Xanthine oxidase inhibitory peptide derived from green and sweet tuna and application of xanthine oxidase inhibitory peptide

Abstract

The invention relates to a xanthine oxidase inhibitory peptide derived from green and sweet tuna and application of the xanthine oxidase inhibitory peptide, and belongs to the technical field of polypeptides, and the amino acid sequence of the xanthine oxidase inhibitory peptide is Trp-Thr-Ala (WTA), Trp-Val-Pro (WVP), Trp-Leu-Pro (WLP) or 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 at least one inhibitory peptide or a composition or functional food 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 a medicine for preventing and treating hyperuricemia or serving as a functional food additive.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptides, and specifically relates to a xanthine oxidase inhibitory peptide derived from bluefin tuna and application thereof. Background Art

[0002] Hyperuricemia (HUA) is a chronic disease caused by abnormally elevated blood uric acid levels due to purine metabolism disorders or reduced uric acid excretion in the body. If the human blood maintains high uric acid levels for a long time, monosodium urate crystals will be deposited in joints, soft tissues, kidneys and other parts, leading to local inflammatory reactions, which may cause edema. The consensus data of multidisciplinary experts on the diagnosis and treatment of hyperuricemia-related diseases in China show that with the changes in dietary patterns and lifestyles, especially the consumption of low-level exercise, high-purine foods, high-sugar diets and alcoholic beverages, hyperuricemia in my country has shown a significant upward and younger development trend, gradually becoming the second largest metabolic disease after type 2 diabetes. In addition, many epidemiological studies have shown that hyperuricemia and gout are associated with the development of hypertension, cardiovascular disease, chronic kidney disease and diabetes, and hyperuricemia promotes the development of these diseases by inducing inflammation, oxidative stress, endothelial dysfunction and insulin resistance. Therefore, controlling high uric acid levels is of great significance for the prevention and treatment of these metabolic diseases.

[0003] There are two main types of drugs for the treatment of hyperuricemia in clinical practice, one is xanthine oxidase (XO) inhibitors, and the other is drugs that promote uric acid excretion. The former is better than the latter because XO inhibitors have relatively fewer side effects. XO is a key enzyme in purine metabolism, responsible for converting xanthine into uric acid. Allopurinol and Febuxostat are common XO inhibitors that reduce the production of uric acid by inhibiting the activity of XO, thereby reducing the level of uric acid in serum. Although there are currently a variety of drugs that can control uric acid levels, these drugs often have certain limitations and side effects, such as liver and kidney damage, allergic reactions, gastrointestinal reactions, Stevens-Johnson Syndrome (SJS), etc. Therefore, seeking natural, low-side-effect XO inhibitors has become an important direction of current research.

[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. They are widely used in drug research for the prevention and improvement of chronic diseases, functional food development, cosmetics, etc. In recent years, the research on the uric acid-lowering effect of protein hydrolysates and peptides has gradually deepened. Studies have shown that some protein hydrolysates and peptides have significant XO inhibitory effects and potential uric acid-lowering effects in vivo. For example, chicken breast hydrolysate can effectively reduce the uric acid level in acute hyperuricemia mice; oyster hydrolysate and the peptides identified from it (sequences: ALSGSW, GGYGIF and MAIGLW) have strong XO inhibitory activity in vitro; bonito hydrolysate and the peptides identified from it (sequences: PGACSN and WML) have strong XO inhibitory activity in vitro.

[0005] Bluefin tuna Thunnus tonggo l) is a tuna ( Thunnus ) is the second smallest species, found in tropical and subtropical waters of the Indo-Pacific region. Compared to some other tunas (such as yellowfin tuna Thunnus albacares and bluefin tuna Thunnus thynnus ) For bluefin tuna, its rough texture and low fat content make its taste and flavor inferior. It is often regarded as low-value tuna in the market and is mainly made into canned, dried, frozen and other products. Bluefin tuna meat contains about 22% protein and has a rich amino acid composition, making it a high-quality source of animal protein. At present, there are few studies on the high-value of bluefin tuna, especially no reports on uric acid-lowering peptides derived from bluefin tuna protein. Summary of the invention

[0006] In view of the above technical problems, the present invention provides an XO inhibitory peptide derived from bluefin tuna and application thereof.

