Antarctic krill-derived xanthine oxidase inhibitory peptide and application thereof

Abstract

This invention discloses a xanthine oxidase inhibitory peptide derived from Antarctic krill and its applications, belonging to the field of biotechnology, specifically relating to bioactive peptide technology. Using Antarctic krill protein as raw material, this invention obtains the xanthine oxidase inhibitory peptide WRIPSRL, with the amino acid sequence shown in SEQ ID NO: 1, through virtual screening, molecular docking, and in vitro activity verification techniques. This invention utilizes the BIOPEP-UWM platform for virtual hydrolysis, and obtains candidate peptides through PeptideRanker activity prediction and ToxinPred toxicity screening; then, molecular docking is performed with the xanthine oxidase crystal structure 1N5X using the Autodock Vina algorithm to screen for high-affinity peptides; in vitro activity assays show that the IC50 of the WRIPSRL peptide is high. 50 The value was 1.04 mM, and the inhibition rate reached 48.68% at a concentration of 1 mM.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to bioactive peptide technology, and more specifically, to a xanthine oxidase inhibitory peptide derived from Antarctic krill, its screening method, and its application in the preparation of drugs or functional foods for the prevention or treatment of hyperuricemia or gout. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Over the past few decades, the incidence and prevalence of gout have increased. Gout is a purine metabolism disorder, the core mechanism of which lies in the excessive production or reduced excretion of uric acid due to purine metabolism disorders. This leads to the deposition of urate crystals in the joints and surrounding tissues, causing severe pain, joint deformities, and even kidney damage. Research has shown that xanthine oxidase (XO) is a key rate-limiting enzyme in the purine metabolism pathway. Its core function is to catalyze the conversion of hypoxanthine to xanthine, and then further oxidize xanthine to uric acid. Therefore, inhibiting XO activity can reduce uric acid synthesis at its source, becoming a core target for clinical intervention in gout.

[0004] Currently, commonly used XO inhibitors such as allopurinol can effectively lower uric acid levels, but long-term use can easily cause adverse reactions such as skin rashes and liver and kidney damage, and some patients develop drug resistance. Against this backdrop, developing naturally derived, safe, low-toxicity XO-inhibiting peptides that combine nutritional value and bioactivity has become an important research direction for replacing or adjuvanting chemical drugs. This approach can meet the long-term intervention needs of gout patients while avoiding the side effects risks of chemical drugs.

[0005] Existing technologies have reported the extraction of XO inhibitory peptides from various marine organisms. For example, one study extracted the tetrapeptide PhePro Ser Val from blue trevally. Another study obtained the hexapeptide PLGPPP from Litopenaeus vannamei and clearly disclosed its IC50 inhibitory activity against xanthine oxidase. 50 The value was 2.356 mM. The XO inhibitory peptides disclosed in the prior art provide valuable candidate molecules for adjunctive intervention in gout. However, inhibitory peptides from different sources and with different structures exhibit differences in activity intensity, and exploring novel and more active XO inhibitory peptides remains an ongoing research direction in this field. In particular, from the perspective of long-term intervention for chronic diseases, obtaining candidate molecules with higher activity is expected to achieve equivalent effects at lower doses, thus providing patients with more diverse options.

[0006] In addition, the traditional technical route for preparing XO inhibitory peptides is mainly based on "enzymatic hydrolysis-stepwise separation-mass spectrometry identification". Although this method can obtain real active peptides, it has disadvantages such as long experimental cycle and high cost, which to some extent restricts the development efficiency of XO inhibitory peptides. In recent years, the rapid development of computer technology and cheminformatics has promoted "virtual screening" to become the core technology for the discovery of natural active peptides. This strategy is based on the "quantitative structure-activity relationship model" and combines machine learning, molecular docking and other technologies to achieve rapid screening and activity prediction of massive peptide sequences. Compared with traditional methods, virtual screening technology has the following potential advantages at the theoretical level: (1) High efficiency: Through virtual hydrolysis and activity prediction, thousands to tens of thousands of peptide sequences can be processed in a short time. (2) Low cost and high accuracy: Virtual screening can eliminate inactive and toxic peptides through computer simulation, reducing the workload of subsequent in vitro experiments; at the same time, combined with molecular docking technology, the binding ability of peptides to XO active pockets can be predicted, and high-affinity candidate peptides can be screened in advance. (3) Synchronicity of mechanism research: Virtual screening can be combined with molecular visualization analysis to clarify the interaction between peptides and XO active sites. However, the results of virtual screening still need to be verified by in vitro experiments to ensure the reliability of the screening results.

