South sea decapterus kurroides uric acid-lowering and antioxidant polypeptide and application thereof

Abstract

The application discloses a South China Sea Decapterus maruadsi polypeptide with uric acid reducing and antioxidant activities and an application thereof, and relates to the technical field of biology. The polypeptide with high uric acid reducing activity and antioxidant activity is screened through activity prediction and molecular docking, and the amino acid sequence is FYDWWK. The polypeptide can be stably combined with the active center of xanthine oxidase, forms multiple hydrogen bonds and hydrophobic interaction, significantly inhibits the generation of uric acid, and has an in-vitro xanthine oxidase inhibition IC 50 of 3.068±0.2547 mmol / L. Meanwhile, the polypeptide has excellent DPPH free radical scavenging capacity, and an IC 50 of 5.033±0.5476 mmol / L. The polypeptide has good stability, still has high efficient inhibitory activity after simulated gastrointestinal digestion, and has oral development potential. The polypeptide is a natural marine small molecule active peptide, is safe and easy to absorb, and can be applied to the preparation of products related to uric acid reducing and / or antioxidant.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a uric acid-lowering and antioxidant polypeptide from the South China Sea finless chub and its applications. Background Technology

[0002] Hyperuricemia (HUA) has many complications, with gout being the most common. Excess uric acid tends to precipitate in the joints, triggering gout. Uric acid is the final product of purine metabolism, and its production requires the participation of two key enzymes: adenosine deaminase and xanthine oxidase (XO). Inhibiting the activity of these two key enzymes can limit uric acid production. During normal physiological metabolism, cells generate energy through pathways such as the mitochondrial respiratory chain, inevitably producing reactive oxygen species (ROS). However, when ROS production exceeds the body's clearance capacity, oxidative stress occurs. In aging or pathological states, the endogenous system's capacity is weakened, thus requiring the supplementation of exogenous antioxidants. Many natural products have been proven to have uric acid-lowering and antioxidant activities. Bioactive peptides have attracted much attention due to their low preparation cost, low sample cost, easy absorption, and high safety. Furthermore, enzymatic hydrolysis for preparing bioactive peptides offers advantages such as mild conditions, controllable safety, and low cost. This method can be used to develop natural, safe, and highly effective uric acid-lowering and antioxidant agents.

[0003] The macarellus (Decapterus macarellus), also known as the red-tailed macarp, is a species of fish belonging to the genus Decapterus in the family Carangidae. It is widely distributed in warm waters worldwide, inhabiting depths of 40-200 meters. Currently, the resource development and utilization of the macarellus is relatively limited, mainly focusing on fresh consumption, salting, and canning, with limited development in advanced processing. Recent studies have found that marine fish protein peptides possess numerous functions such as anti-fatigue, liver protection, and uric acid reduction; however, research on macarellus protein peptides has not yet received widespread attention. Summary of the Invention

[0004] The purpose of this invention is to provide a uric acid-lowering and antioxidant polypeptide from the South China Sea finless chub, to address the problems existing in the prior art. This polypeptide not only has high uric acid-lowering activity but also significant antioxidant activity, and can be used to prepare products related to uric acid lowering and / or antioxidant activity.

[0005] To achieve the above objectives, the present invention provides the following solution: The present invention also provides a uric acid-lowering and antioxidant polypeptide from the South China Sea finless chub, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides a method for preparing the above-mentioned uric acid-lowering and antioxidant peptides of the South China Sea fine-scaled trevally, including the step of preparing the uric acid-lowering and antioxidant peptides of the South China Sea fine-scaled trevally using a solid-phase synthesis method.

[0007] The present invention also provides the application of the above-mentioned uric acid-lowering and antioxidant polypeptides of the South China Sea fine-scaled trevally in the preparation of drugs with uric acid-lowering and / or antioxidant effects.

[0008] The present invention also provides the application of the above-mentioned uric acid-lowering and antioxidant peptides of the South China Sea fine-scaled round trevally in the preparation of health products that help with anti-oxidation.

[0009] The present invention also provides the application of the above-mentioned uric acid-lowering and antioxidant polypeptides of the South China Sea fine-scaled trevally in the preparation of xanthine oxidase inhibitors.

