Uric acid reducing peptide of Bombay duck as well as preparation method and application of uric acid reducing peptide

Abstract

The invention relates to the technical field of polypeptide application, in particular to a uric-acid-reducing peptide of Bombay duck as well as a preparation method and application of the uric-acid-reducing peptide. The invention provides a uric acid reducing peptide for Bombay duck. The uric acid reducing peptide for Bombay duck comprises one or more peptide fragments of LF3, FH3, FF4, LF4, IF6, WY6 and NM7. According to the invention, marine low-value fish Bombay fascia is taken as a raw material to prepare the Bombay fascia uric acid reducing peptide, the Bombay fascia uric acid reducing peptide can achieve the uric acid reducing effect by inhibiting the activity of xanthine dehydrogenase, and meanwhile, the Bombay fascia uric acid reducing peptide has an obvious improvement effect on liver oxidative injury and kidney immune inflammatory response.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide applications, and particularly relates to a Harpadon nehereus hypouricemic peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disease caused by disordered purine metabolism in the body, resulting in elevated serum uric acid (UA). With the changes in dietary patterns and living habits, the incidence of HUA shows an increasing trend. Long-term high uric acid levels are likely to induce metabolic diseases such as gout, kidney inflammation, hypertension, and obesity. Currently, the clinically used hypouricemic drugs have toxic and side effects. In recent years, with the improvement of people's health awareness, finding safe and non-toxic drug alternatives has become a current research and development hotspot.

[0003] Natural active peptides, which are a high-quality resource, have shown great potential in terms of hypouricemic activity. Harpadon nehereus is rich in nutrients, with a protein content as high as 70%, and the calcium and phosphorus contents are much higher than those of other traditional economic fish. However, its extremely high water content (more than 90%) makes the muscle protein of the fish extremely unstable and difficult to store for a long time; moreover, the market products are still mainly fresh fish and their dried products, with a single product category, resulting in a low development and utilization rate and causing waste of resources. Enzymatically hydrolyzing Harpadon nehereus into fish protein hydrolysates and extracting and preparing active peptides is an effective method to improve its bioavailability. Currently, there are reports on the isolation, purification, and preparation of hypouricemic peptides from marine fish, such as Auxis thazard peptide (amino acid sequence: PDL and SVGGAL) and tilapia skin collagen peptide (amino acid sequence: TP). However, there is no report on the preparation of hypouricemic peptides from Harpadon nehereus and the verification of their efficacy. Therefore, if Harpadon nehereus is developed and utilized to prepare hypouricemic active peptides, significant economic and social benefits will be achieved.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to prepare a Harpadon nehereus hypouricemic peptide using the low-value marine fish Harpadon nehereus as a raw material.

[0006] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a Harpadon nehereus hypouricemic peptide, which comprises one or more of the following peptide segments: LF3, FH3, FF4, LF4, IF6, WY6, and NM7;

[0008] The amino acid sequence of LF3 is Leu-Arg-Phe;

[0009] The amino acid sequence of FH3 is Phe-Leu-His;

[0010] The amino acid sequence of FF4 is Phe-Gly-Lys-Phe;

[0011] The amino acid sequence of LF4 is Leu-His-Leu-Phe;

[0012] The amino acid sequence of IF6 is Ile-Trp-His-His-Thr-Phe;

[0013] The amino acid sequence of WY6 is Trp-His-His-Thr-Phe-Tyr;

[0014] The amino acid sequence of NM7 is Asn-Pro-Tyr-Asp-Phe-Pro-Met.

[0015] The present invention also provides a method for preparing the uric acid-lowering peptide from Harpadon nehereus, comprising the following steps:

[0016] (1) Crushing and drying Harpadon nehereus to obtain Harpadon nehereus freeze-dried powder;

[0017] (2) Mixing the Harpadon nehereus freeze-dried powder with water to obtain a suspension; mixing the suspension with papain and enzymolyzing for 4-6 h to obtain an enzymolyzed solution;

[0018] (3) Mixing the enzymolyzed solution with absolute ethanol, performing alcohol precipitation for 12-18 h to obtain a mixture; centrifuging the mixture and taking the supernatant; concentrating the supernatant to a total solid content of 20-30% and drying to obtain Harpadon nehereus protein hydrolysate freeze-dried powder;

[0019] (4) Ultrafiltering the Harpadon nehereus protein hydrolysate freeze-dried powder, taking the filtrate with a molecular weight <1 kDa for chromatography, taking the chromatographic fraction with a xanthine oxidase inhibition rate >80% for elution to obtain Harpadon nehereus oligopeptide; screening the Harpadon nehereus oligopeptide with a functional score >0.8 and a mass spectrometry identification >20 by using a bioinformatics database, which is the uric acid-lowering peptide from Harpadon nehereus.

