Preparation method of xanthine oxidase inhibitory peptide and its application in preparing uric acid-lowering products

Abstract

The present invention belongs to the technical field of active peptide preparation, and specifically relates to a method for preparing a xanthine oxidase inhibitory peptide and its application in the preparation of uric acid-lowering products. The present invention separates and characterizes tea protein hydrolysates with xanthine oxidase inhibitory activity, and uses molecular docking technology to screen out xanthine oxidase inhibitory peptides, whose amino acid sequence is: IAAGLQNTG. The present invention further combines in vitro gastrointestinal simulated digestion to study the biological stability of tea protein XO inhibitory peptides, and evaluates the uric acid-lowering activity of active peptides based on the HK‑2 hyperuricemia cell model. The present invention provides raw materials for the development of drugs with uric acid-lowering activity, xanthine oxidase inhibitors and other products, and provides a new way to achieve high-value utilization of tea protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of active peptide preparation, and particularly relates to a method for preparing a xanthine oxidase inhibitory peptide and its application in preparing a uric acid-lowering product. Background Art

[0002] Hyperuricemia (HUA) has become the fourth highest-risk chronic disease, after hyperlipidemia, hypertension, and hyperglycemia. Hyperuricemia is a metabolic disease caused by excessive purine intake, overproduction of uric acid, or insufficient excretion. It manifests primarily as serum uric acid concentrations exceeding normal limits (≥420 mol / L for men and ≥360 mol / L for women). Excessive uric acid concentrations have multiple adverse effects on the body, the most direct of which is the accumulation of crystals leading to gout and kidney stones. Currently, research on uric acid-lowering activity in China and abroad is primarily focused on the screening of xanthine oxidase inhibitors. Xanthine oxidase (XO), a key enzyme in purine metabolism, catalyzes the oxidation of hypoxanthine to xanthine, which is then further oxidized to uric acid, purines, and pyrimidines. Based on this mechanism, uric acid-lowering drugs such as allopurinol and febuxostat have been developed for the clinical treatment of hyperuricemia. However, long-term use of these drugs can have side effects, such as gastrointestinal discomfort, allergies, and nephrotoxicity. In recent years, the development of safe and effective bioactive peptides with xanthine oxidase inhibitory activity from natural products has become a hot topic of research. Examples include white shrimp protein XO inhibitory peptides (amino acid sequence: AEAQMWR), egg protein XO inhibitory peptides (amino acid sequence: EEK), and nut protein XO inhibitory peptides (amino acid sequences: RPLY and PGPR). These active peptides have a small molecular weight and readily bind to the active site of XO. They also possess advantages such as non-toxicity, high activity, and high tissue accumulation.

[0003] Tea is a traditional cash crop in China, boasting a rich variety of varieties and a vast cultivation area. In recent years, with the rapid development of the tea beverage industry, a large amount of waste tea residue has been generated during processing. However, the development and application of tea residue is still limited to low-value products such as organic fertilizer and animal feed. Tea protein accounts for 18%-20% of the weight of tea residue, and most of it is water-insoluble. Tea protein does not contain cholesterol and has a rich amino acid composition and variety of bioactive peptides. It is a high-quality edible plant protein with promising application prospects in the food industry for its nutritional and functional components. However, current research on the development of tea protein is relatively limited, especially in the field of bioactive peptides. Summary of the Invention

[0004] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a xanthine oxidase inhibitory peptide.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned xanthine oxidase inhibitory peptide.

[0006] Another object of the present invention is to provide the use of the above-mentioned xanthine oxidase inhibitory peptide in the preparation of uric acid-lowering products.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A xanthine oxidase inhibitory peptide having an amino acid sequence of IAAGLQNTG;

[0009] The method for preparing the xanthine oxidase inhibitory peptide comprises the following steps:

[0010] Directly adopting in vitro solid phase synthesis of the above xanthine oxidase inhibitory peptide or tea residue as raw material, extracting by alkali and acid precipitation, hydrolyzing by neutral protease, and purifying to obtain the xanthine oxidase inhibitory peptide;

[0011] The conditions for the neutral protease hydrolysis are preferably: hydrolysis at 39°C and pH 7.5 for 3.4 h;

[0012] The substrate concentration of the neutral protease hydrolysis is preferably 2% (m / V), and the enzyme-substrate ratio is preferably 0.3% (m / m);

[0013] The purification comprises the steps of ultrafiltration separation and liquid chromatography separation;

[0014] The specific operation of the ultrafiltration separation is:

[0015] Tea protein hydrolysate was separated using ultrafiltration tubes to collect fractions with molecular weight <3 kDa;

[0016] The specific operation of the liquid chromatography separation is:

[0017] Ultrafiltration fractions <3 kDa from tea protein hydrolysate were separated and purified by high performance liquid chromatography using a preparative reverse-phase C18 glass column. The active fractions were collected, concentrated by rotary evaporation, and freeze-dried to obtain xanthine oxidase inhibitory peptides.

