Dipeptide with uric acid-lowering activity, encoding gene and application thereof

Abstract

The application discloses a dipeptide with uric acid reducing activity, a coding gene and application thereof. The dipeptide with xanthine oxidase inhibiting activity is named HH, and the amino acid sequence is His-His, wherein His is a histidine residue. Through in-vitro xanthine oxidase inhibiting activity experiments and a high uric acid Caenorhabditis elegans model, it is proved that the dipeptide has significant in-vitro xanthine oxidase inhibiting activity, thereby effectively inhibiting the body from generating uric acid, can be widely applied to preparation of uric acid reducing drugs, can effectively treat and prevent hyperuricemia, and relieve gout symptoms, and has good social and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology, specifically relating to a dipeptide with uric acid-lowering activity, its encoding gene, and its applications. Background Technology

[0002] Hyperuricemia is a metabolic disease, mainly caused by excessive uric acid production and impaired uric acid excretion. When serum uric acid concentration is too high, urate crystals are easily deposited in the joints, causing gout symptoms. Currently, the mechanisms of action and corresponding drugs of three common uric acid-lowering drugs are: 1) inhibiting uric acid production, mainly allopurinol and febuxostat; 2) promoting uric acid excretion, mainly probenecid and benzbromarone; 3) breaking down uric acid, these drugs are enzymes, such as uricase. However, the above drugs have varying degrees of side effects, ranging from mild nausea and rash to severe kidney damage and liver inflammation. Therefore, finding a safe and effective compound is crucial.

[0003] Peptides, as one of the important raw materials in current research on functional active ingredients, possess rich physiological activities, such as antioxidant, antibacterial, and anti-inflammatory activities, and have advantages such as easy absorption and non-toxicity or low toxicity. The uric acid-lowering activity of peptides, especially their xanthine oxidase inhibitory activity, has gradually become a research hotspot worldwide. Weiwei He et al. demonstrated that tuna hydrolysate has in vivo uric acid-lowering activity in rats with potassium oxonate-induced hyperuricemia, and isolated, purified, and identified a dipeptide FH with xanthine oxidase inhibitory activity, whose IC50 value... 50 The concentration was 25.7 mM (IC50 of allopurinol). 50 The concentration was 22.0 μM. FH was evaluated in vivo to lower uric acid in a rat model of hyperuricemia induced by potassium oxonate. Qingyong Li et al. enzymatically hydrolyzed walnut powder and identified seven xanthine oxidase inhibitory peptides: WDD, HCPF, WDQW, PPKNW, WPPKN, ADIYTE, and WSREEQE, with corresponding IC50 values. 50 The concentrations of allopurinol were 2.41 mM, 15.07 mM, 0.95 mM, 2.21 mM, 2.06 mM, 7.37 mM, and 1.88 mM, respectively (IC50 of allopurinol). 50 The concentration was 5.71 μM. Structural analysis revealed that the higher the content of proton donor amino acid residues, the higher the xanthine oxidase inhibitory activity of the peptide. Histidine (His) has a side-chain equilibrium dissociation constant of approximately 7.0, allowing it to act as both a proton donor and acceptor. Therefore, peptides containing His may possess potential xanthine oxidase inhibitory activity.

[0004] Currently, research on obtaining uric acid-lowering active peptides mainly involves enzymatically digesting and purifying dietary proteins, identifying components with high xanthine oxidase inhibitory activity, and then using mass spectrometry to analyze the polypeptide sequence of these components. This method is often indiscriminate, time-consuming, and labor-intensive, focusing only on common food proteins and lacking breadth. Few reports describe a batch virtual screening approach that identifies polypeptide sequences with potential xanthine oxidase inhibitory activity from all possible dipeptide, tripeptide, or longer polypeptide sequences, followed by artificial synthesis or bioengineering to obtain the target sequence polypeptide and perform functional verification. This research approach can more comprehensively, rapidly, and efficiently identify target polypeptide sequences. It can also be combined with protein databases (such as NCBI) and BLAST technology to find dietary proteins containing the target peptide, obtaining the target polypeptide through simulated enzymatic digestion, or using solid-phase synthesis and microbial fermentation methods. Furthermore, this research approach has broad prospects for application in the preparation of drugs for the prevention or treatment of hyperuricemia, or in the preparation of drugs to relieve gout symptoms.