[0007] Antarctic krill ( Euphausia superba Antarctic krill is one of the world's largest marine crustaceans in terms of biomass, with stable and sustainable resources. Its meat contains 60%-70% protein, which readily yields short-chain bioactive peptides upon hydrolysis. Previous studies have shown that hydrolyzed peptides of Antarctic krill protein possess various biological activities, including antioxidant and anti-inflammatory effects, suggesting that its protein sequence may contain a variety of functional bioactive peptides. However, current research on XO-inhibiting peptides from Antarctic krill is insufficient, particularly regarding specific peptides with high activity, which have not been publicly reported. Therefore, the discovery of novel XO-inhibiting peptides from Antarctic krill protein has significant scientific research value and industrial prospects.

[0008] Therefore, developing an XO inhibitory peptide with higher activity, novel sequence, and a clear mechanism of action, and establishing an efficient screening method, is of great practical significance for enriching the means of adjuvant intervention for gout and promoting the high-value utilization of marine biological resources. Summary of the Invention

[0009] This invention aims to overcome the shortcomings of existing technologies and provide a highly active xanthine oxidase inhibitory peptide derived from Antarctic krill, along with its screening method and applications. Specifically, the technical problems to be solved by this invention include: First, the activity of the XO inhibitory peptides disclosed in the existing technology still has room for improvement and cannot yet meet the clinical need to achieve efficient inhibition at lower doses.

[0010] Second, the traditional "enzymatic hydrolysis-stepwise separation-mass spectrometry identification" method for screening active peptides has limitations such as long experimental cycle, high cost and low efficiency. There is an urgent need to establish a highly efficient XO inhibitory peptide mining technology system.

[0011] Third, Antarctic krill, as the marine crustacean with the largest biomass in the world, is rich in protein resources. However, research on XO inhibitory peptides from Antarctic krill is still insufficient, especially the lack of publicly reported specific peptides with high activity, and their potential value remains to be explored.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an Antarctic krill-derived xanthine oxidase inhibitory peptide with the amino acid sequence Trp-Arg-Ile-Pro-Ser-Arg-Leu, as shown in SEQ ID NO: 1, and named WRIPSRL.

[0013] Furthermore, the xanthine oxidase inhibitory peptide is derived from Antarctic krill protein, specifically from the Antarctic krill Timeless2 protein, whose NCBI reference sequence is AUI80375.1.

[0014] In a second aspect, the present invention provides a xanthine oxidase inhibitor comprising the xanthine oxidase inhibitory peptide, which further comprises a pharmaceutically or food-acceptable carrier, diluent or excipient.

[0015] Thirdly, the present invention provides a method for screening xanthine oxidase inhibitory peptides, employing a technical route combining virtual hydrolysis, activity prediction, toxicity screening, molecular docking, and in vitro activity verification, comprising the following steps: (1) Virtual enzymatic digestion: Virtual enzymatic digestion of Antarctic krill protein sequences to obtain polypeptide fragments with a length of 5-8 amino acids; (2) Activity and toxicity prediction: Bioactivity and toxicity prediction were performed on the polypeptide fragments obtained in step (1), and candidate peptides with non-toxicity and predicted activity scores > 0.5 were screened out. (3) Molecular docking: The candidate peptides screened in step (2) are molecularly docked with xanthine oxidase protein to screen out peptides with low binding energy to xanthine oxidase and high Vina fraction. (4) In vitro activity verification: The peptides screened in step (3) were synthesized and subjected to in vitro xanthine oxidase inhibitory activity assay, and the IC50 was calculated. 50 The value was ultimately confirmed to be a highly active xanthine oxidase inhibitory peptide.

[0016] In a preferred embodiment, in step (1), the virtual enzymatic hydrolysis is performed using the BIOPEP-UWM platform, employing 34 different enzymes to virtually hydrolyze the Antarctic krill Timeless2 protein (NCBI reference sequence: AUI80375.1).

[0017] In a preferred embodiment, in step (2), the bioactivity prediction uses the PeptideRanker platform, and the toxicity prediction uses the ToxinPred platform.

[0018] In a preferred embodiment, in step (3), the molecular docking adopts the Autodock Vina algorithm, the acceptor is xanthine oxidase crystal structure 1N5X, the center coordinates of the docking box are set to (96, 54, 39), the box size is set to 15 Å × 15 Å × 15 Å, and the number of docking times is set to 10.

