Anti-inflammatory peptides af and fl and uses thereof

Abstract

This invention relates to the field of plant bioactive peptide technology, specifically to the anti-inflammatory peptides AF and FL and their applications. The amino acid sequences of anti-inflammatory peptides AF and FL are ASHFGLQRQF and FFHLQQQRL, respectively. This invention prepares peptides through enzymatic hydrolysis of red kidney beans, identifies the peptides using ultrafiltration and LC-MS / MS, and screens for anti-inflammatory peptides FL and AF using bioinformatics methods. These peptides are highly anti-inflammatory, non-toxic, non-sensitizing, non-carcinogenic, and have good intestinal absorption capacity. The peptides are artificially synthesized, and their anti-inflammatory activity is verified experimentally. It was found that anti-inflammatory peptides FL and AF have good anti-inflammatory activity, no obvious toxic side effects on RAW264.7 cells, and can significantly inhibit LPS-induced NO release in RAW264.7 cells, as well as inhibit the expression of inflammatory factors iNOS, TNF-α, and IL-6. They can be used in the preparation of anti-inflammatory drugs or products.

Description

Technical Field

[0001] This invention belongs to the field of plant bioactive peptides, specifically relating to anti-inflammatory peptides AF and FL and their applications. Background Technology

[0002] Inflammation is a fundamental pathological process, primarily a defensive response, to various damaging factors. Normally, inflammation is beneficial, serving as the body's automatic defense mechanism. However, excessive inflammation is harmful, leading to decreased immunity and tissue damage. Inflammation is associated with many diseases, such as diabetes, inflammatory bowel disease, and cancer. Therefore, the development of natural drugs with anti-inflammatory activity is urgently needed.

[0003] Bioactive peptides are polypeptides with certain physiological regulatory functions. Anti-inflammatory peptides are a class of bioactive peptides with anti-inflammatory activity, mainly inhibiting the body's inflammatory response by regulating the secretion of cytokines and the synthesis of inflammatory mediators. Numerous studies have demonstrated that food-derived bioactive peptides have a certain alleviating effect on inflammation. Compared with traditional anti-inflammatory drugs, food-derived anti-inflammatory peptides are safe and have no side effects. Therefore, developing safe and highly effective anti-inflammatory peptides from food has promising prospects and significance. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide anti-inflammatory peptides AF and FL and their applications. This invention uses red kidney beans as raw material, prepares active peptides through enzymatic hydrolysis, separates them by ultrafiltration according to molecular weight, and identifies the components with good anti-inflammatory activity by LC-MS / MS to obtain peptide information. Peptides with high scores and anti-inflammatory activity are screened using the BIOPEP database and PeptideRanker; peptides with no acute oral toxicity, no carcinogenicity, and good intestinal absorption are screened using ADMET; peptides without sensitization are screened using the AllergenFP program; molecular docking is then used to perform molecular docking between individual anti-inflammatory peptides and iNOS; finally, the anti-inflammatory activity of the anti-inflammatory peptides is verified experimentally.

[0005] To achieve the above objectives, 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 an anti-inflammatory peptide AF or an anti-inflammatory peptide FL, wherein the amino acid sequence of the anti-inflammatory peptide AF is ASHFGLQRQF and the amino acid sequence of the anti-inflammatory peptide FL is FFHLQQQRL.

[0007] Furthermore, the molecular weights of the anti-inflammatory peptides AF and 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 peptides described in the first aspect in the preparation of anti-inflammatory drugs or products.

[0009] A third aspect of the invention is an anti-inflammatory drug or product comprising the anti-inflammatory peptides described in the first aspect.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0011] This invention uses red kidney beans as raw material to prepare active peptides through enzymatic hydrolysis, and identifies the peptides using ultrafiltration and LC-MS / MS. Anti-inflammatory peptides are screened using bioinformatics methods. The peptides are then artificially synthesized and their anti-inflammatory activity is experimentally verified. The screened anti-inflammatory peptides exhibit good anti-inflammatory activity, show no significant toxic side effects on RAW264.7 cells, and can significantly inhibit LPS-induced NO release from RAW264.7 cells, as well as inhibit the expression of inflammatory factors iNOS, TNF-α, and IL-6. These peptides can be used in the preparation of anti-inflammatory drugs or products. Attached Figure Description

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

[0013] Figure 2 Results of docking of anti-inflammatory peptide AF with iNOS;

[0014] Figure 3 Results of docking of anti-inflammatory peptide FL with iNOS;

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

[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 Implementation

[0020] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0021] Example 1

[0022] Screening of anti-inflammatory peptides

[0023] Active peptides were prepared from red kidney beans via pepsin enzymatic hydrolysis. The peptides were then separated by ultrafiltration according to molecular weight, and fractions with molecular weights less than 3 kDa were identified by LC-MS / MS. PeptideRanker was used to score the potential functions of the peptides; a score greater than 0.5 indicated potential biological activity. The BIOPEP database was used to analyze peptides with scores greater than 0.5 selected by PeptideRanker to predict their anti-inflammatory activity. The ADMET tool was used to assess the oral toxicity, carcinogenicity, and intestinal absorption capacity of the peptides with high scores. The AllergenFP program was used to assess the sensitization potential of the peptides with high scores.

