Marine fish-derived xanthine oxidase inhibiting heptapeptide nplvttp and its application for reducing uric acid

Abstract

The present application belongs to the technical field of bioactive peptides, functional food and biological medicine, and particularly relates to a marine fish-derived xanthine oxidase inhibiting heptapeptide NPLVTPP and its application in reducing uric acid. The present application discloses a marine fish-derived heptapeptide NPLVTPP with xanthine oxidase inhibiting activity, and the amino acid sequence of the heptapeptide is Asn-Pro-Leu-Val-Thr-Pro-Pro. The heptapeptide can be prepared by solid-phase chemical synthesis or marine fish protein enzymatic separation and purification, can significantly inhibit xanthine oxidase activity, reduce uric acid generation, can effectively reduce serum uric acid level in a hyperuricemia animal model, and can improve high uric acid related kidney function damage. The present application also protects the application of the heptapeptide in preparing xanthine oxidase inhibitors, uric acid reducing products, products for preventing or improving hyperuricemia and kidney damage, and a medicine, food or nutritional composition containing the heptapeptide. The polypeptide sequence of the present application is clear, the target is clear, the in vivo and in vitro efficacy is clear, the source is safe, and the polypeptide can be used in the fields of functional food and biological medicine.

Description

Technical Field

[0001] This invention belongs to the fields of bioactive peptides, functional foods and biomedicine, specifically relating to a marine fish-derived xanthine oxidase inhibitory heptapeptide NPLVTPP and its uric acid-lowering application. Background Technology

[0002] Hyperuricemia (HUA) is a metabolic disease caused by abnormal purine metabolism, resulting in excessive uric acid production or reduced excretion. It is an important risk factor for gout, urate deposition, kidney damage, and metabolic syndrome.

[0003] Xanthine oxidase (XOD) is a key rate-limiting enzyme that catalyzes the conversion of hypoxanthine and xanthine into uric acid. Therefore, inhibiting xanthine oxidase activity is one of the important strategies for lowering uric acid.

[0004] While commonly used xanthine oxidase inhibitors such as allopurinol and febuxostat have good uric acid-lowering effects, they may cause certain adverse reactions in some people. Therefore, it is of great significance to develop natural uric acid-lowering active substances that are safe in origin, have clear effects, and have good application potential.

[0005] In recent years, bioactive peptides derived from food proteins have become a hot topic in functional food and natural drug research due to their small molecular weight, easy absorption, wide availability, and potentially high safety. However, existing reports on bioactive peptides with well-defined amino acid sequences, clear target effects, and intracellular uric acid-lowering activity are still relatively limited, especially regarding the short peptide sequence NPLVTPP derived from marine fish proteins and its uric acid-lowering applications, which currently lack systematic publication.

[0006] Therefore, developing a natural bioactive polypeptide with a clear sequence, a clear target action, and in vivo efficacy support in animals has high scientific value and application prospects. Summary of the Invention

[0007] The purpose of this invention is to address existing problems by providing a marine fish-derived heptapeptide NPLVTPP with xanthine oxidase inhibitory activity and uric acid-lowering effect, and to provide its application in the preparation of xanthine oxidase inhibitors, uric acid-lowering products, and products for the prevention or improvement of hyperuricemia.

[0008] This invention is achieved through the following technical solution: The first objective of this invention is to provide a marine fish-derived heptapeptide NPLVTPP with xanthine oxidase inhibitory activity, the amino acid sequence of which is Asn–Pro–Leu–Val–Thr–Pro–Pro (NPLVTPP).

[0009] A second object of the present invention is to provide the use of the aforementioned polypeptide in the preparation of xanthine oxidase inhibitors.

[0010] The three objectives of this invention are to provide the use of the aforementioned polypeptide in the preparation of products that lower uric acid levels.

[0011] A fourth objective of this invention is to provide the use of the aforementioned polypeptide in the preparation of products for the prevention or improvement of hyperuricemia and hyperuricemia-related kidney damage.

[0012] A fifth object of the present invention is to provide a composition comprising the aforementioned polypeptide.

[0013] Furthermore, the composition may be in the form of tablets, capsules, granules, powders, oral liquids, or solid beverages.

[0014] Furthermore, the composition is a pharmaceutical composition.

[0015] Furthermore, the composition is a food composition or a nutritional composition.