[0018] The liquid chromatography conditions are preferably as follows: mobile phase A: ultrapure water + 0.1% TFA; mobile phase B: methanol + 0.1% TFA; injection volume 5 mL; elution flow rate 10 mL / min; detection wavelengths 214 nm and 280 nm; time program: 0-20 min 6%-25% B, 20-40.00 min 25%-70% B, 40-60 min 70%-90% B, all percentages are by volume;

[0019] Use of the xanthine oxidase inhibitory peptide in the preparation of a product for inhibiting xanthine oxidase;

[0020] Use of the xanthine oxidase inhibitory peptide in the preparation of uric acid-lowering products;

[0021] The uric acid-lowering product may be a drug having uric acid-lowering activity;

[0022] A xanthine oxidase inhibitor comprising at least one of the xanthine oxidase inhibitory peptide, a tea protein neutral protease hydrolyzate containing the xanthine oxidase inhibitory peptide, and a hydrolyzate containing the xanthine oxidase inhibitory peptide as an active ingredient;

[0023] The xanthine oxidase inhibitor further comprises a pharmaceutically acceptable carrier or excipient;

[0024] The pharmaceutically acceptable carrier or excipient includes at least one of a cosolvent, a moisturizer, a surfactant, a matrix, an emulsifier, a preservative and a solvent;

[0025] A uric acid-lowering drug comprising at least one of the above-mentioned xanthine oxidase inhibitory peptide, a tea protein neutral protease hydrolyzate containing the above-mentioned xanthine oxidase inhibitory peptide, and a hydrolyzate containing the above-mentioned xanthine oxidase inhibitory peptide as an active ingredient;

[0026] The uric acid-lowering drug further comprises a pharmaceutically acceptable carrier or excipient;

[0027] The pharmaceutically acceptable carrier or excipient includes at least one of a cosolvent, a moisturizer, a surfactant, a matrix, an emulsifier, a preservative and a solvent;

[0028] The present invention has the following advantages and effects compared to the prior art:

[0029] (1) This study isolated and characterized tea protein hydrolysates with xanthine oxidase inhibitory activity. Molecular docking technology was used to screen for active peptides with excellent XO inhibitory activity. The biostability of tea protein XO inhibitory peptides was investigated using simulated in vitro gastrointestinal digestion. The uric acid-lowering activity of the active peptides was evaluated using the HK-2 hyperuricemia cell model. Through these studies, highly active XO inhibitory peptides were screened.

[0030] (2) This study prepared tea protein hydrolysate based on the enzymatic hydrolysis of the previous process parameters, and then used ultrafiltration, RP-HPLC and HPLC-MS / MS techniques to separate, purify, characterize and identify tea protein hydrolysate with xanthine oxidase inhibitory activity. At the same time, molecular docking technology was used to screen out active peptides with good XO inhibitory effect, and the biostability of tea protein XO inhibitory peptides was studied in combination with in vitro gastrointestinal simulated digestion; the uric acid-lowering activity of the target peptide was evaluated based on the HK-2 hyperuricemia cell model, and the uric acid-lowering activity and mechanism of action of the target peptide were deeply studied by transcriptome biotechnology. The results showed that a new peptide IAAGLQNTG (843.4450 Da) obtained by the present invention had good xanthine oxidase (XO) inhibitory activity, and its inhibition rate IC 50 The value was 0.24 mg / mL. After simulated gastrointestinal digestion, IAAGLQNTG maintained a certain degree of stability during gastrointestinal digestion, but the inhibitory activity of its digestion product, XO, decreased. Molecular docking results showed that traditional hydrogen bonding and hydrophobic interactions have a significant influence on the interaction between the active peptide and XO. In addition, in the HK-2 hyperuricemia cell model, 1.0 mg / mL of IAAGLQNTG significantly reduced cellular uric acid levels by 33.33% compared with the model group.

[0031] (3) The present invention provides raw materials for the development of drugs with uric acid-lowering activity, xanthine oxidase inhibitors and other products, and provides a new way to achieve high-value utilization of tea protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the xanthine oxidase inhibition rate analysis of the tea protein hydrolysate and ultrafiltration fractions obtained in Example 1, where different letters indicate significant differences ( p <0.05).

[0033] Figure 2 This is a graph showing the HPLC separation of ultrafiltration fractions (MW < 3 kDa) and the xanthine oxidase inhibition rate analysis of each fraction, where different letters indicate significant differences ( p <0.05).

[0034] Figure 3 This is the LC-MS / MS total ion current graph of the liquid phase purified fraction.

[0035] Figure 4 This is the peptide spectrum characterization and molecular docking analysis diagram of the tea protein XO inhibitory peptide in Example 2, wherein: (A): secondary mass spectrum diagram of IAAGLQNTG; (B): optimal conformation 3D diagram and interaction 2D diagram obtained after molecular docking of IAAGLQNTG and XO (1N5X).

[0036] Figure 5 The following are analysis results of the stability and activity change characteristics of the active peptide IAAGLQNTG in simulated gastrointestinal digestion in vitro, including: (A): chromatogram of IAAGLQNTG after digestion with simulated gastric juice; (B): chromatogram of IAAGLQNTG after digestion with simulated gastrointestinal juice; (C): relative xanthine oxidase inhibition rate of IAAGLQNTG before and after digestion;* p <0.05,** p <0.01.

