A walnut source xanthine oxidase inhibiting peptide and application thereof

Abstract

A walnut-derived xanthine oxidase inhibitory peptide and its application belong to the field of bioactive peptide biotechnology. The amino acid sequence of the xanthine oxidase inhibitory peptide provided by this invention is shown in SEQ ID No. 1. In vitro activity assays showed that the IC50 of this inhibitory peptide... 50 The concentration was 3.34 mg / mL, demonstrating its high inhibitory activity against xanthine oxidase. Furthermore, this inhibitory peptide can reduce the levels of creatinine, blood urea nitrogen, adenosine deaminase, aspartate aminotransferase, and alanine aminotransferase, regulate the expression of renal uric acid reabsorption protein URAT1, uric acid excretion protein GLUT9, and ABCG2, and significantly reduce uric acid production. It can be used to inhibit xanthine oxidase, prevent or improve hyperuricemia, and is applicable to the preparation of drugs for improving hyperuricemia. These drugs, with the active peptide as the sole active ingredient or one of its active ingredients, have promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide biotechnology, specifically relating to a walnut-derived xanthine oxidase inhibitory peptide and its application. Background Technology

[0002] Hyperuricemia (HUA) is a key biochemical basis and direct cause of gout. In recent years, the prevalence of HUA has shown a significant upward trend globally. In Europe and the United States, the prevalence is approximately 20%–30%; in China, with changes in lifestyle and dietary structure, the prevalence of HUA has risen sharply, currently reaching 14.7%, affecting over 167 million people, making it the "fourth high" after hypertension, hyperglycemia, and hyperlipidemia. Furthermore, HUA is closely related to various chronic diseases such as cardiovascular disease, diabetes, and chronic kidney disease, seriously threatening human health and quality of life.

[0003] Xanthine oxidase (XO), a key rate-limiting enzyme in the purine metabolism pathway, catalyzes the conversion of hypoxanthine to xanthine, which further generates uric acid. When xanthine oxidase activity is excessively high, it leads to excessive uric acid production in the body, exceeding the kidneys' excretion capacity. The excess uric acid then deposits as urate crystals in joints, cartilage, and other tissues, causing a series of complications such as gouty arthritis and kidney stones. Furthermore, excessive activation of XO can exacerbate inflammation and endothelial damage, further inducing cardiovascular disease and kidney dysfunction. Therefore, targeting and inhibiting XO activity has become one of the core strategies for regulating uric acid production.

[0004] Commonly used xanthine oxidase inhibitors in clinical practice, such as allopurinol and febuxostat, have many limitations, such as the potential to cause abnormal liver and kidney function or allergic reactions. Furthermore, some patients may develop drug resistance after long-term use, leading to decreased therapeutic efficacy. Bioactive peptides extracted from food have good biocompatibility and safety, and are less likely to cause allergic reactions. At the same time, naturally derived XO inhibitory peptides are more readily accepted by consumers and suitable for long-term consumption to control uric acid levels, possessing high market potential. However, the scarcity of highly active food-derived XO inhibitory peptides is due to multiple factors, including the limitations of natural raw materials, the complexity of extraction processes, and the high difficulty of activity screening. Existing patent CN202411170152.6 discloses small molecule peptides from sunflower heads that have the potential to act as XO inhibitors, with an inhibitory activity of 20.32 mM; patent CN202410527310.2 discloses three whey protein XO inhibitory peptides, with a highest half-maximum inhibitory concentration (HMC) of 4.34 ± 0.91 mM. Compared to chemically synthesized inhibitors, their activity needs further improvement. Therefore, accurately identifying food-derived XO inhibitory peptides with high inhibitory activity and good stability remains one of the core strategies for preventing hyperuricemia. Summary of the Invention

[0005] The purpose of this invention is to provide a walnut-derived xanthine oxidase inhibitory peptide and its application, particularly in the preparation of drugs for the prevention or treatment of hyperuricemia, wherein the drug uses the active peptide as the sole active ingredient or one of the active ingredients.