[0016] The sixth objective of this invention is to provide a method for preparing the aforementioned polypeptide, wherein the method is a solid-phase chemical synthesis method or the polypeptide is obtained by separation and purification from the enzymatic hydrolysis products of marine fish.

[0017] The present invention has the following advantages over the prior art: 1) The sequence is well-defined. This invention provides a marine fish-derived short peptide NPLVTPP with a well-defined amino acid sequence, which facilitates synthesis, preparation, quality control, and subsequent application development.

[0018] 2) It has clear xanthine oxidase inhibitory activity. The polypeptide of this invention can significantly inhibit xanthine oxidase activity, thereby reducing uric acid production, and has the potential to be used as a natural uric acid-lowering active ingredient.

[0019] 3) Has the effect of lowering uric acid in vivo. In animal models of hyperuricemia, the peptides of this invention can significantly reduce serum uric acid levels and improve abnormal renal function indicators associated with hyperuricemia.

[0020] 4) It has strong institutional support. This invention provides systematic support for its uric acid-lowering effect through multiple layers of evidence, including enzyme activity experiments, molecular docking, molecular dynamics simulation, target binding verification, and animal efficacy evaluation, thereby improving its technical credibility and application value.

[0021] 5) Possesses good development potential The polypeptides of this invention are derived from marine fish proteins and have the potential to be developed into functional foods, nutritional preparations, or pharmaceutical candidate active ingredients. Attached Figure Description

[0022] Figure 1 The chemical structural formula of the polypeptide NPLVTPP; Figure 2 The graph shows the identification and purity detection of the peptide NPLVTPP. Figure 3 This is a diagram showing the inhibitory effect of the peptide NPLVTPP on xanthine oxidase (XOD) activity. Figure 4 This is a schematic diagram of the molecular docking of the polypeptide NPLVTPP with xanthine oxidase (XOD). Figure 5 Figure showing the binding verification and activity effect of peptide NPLVTPP to CETSA of xanthine oxidase (XOD); Figure 6 Diagram showing the key interaction sites between the polypeptide NPLVTPP and xanthine oxidase (XOD); Figure 7 Figure showing the binding verification and activity effect of peptide NPLVTPP to CETSA of xanthine oxidase (XOD); Figure 8 The effect of the peptide NPLVTPP on xanthine oxidase (XOD) in an animal model of hyperuricemia is shown in the figure. Figure 9 The effect of the polypeptide NPLVTPP on renal function indicators (Cr, BUN) is shown in the figure. Figure 10 This is a diagram illustrating the effect of the peptide NPLVTPP on improving pathological damage in renal tissue. Detailed Implementation

[0023] To further explain the present invention, the following specific embodiments are described.

[0024] Example 1: Method for isolating and purifying the polypeptide NPLVTPP from marine fish protease hydrolysates This embodiment provides a method for isolating and purifying the heptapeptide NPLVTPP from the enzymatic hydrolysis products of marine fish.

[0025] (1) Preparation of marine fish protein hydrolysate Take 100 g of marine fish protein raw material (such as fish paste or fish meat), add 1000 mL of deionized water, homogenize, adjust the pH to 8.0, add alkaline protease (enzyme addition amount is 2% of substrate weight), and enzymatically hydrolyze at 50℃ for 4 h. After the reaction is completed, heat the hydrolysate to 95℃ and hold for 10 min to inactivate the enzyme, cool and centrifuge (8000 rpm, 15 min), and collect the supernatant to obtain marine fish protein hydrolysate (MFPP).

[0026] (2) Ultrafiltration stage The above enzymatic hydrolysate was fractionated sequentially through 10 kDa and 3 kDa ultrafiltration membranes, and the fraction with a molecular weight less than 3 kDa was collected. This fraction is rich in low molecular weight peptides (mainly in the range of 600-800 Da), which is the main source of the target active peptides.

[0027] (3) Gel filtration separation Fractions <3 kDa were loaded onto a Sephadex G-15 gel column (2.6 cm × 60 cm), eluted with deionized water at a flow rate of 0.5 mL / min, and the absorbance of each fraction was measured (220 nm). Low molecular weight elution peaks were collected, combined, and lyophilized to obtain crude peptides.

