A xanthine oxidase inhibitory peptide derived from Thunnus tonggol and its application

Abstract

The present invention relates to a xanthine oxidase inhibitory peptide derived from Thunnus tonggol and its applications, belonging to the technical field of polypeptides. Its amino acid sequences are Trp-Thr-Ala (WTA), Trp-Val-Pro (WVP), Trp-Leu-Pro (WLP), or Trp-Gly-Phe (WGF). The present invention also provides the application of the inhibitory peptide in being or preparing a xanthine oxidase inhibitor, and its application in being or preparing a drug with the efficacy of inhibiting uric acid levels. The present invention also provides a xanthine oxidase inhibitor or a composition for inhibiting uric acid containing at least one of the inhibitory peptides; the selected inhibitory peptide is a novel food-derived XO inhibitory peptide, which has good water solubility, is non-toxic, has good intestinal absorption and blood-brain barrier permeability, has the potential to be a xanthine oxidase inhibitor and reduce uric acid levels, and can be used to prepare drugs for preventing and treating hyperuricemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides, and particularly relates to a xanthine oxidase inhibitory peptide derived from Thunnus tonggol and its application. Summary of the Invention

[0002] 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, monosodium urate crystals will deposit 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 consumption of alcoholic beverages, 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 blood uric acid levels is of great significance for the prevention and treatment of these metabolic diseases.

[0003] Clinically, there are mainly two types of drugs for the treatment of hyperuricemia. One type is xanthine oxidase (XO) inhibitory 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 inhibitory drugs, which reduce uric acid production by inhibiting the activity of XO, thereby reducing the blood uric acid level in serum. Although there are currently various drugs that can control blood uric acid levels, 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.

[0004] Bioactive peptides 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 effect 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 therefrom (sequences: ALSGSW, GGYGIF, and MAIGLW) have strong XO inhibitory activity in vitro; skipjack hydrolysate and the polypeptides identified therefrom (sequences: PGACSN and WML) have strong XO inhibitory activity in vitro.

[0005] The frigate 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 frigate tuna has a relatively inferior 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, mainly made into products such as canned food, dried products, and frozen products. The frigate tuna meat contains about 22% protein, and its amino acid composition is rich, making it a high-quality source of animal protein. Currently, there is less research on the high-value utilization of frigate tuna, especially no reports on uric acid-lowering peptides derived from frigate tuna protein. Summary of the Invention

[0007] The present invention provides an XO inhibitory peptide derived from frigate tuna and its application in view of the above technical problems.

[0008] The present invention is realized through the following technical solutions:

[0009] An XO inhibitory peptide derived from frigate tuna, its amino acid sequence is Trp-Thr-Ala (WTA), Trp-Val-Pro (WVP), Trp-Leu-Pro (WLP), or Trp-Gly-Phe (WGF).

[0010] The present invention also provides the application of the inhibitory peptide in being or preparing a xanthine oxidase inhibitor.

[0011] 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.

[0012] The present invention also provides a xanthine oxidase inhibitor, and the inhibitor contains at least one of the inhibitory peptides.

[0013] The present invention also provides a composition for inhibiting uric acid, which contains at least one of the inhibitory peptides.

[0014] 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:

[0015] Mix the minced 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, with the enzyme addition amount of 1 - 1.5% (substrate calculated based on the wet weight of fish flesh), the enzymatic hydrolysis temperature of 50 - 55°C, and the enzymatic hydrolysis time of 3 - 5 h. After the enzymatic hydrolysis is completed, inactivate the enzyme in 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;

[0016] 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-tandem mass spectrometry (LC-MS / MS) method, and then obtain the amino acid sequence of the XO inhibitory peptide through database search (DB search) and molecular docking.

[0017] Using the above method, a total of 11 potential XO inhibitory peptides were screened out, and then they were solid-phase synthesized, and the XO inhibitory rate was measured by spectrophotometry. The results showed that a total of 4 peptides showed strong XO inhibitory effects.

[0018] Evaluate the toxicity of the screened peptide fragments through the ToxinPred program. The screened peptides are novel food-derived XO inhibitory peptides, which have good water solubility, no toxicity, good small intestine 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

[0019] Figure 1 It is a bar graph showing the effects of enzyme type, addition amount, and enzymatic hydrolysis time on the XO inhibitory rate; among them, A is the effect of enzyme type on the XO inhibitory rate, B is the effect of enzyme addition amount on the XO inhibitory rate, C is the effect of enzymatic hydrolysis time on the XO inhibitory rate, there are significant differences between groups with different letters (a, b, c, etc.), and the p value is less than 0.05;

[0020] Figure 2 Total ion chromatogram;

[0021] Figure 3 It is the secondary mass spectra of four polypeptides, where A is WTA, B is WVP, C is WLP, and D is WGF;

[0022] Figure 4Graph showing the relationship between the mass concentration of polypeptide WTA and the inhibition rate of XO;

[0023] Figure 5 Visualization 3D graph of the mechanism of action between polypeptide and XO; A is WLP, B is WGF, C is WTA, D is WVP;

[0024] Figure 6 Visualization 2D graph of the mechanism of action between polypeptide and XO. A is WLP, B is WGF, C is WTA, D is WVP;

[0025] Figure 7 Planetary graph of the binding sites between XO and febuxostat.

[0026] Specific implementation:

[0027] 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.

[0028] Example 1: Preparation of XO inhibitory peptide from Thunnus tonggol

[0029] The head, tail, bones, skin and viscera of Thunnus tonggol were removed and minced into fish meat paste.

