A mussel peptide and its application in preparing uric acid-lowering products

Abstract

The present invention relates to the preparation and application of biological peptides, and in particular to a mussel peptide and its application in the preparation of uric acid-lowering products. The amino acid sequence is shown in SEQ ID NO.1‑4. The present invention prepares mussel polypeptides by composite enzyme-directed enzymatic hydrolysis and membrane separation and purification technology, and screens out four novel polypeptides with potential biological activity by combining LC‑MS / MS analysis and activity prediction. Molecular docking and cell experiments have confirmed that these four polypeptides can specifically inhibit GLUT9 protein activity and reduce uric acid levels; animal experiments have further verified their efficacy in lowering uric acid, providing a scientific basis for the high-value development of mussel resources and the development of functional uric acid-lowering products.

Description

Technical Field

[0001] The present invention relates to the preparation and application fields of biological peptides, and in particular to a mussel peptide and its application in the preparation of uric acid-lowering products. Background Art

[0002] Hyperuricemia is a metabolic disease characterized by abnormally elevated serum uric acid concentrations. Its background involves complex physiological mechanisms and multiple pathogenic factors. Traditional medications such as allopurinol and benzbromarone can lower uric acid, but long-term use can lead to liver and kidney toxicity and gastrointestinal side effects. Traditional Chinese medicine formulas, while relatively safe, are difficult to standardize due to their complex composition and unclear mechanisms. Therefore, there is an urgent need to develop safe and effective food-derived natural ingredients to alleviate hyperuricemia.

[0003] Bioactive peptides, low-molecular-weight fragments produced by protein enzymatic hydrolysis, combine high safety, multi-target synergy, and resource sustainability, attracting significant attention in the development of functional foods. Mussels are the most important commercial shellfish cultured along my country's coast. Mussels are rich in protein and peptides, and recent studies have demonstrated significant efficacy in antibacterial, antioxidant, bone repair, and antithrombotic activities. Chinese patent CN118496307A discloses a mussel xanthine oxidoreductase inhibitory peptide for the treatment of hyperuricemia, as well as its preparation method and application. The active peptide YQMCGW prepared in this application significantly reduces xanthine oxidoreductase activity and serum uric acid levels, demonstrating therapeutic efficacy for hyperuricemia and gout. YQMCGW inhibits xanthine oxidoreductase activity primarily through hydrogen bonding, electrostatic forces, and hydrophobic interactions. Therefore, YQMCGW is promising for development as a therapeutic agent and adjunctive therapy for hyperuricemia and gout. However, only one peptide segment cannot meet the huge market demand. More mussel peptides need to be further developed to meet the diversification of raw materials and open up a broader market space for the efficient development and utilization of marine shellfish resources. Summary of the Invention

[0004] The technical problem to be solved by the present invention is that the current research on the application of mussel peptides in lowering uric acid is relatively single, and more mussel peptides need to be further developed to meet the diversification of raw materials and open up a broader market space for the efficient development and utilization of marine shellfish resources.

[0005] To address these issues, the present invention prepared mussel polypeptides using a composite enzyme-directed hydrolysis method and membrane separation and purification. Four novel polypeptides with potential bioactivity were screened using LC-MS / MS analysis and activity prediction. Molecular docking and cell-based experiments confirmed that these four polypeptides specifically inhibited GLUT9 protein activity and reduced uric acid levels. Animal experiments further validated their uric acid-lowering efficacy, providing a scientific basis for the high-value development of mussel resources and the development of functional uric acid-lowering products.

[0006] To achieve the above object, the present invention is achieved by the following technical means: a use of a mussel peptide in the preparation of a uric acid-lowering product, comprising a polypeptide with an amino acid sequence as shown in SEQ ID NO.1-4:

[0007] SEQ ID NO.1:

[0008] WTFFGA.

[0009] SEQ ID NO.2:

[0010] PDPFYK.

[0011] SEQ ID NO.3:

[0012] SPFFKV.

[0013] SEQ ID NO.4:

[0014] TSPFFK.

