Anti-inflammatory peptides AF and FL and application thereof

Abstract

The invention relates to the technical field of plant active peptides, in particular to anti-inflammatory peptides AF and FL and application thereof. The amino acid sequences of the anti-inflammatory peptide AF and the anti-inflammatory peptide FL are ASHFGLQRQF and FFHLQQQRL respectively. The polypeptide is prepared through enzymolysis of red kidney bean protein, and the polypeptide is identified by utilizing ultrafiltration separation and LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry); the anti-inflammatory peptides FL and AF which are high in anti-inflammatory performance, free of toxicity, allergenicity and carcinogenicity and good in human intestinal absorption capacity are obtained through screening with a bioinformatics method. Polypeptides are artificially synthesized, the anti-inflammatory activity of the anti-inflammatory peptides is verified through experiments, and it is found that the anti-inflammatory peptides FL and AF have good anti-inflammatory activity, have no obvious toxic or side effect on RAW264.7, can obviously inhibit the release amount of RAW264.7 cell NO induced by LPS and inhibit expression of inflammatory factors iNOS, TNF-alpha and IL-6, and can be used for preparing anti-inflammatory drugs or products.

Description

Technical Field

[0001] The invention belongs to the field of plant active peptides, and in particular relates to anti-inflammatory peptides AF and FL and applications thereof. Background Art

[0002] Inflammation is a basic pathological process in which the body reacts to various damaging factors as a defensive response. Normally, inflammation is beneficial and is an automatic defensive response of the human body, but excessive inflammation is harmful to the body, causing a decrease in the body's resistance and tissue damage. Inflammation is associated with a variety of diseases, such as diabetes, inflammatory bowel disease, cancer, and so on. Therefore, it is urgent to develop natural drugs with anti-inflammatory activity.

[0003] Bioactive peptides are polypeptides with certain physiological regulatory functions. Anti-inflammatory active peptides are a class of bioactive peptides with anti-inflammatory activity, which mainly inhibit the body's inflammatory response by regulating the secretion of cytokines and the synthesis of inflammatory mediators. Numerous studies have shown that food-derived active peptides have a certain alleviating effect on inflammation. Compared with traditional anti-inflammatory drugs, food-derived anti-inflammatory active peptides are safe and have no side effects. Therefore, the development of anti-inflammatory peptides with good safety and high anti-inflammatory properties from food has good prospects and significance. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide anti-inflammatory peptides AF and FL and their applications. The present invention uses red kidney beans as raw materials, prepares active peptides by enzymatic hydrolysis, performs ultrafiltration separation according to molecular weight, and performs LC-MS / MS identification on the components with good anti-inflammatory activity after ultrafiltration separation to obtain peptide information in the components; uses BIOPEP database and PeptideRanker to screen polypeptides with high scores and anti-inflammatory activity; uses ADMET to screen polypeptides with no acute oral toxicity, no carcinogenicity and good intestinal absorption capacity; uses the AllergenFP program to screen non-sensitizing polypeptides; uses molecular docking again to perform molecular docking on a single anti-inflammatory peptide and iNOS; and finally verifies the anti-inflammatory activity of the anti-inflammatory polypeptide through experiments.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] The first aspect of the present invention is an anti-inflammatory peptide, which is anti-inflammatory peptide AF or anti-inflammatory peptide FL. The amino acid sequence of anti-inflammatory peptide AF is ASHFGLQRQF, and the amino acid sequence of anti-inflammatory peptide FL is FFHLQQQRL.

[0007] Furthermore, the molecular weights of anti-inflammatory peptide AF and anti-inflammatory peptide FL are 1190.3 Da and 1216.4 Da, respectively.

[0008] The second aspect of the present invention is the use of the anti-inflammatory peptide described in the first aspect in the preparation of anti-inflammatory drugs or products.

