] Table 1 Formulation of storage solution

[0050]

[0051] Note: The aqueous CaCl2 solution is added before use.

[0052] Example 3, determination of pepsin enzyme activity (spectrophotometry)

[0053] 1. Preparation of 2% bovine hemoglobin solution

[0054] 0.5 g of bovine hemoglobin was dissolved in 20 mL of ultrapure water, and the pH was adjusted to 2 with 300 mM HCl solution, and the volume was adjusted to 25 mL.

[0055] 2. Preparation of pepsin diluent.

[0056] Pepsin (commercially available solid preparation) was taken and a solution of pepsin with a concentration of 1 mg / mL was prepared using a buffer containing 10 mM Tris and 150 mM NaCl (pH 6.5) as solvent, i.e. a pepsin enzyme solution.

[0057] Before use, the pepsin enzyme solution was diluted with a 10 mM HC1 solution so that the pepsin concentration was 5, 10, 15, 20, 25, 30 or 35 μg / mL, i.e. a pepsin diluent, which was stored on ice.

[0058] 3. Enzyme reaction tubes (denoted Test) and blank tubes (denoted Blank) were prepared, and 500 μL of a 2% bovine hemoglobin solution was added to each tube, which was then placed in a 37°C water bath for 3-4 min.

[0059] 4. After step 3, 100 μL of the pepsin diluent was added to each of the enzyme reaction tubes, which was then placed in a 37°C water bath for 10 min, and then 1 mL of a 5% TCA (w / v) solution was added to each tube to terminate the reaction. After step 3, 1 mL of a 5% TCA (w / v) solution was added to each of the blank tubes, and then 100 μL of the pepsin diluent was added to each tube.

[0060] 5. After step 4, all the reaction tubes were centrifuged at 6000 g for 30 min, and the supernatant was collected.

[0061] 6. The supernatant obtained in step 5 was taken and the absorbance at 280 nm was measured after equilibration at room temperature for 5 min.

[0062] 7. The pepsin enzyme activity was calculated according to formula (1).

[0063]

[0064] Δt: reaction duration, i.e. 10 min;

[0065] X: the amount of pepsin contained in each mL of the enzyme solution to be measured, i.e. 5, 10, 15, 20, 25 or 30 μg;

[0066] 1000: dilution factor for converting μg to mg;

[0067] 0.001: the absorbance change value (ΔA280) caused by each unit of pepsin.

[0068] Example 4, determination of trypsin enzyme activity

[0069] 1. Take pancreatin (commercially available solid preparation), and prepare pancreatin solutions with pancreatin contents of 0.25, 0.5, or 1 mg / mL using 1 mM HCl solution as solvent, and place on ice.

[0070] 2. Prepare enzyme reaction tubes (denoted as Test) and blank tubes (denoted as Blank), and 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.

[0071] Substrate solution: 10 mM TAME solution.

[0072] Working solution: Tris-HCl buffer (pH 8.1, 46 mM) containing 11.5 mM CaCl2.

[0073] 3. After completing step 2, add 100 μL of pancreatin solution to each enzyme reaction tube, and add 100 μL of working solution to each blank tube, mix quickly, and then measure the absorbance change at 247 nm within 10 min (measure once every 10 s).

[0074] 4. Calculate the trypsin activity according to formula (2).

[0075]

[0076] ΔA247: slope, unit absorbance / min;

[0077] 1000: factor for converting mL to μL;

[0078] 3: total reaction volume (mL), 3 mL;

[0079] 540: molar extinction coefficient of TAME at 247 nm (L / (mol×cm));

[0080] X: corresponding enzyme amount (mg) in 100 μL of enzyme solution.

[0081] Example 5, Bile acid content determination

[0082] A bile acid determination kit is used (each component of the working solution is provided by the kit).

[0083] Internal standard solution: 80 µM sodium cholate solution.

[0084] Pig bile solution: take pig bile extract (commercially available solid preparation), and prepare a 1 mg / mL solution using ultrapure water as solvent, which is the pig bile solution. Sample solution: take the pig bile solution, and dilute to 25 times the volume using ultrapure water, which is the sample solution.