[0019] In a preferred embodiment, in step (4), the in vitro activity assay is performed using an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance change at a wavelength of 290 nm, and the inhibition rate is calculated according to the following formula: XO inhibition rate (%) = (V0 - Vs) / V0 × 100%, where Vs represents the initial reaction rate of the sample and V0 represents the initial reaction rate of the blank control.

[0020] Fourthly, the present invention provides the use of the xanthine oxidase inhibitory peptide in the preparation of pharmaceuticals or functional foods for inhibiting xanthine oxidase activity.

[0021] Fifthly, the present invention provides the use of the xanthine oxidase inhibitory peptide in the preparation of a medicament or functional food for the prevention and / or treatment of diseases related to xanthine oxidase activity, wherein the diseases are preferably hyperuricemia or gout.

[0022] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) The heptapeptide WRIPSRL screened from Antarctic krill in this invention has an in vitro xanthine oxidase inhibitory activity of IC50. 50 The value was 1.04 mM. This is similar to the IC50 of the hexapeptide PLGPPP from Litopenaeus vannamei disclosed in the prior art. 50 Compared to a concentration of 2.356 mM, the activity of the peptide of this invention is 2.3 times that of the prior art. This significant increase in activity obtained under the same in vitro activity evaluation system is not obvious to those skilled in the art, indicating that the present invention produces unexpected technical effects compared to existing technologies.

[0023] (2) Compared with other marine-derived XO inhibitory peptides reported in the literature, the IC50 of the WRIPSRL peptide of this invention is significantly higher. 50The value was 1.04 mM, and the IC50 of the skipjack tuna-derived peptide ACECD was [value missing]. 50 The value was 13.40 mM, and the IC50 of the tuna-derived peptide FH was [value missing]. 50 The value is 25.70 mM.

[0024] (3) Using molecular docking technology, this invention analyzed the binding mode of the heptapeptide with the sequence WRIPSRL to the XO enzyme.

[0025] Regarding binding sites, the peptide WRIPSRL binds to 1N5X at residues such as GLU879, PHE1013, THR1010, PHE1009, and PHE914. Literature reports indicate that amino acid residues GLU802, SER876, ARG880, PHE914, PHE1009, and THR1010 are XO activity-related sites, and the binding sites of the peptide in this invention overlap with these sites.

[0026] Regarding binding forces, the binding forces between peptide WRIPSRL and 1N5X include: THR1010 and SER876 forming hydrogen bonds with the peptide; PHE914 and PHE1009 forming π-π interactions with the peptide; PHE1013, VAL1011, ALA1078, ALA1079, and PHE1142 forming π-alkyl interactions with the peptide; and GLU879 forming electrostatic interactions.

[0027] Regarding the binding mode, the benzene ring structure located at the N-terminus Trp of the peptide is embedded in the active pocket of 1N5X.

[0028] (4) This invention employs a screening system of “virtual hydrolysis-activity prediction-toxicity screening-molecular docking-in vitro verification”. A total of 2224 peptides with lengths of 5-8 were obtained from the Antarctic krill protein sequence, and 34 candidate peptides were obtained after activity prediction and toxicity screening. The WRIPSRL peptide was obtained by synthesizing and verifying 6 high-affinity peptides in vitro.

[0029] (5) This invention has discovered and verified a highly active XO inhibitory peptide from the huge marine biological resource of Antarctic krill. Its amino acid sequence WRIPSRL is different from other known peptides in the prior art. It is a brand new active peptide molecule with independent intellectual property rights.

[0030] (6) The xanthine oxidase inhibitory peptide WRIPSRL provided by this invention can be prepared by solid-phase synthesis. In pharmaceuticals, this peptide can be used to prepare drugs that inhibit xanthine oxidase activity for the intervention of diseases related to xanthine oxidase activity. In functional foods, this peptide can be used as a raw material for functional foods. In terms of resource utilization, this invention obtains an active peptide from Antarctic krill protein, providing a new direction for the utilization of Antarctic krill resources. Attached Figure Description

[0031] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 The diagram shows the molecular docking results of the peptide WIMRFF and xanthine oxidase (1N5X) of this invention, where A is a three-dimensional interaction diagram and B is a two-dimensional interaction diagram.

[0033] Figure 2 The diagram shows the molecular docking results of the peptide EMFWRI and xanthine oxidase (1N5X) of this invention, where A is a three-dimensional interaction diagram and B is a two-dimensional interaction diagram.