[0005] Existing literature reports that the peptide FH has xanthine oxidase inhibitory activity, but its IC50 value is low. 50 The concentration was 25.7 mM (IC50 of allopurinol). 50 The concentration was 22.0 μM, indicating weak xanthine oxidase inhibitory activity (He W, Su G, Sun-Waterhouse D, et al. In vivo anti-hyperuricemic and xanthine oxidase inhibitory properties of tuna protein hydrolysates and its isolated fractions[J]. Food Chemistry, 2019, 272: 453-461.).

[0006] Existing literature reports the isolation and identification of seven xanthine oxidase inhibitory peptides based on their xanthine oxidase inhibitory activity by enzymatic hydrolysis of walnut protein. However, the peptide chains are generally longer than three amino acid residues, and experiments have shown that some peptides are structurally unstable after gastrointestinal digestion, affecting their activity. In addition, this experiment follows traditional research methods, which is time-consuming, labor-intensive, and somewhat unreliable, and lacks breadth by focusing only on common food proteins (Li Qingyong. Structure-activity mechanism study of walnut-derived uric acid-lowering peptides targeting xanthine oxidase activity [D]. South China University of Technology, 2018). Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention aims to provide a dipeptide with uric acid-lowering activity, its encoding gene, and its applications. This dipeptide can inhibit xanthine oxidase activity, thereby effectively inhibiting the body's production of uric acid. It can be widely used in the preparation of uric acid-lowering drugs, effectively treating and preventing hyperuricemia, and relieving gout symptoms.

[0008] The purpose of this invention is to provide a dipeptide encoding the aforementioned activity that lowers uric acid.

[0009] The purpose of this invention is to provide a gene encoding a dipeptide having uric acid-lowering activity.

[0010] The purpose of this invention is to provide the application of the dipeptide with uric acid-lowering activity.

[0011] The objective of this invention is achieved through the following technical solution.

[0012] This invention provides a dipeptide with uric acid-lowering activity, the dipeptide being named HH and having an amino acid sequence of His-His; His is a histidine residue.

[0013] Furthermore, the preparation methods of the dipeptide with uric acid-lowering activity include, but are not limited to, chemical synthesis, enzymatic hydrolysis, or genetic engineering.

[0014] Furthermore, the screening method for dipeptides with uric acid-lowering activity is based on four dimensions, including xanthine oxidase binding stability, polypeptide molecular physical characteristics, polypeptide molecular structural similarity, and intermolecular force analysis. High-throughput virtual screening is performed on polypeptides (including but not limited to dipeptides) with D / L conformation that may inhibit xanthine oxidase activity, and the target polypeptide sequence is finally obtained.

[0015] Furthermore, the in vivo uric acid-lowering activity of the dipeptide with uric acid-lowering activity is verified by comparing the concentration of uric acid produced by a unit of nematode based on a high-uric acid Caenorhabditis elegans model, thereby verifying the in vivo uric acid-lowering function of the dipeptide.

[0016] This invention provides a gene encoding the dipeptide with the above-mentioned uric acid-lowering activity, wherein the base sequence of the encoding gene is CACCAC, CAUCAC, CACCAU or CAUCAU; and the gene length is 6 bases. CAC or CAU is a histidine codon.

[0017] The present invention also provides an application of the above-mentioned dipeptide with uric acid-lowering activity, wherein the dipeptide with uric acid-lowering activity is used in the preparation of drugs for the prevention or treatment of hyperuricemia, or in the preparation of drugs for relieving gout symptoms.

[0018] Furthermore, the preparation method of the drug includes the following steps: mixing a dipeptide with uric acid-lowering activity as described in this invention with a food matrix homogenate, adding excipients and mixing evenly, and then obtaining a powder by spray drying, thereby preparing a drug containing a dipeptide with uric acid-lowering activity.

[0019] Furthermore, the food matrix homogenate includes one or more of food puree and food concentrate.

[0020] Furthermore, the excipients include one or more of corn starch and microcrystalline cellulose.

[0021] Furthermore, the dosage form of the drug includes tablets, capsules, granules, pills, ointments, powders, syrups, or oral liquids.

[0022] Furthermore, the drug can be delivered through the skin by means of application, patching, or microneedle puncture.