[0007] The present invention is achieved through the following technical solutions: A XO inhibitory peptide derived from bluefin tuna, whose amino acid sequence is Trp-Thr-Ala (WTA), Trp-Val-Pro (WVP), Trp-Leu-Pro (WLP) or Trp-Gly-Phe (WGF).

[0008] The present invention also provides the use of the inhibitory peptide as or in the preparation of a xanthine oxidase inhibitor.

[0009] The present invention also provides the use of the inhibitory peptide as or in the preparation of a drug having the efficacy of inhibiting uric acid levels.

[0010] The present invention also provides a xanthine oxidase inhibitor, wherein the inhibitor contains at least one inhibitory peptide.

[0011] The present invention also provides a composition for inhibiting uric acid, wherein the composition contains at least one of the inhibitory peptides.

[0012] The present invention also provides a functional food, which contains at least one inhibitory peptide.

[0013] The preparation method of the inhibitory peptide is to extract it from the meat of bluefin tuna, and the specific extraction process is as follows: The minced Qinggan tuna and water are mixed at a material-liquid ratio of 1:2-4 to form a mixed solution, and protease is added for enzymolysis, and the amount of enzyme added is 1-1.5% (the substrate is calculated based on the wet basis of fish meat), the enzymolysis temperature is 50-55°C, and the enzymolysis time is 3-5h. After the enzymolysis is completed, the enzyme is inactivated in boiling water for 10-20 min, and then the supernatant is taken and freeze-dried after centrifugation at 4°C for 20-30 min, and stored at -20°C for later use; the protease is one of neutral protease, papain, alkaline protease, animal protease, and flavor protease; Sephadex G-15 gel column was used to separate and purify the enzymatic hydrolysate, and the components with the strongest XO inhibition rate were collected. The amino acid sequence with the strongest XO inhibition rate was identified by liquid chromatography-mass spectrometry (LC-MS / MS), and then the amino acid sequence of the XO inhibitory peptide was obtained by DB search and molecular docking.

[0014] Using the above method, a total of 11 potential XO inhibitory peptides were screened, then solid-phase synthesized, and the XO inhibition rate was determined by spectrophotometry. The results showed that a total of 4 peptides showed strong XO inhibitory effects.

[0015] The toxicity of the screened peptide fragments was evaluated using the ToxinPred program. The screened peptides are novel food-borne XO inhibitory peptides with good water solubility, non-toxicity, good small intestinal absorption and blood-brain barrier permeability. They have the potential to act as xanthine oxidase inhibitors and lower uric acid levels, and can be used to prepare drugs for the prevention and treatment of hyperuricemia, or as functional food additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is a bar graph showing the effects of enzyme type, addition amount and enzymolysis time on XO inhibition rate; A is the effect of enzyme type on XO inhibition rate, B is the effect of enzyme addition amount on XO inhibition rate, and C is the effect of enzymolysis time on XO inhibition rate. There are significant differences between groups with different letters (a, b, c, etc.), and p Value is less than 0.05; Figure 2 Total ion current; Figure 3 The secondary mass spectra of four peptides, A is WTA, B is WVP, C is WLP, and D is WGF; Figure 4 This is the relationship between the mass concentration of peptide WTA and the XO inhibition rate; Figure 5 3D diagram for visualization of the mechanism of action between peptides and XO; A is WLP, B is WGF, C is WTA, and D is WVP; Figure 6 2D diagram for visualizing the mechanism of action between peptides and XO. A is WLP, B is WGF, C is WTA, and D is WVP; Figure 7 This is a planetary diagram of the action sites of XO and febuxostat. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further explained below by way of embodiments, but the protection scope of the present invention is not limited in any form by the embodiments.

[0018] Example 1: Preparation of Bluefin Tuna XO Inhibitory Peptide Remove the head, tail, bones, skin and internal organs of the bluefin tuna and mince it into minced fish.