[0006] The present invention discloses a walnut-derived xanthine oxidase inhibitory peptide with the amino acid sequence Asp-Val-Trp-Asp-Pro-Phe (DVWDPF), and its amino acid sequence is shown in SEQ ID No. 1.

[0007] This invention discloses a walnut-derived xanthine oxidase inhibitory peptide (DVWDPF) and its applications. The IC50 of this inhibitory peptide was determined by in vitro activity assay. 50 The concentration was 3.34 mg / mL, demonstrating its high inhibitory activity against xanthine oxidase. Furthermore, this inhibitory peptide can reduce the levels of creatinine, blood urea nitrogen, adenosine deaminase, aspartate aminotransferase, and alanine aminotransferase, regulate the expression of renal uric acid reabsorption protein URAT1, uric acid excretion protein GLUT9, and ABCG2, and significantly reduce uric acid production. It can be used to inhibit xanthine oxidase, prevent or improve hyperuricemia, and has promising applications in the development of drugs to improve hyperuricemia. Attached Figure Description

[0008] Figure 1 This is a docking diagram of the inhibitory peptide and XO molecule provided in Example 3 of the present invention;

[0009] Figure 2 This is a graph of XO index in mouse serum provided in Example 4 of the present invention;

[0010] Figure 3 This is a graph of uric acid levels in mouse serum provided in Example 4 of the present invention;

[0011] Figure 4 This is a graph showing the creatinine index in mouse serum provided in Example 4 of the present invention;

[0012] Figure 5 This is a graph of blood urea nitrogen levels in mouse serum provided in Example 4 of the present invention;

[0013] Figure 6 This is a graph showing the adenosine deaminase index in mouse serum provided in Example 4 of the present invention;

[0014] Figure 7 This is a graph showing the aspartate aminotransferase (AST) level in mouse serum provided in Example 4 of the present invention.

[0015] Figure 8 This is a graph showing the alanine aminotransferase (ALT) index in mouse serum provided in Example 4 of the present invention.

[0016] Figure 9 This is a mouse kidney protein WB imaging image provided in Example 5 of the present invention;

[0017] Figure 10 This is a graph showing the expression level of mouse kidney protein ABCG2 provided in Example 5 of the present invention;

[0018] Figure 11 This is a graph showing the expression level of the mouse kidney protein NPT1 provided in Example 5 of the present invention;

[0019] Figure 12 This is a graph showing the expression level of mouse kidney protein OAT1 provided in Example 5 of the present invention;

[0020] Figure 13 This is a PAS staining image of mouse kidneys provided in Example 6 of the present invention;

[0021] Figure 14 This is a graph showing the expression level of the mouse kidney protein GLUT9 provided in Example 7 of the present invention.

[0022] Figure 15 This is a graph showing the expression level of the mouse kidney protein URAT1 provided in Example 7 of the present invention. Detailed Implementation

[0023] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0024] Example 1:

[0025] A solid-phase synthesis method for walnut-derived xanthine oxidase inhibitory peptide (DVWDPF) includes the following steps:

[0026] 2-CTC resin was weighed and swollen in dichloromethane (DCM) for 1 hour, then washed three times with N,N-dimethylformamide (DMF). Subsequently, in DMF solvent, 1 eq of the first Fmoc-protected amino acid and 1.5 eq of N,N-diisopropylethylamine (DIEA) were coupled to the 2-CTC resin for 2 hours to immobilize the amino acid on the resin. After the reaction, the solvent was dried and the resin was washed with DMF, followed by a 1 hour end-capping reaction with a methanol solution containing DIEA to block unreacted active sites. After end-capping, the Fmoc protecting group was removed using a 20% piperidine / DMF solution (reaction 10 min, repeated twice), and the resin was washed with DMF. Subsequent amino acid coupling was then performed: in each round of reaction, 3 eq of Fmoc-protected amino acid, 3 eq of 1-hydroxybenzotriazole, and 3 eq of N,N'-diisopropylcarbodiimide were reacted with the free amino group on the resin in DMF for 1.5 hours. By repeating the "deprotection-coupling" cycle, all pre-defined amino acid sequences were sequentially linked, and the N-terminal Fmoc protecting group was removed after the last amino acid was linked. After synthesis, the resin was thoroughly washed and dried. Then, the resin was reacted with 95% trifluoroacetic acid, 2% triisopropylsilane, 2% ethylenedithiol, and 1% water for 2 hours to cleave the peptide from the resin and simultaneously remove the side-chain protecting groups. After filtration of the reaction solution, the filtrate was collected, and the peptide was precipitated with ice-cold diethyl ether. The crude peptide was obtained by centrifugation. Finally, the crude peptide was purified by preparative high-performance liquid chromatography and lyophilized to obtain the final product, purified walnut-derived xanthine oxidase inhibitory peptide (DVWDPF).

[0027] Example 2:

[0028] Assay for xanthine oxidase inhibitory activity:

[0029] (1) Assay for inhibition of xanthine oxidase:

[0030] Preparation of 0.2 mol / L, pH=7.4 phosphate buffer solution: Weigh 7.16 g sodium dihydrogen phosphate and 3.12 g disodium hydrogen phosphate and dilute to 100 mL of distilled water respectively, and mix well at a volume ratio of 81:19.

[0031] 0.48mM xanthine solution: Weigh 7.30mg xanthine powder, dissolve it in 400μL of 1mol / L NaOH solution, and then bring the volume to 100mL with PBS buffer.

[0032] 0.02 U / mL xanthine oxidase solution: Weigh 1 mg of xanthine oxidase and dissolve it in 1 mL of distilled water to obtain the stock solution. Then take 21.7 μL of the stock solution and dilute it in 9.9783 mL of distilled water.

[0033] Different concentrations of DVWDPF solutions: Weigh 0.5 mg, 1 mg, 1.5 mg, 2 mg, and 2.5 mg of DVWDPF respectively, and add phosphate buffer solution to 1 mL;

[0034] Add 50 μL of DVWDPF solution at different concentrations (0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL) and 50 μL of xanthine oxidase solution at a concentration of 0.02 U / mL to each well of a 96-well plate. Shake for 30 s, incubate at 25°C for 5 min, then add 150 μL of 0.48 mM xanthine solution. Shake for 30 s, incubate at 25°C for 25 min, and measure the absorbance at 290 nm. The formula for calculating the xanthine oxidase inhibitory activity is as follows:

[0035] .

[0036] In the formula, A l A1 represents the absorbance of DVWDPF solution with xanthine oxidase solution added; A2 represents the absorbance of DVWDPF solution without xanthine oxidase solution added; A3 represents the absorbance of phosphate buffer solution with xanthine oxidase solution added; A4 represents the absorbance of phosphate buffer solution without xanthine oxidase solution added. The results show that the IC50 of DVWDPF... 50 The lowest result was 3.34 mg / mL.

[0037] Example 3:

[0038] DVWDPF docking with XO protein molecules:

[0039] The XO protein crystal structure used for docking was obtained from PDB, PDB ID 2E1Q. The short peptide 3D structure was drawn using PyMol 2.5.21 and energy was minimized under the MMFF94 force field.

[0040] This embodiment uses AutoDockVina 1.2.3 software for molecular docking. Before docking, the XO protein was processed using PyMol 2.5.21, including the removal of water molecules, salt ions, and short peptides. A docking box was then set, with the center of the box aligned with the centroid of the ligand in the original crystal, and a size of 25*25*25 cubic angstroms. Furthermore, ADFRsuite 1.03 was used to convert all processed short peptides and receptor proteins into the PDBQT format required for AutoDockVina 1.2.3 docking. During docking, the global search detail was set to 32, while other parameters remained at their default settings. The docking conformation with the highest score was considered the binding conformation. Finally, the docking results were visualized and analyzed using PyMol 2.5.2.