[0028] Example 2: Method for solid-phase synthesis of heptapeptide Asn–Pro–Leu–Val–Thr–Pro–Pro (NPLVTPP) using CTC resin This embodiment employs the Fmoc solid-phase peptide synthesis method, using 2-chlorotrityl chloride resin (CTCresin) as the solid-phase support. Amino acids are sequentially coupled from the C-terminus to the N-terminus to synthesize the heptapeptide NPLVTPP with a purity of 95%. The specific steps are as follows: (1) Crude peptide dissolution Weigh 100 mg of each of the crude heptapeptide obtained in Example 1 or Example 2, add 10 mL of aqueous solution containing 0.1% formic acid, and allow it to dissolve completely. If it does not dissolve completely, add a small amount of 1-2 mL of acetonitrile to aid dissolution, so that the crude peptide mass concentration is 10 mg / mL. After dissolution, filter through a 0.45 μm microporous membrane to obtain the sample solution to be purified.

[0029] (2) Preparative high performance liquid chromatography purification Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) with an acidic aqueous solution and acetonitrile as the mobile phase, using gradient elution. The main peak was collected, and the target peptide was obtained by freeze-drying with a purity greater than 95%. Preparative RP-HPLC was also used for purification under the following chromatographic conditions: Chromatographic column: C18 preparative column (250 mm × 21.2 mm, 10 μm), mobile phase A: water + 0.1% formic acid, mobile phase B: acetonitrile + 0.1% formic acid, flow rate: 3 mL / min, detection wavelength: 220 nm, column temperature: 30℃, injection volume: 0.5 mL, gradient elution 0-5 min: 5% B, 5-25 min: 5%→35% B, 25-30 min: 35%→60% B, collect the main peak eluent.

[0030] (3) Freeze-drying The collected main peak eluates were combined, and after removing acetonitrile by rotary evaporation, the eluates were freeze-dried for 48 h to obtain a white powdery heptapeptide. The purity was greater than 95% as determined by HPLC.

[0031] (4) Identification The heptapeptide was identified using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). The chromatographic conditions were as follows: column: C18 nanocolumn (150 μm × 15 cm); mobile phase: 0.1% formic acid / water / 80% acetonitrile; flow rate: 300 nL / min; scan range: m / z 350–1500. The calculated [M+H]⁺ = 737.4192 (theoretical molecular weight 736.4119) Figure 2 This proves that the prepared product is the target heptapeptide NPLVTPP.

[0032] Example 4: Inhibitory effect of peptide NPLVTPP on xanthine oxidase activity The inhibitory activity of the peptide NPLVTPP was evaluated using a hyperuricemic cell model and a xanthine oxidase activity assay.

[0033] Experimental methods A hyperuricemia model was established using HepG2 cells. The induction conditions were: hypoxanthine 200 μM and XOD 0.005 U / mL. After model establishment, the cells were treated with NPLVTPP for 24 h, and uric acid levels and XOD activity were measured. The NPLVTPP concentrations investigated were 40 μM, 80 μM, and 160 μM, while the positive control concentration of allopurinol was 10 μM.

[0034] Experimental results The results showed that the peptide NPLVTPP significantly inhibited xanthine oxidase in a dose-dependent manner. In a unified comparison experiment of candidate peptides, NPLVTPP exhibited significant XOD inhibitory activity at 160 μM, with an inhibition rate of approximately 72.1% ± 3.2%, and its inhibition rate on uric acid production was approximately 68.3%. Figure 3The results suggest that NPLVTPP can reduce uric acid production by inhibiting xanthine oxidase activity.

[0035] Example 5: Interaction analysis between peptide NPLVTPP and xanthine oxidase To investigate the interaction mechanism between the peptide NPLVTPP and xanthine oxidase, molecular docking and molecular dynamics simulations were used for analysis.

[0036] Experimental methods The xanthine oxidase protein crystal structure was selected as the receptor, and the receptor source was XOD (PDB: 2E1Q). After constructing the molecular structure of the peptide NPLVTPP, molecular docking analysis was performed, and molecular dynamics simulations were further used to evaluate its stability with the XOD complex. The molecular dynamics simulation time was 100 ns.

[0037] Experimental results The results showed that the peptide NPLVTPP could form a stable binding with the active site of xanthine oxidase and enter its active pocket, forming hydrogen bonds, hydrophobic interactions, and other non-covalent interactions with surrounding amino acid residues. Figure 4 , Figure 6 Molecular dynamics results further support the good stability of the NPLVTPP-XOD complex, suggesting that the peptide has a well-defined target binding basis.