[0020] Preferably, in step (1), the drying is freeze-drying; the drying includes precooling, primary drying and secondary drying;

[0021] The temperature of the precooling is -70 to -90 °C; the time of the precooling is 5 to 7 h;

[0022] The temperature of the primary drying is 35 to 45 °C; the time of the primary drying is 10 to 14 h; the pressure of the primary drying is 8 to 12 Pa;

[0023] The temperature of the second - stage drying is 25 - 35°C; the time of the second - stage drying is 22 - 26 h; the pressure of the second - stage drying is 8 - 12 Pa.

[0024] Preferably, in step (2), the mass - to - volume ratio of the frozen powder of Harpadon nehereus and water is 1 g:90 - 100 mL.

[0025] The addition amount of the papain is 10000 - 14000 U / g.

[0026] The temperature of the enzymatic hydrolysis is 50 - 60°C.

[0027] Preferably, in step (3), the volume ratio of the enzymatic hydrolysate and absolute ethanol is 18 - 25:75 - 82.

[0028] The rotation speed of the centrifugation is 10000 - 14000 rpm; the time of the centrifugation is 8 - 12 min.

[0029] The concentration is vacuum concentration; the pressure of the concentration is - 0.05 - - 0.15 MPa; the temperature of the concentration is 50 - 60°C.

[0030] The drying is freeze - drying; the drying includes precooling, primary drying and second - stage drying.

[0031] The temperature of the precooling is - 70 - - 90°C; the time of the precooling is 5 - 7 h.

[0032] The temperature of the primary drying is 35 - 45°C; the time of the primary drying is 10 - 14 h; the pressure of the primary drying is 8 - 12 Pa.

[0033] The temperature of the second - stage drying is 25 - 35°C; the time of the second - stage drying is 22 - 26 h; the pressure of the second - stage drying is 8 - 12 Pa.

[0034] Preferably, in step (4), the chromatography is Sephadex gel column chromatography.

[0035] The specific conditions of the chromatography are: the sample loading volume is 4 - 6 mL, the flow rate is 0.5 - 1.5 mL / min, and the detection wavelength is 200 - 240 nm.

[0036] Preferably, in step (4), the bioinformatics database is the PeptideRanker database.

[0037] The present invention also provides the application of the Harpadon nehereus - derived uric - acid - lowering peptide or the Harpadon nehereus - derived uric - acid - lowering peptide prepared by the preparation method in the preparation of drugs or health products for reducing uric acid.

[0038] The present invention also provides the application of the Harpadon nehereus uric acid-lowering peptide or the Harpadon nehereus uric acid-lowering peptide prepared by the preparation method in the preparation of a drug or health product for inhibiting the activity of xanthine oxidase.

[0039] The present invention also provides the application of the Harpadon nehereus uric acid-lowering peptide or the Harpadon nehereus uric acid-lowering peptide prepared by the preparation method in the preparation of a drug or health product for treating liver and kidney injuries.

[0040] Advantages of the present invention:

[0041] The present invention uses the marine low-value fish Harpadon nehereus as a raw material, effectively avoiding environmental pollution and improving resource utilization rate; a single enzyme hydrolysis technology, ethanol precipitation, ultrafiltration, gel column chromatography, and linear gradient elution technology are used in combination to prepare Harpadon nehereus oligopeptides. The preparation method is simple and easy to control, and the enzymatic hydrolysis efficiency is high; then, a bioinformatics database is used to accurately screen for Harpadon nehereus uric acid-lowering peptides with high xanthine oxidase (XOD) inhibitory activity and no toxicity in Harpadon nehereus oligopeptides. This method simplifies the screening process and greatly saves experimental costs.

