A walnut meal cyclooxygenase-2 (COX-2) inhibitory peptide and its application

Abstract

This invention discloses a walnut meal cyclooxygenase-2 (COX-2) inhibitory peptide and its application, belonging to the field of food deep processing technology. The COX-2 inhibitory peptide is AGFP, FPGA, LFPD, or VGFP; its amino acid sequences are shown in SEQ ID NO. 1–4, respectively. This invention prepares COX-2 inhibitory peptides from walnut meal through enzymatic hydrolysis, purifies them using ultrafiltration, identifies the peptide sequences, and uses virtual screening and molecular docking techniques to screen for COX-2 inhibitory active peptides. Peptides with high scores are artificially synthesized and their COX-2 inhibitory activity is verified. AGFP was found to have COX-2 inhibitory activity, and the inhibition kinetics show that AGFP inhibits COX-2 in a mixed manner. Molecular docking results indicate that AGFP and COX-2 mainly interact through hydrogen bonds and hydrophobic interactions.

Description

Technical Field

[0001] This invention relates to a walnut meal cyclooxygenase-2 (COX-2) inhibitory peptide and its application, belonging to the field of food deep processing technology. Background Technology

[0002] Cyclooxygenase-2 (COX-2) is an important enzyme with multiple biological functions. In recent years, it has attracted much attention due to its diverse functions and wide applications in the food and medical industries. COX-2 is a key mediator of inflammation and has been identified as an important therapeutic target for inflammation, diabetes, and pain management. Although COX-2 inhibitors have been extensively studied, considerable research evidence suggests that they have significant side effects. Therefore, the production of COX-2 inhibitors from natural products is crucial.

[0003] Walnut meal, a byproduct of walnut oil production, boasts a protein content as high as 40-50%, is rich in essential amino acids, and possesses high nutritional value and potential for related product development. However, the utilization of this byproduct from walnut processing is currently insufficient. Multiple studies have shown that not only does walnut protein have high nutritional value, but walnut-derived peptides also exhibit high activity, including anti-inflammatory, antioxidant, anti-osteoporosis, and dipeptidyl peptidase IV (DPP-IV) activity inhibition. However, no studies have reported the release of bioactive peptides with COX-2 inhibitory activity from plant proteins.

[0004] Virtual screening is a method for targeted searching of bioactive small molecules from a database. Compared with traditional methods for isolating bioactive peptides from proteins (such as ultrafiltration, gel size exclusion chromatography, and reverse HPLC), it is flexible, reliable, rapid, and efficient. Furthermore, the combination of virtual screening and in vitro experiments has been proven to effectively screen and identify potential bioactive peptides in proteins. Summary of the Invention

[0005] To address the above issues, this application uses walnut meal as raw material, prepares active peptides via enzymatic hydrolysis, purifies them using ultrafiltration, identifies the peptide sequences using nano-LC-MS / MS, and screens COX-2 inhibitory fragments using virtual screening and molecular docking techniques. Molecular docking is then used to dock the COX-2 inhibitory peptides with the receptor protein COX-2. Peptides with high scores are then artificially synthesized and their COX-2 inhibitory activity is verified. The key amino acids and interactions are analyzed. This invention provides a COX-2 inhibitory peptide extracted from walnut meal, and its application in the preparation of COX-2 inhibitory drugs or products.

[0006] The present invention provides a COX-2 inhibitory peptide, wherein the COX-2 inhibitory peptide is AGFP, FPGA, LFPD or VGFP; and its amino acid sequence is shown in SEQ ID NO.1 to 4 respectively.

[0007] The COX-2 inhibitory peptide is a tetrapeptide with an amino acid sequence of Ala-Gly-Phe-Pro from the C-terminus to the N-terminus, named AGFP (SEQ ID NO.1).

[0008] The COX-2 inhibitory peptide is a tetrapeptide with an amino acid sequence of Phe-Pro-Gly-Ala from the C-terminus to the N-terminus, named FPGA (SEQ ID NO.2).