[0010] The present invention also provides a drug with uric acid-lowering and / or antioxidant effects, the active ingredients of which include the above-mentioned uric acid-lowering and antioxidant polypeptides of the South China Sea fine-scaled round trevally.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] The present invention also provides a health product that helps with anti-oxidation, the active ingredients of which include the above-mentioned South China Sea fine-scaled round trevally that lowers uric acid and antioxidant peptides.

[0013] Furthermore, the health product also includes excipients for its preparation.

[0014] The present invention also provides an inhibitor of xanthine oxidase, characterized in that the active ingredients include the above-mentioned uric acid-lowering and antioxidant polypeptides from the South China Sea fine-scaled chub.

[0015] The present invention discloses the following technical effects: This invention screens a polypeptide with high uric acid-lowering and antioxidant activities through activity prediction and molecular docking. The amino acid sequence is FYDWWK. This polypeptide can stably bind to the active site of xanthine oxidase, forming multiple hydrogen bonds and hydrophobic interactions, significantly inhibiting uric acid production. In vitro, xanthine oxidase inhibits IC50 by [missing value]. 50 It reached 3.068±0.2547 mmol / L, and also exhibited excellent DPPH free radical scavenging ability, IC50... 50 The concentration was 5.033 ± 0.5476 mmol / L. This peptide exhibits good stability at pH 3-10, 4-100 °C, and under common metal ion environments. It retains high inhibitory activity even after simulated gastrointestinal digestion, demonstrating potential for oral development. This peptide is a small-molecule bioactive peptide derived from natural marine sources, is safe and easily absorbed, and can be used to prepare products related to lowering uric acid and / or antioxidation. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A statistical graph showing the XO enzyme inhibition rate and DPPH activity inhibition rate of the hydrolysates of five proteases; Figure 2 Statistical graphs of XO enzyme inhibition rate and DPPH activity inhibition rate of enzymatic hydrolysate prepared under different conditions; where (A)-(C) are statistical graphs of XO enzyme inhibition rate and DPPH activity inhibition rate of enzymatic hydrolysate prepared under different enzymatic hydrolysis time, enzymatic hydrolysis temperature and acidic protease activity conditions, respectively. Figure 3 The response surface plots show the effects of interactions among various factors on the enzymatic hydrolysis extraction process of *Siniperca scutellarioides*. A and B are the surface plots and contour plots for hydrolysis time and temperature, respectively; C and D are the surface plots and contour plots for hydrolysis temperature and enzyme activity, respectively; and E and F are the surface plots and contour plots for hydrolysis time and enzyme activity, respectively. Figure 4 Chromatogram of molecular weight distribution of enzymatic hydrolysate of scaly trevally; Figure 5 A statistical graph showing the inhibition rates of XO enzyme and DPPH activity of different components after dextran gel column chromatography separation; Figure 6 The optimal conformation (3D) and interaction (2D) diagrams of FYDWWK and XO enzyme molecules after docking are shown in Figure 3 (A). Figure 7 IC50 of the XO enzyme inhibitory activity of FYDWWK peptide 50 Value measurement results graph; Figure 8 IC50 for DPPH inhibitory activity of FYDWWK peptide 50 Value measurement results graph; Figure 9 The figure shows the results of measuring the effects of different pH values ​​(A), temperatures (B), metal ions (C), and simulated gastrointestinal digestion (D) on the stability of FYDWWK peptides. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The method for detecting XO enzyme inhibition rate and DPPH activity inhibition rate involved in this invention is as follows: XO enzyme inhibition rate: Prepare a chromogenic solution by diluting 0.2061 g of 4-aminoantipyrine, 0.018 g of horseradish peroxidase, and 0.5594 g of phenol to 1 L with Tris-HCl buffer. Add 50 μL of xanthine oxidase (0.52 U / mL) solution and 50 μL of the sample to each well of a 96-well plate, shake for 30 s, and incubate at 37°C for 10 min. After incubation, add 100 μL of xanthine solution (2.0 mmol / L) to 100 μL of the chromogenic solution and label this mixture A1. Replace the xanthine solution with buffer and label this A0. Replace the sample with buffer and label this A2. Then, incubate the 96-well plate at 37°C for 15 min. Terminate the reaction by adding 30 μL of NaOH (1.0 mol / L) and measure the absorbance at 508 nm. The XO enzyme inhibition rate is calculated using the following formula: Inhibition rate (%) = [A2 - (A1 - A0)] / A2 × 100%.