[0041] The results are as follows Figure 1 As shown, the left image presents the overall binding of the peptide to the protein, while the magnified image shows in detail the specific interactions with amino acid residues near the XO active site. DVWDPF can form hydrogen bonds with THR-1011, SER-877, ASN-769, and TYR-1141 in the XO protein, making the protein bind more tightly to the short peptide; at the same time, it also interacts with 16 amino acids, including PHE-91, PHE-1010, and LEU-874, providing strong van der Waals forces for the molecule.

[0042] Example 4:

[0043] Serum markers in hyperuricemic mice:

[0044] Animal research was approved by the Ethics Committee of Jilin Agricultural University and followed the European Commission's Guidelines for the Care and Use of Laboratory Animals. Male C57BL / 6J mice, 4 weeks old and weighing 20±2g, were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China). Experiments were conducted at the Experimental Animal Center of Jilin Agricultural University (temperature 20±2℃, humidity 55±5%), with a 12h light-12h dark cycle. Walnut active peptide DVWDPF (purity ≥95%, obtained by the method described in Example 1) was used.

[0045] After one week of acclimatization, male C57BL / 6J mice were randomly divided into four groups: blank control group (Con), model group (Mod), peptide intervention group (Pep), and positive group (Pos).

[0046] Blank control group (Con): Normal feeding, no intervention, and daily gavage administration of 200 μL of physiological saline for 21 days;

[0047] Model group (Mod): Potassium oxanoate and adenine were dissolved in physiological saline and administered to mice by gavage at a dose of 50 mg / kg potassium oxanoate combined with 250 mg / kg adenine for 7 consecutive days to establish the HUA model; then, 200 μL of physiological saline was administered by gavage daily for 14 days.

[0048] Peptide intervention group (Pep): The modeling method was the same as that of the model group, that is, mice were continuously gavaged for 7 days with potassium oxonate 50 mg / kg combined with adenine 250 mg / kg, and then gavaged daily with 100 mg / kg of DVWDPF (prepared with physiological saline) for 14 days.

[0049] Positive group (Pos): The modeling method was the same as that of the model group, that is, mice were continuously gavaged for 7 days with potassium oxonate 50 mg / kg combined with adenine 250 mg / kg, and then benzbromarone (prepared with physiological saline) was administered by gavage daily for 14 days.

[0050] At the end of the experiment, all mice were euthanized. Before euthanasia, blood samples were collected via ocular sampling. The blood samples were placed in an ice pack and left to stand for 10 minutes, then centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected as serum for later use.

[0051] Potassium oxazine and adenine were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China), and benzbromarone was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). The kits for xanthine oxidase, uric acid, blood urea nitrogen, creatinine, adenosine deaminase, aspartate aminotransferase, and alanine aminotransferase were all purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China). The tests were performed according to the instructions of the test kits.

[0052] Serum marker test results as follows Figures 2-8 As shown, compared to the model group, the peptide intervention group showed a decrease in xanthine oxidase levels from 20.46 U / L to 18.70 U / L; uric acid levels from 49.28 μmol / L to 40.65 μmol / L; creatinine levels from 55.46 μmol / L to 34.70 μmol / L; blood urea nitrogen levels from 13.07 mmol / L to 10.68 mmol / L; adenosine deaminase levels from 4.40 U / L to 2.75 U / L; aspartate aminotransferase levels from 18.86 U / L to 12.42 U / L; and alanine aminotransferase levels from 7.98 U / L to 5.09 U / L.