[0037] Figure 6 The following are analysis results of the cytotoxicity and uric acid-lowering activity of the active peptide IAAGLQNTG, including (A) cytotoxic effects of IAAGLQNTG, febuxostat and allopurinol on HK-2 cells; (B): HPLC chromatography (from top to bottom: spectra of 5 standard substances, spectra of cell samples in the optimal model group, spectra of cell samples in the 1.0 mg / mL IAAGLQNTG-treated group, spectra of cell samples in the allopurinol-treated group, and spectra of cell samples in the febuxostat-treated group); (C): Effects of different XO addition amounts and enzyme-catalyzed reaction times on cellular uric acid content in the hyperuricemia cell model establishment experiment; (D): Uric acid-lowering activity of IAAGLQNTG, febuxostat and allopurinol in the hyperuricemia cell model; different letters indicate significant differences ( p < 0.05). DETAILED DESCRIPTION

[0038] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0039] Biological materials and chemical reagents involved in the examples: neutral protease (1×10 5 U / g), Nanning Pangbo Bioengineering Co., Ltd.; xanthine oxidase (XO), Beijing Solaibao Technology Co., Ltd.; febuxostat, allopurinol, xanthine, uric acid, adenosine, hypoxanthine, inosine, methythiazolylthiocyanate (MTT), and DMSO, Shanghai MacLean Biochemical Technology Co., Ltd.; HK-2 cells, purchased from the Cell Bank of the Chinese Academy of Sciences; RPMI 1640 medium, fetal bovine serum, and double-streptomycin antibodies (penicillin / streptomycin) were purchased from Gibco, USA; acetonitrile (chromatographic grade) and potassium dihydrogen phosphate (chromatographic grade) were purchased from Tianjin Komiou Chemical Reagent Co., Ltd.; sodium pentanesulfonate was purchased from Shanghai Rui'an Biotechnology Co., Ltd. All other reagents were of analytical grade.

[0040] Statistical analysis: All experimental data are presented as mean ± standard deviation. Origin 2018 was used to plot data graphs. IC was calculated using SPSS 26.0 software. 50The data were analyzed by ANOVA and Duncan's method. P <0.05, the difference was statistically significant.

[0041] Example 1 Isolation and Purification of Tea Protein Xanthine Oxidase (XO) Inhibitory Peptide

[0042] 1. Experimental Methods

[0043] 1. Preparation of Tea Enzyme Hydrolysate

[0044] Yinghong No. 9 black tea residue was used as raw material, and the tea protein crude extract and tea protein hydrolysate were prepared according to the reference (Ye Haoduo, Guan Xiaosheng, Ma Feng, et al. Enzymatic preparation of uricosuric peptides from Yinghong No. 9 tea protein and comparison of activity of different molecular weight components [J]. Modern Food Science and Technology, 2023, 39(3):147-155. 10.13982 / j.mfst.1673-9078.2023.3.0401.). The enzymatic hydrolysis process was as follows: the tea protein crude extract was hydrolyzed by neutral protease at a substrate concentration of 2% (m / V), an enzyme-substrate ratio of 0.3% (m / m), 39 ℃, pH 7.5, and 3.4 h; after hydrolysis, the extract was heated at 95 ℃ for 10 min, cooled to room temperature, and centrifuged at 4000 r / min for 20 min, collect the supernatant (i.e., tea enzyme hydrolysate), and take part of the supernatant for lyophilization; both the supernatant and lyophilized powder were stored at -20 °C for future use.

[0045] 2. Purification and Enrichment of Tea Protein XO Inhibitory Peptides

[0046] (1) Ultrafiltration separation

[0047] Ultrafiltration is a membrane separation technique commonly used in laboratory and commercial settings to separate, purify, and concentrate proteins and peptides. The tea protein hydrolysate prepared in step 1 was separated through a 3 kDa ultrafiltration tube (Merck Millipore, Billerica, MA, USA) to obtain two ultrafiltration fractions, one with a concentration of >3 kDa and one with a concentration of <3 kDa. After freeze-drying, the xanthine oxidase inhibition rate was determined.

[0048] (2) RP-HPLC separation

[0049] Ultrafiltration fractions <3 kDa from tea protein hydrolysate were separated by high-performance liquid chromatography using a preparative reversed-phase C18 glass column (30 mm × 250 mm, 15 μm). Mobile phase A: ultrapure water + 0.1% TFA; mobile phase B: methanol + 0.1% TFA. The injection volume was 5 mL, the elution flow rate was 10 mL / min, and the detection wavelengths were 214 nm and 280 nm. The time program was: 0–20 min (6%–25% B), 20–40.00 min (25%–70% B), and 40–60 min (70%–90% B). Percentages are by volume. After elution, the fractions were collected, concentrated by evaporation, and freeze-dried. The XO inhibition rate of each fraction (1 mg / mL) was determined, and the most active fraction was selected for mass spectrometry structural identification.

[0050] 3. Determination of XO Inhibitory Activity

[0051] Referring to the team's previous research method (Chen Binbin, Xia Zhen, Ye Haoduo, et al. Response surface optimization of selenium-enriched Moringa oleifera seedpeptides with antioxidant, ACEI and XOI activities[J]. Journal of Food Measurement and Characterization, 2023, 17(2): 1289-1299. https: / / doi.org / 10.1007 / s11694-022-01690-x.), the inhibitory activity of the samples against XO was analyzed by measuring the production rate of uric acid in the enzymatic reaction, with slight modifications. The specific method is as follows: All reagents and sample solutions were prepared using pH 7.4 phosphate buffer. 50 μL of the sample to be tested and 50 μL of XO with a concentration of 0.02 U / mL were added to a 96-well plate in sequence, and then 50 μL of 0.48 mmol / L xanthine solution was added. Buffer was used instead of the sample as a blank control group. After the addition of the sample, the 96-well plate was shaken for 30 seconds, reacted at 25°C for 25 minutes, and the absorbance of the solution at 290 nm was measured. (1)

[0052] Where: A1 is the absorbance value of the added sample solution; A2 is the absorbance value of the sample solution without XO; A3 is the absorbance value of the sample solution replaced by the buffer solution; A4 is the absorbance value of the sample solution replaced by the buffer solution.