[0024] DPPH activity inhibition rate: A DPPH solution with a concentration of 0.2 mg / mL was prepared using anhydrous ethanol. 0.5 mL of the test solution was reacted with the DPPH solution in the dark for 30 min, centrifuged at 8000 r / min for 5 min, and 200 μL of the solution was transferred to a 96-well plate. The absorbance A1 of the solution was measured at 517 nm. Simultaneously, the absorbance A2 of a mixture of 1 mL of the test solution and 2 mL of anhydrous ethanol solution, and the absorbance A3 of a mixture of 1 mL of distilled water and 2 mL of DPPH solution were measured. The DPPH activity inhibition rate was calculated using the following formula: Inhibition rate (%) = 1 - (A1 - A2) / A3 × 100%.

[0025] Example 1: Optimization of the enzymatic hydrolysis process for fine-scaled round trevally 1. Basic enzymatic hydrolysis process for fine-scaled round trevally: (1) Pre-treatment of fine-scaled round mackerel meat After mincing the fish meat, it was mixed with pure water at a ratio of 1:4 (w / v), stirred at 40℃ for 20 minutes, and then centrifuged at 8000 rpm for 10 minutes to separate the upper layer of oil. This process was repeated twice to obtain defatted fish paste, which was then mixed with water to obtain a fish paste solution. (2) Preparation of crude enzymatic hydrolysis product Adjust the pH of the fish paste solution, add enzymes, and perform enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzymes to obtain the enzymatic hydrolysate. After cooling the enzymatic hydrolysate, centrifuge, collect the supernatant, purify, filter, and freeze-dry.

[0026] 2. Single-factor experiment Based on the basic enzymatic hydrolysis process of *Sinibotia scaberne*, the effects of enzyme type, hydrolysis time, hydrolysis temperature, and enzyme activity on the hydrolysis products of *Sinibotia scaberne* were investigated sequentially. The hydrolysis effects of each protease were compared using XO enzyme inhibition rate and DPPH activity inhibition rate as indicators.

[0027] 2.1 Optimal enzyme screening The proteases listed in Table 1 were selected for enzymatic hydrolysis. The enzyme dosage (6000 U / g), time (4 h), and the ratio of defatted fish paste to water (1:3) were fixed. Enzymatic hydrolysis was carried out under their respective optimal pH and temperature conditions. After 4 h of hydrolysis, the enzymes were inactivated at 100℃ for 15 min, cooled to room temperature, and centrifuged at 10200 r / min, 4℃ for 10 min. The supernatant was collected, and the enzymatic hydrolysis effects of each protease were compared using XO enzyme inhibition rate and DPPH activity inhibition rate as indicators.

[0028] Table 1. Optimal enzymatic hydrolysis conditions for different proteases 2.2 Single-factor experimental design The effects of enzymatic hydrolysis temperature (°C), hydrolysis time (h), and enzyme dosage (U / mL) on the enzymatic hydrolysis efficiency of *Sinibrachium scutellarioides* were investigated. Using XO activity inhibition rate as the main indicator and DPPH activity inhibition rate as a reference, single-factor experiments were conducted with fixed hydrolysis times of 4 h, hydrolysis temperatures of 35°C, and enzyme dosages of 8000 U / g. Single-factor experiments were conducted to explore the effects of different temperatures (25, 30, 35, 40, 45 °C), different enzyme dosages (5000, 6000, 7000, 8000, 9000, 10000, 12000 U / mL), and different hydrolysis times (1, 2, 3, 4, 5, 6, 7, 8 h) on the enzymatic hydrolysis process of *Sinibrachium scutellarioides* flesh.

[0029] 3. Optimization experiment using response surface methodology Based on the results of single-factor experiments, the independent variable levels of enzyme activity (A), hydrolysis time (B), and hydrolysis temperature (C) were determined. XO activity inhibition rate (Y1) and DPPH activity inhibition rate (Y2) were used as response values, and a Box-Behnken model was employed for response surface optimization. The two response values ​​were assigned equal weights to predict the optimal preparation conditions for the enzymatic hydrolysate of *Siniperca scutellarioides*. The design of the response surface factor levels is shown in Table 2.