[0034] Figure 3 The diagram shows the molecular docking results of the peptide CHRIGF and xanthine oxidase (1N5X) of this invention, where A is a three-dimensional interaction diagram and B is a two-dimensional interaction diagram.

[0035] Figure 4 The diagram shows the molecular docking results of the peptide FFLEFNR and xanthine oxidase (1N5X) of this invention, where A is a three-dimensional interaction diagram and B is a two-dimensional interaction diagram.

[0036] Figure 5 The diagram shows the molecular docking results of the peptide WRIPSRL and xanthine oxidase (1N5X) of this invention, where A is a three-dimensional interaction diagram and B is a two-dimensional interaction diagram.

[0037] Figure 6 The diagram shows the molecular docking results of the peptide SGAYNF and xanthine oxidase (1N5X) of this invention, where A is a three-dimensional interaction diagram and B is a two-dimensional interaction diagram.

[0038] Figure 7 This is a statistical diagram showing the binding sites of the six bioactive peptides of this invention with 1N5X.

[0039] Figure 8 This is a statistical diagram showing the binding forces between the six active peptides of this invention and 1N5X.

[0040] Figure 9This is a standard curve showing the relationship between the concentration of the peptide WIMRFF and the inhibition rate of the present invention.

[0041] Figure 10 This is a standard curve showing the relationship between the concentration of the peptide EMFWRI and the inhibition rate of the present invention.

[0042] Figure 11 This is a standard curve showing the relationship between the concentration of the peptide WRIPSRL and the inhibition rate of the present invention. Detailed Implementation

[0043] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0045] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0046] The xanthine oxidase inhibitory peptide of this invention has the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the sequences of representative peptides WIMRFF, EMFWRI, CHRIGF, FFLEFNR, and SGAYNF obtained during the screening process are shown in SEQ ID NO: 2-6, respectively.

[0047] This invention focuses on Antarctic krill protein (NCBI reference sequence: AUI80375.1) and utilizes virtual screening, molecular docking, and in vitro activity verification techniques to discover novel xanthine oxidase inhibitory peptides. Specifically, virtual hydrolysis was first performed using the BIOPEP-UWM platform. Following PeptideRanker activity prediction and ToxinPred toxicity screening, 34 non-toxic candidate peptides with a bioactivity Rank score > 0.5 were obtained. Then, molecular docking with the XO crystal structure (1N5X) was performed using the Autodock Vina algorithm to screen for 6 high-affinity peptides. Further in vitro XO inhibition rate determination revealed that the WRIPSRL peptide exhibited the best inhibitory activity, with an IC50 value of [missing information]. 50The value was 1.04 mM, and the inhibition rate reached 48.68% at a concentration of 1 mM. The following examples will describe in detail the above screening process, molecular mechanism analysis, and activity verification results.

[0048] Main materials and reagents used in the examples: Allopurinol (Beijing Solarbio Science & Technology Co., Ltd.), xanthine oxidase (Beijing Solarbio Science & Technology Co., Ltd.), xanthine (Sinopharm Chemical Reagent Co., Ltd.), potassium chloride (Sinopharm Chemical Reagent Co., Ltd.), sodium chloride (Sinopharm Chemical Reagent Co., Ltd.), sodium dihydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd.), potassium dihydrogen phosphate (Sinopharm Chemical Reagent Co., Ltd.).

[0049] The main instruments and equipment used in the embodiments are as follows: DK-98-1 constant temperature water bath (Tess Instruments Co., Ltd.), PL202-S electronic balance (Metteler Toledo), DHG-9075A electric thermostatic drying oven (Shanghai Shuli Instrument Co., Ltd.), MQX20R2 microplate reader (Thermo Fisher Scientific), 5804R refrigerated centrifuge (Eppendorf, Germany), BC / BD-218SHT refrigerator (Qingdao Haier Co., Ltd.).

[0050] Example 1: Virtual enzymatic hydrolysis of Antarctic krill-derived protein and prediction of its polypeptide properties (1) Protein sequence selection and virtual enzymatic digestion Search at https: / / www.uniprot.org Euphausia superba Existing Antarctic krill peptides are generally short in length. Therefore, the 1364 AA peptide Timeless2 (NCBI reference sequence: AUI80375.1) was selected as the target for virtual enzymatic hydrolysis. Using BIOPEP-UWM – Katedra Biochemii Żywności, 34 different enzymes were used to virtually hydrolyze the selected protein. Because excessively long peptide chains result in large spatial configurations, making it difficult to bind to key sites in the XO active pocket and block substrate channels, the inhibitory effect was poor. Therefore, peptides with lengths between 5 and 8 AA (a total of 2224 non-repeating peptides) were selected for this study.