[0023] A screening method for the above-mentioned uric acid-lowering peptides specifically involves high-throughput virtual screening of peptides (including but not limited to dipeptides) with potential xanthine oxidase-inhibiting activity containing D / L configurations, based on factors such as xanthine oxidase binding stability, peptide molecular physical characteristics, peptide molecular structural similarity, and intermolecular force analysis, to ultimately obtain the target peptide sequence.

[0024] A method for verifying the function of the aforementioned dipeptide with uric acid-lowering activity is as follows: based on a high-uric acid Caenorhabditis elegans model, the concentration of uric acid produced by a unit nematode is compared to verify the in vivo uric acid-lowering function of the dipeptide.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention provides a dipeptide with uric acid-lowering activity. In vitro xanthine oxidase inhibition activity experiments and a Caenorhabditis elegans model with high uric acid have demonstrated that the dipeptide has a significant uric acid-lowering effect.

[0026] 2. The dipeptide provided by this invention, which inhibits xanthine oxidase activity, can be widely used in the preparation of uric acid-lowering drugs, effectively treating and preventing hyperuricemia and relieving gout symptoms, thus having good social and economic benefits. Attached Figure Description

[0027] Figure 1 Contribution diagram to the structural similarity between HH and febuxostat.

[0028] Figure 2a This is the high performance liquid chromatogram of HH.

[0029] Figure 2b This is the HH mass spectrum.

[0030] Figure 3a The bar chart shows the inhibition rate of HH on xanthine oxidase at different concentrations.

[0031] Figure 3b The IC50 of allopurinol and HH on xanthine oxidase is... 50 Bar chart.

[0032] Figure 4a Bar chart showing the effect of allopurinol on the total uric acid production per unit of nematode.

[0033] Figure 4b A bar chart evaluating the in vivo uric acid-lowering activity of a synthetic dipeptide in a hyperuricemic Caenorhabditis elegans model.

[0034] Figure 4c This is a bar chart comparing the in vivo uric acid-lowering activity of HH with anserine and carnosine based on a hyperuricemic Caenorhabditis elegans model.

[0035] Figure 5 MTT assay to evaluate the effect of different concentrations of HH on the survival rate of LO-2 cells (bar chart). Detailed Implementation

[0036] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the specific implementation and protection scope of the present invention are not limited thereto.

[0037] The dipeptide of the present invention having uric acid-lowering activity is named HH, and its amino acid sequence is His-His; His is a histidine residue.

[0038] The structural formula of the dipeptide with uric acid-lowering activity of this invention is as follows:

[0039] The gene encoding the dipeptide with uric acid-lowering activity mentioned above has the base sequence CACCAC, CAUCAC, CACCAU, or CAUCAU; the gene length is 6 bases. CAC or CAU is a histidine codon.

[0040] In a specific embodiment, the uric acid-lowering dipeptide of the present invention is applied by first screening 1521 dipeptides with D / L configurations using high-throughput virtual screening technology based on factors such as binding energy of key action sites of xanthine oxidase, molecular structural similarity, intermolecular forces, and molecular structure and physical characteristics, to obtain potential xanthine oxidase peptide inhibitors; secondly, in vitro xanthine oxidase inhibition experiments and a high-uric acid Caenorhabditis elegans model are used to verify the in vivo and in vitro uric acid-lowering function of the above-mentioned potential xanthine oxidase peptide inhibitors; finally, the effect of the dipeptide on the survival rate of LO-2 cells is verified by MTT assay, proving that the dipeptide of the present invention has xanthine oxidase inhibitory activity, thereby effectively reducing uric acid production, while also exhibiting low cytotoxicity.

[0041] Example 1 High-throughput virtual screening of dipeptides with potential uric acid-lowering activity 1. Establishment of the dipeptide dataset First, a matrix of 39 amino acids with D / L configurations is set up, using uppercase letters to represent L-amino acids and lowercase letters to represent D-amino acids (e.g., A represents L-Ala, a represents D-Ala). Using Python, the matrix is ​​processed by Cartesian product to obtain 1521 possible peptide sequences. Then, by reading this table and performing operations to generate peptide structures, 1521 dipeptide structure files are obtained. Based on this method, a dataset of 1521 dipeptides with D / L configurations can be quickly generated.