[0030] Y = Y1 × 0.5 + Y2 × 0.5.

[0031] Table 2 Response Surface Experiment Factor Level Design Table 4. Experimental Results 4.1 Results of Single-Factor Experiment 4.1.1 Effect of protease on the enzymatic hydrolysis effect of wrasse. Depend on Figure 1 It was found that after degradation reactions with different enzymes under the same conditions, the combined ranking of XO activity inhibition rate and degree of hydrolysis in the *Siniperca scutellarioides* reaction system was: acidic protease > alkaline protease > neutral protease > papain > flavor protease. Among them, the acidic protease hydrolysis product showed the highest XO activity inhibition rate (60.06%) and the highest DPPH activity inhibition rate (61.98%). This indicates the best degradation effect. Therefore, acidic protease was selected as the protease for modifying *Siniperca scutellarioides*.

[0032] 4.1.2 Effect of enzymatic hydrolysis time on the enzymatic hydrolysis effect of *Siniperca scutellarioides* like Figure 2 As shown in Figure A, the combined inhibition rates of XO and DPPH activities in the *Siniperca scutellarioides* reaction system are ranked as follows: 3 h > 4 h > 2 h > 1 h > 5 h > 6 h > 8 h > 7 h. The maximum value is reached at a hydrolysis time of 3 h. Therefore, 3 h is selected as the optimal hydrolysis time.

[0033] 4.1.3 Effect of enzymatic hydrolysis temperature on the enzymatic hydrolysis effect of *Siniperca scutellarioides* Enzyme activity has an optimal temperature range; below or above this temperature, enzyme activity will be affected. For example... Figure 2 As shown in Figure B, the combined inhibition rates of XO and DPPH activities in the *Siniperca scutellarioides* reaction system are ranked as follows: 35℃ > 25℃ > 30℃ > 40℃ > 45℃. Therefore, 35℃ is selected as the optimal enzymatic hydrolysis temperature.

[0034] 4.1.4 Effect of enzyme activity on the enzymatic hydrolysis effect of *Siniperca scutellarioides* Depend on Figure 2 As shown in Figure C, within a certain range, the inhibition rates of XO and DPPH activities are positively correlated with enzyme activity, reaching their maximum at an enzyme activity of 8000 U / mL. Therefore, 8000 U / mL is selected as the optimal enzyme activity value.

[0035] 4.2 Optimization Experiment Results Using Response Surface Method 4.2.1 Optimization Experiment Results Using Response Surface Method Based on the single-factor experiments, the results of the optimization of the enzymatic hydrolysis process of *Siniperca scutellarioides* using response surface methodology are shown in Tables 3 and 4. Table 3 presents the response surface methodology experiments and results; Table 4 presents the regression equations and analysis of variance established for the optimized enzymatic hydrolysis process of *Siniperca scutellarioides*.

[0036] Table 3 Response Surface Design Experiments and Results Table 4. Analysis of variance of the regression equation established for the optimized enzymatic hydrolysis process of *Siniperca scutellarioides*. Note: P<0.05 indicates a significant difference; P<0.01 indicates an extremely significant difference; P<0.001 indicates a highly significant difference.

[0037] By fitting a binomial equation using multiple linear regression, the equation is obtained as follows: Y = -464.307 + 0.0428027 × A + 28.0059 × B + 16.6118 × C + -0.0015875 × AB + 0.000278 × AC + 0.43125 × BC + -2.84837e⁻⁶ × A 2 +-4.62212×B 2 +-0.283485×C 2As shown in Table 4, the p-value of the model is less than 0.05, indicating that the experimental factors have a significant impact on the experimental results, and the model has good analytical and predictive significance. The p-value of the lack-of-fit term is 0.5997, which is greater than 0.05, indicating that its impact on the experimental results is not significant, suggesting that the equation fits well and can be used to predict experimental results. The results of the analysis of variance for each factor show that for the linear and quadratic terms of the equation: each factor and its quadratic term have a significant impact on the weighted value ( P The F-value <0.05 indicates that the relationship between the factors and the weighted value is not a simple linear one. The F-value shows that the order of influence of the three factors on the weighted value is: hydrolysis time > enzyme activity > hydrolysis temperature. The optimal conditions are a hydrolysis time of 3.058 h, a hydrolysis temperature of 35.95℃, and an enzyme activity of 7995.06 U / mL; under these conditions, the expected value of the weighted value is 59.29%.

