Camellia meal polypeptide and application thereof in synergistic reduction of uric acid with flavone

Abstract

The invention belongs to the technical field of biology, and particularly relates to a xanthine oxidase inhibitory peptide as well as a preparation method and application thereof. The preparation method comprises the following steps: extracting protein by taking oil tea meal as a raw material, preparing oil tea meal active peptide by a biological enzymolysis method, analyzing and screening out the xanthine oxidase inhibitory peptide HYIG by utilizing bioinformatics, and ensuring that the IC50 value of the xanthine oxidase inhibitory peptide HYIG reaches 110.18 + / -8.29 mu mol / L. The xanthine oxidase inhibitory peptide HYIG and the kaempferol KAE are combined, the inhibition effect of the combination of the xanthine oxidase inhibitory peptide HYIG and the kaempferol KAE on the activity of the xanthine oxidase is further explored, and when the HYIG with the concentration of 200 mu mol / L and the KAE with the concentration of 20 mu mol / L are combined, namely when the molar ratio is 10: 1, the CI value is 0.61, and the inhibition rate on the xanthine oxidase reaches 80.00 + / -0.09%. The invention also provides a preparation method of ovalbumin OVA-embedded OVA-HYIG / KAE NPs particles, the OVA-HYIG / KAE NPs has better slow release performance and stability compared with a non-embedded HYIG / KAE composition under an in-vitro simulated digestion condition, and the OVA-HYIG / KAE NPs is applied to preparation of products for inhibiting xanthine oxidase activity or preparation of products for reducing uric acid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a xanthine oxidase inhibiting peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorder. If the related indicators are abnormal twice on different dates, it can be diagnosed as hyperuricemia. When the blood uric acid level rises above the saturation point, sodium urate crystals may be generated and accumulated at the joint site, thereby triggering local inflammatory response and tissue damage, and further inducing gout. Hyperuricemia can cause various chronic diseases and is closely related to the occurrence of cardiovascular diseases such as coronary heart disease and heart failure, and may increase the risk of cardiovascular diseases through mechanisms such as promoting atherosclerosis and damaging vascular endothelial function. The formation of hyperuricemia is relatively complex, mainly involving the combined action of excessive uric acid synthesis, uric acid excretion disorder and diet-related factors.

[0003] The clinical treatment drugs for hyperuricemia are mainly divided into three categories, among which the main category is represented by allopurinol and febuxostat, which are uric acid synthesis inhibitors, and play a therapeutic role by targeting the activity of xanthine oxidase (XO). As the core catalytic enzyme of uric acid biosynthesis, xanthine oxidase assumes a dual catalytic function in the purine metabolic pathway. When the activity of xanthine oxidase is inhibited, the amount of uric acid generated will decrease, and the serum uric acid level will decrease, thus becoming an effective target. However, these drugs may have some adverse reactions in the treatment of hyperuricemia, such as gastrointestinal discomfort, and the occurrence of these side effects has prompted the research on natural enzyme activity inhibitors to find effective products for treating hyperuricemia disease.

[0004] Camellia oleifera contains rich high-quality plant proteins, tea saponin, flavonoids, polyphenols, and polysaccharides, and other active ingredients. Camellia meal is a by-product of Camellia oleifera processing, which contains 10% to 20% of protein and a high content of functional flavonoids such as Kaempferol (KAE), about 3%, which has important application value due to its antioxidant and antibacterial activities. At present, Camellia meal is mostly used as a new type of fertilizer for soil improvement or fermented protein feed, but its high-value utilization is insufficient, resulting in a large amount of resource waste. Extraction and application of functional components in Camellia meal is a key measure to improve the utilization of Camellia resources. In the present application, bioactive peptides are extracted from Camellia meal to obtain natural uric acid-lowering active ingredients, and the synergistic effect of active peptides and Kaempferol in Camellia on uric acid reduction is further explored. Ovalbumin (OVA) is used to embed the two to prepare polypeptide / Kaempferol-ovalbumin nanoparticles to improve the bioavailability of active ingredients in vivo. SUMMARY

[0005] In order to solve the above technical problems, the present application extracts protein from oil tea meal as raw material, prepares oil tea meal peptide by biological enzymatic hydrolysis, screens xanthine oxidase inhibitory peptide by bioinformatics analysis, analyzes the interaction between xanthine oxidase inhibitory peptide from oil tea meal and kaempferol by molecular docking and CompuSyn 2.0 software, explores the inhibitory effect of xanthine oxidase inhibitory peptide and kaempferol combination on xanthine oxidase activity, and further prepares ovalbumin nanoparticles and simulates gastrointestinal digestion to explore the inhibitory effect of nanoparticles on xanthine oxidase activity.