[0042] 2. Calculation of dipeptide-related physical characteristics and structural descriptors Using Python's computational chemistry library, the relative molecular mass (MolWt), number of hydrogen bond acceptors (NumHAcceptors), number of hydrogen bond donors (NumHDonors), molecular oil-water partition coefficient (MollogP), number of rotatable bonds (NumRotatableBonds), number of aromatic rings (NumAromaticRings), number of aliphatic rings (NumAliphaticRings), topological polar surface area (TPSA), number of saturated heterocycles (NumSaturatedHeterocycles), and number of saturated carbocyles (NumSaturatedCarbocyles) were calculated for each dipeptide molecule by reading the dipeptide sequence from an Excel file. These physical and structural features were used as descriptors, and mathematically, these values ​​were converted into a ten-dimensional vector and associated with the dipeptide sequence for subsequent analysis. After calculating the physical structural features, the molecular fingerprint information of each dipeptide molecule needed to be calculated. Since physical structural features were previously used to describe the local molecular structure, a topological or path structure descriptor was chosen to measure the overall molecular structure.

[0043] Considering that existing descriptors are insufficient to characterize the differences in chiral molecules, an improvement is made by adding a correction term after the similarity calculation. Here, L-amino acids are used as standard amino acids, and D-amino acids are the corresponding chiral amino acids. Excluding cases where molecules are completely identical, a statistical term is added to count the frequency of chiral amino acid occurrences. Dividing this by the total number of atoms yields a small correction value W, which can distinguish the chiral differences in the structure of dipeptides containing chiral amino acids. The specific formula is as follows:

[0044] In the formula: A, B — Vector elements of the dipeptide, i.e., the dipeptide molecular structure represented by the molecular descriptor; A*B — the product of two vectors; ||A|| 2 —The magnitude of vector A; ||B|| 2 —The magnitude of vector B.

[0045] 3. Acquisition, analysis and preprocessing of xanthine oxidase files This invention uses a xanthine oxidase structural file with a resolution of 1.6 Å and PDB number 3NVZ. The active site, file number 6, is used as the molecular docking site, with coordinates (37.7825, 21.6461, 18.0794) and a radius of 8.9. After determining the docking active site, the xanthine oxidase structural file needs to be preprocessed, including completing missing residues, molecular hydrogenation, removing water molecules, removing redundant protein chains, adding a force field environment, and deleting the indole-3-aldehyde occupying site 6.

[0046] 4. Molecular docking First, the batch energy of 1521 dipeptide molecules was minimized using the force field CHARMm. Then, the molecular docking module of Discovery Studio 2019 was used to evaluate the binding stability of the 1521 polypeptide sequences with xanthine oxidase at the molybdenum pterin center, and the Libdock semi-flexible docking method was adopted.

[0047] 5. Screening to obtain the target dipeptide sequence By considering four evaluation parameters—molecular docking results, molecular structural similarity, intermolecular forces, and molecular structure and physical characteristics—a dipeptide with potential inhibitory activity against xanthine oxidase, HH (L-His-L-His), was identified. Figure 1 It is known that HH is structurally similar to febuxostat, and therefore has the potential to inhibit xanthine oxidase activity.

[0048] Example 2 Solid-phase synthesis of dipeptides with uric acid-lowering activity 1. Synthesis sequence: From the C-terminus to the N-terminus, the steps are as follows: First, weigh 0.05 mol of resin into the reactor, add 10 mL of DCM (dichloromethane) to swell for 30 min, remove the DCM, add 0.10 mol of the first amino acid in the sequence, then add 0.1 mol of DIEA (diisopropylethylamine), 15 mL of DMF (dimethylformamide), and bubble under nitrogen for 60 min. Then add 0.25 mol of methanol and react for 30 min. Remove the reaction solution and wash with DMF and methanol. Second, add 0.10 mol of the second amino acid in the sequence to the reactor, then add 0.10 mol of HBTU (1-hydroxybenzo[a]trichloroazole]tetramethylhexafluorophosphate) and DIEA. Bubble under nitrogen for 30 min, detect ninhydrin, then cap with pyridine and acetic anhydride, finally wash, and add to the decapping solution to react for 2 minutes. h removes the Fmoc (9-fluorenylmethoxycarbonyl) protecting group. The decapping solution is a mixture of trifluoroacetic acid, ethylenediamine tartrate, distilled water, and trypsin inhibitor (TIS), wherein the volume ratio of trifluoroacetic acid:ethylenediamine tartrate:distilled water:trypsin inhibitor (TIS) is 94.5:2.5:2:1. The solution is washed and ninhydrin is detected. The resin is then dried with nitrogen and removed from the reaction column, transferred to a flask, and 15 mL of a cutting solution is added. This cutting solution is a mixture of trifluoroacetic acid, ethylenedithiol, triisopropylsilane, and distilled water, wherein the volume ratio of trifluoroacetic acid:ethylenedithiol:triisopropylsilane:distilled water is 95:2:2:1. The solution is shaken, and the resin is filtered off. Finally, 50 mL of diethyl ether is added to the filtrate to precipitate the crude product. The crude product is purified to a purity of over 95% using high-performance liquid chromatography and then freeze-dried into powder.