[0009] The COX-2 inhibitory peptide is a tetrapeptide with an amino acid sequence of Leu-Phe-Pro-Asp from the C-terminus to the N-terminus, named LFPD (SEQ ID NO.3).

[0010] The COX-2 inhibitory peptide is a tetrapeptide with an amino acid sequence of Val-Gly-Phe-Pro from the C-terminus to the N-terminus, named VGFP (SEQ ID NO.4).

[0011] The present invention also provides a medicine in which the above-mentioned COX-2 inhibitory peptide is present.

[0012] In one embodiment of the present invention, the pharmaceutical product further contains any pharmaceutical carrier and / or pharmaceutical excipients that are permissible or acceptable for pharmaceutical (pharmacological) production.

[0013] In one embodiment of the present invention, when the COX-2 inhibitory peptide is AGFP, the amount of AGFP added to the drug is at least 3.69 g; when the COX-2 inhibitory peptide is FPGA, the amount of FPGA added to the drug is at least 0.76 g; when the COX-2 inhibitory peptide is LFPD, the amount of LFPD added to the drug is at least 0.88 g; and when the COX-2 inhibitory peptide is VGFP, the amount of VGFP added to the drug is at least 4.58 g.

[0014] The present invention also provides the use of the above-mentioned COX-2 inhibitory peptide in the preparation of anti-inflammatory and / or analgesic pharmaceutical products.

[0015] In one embodiment of the present invention, the pharmaceutical product further contains any pharmaceutical carrier and / or pharmaceutical excipients that are permissible or acceptable for pharmaceutical (pharmacological) production.

[0016] In one embodiment of the present invention, when the COX-2 inhibitory peptide is AGFP, the amount of AGFP added to the drug is at least 3.69 g; when the COX-2 inhibitory peptide is FPGA, the amount of FPGA added to the drug is at least 0.76 g; when the COX-2 inhibitory peptide is LFPD, the amount of LFPD added to the drug is at least 0.88 g; and when the COX-2 inhibitory peptide is VGFP, the amount of VGFP added to the drug is at least 4.58 g.

[0017] The present invention provides a feed additive containing the COX-2 inhibitory peptide.

[0018] In one embodiment of the present invention, when the COX-2 inhibitory peptide is AGFP, the amount of AGFP added to the drug is at least 3.69 g; when the COX-2 inhibitory peptide is FPGA, the amount of FPGA added to the drug is at least 0.76 g; when the COX-2 inhibitory peptide is LFPD, the amount of LFPD added to the drug is at least 0.88 g; and when the COX-2 inhibitory peptide is VGFP, the amount of VGFP added to the drug is at least 4.58 g.

[0019] The present invention also provides the application of the above-mentioned COX-2 inhibitory peptide in the preparation of anti-inflammatory and / or analgesic feed additives.

[0020] Beneficial effects

[0021] (1) This invention extracts walnut protein from walnut meal, prepares high COX-2 inhibitory peptides by enzymatic hydrolysis with six proteases, purifies them by ultrafiltration, identifies the peptide sequences by nano-LC-MS / MS, and screens COX-2 inhibitory fragments using virtual screening and molecular docking technology. Molecular docking technology is used to perform molecular docking between COX-2 inhibitory peptides and receptor protein COX-2. Peptides with high scores are artificially synthesized and their COX-2 inhibitory activity is verified. The key amino acids and forces of their interaction are analyzed, laying a theoretical foundation for the in-depth development and utilization of walnut meal.

[0022] (2) After artificial synthesis, the COX-2 inhibitory activity was verified, and it was found that AGFP has COX-2 inhibitory activity. The inhibition kinetics showed that the inhibition of COX-2 by AGFP is a mixed type of inhibition. Molecular docking results showed that AGFP and COX-2 mainly interact through hydrogen bonds and hydrophobic interactions. Attached Figure Description

[0023] Figure 1A represents the degree of hydrolysis of walnut meal protein obtained by enzymatic hydrolysis of alkaline protease, trypsin, papain, flavor protease, chymotrypsin, and neutral protease; B represents the COX-2 enzyme inhibitory activity of the six enzyme hydrolysates; and C represents the inhibitory activity of the six enzyme hydrolysates on LPS-induced NO release from RAW264.7.