[0038] 4.2.2 Response Surface Analysis The results of the interaction of the average weighted values ​​of various factors on the inhibition rate of XO and the inhibition rate of DPPH activity in the enzymatic hydrolysate of *Siniperca scutellarioides* are shown in the figure. Figure 3 Surface plots of enzymatic hydrolysis temperature and enzyme activity ( Figure 3 (B) The steep slope indicates that the interaction between enzymatic hydrolysis temperature and enzyme activity has a strong influence on the average weighted value of the XO inhibition rate and DPPH activity inhibition rate of the *Sinibrachium scutum* hydrolysate. Based on the response surface methodology to optimize the enzymatic hydrolysis process of *Sinibrachium scutum*, and considering ease of implementation, the optimized conditions were modified to an enzymatic hydrolysis time of 3 h, an enzymatic hydrolysis temperature of 35 °C, and an enzyme activity of 8000 U / mL. The experiment was repeated three times, and the average weighted value of the XO inhibition rate and DPPH activity inhibition rate was 58.43%, with a relative error of less than 5% compared to the predicted value of 59.29%, indicating good agreement. Therefore, this method can effectively optimize the enzymatic hydrolysis conditions of *Sinibrachium scutum* and has good reliability.

[0039] Example 2: Molecular weight distribution analysis of enzymatic hydrolysis products of *Siniperca scutellarioides* 1. Preparation of enzymatic hydrolysates of *Siniperca scutellarioides* The enzymatic hydrolysis product of *Siniperca scutellarioides* was prepared according to the optimal process modified in Example 1.

[0040] 2. Molecular weight distribution analysis of enzymatic hydrolysis products of *Siniperca scutellarioides* Referring to the high-performance gel filtration chromatography method in "Marine Fish Oligopeptide Powder" (GB / T 22729-2008), the enzymatic hydrolysate sample with a mass concentration of 10 mg / mL was passed through a membrane, and the molecular weight of the enzymatic hydrolysate was determined.

[0041] All experimental data are the average of three replicates. Design-Expert V8.0.6.1, GraphPad Prism, and Excel were used for data analysis.

[0042] 3. Experimental Results Depend on Figure 4 Table 5 shows that the molecular weight of protein peptides in the enzymatic hydrolysate of *Siniperca scaberne* is mainly below 1 kDa, accounting for 92.09%; peptides with a molecular weight greater than 3 kDa account for only 0.94% after enzymatic hydrolysis. It is speculated that peptides with a molecular weight less than 1 kDa are the main components in the enzymatic hydrolysate that exert XO inhibitory activity.

[0043] Table 5 Molecular weight distribution of enzymatic hydrolysates of scaly trevally Example 3: Isolation, purification, and screening of high-activity peptides from enzymatic hydrolysis products of *Siniperca scutellarioides* 1. Isolation and purification of enzymatic hydrolysis products of *Siniperca scutellarioides* The enzymatic hydrolysis product of *Sinibotia scutellarioides*, prepared according to the optimized process modified in Example 1, was diluted with ultrapure water to a concentration of 10 mg / mL for loading. After sterilization via a 0.22 μm sterile filter membrane, the solution was loaded onto an ultrafiltration membrane. The filtrate with a molecular weight less than 1 kDa was collected and freeze-dried to obtain a second lyophilized powder of the enzymatic hydrolysis product. This second lyophilized powder was then subjected to dextran gel column chromatography using a Sephadex G-15 dextran gel column. The specific conditions were: loading volume 3 mL, loading concentration 50 mg / mL, detection wavelength 220 nm, and flow rate 0.5 mL / min. The fractions obtained from the dextran gel column chromatography were collected and named F1-F4.

[0044] like Figure 5 As shown, comparing the XO inhibitory and antioxidant activities of different fractions, it can be seen that the inhibitory activity of F2 is far superior to that of other fractions. Therefore, F2 was selected for subsequent peptide identification.