[0051] (2) Activity prediction The bioactivity of selected peptides was predicted using PeptideRanker (https: / / distilldeep.ucd.ie / PeptideRanker / ), and peptides with an activity greater than 0.5 were retained. Peptides with a Ranker score higher than 0.5 were considered to have potential bioactivity.

[0052] (3) Toxicity prediction and water solubility calculation Toxicity and water solubility are also two important indicators for evaluating the application value of peptides. Toxicity prediction can be performed on the ToxinPred website using the "Designing of peptides for determined toxicity" module. This module can not only predict peptide toxicity but also assess changes in peptide toxicity due to amino acid mutations. Furthermore, the relevant properties of the obtained peptides, including molecular weight, hydrophobicity, and isoelectric point, can be calculated using PepCalc.com - Peptide calculus.

[0053] (4) Through the above screening, 34 candidate peptides with non-toxicity and bioactivity Ranger scores > 0.5 were obtained, and their details are shown in Table 1.

[0054] Table 1. Ranker scores and related properties of each peptide segment.

[0055] The target peptide WRIPSRL of this invention has a Ranker score of 0.8079, a molecular weight of 927.1, a hydrophobicity of 7.66 kcal / mol, and an isoelectric point of 12.1, indicating that it has good potential biological activity and physicochemical properties.

[0056] Example 2: Study on the inhibitory mechanism of peptides with xanthine oxidase inhibitory activity (1) Ligand treatment The molecular structures of amino acids and peptides were drawn using ChemDraw 23.1 software, and the 3D structures of amino acids or peptides were obtained using ChemDraw 3D 23.1. The specific steps were as follows: input and select the target peptide sequence; use the FASTA peptide function in Edit (Paste special) to obtain the abbreviated amino acid or peptide sequence; and use the Expand Lebel function in Structure to obtain the structural formula of the amino acid or peptide. The structural formula was then copied and pasted into the ChemDraw 3D 23.1 drawing interface, optimized using the MM2 force field, and saved as a PDB file.

[0057] (2) Receptor processing The 3D structure of 1N5X was downloaded from the PDB database (https: / / www1.rcsb.org / ). The B chain in 1N5X was deleted using PyMOL software, along with any febuxostat (TEI) ligand molecules in a bound state. The processed molecule was saved in PDBQT format for later use. Auldock 1.5.7 was then used to perform dehydration and hydrogenation of the acceptor 1N5X, and the gasteiger charge was calculated. Finally, all atoms were classified as AD4 and saved in PDBQT format.

[0058] (3) Molecular docking The Autodock Vina algorithm in PyRx was used to perform molecular docking between the processed receptor 1N5X and the ligand peptide to simulate the interaction between the receptor and ligand. Based on the position of febuxostat (TEI) in the 1N5X bound state, the center coordinates of the docking box were set to (96, 54, 39), the box size was set to 15×15×15, the number of dockings was set to 10, and other parameters were set to default values. The peptide ligand was docked with 1N5X one by one to obtain the Vina score of each peptide.

[0059] (4) Molecular docking results The Vina fractions of the six peptides were: WIMRFF (-7.3), EMFWRI (-6.3), CHRIGF (-7.8), FFELFNR (-6.2), WRIPSRL (-7.0), and SGAYNF (-7.2). Molecular docking results showed that all six peptides could bind to 1N5X and had good Vina fractions, indicating that these six peptides have potential inhibitory activity.

[0060] (5) Visualization analysis The top six peptides with the highest Rank scores were selected for visualization analysis. Specifically, the following steps were taken: The PyRx integration docking software output a file in peptide-out format. This output file, along with the modified 1N5X file, was simultaneously opened in Pymol software and exported as ligand-receptor bound molecules. This file was saved as peptide-1N5X in pdb format. The bound molecule file was then opened in Discovery Studio Client software. The small molecule ligand was located and defined as a Ligand. The Ligand interaction function was used to view the 3D interaction between the small molecule ligand and the large molecule receptor. The Show 2D diagram function was used to represent the 2D interaction diagram between peptides 1N5X.