[0024] Figure 2 The study investigated the COX-2 enzyme inhibitory activity and the inhibitory activity of ultrafiltration fractions on LPS-induced NO release from RAW264.7. A represents the COX-2 enzyme inhibitory activity of the ultrafiltration fractions (<1kDa, 1-3kDa, 3-5kDa, 5-10kDa); B represents the inhibitory activity of the ultrafiltration fractions (<1kDa, 1-3kDa, 3-5kDa, 5-10kDa) on LPS-induced NO release from RAW264.7.

[0025] Figure 3 Results of screening of walnut meal peptides for docking with COX-2.

[0026] Figure 4 Secondary mass spectra for identification of peptides AGFP, FPGA, LFPD, and VGFP.

[0027] Figure 5 : Primary mass spectrum and liquid chromatography chromatogram of peptide AGFP synthesis; where A is the primary mass spectrum and liquid chromatography chromatogram of peptide AGFP synthesis; B is the primary mass spectrum and liquid chromatography chromatogram of peptide FPGA synthesis; C is the primary mass spectrum and liquid chromatography chromatogram of peptide LFPD synthesis; D is the primary mass spectrum and liquid chromatography chromatogram of peptide VGFP synthesis.

[0028] Figure 6 COX-2 enzyme inhibitory activity; where A is the enzyme inhibitory activity of AGFP, B is the enzyme inhibitory activity of FPGA, C is the enzyme inhibitory activity of LFPD and D is the enzyme inhibitory activity of VGFP; the error bars represent the standard deviation of three samples.

[0029] Figure 7 The inhibitory activity of COX-2 inhibitory peptides on LPS-induced NO release from RAW264.7; wherein, A is an AGFP inhibitory peptide, B is an FPGA inhibitory peptide, C is an LFPD inhibitory peptide, and D is a VGFP inhibitory peptide on the inhibitory activity of COX-2 inhibitory peptides on LPS-induced NO release from RAW264.7.

[0030] Figure 8 : Interaction diagrams of COX-2 inhibitory peptides with COX-2; where A is the interaction diagram of AGFP with COX-2; B is the interaction diagram of FPGA with COX-2; C is the interaction diagram of LFPD with COX-2; and D is the interaction diagram of VGFP with COX-2.

[0031] Figure 9 : COX-2 inhibitory peptides inhibit COX-2 kinetics; where A is the COX-2 inhibitory kinetics of AGFP; where B is the COX-2 inhibitory kinetics of FPGA; where C is the COX-2 inhibitory kinetics of LFPD; where D is the COX-2 inhibitory kinetics of VGFP. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0033] Those skilled in the art can obtain the COX-2 inhibitory peptide provided by the present invention through artificial synthesis or enzymatic hydrolysis of walnut meal.

[0034] The sources of Alcalase, Neutrase, Trypsin, Chymotrypsin, Papain, and Flavorzyme involved in the following examples are as follows: Alcalase: purchased from Solarbio (B8360), obtained by fermentation of Bacillus licheniformis; Neutrase: purchased from Solarbio (Z8031), obtained by deep fermentation of Bacillus subtilis; Trypsin: purchased from Aladdin (R141080), derived from porcine pancreas; Chymotrypsin: purchased from Aladdin (C106197), derived from porcine pancreas; Papain: purchased from Solarbio (G8431), extracted from immature papaya fruit using bioengineering technology; Flavorzyme: purchased from Solarbio (F8271), obtained by fermentation with Aspergillus oryzae, and refined using advanced extraction processes such as microfiltration, ultrafiltration, and vacuum freeze-drying.

[0035] The detection methods involved in the following embodiments are as follows:

[0036] (1) Determination of degree of hydrolysis

[0037] The degree of hydrolysis of the samples was determined using the o-phthalaldehyde (OPA) method. A standard curve was plotted using L-leucine as the standard solution. 10 μL of sample solution was added to 200 μL of OPA solution and reacted at room temperature for 2 min. The absorbance was measured at 340 nm. The calculation formula is as follows:

[0038]

[0039] Note: N Pb The content of peptide bonds (N) in the protein substrate is 7.6 mmol / g protein;

[0040] AN2 The amino nitrogen (N) content after hydrolysis (mmol / g protein);

[0041] A N1 The content of amino nitrogen (N) before hydrolysis (mmol / g protein).