[0045] 2. Screening and identification of fine-scaled scad protein peptides with high XO inhibition and antioxidant activity based on molecular docking. The high XO inhibitory activity fraction F2 was determined by LC-MS / MS. The analytical conditions for LC-MS / MS were as follows: UPLC-QTOF high-resolution LC-MS / MS was used for sample separation and detection. The mobile phase consisted of 0.1% (v / v) formic acid aqueous solution (A) and acetonitrile (B). The elution method was as follows: 0-4.00 min 5.0% B, 4.00-6.00 min 5.0-10.0% B, 6.00-30.00 min 10.0-40.0% B, 30.00-34.00 min 40.0-90.0% B, 34.00-40.00 min 90% B, 40.00-42.00 min 90.0-5.0% B, 42.00-55.00 min 5.0% B, flow rate 0.2%. The mass spectrometry parameters were: mL / min, injection volume 2 μL, column temperature 40 ℃; mass spectrometry detection method: positive ion mode, acquisition strategy Auto MS / MS, acquisition frequency 5 Hz, mass-to-charge ratio acquisition range 50-1500 m / z, TopN set to 6, and dynamic exclusion of precursor ions enabled.

[0046] Due to the large size of the F2 peptide sample, the toxicity of key sequences was first assessed using toxinpred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) and the Peptide Property Calculator (https: / / www.innovagen.com / services / ). Dangerous sequences were screened out, resulting in peptides with a score greater than 0.8 and a hydrophobic amino acid at the N-terminus. The antioxidant properties of the peptides were then predicted online using AnOxPePred (https: / / services.healthtech.dtu.dk / services / AnOxPePred-1.0 / ). A total of 27 peptide sequences were selected.

[0047] Molecular docking was performed using AutoDock Vina, and the docking results were visualized using Discovery Studio 2019 Client (DS2019). This allowed for the selection of the optimal sequence, FYDWWK (SEQ ID NO.1). Molecular docking results ( Figure 6This indicates that FYDWWK can bind well to the hydrophobic channels on the XO surface, preventing substrates from entering the active site of XO and thus effectively exerting XO inhibitory activity. The *Siniperca scaly scops* protein peptide forms five conventional hydrogen bonds with the xanthine oxidases Ser928, Asp984, Val932, Arg1279, and Gly935, and one C-H bond with Asn988; in addition, it forms a cation-π interaction with Lys981 and an anion-π interaction with Asp1276; the xanthine oxidases Lys989, Ile998, Cys999, Leu1290, Ala1283, Glu939, Glu936, and Gln1284 bind to the *Siniperca scaly scops* protein peptide through hydrophobic interactions. Based on these results, we believe that hydrogen bonds and hydrophobic interactions are crucial for the binding of the active peptide to XO.

[0048] Example 4: Synthesis and Activity Verification of FYDWWK Peptide Nanjing Jietai Biotechnology Co., Ltd. was commissioned to synthesize the FYDWWK polypeptide using a solid-phase synthesis method.

[0049] The inhibitory activities of XO enzyme and DPPH were detected using different concentrations of FYDWWK peptide.

[0050] like Figures 7-8 As shown, the IC50 of the FYDWWK polypeptide against XO enzymes... 50 The IC50 for DPPH was 3.068 ± 0.2547 mmol / L. 50 The concentration was 5.033 ± 0.5476 mmol / L. In this experiment, the IC50 concentration was... 50 This refers to the sample concentration at which the inhibition rate reaches 50%.

[0051] Example 5: Stability determination of FYDWWK peptide 1. Effects of different pH values, temperatures, and metal ion environments on the stability of enzymatic hydrolysis products. FYDWWK lyophilized peptide powder was prepared into a solution with a mass concentration of 1 mg / mL to investigate its efficacy under different pH values ​​(3, 4, 5, 6, 7, 8, 9, 10), different temperatures (4, 30, 40, 50, 60, 70, 100 ℃), and different metal ion environments (K). + Na + Mg 2+ Ca 2+ Fe 2+ NH4 + Zn 2+ The inhibitory effect of the concentration of XO enzyme (all at 10 mmol / mL) on XO enzyme.

[0052] 2. Effect of gastrointestinal simulated digestion on the XO inhibitory activity of FYDWWK peptide. 2.1 Preparation of gastrointestinal fluid Preparation of artificial gastric fluid: Take 0.2g NaCl, add 70mL of ultrapure water and 730μL of hydrochloric acid, adjust the pH to 1.2 with hydrochloric acid, and make up to 100mL. Prepare fresh before use.

