Xanthine oxidase inhibitory peptide WGF and application thereof

Abstract

The invention relates to a xanthine oxidase inhibitory peptide WGF and application thereof, and belongs to the technical field of polypeptides, and the amino acid sequence of the xanthine oxidase inhibitory peptide WGF is Trp-Gly-Phe (WGF). The invention also provides an application of the inhibitory peptide as or in preparation of a xanthine oxidase inhibitor and an application of the inhibitory peptide as or in preparation of a medicine with an effect of inhibiting uric acid level. The invention also provides a xanthine oxidase inhibitor containing the inhibitory peptide or a composition for inhibiting uric acid. The selected inhibitory peptide is a novel food-borne XO inhibitory peptide, is good in water solubility, non-toxic and good in small intestine absorption and blood brain barrier permeability, has the potential of serving as a xanthine oxidase inhibitor and reducing the uric acid level, and can be used for preparing the medicine for preventing and treating hyperuricemia.

Description

[0001] This patent is a divisional case based on the patent with the application number 202411924370.4 and the patent title "A Xanthine Oxidase Inhibitory Peptide Derived from Thunnus tonggol and Its Application" applied on December 25, 2024. The divisional case was made due to the issue of lack of unity pointed out in the "First Examination Opinion" notice (document serial number: 2025012500232830) issued by the National Intellectual Property Administration on January 25, 2025. Technical Field

[0002] The present invention belongs to the technical field of polypeptides, and specifically relates to a xanthine oxidase inhibitory peptide WGF and its application. Background Art

[0003] Hyperuricemia (HUA) is a chronic disease caused by disorders of purine metabolism or reduced uric acid excretion in the body, resulting in abnormally elevated blood uric acid levels. If the blood uric acid level in the human body remains high for a long time, it will cause the deposition of monosodium urate crystals in joints, soft tissues, kidneys and other parts, leading to local inflammatory reactions and potentially triggering gout. Data from the Multidisciplinary Expert Consensus on the Diagnosis and Treatment of Hyperuricemia-Related Diseases in China shows that with the changes in dietary patterns and lifestyles, especially low-level exercise, high-purine foods, high-sugar diets and the consumption of alcoholic beverages, the incidence of hyperuricemia in China shows an obvious upward and younger trend, gradually becoming the second most common metabolic disease after type 2 diabetes. In addition, many epidemiological studies have shown that hyperuricemia and gout are related to the development of hypertension, cardiovascular diseases, chronic kidney diseases and diabetes. Hyperuricemia promotes the development of these diseases through mechanisms such as inducing inflammation, oxidative stress, vascular endothelial dysfunction and insulin resistance. Therefore, controlling the high uric acid level is of great significance for the prevention and treatment of these metabolic diseases.

[0004] There are mainly two types of drugs for the clinical treatment of hyperuricemia. One type is xanthine oxidase (XO) inhibitor drugs, and the other type is drugs that promote uric acid excretion. The former is superior to the latter because the side effects of XO inhibitors are relatively small. XO is a key enzyme in purine metabolism, responsible for converting xanthine into uric acid. Allopurinol and Febuxostat are common XO inhibitor drugs, which reduce the production of uric acid by inhibiting the activity of XO, thereby reducing the uric acid level in the serum. Although there are currently various drugs that can control the uric acid level, these drugs often have certain limitations and side effects, such as liver and kidney function damage, allergic reactions, gastrointestinal reactions, Stevens-Johnson Syndrome (SJS), etc. Therefore, seeking natural XO inhibitors with low side effects has become an important research direction.

[0005] Bioactive peptides derived from food sources have attracted much attention. They have the advantages of strong biological activity, wide sources, easy absorption, multiple targets, and low toxicity, and are widely used in the fields of drug research and cosmetics for the prevention and improvement of chronic diseases. In recent years, the research on the uric acid-lowering effects of protease hydrolysates and polypeptides has been gradually deepened. Existing research has shown that some protease hydrolysates and polypeptides have significant XO inhibitory effects and potential uric acid-lowering effects in vivo. For example, chicken breast meat hydrolysate can effectively reduce the uric acid level in acute hyperuricemic mice; oyster hydrolysate and the polypeptides identified from it (sequences: ALSGSW, GGYGIF, and MAIGLW) have strong XO inhibitory activity in vitro; skipjack tuna hydrolysate and the polypeptides identified from it (sequences: PGACSN and WML) have strong XO inhibitory activity in vitro.