[0085] 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.

[0086] Take a black enzyme plate, add 20 μL of sample solution, 5 μL of internal standard solution and 80 μL of working solution 1 to each well of the internal standard group (indicated by Internal standard), add 20 μL of sample solution, 5 μL of ultrapure water and 80 μL of working solution 1 to each well of the sample group (indicated by Test), and add 20 μL of sample solution, 5 μL of ultrapure water and 80 μL of working solution 2 to each well of the blank group (indicated by Blank), then incubate in the dark for 20 min. Then, detect the fluorescence value (λ ex = 530 nm / λ em = 585 nm) of each well, i.e. F value, and then calculate the bile acid content of the pig bile solution according to formula (3).

[0087]

[0088] n: the dilution factor of the sample solution prepared from the pig bile solution, which is 25.

[0089] 20: the addition volume (µL) of the sample solution, which is 20 μL;

[0090] 1000: the dilution factor for converting µM to mM.

[0091] Example 6, in vitro simulated digestion

[0092] The entire in vitro simulated digestion process includes three consecutive stages: oral digestion, gastric digestion and intestinal digestion.

[0093] Take the freeze-dried powder obtained in Example 1, reconstitute it with ultrapure water to make the protein concentration 60 mg / mL, i.e. the sample to be digested.

[0094] Oral digestion stage: first, preheat the SSF stock solution in a 37℃ 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 then incubate in a 37℃ water bath for 2 min (magnetic stirring is performed during the process), to obtain the digestion product simulating oral digestion. Since the quinoa protein beverage has been subjected to enzymatic treatment with two enzymes during preparation, its carbohydrate content has been significantly reduced, and the quinoa protein purity of the quinoa protein beverage is higher than 90%, so no salivary amylase is added in this stage.

[0095] Gastric digestion stage: porcine pepsin was prepared into a 40000 U / mL solution (enzyme activity determination method see Example 3) with ultrapure water, namely porcine pepsin solution; 10 mL of simulated oral digestion digestion product was taken, 8 mL of 37℃ preheated SGF stock solution was added, then the pH of the system was adjusted to 3.0 with 1 M HC1 solution, then 1 mL of 37℃ preheated porcine pepsin solution was added, and then ultrapure water was added to a total system volume of 20 mL; finally, the whole system was incubated in a 37℃ water bath for 2 h (magnetic stirring was carried out during the process), and the simulated gastric digestion digestion product was obtained.

[0096] Intestinal digestion stage: porcine pancreatin was prepared into an 800 U / mL solution (enzyme activity determination method see Example 4) with SIF stock solution, namely porcine pancreatin solution; porcine bile extract was prepared into a solution containing 160 mM bile acid (bile acid content determination method see Example 5) with SIF stock solution, namely porcine bile solution; 20 mL of simulated gastric digestion digestion product was taken, 8.5 mL of 37℃ preheated SIF stock solution was added, then the pH of the system was adjusted to 7.0 with 1 M HC1 solution or 1 M NaOH solution, then 2.5 mL of 37℃ preheated porcine bile solution and 5 mL of 37℃ preheated porcine pancreatin solution were added, and then ultrapure water was added to a total system volume of 40 mL; finally, the whole system was incubated in a 37℃ water bath for 2 h (magnetic stirring was carried out during the process), and the in vitro simulated digestion product was obtained.

[0097] The in vitro simulated digestion product was freeze-dried to obtain a dry powder substance, which was named digestion product and stored at -20℃.

[0098] 5 mL of ultrapure water was used instead of the sample to be digested, and the above-mentioned oral digestion stage, gastric digestion stage and intestinal digestion stage were carried out in turn, and then freeze-drying was carried out to obtain a dry powder substance, which was named digestion control and stored at -20℃.

[0099] Example 7, Effect of in vitro simulated digestion product on cells

[0100] Complete culture medium: DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0101] Incubator conditions: 37℃, 5% CO2.

[0102] I. Determination of cell viability

[0103] Test samples were: digestion product or digestion control prepared in Example 6.