[0053] Example 5:

[0054] Western blot analysis of kidney proteins in hyperuricemic mice:

[0055] The experimental animals were grouped and treated as in Example 4. Total protein was extracted from the tissues: A small amount of mouse kidney tissue was placed in a homogenizer, lysis buffer containing PMSF was added, and homogenization was performed rapidly on ice. After standing for 2 minutes, homogenization was performed again on ice. This process was repeated several times to thoroughly break down the tissue. The homogenizer was then shaken at 4°C for 40 minutes. The lysis buffer was transferred to a 1.5 mL centrifuge tube using a pipette, and then centrifuged at 10,000 rpm for 10 minutes at 4°C to obtain a supernatant containing protein. The protein concentration was determined using a BCA kit.

[0056] Clean and dry the perforated glass plate, then clamp it vertically onto the clip. Pour in the separating gel and stacking gel of the corresponding antibody molecular weight concentration. Add 20 μg of supernatant containing protein to each well. Before loading the sample, heat the protein in a metal bath for 10 min to denature the protein. The PVDF membrane was run at a constant voltage of 80V for 20 minutes, then increased to 120V for 90 minutes for transfer. The membrane was then activated with methanol for 15 seconds and equilibrated in wet transfer buffer for 30 minutes. The gel was cut according to the molecular weight of the target protein, and the gel was arranged in the following order from bottom to top: filter paperboard-PVDF membrane-gel-filter paperboard. The entire process was performed in wet transfer buffer, continuously removing air bubbles. The membrane was then transferred to a wet transfer apparatus and transferred at 400mA for 25 minutes. The membrane was removed and washed three times with TBST for 5 minutes each time. It was then placed in blocking buffer and blocked at room temperature for 1 hour. The PVDF membrane was then incubated overnight at 4°C with diluted primary antibodies ABCG2 (ATP-binding cassette subfamily G member 2), NPT1 (sodium-phosphorus cotransporter-1), OAT1 (organic anion transporter-1), and internal control β-Action. The membrane was then incubated with secondary antibody goat anti-rabbit IgG (Goat Anti-Rabbit IgG). Incubate at room temperature for 1 hour, wash three times with TBST for 5 minutes each time, and perform chemiluminescence reaction; expose the PVDF membrane with ECL luminescent solution, develop and photograph it using a Bio-rad gel imaging system, and perform protein quantification analysis using ImageJ software.

[0057] Protein immunoblotting results as follows Figure 9 , Figure 10 , Figure 11 and Figure 12 As shown, ABCG2, NPT1, and OAT1 are involved in the excretion, reabsorption, and transport of uric acid in the kidneys, and their abnormal expression affects the balance of uric acid.

[0058] Compared to the model group, the protein expression level in the peptide intervention group was significantly increased. P <0.05); the relative expression levels of ABCG2, NPT1, and OAT1 proteins in the model group decreased to 0.28±0.08, 0.30±0.12, and 0.33±0.08, respectively, while the expression of all three proteins was significantly upregulated after DVWDPF intervention. P <0.05), with ABCG2 increasing to 0.66±0.11, NPT1 increasing to 0.81±0.12, and OAT1 increasing to 0.68±0.14, indicating that the expression levels of the three proteins were significantly restored after DVWDPF intervention.

[0059] Example 6:

[0060] Periodic acid-Schiff (PAS) staining of kidneys in hyperuricemic mice:

[0061] The experimental animals were grouped and treated as in Example 4. They were fixed with 4% paraformaldehyde, dehydrated using a gradient of ethanol, cleared with xylene, and then embedded in paraffin. The kidney tissue was then sectioned using a paraffin microtome to a thickness of approximately 5 μm. The sections were dewaxed and hydrated. After being removed from xylene and slightly dried, they were mounted with neutral resin; microscopic examination and image acquisition and analysis were then performed.

[0062] PAS staining results are as follows Figure 13 As shown, compared with the model group, the peptide intervention group exhibited reduced widening of the glomerular mesangial area, decreased deposition of PAS-positive substances, and a more regular distribution. The lesions of renal tubular epithelial cells also improved, with a reduction in abnormal deposits within the lumen. This indicates that DVWDPF treatment has a certain alleviating effect on renal polysaccharide metabolism disorders and tissue damage caused by hyperuricemia, and can alleviate pathological changes in the mesangial area.