[0049] All of the above processes were completed in a SYMPHONY 12-channel peptide synthesizer. The synthesized peptides were purified by SHIMADZU high-performance liquid chromatography with a purity of over 99%, and qualitative analysis was performed by HPLC-MS to determine their amino acid sequences.

[0050] High-performance liquid chromatography and mass spectra of synthesized dipeptides with potential uric acid-lowering activity are shown below. Figure 2a and Figure 2b As shown, the primary amino acid sequence of the synthesized dipeptide is His-His, which means that the target dipeptide with uric acid-lowering activity was obtained.

[0051] Example 3 In vitro xanthine oxidase inhibitory activity assay of synthesized dipeptides with uric acid-lowering activity Xanthine oxidase is a key enzyme in the production of uric acid in vivo. This invention is based on inhibiting xanthine oxidase (XO) activity to reduce uric acid production. An in vitro xanthine oxidase inhibition experiment can preliminarily determine the uric acid-lowering activity of the dipeptide. The specific procedures are as follows: The dipeptide powder was dissolved in 1×PBS to prepare dipeptide solutions with concentrations of 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 6.0 mM, 9.0 mM, and 12.0 mM. 1.1 mg of allopurinol powder was weighed and dissolved in 1×PBS to obtain a 20 mM stock solution. This stock solution was then diluted with 1×PBS to prepare 10 μM, 20 μM, 30 μM, 40 μM, and 80 μM allopurinol solutions as control groups. The prepared solution was incubated with XO at 37°C for 10 min, and simultaneously, a xanthine solution with a final concentration of 0.4 mM was added. The enzymatic reaction was initiated at 37°C, and the kinetic changes in absorbance at 290 nm were recorded. The cycle interval was 30 s. The initial rate of the enzymatic reaction was denoted as V0, and the rate of the enzymatic reaction in the presence of the dipeptide was denoted as V0. S V0 and V S The units are all mol / (L·min). The inhibition rate of the sample against xanthine oxidase is calculated according to the following formula:

[0052] Figure 3a The inhibition rate of HH on xanthine oxidase at different concentrations (0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 6.0 mM, 9.0 mM, 12.0 mM) was shown, revealing a dose-effect relationship between the inhibition rate of HH on xanthine oxidase. The logarithm of each concentration (base 10) was used as x, and the corresponding xanthine oxidase inhibition rate was used as y. A linear equation was fitted to obtain the relationship between the logarithm of the dipeptide concentration and the inhibition rate, from which the half-maximal inhibitory concentration (IC50) of HH on xanthine oxidase was calculated. 50 )get Figure 3b Allopurinol was used as a positive control. Currently disclosed peptides with uric acid-lowering activity, such as dipeptide FH, have IC50... 50 The value was 25.7 mM (allopurinol IC50). 50 The value is 2.2 × 10 -2 mM), IC50 of tetrapeptide HCPF 50 The value was 15.07 mM (allopurinol IC50). 50 The value is 5.71 × 10 -3 mM), IC50 of the dipeptide HH of the present invention 50 The value was 5.60 ± 0.18 mM (allopurinol IC50). 50 The value is 3.02 × 10 -2In comparison, the dipeptide HH of this invention has stronger xanthine oxidase inhibitory activity, and therefore has better application prospects.