[0042] The present invention also provides a Harpadon nehereus uric acid-lowering peptide, which includes one or several peptide segments of LF3 (LRF), FH3 (FLH), FF4 (FGKF), LF4 (LHLF), IF6 (IWHHTF), WY6 (WHHTFY), and NM7 (NPYDFPM); the Harpadon nehereus uric acid-lowering peptide of the present invention can achieve the effect of reducing uric acid by inhibiting the activity of xanthine oxidase (XOD), and at the same time has an obvious improvement effect on liver oxidative damage and kidney immune inflammatory response. Therefore, the Harpadon nehereus uric acid-lowering peptide has good application prospects. Description of the drawings

[0043] Figure 1 is the inhibition rate of the filtrate in different molecular weight ranges on the XOD activity;

[0044] Figure 2 is the ultraviolet spectrum of gel chromatography;

[0045] Figure 3 is the inhibition rate of different chromatography fractions on the XOD activity;

[0046] Figure 4 is the inhibition rate of the Harpadon nehereus uric acid-lowering peptide on the XOD activity before and after digestion;

[0047] Figure 5 is the effect of the Harpadon nehereus uric acid-lowering peptide FF4 on the body weight change of hyperuricemia mice;

[0048] Figure 6 is the effect of the Harpadon nehereus uric acid-lowering peptide FF4 on the serum uric acid level of hyperuricemia mice;

[0049] Figure 7 To study the effect of the uric acid-lowering peptide FF4 from Harpadon nehereus on the kidneys of hyperuricemic mice;

[0050] Figure 8 To study the effect of the uric acid-lowering peptide FF4 from Harpadon nehereus on the liver of hyperuricemic mice. Specific embodiments

[0051] The present invention provides a uric acid-lowering peptide from Harpadon nehereus, which comprises one or more of the following peptide segments: LF3, FH3, FF4, LF4, IF6, WY6, and NM7;

[0052] The amino acid sequence of LF3 is Leu-Arg-Phe (LRF);

[0053] The amino acid sequence of FH3 is Phe-Leu-His (FLH);

[0054] The amino acid sequence of FF4 is Phe-Gly-Lys-Phe (FGKF, SEQ ID NO.1);

[0055] The amino acid sequence of LF4 is Leu-His-Leu-Phe (LHLF, SEQ ID NO.2);

[0056] The amino acid sequence of IF6 is Ile-Trp-His-His-Thr-Phe (IWHHTF, SEQ ID NO.3);

[0057] The amino acid sequence of WY6 is Trp-His-His-Thr-Phe-Tyr (WHHTFY, SEQ ID NO.4);

[0058] The amino acid sequence of NM7 is Asn-Pro-Tyr-Asp-Phe-Pro-Met (NPYDFPM, SEQ ID NO.5).

[0059] The present invention also provides a method for preparing the uric acid-lowering peptide from Harpadon nehereus, comprising the following steps:

[0060] (1) Crushing and drying Harpadon nehereus to obtain freeze-dried powder of Harpadon nehereus;

[0061] (2) Mixing the freeze-dried powder of Harpadon nehereus with water to obtain a suspension; mixing the suspension with papain and enzymatically hydrolyzing for 4-6 h to obtain a hydrolysate;

[0062] (3) Mix the enzymatic hydrolysate with absolute ethanol, perform alcohol precipitation for 12 - 18 h to obtain a mixture; centrifuge the mixture and take the supernatant; concentrate the supernatant to a total solid content of 20 - 30%, and dry it to obtain freeze-dried powder of Lophiomus setigerus protein hydrolysate;

[0063] (4) Ultrafilter the freeze-dried powder of Lophiomus setigerus protein hydrolysate, take the filtrate with a molecular weight < 1 kDa for chromatography, take the chromatographic fraction with a xanthine oxidase inhibition rate > 80% for elution to obtain Lophiomus setigerus oligopeptides; screen Lophiomus setigerus oligopeptides with a functional score > 0.8 and a mass spectrometry identification > 20 using a bioinformatics database, which are the Lophiomus setigerus peptides for reducing uric acid.