[0009] The third aspect of the present invention is an anti-inflammatory drug or product, which comprises the anti-inflammatory peptide described in the first aspect.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention uses red kidney beans as raw materials, prepares active polypeptides by enzymatic hydrolysis, uses ultrafiltration separation and LC-MS / MS to identify polypeptides; uses bioinformatics methods to screen anti-inflammatory peptides; artificially synthesizes polypeptides and verifies the anti-inflammatory activity of anti-inflammatory peptides by experiments. The anti-inflammatory peptides obtained by screening have good anti-inflammatory activity, have no obvious toxic side effects on RAW264.7, can significantly inhibit the NO release of RAW264.7 cells induced by LPS, and inhibit the expression of inflammatory factors iNOS, TNF-α and IL-6, and can be used for the preparation of anti-inflammatory drugs or products. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The structures of the anti-inflammatory peptides AF and FL;

[0013] Figure 2 Docking results of anti-inflammatory peptide AF and iNOS;

[0014] Figure 3 Docking results of anti-inflammatory peptide FL and iNOS;

[0015] Figure 4 Effects of anti-inflammatory peptides on RAW264.7 cell viability;

[0016] Figure 5 Effects of anti-inflammatory peptides on LPS-induced NO release in RAW264.7 cells.

[0017] Figure 6 Effects of anti-inflammatory peptides on LPS-induced iNOS expression in RAW264.7 cells.

[0018] Figure 7 Effects of anti-inflammatory peptides on LPS-induced TNF-α expression in RAW264.7 cells.

[0019] Figure 8 Effects of anti-inflammatory peptides on LPS-induced IL-6 expression in RAW264.7 cells. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0021] Example 1

[0022] Screening of anti-inflammatory peptides

[0023] Using red kidney beans as raw materials, active peptides were prepared by pepsin hydrolysis, separated by ultrafiltration according to molecular weight, and the components with molecular weight less than 3KD were identified by LC-MS / MS. PeptideRanker was used to score the potential functions of the peptides, and a score greater than 0.5 indicated that the peptide had potential biological activity. The BIOPEP database was used to analyze the peptides with a score greater than 0.5 screened by PeptideRanker, and a score prediction was made as to whether they had anti-inflammatory activity. The tool ADMET was used to evaluate the oral toxicity, carcinogenicity and human intestinal absorption capacity of the above-mentioned high-scoring peptides. The AllergenFP program was used to evaluate the sensitization of the above-mentioned high-scoring peptides.

[0024] Table 1 shows the sequences and functional properties of the anti-inflammatory peptides AF and FL obtained by screening. Figure 1 The structures of the anti-inflammatory peptides AF and FL.

[0025] Table 1

[0026]

[0027] Example 2

[0028] Docking of anti-inflammatory peptides with inducible nitric oxide synthase (iNOS) molecules

[0029] The secondary structures of anti-inflammatory peptides AF and FL were drawn using Chemdraw3D software. The x-ray crystal structure of iNOS (PDB ID: 3NW2) was downloaded from the RCSB protein database. In Discovery studio 2.5 software, molecular docking of anti-inflammatory peptides AF and FL with iNOS was performed. The higher the molecular docking score, the tighter the binding between the ligand and the receptor protein.

[0030] The results of the docking between AF and iNOS are as follows Figure 2 As shown, the anti-inflammatory peptide AF docks in the cavity of iNOS. The anti-inflammatory peptide AF binds to the Glu335, Arg391 and Asn384 residues of the iNOS protein through hydrogen bonds and to the Arg272 residue of the iNOS protein through hydrophobic interactions. The "LibDockScore" between peptide AF and iNOS is 191, indicating that it binds tightly to the receptor protein.

[0031] The docking results of FL and iNOS are as follows Figure 3As shown, the anti-inflammatory peptide FL is docked in the cavity of iNOS. The anti-inflammatory peptide FL binds to the Arg381, Thr380, Gly85 and Glu388 residues of the iNOS protein through hydrogen bonds, and to the Tyr383 and Ile385 residues of the iNOS protein through hydrophobic interactions. The "LibDockScore" between the peptide FL and iNOS is 186, indicating that it is tightly bound to the receptor protein.

[0032] Example 3

[0033] Effects of anti-inflammatory peptides on the survival of RAW264.7 cells

[0034] The peptide was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. with a purity of ≥95%, meeting the requirements of activity detection related experiments.