[0053] The active peptide freeze-dried powder was prepared into a concentration gradient solution, and its XO inhibition rate was determined. SPSS 26.0 software was used to fit the data, and the half-inhibitory concentration (IC50) of the XO inhibitory peptide was calculated by the regression equation. 50 ).

[0054] 2. Results Analysis

[0055] Previous studies have determined the process conditions for preparing tea protein hydrolysate (Ye Haoduo, Guan Xiaosheng, Ma Feng, et al. Enzymatic preparation of uric acid-lowering peptides from Yinghong No. 9 tea protein and comparison of the activities of different molecular weight components [J]. Modern Food Science and Technology, 2023, 39(3):147-155. 10.13982 / j.mfst.1673-9078.2023.3.0401.). The molecular weight of the peptide fragment is crucial to its biological activity. The XO inhibitory activity results of different ultrafiltration components of tea protein hydrolysate are shown in Figure 1 At a concentration of 1 mg / mL, the ultrafiltration fraction <3 kDa (60.56%) exhibited superior xanthine oxidase inhibitory activity compared to the tea protein hydrolysate (54.24%) and ultrafiltration fractions >3 kDa (45.89%). Therefore, the <3 kDa fraction was selected for the next purification step.

[0056] The ultrafiltration fraction <3 kDa was separated by RP-HPLC using a reverse phase C18 column to obtain four purified fractions, which were named F1, F2, F3, and F4 ( Figure 2 After concentration and freeze-drying, the xanthine oxidase inhibition rate of each component was determined at a concentration of 1.0 mg / mL. Figure 2 As shown in Table 1, the XO inhibition rates of F2 (88.71%) and F4 (89.4%) were significantly higher than those of F1 (18.96%) and F3 (36.28%), but there was no significant difference in the activity of the two components F2 and F4. In order not to miss the possible high-activity peptide components, the present invention mixed F2 and F4. As shown in Table 1, the XO inhibition rates of F2 (88.71%) and F4 (89.4%) were significantly higher than those of F1 (18.96%) and F3 (36.28%). 50 =0.72 mg / mL) and <3 kDa ultrafiltration fraction (IC 50 =0.41 mg / mL) compared to the mixture F2+F4 (IC 50 =0.29 mg / mL) showed stronger XO inhibitory activity. Therefore, the present invention will subsequently identify the active peptide composition of the F2+F4 mixed component.

[0057] Table 1 IC values ​​of xanthine oxidase inhibitory activity of different components 50 value

[0058] sample <![CDATA[IC 50 Value (mg / mL)]]> Tea protein hydrolysate 0.72 >3 kDa 1.79 <3 kDa 0.41 F2+F4 0.29 IAAGLQNTG 0.24 Allopurinol 0.032

[0059] Example 2 Peptide spectrum characterization and molecular docking simulation of tea protein XO inhibitory peptide

[0060] 1. Experimental Methods

[0061] 1. Structural identification of tea protein XO inhibitory peptide

[0062] The highly active fractions (mixture F2 + F4) from the purified tea protein XO inhibitory peptides were desalted on a C18 desalting column (Acclaim PepMap100, 75 μm × 2 cm). The amino acid sequences of the active peptides were analyzed by LC-MS / MS equipped with an online nanospray source. The system consisted of a Q Exactive™ Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) coupled to an EASY-nanoLC1200. A total of 5 μL of sample was loaded onto an Acclaim PepMap C18 column (75 μm × 25 cm). The sample was separated using a 60-min gradient with a controlled flow rate of 300 nL / min, a column temperature of 40°C, and an electrospray voltage of 2 kV. The gradient started with 2% phase B (80% acetonitrile + 0.1% formic acid) and was increased nonlinearly to 35% at 47 min, then to 100% over 1 min, where it was maintained for 12 min. All percentages are by volume. 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): 200–1800; resolution: 70,000; AGC target: 3e6; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 17,500; AGC target: 1e5; maximum injection time: 45 ms; collision energy: 28; dynamic exclusion time: 30 s. Tandem mass spectra were analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). PEAKS DB was searched against the uniprot-Camellia_sinensis (version 202012, 30052 entries) database, with the enzyme digest set to none. Protein card values ​​were: −101gP ≥ 0, with at least one unique peptide; peptide card values ​​were: −101gP ≥ 20.

[0063] 2. Molecular docking simulation analysis of tea protein XO inhibitory peptide

[0064] Molecular docking was used to investigate the interaction between the XO receptor protein and the active peptide, following the method of Zhao et al. (Zhao Qiang, Meng Ying, Liu Juncai, et al. Separation, identification, and docking analysis of xanthine oxidase inhibitory peptides from pacific cod bone-flesh mixture [J]. LWT, 2022, 167:113862. https: / / doi.org / 10.1016 / j.lwt.2022.113862.). The X-ray crystal structure of XO (PDBID: 1N5X) was downloaded from the RCSB Protein Data Bank (https: / / www.rcsb.org / ). The inhibitory ligand TEI-6720 and the symmetric B chain in 1N5X were removed in Pymol and saved in PDB format. The active peptide ligand was drawn using MarvinSketch software and saved in PDB format as the lowest energy conformation. The processed PDB receptor protein and ligand were then opened using Autodock Tools 1.5.6 software. Dehydration and hydrogenation modifications were performed, and gasteiger charges and bond rotations were calculated for the ligand. All data were saved in PDBQT format. Receptor-ligand docking was performed using AutoDock Vina software, with docking center coordinates of 96, 54, and 39 (x, y, z), a box size of 40 × 40 × 40, and a grid spacing of 0.375 Å. All other parameters were set to the software defaults. Finally, the docked binding energy was used as the predicted affinity (calculated in kJ / mol) for the binding of the active peptide to the receptor protein structure. The conformation with the lowest binding energy was selected as the optimal binding site, and the docking results were visualized and analyzed using Discovery Studio 2017.