[0006] One of the technical solutions provided by the present application is a xanthine oxidase inhibitory peptide, the amino acid sequence of which is His-Tyr-Ile-Gly, abbreviated as HYIG. The xanthine oxidase inhibitory peptide HYIG is prepared by biological enzymatic hydrolysis of oil tea meal peptide extracted from oil tea meal as raw material, and the IC50 value of the xanthine oxidase reaches 110.18±8.29 μmol / L.

[0007] The second technical solution provided by the present application is the application of the xanthine oxidase inhibitory peptide HYIG in technical solution one, particularly in the application of inhibiting xanthine oxidase activity, more particularly in the application of preparing a product for inhibiting xanthine oxidase activity, or in the application of preparing a product for reducing uric acid.

[0008] The product includes but is not limited to food, health care product or medicine.

[0009] The third technical solution provided by the present application is a composition containing xanthine oxidase inhibitory peptide HYIG.

[0010] Further, the composition takes inhibitory peptide HYIG as an active ingredient.

[0011] Further, the composition contains inhibitory peptide HYIG and KAE.

[0012] Further, the mass ratio of inhibitory peptide HYIG and KAE in the composition is 1-25:1.

[0013] Preferably, when the concentration of xanthine oxidase inhibitory peptide HYIG in the composition is 200 μmol / L and the concentration of KAE is 20 μmol / L, the inhibition rate of HYIG / KAE on xanthine oxidase is 80.00%±0.09%, and the synergistic inhibitory effect of the two on xanthine oxidase is the best, with a CI value of 0.61.

[0014] The fourth technical solution of the present application is to obtain the composition of the third technical solution by embedding the egg white OVA.

[0015] Further, the preparation method is as follows:

[0016] OVA (CAS: 9006-59-1) is dissolved in a buffer solution, heated and stirred to obtain OVA NPs, and then a polypeptide HYIG / KAE solution is added to the OVA NPs after cooling to room temperature, and the HYIG, KAE and OVA are fully combined under stirring at room temperature. The solution is centrifuged, and the precipitate is vacuum freeze-dried to obtain OVA-HYIG / KAE NPs.

[0017] The fifth technical solution of the present application is the application of the composition of the third technical solution and the fourth technical solution, in particular, the application in inhibiting the activity of xanthine oxidase, more particularly, the application in preparing a product for inhibiting the activity of xanthine oxidase, or the application in preparing a product for reducing uric acid.

[0018] The product includes but is not limited to food, health care product or medicine.

[0019] Beneficial effects:

[0020] (1) The present application uses oil tea meal as raw material, and prepares and separates xanthine oxidase inhibiting peptide HYIG by biological enzymatic hydrolysis, and the IC50 value of the xanthine oxidase inhibiting peptide HYIG for xanthine oxidase reaches 110.18±8.29 μmol / L.

[0021] (2) The present application explores the interaction of xanthine oxidase inhibiting peptide HYIG and KAE for xanthine oxidase. The IC50 values of HYIG and KAE for xanthine oxidase are 110.18±8.29 μmol / L and 40.67±0.67 μmol / L respectively. When the concentration of HYIG is 200 μmol / L and the concentration of KAE is 20 μmol / L, that is, the molar ratio is 10:1, the synergistic inhibition rate of HYIG / KAE for xanthine oxidase is 80.00%±0.09%, and the synergistic uric acid reducing effect of the two is the best, and the CI value is 0.61.

[0022] (3) The present application provides a new type of xanthine oxidase inhibiting peptide and KAE composition nanoparticles, which show more excellent sustained release performance and stability compared with the composition without embedding. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The inhibition of xanthine oxidase by different molecular weight components after ultrafiltration.

[0024] Figure 2 LC-MS / MS total ion chromatogram of the active peptide.