[0035] RAW264.7 cells in logarithmic growth phase were cultured at 1×10 4 Each well was transferred into a 96-well culture plate. After incubation for 24 hours at 37°C in a 5% CO2 incubator, anti-inflammatory peptides AF and FL were added to a final concentration of 0, 25, 50, 100 and 200 μg / mL, respectively. Six replicate wells were set for each concentration, and a culture medium containing the same volume of Tris-Hcl buffer was used as a control. After incubation for 24 hours, 20 μL of MTT solution (5 mg / mL) was added to each well, and the culture was terminated after incubation for another 4 hours. The supernatant was carefully aspirated and discarded, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 minutes, and then the light absorbance of each well was measured at a wavelength of 570 nm on an ELISA reader.

[0036] The results are as follows Figure 4 As shown, the anti-inflammatory peptides AF and FL had no significant effect on the growth of RAW264.7 cells in the concentration range of 0-200 μg / mL.

[0037] Example 4

[0038] Analysis of anti-inflammatory activity of anti-inflammatory peptides

[0039] The peptide was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. with a purity of ≥95%, meeting the requirements of activity detection related experiments.

[0040] 1. Effect of anti-inflammatory peptides on NO release induced by LPS in RAW264.7 cells

[0041] RAW264.7 cells in logarithmic growth phase were cultured at 1×10 6Each well was transferred into a 30 mm culture dish. After culturing at 37°C for 24 h, anti-inflammatory peptides AF and FL were added to a final concentration of 25 μg / mL and 50 μg / mL, respectively. After 4 h, LPS was added to a final concentration of 0.5 μg / mL, and three replicate wells were set for each concentration. After incubation for 18 h, the supernatant was collected and the NO content was detected by the Griess method, that is, 25 μL of supernatant was added with 25 μL of GriessI and 25 μL of GriessII, and the light absorption value of each well was measured at a wavelength of 540 nm on the microplate reader, and the NO concentration was calculated according to the standard curve.

[0042] The results are as follows Figure 5 As shown, the anti-inflammatory peptides AF and FL could inhibit the excessive NO production induced by LPS in RAW264.7 cells.

[0043] 2. Effects of anti-inflammatory peptides on the expression of iNOS, TNF-α and IL-6 in RAW264.7 cells induced by LPS

[0044] RAW264.7 cells in logarithmic growth phase were cultured at 1×10 6 Each well was transferred into a 30 mm culture dish. After culturing at 37°C for 24 h, anti-inflammatory peptides AF and FL were added to a final concentration of 25 μg / mL and 50 μg / mL, respectively. After 4 h, LPS was added to a final concentration of 0.5 μg / mL. After 20 h, a cell suspension was prepared and RNA was extracted using Trizol reagent. Then, cDNA was synthesized by reverse transcription, and the mRNA expression levels of iNOS, TNF-α, and IL-6 were detected using SYBRGreen fluorescent dye and qRT-PCR.

[0045] Figure 6 The results showed that the anti-inflammatory peptides AF and FL could inhibit the upregulation of iNOS mRNA expression in RAW264.7 cells induced by LPS. Figure 7 and Figure 8 The results showed that the anti-inflammatory peptides AF and FL could also inhibit the upregulation of TNF-α and IL-6 mRNA expression levels in RAW264.7 cells induced by LPS.

[0046] The above description is only for better explaining the embodiments of the present invention, and is not intended to limit the present invention. Any modification or equivalent substitution that does not depart from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims

1. An anti-inflammatory peptide, characterized in that It is anti-inflammatory peptide AF or anti-inflammatory peptide FL. The amino acid sequence of anti-inflammatory peptide AF is ASHFGLQRQF, and the amino acid sequence of anti-inflammatory peptide FL is FFHLQQQRL.

2. The anti-inflammatory peptide according to claim 1, characterized in that The molecular weights of anti-inflammatory peptide AF and anti-inflammatory peptide FL are 1190.3 Da and 1216.4 Da, respectively.

3. Use of the anti-inflammatory peptide according to claim 1 in the preparation of anti-inflammatory drugs or products.

4. An anti-inflammatory drug or product, characterized in that: The invention comprises the anti-inflammatory peptide according to claim 1.

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