[0065] 2. Results Analysis

[0066] 1. Peptide mapping of tea protein XO inhibitory peptides

[0067] The active peptide composition and amino acid sequence of the highly active fraction (F2+F4) were identified by LC-MS / MS. The total ion current is shown in Figure 3 Using −10 lg P>35.0 and peptide chain length ≤10 as screening criteria, a number of peptides were obtained. Among them, the secondary mass spectrum of the peptide IAAGLQNTG is shown in Figure 4(A). The toxicity and sensitization properties of the active peptide were predicted using the online tools Toxinpred (http: / / crdd.osdd.net / raghava / toxinpred / ) and AllerTOP (https: / / www.ddg-pharmfac.net / AllerTOP / ), respectively, and the peptide was found to be non-toxic and non-sensitizing (Table 2). Molecular docking can be used to predict the interaction between structural ligands and receptors. Active peptides with lower docking binding energy values ​​are more likely to dock with XO, and the resulting complexes are more stable. The peptide was molecularly docked with the receptor protein using Autodock vina software. As shown in Table 2, the peptide has a low docking binding energy, indicating that it can dock well with XO.

[0068] As shown in Table 1, the XO inhibitory peptide IAAGLQNTG (843.4450 Da, IC 50 =0.24 mg / mL) 50 The value is lower than that of tea enzyme hydrolysate (IC 50 =0.72 mg / mL), >3 kDa (IC 50 =1.79 mg / mL), <3 kDa (IC 50 =0.41 mg / mL) and liquid-phase purified fractions F2+F4 (IC 50 =0.29 mg / mL). This indicates that it is feasible to screen for highly active XO inhibitory peptides using molecular docking technology. A literature search revealed that the peptide sequence of IAAGLQNTG had not been previously reported, making it the first novel peptide discovered and proven to have biological activity. Notably, the peptide IAAGLQNTG has a hydrophobic amino acid at its N-terminus, confirming the importance of hydrophobic amino acids in XO inhibitory peptides. Furthermore, the peptide was compared with WPPKN (IC 50 = 17.75 mg / mL) and ADIYTE (IC 50 =19.01 mg / mL), the peptide IAAGLQNTG exhibited stronger XO inhibitory activity. Online computer tools predicted that the peptide IAAGLQNTG was non-toxic and non-allergenic, potentially indicating that the bioactive peptide is safer than the drug allopurinol.

[0069] Table 2 Docking binding energy and active peptide characteristics of peptide IAAGLQNTG

[0070]

[0071] 2. Molecular docking simulation of tea protein XO inhibitory peptide

[0072] Molecular docking can be used to predict the interaction between substrates and target proteins, as well as to determine the binding affinity between small compounds and biological target molecules. It is reported that XO is a key enzyme in purine metabolism, and its molybdate domain is the key active site of catalysis, containing 13 key amino acid residues that affect activity (G1u1261, Phe649, Thr1010, Arg880, Phe914, Phe1009, Asn768, Lys771, Val1011, Glu802, Ser876, Leu783, Leu1014). In order to predict the inhibitory mechanism of active peptides on XO, the interaction between active peptides and XO was analyzed by molecular docking and visualization. Molecular docking results ( Figure 4 (B) shows that the N-terminus and C-terminus of IAAGLQNTG are both at the surface entrance of the XO hydrophobic channel, which can prevent the substrate from entering the active center of XO, thereby effectively exerting the inhibitory activity of XO. Figure 4 As shown in (B), the peptide IAAGLQNTG forms six conventional hydrogen bonds with Glu879, His875, Val1011, and Thr1010 of XO, as well as one carbon-hydrogen bond with His875. The hydrogen bonds formed between the active peptide and Glu879, His875, and Thr1010 are crucial for stabilizing the complex. Furthermore, Pro1012, Val1011, Tyr1140, and Phe142 of XO bind to IAAGLQNTG through hydrophobic interactions. Based on these results, we conclude that hydrogen bonds and hydrophobic interactions are crucial for the binding of the active peptide to XO.

[0073] Example 3

[0074] 1. Experimental Methods

[0075] 1. Solid-Phase Synthesis

[0076] The xanthine oxidase inhibitory peptide IAAGLQNTG, screened by molecular docking, was synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. and analyzed by high-performance liquid chromatography (HPLC), with a purity greater than 98%.