[0006] The tonggol tuna (Thunnus tonggol) is the second smallest species in the genus Thunnus and is distributed in the tropical and subtropical waters of the Indo-Pacific region. Compared with some other tunas (such as the yellowfin tuna Thunnus albacares and the bluefin tuna Thunnus thynnus), the tonggol tuna has a relatively poor taste and flavor due to its rough meat fibers and low fat content, and is often regarded as a low-value tuna in the market and is mainly made into canned, dried, and frozen products. The flesh of the tonggol tuna contains about 22% protein, and its amino acid composition is rich, making it a high-quality source of animal protein. Currently, there is little research on the high-value utilization of the tonggol tuna, especially no reports on the uric acid-lowering peptides derived from the protein of the tonggol tuna. Summary of the Invention

[0008] The present invention provides an XO inhibitory peptide derived from Thunnus tonggol and its application in view of the above technical problems.

[0009] The present invention is realized through the following technical solutions:

[0010] A xanthine oxidase inhibitory peptide WGF, whose amino acid sequence is Trp-Gly-Phe ((WGF).

[0011] The present invention also provides the application of the inhibitory peptide in being or preparing a xanthine oxidase inhibitor.

[0012] The present invention also provides the application of the inhibitory peptide in being or preparing a drug with the efficacy of inhibiting uric acid level.

[0013] The present invention also provides a xanthine oxidase inhibitor, and the inhibitor contains the inhibitory peptide.

[0014] The present invention also provides a composition for inhibiting uric acid, and the composition contains the inhibitory peptide.

[0015] The preparation method of the inhibitory peptide is to extract it from the flesh of Thunnus tonggol, and the specific extraction process is as follows:

[0016] Mix the flesh of Thunnus tonggol with water at a solid-liquid ratio of 1:2 - 4 to form a mixed solution, add protease for enzymatic hydrolysis, the added enzyme amount is 1 - 1.5% (substrate calculated based on the wet basis of fish meat), the enzymatic hydrolysis temperature is 50 - 55 °C, and the enzymatic hydrolysis time is 3 - 5 h. After the enzymatic hydrolysis ends, inactivate the enzyme with boiling water for 10 - 20 min, then centrifuge at 4 °C for 20 - 30 min with a refrigerated centrifuge, take the supernatant and perform freeze-drying, and store it at -20 °C for standby; the protease is one of neutral protease, papain, alkaline protease, animal protease, and flavor protease;

[0017] Use a Sephadex G-15 gel column to separate and purify the enzymatic hydrolysate, and collect the fraction with the strongest XO inhibitory rate. Identify the amino acid sequence with the strongest XO inhibitory rate by liquid chromatography-mass spectrometry (LC-MS / MS) method, and then obtain the amino acid sequence of the XO inhibitory peptide through database search and molecular docking.

[0018] Using the above method, a total of 11 potential XO inhibitory peptides were screened, and then solid-phase synthesis was carried out on them, and the XO inhibitory rate was measured by spectrophotometry. The results showed that a total of 4 peptides showed strong XO inhibitory effects.

[0019] The toxicity of the screened peptide fragments was evaluated by the ToxinPred program. The screened peptides are novel food-derived XO inhibitory peptides with good water solubility, non-toxicity, good intestinal absorption and blood-brain barrier permeability, and have the potential to be used as xanthine oxidase inhibitors and reduce uric acid levels, and can be used to prepare drugs for preventing and treating hyperuricemia. Description of the Drawings

[0020] Figure 1 It is a bar graph showing the effects of enzyme type, addition amount and enzymolysis time on the XO inhibition rate; among them, A is the effect of enzyme type on the XO inhibition rate, B is the effect of enzyme addition amount on the XO inhibition rate, C is the effect of enzymolysis time on the XO inhibition rate, and there are significant differences between groups with different letters (a, b, c, etc.), and the p value is less than 0.05;

[0021] Figure 2 Total ion chromatogram;

[0022] Figure 3 It is the secondary mass spectrum of four polypeptides, where A is WTA, B is WVP, C is WLP, and D is WGF;

[0023] Figure 4 It is a graph showing the relationship between the mass concentration of polypeptide WTA and the XO inhibition rate;

[0024] Figure 5 It is a visualized 3D map of the interaction mechanism between polypeptide and XO; A is WLP, B is WGF, C is WTA, and D is WVP;

[0025] Figure 6 It is a visualized 2D map of the interaction mechanism between polypeptide and XO. A is WLP, B is WGF, C is WTA, and D is WVP;

[0026] Figure 7 It is a planetary map of the binding sites of XO and febuxostat.

[0027] Specific Embodiments:

[0028] The technical solution of the present invention will be further explained below through examples, but the protection scope of the present invention is not limited by any form of the examples.