[0061] (5-1) Analysis of Active Peptide Molecular Docking Results: Analysis of Molecular Docking between Peptide WIMRFF and 1N5X from Figure 1 The 3D interaction diagram shows that the Phe at the N-terminus of WIMRFF falls into the channel of the active pocket of 1N5X. The 2D interaction diagram shows that 802GLU and 1010THR form conventional hydrogen bond interactions with the peptide; 1011VAL, 1078ALA, and 1079ALA around the active pocket of 1N5X form π-alkyl interactions; 914PHE and 1009PHE around the active pocket of 1N5X form π-π interactions; in addition to the above interactions, 1011VAL on 1N5X forms alkyl interactions with -CH3 on the peptide, and 879GLU forms electrostatic interactions.

[0062] (5-2) Molecular docking analysis of peptide EMFWRI and 1N5X from Figure 2 The 3D interaction diagram shows that the benzene ring structure in the EMFWRI peptide is completely embedded in the active pocket of 1N5X to restrict the contact between the substrate and the enzyme's active site. The 2D interaction diagram shows that 875HIS, 876SER, and 1010THR all form conventional hydrogen bond interactions with the peptide; 1012PRO around the active pocket of 1N5X forms π-alkyl interactions; in addition to the above interactions, 1009PHE, 1011VAL, and 1014LEU on 1N5X form alkyl interactions with the -CH3 group on the peptide.

[0063] (5-3) Molecular docking analysis of peptide CHRIGF and 1N5X from Figure 3 The 3D interaction diagram shows that the N-terminal Phe benzene ring structure of the CHRIGF peptide is completely embedded in the active pocket of 1N5X to restrict the contact between the substrate and the enzyme's active site. The 2D interaction diagram shows that 876SER, 879GLU, and 1010THR all form conventional hydrogen bond interactions with the peptide; 1079ALA around the active pocket of 1N5X forms a π-alkyl interaction with the peptide; 914PHE and 1009PHE around the active pocket of 1N5X form π-π interactions; in addition to the above interactions, 649PHE, 1011VAL, and 1013PHE on 1N5X form alkyl interactions with the -CH3 group on the peptide.

[0064] (5-4) Molecular docking analysis of peptide FFLEFNR and 1N5X from Figure 4The 3D interaction diagram shows that the C-terminal benzene ring structure of the peptide FFLEFNR is completely embedded in the active pocket of 1N5X to restrict the contact between the substrate and the enzyme's active site. The 2D interaction diagram shows that 879GLU and 1140TYR form conventional hydrogen bonds with the peptide; 648LEU forms C-H bonds with the peptide molecule; 1011VAL forms π-σ interactions with the benzene ring; 648LEU, 771LYS, and 775PHE form alkyl interactions with the peptide; 648LEU, 1014LEU, and 1076PRO around the 1N5X active pocket form π-alkyl interactions with the peptide; 648LEU, 771LYS, and 775PHE form alkyl interactions with the peptide; and 875HIS forms a π-donor hydrogen bond with the peptide. In addition to the above interactions, 879GLU also forms a salt bridge with the N-terminus of the peptide.

[0065] (5-5) Molecular docking analysis of peptide WRIPSRL and 1N5X from Figure 5 The 3D interaction diagram shows that the benzene ring structure located at the N-terminal Trp of the peptide is completely embedded in the active pocket of 1N5X to restrict the contact between the substrate and the enzyme's active site. The 2D interaction diagram shows that 1010THR and 876SER form conventional hydrogen bond interactions with the peptide; 914PHE and 1009PHE around the 1N5X active pocket form π-π interactions with the peptide; 1011VAL, 1013PHE, 1078ALA, 1079ALA, and 1142PHE form π-alkyl interactions with the peptide; and 879GLU forms an electrostatic interaction.

[0066] (5-6) Molecular docking analysis of peptide SGAYNF and 1N5X from Figure 6 The 3D interaction diagram shows that the C-terminal benzene ring structure of the SGAYNF peptide is completely embedded in the active pocket of 1N5X to restrict the contact between the substrate and the enzyme's active site. The 2D interaction diagram shows that 768ASN, 879GLU, and 1140TYR form conventional hydrogen bonds with the peptide; while 771LYS and 1010THR form carbon-hydrogen bonds; 1014LEU forms a π-σ interaction with the C-terminal benzene ring structure; and 1013PHE of 1N5X forms a π-π stacking interaction with the Phe benzene ring structure at the C-terminus of the peptide; furthermore, 648LEU also forms a π-π stacking interaction with the benzene ring structure of Tyr in the peptide.