[0053] Preparation of artificial intestinal fluid: Weigh 0.7 g of potassium dihydrogen phosphate and dissolve it in 25 mL of ultrapure water and shake. Add 19 mL of NaOH (0.2 mol / L) and 40 mL of ultrapure water. Adjust the pH to 7.5 with NaOH and bring the volume to 100 mL. Prepare and use immediately.

[0054] 2.2 Stability determination Take 40 mL of gastric juice and prepare a sample solution of FYDWWK peptide with a mass concentration of 1 mg / mL using simulated gastric juice. Add the sample and pepsin (mass ratio 50:1), and digest in a 37℃ water bath. Take 5 mL of digestion solution every 20 min and inactivate the enzyme in boiling water. Then store at 4℃ for a total of 1 h of digestion. Then adjust the pH of the remaining 25 mL of digestion solution to 6.5 with 1 mol / L sodium bicarbonate. Subsequently, add the pH 6.5 gastric juice to 25 mL of simulated intestinal juice to simulate duodenal digestion. Add trypsin (mass ratio 25:1) and digest at 37℃. Take 10 mL of digestion solution every 30 min and inactivate the enzyme in boiling water. Digest for 2 h. Then measure the XO inhibition rate of the protein peptide after digestion with gastric juice and intestinal juice to determine the effect of simulated digestion on peptide.

[0055] All experimental data are the average of three replicates. Design-Expert V8.0.6.1, GraphPad Prism, and Excel were used for data analysis.

[0056] 3. Experimental Results 3.1 Effects of different pH values, temperatures, and metal ions on the stability of FYDWWK peptides Depend on Figure 9 As shown in AC, the FYDWWK polypeptide exhibits good stability to acids, alkalis, and heat. Different metal ions have varying effects on the stability of the FYDWWK polypeptide, with Ca... 2+ Mg 2+ K + Na + Fe 2+ The ions had no significant effect on the stability of the FYDWWK peptide, and the addition of Zn... 2+ NH4 + It can significantly increase the XO inhibitory activity of the enzymatic hydrolysate.

[0057] 3.2 Effects of simulated gastrointestinal digestion on the stability of protein peptides in scaly trevally. Depend on Figure 9 As shown in Figure D, the FYDWWK peptide exhibits relatively stable XO inhibition activity in gastric juice, with no significant change in its inhibitory effect on XO after 60 minutes of gastric digestion. However, during intestinal digestion, the inhibitory activity of the FYDWWK peptide on XO is significantly enhanced, although its inhibitory activity does not show a significant difference with prolonged digestion time.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A uric acid-lowering and antioxidant polypeptide from the South China Sea fin-scaled round trevally, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing the uric acid-lowering and antioxidant polypeptides from the South China Sea fine-scaled round trevally as described in claim 1, characterized in that, The method includes the steps of preparing the uric acid-lowering and antioxidant peptides of the South China Sea fine-scaled trevally using a solid-phase synthesis method.

3. The use of the uric acid-lowering and antioxidant polypeptide of the South China Sea fine-scaled trevally as described in claim 1 in the preparation of a drug with uric acid-lowering and / or antioxidant effects.

4. The application of the uric acid-lowering and antioxidant polypeptides of the South China Sea fine-scaled round trevally as described in claim 1 in the preparation of health products that help with antioxidation.

5. The application of the uric acid-lowering and antioxidant polypeptide of the South China Sea fine-scaled trevally as described in claim 1 in the preparation of an inhibitor of xanthine oxidase.

6. A drug having uric acid-lowering and / or antioxidant effects, characterized in that, The active ingredients include the uric acid-lowering and antioxidant peptides from the South China Sea fine-scaled round trevally as described in claim 1.

7. The drug according to claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. A health supplement that helps with antioxidation, characterized in that, The active ingredients include the uric acid-lowering and antioxidant peptides from the South China Sea fine-scaled round trevally as described in claim 1.

9. The health product according to claim 8, characterized in that, The health products also include excipients for their preparation.

10. An inhibitor of xanthine oxidase, characterized in that, The active ingredients include the uric acid-lowering and antioxidant peptides from the South China Sea fine-scaled round trevally as described in claim 1.

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