[0015] The preparation method of the above-mentioned mussel peptide comprises the following steps:

[0016] Fresh mussel meat after shelling was taken, added with distilled water for homogenization, 3% composite protease was added and stirred evenly, and the pH value was adjusted to 7.5 with HCL and NaOH, and hydrolyzed at 50°C in a water bath constant temperature oscillator for 5 h; after enzymatic hydrolysis, the enzyme was inactivated and cooled, and centrifuged at 8000 rpm at 4°C for 15 min to collect the supernatant; the supernatant was further ultrafiltered at 0.1 MPa and 25°C, and an ultrafiltration tube with a molecular weight cutoff of 3 kDa was selected. The filtrate was collected, desalted and freeze-dried to obtain mussel peptide powder.

[0017] Furthermore, distilled water was added at a material-liquid ratio of 1:5.

[0018] Furthermore, the concentrations of the HCL solution and the NaOH solution are both 1 mol / L.

[0019] Furthermore, the composite protease (S10155) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0020] Furthermore, the enzyme was inactivated by boiling water bath for 10 min.

[0021] The above amino acid sequence is the mussel peptide shown in SEQ ID NO.1-4.

[0022] Furthermore, the mussel peptides with amino acid sequences shown in SEQ ID NO. 1-4 are artificially synthesized.

[0023] This active peptide can inhibit the uric acid transporter GLUT9, reduce uric acid reabsorption, and reduce the uric acid content in the culture medium in a cell model. It has also been proven in an animal model that it can effectively reduce serum uric acid and urea nitrogen levels, inhibit xanthine oxidase activity, and has uric acid-lowering activity.

[0024] The beneficial effects of the present invention are:

[0025] Mussel peptides were prepared using a combination of enzyme-directed hydrolysis and membrane separation and purification techniques. Four novel peptides with potential bioactivity were screened using LC-MS / MS analysis and activity prediction. Molecular docking and cell-based experiments confirmed that these four peptides specifically inhibited GLUT9 protein activity and reduced uric acid levels. Animal experiments further validated their uric acid-lowering efficacy, providing a scientific basis for the high-value development of mussel resources and the development of functional uric acid-lowering products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : The docking results of the peptide segment of SEQ ID NO.1 and the XOD receptor protein molecule. A is the three-dimensional structure diagram, and B is the two-dimensional structure diagram.

[0027] Figure 2 : The docking results of the peptide segment of SEQ ID NO. 2 and the XOD receptor protein molecule. A is the three-dimensional structure diagram, and B is the two-dimensional structure diagram.

[0028] Figure 3 : The docking result diagram of the peptide segment of SEQ ID NO.3 and the XOD receptor protein molecule. Among them, A is the three-dimensional structure diagram, and B is the two-dimensional structure diagram.

[0029] Figure 4 : The docking result of the peptide segment of SEQ ID NO. 4 and the XOD receptor protein molecule. A is the three-dimensional structure diagram, and B is the two-dimensional structure diagram.

[0030] Figure 5 : Effects of mussel active peptides on uric acid levels in the supernatant of HK-2 cell model.

[0031] Figure 6 : Effects on renal indices in hyperuricemic rats.

[0032] Figure 7 : Effects on serum biochemical parameters in hyperuricemia rats; A is uric acid level, B is creatinine level, and C is urea nitrogen level.

[0033] Figure 8 : Effects on urine biochemical indices and uric acid excretion in hyperuricemic rats; A is uric acid level, B is creatinine level, and C is uric acid excretion fraction. DETAILED DESCRIPTION

[0034] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] In addition, all materials used in the examples of the present invention, unless otherwise specified, were purchased from the market.

[0036] Example 1:

[0037] This example provides a screening and identification technology for mussel-derived uric acid-lowering functional polypeptides, which specifically includes the following steps:

[0038] Preparation of mussel peptides: 100 g of fresh, shelled mussel meat was homogenized with distilled water at a ratio of 1:5. 3% composite protease was added and stirred thoroughly. The pH was adjusted to 7.5 with 1 mol / L HCl and 1 mol / L NaOH. Hydrolysis was performed in a water bath in a constant-temperature shaker at 50°C for 5 h. After enzymatic hydrolysis, the enzyme was inactivated in a boiling water bath for 10 min, cooled, and centrifuged at 8000 rpm for 15 min at 4°C. The supernatant was collected. The supernatant was further ultrafiltered at 0.1 MPa and 25°C using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The filtrate was collected, desalted, and lyophilized to obtain mussel peptide powder. The composite protease (1.2 million U / mg, Catalog No. S10155) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0039] Identification of active peptides from mussels: An Ultimate 3000 UPLC system (Thermo, Waltham, MA, USA) was connected to a Q Exactive mass spectrometer (Thermo, Waltham, MA, USA). Proteome Discoverer 2.4 was used to search the raw data files against the Uniport mussel protein database downloaded from NCBI. Peptide Ranker was used to predict the activity of the identified peptides and select peptide sequences with high scores.