[0042] (2) Cytotoxicity assay of mouse macrophages RAW264.7

[0043] All cell experiments were performed in a sterile operating room. RAW264.7 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% carbon dioxide. Logarithmically growing RAW264.7 mouse macrophages were seeded at 2×10⁵ cells / mL, 200 μL / well in 96-well plates and cultured for 12 h to allow adherence. Different concentrations of sample solutions were added and cultured for another 24 h. Each group was divided into 6 replicates, and cell viability was determined by the MTT assay.

[0044] (3) NO emission measurement

[0045] Two x 10⁵ RAW264.7 cells per well were seeded into 96-well plates. After cell attachment, the sample was added and the cells were co-cultured for 24 h. 50 μL of cell culture supernatant was aspirated, followed by 50 μL of Griess Resgent I and 50 μL of Griess Resgent II. After the reaction was carried out in the dark, the absorbance was measured at 550 nm.

[0046] (4) Inhibit activity with COX-2

[0047] The in vitro inhibitory effect of the samples was evaluated using a cyclooxygenase-2 inhibitor screening kit (Beyotime, Shanghai, China). Specifically, COX-2 Cofactor working solution and COX-2 working solution were added to COX-2 Assay Buffer, followed by sample solutions of different concentrations. After incubation at 37°C for 10 min, COX-2 Probe and COX-2 Substrate working solutions were added, and the mixture was incubated at 37°C in the dark for 5 min. Fluorescence was then measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Each sample was tested in triplicate. The average fluorescence value of each sample was calculated, and the inhibition percentage of each sample was calculated using the following formula:

[0048] Inhibition rate (%) = (RFU 100% enzyme activity control - RFU sample) / (RFU 100% enzyme activity control - RFU blank control) × 100%.

[0049] (5) COX-2 inhibition mode (type and constant of inhibition of cyclooxygenase by peptide)

[0050] Lineweaver-Burk plots were used to determine the type and constant of inhibition of cyclooxygenase by the peptide.

[0051] The assay method is the same as for inhibitory activity; the assay concentration of the peptide is IC50. 50 and 1 / 2 IC 50 Substrate concentrations (12.5×, 25×, 50× COX⁻²Substrate) were plotted. A curve was constructed with the reciprocal of the substrate concentration (1 / [S]) on the x-axis and the reciprocal of the enzyme reaction initiation rate (1 / V₀) on the y-axis. The type of inhibition of cyclooxygenase by the peptide was then determined based on the intersection of the curves. The inhibition constant (Ki) was obtained from a quadratic plot of the slope of the Lineweaver-Burk line against peptide concentration.

[0052] (6) Molecular docking

[0053] The peptide structure was constructed using ChemBioDraw Ultra 14.0, and then imported into ChemBio3D Ultra 14.0 for energy minimization. The Minimum RMS Gradient was set to 0.001, and the small molecule was saved in mol2 format. The optimized small molecule was then imported into AutodockTools-1.5.6 for hydrogenation, charge calculation, charge assignment, and setting of rotatable bonds before being saved in "pdbqt" format. The COX-2 structure (PDB ID: 3LN1) was downloaded from the PDB database; Pymol 2.3.0 was used to remove protein water of crystallization and primitive ligands, and the protein structure was imported into AutoDocktools (v1.5.6) for hydrogenation, charge calculation, charge assignment, and specification of atom types before being saved in "pdbqt" format. AutoDock Vina 1.1.2 was used for docking. The relevant parameters for 3LN1 were set as follows: center_x = 30.179, center_y = -23.19, center_z = -14.527; search space: size_x: 27.75, size_y: 27.75, size_z: 28.5 (the spacing between each grid point is...). The exhaustiveness setting was 10, and the remaining parameters were set to default. Interaction mode analysis of the docking results was performed using PyMOL 2.3.0 and LIGPLOT V 2.2.4.