[0063] Example 7:

[0064] Measurement of GLUT9 and URAT1 renal proteins in hyperuricemic mice:

[0065] The experimental animals were grouped and treated as in Example 4. Paraffin sections were dewaxed to water using xylene and graded ethanol, while cell slides or frozen sections were rinsed directly with tap water. Antigen retrieval was then performed in a microwave oven using citrate antigen retrieval buffer (pH 6.0). The mixture was heated to boiling over medium heat and then kept at that temperature, followed by maintenance over medium-low heat. Excessive evaporation of the liquid during the retrieval process was avoided. After retrieval, the tissue was allowed to cool naturally and washed three times with PBS. Next, the tissue was incubated with 3% hydrogen peroxide at room temperature in the dark to block endogenous peroxidase, followed by washing with PBS again. Then, a histochemical circle was drawn around the tissue with a histochemical pen, and 3% BSA or serum was added for blocking at room temperature. After blocking, the proportionally diluted primary antibodies GLUT9 (glucose transporter-9) and URAT1 (urate transporter-1) were added, and the tissue was incubated overnight in a humidified chamber at 4°C. The following day, after washing with PBS, HRP-labeled secondary antibody was added (same as in Example 5), and the cells were incubated at room temperature for 50 minutes. After washing again, freshly prepared DAB chromogenic solution was added, and the degree of color development was controlled under a microscope. The reaction was terminated with tap water. Subsequently, hematoxylin was used for nuclear counterstaining, followed by differentiation with differentiation solution and treatment with blueing solution, and then rinsing with running water. Finally, the cells were dehydrated and cleared using a gradient of ethanol and xylene, and after slight drying, they were mounted with neutral resin.

[0066] Immunohistochemical results as follows Figure 14 and Figure 15As shown, GLUT9 is a protein mainly involved in the transmembrane transport of uric acid and glucose, while URAT1 is a core molecule regulating uric acid metabolism and a key transporter protein responsible for uric acid reabsorption in the kidneys. In the model group, the positive staining intensity of GLUT9 was significantly reduced, and its distribution became scattered. In the peptide intervention group, the positive staining intensity and uniformity of GLUT9 were significantly improved compared to the model group. Although it did not completely recover to the control group level, the number of positive staining areas increased and the distribution became more orderly. In the model group, the positive staining intensity of URAT1 in the kidney tissue was significantly enhanced, and its distribution was more widespread and dense. In the peptide intervention group, the positive staining intensity of URAT1 was significantly reduced compared to the model group, and its distribution tended to be normal, approaching the control group level. This indicates that peptide treatment can effectively inhibit the upregulation of GLUT9 and URAT1 expression caused by hyperuricemia, reduce excessive reabsorption of uric acid by the kidneys, promote uric acid excretion, and thus help lower blood uric acid levels and alleviate the symptoms of hyperuricemia.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A walnut-derived xanthine oxidase inhibitory peptide, characterized in that: Its amino acid sequence is Asp-Val-Trp-Asp-Pro-Phe, and the amino acid sequence is shown in SEQ ID No.

1.

2. The use of the walnut-derived xanthine oxidase inhibitory peptide according to claim 1 in the preparation of drugs for the prevention or treatment of hyperuricemia.

3. The application of the walnut-derived xanthine oxidase inhibitory peptide as described in claim 2 in the preparation of drugs for the prevention or treatment of hyperuricemia, characterized in that: Its use in the preparation of drugs for the prevention or treatment of hyperuricemia caused by potassium oxonate.

4. The application of the walnut-derived xanthine oxidase inhibitory peptide as described in claim 2 in the preparation of drugs for the prevention or treatment of hyperuricemia, characterized in that: The drug uses walnut-derived xanthine oxidase inhibitory peptide as its sole active ingredient or one of its active ingredients.

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