[0067] (5-7) Analysis of peptide binding sites and binding interactions with 1N5X The statistical analysis of the binding sites of six bioactive peptides to the receptor 1N5X is as follows: Figure 7As shown in the figure, the binding sites of the six bioactive peptides to 1N5X are mainly GLU879, PHE1013, VLA1011, THR1010, PHE1009, PHE914, and HIS875, with GLU879 being the most predominant amino acid residue. In studies on molecular docking of ligands with 1N5X, amino acid residues GLU802, SER876, ARG880, PHE914, PHE1009, and THR1010 are considered key sites for inhibiting XO activity. The amino acid residues in this experiment share the same PHE and THR values.

[0068] The statistical analysis of the binding forces of six bioactive peptides to receptor 1N5X is as follows: Figure 8 As shown in the figure, the binding forces between the six active peptides and 1N5X are mainly conventional hydrogen bonds, π-alkyl interactions, π-hydrophobic interactions, and alkyl interactions, with conventional hydrogen bonds being the most dominant type of interaction. CHRIGF, EMFWRI, WIMRFF, and WRIPSRL bind to 1N5X through hydrogen bonds and π-hydrophobic interactions. This multi-mode, multi-valent interaction mechanism collectively constructs a highly stable inhibitory complex system. Among them, CHRIGF and WIMRFF bind to 1N5X through π-hydrophobic interactions. π-hydrophobic interactions, as a relatively rare but highly selective binding mode, provide a unique binding advantage for the inhibitory peptides, effectively distinguishing them from the binding modes of natural substrates. All of the above-mentioned different binding forces enhance the stability of the inhibitory peptides binding to XO, achieving highly efficient inhibition of XO enzyme activity.

[0069] Example 3: Synthesis of peptides The top six peptides with the highest Ranker scores (WIMRFF, EMFWRI, CHRIGF, FFLEFNR, WRIPSRL, and SGAYNF) were selected for synthesis. They were synthesized by Shanghai Sangon Biotech Co., Ltd. using the Fmoc solid-phase synthesis method, achieving a purity of 95%. 10 mg of each peptide was synthesized for later use.

[0070] Example 4: Determination of xanthine oxidase XO inhibition rate (1) Solution preparation A 1.5 mM xanthine solution and a 0.1 U / mL XO solution were prepared using PBS buffer.

[0071] (2) Measurement method Mix 50 μL of sample solution with 50 μL of XO in a 96-well microplate and incubate at 37°C for 20 min. After incubation, quickly add 100 μL of 1.5 mM xanthine solution to each well and mix thoroughly with a shaker. Then, measure the absorbance at 290 nm using a microplate reader for 3 min, recording data every 10 s. Use PBS buffer as a blank control.

[0072] (3) Inhibition rate calculation The XO inhibition rate is calculated as follows: ; In the formula: Indicates the initial reaction rate of the sample; This indicates the initial reaction rate of the blank control.

[0073] (4) Active peptide IC 50 Measurement 50% inhibitory concentration (IC50) of active peptides on XO activity 50 The XO inhibition percentage versus concentration curve was calculated. Six peptides were dissolved in PBS to prepare solutions of 4 mM, 2 mM, 1 mM, and 0.5 mM concentrations. The in vitro XO inhibitory activity of the synthesized peptides was determined using the method described above, and the IC50 was calculated. 50 value.

[0074] (5) Measurement results Analysis of the inhibition rate of active peptide XO: The inhibition rates of six peptides and allopurinol against XO at different concentrations are detailed in Table 2. The IC50 fitting curves of WRIPSRL (with the best activity) and two representative peptides, WIMRFF and EMFWRI, are presented. Data for the remaining peptides are shown in Table 2.

[0075] Table 2. Inhibition rate of xanthine by various substances at different concentrations

[0076] (5-1) Analysis of the relationship between peptide WIMRFF concentration and inhibition rate A standard curve was plotted based on the linear relationship between WIMRFF concentration and inhibition rate, as shown below. Figure 9 As shown. WIMRFF IC can be obtained through calculation using the standard curve. 50 = 1.30 mM.

[0077] (5-2) Analysis of the relationship between peptide EMFWRI concentration and inhibition rate A standard curve was plotted based on the linear relationship between EMFWRI concentration and inhibition rate, as shown below. Figure 10As shown. The EMFWRI IC can be calculated using the standard curve. 50 = 1.25 mM.

[0078] (5-3) Analysis of the relationship between WRIPSRL peptide concentration and inhibition rate A standard curve was plotted based on the linear relationship between WRIPSRL concentration and inhibition rate, as shown below. Figure 11 As shown. The WRIPSRL IC can be calculated using the standard curve. 50 = 1.04 mM.