[0024] Table 1 shows the sequences and functional characteristics of the anti-inflammatory peptides AF and FL obtained through screening. Figure 1 The structures of the anti-inflammatory peptides AF and FL are shown.

[0025] Table 1

[0026]

[0027] Example 2

[0028] Anti-inflammatory peptides docked with inducible nitric oxide synthase (iNOS)

[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. Molecular docking of anti-inflammatory peptides AF and FL with iNOS was performed using Discovery Studio 2.5 software. A higher molecular docking score indicates a tighter binding between the ligand and receptor protein.

[0030] The docking results of AF and iNOS are as follows Figure 2 As shown, the anti-inflammatory peptide AF is docked to the cavity of iNOS. AF binds to Glu335, Arg391, and Asn384 residues of the iNOS protein via hydrogen bonds and to Arg272 residue via hydrophobic interactions. The "LibDockScore" between peptide AF and iNOS is 191, indicating its tight binding 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 to the cavity of iNOS. FL binds to iNOS protein residues Arg381, Thr380, Gly85, and Glu388 via hydrogen bonds, and to iNOS protein residues Tyr383 and Ile385 via hydrophobic interactions. The "LibDockScore" between peptide FL and iNOS is 186, indicating its tight binding to the receptor protein.

[0032] Example 3

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

[0034] The peptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity of ≥95%, meeting the requirements of activity detection experiments.

[0035] RAW264.7 cells in logarithmic growth phase were subjected to a 1×10⁻⁶ thiocyanate incubation. 4 Each sample was transferred to a 96-well plate. After incubation at 37°C in a 5% CO2 incubator for 24 h, anti-inflammatory peptides AF and FL were added to final concentrations of 0, 25, 50, 100, and 200 μg / mL, respectively, with six replicates for each concentration. Medium containing the same volume of Tris-HCl buffer was used as a control. After 24 h of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for another 4 h before termination. The supernatant was carefully aspirated, and 150 μL of DMSO was added to each well. The plate was shaken for 10 min, and the absorbance of each well was measured at 570 nm using a microplate 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] Anti-inflammatory activity analysis of anti-inflammatory peptides

[0039] The peptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity of ≥95%, meeting the requirements of activity detection experiments.

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

[0041] RAW264.7 cells in logarithmic growth phase were subjected to a 1×10⁻⁶ thiocyanate incubation. 6Each well was transferred to a 30 mm culture dish. After incubation at 37°C for 24 h, anti-inflammatory peptides AF and FL were added to final concentrations of 25 μg / mL and 50 μg / mL, respectively. After 4 h, LPS was added to a final concentration of 0.5 μg / mL. Each concentration was tested in triplicate. After 18 h of incubation, the supernatant was collected, and the NO content was determined using the Griess method: 25 μL of supernatant was mixed with 25 μL of Griess I and 25 μL of Griess II, and the absorbance of each well was measured at 540 nm using a microplate reader. The NO concentration was calculated based on the standard curve.

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

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

[0044] RAW264.7 cells in logarithmic growth phase were subjected to a 1×10⁻⁶ thiocyanate incubation. 6 Cells were transferred per well into 30 mm culture dishes. After incubation at 37°C for 24 h, anti-inflammatory peptides AF and FL were added to final concentrations 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, cell suspensions were prepared, and RNA was extracted by lysis using Trizol reagent. cDNA was then synthesized by reverse transcription, and the mRNA expression levels of iNOS, TNF-α, and IL-6 were detected using SYBR Green fluorescent dye and qRT-PCR.

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

[0046] The above description is only for better explaining the embodiments of the present invention and is not intended to limit them. Any modifications or equivalent substitutions that do 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, the amino acid sequence of the anti-inflammatory peptide AF is ASHFGLQRQF.

2. The anti-inflammatory peptide of claim 1, wherein, The molecular weight of the anti-inflammatory peptide AF is 1190.3 Da.

3. The use of the anti-inflammatory peptide of claim 1 in the preparation of an anti-inflammatory drug.

4. An anti-inflammatory drug, characterized in that, The anti-inflammatory peptide of claim 1 is included.

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