[0029] Example 1: Preparation of XO Inhibitory Peptide from Thunnus tonggol

[0030] The head, tail, bones, skin and viscera of Thunnus tonggol were removed, and the fish meat was minced.

[0031] Mix the flesh paste of Thunnus tonggol with water at a material-liquid ratio of 1:3 to form a mixed solution, add flavor protease for enzymatic hydrolysis, with an enzyme addition amount of 1% (substrate calculated based on the wet basis of fish meat), an enzymatic hydrolysis temperature of 50 °C, and an enzymatic hydrolysis time of 3 h. After the enzymatic hydrolysis, inactivate the enzyme in boiling water for 10 min, then centrifuge at 4 °C for 30 min using a refrigerated centrifuge, take the supernatant and perform freeze-drying, and store it at -20 °C for standby.

[0032] The results show that: as Figure 1 shown, the XO inhibitory peptide of Thunnus tonggol with a strong in vitro XO inhibitory rate can be obtained according to the above preparation conditions.

[0033] Example 2 Isolation and purification of the XO inhibitory peptide of Thunnus tonggol

[0034] Use size exclusion chromatography (Gel Filtration Chromatography, GFC) to purify and separate the sample of Example 1. The specifications of the chromatographic column are 1.6 cm * 61 cm, the packing material is Sephadex G-15, the mobile phase is ultrapure water, and the elution conditions are: detection wavelength 214 nm, flow rate 1 mL / min, sample loading concentration 50 mg / mL, and sample loading volume 2 mL. Use an automatic collector to collect, 10 mL per tube, and collect the eluent of the same elution peak as the same component. Four components (F1, F2, F3, F4) are obtained in sequence. After vacuum freeze-drying respectively, measure the XO inhibitory rate at 20 mg / mL, and use allopurinol as the positive control.

[0035] The results show that: as shown in Table 1, the XO inhibitory rate of the F4 component at 20 mg / mL is significantly higher than that of the other three components and significantly higher than that of the enzymatic hydrolysate. At the same time, the IC50 value of F4 is measured to be 4.56 ± 0.34 mg / mL. Therefore, the active peptide composition of the F4 component will be identified subsequently;

[0036] Table 1: XO inhibitory rate / % and IC of different samples at 20 mg / mL 50

[0037]

[0038]

[0039] Note: There are significant differences between groups with different letters (a, b, c, etc.), and the p value is less than 0.05. "-" is recorded as no activity detected or not measured.

[0040] Example 3 Peptide mapping characterization and molecular docking simulation of the XO inhibitory peptide of Thunnus tonggol

[0041] The amino acid sequence of the fourth component was analyzed using LC-MS / MS. The specific operations and parameters are as follows: The sample was analyzed by LC-MS / MS equipped with an online nano-spray ion source. The whole system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) in tandem with an EASY-nanoLC 1200. A total of 5 μL of the sample was loaded (C18 chromatographic column: 20 cm × 75 μm i.d, 1.9 μm particle size), and the sample was separated with a 60-min gradient. The column flow rate was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 4% of phase B and increased to 50% in a non-linear gradient at 53 min 40 s, and increased to 95% in 40 s and maintained for 5 min 40 s.

[0042] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisitions. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 100 - 1500; resolution: 120,000; Normalized AGC target: 200%; maximum injection time: Custom; (2) HCD-MS / MS: resolution: 50,000; Normalized AGC target: 200%; maximum injection time: 86 ms; collision energy 25%, 30%, 35%; dynamic exclusion time: 30 s.

[0043] The tandem mass spectra were analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database was uniprot-Thunnus tonggol_2024 (version 2024, 73 entries), and no enzyme digestion was set. The search parameters were as follows: fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm, variable modifications: Oxidation(M)15.99, Deamidation(NQ)0.98. The protein score threshold was at least 1 unique peptide; the peptide score threshold was -10lgP ≥ 20.

[0044] Use Kingdraw software to draw the molecular structural formula of polypeptides, and obtain the crystal structure of the complex of XO and the inhibitor febuxostat (TEI) (PDB ID: 1N5X) from the PDB database (https: / / www1.rcsb.org / ). Use PyMOL software to delete chain B in 1N5X, and at the same time delete the bound febuxostat ligand in its sequence. Then continue to use Autdock 1.5.7 to dehydrate and hydrogenate the receptor 1N5X and the small molecules present in it. Use Autodock4.2 (Vina version) to perform molecular docking of the processed receptor 1N5X with the ligand polypeptide. The center coordinates of the docking box are (95.9626, 54.1077, 39.0105) (x, y, z), the box size is 15×15×15, and other parameters are set to default values. After the calculation is completed, determine the binding degree between the receptor 1N5X and the ligand polypeptide through the value of Binding Energy, and screen out 11 unreported polypeptides with a binding energy less than -8.5.