[0077] 2. Gastrointestinal stability of monomeric peptides

[0078] The in vitro gastrointestinal digestion simulation study was conducted based on the team's previous experimental method (Liao W, Chen H, Jin W, et al. Three Newly Isolated Calcium-Chelating Peptides from Tilapia BoneCollagen Hydrolysate Enhance Calcium Absorption Activity in Intestinal Caco-2 Cells[J]. Journal of agricultural and food chemistry, 2020, 68(7):2091-2098. https: / / doi.org / 10.1021 / acs.jafc.9b07602.) with slight modifications. The specific method is as follows:

[0079] Simulated gastric fluid was prepared by dissolving 40 mg of pepsin in 1 mL of 0.1 mol / L HCl and adjusting the pH to 2.0. Simulated intestinal fluid was prepared by adding 60 mg of bile salts and 10 mg of pancreatin to 5 mL of 0.1 mol / L NaHCO₃ and adjusting the pH to 7.5. The xanthine oxidase inhibitory peptide IAAGLQNTG was dissolved in simulated gastric fluid at a concentration of 1.0 mg / mL and incubated at 37°C for 1 h. The reaction was terminated by heating in a boiling water bath for 10 min. This product was obtained after a single simulated gastric digestion of the xanthine oxidase inhibitory peptide IAAGLQNTG. Subsequently, three volumes of simulated intestinal fluid were added to the simulated gastric digestion solution, and the pH was adjusted to 7.5 with 1 mol / L NaOH. The solution was incubated at 37°C for 2 h. The reaction was terminated by heating in a boiling water bath for 10 min. This product was obtained after a complete simulated gastrointestinal digestion of the xanthine oxidase inhibitory peptide IAAGLQNTG. In addition, a xanthine oxidase inhibitory peptide IAAGLQNTG dissolved in ultrapure water was used as a positive control, and simulated gastric and intestinal fluids were used as negative controls. After digestion, the biostability of the xanthine oxidase inhibitory peptide IAAGLQNTG and the changes in its activity before and after digestion were evaluated.

[0080] The digested samples were analyzed by HPLC using an Ultimate LP-C18 column (4.6 × 250 mm). Chromatographic conditions were as follows: mobile phase A: ultrapure water + 0.1% TFA; mobile phase B: acetonitrile + 0.1% TFA; elution flow rate: 1 mL / min; detection wavelengths: 214 nm and 280 nm; time program: 0–20 min (5%–25% B), 20–40 min (25%–70% B), and 40–60 min (70%–90% B). All percentages are by volume.

[0081] 3. Cell experiments

[0082] 3.1 HK-2 cell culture and viability assay

[0083] HK-2 cell culture was performed with slight modifications based on the method of HOU et al. (Hou Chuanli, Sha Wangqian, Li Yujuan, et al. A modified xanthine oxidase cell model for screening of antihyperuricemicfunctional compounds [J]. Food & Function, 2022, 13(20): 10546-10557. https: / / doi.org / 10.1039 / D2FO00297C.). The specific method is as follows:

[0084] (1) HK-2 cells were inoculated into RPMI 1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in an incubator containing 5% CO2 and 37°C. The medium was changed every 2 days, and subculture was performed when the cell growth density reached 80%.

[0085] (2) 100 μL cell suspension (5×10 4 Cells were seeded in 96-well plates at 100 μL (0.025, 0.05, 0.1, 0.25, 0.5, and 1.0 mg / mL) per well and cultured for 24 hours. The blank control group received 100 μL of complete RPMI 1640 medium. The xanthine oxidase inhibitory peptide group received 100 μL of sample solution (at concentrations of 0.025, 0.05, 0.1, 0.25, 0.5, and 1.0 mg / mL). The positive drug group received 100 μL of febuxostat and allopurinol (at concentrations of 0.025, 0.05, 0.1, 0.25, 0.5, and 1.0 mg / mL). After an additional 24 hours of culture, the medium was discarded, the cells were washed twice with PBS, and 100 μL of 0.5 mg / mL MTT solution was added to each well. The cells were incubated in the dark for 4 hours. The MTT solution was discarded, and 100 μL of DMSO was added. The cells were shaken for 10 minutes to dissolve the blue-purple crystals. The absorbance at 490 nm was measured using a microplate reader.

[0086] (2)

[0087] Where A t is the absorbance value of the experimental group, and A0 is the absorbance value of the blank control group.

[0088] 3.2 Establishment of a hyperuricemia cell model

[0089] Based on the construction method of HOU et al. (Hou Chuanli, Sha Wangqian, Li Yujuan, et al. A modifiedxanthine oxidase cell model for screening of antihyperuricemic functional compounds[J]. Food & Function, 2022, 13(20): 10546-10557. https: / / doi.org / 10.1039 / D2FO00297C.) and optimized. Adenosine was selected as an inducer to stimulate cells to synthesize uric acid precursors, and further combined with exogenously added XO for enzyme catalysis reaction to construct an HK-2 hyperuricemia cell model. The amount of XO added and the enzyme catalysis reaction time were optimized. Because fetal bovine serum has complex components and may contain some enzymes that can oxidize adenosine to inosine and hypoxanthine, adenosine was dissolved in RPMI1640 basal culture medium without fetal bovine serum for the construction of the cell model. The specific method is as follows:

[0090] 1.0 mL of HK-2 cell suspension (1×10 5cells / mL) were inoculated in a 24-well plate and cultured for 48 h. After washing with PBS, the cells were randomly divided into a blank group and a model group: (1) Blank group: cells continued to be cultured in RPMI 1640 basal medium; (2) Model group: cells were cultured in RPMI 1640 basal medium containing 2.5 mmol / L adenosine for 30 h, after which XO was added to the wells to a final addition amount of 0.005 U / mL and 0.01 U / mL. Subsequently, the cells were cultured and after a certain period of XO reaction (10 h, 12 h, 14 h and 16 h), the uric acid content in the cell supernatant was determined by HPLC. Quantitative HPLC separation was performed using an Ultimate LP-C18 column (4.6×250 mm) and detection was performed using a photodiode array detector (PDA) at a wavelength of 254 nm. Mobile phase A: 0.2 mol / L KH2PO4, 0.52 mmol / L sodium pentanesulfonate, pH 4.0; mobile phase B: 0.2 mol / L KH2PO4, 0.52 mmol / L sodium pentanesulfonate, 10% acetonitrile, pH 3.7; flow rate 1 mL / min, detection wavelength 254 nm; elution gradient: 0-6 min (0% B), 6-14 min (0%-70% B), 14-17.4 min (70% B), 17.4-17.5 min (70%-0% B), 17.5-25 min (0% B); all percentages are by volume. Uric acid standards were prepared into gradient solutions, and a standard curve related to uric acid content was generated by HPLC: Y = 0.039X + 0.2528, R 2 =0.9996.

[0091] 3.3 Evaluation of xanthine oxidase inhibitory peptide activity at the cellular level

[0092] 1 mL of HK-2 cells (1 × 10 5Cells were inoculated into 24-well plates at 400 μg / mL and cultured for 24 h. After washing with PBS, the cells were randomly divided into four groups: (1) blank group: cells were cultured in RPMI 1640 complete medium; (2) model group: cells were cultured in RPMI 1640 complete medium; (3) positive drug group: cells were cultured in complete medium containing 0.05 mg / mL febuxostat and allopurinol, respectively; (4) IAAGLQNTG group: cells were cultured in complete medium containing low-dose (0.25 mg / mL), medium-dose (0.5 mg / mL), and high-dose (1.0 mg / mL) IAAGLQNTG, respectively. After culturing the cells for 24 h, the supernatant of all groups was discarded. Except for the blank group, which continued to be cultured in RPMI 1640 basal medium, all other groups were cultured in RPMI 1640 basal medium containing adenosine (2.5 mmol / L) for an additional 30 hours. Exogenous XO was then added for the enzyme-catalyzed reaction. After the reaction time, the cell supernatant was collected from each group. Cellular uric acid levels were measured using HPLC.

[0093] 2. Results Analysis

[0094] 1. In vitro simulation of gastrointestinal digestion

[0095] In order to investigate the stability and activity change characteristics of the monomeric peptide before and after digestion in the gastric and intestinal environments, we used pepsin and trypsin to simulate in vitro digestion. The results of the stability and activity change of the peptide before and after digestion are shown in Figure 5 IAAGLQNTG showed a certain degree of stability during single gastric digestion, and two new absorption peaks appeared at 11.09 min and 12.35 min. Then, IAAGLQNTG still showed a certain degree of stability after complete gastrointestinal digestion, and a new absorption peak appeared at 13.85 min, indicating that intestinal digestion promoted the decomposition of IAAGLQNTG ( Figure 5 (A) and 5 (B)). This indicates that the peptide bonds of active peptides are easily affected by gastrointestinal digestion, resulting in peptide fragment decomposition, and active peptides from the same protein source have different digestion characteristics.

[0096] Depend on Figure 5 (C) As shown, the XO inhibition rate of IAAGLQNTG increased after gastric digestion, which may be related to the appearance of two new absorption peaks. However, after complete gastrointestinal digestion, the relative inhibitory activity of IAAGLQNTG decreased, which may be related to the reduced stability of IAAGLQNTG. Therefore, in future studies, we will further investigate the mechanism of IAAGLQNTG transport and absorption in vivo and explore the relationship between its activity and changes in the peptide segment.

[0097] 2. Effects of Xanthine Oxidase Inhibitory Peptides on HK-2 Cells

[0098] 2.1 Cytotoxicity of xanthine oxidase inhibitory peptides

[0099] In order to investigate the effects of different concentrations of xanthine oxidase inhibitory peptide IAAGLQNTG on HK-2 cell viability, the MTT assay was used to determine HK-2 cell viability using febuxostat and allopurinol as positive drugs. Figure 6 As shown in (A), HK-2 cell viability after treatment with IAAGLQNTG exceeded 100% within the concentration range of 0.025-1.0 mg / mL, indicating that this xanthine oxidase inhibitory peptide has no toxic effects on HK-2 cells. Within the concentration range of 0.025-0.1 mg / mL, HK-2 cell viability after treatment with febuxostat and allopurinol exceeded 98%. However, when the concentration increased to 0.25-1.0 mg / mL, HK-2 cell viability decreased to below 60%, indicating that the active drug had deleterious effects on cells within this concentration range. Based on these results, the xanthine oxidase inhibitory peptide IAAGLQNTG was divided into low-dose (0.25 mg / mL), medium-dose (0.5 mg / mL), and high-dose (1.0 mg / mL) groups for subsequent experiments. For the active drug groups, the optimal sample concentration for both febuxostat and allopurinol was 0.05 mg / mL.

[0100] 2.2 Evaluation of the uric acid-lowering activity of active peptides using a hyperuricemia cell model

[0101] It has been reported that the blood uric acid level in adult males with hyperuricemia is ≥ 420 μmol / L, and in females it is ≥ 360 μmol / L (She Dunmin, Wang Yongliang, Liu Jing, et al. Changes in the prevalence of hyperuricemia in clients of health examination in Eastern China, 2009 to 2019[J]. BMC Endocrine Disorders, 2022, 22(1): 1-11. https: / / doi.org / 10.1186 / s12902-022-01118-Z.). Therefore, the uric acid level in cells ≥ 420 μmol / L was used as the modeling standard, and the uric acid content in cell samples was determined by HPLC.