[0053] Example 4 Verification of the in vivo uric acid-lowering activity of the synthesized dipeptide using a hyperuricemic Caenorhabditis elegans model Animal models of high uric acid include rodents and birds. However, rodents contain uricase, which can further oxidize uric acid to produce allantoin. Therefore, uricase gene knockout mice can be used as research subjects. However, defective mice are more prone to death than normal mice, making modeling difficult. Although birds do not contain uricase, their genes have low homology with humans, making them inconvenient for laboratory breeding. However, there are reports that Caenorhabditis elegans can create a high uric acid model through simple means, and has a short reproductive cycle and high gene homology (Li Z, Xue Y, Wang N, et al. High uric acid model in Caenorhabditis elegans[J]. Food Science and Human Wellness, 2019, 8(1): 63-66. DOI: 10.1016 / j.fshw.2019.02.003). Therefore, this invention uses the high uric acid Caenorhabditis elegans model to verify the in vivo uric acid-lowering activity of the synthesized dipeptide. The specific operation is as follows: 1. Nematode culture Prepare a solid culture medium NGM for nematodes, and spread an OP50 bacterial suspension in the center of the NGM for wild-type N2 nematodes to feed on and reproduce.

[0054] 2. Nematode synchronization When L4 stage nematodes and eggs occupy more than 3 / 4 of the culture dish area, rinse them with M9 buffer into a 1.5 mL centrifuge tube and centrifuge at 1000 rpm and 20℃ for 1 min. Repeat the operation twice. After discarding the supernatant, add 600 μL of M9 buffer, then add 300 μL of pre-prepared nematode lysis buffer (equal volumes of 5 M NaOH aqueous solution and 50 mg / mL NaClO aqueous solution). Shake well for 5 min. You can observe whether the nematodes are completely lysed under light. Centrifuge at 2000 rpm and 20℃ for 1 min, then rinse twice with M9 buffer. Add 1.5 mL of S-Medium and incubate overnight at 20℃. Once the eggs hatch, you can obtain L1 stage larvae with consistent growth stages.

[0055] 3. Construction of a hyperuricemic Caenorhabditis elegans model Synchronized and hatched L1-stage larval suspensions were transferred in equal volumes to NGM medium containing OP50 and cultured at 20°C for 72 h to obtain L4-stage adults. The larvae were then washed with M9 buffer into 1.5 mL centrifuge tubes and centrifuged at 1000 rpm for 1 min at 20°C. This process was repeated twice. After discarding the supernatant, the larvae were resuspended in 900 μL of S-Medium and homogenized. 300 μL of each resuspended larvae were then transferred to 24-well plates, photographed under a stereomicroscope, and counted using ImageJ software. The control group was incubated with 1 mL of S-Medium at 20℃ for 24 h, in triplicate. The model group was first incubated with 900 μL of S-Medium at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) at 20℃ for 18 h, in triplicate. The allopurinol group was first incubated with 880 μL of S-Medium and 20 μL of 0.02 mg / mL allopurinol solution (S-Medium containing 1.5% DMSO) at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) at 20℃ for 18 h, in triplicate. Nematode bodies and supernatants were collected, and the uric acid content was determined by HPLC. The total amount of uric acid produced per unit nematode was calculated.

[0056] like Figure 4a As shown, the total amount of uric acid produced per unit of nematode in the allopurinol group was not significantly different from that in the control group. p >0.05), and significantly lower than the model group ( p The value <0.05 indicates that the hyperuricemic Caenorhabditis elegans model was successfully established. One-way ANOVA was used for significance analysis, where a and b showed a significant difference. p <0.05, the same letter indicates no significant difference between groups. p >0.05.

[0057] 4. Evaluation of the in vivo uric acid-lowering activity of the synthetic dipeptide based on a hyperuricemic Caenorhabditis elegans model. An equal volume of the synchronized and L1-stage larval suspension was transferred to NGM medium containing OP50 and cultured at 20°C for 72 h to obtain L4-stage adults. The larvae were then washed with M9 buffer into 1.5 mL centrifuge tubes and centrifuged at 1000 rpm for 1 min at 20°C. This process was repeated twice. After discarding the supernatant, 900 μL of S-Medium was added to resuspend the larvae, and 300 μL of each tube was transferred to a 24-well plate. The plates were photographed under a stereomicroscope, and the larvae were counted using ImageJ software. The control group was incubated with 1 mL of S-Medium at 20℃ for 24 h, in triplicate. The model group was first incubated with 900 μL of S-Medium at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) at 20℃ for 18 h, in triplicate. The HH group was first incubated with 890 μL of S-Medium and 10 μL of 80.0 mM HH solution (S-Medium containing 1.5% DMSO) at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) at 20℃ for 18 h, in triplicate. Nematode bodies and supernatants were collected, and the uric acid content was determined by HPLC. The total amount of uric acid produced per unit nematode was calculated.