[0025] Figure 3 Figure 6 is a mass spectrum analysis chart of HYIG.

[0026] Figure 4 Figure 7 is a docking chart of the interaction between HYIG and KAE.

[0027] Figure 5 Figure 8 is a curve of the inhibition rate of HYIG on xanthine oxidase.

[0028] Figure 6 Figure 9 is a curve of the inhibition rate of KAE on xanthine oxidase.

[0029] Figure 7 Figure 10 is an equivalent effect chart of HYIG and KAE used alone and in combination.

[0030] Figure 8 Figure 11 is a combination index chart of different concentrations of HYIG and KAE used in combination.

[0031] Figure 9 Figure 12 is a particle size distribution chart of OVA-HYIG / KAE NPs.

[0032] Figure 10 Figure 13 is a Zeta potential chart of OVA-HYIG / KAE NPs.

[0033] Figure 11 Figure 14 is an X-ray diffraction analysis chart of HYIG, KAE, OVA, OVA-HYIG / KAE NPs.

[0034] Figure 12 Figure 15 is a Fourier transform infrared spectroscopy analysis chart of HYIG, KAE, OVA, OVA-HYIG / KAE NPs.

[0035] Figure 13 Figure 16 is a curve of the inhibition rate of HYIG / KAE and OVA-HYIG / KAE NPs on xanthine oxidase in simulated gastrointestinal digestion. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the patent more clear and explicit, the following will further describe the patent in detail with specific examples. It should be understood that the specific examples described herein are only used to explain the patent, and are not used to limit the patent.

[0037] The xanthine oxidase inhibition activity determination method involved in the present application is as follows:

[0038] The principle of using the product of xanthine hydrolysis by xanthine oxidase, uric acid, which has a specific absorption peak at 290 nm, is used to evaluate the inhibitory effect of the sample to be tested on xanthine oxidase activity. Take an appropriate amount of reaction termination liquid to measure the OD value at 290 nm, and calculate the sample inhibition rate. Each sample is tested at least 3 times in parallel. The specific experimental steps are as follows:

[0039] As shown in Table 1, 50 μL of the sample to be tested (10 mg / mL) and 50 μL of 0.1 U / mL xanthine oxidase solution were added to a 96-well plate, and incubated at 37°C for 10 min. Then, 200 μL of 0.30 mmol / L xanthine solution was added to start the enzyme reaction, and the absorbance value of the reaction system was recorded at 290 nm after 10 min. The inhibitory activity of the sample on xanthine oxidase was calculated according to the formula.

[0040]

[0041] In the formula, A1 is the sample solution with enzyme; A2 is the sample solution without enzyme; A3 is the blank group with PBS buffer instead of the sample solution; and A4 is the blank group without enzyme.

[0042] Table 1 Xanthine oxidase inhibitory activity determination system

[0043]

[0044] The present application will be further explained and described by specific examples.

[0045] Example 1 Preparation of polypeptide HYIG

[0046] (1) Extraction of oil tea meal protein by alkali dissolution and acid precipitation method

[0047] The defatted oil tea meal was dissolved in distilled water at a ratio of 1:10 (w / v), stirred uniformly, and the pH was adjusted to 11 with 0.1 mol / L NaOH solution. The extraction was carried out at room temperature 25°C for 90 min, and the lower precipitate was removed by centrifugation at 6000 rpm for 20 min. The pH of the collected supernatant was adjusted to 3.5 with 0.1 mol / L hydrochloric acid solution, and the protein was precipitated by standing for 30 min. The precipitate was collected by centrifugation at 6000 rpm for 20 min. All the extracted proteins were vacuum frozen for 48 h to obtain dry powder of oil tea meal protein.

[0048] (2) Obtaining oil tea meal polypeptide by protease hydrolysis

[0049] The freeze-dried oil tea dregs protein was mixed with distilled water at a ratio of 1:10 (w / v), and then the mixture was incubated at 56°C and pH 6.9. Papain was added at a dosage of 2% of the substrate mass, i.e., 20 mg of the enzyme (enzyme activity: 1000 U / mg) per gram of the substrate. The reaction was performed for 4 h, and then the enzyme was inactivated by heating in a boiling water bath for 5 min. The mixture was centrifuged at 6000 rpm for 15 min to obtain a supernatant, and then the oil tea dregs polypeptide hydrolysate was obtained.