[0079] (6) Activity comparison analysis The results showed that all six peptides obtained from enzymatic hydrolysis possessed in vitro XO inhibitory activity. Among them, the WRIPSRL peptide showed the highest IC50 value. 50 The concentration was as low as 1.04 mM, and the inhibition rate reached 48.68% at a concentration of 1 mM, exhibiting the best in vitro inhibitory effect among its peers. Currently, XO inhibitory peptides with the sequence ACECD (ICP-1) extracted from skipjack tuna hydrolysates have been reported. 50 = 13.40 mM), and an XO repressor peptide with the sequence FH obtained from tuna (IC50). 50 = 25.70 mM); Compared with the peptides with the same activity reported above, the six peptide sequences screened in this study all have stronger XO inhibitory activity.

[0080] It is worth noting that, compared with the hexapeptide PLGPPP (IC) from Litopenaeus vannamei reported in the prior art, 50 Compared to (2.356 mM), the IC50 of the WRIPSRL heptapeptide obtained by screening in this invention is significantly higher. 50 The value was 1.04 mM, and its xanthine oxidase inhibitory activity was approximately 2.3 times that of the former. This indicates that WRIPSRL is a novel xanthine oxidase inhibitory peptide with significantly enhanced activity, providing a more promising candidate molecule for adjunctive intervention in gout or hyperuricemia.

[0081] Example 5: Data Analysis Method Data in this invention are expressed as mean ± standard deviation. SPSS 17.0 statistical software package (SPSS Inc. Chicago, Illinois, USA) and Origin 2021 (OriginLab, Northampton, MA, USA) were used to perform one-way ANOVA and plotting, respectively. p <0.05 indicates a statistically significant difference.

[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A xanthine oxidase inhibitory peptide derived from Antarctic krill, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The xanthine oxidase inhibitory peptide according to claim 1, characterized in that, It is derived from Antarctic krill protein.

3. The xanthine oxidase inhibitory peptide according to claim 2, characterized in that, The Antarctic krill protein is the Antarctic krill Timeless2 protein, whose NCBI reference sequence is AUI80375.

1.

4. A xanthine oxidase inhibitor comprising the xanthine oxidase inhibitory peptide according to any one of claims 1-3.

5. The xanthine oxidase inhibitor according to claim 4, characterized in that, It also includes pharmaceutically or food-grade carriers, diluents, or excipients.

6. A method for screening xanthine oxidase inhibitory peptides according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Virtual enzymatic digestion: Virtual enzymatic digestion of Antarctic krill protein sequences to obtain polypeptide fragments with a length of 5-8 amino acids; (2) Activity and toxicity prediction: Bioactivity and toxicity prediction were performed on the polypeptide fragments obtained in step (1), and candidate peptides with non-toxicity and predicted activity scores > 0.5 were screened out. (3) Molecular docking: The candidate peptides screened in step (2) are molecularly docked with xanthine oxidase protein to screen out peptides with low binding energy to xanthine oxidase and high Vina fraction. (4) In vitro activity verification: The peptides screened in step (3) were synthesized and subjected to in vitro xanthine oxidase inhibitory activity assay, and the IC50 was calculated. 50 The value was ultimately confirmed to be a highly active xanthine oxidase inhibitory peptide.

7. The method according to claim 6, characterized in that, The virtual enzymatic hydrolysis in step (1) uses the BIOPEP-UWM platform and employs 34 different enzymes to perform virtual hydrolysis of Antarctic krill Timeless2 protein.

8. The method according to claim 6, characterized in that, In step (2), the bioactivity prediction was performed using the PeptideRanker platform, and the toxicity prediction was performed using the ToxinPred platform. In step (3), the molecular docking was performed using the AutodockVina algorithm, and the acceptor was xanthine oxidase crystal structure 1N5X. In step (4), the in vitro activity assay was performed using an enzyme-linked immunosorbent assay (ELISA) reader, measuring the absorbance change at a wavelength of 290 nm and calculating the IC50. 50 value.

9. The use of the xanthine oxidase inhibitory peptide according to any one of claims 1-3 in the preparation of a pharmaceutical or functional food for inhibiting xanthine oxidase activity.

10. The use of the xanthine oxidase inhibitory peptide according to any one of claims 1-3 in the preparation of a medicament or functional food for the prevention and / or treatment of diseases related to xanthine oxidase activity, wherein the disease is hyperuricemia or gout.

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