[0058] like Figure 4b As shown, the total amount of uric acid produced by each nematode in the HH group was not significantly different from that in the control group. p >0.05), and significantly lower than the model group ( p <0.05), indicating that HH exhibits strong in vivo uric acid-lowering activity in the hyperuricemic Caenorhabditis elegans model. One-way ANOVA was used for significance analysis, where *, p <0.05.

[0059] Example 5 Effect of synthesized dipeptides with uric acid-lowering activity on LO-2 cell survival LO-2 cells in logarithmic growth phase were lysed with trypsin at 37°C for 1 min to eliminate intercellular adhesions and allow them to form a single-cell suspension. Complete cell culture medium was then added to terminate the lysis process. Cells were then transferred into 96-well plates to achieve a cell count of 5 × 10⁶ cells per well. 3The cells were incubated at 37°C for 24 h until they adhered to the culture vessel. During the incubation period, cell culture media containing HH were prepared at concentrations of 1.0 mM and 0.1 mM. The solution in the 96-well plate was removed, HH-containing cell culture media was added, and the plate was incubated at 37°C for 24 h. After removing the HH-containing medium, MTT solution (containing 180 µL of complete medium and 20 µL of MTT) was added, and the plate was incubated at 37°C for 4 h. The solution in the wells was then discarded, and 150 μL of dimethyl sulfoxide was added to dissolve the HH-containing medium. The plate was then shaken for 10 min, and the absorbance at 490 nm was read using a microplate reader. The complete medium served as the blank control group, and the HH-containing complete medium served as the experimental group. The LO-2 cell viability was calculated using the following formula:

[0060] Depend on Figure 5 It can be seen that HH at a concentration of 0.1 mM had no significant effect on the survival rate of LO-2 cells. p >0.05), while at a drug concentration of 1.0 mM, it can significantly reduce the survival rate of LO-2 cells to 80% ( p The result of <0.01 indicates that the dipeptide HH of the present invention has no cytotoxicity at low doses, but exhibits slight cytotoxicity at high doses. Therefore, attention should be paid to the dosage when it is put into practical application.

[0061] Comparative Example 1 Comparing the in vivo uric acid-lowering activity of the dipeptide of this invention with similar structural dipeptides or amino acids. An equal volume of the synchronized and L1-stage larval suspension was transferred to NGM medium containing OP50 and cultured at 20°C for 72 h to obtain L4-stage adults. The larvae were then washed with M9 buffer into 1.5 mL centrifuge tubes and centrifuged at 1000 rpm for 1 min at 20°C. This process was repeated twice. After discarding the supernatant, 900 μL of S-Medium was added to resuspend the larvae, and 300 μL of each tube was transferred to a 24-well plate. The plates were photographed under a stereomicroscope, and the larvae were counted using ImageJ software. The control group received only 1 mL of S-Medium and cultured at 20℃ for 24 h, in triplicate. The model group received 900 μL of S-Medium and cultured at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) and cultured at 20℃ for 18 h, in triplicate. The HH group received 890 μL of S-Medium and 10 μL of 80.0 mM HH solution (S-Medium containing 1.5% DMSO) and cultured at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) and cultured at 20℃ for 18 h, in triplicate. The HY group received 890 μL of S-Medium and 10 μL of 80.0 mM HH solution (S-Medium containing 1.5% DMSO) and cultured at 20℃ for 18 h, in triplicate. Group HY was cultured in 20℃ for 6 h in solution (S-Medium containing 1.5% DMSO by volume), followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO by volume) for 18 h at 20℃, with three replicates. Group H was first cultured in 880 μL of S-Medium and 20 μL of 80.0 mM free histidine solution (S-Medium containing 1.5% DMSO by volume) at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO by volume) for 18 h at 20℃, with three replicates. Nematode bodies and supernatants were collected, and uric acid content was determined by HPLC. The total amount of uric acid produced per unit nematode was calculated.