[0050] (3) Ultrafiltration

[0051] The oil tea dregs polypeptide hydrolysate was centrifuged at 6000 rpm for 15 min to obtain a supernatant, which was then filtered through a 0.22-μm microfiltration membrane. Then, the filtered solution was separated by a 3-KDa ultrafiltration membrane to obtain a residue and a filtrate. The filtrate was further separated by a 1-KDa ultrafiltration membrane to obtain a residue and a filtrate. Finally, oil tea dregs polypeptide solutions with different molecular weights (<1 KDa, 1-3 KDa, and >3 KDa) were obtained. The three polypeptide solutions with different molecular weights were vacuum freeze-dried and stored. The effects of 10 mg / mL of the three polypeptide components with different molecular weights on the inhibition of xanthine oxidase were determined.

[0052] The results are shown in Table 1. Figure 1 As can be seen from the results, the effects of the polypeptide components on the activity of xanthine oxidase are related to the molecular weight. The polypeptide components with different molecular weights all have inhibitory effects on the activity of xanthine oxidase, but the shorter peptides with lower molecular weights have stronger inhibitory effects on the activity of xanthine oxidase. The xanthine oxidase inhibition rate of the polypeptide component with a molecular weight of <1 KDa is 39.90% ± 1.31%, which is significantly higher than that of the polypeptide components with a molecular weight of 1-3 KDa and >3 KDa.

[0053] Therefore, the oil tea dregs polypeptide component with a molecular weight of <1 KDa was further separated and purified to screen out a peptide segment with a uric acid-lowering activity for subsequent experiments.

[0054] (4) LC-MS / MS identification

[0055] The oil tea dregs polypeptide component with a molecular weight of <1 KDa was subjected to chromatographic separation and mass spectrometric identification according to the relevant operations, and then the results were analyzed and identified by using the MaxQuant database to obtain the corresponding polypeptide sequences. A total ion current diagram of 413 polypeptide sequences obtained through liquid chromatography-mass spectrometry identification is shown in FIG. 2. Figure 2

[0056] (5) Bioinformatics analysis of non-toxic and non-allergenic active peptides

[0057] ​413 polypeptide sequences were obtained by LC-MS / MS sequencing. The peptides were analyzed for non-toxicity, hydrophobicity or hydrophilicity by ToxinPred and AllerTOP v.2.0 online websites. The ADME properties of the polypeptides were predicted by SwissAdme website. The results were mainly based on Human Intestinal Absorption (HIA), Blood-Brain Barrier Penetration (BBB) and toxicity. HIA is the polypeptide absorption index in human gastrointestinal tract, BBB is the index of whether the active peptide can penetrate the human blood-brain barrier; if the amphiphilicity is negative, it represents that the peptide segment is hydrophilic, and the smaller the value, the better the hydrophilicity. The peptide segment was compared with the amino acid sequence of the existing functional peptide in the BIOPEP-UWN database to ensure the novelty of the research peptide segment.

[0058] (6) Molecular docking simulation screening of inhibitory peptides

[0059] ① Construction of small molecule ligands

[0060] Draw the peptide structure and optimize it using the MM2 force field, and save the polypeptide molecule as a pdbqt format.

[0061] ② PyRx screening of peptides with xanthine oxidase inhibitory activity

[0062] PyRx is a virtual screening software for drug discovery, which integrates molecular docking tools such as AutoDock, can screen out compounds with high binding affinity in advance, and improve screening efficiency and accuracy. The peptide segments with high absolute score screened by PyRx are reserved for subsequent molecular docking.

[0063] ③ Preparation of xanthine oxidase receptor

[0064] The protein crystal structure of xanthine oxidase (PDB ID: 1N5X) was searched and downloaded from the PDB database (http: / / www.rcsb.org / pdb). 1N5X was pretreated using Pymol software, the B chain in 1N5X was deleted, the A chain was retained, then the water molecules were deleted, the polar hydrogen atoms were added, the gasteiger charge was calculated, and all atoms of 1N5X were assigned to AD4 type. The structure of XO contains a molybdenum atom, so the molybdenum atom parameters need to be manually added during docking. The processed receptor is saved as a pdbqt format for standby.