[0104] 1. Collect RAW264.7 cells in logarithmic growth phase, wash with PBS buffer, then resuspend with complete culture medium to obtain a cell suspension of 4 x 10 5

[0105] 2. Take a 96-well cell culture plate, add 200 μL of the cell suspension prepared in step 1 to each well of the test wells and negative control wells, and add 200 μL of complete culture medium to each well of the blank control wells, then place the cell culture plate in an incubator and culture for 24 h. Three replicate wells are set for the blank control wells and the negative control wells.

[0106] 3. After step 2 is completed, take the cell culture plate, discard the supernatant, add 200 μL of complete culture medium containing the test sample to each well of the test wells (different polypeptide contents are set for each test sample, i.e. 0.625, 1.25, 2.5 or 5 mg / mL, and three replicate wells are set for each polypeptide content of each test sample; polypeptide concentration is measured by Ortho-Phthaldehyde method), add 200 μL of complete culture medium to each well of the negative control wells, and add 200 μL of complete culture medium to each well of the blank control wells, then place the cell culture plate in an incubator and culture for 24 h.

[0107] 4. After step 3 is completed, take the cell culture plate, add 10 μL of CCK solution to each well, then place the cell culture plate in an incubator and culture for 1 h.

[0108] 5. After step 4 is completed, measure the absorbance of each well at 450 nm, and calculate the cell activity according to formula (4).

[0109]

[0110] A s : absorbance of test wells; A b : absorbance of blank control wells; A c : absorbance of negative control wells.

[0111] The results are shown in Figure 1 . The results show that the cell activity of RAW264.7 cells treated with the digestion product at a polypeptide concentration of 2.5 mg / mL is 96.46%, which is generally considered to be non-toxic in the field if the cell activity is higher than 85%.

[0112] II. Determination of NO production, IL-6 content and TNF-α content

[0113] The test samples are the digestion product prepared in Example 6 or the digestion control.

[0114] 1. Collect RAW264.7 cells in logarithmic growth phase, wash with PBS buffer, then resuspend with complete culture medium to obtain a cell suspension of 4 x 10​5 a cell suspension of 1 x 106cells / mL.

[0115] 2. Take 96-well cell culture plates, add 200 μL of the cell suspension prepared in step 1 to each well, and then place the cell culture plates in an incubator for 24 h.

[0116] 3. After step 2 is completed, take the cell culture plates, discard the supernatant, add 200 μL of complete medium to each well of the negative control wells and the positive control wells, and add 200 μL of complete medium containing the test sample to each well of the sample wells (set different polypeptide contents for each test sample, i.e. 0.625, 1.25 or 2.5 mg / mL, and set three replicate wells for each polypeptide content of each test sample), and then place the cell culture plates in an incubator for 2 h. Set three replicate wells for each of the negative control wells and the positive control wells.

[0117] 4. After step 3 is completed, take the cell culture plates, add 10 μL of complete medium containing 20 μg / mL LPS to each well of the positive control wells and the sample wells, and add 10 μL of complete medium to each well of the negative control wells, and then place the cell culture plates in an incubator for 24 h.

[0118] 5. After step 4 is completed, take the cell culture plates, collect the supernatant of each well, and detect the amount of NO production, the content of IL-6 and the content of TNF-α in the supernatant.

[0119] The results of the amount of NO production are shown in Table 1. Figure 2 The results of the content of IL-6 are shown in Table 2. Figure 3 The results of the content of TNF-α are shown in Table 3. Figure 4 Figures 2 to 4 In Table 1, the negative control wells are represented by CK, and the positive control wells are represented by LPS.