[0040] Molecular docking screening of active peptides: Using uric acid transporter (GLUT9) as the target protein, we selected the top five peptides with the highest predicted activity for homology modeling. These peptides were docked to the primary binding active sites of the two proteins, using their own ligand, uric acid (UA), as a negative control. Peptide sequence homology modeling: 3D modeling was performed using Chemdraw 18.1 software, inputting the peptide sequence to generate the peptide structure. Energy minimization of the peptide structure was performed using the CHARMm program in Discover Studio 2019.

[0041] Receptor pre-processing: The X-ray crystal structure of the GLUT9 protein (PDBID: 8Y65) was downloaded from the PDB database. After removing the existing ligand, chemical bond correction and charge addition were performed. Active pockets were further identified and covered. The receptor and peptide were molecularly docked using the CDOCKER program. Binding free energy, hydrogen bonds, hydrophobic groups, and charged groups were analyzed based on the highest CDOCKER Energy score, and the docking score was calculated.

[0042] The amino acid sequence of the mussel peptide was determined using Q-Exactive plus. ProteomeDiscover 2.4 software was used for analysis and the Uniport mussel peptides database was searched. A total of 1,315 different peptide sequences were matched. Further screening and identification revealed four peptides with residual local confidence greater than 80% and peptide sequence Peptide Ranker scores greater than 0.9: WTFFGA, PDPFYK, SPFFKV, and TSPFFK. Comparison with previously discovered active peptides in the BIOPEP database revealed that none of the four peptides had been previously reported and were considered novel active peptide sequences. The results are shown in Table 1.

[0043] Table 1 Identification and bioactivity prediction of active peptides from mussels:

[0044] .

[0045] The above four peptides were selected for homology modeling and molecular docking with GLUT9 protein to analyze the interaction between the peptides and the amino acid residues in the active center of GLUT9. The docking results are as follows: Figure 1 Middle AB to Figure 4As shown in Figures AB, the target protein GLUT9 used in this experiment has its own uric acid ligand, with a binding energy of -107.60. The following peptides were docked to the uric acid transporter, representing the optimal poses after simulation. The docking scores of the four peptides to the XOD protein were: WTFFGA (-232.48), PDPFYK (-273.87), SPFFKV (-238.90), and TSPFFK (-234.40). The structures of these four peptides are expected to fit into the active pocket of GLUT9, interacting with key amino acid residues through conventional hydrogen bonds and hydrophobic interactions, thereby blocking GLUT9 binding to uric acid. These results demonstrate that these four mussel-derived active peptides are promising GLUT9 inhibitors based on molecular docking and have the potential to be developed as multi-target uric acid-lowering active ingredients.

[0046] Example 2:

[0047] This example provides an investigation into the effects of mussel-derived uric acid-lowering functional peptides on an in vitro cell model, specifically including the following steps:

[0048] Artificial synthesis of mussel active peptides: synthesized by Fmoc-peptide solid phase synthesis method and analyzed by HPLC and MS sequence, the purity of the four peptides WTFFGA, PDPFYK, SPFFKV and TSPFFK were all greater than 98.89%.

[0049] In vitro uric acid-lowering activity assay of active peptides: HK-2 cells 3x10 4 Cells were seeded at a density of 100 μg / mL into 96-well plates and divided into four groups: control group (Control), model group (Model), each active peptide group (100 μg / mL), and allopurinol (100 μmol / L) positive control group (Positive). Each group had six replicate wells. After pre-incubation at 37°C and 5% CO2 for 24 hours, the culture medium in the wells was aspirated and washed with PBS. 250 μL of 4 mmol / L adenosine serum-free medium was added to each well of the model group, positive control group, and each peptide group. The blank group was incubated with serum-free medium for 24 hours. 50 μL of 0.01 U / mL xanthine oxidase was added to each well and the treatment continued for 8 hours. The uric acid content in the culture supernatant was determined strictly according to the instructions of the uric acid kit.