[0019] The minced Qinggan tuna was mixed with water at a material-liquid ratio of 1:3 to form a mixed solution, and flavor protease was added for enzymolysis, with an enzyme amount of 1% (substrate based on the wet basis of fish meat), an enzymolysis temperature of 50°C, and an enzymolysis time of 3h. After the enzymolysis, the enzyme was inactivated in boiling water for 10 minutes, and then centrifuged in a refrigerated centrifuge at 4°C for 30 minutes, the supernatant was taken and freeze-dried, and stored at -20°C for later use.

[0020] The results show that: Figure 1 As shown, the bluefin tuna XO inhibitory peptide with a strong in vitro XO inhibition rate can be obtained according to the above preparation conditions.

[0021] Example 2 Isolation and purification of XO inhibitory peptide from bluefin tuna The sample of Example 1 was purified and separated by size exclusion chromatography (Gel Filtration Chromatography, GFC), the chromatographic column size was 1.6 cm × 61 cm, the filler was Sephadex G-15, the mobile phase was ultrapure water, and the elution conditions were: detection wavelength 214nm, flow rate 1 mL / min, loading concentration 50 mg / mL, and loading volume 2 mL. An automatic collector was used to collect, 10 mL per tube, and the eluate of the same elution peak was collected as the same component, and four components (F1, F2, F3, F4) were obtained in sequence. After vacuum freeze-drying, the XO inhibition rate at 20 mg / mL was determined respectively, and allopurinol was used as a positive control.

[0022] The results showed that as shown in Table 1, the XO inhibition rate of the F4 component at 20 mg / mL was significantly higher than that of the other three components and significantly higher than that of the enzymatic solution. The IC50 value of F4 was also determined to be 4.56±0.34 mg / mL. Therefore, the active peptide composition of the F4 component will be identified in the future; Table 1: XO inhibition rate / % and IC of different samples at 20 mg / mL 50 ; Note: There are significant differences between groups with different letters (a, b, c, etc.), and the p value is less than 0.05. "-" means no activity was detected or not measured.

[0023] Example 3 Peptide spectrum characterization and molecular docking simulation of bluefin tuna XO inhibitory peptide The amino acid sequence of the fourth component was analyzed by LC-MS / MS. The specific operation and parameters were as follows: the samples were analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was an Orbitrap FusionLumos mass spectrometer (Thermo Fisher Scientific, MA, USA) connected to an EASY-nanoLC 1200. A total of 5 μL of sample was loaded (C18 column: 20 cm × 75 μm id, 1.9 μm particle size). The sample was separated by a gradient of 60 min, the column flow was controlled at 300 nL / min, the column temperature was 40°C, the electrospray voltage was 2 kV, and the gradient started from 4% B phase, increased to 50% in 53 min 40 s, increased to 95% in 40 s, and maintained for 5 min 40 s.

[0024] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. 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 AGCtarget: 200%; maximum injection time: 86 ms; collision energy 25%, 30%, 35%; dynamic exclusion time: 30 s.

[0025] Tandem mass spectra were analyzed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database was uniprot-Thunnus tonggol_2024 (version2024, 73 entries), and the enzyme digestion was set to None. The search parameters were fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm, variable modification: Oxidation (M) 15.99, Deamidation (NQ) 0.98. The protein card value was at least 1 unique peptide; the peptide card value was -10lgP ≥ 20.

[0026] The molecular structure of the peptide was drawn using ChemDraw 21.0 software, and the 3D structure of the peptide was obtained using ChemDraw 3D 21.0. 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 / ). The B chain in 1N5X was deleted using PyMOL software, and the febuxostat ligand in the bound state in its sequence was deleted. The receptor 1N5X was dehydrated and hydrogenated and the small molecules existing in it were continued using Autdock 1.5.7. The processed receptor 1N5X was molecularly docked with the ligand peptide using Autodock 4.2 (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 was completed, the binding degree between the receptor 1N5X and the ligand peptide was determined by the Binding Energy value, and 11 unreported peptides with a binding energy less than -8.5 were screened out.