[0065] ④ Molecular docking method and evaluation

[0066] The ligand peptide and the receptor xanthine oxidase protein were docked by AutoDock Vina, and the PDBQT files of the xanthine oxidase protein and the ligand peptide were opened respectively. The specific parameters such as the binding site of the GridBox, the docking algorithm and the docking parameters were set according to the steps of molecular docking. The final docking results were obtained by running AutoDock. According to the binding energy of the docking results, the best binding conformation of the peptide and the xanthine oxidase was screened, and the 3D image of the best conformation was viewed by using PyMol software and the best conformation was further analyzed.

[0067] The results are as follows:

[0068] The computer molecular simulation docking showed that the peptide to be studied could be docked with the xanthine oxidase. The negative binding energy indicated that the target bioactive peptide could form a stable complex with the xanthine oxidase. The lowest binding energy was HYIG (4 peptide), the water solubility of the peptide HYIG was good, the HIA value indicated that the gastrointestinal absorption efficiency of HYIG was good, the BBB value indicated that the blood-brain barrier penetration rate was low, and HYIG was a non-toxic and non-allergenic peptide. The amino acid sequence composition of the polypeptide HYIG and the binding energy of the polypeptide HYIG and the xanthine oxidase are shown in Table 2, and the mass spectrum analysis chart of HYIG is shown in Figure 3 .

[0069] Table 2 Related properties of polypeptide HYIG

[0070]

[0071] The 3D image results of the best conformation by computer molecular simulation are shown in Figure 4 , and the polypeptide HYIG tends to bind to the flexible region near the coenzyme flavin adenine dinucleotide (FAD) active site cavity of XO. It is shown that the compound can interact with a specific region of XO, and the flexibility of the region is conducive to their binding. HYIG can bind to the amino acid residues GLN1088, TYR1254, ALA1252 and LYS1250 around the FAD active site through hydrogen bonds, and can also bind to ALA1255, LYS1250, TYR1254, TYR1091, CLN1088 and LYS1251 through hydrophobic interaction. Hydrogen bond is a strong non-covalent interaction, which can provide relatively stable binding; although the hydrophobic interaction is weak, it can also significantly enhance the binding stability under the joint action of multiple residues. This binding will change the molecular conformation of XO, hinder the entry of the substrate, and prevent the electron transfer in the FAD unit, ultimately reducing the catalytic activity of the enzyme. The docking results show that the binding energy of HYIG and the xanthine oxidase is the lowest, and the binding ability is the strongest, so HYIG is selected for artificial synthesis and further study.

[0072] Example 2 Determination of the xanthine oxidase inhibitory activity of polypeptide HYIG

[0073] (1) Nanjing Yuanpeide Biotechnology Co., Ltd. was commissioned to synthesize polypeptide HYIG according to the amino acid sequence of HYIG.

[0074] (2) The effect of xanthine oxidase inhibitory peptide HYIG at concentrations of 50, 100, 200, 400, and 800 μmol / L, respectively, on the activity of xanthine oxidase was determined according to the aforementioned method for determining the xanthine oxidase inhibitory activity.

[0075] The results are shown in Table 1. Figure 5 As can be seen from Table 1, the inhibitory effect of HYIG on xanthine oxidase increased with increasing concentration. The logarithm of the added concentration (X) of HYIG was taken, and a linear regression equation Y = 19.4542lnX - 27.7471 between the inhibition rate (Y) and the logarithm of the added concentration (X) of HYIG was established, with R 2 = 0.9923. The IC50value of HYIG for xanthine oxidase was calculated. The IC50value of HYIG for xanthine oxidase was 110.18 ± 8.29 μmol / L.

[0076] Example 3 Determination of the xanthine oxidase inhibitory activity of kaempferol

[0077] The effect of KAE at concentrations of 20, 30, 40, 50, and 60 μmol / L, respectively, on the activity of xanthine oxidase was determined according to the aforementioned method for determining the xanthine oxidase inhibitory activity. Before determination, KAE was dissolved in ethanol and then diluted in PBS buffer to control the ethanol content in the system to be below 0.3%.