[0030] The fish meat paste of Thunnus tonggol was mixed with water at a material-liquid ratio of 1:3 to form a mixed solution, and flavor protease was added for enzymatic hydrolysis. The enzyme addition amount was 1% (substrate calculated based on the wet basis of fish meat), the enzymatic hydrolysis temperature was 50 °C, and the enzymatic hydrolysis time was 3 h. After the enzymatic hydrolysis was completed, the enzyme was inactivated by boiling water for 10 min, and then centrifuged at 4 °C for 30 min by a refrigerated centrifuge. The supernatant was taken and freeze-dried, and stored at -20 °C for later use.

[0031] The results showed that: as Figure 1 shown, XO inhibitory peptide from Thunnus tonggol with strong in vitro XO inhibitory rate can be obtained according to the above preparation conditions.

[0032] Example 2 Separation and purification of XO inhibitory peptide from Thunnus tonggol

[0033] The sample of Example 1 was purified and separated by size exclusion chromatography (Gel Filtration Chromatography, GFC). The specifications of the chromatographic column were 1.6 cm * 61 cm, the packing material was Sephadex G-15, the mobile phase was ultrapure water, and the elution conditions were: detection wavelength 214 nm, flow rate 1 mL / min, sample loading concentration 50 mg / mL, and sample loading volume 2 mL. An automatic collector was used for collection, 10 mL per tube. The eluents of the same elution peak were collected as the same component, and four components (F1, F2, F3, F4) were obtained in sequence. After vacuum freeze-drying respectively, the XO inhibitory rate at 20 mg / mL was measured respectively, with allopurinol as the positive control.

[0034] The results showed that, as shown in Table 1, the XO inhibitory rate of the F4 fraction at 20 mg / mL was significantly higher than those of the other three fractions and significantly higher than that of the enzymatic hydrolysate. At the same time, the IC50 value of F4 was determined to be 4.56 ± 0.34 mg / mL. Therefore, the active peptide composition of the F4 fraction will be identified subsequently;

[0035] Table 1: XO inhibitory rate / % and IC of different samples at 20 mg / mL 50

[0036]

[0037] Note: There were significant differences between groups with different letters (a, b, c, etc.), and the p-value was less than 0.05. "-" was recorded as no activity detected or not measured.

[0038] Example 3 Peptide Mapping Characterization and Molecular Docking Simulation of XO Inhibitory Peptides from Thunnus tonggol

[0039] The amino acid sequence of the fourth fraction was analyzed using LC-MS / MS. The specific operations and parameters were as follows: The sample was analyzed by LC-MS / MS equipped with an online nano-ESI ion source. The whole system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) in tandem with 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, then increased to 95% in 40 s and maintained for 5 min 40 s.

[0040] 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.

[0041] The tandem mass spectrum was analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database was uniprot - Thunnus tonggol_2024 (version 2024, 73 entries), and the digestion enzyme was set to None. 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 threshold was at least 1 unique peptide; the peptide threshold was -10lgP≥20.

[0042] The molecular structural formula of the polypeptide was drawn using software to obtain the 3D structural formula of the polypeptide. The crystal structure of the complex of XO and the inhibitor febuxostat (TEI) (PDB ID: 1N5X) was obtained from the PDB database (https: / / www1.rcsb.org / ). Using the PyMOL software, chain B in 1N5X was deleted, and at the same time, the bound febuxostat ligand in its sequence was deleted. Then, the receptor 1N5X was dehydrated, hydrogenated, and the existing small molecules were processed using Autdock 1.5.7. The processed receptor 1N5X and the ligand polypeptide were subjected to molecular docking using Autodock4.2 (Vina version). The center coordinates of the docking box were (95.9626, 54.1077, 39.0105) (x, y, z), the box size was 15×15×15, and other parameters were set to default values. After the calculation, the binding degree between the receptor 1N5X and the ligand polypeptide was determined by the value of Binding Energy, and 11 unreported polypeptides with a binding energy less than -8.5 were screened out.

[0043] The toxicity of the polypeptide was calculated and verified through https: / / webs.iiitd.edu.in / raghava / toxinpred / . The 11 screened polypeptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid - phase synthesis method.

[0044] 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. The absorbance value is continuously detected at 290 nm using an enzyme - linked immunosorbent assay reader. Allopurinol was used as a positive control. The XO inhibition rate can be expressed as:

[0045]

[0046] Among them, V sample and V blank respectively represent the reaction rates in the presence and absence of an inhibitor.

[0047] The results showed that the active peptide composition and amino acid sequence of fraction F4 were identified by 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 .

[0048] 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 selected for solid-phase synthesis (purity greater than 95%). The XO inhibitory activity was determined by spectrophotometry, and the results are shown in Table 2.

[0049] 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;

[0050] Table 2: Information of the selected polypeptides

[0051]

[0052]

[0053] Note: "-" is recorded as no activity detected or not determined.

[0054] 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 .

[0055] 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 Figures 5 - 6 shown. The results indicate 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 interaction sites of XO and febuxostat (see Figure 7 , generation method: download the PDB Format of 1N5X from the RCSB database and import it into the corresponding program for automatic generation) shows that febuxostat mainly binds to Phe914, Arg880, Glu802, Leu648, Thr1010, and Phe1009 in the active center of XO, and 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 interaction between WTA and XO, while it shows van der Waals forces in the binding of the other three peptides to XO.

Claims

1. Use of a xanthine oxidase inhibitory peptide derived from bluefin tuna in the preparation of a xanthine oxidase inhibitor, characterized in that: The amino acid sequence of the inhibitory peptide is Trp-Thr-Ala.

2. Use of a xanthine oxidase inhibitory peptide derived from bluefin tuna in the preparation of a drug having the effect of inhibiting uric acid levels, characterized in that: The amino acid sequence of the inhibitory peptide is Trp-Thr-Ala.

Images