[0045] The toxicity of the polypeptides was calculated and verified through https: / / webs.iiitd.edu.in / raghava / toxinpred / . The 11 selected polypeptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method.

[0046] The inhibition rate of XO was determined by spectrophotometry: Take the polypeptide solution or blank reagent (pH 7.4 phosphate buffer solution) and incubate it with 0.026 U / mL XO solution at 37 °C for 3 minutes to allow the solution to reach equilibrium. Then add 0.48 mM xanthine solution, and the reaction starts. Continuously detect the absorbance value at 290 nm using an enzyme-linked immunosorbent assay reader. Use allopurinol as a positive control. The XO inhibition rate can be expressed as:

[0047]

[0048] where V sample and V blank represent the reaction rates in the presence and absence of the inhibitor, respectively.

[0049] The results showed that: The active peptide composition and amino acid sequence of the F4 fraction were identified by liquid chromatography-mass spectrometry (LC-MS). The total ion chromatogram (TIC) is shown in Figure 2 . The secondary mass spectra of the peptide segments Trp-Thr-Ala (WTA), Trp-Val-Pro (WVP), Trp-Leu-Pro (WLP), and Trp-Gly-Phe (WGF) are shown in Figure 3 .

[0050] Taking the protein card value as 1 unique peptide and the peptide segment card value as -10lgP≥20, a total of 199 polypeptide sequences were obtained by database searching. Molecular docking can predict the interaction between the XO receptor and the polypeptide receptor. Usually, the bioactive polypeptides with lower binding energy are more likely to bind to XO, making the structure of the generated complex more stable. Eleven peptides with a binding energy less than -8.5 were screened for solid-phase synthesis (purity greater than 95%), and the XO inhibitory activity was determined by spectrophotometry. The results are shown in Table 2.

[0051] The toxicity of the polypeptides was calculated and verified through https: / / webs.iiitd.edu.in / raghava / toxinpred / , and it was found that none of the peptide segments were toxic. The results are shown in Table 2;

[0052] Table 2: Information of the screened polypeptides

[0053]

[0054] Note: "-" is recorded as no activity detected or not measured.

[0055] Among them, four polypeptides with good in vitro XO inhibition rates were found. The IC 50 value of the polypeptide sequence WGF was 7.56±0.64 mM, the IC 50 value of the polypeptide sequence WLP was 8.16±1.02 mM, the IC 50 value of the polypeptide sequence WVP was 5.98±0.23 mM, and the IC 50 value of the polypeptide sequence WTA was 5.56±0.25 mM. Among them, for WTA with the best inhibitory effect, the relationship diagram between its mass concentration and the XO inhibition rate is shown in Figure 4 .

[0056] To further explore the reasons for the interaction between the XO inhibitory peptide and the polypeptide, the active sites of these four peptides were analyzed, and the docking results are as shown in Figures 5-6 . The results show that there are interactions such as traditional hydrogen bonds, van der Waals forces, and hydrophobic forces such as π-π stacking, π-π T-shaped, and π-alkyl between the XO receptor and the four peptides. Further comparative analysis of the planetary map of the binding sites of XO and febuxostat (see Figure 7, Generation method: Download the PDB Format of 1N5X from the RCSB database and generate it through https: / / pca.mbgroup.bio / . It can be seen that febuxostat mainly binds to Phe914, Arg880, Glu802, Leu648, Thr1010, and Phe1009 in the active center of XO. It is found that these binding sites also exist in the interaction forces between the four synthetic peptides and XO. The Phe914 and Phe1009 residues show hydrophobic interactions in the interaction between the four synthetic peptides and XO; the Arg880 and Leu648 residues show van der Waals forces in the interaction between the four synthetic peptides and XO; the Thr1010 residue shows hydrogen bond forces in the interaction between the four synthetic peptides and XO; the Glu802 residue participates in the hydrogen bond force binding between WTA and XO, while showing van der Waals forces in the binding of the other three peptides to XO.

Claims

1. Use of xanthine oxidase inhibitory peptide WGF in the preparation of a xanthine oxidase inhibitor, characterized in that, The amino acid sequence of the inhibitory peptide is Trp-Gly-Phe.

2. Use of xanthine oxidase inhibitory peptide WGF in the preparation of a drug with the efficacy of inhibiting uric acid level, characterized in that, The amino acid sequence of the inhibitory peptide is Trp-Gly-Phe.

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