[0027] The toxicity of the peptides was calculated and verified by https: / / webs.iiitd.edu.in / raghava / toxinpred / . The 11 selected peptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid phase synthesis method.

[0028] The XO inhibition rate was determined by spectrophotometry: the peptide solution or blank reagent (pH 7.4 phosphate buffer solution) was incubated with 0.026 U / mL XO solution at 37°C for 3 minutes to allow the solution to reach equilibrium, and then 0.48 mM xanthine solution was added. The reaction began, and the absorbance was continuously detected at 290 nm using an ELISA reader. Allopurinol was used as a positive control. The XO inhibition rate can be expressed as: ; Among them, V sample and V blank represent the reaction rates in the presence and absence of inhibitors, respectively.

[0029] The results showed that the active peptide composition and amino acid sequence of F4 component were identified by LC-MS, and the total ion map (TIC) was shown in Figure 2 The secondary mass spectra of peptides Trp-Thr-Ala (WTA), Trp-Val-Pro (WVP), Trp-Leu-Pro (WLP), and Trp-Gly-Phe (WGF) are shown in Figure 3 .

[0030] With the protein card value as 1 unique peptide and the peptide card value as -10lgP≥20, a total of 199 peptide sequences were obtained from the search library. Molecular docking can predict the interaction between XO receptor and peptide receptor. Usually, active peptides with lower binding energy are more likely to bind to XO, making the resulting complex structure more stable. 11 peptides with 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.

[0031] The toxicity of the peptides was verified by calculation using https: / / webs.iiitd.edu.in / raghava / toxinpred / , and it was found that none of the peptides were toxic. The results are shown in Table 2; Table 2: Information of the peptides screened ; Note: “-” indicates that the activity was not detected or not determined.

[0032] Among them, four peptides with good in vitro XO inhibition rates were found, and the IC of peptide sequence WGF 50 The value is 7.56±0.64mM, and the IC of the peptide sequence WLP 50 The value was 8.16±1.02 mM, and the IC of the peptide sequence WVP 50 The value was 5.98±0.23 mM, and the IC 50The value is 5.56±0.25 mM. Among them, WTA has the best inhibitory effect. The relationship between its mass concentration and XO inhibition rate is shown in Figure 4 .

[0033] In order to further explore the reasons for the interaction between XO inhibitory peptides and polypeptides, the active sites of these four peptides were analyzed, and the docking results are as follows Figures 5 and 6 The results show that there are traditional hydrogen bonding, van der Waals forces, hydrophobic forces such as π-π stacking, π-π T-type, π-alkyl and other interactions between XO receptor and the four peptides. Further comparative analysis of the planetary map of XO and febuxostat action sites (see Figure 7 , Generation method: Download the PDB Format of 1N5X from the RCSB database, import it into the Protein Contacts Atlas program to automatically generate), it can be seen 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 between the four synthetic peptides and XO. Phe914 and Phe1009 residues show hydrophobic interactions in the interaction between the four synthetic peptides and XO; Arg880 and Leu648 residues show van der Waals forces in the interaction between the four synthetic peptides and XO; Thr1010 residues show hydrogen bonding forces in the interaction between the four synthetic peptides and XO; Glu802 residue participates in the hydrogen bonding force between WTA and XO, and shows van der Waals forces in the binding between the other three peptides and XO.

Claims

1. A xanthine oxidase inhibitory peptide derived from bluefin tuna, characterized in that: The amino acid sequence of the inhibitory peptide is Trp-Thr-Ala, Trp-Val-Pro, Trp-Leu-Pro or Trp-Gly-Phe.

2. Use of the inhibitory peptide according to claim 1 as or in the preparation of a xanthine oxidase inhibitor.

3. Use of the inhibitory peptide according to claim 1 as or in the preparation of a drug having the effect of inhibiting uric acid levels.

4. A xanthine oxidase inhibitor, characterized in that The inhibitor contains at least one inhibitory peptide according to claim 1.

5. A composition for inhibiting uric acid, characterized in that: The composition contains at least one inhibitory peptide according to claim 1.

6. A functional food, characterized in that: The functional food contains at least one inhibitory peptide according to claim 1.

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