[0102] Depend on Figure 6(B) It can be seen that the HPLC separation and analysis procedure can effectively separate uric acid and its precursors (adenosine, inosine, hypoxanthine, and xanthine), so this method can accurately determine the uric acid content in cells. Figure 6 (C) It can be seen that the amount of exogenous XO added and the duration of the enzyme-catalyzed reaction significantly affect the uric acid concentration in the cells. When the XO addition amount was 0.005 U / mL, the enzyme-catalyzed reaction time was extended from 10 h to 16 h, but the cellular uric acid concentration did not exceed 420 μmol / L. However, when the XO addition amount was increased to 0.01 U / mL, the cellular uric acid concentration reached 422.36 μmol / L after 12 h of enzyme-catalyzed reaction. After continuing the reaction for 14 h, the cellular uric acid concentration reached 486.35 μmol / L and stabilized. Therefore, the exogenous XO addition amount was determined to be 0.01 U / mL, and the enzyme-catalyzed reaction duration was set to 14 h as the induction condition for constructing the hyperuricemia cell model.

[0103] according to Figure 6 The results in (D) show that under these induction conditions, the uric acid concentration in the model group cells reached 486.35 μmol / L, significantly higher than the 4.08 μmol / L in the blank control group. This indicates that it is feasible to construct a hyperuricemia cell model by combining adenosine with exogenous XO.

[0104] The trend of uric acid levels in hyperuricemic cells after treatment with different concentrations of xanthine oxidase inhibitory peptide is shown in the figure. Figure 6 (D). Cellular uric acid levels in the positive drug groups were significantly lower than those in the model group. Cellular uric acid levels after treatment with febuxostat and allopurinol were 4.29 μmol / L and 322.48 μmol / L, respectively. This suggests that this hyperuricemia cell model can be used to effectively screen for substances with uric acid-lowering activity. Treatment with different concentrations of the xanthine oxidase inhibitory peptide IAAGLQNTG significantly reduced cellular uric acid levels compared to the model group, showing a clear dose-dependent effect. At the high dose of 1.0 mg / mL, cellular uric acid levels in the IAAGLQNTG-treated group were reduced by 33.33% compared to the model group. Although the uric acid-lowering activity of the active peptide was not as significant as that of the positive drug febuxostat, the high-dose group of IAAGLQNTG exhibited uric acid-lowering activity comparable to that of the positive drug allopurinol, indicating that IAAGLQNTG has excellent efficacy against hyperuricemia at the cellular level.

[0105] This study isolated and identified tea protein hydrolysates with xanthine oxidase (XO) inhibitory activity, and screened a novel, highly active XO inhibitory peptide, IAAGLQNTG, through molecular docking technology. This active peptide can access the hydrophobic channel of XO and has the potential to bind to the XO active site at the molecular level, effectively preventing substrate binding to XO. Results from simulated in vitro gastrointestinal digestion demonstrated that IAAGLQNTG exhibited a certain degree of tolerance to gastrointestinal digestion. Furthermore, this active peptide demonstrated significant uric acid-lowering activity in the HK-2 hyperuricemia cell model. These findings will provide new insights and theoretical insights for the development of novel natural XO inhibitors and the high-value utilization of tea proteins.

[0106] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A xanthine oxidase inhibitory peptide, characterized in that Its amino acid sequence is IAAGLQNTG.

2. The method for preparing the xanthine oxidase inhibitory peptide according to claim 1, characterized in that The following steps are included: The xanthine oxidase inhibitory peptide is directly synthesized in vitro solid phase or is obtained by using tea residue as raw material, extracting tea protein through alkali extraction and acid precipitation, and hydrolyzing and purifying with neutral protease.

3. The method for preparing the xanthine oxidase inhibitory peptide according to claim 2, wherein: The purification comprises ultrafiltration separation and liquid chromatography separation steps.

4. The method for preparing a xanthine oxidase inhibitory peptide according to claim 3, wherein: The specific operation of the ultrafiltration separation is: Tea protein hydrolysate was separated using ultrafiltration tubes to collect fractions with molecular weight <3 kDa; The specific operation of the liquid chromatography separation is: The ultrafiltration fractions of tea protein hydrolysate <3 kDa were separated and purified by high performance liquid chromatography using a preparative reversed-phase C18 glass column. The active fractions were collected, concentrated by rotary evaporation and freeze-dried to obtain xanthine oxidase inhibitory peptides.

5. Use of the xanthine oxidase inhibitory peptide according to claim 1 in the preparation of uric acid-lowering drugs.

6. A xanthine oxidase inhibitor, characterized in that The invention comprises at least one of the xanthine oxidase inhibitory peptide according to claim 1, a tea protein neutral protease hydrolyzate containing the xanthine oxidase inhibitory peptide according to claim 1, and a hydrolyzate containing the xanthine oxidase inhibitory peptide according to claim 1 as an active ingredient.

7. A uric acid-lowering drug, characterized in that The invention comprises at least one of the xanthine oxidase inhibitory peptide according to claim 1, a tea protein neutral protease hydrolyzate containing the xanthine oxidase inhibitory peptide according to claim 1, and a hydrolyzate containing the xanthine oxidase inhibitory peptide according to claim 1 as an active ingredient.

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