[0120] Example 8. Discovery of Anti-inflammatory Activity Peptides

[0121] I. Mass Spectrometry Identification of Polypeptide Sequences

[0122] ​The digested products prepared in Example 6 were subjected to identification analysis by a nano liquid chromatography (Nano LC1000, Thermo Fisher Scientific) coupled with an electrostatic field orbitrap high-resolution mass spectrometer (Orbitrap Q Exactive, Thermo Fisher Scientific, San Jose, CA) system (Shevchenko et al., 2006). The analytical column was a C18 reversed-phase chromatographic column (75 μm x 15 cm, 3 μm ReproSil-Pur C18-AQ packing), and the sample column was a C18 column (150 μm x 3 cm, 5 μm ReproSil-Pur C18-AQ packing). The mobile phase A was 0.5% formic acid aqueous solution, and the mobile phase B was 0.5% formic acid acetonitrile solution. Based on the nano flow rate optimization gradient of 300 nL / min, the target polypeptides were effectively separated. The mass spectrometry acquisition was performed in a data-dependent scanning mode. The data were analyzed by Proteome Discoverer (version 2.1, Thermo Fisher Scientific), and the MS2 spectrum was searched by the SEQUEST search engine against the uniprotkb_Chenopodium_quinoa.fasta database 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 as cysteine alkylation, and variable modification as methionine oxidation. The search results were filtered by the Percolator algorithm (http: / / percolator.org / ) with the value <1% and FDR ≤ 1%. q

[0123] II. In silico screening of potential anti-inflammatory activity peptides

[0124] Firstly, the potential toxicity of all polypeptides was predicted by the ToxinPred tool (http: / / webs.iiitd.edu.in / raghava / toxinpred / ). After excluding the polypeptides with toxicity, the PeptideRanker tool (http: / / distilldeep.ucd.ie / PeptideRanker / ) was further used to screen the 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 the potential bioactive peptides that might have anti-inflammatory activity, so as to screen the potential anti-inflammatory activity peptides.

[0125] III. Molecular docking

[0126] ​First, the tertiary structure of the receptor TLR4 / MD2 / LPS (PDB ID: 3FXI) complex was downloaded from the protein data (https: / / www.rcsb.org / ) and observed using PyMOL (version 2.6.0a0) software. Based on the potential anti-inflammatory activity peptide sequence screened, its three-dimensional structure was predicted using PEP-FOLD 4 (https: / / mobyle2.rpbs.univ-paris-diderot.fr / cgi-bin / portal.py#forms::PEP-FOLD4). In order to carry out molecular docking analysis, water and redundant ligands in the TLR4 / MD2 complex were removed, and Autodock Vina 1.5.6 software was used for docking analysis with the potential anti-inflammatory activity peptide. The docking conditions are as follows: for the TLR4 / MD2 complex, the box spacing is set to 1 Å; the box grid size is 33, 40.5 and 35.25 Å (x, y, z), respectively; and the box center coordinates are 25.692, -5.342 and 14.883 (x, y, z), respectively. The docking results were visualized and analyzed using Discovery Studio 2021 Client software.

[0127] The active peptide discovered based on the above steps is named polypeptide SLSALLQLIR. The polypeptide SLSALLQLIR is mainly connected with the TLR4 / MD2 complex through hydrogen bonds and hydrophobic interactions, etc.

[0128] The amino acid sequence of the polypeptide SLSALLQLIR (SEQ ID NO: 1) is SLSALLQLIR.

[0129] Example 9, synthesis and functional verification of active peptide

[0130] Complete medium: DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0131] Incubator conditions: 37°C, 5% CO2.

[0132] I. Synthesis of active peptide

[0133] The polypeptide SLSALLQLIR (i.e., the polypeptide shown in SEQ ID NO: 1) was artificially synthesized.

[0134] II. Determination of cell viability

[0135] Test sample: polypeptide SLSALLQLIR prepared in step I.

[0136] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash with PBS buffer, then resuspend with complete medium to obtain 4x105 a cell suspension of 4 x 105cells / mL.

[0137] 2. Take a 96-well cell culture plate, and add 200 μL of the cell suspension prepared in step 1 to each well of the test wells and the negative control wells, and add 200 μL of complete culture medium to each well of the blank control wells, and then place the cell culture plate in an incubator for 24 h. Three replicate wells are set for the blank control wells and the negative control wells.