[0054] The present invention will be further explained and described below through specific embodiments.

[0055] Example 1: Preparation, structural identification, and molecular docking of COX-2 inhibitory peptide from walnut meal

[0056] 1. Preparation of different protease hydrolysates

[0057] (1) Walnut protein extraction:

[0058] Walnut meal was ground into flour and defatted with petroleum ether at a ratio of 1:10 (w / v) for 4 hours. After defatting, the petroleum ether was removed by filtration, and the flour was dried and ground into fine powder to obtain defatted powder.

[0059] Then, the defatting powder was dispersed in 1M NaOH solution at a ratio of 1:15 (w / v), and the pH was adjusted to 9.0. Extraction was carried out by stirring at 45°C for 1 h. After adjusting the pH of the supernatant to 4.5 with 1.0M HCl, the precipitate was obtained by centrifugation at 4000 r / min for 20 min. Finally, the precipitate was washed with distilled water until pH = 7.0, lyophilized, and stored at -20°C.

[0060] (2) Preparation of enzymatic hydrolysates (WDPHs):

[0061] Prepare a 5% protein solution from step (1) using distilled water, stir thoroughly, and place in a 90℃ water bath for 15 minutes to denature the protein. Adjust the pH to the optimal range for the protease, add different proteases, and maintain the optimal temperature for the protease in a constant temperature water bath. The enzymatic hydrolysis conditions for different proteases are shown in Table 1. After 4 hours of enzymatic hydrolysis, take a sample and place the reaction solution in a 100℃ water bath for 10 minutes to inactivate the enzyme. After cooling, adjust the pH of the solution to neutral, centrifuge at 4000 rpm for 20 minutes, and freeze-dry the supernatant for later use.

[0062] Table 1: Enzymatic hydrolysis conditions for different proteases

[0063]

[0064] (3) Degree of hydrolysis of different protease hydrolysates

[0065] Walnut meal protein was enzymatically hydrolyzed for 4 hours using six proteases at their optimal pH and temperature to obtain six hydrolysates: alkaline protease hydrolysate (Al-WDPH), papain hydrolysate (Pa-WDPH), neutral protease hydrolysate (Ne-WDPH), flavor protease hydrolysate (Fl-WDPH), trypsin hydrolysate (Tr-WDPH), and chymotrypsin hydrolysate (Ch-WDPH). The degree of hydrolysis of the six WDPHs is as follows: Figure 1As shown in Figure A, Al-WDPH and Ne-WDPH exhibited the highest degrees of hydrolysis, at 26.80±1.28% and 26.40±0.68%, respectively, which were significantly different from other WDPHs (P<0.05). The degree of hydrolysis of Fl-WDPH reached 20.00±2.00%, followed by Tr-WDPH and Ch-WDPH, with degrees of hydrolysis of 13.20±1.11% and 14.20±0.49%, respectively. The lowest degree of hydrolysis was Pa-WDPH (6.00±0.71%).

[0066] (4) Effects of different protease hydrolysates on COX-2 enzyme

[0067] Different protease hydrolysates showed varying degrees of COX-2 enzyme inhibition, as shown in the following results. Figure 1 As shown in B, Al-WDPH and Pa-WDPH achieved the highest COX-2 enzyme inhibition. Al-WDPH exhibited the highest degree of hydrolysis and COX-2 enzyme inhibition.

[0068] (5) Inhibitory effect of different protease hydrolysates on NO release from mouse macrophages RAW264.7

[0069] The effects of different enzymatic hydrolysates on LPS-induced NO production in macrophages. Results are as follows: Figure 1 As shown in C, Al-WDPH, Pa-WDPH, and Tr-WDPH significantly inhibited LPS-induced macrophage release, with inhibition rates of 35.00±1.00%, 34.00±2.52%, and 32.33±1.20%, respectively.

[0070] 2. Preparation of peptides with different molecular weights

[0071] (1) Ultrafiltration:

[0072] Based on the results of step 1, the alkaline protease hydrolysate Al-WDPH was selected for subsequent experiments.