[0038] Example 6: Verification of direct binding of peptide NPLVTPP to xanthine oxidase Cellular thermal displacement analysis (CETSA) was used to verify the direct interaction between the peptide NPLVTPP and xanthine oxidase.

[0039] Experimental methods HepG2 cells were co-incubated with NPLVTPP at a concentration of 160 μM for 24 h. Cell lysates were then treated at a temperature gradient of 37–75°C, and changes in the thermostability of XOD protein were detected by Western blot.

[0040] Experimental results The results showed that the thermal stability of XOD protein changed after NPLVTPP treatment, suggesting a direct interaction between NPLVTPP and XOD. Figure 5 , Figure 7 The results of molecular docking and molecular dynamics simulations further support the view that this peptide exerts its uric acid-lowering effect by directly targeting XOD.

[0041] Example 7: Uric acid-lowering effect of peptide NPLVTPP on an animal model of hyperuricemia A mouse model of hyperuricemia was established by inducing hyperuricemia with potassium oxonate and hypoxanthine to evaluate the in vivo uric acid-lowering effect of the peptide NPLVTPP.

[0042] laboratory animals The experimental animals were male Balb / c mice, 7-8 weeks old, 24-25 g.

[0043] Modeling methods Except for the normal group, the other groups were given potassium oxonate 300 mg / kg and hypoxanthine 300 mg / kg by gavage daily for 14 consecutive days.

[0044] Dosing group The experimental groups included: a normal control group, a model group, a positive control group (allopurinol), a low-dose peptide group, a medium-dose peptide group, and a high-dose peptide group. The allopurinol dose was 5 mg / kg, the low-dose peptide dose was 140 mg / kg, the medium-dose peptide dose was 285 mg / kg, and the high-dose peptide dose was 550 mg / kg. All doses were administered by gavage for 21 days.

[0045] Experimental results The results showed that, compared with the model group, the serum uric acid level of mice in the NPLVTPP-treated group was significantly reduced, and the decrease showed a dose-dependent trend; the high-dose group showed a more significant uric acid-lowering effect. Furthermore, NPLVTPP significantly inhibited XOD activity in the serum, liver, and kidneys of hyperuricemic model mice, and downregulated the gene and protein expression levels of XDH / XOD in the liver and kidneys. Figure 8 ).

[0046] Example 8: The effect of peptide NPLVTPP on improving hyperuricemia-related renal function injury Based on the above animal experiments, further tests were conducted on renal function-related indicators, including blood urea nitrogen (BUN), creatinine (Cr), and renal tissue pathological changes.

[0047] Experimental results The results showed that, compared with the model group, the levels of BUN and Cr in mice in the NPLVTPP administration group were significantly decreased. Figure 9 This suggests that it can improve hyperuricemia-related renal dysfunction. This improvement was observed in the low, medium, and high dose groups, showing a dose-dependent trend. Further pathological observation showed that the model group's renal tissue exhibited pathological changes such as glomerular atrophy, structural disorder, swelling and vacuolar degeneration of renal tubular epithelial cells, and interstitial inflammatory cell infiltration; however, after NPLVTPP intervention, the pathological damage to renal tissue was significantly improved, especially in the high-dose group where the recovery was most significant. Figure 10 ).

[0048] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A marine fish-derived xanthine oxidase inhibitory heptapeptide NPLVTPP, characterized in that, Its amino acid sequence is Asn–Pro–Leu–Val–Thr–Pro–Pro (NPLVTPP).

2. The use of the polypeptide according to claim 1 in the preparation of xanthine oxidase inhibitors.

3. The use of the polypeptide according to claim 1 in the preparation of products that lower uric acid levels.

4. The use of the polypeptide of claim 1 in the preparation of products for preventing or improving hyperuricemia and hyperuricemia-related kidney damage.

5. A composition, characterized in that, It contains the polypeptide as described in claim 1.

6. The composition according to claim 5, characterized in that, The composition is in the form of tablets, capsules, granules, powders, oral liquids, or solid beverages.

7. The composition according to claim 6, characterized in that, The composition is a pharmaceutical composition.

8. The composition according to claim 6, characterized in that, The composition is a food composition or a nutritional composition.

9. A method for preparing the polypeptide of claim 1, characterized in that, The method is a solid-phase chemical synthesis method, or it can be obtained by separating and purifying from the protein hydrolysate of marine fish.

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