[0078] The results are shown in Table 2. Figure 6 As can be seen from Table 2, the inhibitory effect of KAE on xanthine oxidase increased with increasing concentration. The logarithm of the added concentration (X) of KAE was taken, and a linear regression equation Y = 24.8442lnX - 11.9963 between the inhibition rate (Y) and the logarithm of the added concentration (X) of KAE was established, with R 2 = 0.9904. The IC50value of KAE for xanthine oxidase was calculated. The IC50value of KAE for xanthine oxidase was 40.67 ± 0.67 μmol / L.

[0079] Example 4 Determination of the xanthine oxidase inhibitory activity of polypeptide HYIG and KAE in combination

[0080] (1) HYIG and KAE were used in combination, the concentration of HYIG was 400, 200, 100, 50, 25 μmol / L, and the concentration of KAE was 40, 20, 10, 5, 2.5 μmol / L, respectively. HYIG and KAE were set in different concentration combinations, a total of 5 groups (see Table 3), and the effect of the combination of HYIG and KAE in different concentration combinations on xanthine oxidase activity was determined.

[0081] (2) The Chou-Talalay method was used to calculate the combination index (CI) to evaluate the interaction between HYIG and KAE. The CI value of the inhibitory effect of HYIG and KAE on xanthine oxidase was calculated by CompuSyn 2.0 software. CI < 1 indicates that HYIG and KAE have synergistic effects, CI = 1 indicates that HYIG and KAE have additive effects, and CI > 1 indicates that HYIG and KAE have antagonistic effects.

[0082] Figure 7 The isobologram is an equivalent line graph used to evaluate the interaction between the two. The results show that the dose combination points of polypeptide HYIG and KAE to achieve the above inhibition rate are all below the isobologram, indicating that the two have synergistic inhibition effects. Figure 8 Table 3 shows the CI values of different concentrations of HYIG and KAE in combination, and the results show that the CI values of different concentration combinations are all between 0.61 and 0.98, all having synergistic effects. When HYIG (200 μmol / L) is combined with KAE (20 μmol / L), the CI value is 0.61, which is significantly lower than that of other concentration combinations, confirming that the synergistic effect of the two is the strongest at this ratio.

[0083] Table 3 Inhibitory effect of HYIG and KAE in combination on xanthine oxidase

[0084]

[0085]

[0086] Example 5 Polypeptide / Kaempferol-ovalbumin nanoparticle

[0087] (1) Preparation of OVA-HYIG / KAE NPs

[0088] 0.3g of whey protein (OVA) was dissolved in 10mM PBS buffer and heated on a 90°C magnetic stirrer for 15 minutes to obtain a 3% (w / v) OVA solution. HYIG and 20μmol / L KAE were mixed to obtain a HYIG / KAE mixture at a final concentration of 200μmol / L. This mixture was then stirred with a 3% (w / v) OVA solution at a ratio of 1:2 (w / w) at room temperature for 2 hours to allow for full binding. After passing through a 0.22μm microfiltration membrane, the solution was centrifuged at 4500rpm for 15 minutes to remove free HYIG and KAE. The precipitate was then freeze-dried in a vacuum to obtain OVA-HYIG / KAE NPs, which were then refrigerated for later use.

[0089] (2) Determination of OVA-HYIG / KAE NPs encapsulation efficiency

[0090] Entrapment Efficiency (EE) generally refers to the percentage of the mass of active substance encapsulated in the composite particles to the added amount. In this study, EE was calculated using the following formula:

[0091]

[0092] Wherein, m1 is the mass of added HYIG or KAE, mg; m2 is the amount of unencapsulated HYIG or KAE added, mg.

[0093] The encapsulation rates of HYIG and KAE in OVA-HYIG / KAE NPs were 89.30%±0.15% and 82.60%±0.09%, respectively.

[0094] (3) Determination of OVA-HYIG / KAE NPs Particle Size, Particle Size Distribution, and Zeta Potential

[0095] The sample was reconstituted and uniformly dispersed to avoid agglomeration. Particle size and polydispersity index (PDI) were determined using dynamic light scattering. Zeta potential was measured using a zeta potential analyzer, and the zeta potential values ​​and distribution were analyzed to assess sample stability.