[0073] Al-WDPH was ultrafiltered in a chromatography cabinet at 4°C using an ultrafiltration unit (MSC300, Mosu Technology, China). The ultrafiltration conditions were: inlet pressure 10–15 MPa and outlet pressure 0.10–0.22 MPa. In the ultrafiltration system, the nylon tubing was first connected to the ultrafiltration cup. The base, ultrafiltration cup, O-ring, and silicone rubber were cleaned with UP water. The Al-WDPH to be separated was added through the feed port. The hose of the nitrogen cylinder was connected to the hose connector of the ultrafiltration cup. The ultrafiltration cup was placed on a magnetic stirrer, and the stirrer was turned on. The sample was passed sequentially through ultrafiltration membranes of 10 kDa, 5 kDa, 3 kDa, and 1 kDa. The filtrate was collected to obtain four components with different molecular weights: <1 kDa, 1–3 kDa, 3–5 kDa, and 5–10 kDa. All components were freeze-dried and stored at -20°C.

[0074] (2) Effects of different molecular weight peptides on COX-2 enzyme

[0075] The results are as follows Figure 2 As shown in A, compared with other components, the COX-2 inhibition rate of the component with a molecular weight <1kDa is significantly higher than that of other components, and the inhibition rate is the strongest, reaching 73.33±2.33%.

[0076] (3) Inhibitory effect of different molecular weight peptides on NO release from mouse macrophages RAW264.7

[0077] The effects of peptides of different molecular weights on LPS-induced NO production in macrophages were determined. Results are as follows: Figure 2 As shown in B, compared with other components, the component with a molecular weight of less than 1 kDa had a significantly higher inhibitory effect on NO release, reaching 25.75 ± 1.18%.

[0078] 3. Polypeptide identification

[0079] The <1kDa fraction was identified by nano-LC-MS / MS, and a total of 1262 amino acid sequences were obtained through analysis and database search comparison.

[0080] To screen for amino acid sequences with COX-2 inhibitory activity, small peptide molecules were virtually screened and docked with COX-2 to identify small molecule anti-inflammatory peptide sequences with high activity. Figure 3 The docking energies and amino acid sequences of the top ten small molecule peptides are listed.

[0081] Four COX-2 inhibitory peptides were finally obtained, namely:

[0082] The amino acid sequence from the C-terminus to the N-terminus is a tetrapeptide of Ala-Gly-Phe-Pro, named AGFP (SEQ ID NO.1).

[0083] The amino acid sequence from the C-terminus to the N-terminus is a tetrapeptide of Phe-Pro-Gly-Ala, named FPGA (SEQ ID NO.2).

[0084] The amino acid sequence from the C-terminus to the N-terminus is a tetrapeptide of Leu-Phe-Pro-Asp, named LFPD (SEQ ID NO.3).

[0085] The amino acid sequence from the C-terminus to the N-terminus is a tetrapeptide of Val-Gly-Phe-Pro, named VGFP (SEQ ID NO.4).

[0086] Secondary mass spectra of four COX-2 repressor peptides are shown below. Figure 4 As shown.

[0087] Example 2: COX-2 inhibitory activity of COX-2 inhibitory peptides in walnut meal

[0088] The specific steps are as follows:

[0089] (1) Chemical synthesis of the four polypeptides obtained in Example 1

[0090] The peptides AGFP (Ala-Gly-Phe-Pro, molecular weight 390.2 Da), FPGA (Phe-Pro-Gly-Ala, molecular weight 390.2 Da), LFPD (Leu-Phe-Pro-Asp, molecular weight 490.2 Da), and VGFP (Val-Gly-Phe-Pro, molecular weight 418.2 Da) were artificially synthesized to verify their COX-2 inhibitory activity. The synthesis was commissioned to Sangon Biotech Co., Ltd. for solid-phase synthesis, achieving a purity >98%, meeting the requirements for activity detection experiments. The mass spectra and chromatograms of peptides AGFP, FPGA, LFPD, and VGFP are shown below. Figure 5 As shown.