[0138] 3. After step 2 is completed, take the cell culture plate, discard the supernatant, add 200 μL of complete culture medium containing the test sample (set different test sample concentrations, 0.1, 0.5, 1 or 2 mg / mL, and three replicate wells are set for each concentration) to each well of the test wells, add 200 μL of complete culture medium to each well of the negative control wells, and add 200 μL of complete culture medium to each well of the blank control wells, and then place the cell culture plate in an incubator for 24 h.

[0139] 4. After step 3 is completed, take the cell culture plate, and add 10 μL of CCK solution to each well, and then place the cell culture plate in an incubator for 1 h.

[0140] 5. After step 4 is completed, measure the absorbance of each well at 450 nm, and calculate the cell viability according to formula (4).

[0141] The results are shown in Table 1. Figure 5 (CK corresponds to the negative control wells). The results show that the cell viability of RAW264.7 cells treated with the polypeptide SLSALLQLIR at a concentration of 1 mg / mL is 94.86%, and it is generally considered in the field that a cell viability higher than 85% is interpreted as non-toxic.

[0142] III. Determination of NO production amount, IL-6 content and TNF-α content

[0143] Test sample: the polypeptide SLSALLQLIR prepared in step 1.

[0144] 1. Collect RAW264.7 cells in the logarithmic growth phase, wash with PBS buffer, and then resuspend with complete culture medium to obtain a cell suspension of 4 x 105cells / mL. 5 a cell suspension of 4 x 105cells / mL.

[0145] 2. Take a 96-well cell culture plate, and 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.

[0146] 3、After step 2, take the cell culture plate, suck off the supernatant, add 200 μL complete culture medium to each well of the negative control wells and the positive control wells, and add 200 μL complete culture medium containing the test sample to each well of the sample wells (set different test sample concentrations, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75 or 1 mg / mL, and set three replicate wells for each concentration), and then place the cell culture plate in an incubator for 2 h. Set three replicate wells for each of the negative control wells and the positive control wells.

[0147] 4、After step 3, take the cell culture plate, add 10 μL complete culture medium containing 20 μg / mL LPS to each well of the positive control wells and the sample wells, and add 10 μL complete culture medium to each well of the negative control wells, and then place the cell culture plate in an incubator for 24 h.

[0148] 5、After 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.

[0149] The results of the NO production are shown in Table Figure 6 The results of the IL-6 content are shown in Table Figure 7 The results of the TNF-α content are shown in Table Figure 8 . Figures 6 to 8 In Table 1, the negative control wells are represented by CK, and the positive control wells are represented by LPS. The inhibition rates of the polypeptide SLSALLQLIR at a concentration of 1 mg / mL on the production of the three inflammatory mediators (NO, IL-6 and TNF-α) are 70.6 ± 3.15%, 94.76 ± 0.24% and 82.65 ± 1.16%, respectively. Inhibition rate = ((content of positive control wells - content of sample wells) / (content of positive control wells - content of negative control wells)) x 100%.

[0150] The above has described the present application in detail. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application, and without performing unnecessary experiments. Although the present application gives a specific example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, the present application is intended to include any changes, uses or improvements of the present application, including changes made by using conventional techniques known in the art, which are out of the scope disclosed in the present 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 pharmaceutical salt of the polypeptide of claim 1.

3. Use of the polypeptide of claim 1 or the pharmaceutical salt of claim 2 in the preparation of a medicament for inhibiting inflammation; the inhibiting inflammation is embodied as inhibiting immune cells from secreting inflammatory mediators.

4. Use of the polypeptide of claim 1 or the pharmaceutical salt of claim 2 in the preparation of a reagent for inhibiting inflammation; the inhibiting inflammation is embodied as inhibiting immune cells from secreting inflammatory mediators.

5. A medicament for inhibiting inflammation, characterized by: The medicament contains the polypeptide of claim 1 or the pharmaceutical salt of claim 2.

6. An agent for inhibiting inflammation, characterized by: The reagent contains the polypeptide of claim 1 or the pharmaceutical salt of claim 2.

7. A pharmaceutical compound, characterized by: The active ingredient of the pharmaceutical compound is the polypeptide of claim 1 or the pharmaceutical salt of claim 2.

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