[0096] like Figure 9 As shown in the figure, the particle size of OVA-HYIG / KAE NPs after rehydration was 253.8±3.5nm and the PDI was 0.421. The smaller PDI value indicates that the particle size distribution is more uniform and the particle size is moderate. Figure 10 The potential of OVA-HYIG / KAE NPs is -28.21±1.68. OVA, KAE and HYIG are all negatively charged, and the surface of OVA-HYIG / KAE NPs is also negatively charged. These charges ensure the stability of the system.

[0097] (4) X-ray diffraction analysis of HYIG / KAE-OVA NPs

[0098] The crystal structures of HYIG, KAE, OVA and OVA-HYIG / KAE NPs were analyzed by X-ray diffraction method. The current and voltage were 40 mA and 40 kV, respectively, the 2θ angle was set to 1° to 70°, and the scanning speed was 2° / min.

[0099] Figure 11 The KAE showed multiple sharp diffraction peaks, corresponding to specific crystal planes of its crystal structure, such as 2θ ≈ 15°, 25°, 30°, etc. The sharp peaks indicated that KAE existed in a highly ordered crystalline state. The OVA spectrum showed a broad amorphous "steamed bun peak", usually concentrated near 2θ ≈ 20°, without sharp peaks, indicating that OVA itself was in an amorphous state. Compared with pure KAE, there were no such sharp peaks in the XRD patterns of OVA and OVA-HYIG / KAE NPs. This indicated that in OVA-HYIG / KAE NPs, KAE did not form a crystal structure, but existed in an amorphous or non-crystalline state. After embedding into the nanoparticles, the crystal structure of KAE was completely destroyed and converted into an amorphous state. This transformation was due to the physical constraints of the OVA matrix and intermolecular interactions such as hydrogen bonding and hydrophobic interaction.

[0100] (5) Fourier transform infrared spectroscopy analysis of OVA-HYIG / KAE NPs

[0101] The structures of HYIG, KAE, OVA and OVA-HYIG / KAE NPs were analyzed and identified by Nicolet iS50 Fourier infrared spectrometer.

[0102] As shown in Figure 12 , the absorption peaks of OVA and HYIG in OVA-HYIG / KAE NPs at 3306.25 cm -1 and 3307.58 cm -1 , related to the C-H bond of alkyne and the O-H bond of hydroxyl, shifted to 3301.5 cm -1 , i.e. the absorption peaks moved to lower wave numbers, which may be related to the formation of hydrogen bonds, causing changes in the position of the absorption peaks. The main absorption band of OVA at 1658 cm -1 , related to the stretching vibration of C=O bond, changed slightly to 1657 cm -1 , 1236 cm -1 , related to the stretching vibration of N-H bond and the bending vibration of C-N bond, to 1238 cm -1 , and 1536 cm-1 The absorption wavelength of amide II (stretching vibration of N-H bond and bending vibration of C-N bond) did not change, and the shift of amide band could indicate the change of protein secondary structure, such as the proportion change of structures such as α-helix, β-sheet and random coil, and the secondary structure of protein changed after KAE and HYIG combined with OVA. In OVA-HYIG / KAE NPs, the characteristic peak of KAE at 1087 cm -1 appeared, which indicated that KAE also changed the protein structure by reacting with the C-N, N-H and COO- groups of the protein, and the successful embedding of KAE further proved the formation of complex particles.

[0103] (6) In vitro simulation of OVA-HYIG / KAE NPs digestion

[0104] The prepared OVA-HYIG / KAE NPs were subjected to in vitro simulation of digestion, and a non-covalent complex in which KAE and HYIG were combined together in a non-covalent form was used as a control to explore the protective effect of OVA embedding on active substances.

[0105] Simulation of gastric phase: 0.32% (w / v) pepsin and 0.20% NaCl were dissolved in ultrapure water, and the pH value of the solution was adjusted to 2.5 to prepare simulated gastric juice. OVA-HYIG / KAE NPs were dissolved in distilled water at 1:10 (w / v) and 10 mL of the mixture was mixed with 10 mL of simulated gastric juice, and then the mixture was placed in a 37°C water bath and incubated with shaking at 100 rpm for 2 h.