[0091] Depend on Figure 5As shown in the AD spectrum, peptide AGFP exhibits an absorption peak with a height of 98.897% and a peak area of ​​99.809%. Furthermore, the primary mass spectrum shows a peak at 390.10 m / z, indicating that the purity of the synthesized peptide AGFP is 99.809%, and its molecular weight is 390.1 Da. Peptide FPGA exhibits an absorption peak with a height of 95.668% and a peak area of ​​98.687%. Furthermore, the primary mass spectrum shows a peak at 390.10 m / z, indicating that the purity of the synthesized peptide FPGA is 98.687%, and its molecular weight is 390.1 Da. Peptide LFPD exhibits an absorption peak with a height of 99.298% and a peak area of ​​99.704%. Furthermore, the primary mass spectrum shows a peak at 490.10 m / z, indicating that the purity of the synthesized peptide LFPD is 99.704%, and its molecular weight is 490.1 Da. The peptide VGFP showed an absorption peak with a height of 99.325% and a peak area of ​​99.582%. In addition, the primary mass spectrum showed that the peptide VGFP had a peak at 418.15 m / z, indicating that the purity of the synthesized peptide VGFP reached 99.582% and its molecular weight was 418.2 Da.

[0092] (2) COX-2 inhibitory activity

[0093] Preparation of sample solutions: Take 5 mg of the polypeptide powder synthesized in step (1), add an appropriate amount of UP water to make its concentration reach 10 mM, and prepare sample solutions of 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM, 3.2 mM and 6.4 mM by gradient dilution.

[0094] The in vitro inhibitory effect of the samples was evaluated using a cyclooxygenase-2 inhibitor screening kit (Beyotime, Shanghai, China). COX-2 Cofactor working solution and COX-2 working solution were added to COX-2 Assay Buffer, followed by sample solutions at final concentrations of 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM, 3.2 mM, and 6.4 mM, respectively. After incubation at 37°C for 10 min, COX-2 Probe and COX-2 Substrate working solutions were added, and the mixture was incubated at 37°C in the dark for 5 min. Fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Each sample was tested in triplicate. The average fluorescence value and inhibition percentage of each sample were calculated. The results are shown below. Figure 5 As shown in Table 2.

[0095] Table 2: Inhibition rate of different peptides on cyclooxygenase-2 at different concentrations

[0096]

[0097]

[0098] The results showed that, Figure 6 As shown, the COX-2 inhibitory activity of peptide AGFP gradually increased with increasing concentration, reaching a maximum of 62.00 ± 2.309% at a concentration of 6.4 mM; IC50 50 It reached 2.308mM.

[0099] The COX-2 inhibitory activity of peptide FPGA gradually increased with increasing concentration, reaching a maximum of 84.00±4.04% at a concentration of 3.2 mM; IC 50 It reached 0.495mM.

[0100] The COX-2 inhibitory activity of peptide LFPD gradually increased with increasing concentration, reaching a maximum of 87.67 ± 2.73% at a concentration of 6.4 mM; IC50 50 It reached 0.455mM.

[0101] The COX-2 inhibitory activity of peptide VGFP gradually increased with increasing concentration, reaching a maximum of 62.33 ± 2.40% at a concentration of 6.4 mM; IC50 50 It reached 2.777mM.

[0102] (3) NO inhibitory activity

[0103] Preparation of sample solution: Take 5 mg of the polypeptide powder synthesized in step (1) and add an appropriate amount of UP water to make the sample solution with a concentration of 50 mM.

[0104] The inhibitory effect of samples on NO release from RAW264.7 cells was evaluated using the Griess Reagent System Nitric Oxide Assay Kit (Promega, USA). 2 × 10⁻⁶ samples were used. 5 RAW264.7 cells were seeded per well in 96-well plates. The sample concentration was diluted to 50 μM using high-glucose DMEM medium containing 1 mg / mL LPS. After cell attachment, the sample was added, and the plates were co-cultured at 37°C. After 24 h, the plates were removed, and 50 μL of cell culture supernatant was aspirated, followed by 50 μL of Griess Reagent I and 50 μL of Griess Reagent II. The reaction was carried out in the dark, and the absorbance was measured at 550 nm.