[0050] like Figure 5 As shown, the uric acid content in the cell culture supernatant of the model group was significantly higher than that of the blank group (P < 0.01), confirming successful modeling. Furthermore, the uric acid content in the allopurinol group was significantly lower than that in the model group, indicating that 100 μmol / L allopurinol can serve as a positive control. After intervention with mussel active peptides, uric acid content was significantly reduced compared to the model group. All five peptides exhibited uric acid-lowering activity, with WTFFGA showing the highest activity.

[0051] Example 3:

[0052] This example provides an investigation into the effects of mussel-derived uric acid-lowering functional polypeptide on hyperuricemia rats, specifically including the following steps:

[0053] Animal Grouping and Treatment: After one week of adaptive feeding, 36 male Sprague-Dawley rats were randomly divided according to body weight into a control group (control), a model group (model), low-, medium-, and high-dose WTFFGA (WA6, 80, 100, and 120 mg / kg body weight) groups, and an allopurinol group (positive, 25 mg kg-1), with six rats in each group. All groups, except the control group, were gavaged daily with a 5% CMC-Na diluted hypoxanthine and potassium oxalate suspension (adenine 0.1 g / kg and potassium oxalate 1.5 g / kg). The control group received an equal volume of 5% CMC-Na solution daily, followed by the corresponding test substance 1 hour after gavage. The control and model groups received an equal volume of 5% CMC-Na solution for 21 consecutive days. On day 20 of the experiment, the rats were placed in metabolic cages with free access to food and water, and urine was collected for 24 hours. After the experiment, the rats were fasted for 6 h but not water. Blood was collected from the abdominal aorta and the rats were killed by cervical dislocation. After killing, the kidneys and livers were quickly separated on ice, rinsed with physiological saline, dried with filter paper, weighed, and stored in a -80℃ refrigerator for later use.

[0054] Determination of organ index: The whole kidney of the experimental rat was weighed and the organ index of the rat was determined. Organ index (mg / g) = organ (mg) / body weight (g);

[0055] Serum and urine biochemical index detection: Serum uric acid (SUA), creatinine (SCr), and blood urea nitrogen (BUN) levels were detected strictly according to the kit instructions. Urine uric acid (UUA) and creatinine (UCr) levels were detected. Uric acid excretion fraction (EUA, %) was calculated as UUA × SCr / (SUA × UCr) × 100%.

[0056] Liver xanthine oxidase (XOD) activity assay: 0.1 g of liver tissue was added to 900 μl of normal saline (1% PMSF and phosphatase inhibitors) and homogenized at 4°C. The supernatant was collected by centrifugation and XOD activity was determined strictly according to the kit instructions.

[0057] like Figure 6 As shown in the results, the renal index of the model group was significantly higher than that of the control group (P<0.05). After the intervention of mussel active peptides, the renal index was significantly improved and showed a dose-dependent effect, but there was no statistical significance.

[0058] like Figure 7As shown, 3 weeks after modeling, the SUA, SCr, and BUN concentrations of the model group rats were significantly increased compared with the control group. The SUA levels of the low, medium, and high dose groups of mussel active peptide WA were significantly lower than those of the model group (P<0.01, P<0.001, P<0.001). The UCr levels of the medium and high dose groups of mussel active peptide WA were significantly lower than those of the model group (P<0.05). Low-dose WA gavage also reduced the UCr level, but there was no significant difference (P>0.05). The BUN levels of the low, medium, and high dose groups of mussel active peptide WA were significantly lower than those of the model group (P<0.001, P<0.05, P<0.001).

[0059] like Figure 8 As shown, compared with the control group, the UUA, UCr levels, and FEUA scores of rats in the model group were significantly reduced. The UUC and UCr concentrations of the mussel active peptide WA low-, medium-, and high-dose groups were higher than those in the model group, but not statistically significant (P>0.05). The FEUA score was significantly higher than that in the model group, and the effect was comparable to that of the positive control group. This suggests that the test substance targeted and inhibited GLUT9-mediated uric acid reabsorption.

[0060] Finally, it should be noted that while the above embodiments describe specific implementations of the present invention, they are not intended to limit the present invention. Those skilled in the art will understand that these are merely illustrative and that the scope of the present invention is defined by the appended claims. All modifications and equivalent substitutions are intended to be within the scope of the present invention.

Claims

1. A mussel peptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the mussel peptide according to claim 1 in the preparation of uric acid-lowering products.

3. The mussel peptide according to claim 1, wherein: The mussel peptide is artificially synthesized.

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