[0062] like Figure 4b As shown, compared with the dipeptide HY group with replaced N-terminal amino acid residues and its free amino acid H group, the HH group of the present invention had the lowest total amount of uric acid produced per unit of nematode, and significantly reduced the total amount of uric acid produced per unit of nematode compared with the model group. p <0.05%, the total amount of uric acid produced by nematodes in groups HY and H was not significantly reduced compared with that in the model group (p >0.05), indicating that the dipeptide HH of the present invention has structural advantages and thus exhibits optimal uric acid-lowering activity. One-way ANOVA was used for significance analysis, where * indicates a significant difference compared to the control group. p <0.05; # indicates that there is a statistical significance compared with the model group. p <0.05.

[0063] Comparative Example 2 Comparison of the in vivo uric acid-lowering activity of the dipeptide of this invention with that of chemoresin and carnosin. An equal volume of the synchronized and L1-stage larval suspension was transferred to NGM medium containing OP50 and cultured at 20°C for 72 h to obtain L4-stage adults. The larvae were then washed with M9 buffer into 1.5 mL centrifuge tubes and centrifuged at 1000 rpm for 1 min at 20°C. This process was repeated twice. After discarding the supernatant, 900 μL of S-Medium was added to resuspend the larvae, and 300 μL of each tube was transferred to a 24-well plate. The plates were photographed under a stereomicroscope, and the larvae were counted using ImageJ software. The control group received only 1 mL of S-Medium and cultured at 20℃ for 24 h, in triplicate. The model group received 900 μL of S-Medium and cultured at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) and cultured at 20℃ for 18 h, in triplicate. The HH group received 890 μL of S-Medium and 10 μL of 80.0 mM HH solution (S-Medium containing 1.5% DMSO) and cultured at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) and cultured at 20℃ for 18 h, in triplicate. The carnosine group received 890 μL of S-Medium and 10 μL of 80.0 mM HH solution (S-Medium containing 1.5% DMSO) and cultured at 20℃ for 18 h, in triplicate. The nematode group was cultured in mM carnosine solution (S-Medium containing 1.5% DMSO) at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) at 20℃ for 18 h, with three replicates. The anserine group was first cultured in 890 μL of S-Medium and 10 μL of 80.0 mM anserine solution (S-Medium containing 1.5% DMSO) at 20℃ for 6 h, followed by 100 μL of 1.0 mg / mL xanthine solution (S-Medium containing 1.5% DMSO) at 20℃ for 18 h, with three replicates. Nematode bodies and supernatants were collected, and uric acid content was determined by HPLC. The total amount of uric acid produced per unit nematode was calculated.

[0064] like Figure 4c As shown, the total amount of uric acid produced by each unit of nematode was not significantly reduced compared to the total amount of uric acid produced by each unit of nematode in the model group, compared to the currently confirmed dipeptides with uric acid-lowering activity—angiosin and carnosin. p Compared to >0.05, HH intervention significantly reduced the total amount of uric acid produced per unit of nematode compared to the model group. p<0.05), indicating that, based on the hyperuricemic Caenorhabditis elegans model, the uric acid-lowering activity of the dipeptide HH of this invention is superior to that of currently recognized uric acid-lowering peptides.

Claims

1. The application of a dipeptide with uric acid-lowering activity, characterized in that, The dipeptide with uric acid-lowering activity is used in the preparation of drugs for the prevention or treatment of hyperuricemia, or in the preparation of drugs for relieving gout symptoms; the dipeptide is named HH, and its amino acid sequence is His-His, wherein His is a histidine residue.

2. The application according to claim 1, characterized in that, The method for preparing the drug, The process includes the following steps: mixing the dipeptide with uric acid-lowering activity as described in claim 1 with a food matrix homogenate, adding excipients and mixing well, and then spray drying to obtain a powder, thereby preparing a drug containing the dipeptide with uric acid-lowering activity.

3. The application according to claim 2, characterized in that, The dosage forms of the drug include tablets, capsules, granules, pills, ointments, powders, syrups, or oral liquids.

4. The application according to claim 1, characterized in that, The drug is delivered through the skin by application, patching, or microneedle puncture.

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