[0105] The results showed that peptide AGFP inhibited NO release by 17.00±1.472%, peptide FPGA by 19.25±1.931%, peptide LFPD by 21.75±0.4787%, and peptide VGFP by 18.50±0.5000%. Figure 7 ).

[0106] (4) Molecular docking of peptides with COX-2

[0107] To further verify the COX-2 inhibition mechanism of peptide AGFP, it was docked with the receptor protein COX-2. The docking results are as follows: Figure 8 As shown, the peptide AGFP is docked into the cavity of COX-2, and hydrophobic interactions and hydrogen bonds are the main interaction forces between it and COX-2.

[0108] Docking results showed that the binding energies between peptide AGFP and COX-2 were -9.1 kcal / mol, -8.1 kcal / mol, -8.4 kcal / mol, and -9.2 kcal / mol, respectively, indicating that peptides AGFP, FPGA, LFPD, and VGFP bind tightly to the receptor protein. The active site Tyr341 (3.30, ...) within the COX-2 pocket of AGFP... ), Tyr371 Ser516 It forms 4 hydrogen bonds and 13 hydrophobic interactions with residues. The active site Tyr341 within the COX-2 pocket of the FPGA... It forms one hydrogen bond with Arg106 (3.54, 2.81, ...). Ser105 It forms 4 hydrogen bonds and 12 hydrophobic interactions with the residues. LFPD interacts with the active site Tyr341 (3.20, 2.74, ...) within the COX-2 pocket. ) and Val509 It forms four hydrogen bonds, an electrostatic interaction with Arg106, and 16 hydrophobic interactions with residues. VGFP interacts with the active site Tyr371. Ser516 (3.06, His75 It forms four hydrogen bonds and 15 hydrophobic interactions with the residues (Table 3).

[0109] Table 3: Interaction binding sites between peptides and COX-2

[0110]

[0111] (5) COX-2 inhibition kinetics

[0112] To further analyze the interaction between the peptide and COX-2, the exact type of enzyme inhibition was determined using a Lineweaver-Burk plot (LB plot). Figure 9 The rate constants (km and Vmax) were determined using Graphpad Prism software via LB plots (Table 4).

[0113] Table 4: Kinetics of AGFP inhibition of COX-2

[0114]

[0115]

[0116]

[0117]

[0118] The results showed that the double reciprocal plots of LFPD, FPGA, and AGFP peptides revealed that the fitted curves for different inhibitor concentrations intersected in the second quadrant. The kinetic parameter Vmax decreased with increasing inhibitor concentration, indicating that LFPD, FPGA, and AGFP exhibited a mixed inhibitory mode for COX-2. The fitted curves for different concentrations of VGFP intersected on the X-axis, with the kinetic parameter Km remaining almost unchanged. Vmax decreased with increasing inhibitor concentration, indicating that VGFP exhibited a non-competitive inhibitory mode for COX-2.

[0119] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A medicine, characterized in that, The drug contains a COX-2 inhibitory peptide; the COX-2 inhibitory peptide is AGFP or VGFP; its amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.4, respectively.

2. The medicine according to claim 1, characterized in that, The drug also contains a drug carrier and / or pharmaceutical excipients.

3. The medicine according to claim 1 or 2, characterized in that, When the COX-2 inhibitory peptide is AGFP, the amount of AGFP added to the drug is at least 3.69g; when the COX-2 inhibitory peptide is VGFP, the amount of VGFP added to the drug is at least 4.58g.

4. Application of COX-2 inhibitory peptides AGFP or VGFP in the preparation of anti-inflammatory and / or analgesic drugs.

5. The application according to claim 4, characterized in that, The drug also contains a drug carrier and / or pharmaceutical excipients.

6. The application according to claim 4 or 5, characterized in that, When the COX-2 inhibitory peptide is AGFP, the amount of AGFP added to the drug is at least 3.69g; when the COX-2 inhibitory peptide is VGFP, the amount of VGFP added to the drug is at least 4.58g.

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