[0106] Simulation of intestinal phase: simulated intestinal fluid containing 150 mM NaCl and 30 mM CaCl2 was prepared, and 20 mL was prepared for standby. 20 mL of the sample digested in the simulated gastric phase was taken, the pH value was adjusted to 7 with 10 mM phosphate buffer, mixed with the simulated intestinal fluid, and then 200 mg of bile salt extract and 64 mg of trypsin were added, and the pH was continuously adjusted to 7, and placed in a 37°C water bath and continuously shaken for 4 h.

[0107] The samples at different digestion stages were centrifuged in 1 KDa ultrafiltration centrifuge tubes at 4500 rpm for 15 min, and the filtrate was used to determine the xanthine oxidase inhibition activity. The results of simulated gastrointestinal digestion are shown in Figure 13As shown, the inhibition rate of HYIG / KAE complex decreased from 70.63% ± 0.59% in two hours of gastric juice digestion to about 55.15% ± 0.48%, which indicated that the polypeptide was significantly degraded under the action of gastric acid and pepsin, leading to the loss of activity and the decrease of enzyme activity inhibition ability. KAE was relatively stable under acidic conditions, but the solubility was low, and the inhibition activity was only partially retained. The inhibition rate of OVA-HYIG / KAE NPs on xanthine oxidase increased from 66.19% ± 0.71% to 77.71% ± 0.67% in two hours of gastric juice digestion. This indicated that OVA effectively protected the active substance from pepsin attack, and HYIG and KAE were slowly released from OVA-HYIG / KAE NPs, gradually enhancing the inhibition of the enzyme.

[0108] In the intestinal fluid 2-6h stage, the inhibition rate of HYIG / KAE complex further decreased to 40.73% ± 0.57%, and the inhibition rate of OVA-HYIG / KAE NPs decreased to 52.98% ± 0.62%, but the decrease was much smaller than that of HYIG / KAE complex, which was attributed to the gradual degradation of OVA by pancreatic enzymes in intestinal fluid, the slow release of active substances, and the slow degradation rate of HYIG due to the slow release of pancreatic enzymes. The enzyme inhibition rate of OVA-HYIG / KAE NPs increased to 54.93% ± 0.65% at 6h, which may be due to the increase in solubility and inhibition activity of KAE in intestinal fluid, partially offsetting the loss of HYIG degradation. These results show that the OVA-based nanodelivery system has excellent slow-release performance and stability in simulated gastrointestinal fluid, and is suitable for the delivery of hydrophilic and hydrophobic active substances.

[0109] The above-described embodiments only express several embodiments of the present application, which are described in more detail and in detail, but should not be understood as limiting the scope of the patent. It should be noted that for those skilled in the art, the above embodiments can be made in several ways, combinations and improvements without departing from the concept of the present patent, which are within the scope of protection of the present patent. Therefore, the scope of protection of the present patent should be subject to the claims.

Claims

1. A xanthine oxidase inhibitory peptide, characterized in that The inhibitory peptide is HYIG, and the amino acid sequence is: His-Tyr-Ile-Gly.

2. Use of the inhibitory peptide HYIG according to claim 1 in inhibiting xanthine oxidase activity, or in preparing a product for inhibiting xanthine oxidase activity, or in preparing a product for lowering uric acid.

3. A composition comprising the xanthine oxidase inhibitory peptide HYIG, characterized in that: The composition uses inhibitory peptide HYIG and kaempferol KAE as active ingredients. The composition contains inhibitory peptide HYIG and KAE, and the mass ratio of the inhibitory peptide HYIG to KAE in the composition is 1-25:

1.

4. The method for preparing ovalbumin nanoparticles of the HYIG / KAE composition according to claim 3, characterized in that: The procedure is as follows: 0.3 g of ovalbumin (OVA) was dissolved in 10 mM PBS buffer and heated at 90°C on a magnetic stirrer for 15 minutes to obtain a 3% (w / v) OVA solution. After cooling to room temperature, HYIG / KAE was added and stirred at room temperature for 2 hours to allow for complete binding. After filtering through a 0.22 μm microfiltration membrane, the solution was centrifuged at 4500 rpm for 15 minutes, and the precipitate was freeze-dried in vacuo to obtain OVA-HYIG / KAE NPs.

5. Use of the OVA-HYIG / KAE NPs according to claim 3 in inhibiting xanthine oxidase activity, or in preparing a product for inhibiting xanthine oxidase activity, or in preparing a product for lowering uric acid.

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