[0064] In the present invention, in step (1), the Lophiomus setigerus needs to be processed before being crushed, and the specific steps are as follows: Wash the Lophiomus setigerus, remove the head, tail, internal organs and skin;

[0065] In the present invention, in step (1), the drying is freeze-drying; the drying includes precooling, primary drying and secondary drying;

[0066] The temperature of the precooling is -70 - -90 °C, preferably -75 - -85 °C, more preferably -80 °C; the time of the precooling is 5 - 7 h, preferably 5.5 - 6.5 h, more preferably 6 h;

[0067] The temperature of the primary drying is 35 - 45 °C, preferably 38 - 42 °C, more preferably 40 °C; the time of the primary drying is 10 - 14 h, preferably 11 - 13 h, more preferably 12 h; the pressure of the primary drying is 8 - 12 Pa, preferably 9 - 11 Pa, more preferably 10 Pa;

[0068] The temperature of the secondary drying is 25 - 35 °C, preferably 28 - 32 °C, more preferably 30 °C; the time of the secondary drying is 22 - 26 h, preferably 23 - 25 h, more preferably 24 h; the pressure of the secondary drying is 8 - 12 Pa, preferably 9 - 11 Pa, more preferably 10 Pa.

[0069] In the present invention, in step (2), the mass-volume ratio of the freeze-dried powder of Lophiomus setigerus to water is 1 g : 90 - 100 mL, preferably 1 g : 93 - 97 mL, more preferably 1 g : 95 mL;

[0070] The addition amount of the papain is 10000 - 14000 U / g, preferably 11000 - 13000 U / g, more preferably 12000 U / g;

[0071] The temperature of the enzymatic hydrolysis is 50 - 60 °C, preferably 53 - 57 °C, more preferably 55 °C;

[0072] The time of enzymatic hydrolysis is preferably 4.5 to 5.5 h, more preferably 5 h;

[0073] The pH of the enzymatic hydrolysis is 6 to 8, preferably 7.

[0074] In the present invention, in step (3), before the enzymatic hydrolysate is mixed with absolute ethanol, enzyme inactivation is required. The specific steps are as follows: The enzymatic hydrolysate is placed in a water bath at 93 to 97 °C and heated for 13 to 17 min; the temperature of enzyme inactivation is preferably 94 to 96 °C, more preferably 95 °C; the time of enzyme inactivation is preferably 14 to 16 min, more preferably 15 min;

[0075] In step (3), the volume ratio of the enzymatic hydrolysate to absolute ethanol for mixing is 18 to 25:75 to 82, preferably 20 to 23:77 to 80, more preferably 21.5:78.5;

[0076] The time of alcohol precipitation is preferably 14 to 16 h, more preferably 15 h;

[0077] The rotation speed of centrifugation is 10000 to 14000 rpm, preferably 11000 to 13000 rpm, more preferably 12000 rpm; the time of centrifugation is 8 to 12 min, preferably 9 to 11 min, more preferably 10 min;

[0078] The concentration is vacuum concentration; the pressure of concentration is -0.05 to -0.15 MPa, preferably -0.08 to -0.12 MPa, more preferably -0.10 MPa; the temperature of concentration is 50 to 60 °C, preferably 53 to 57 °C, more preferably 55 °C;

[0079] The preference is: The supernatant is concentrated to a total solid content of 23 to 27%, more preferably 25%.

[0080] The drying is freeze-drying; the drying includes precooling, primary drying and secondary drying;

[0081] The temperature of precooling is -70 to -90 °C, preferably -75 to -85 °C, more preferably -80 °C; the time of precooling is 5 to 7 h, preferably 5.5 to 6.5 h, more preferably 6 h;

[0082] The temperature of primary drying is 35 to 45 °C, preferably 38 to 42 °C, more preferably 40 °C; the time of primary drying is 10 to 14 h, preferably 11 to 13 h, more preferably 12 h; the pressure of primary drying is 8 to 12 Pa, preferably 9 to 11 Pa, more preferably 10 Pa;

[0083] The temperature of the second drying step is 25-35°C, preferably 28-32°C, and more preferably 30°C; the time of the second drying step is 22-26 h, preferably 23-25 h, and more preferably 24 h; the pressure of the second drying step is 8-12 Pa, preferably 9-11 Pa, and more preferably 10 Pa.

[0084] In the present invention, in step (4), the chromatography is Sephadex gel column chromatography;

[0085] The specific conditions of the chromatography are as follows: the sample loading volume is 4-6 mL, preferably 5 mL, the flow rate is 0.5-1.5 mL / min, preferably 0.8-1.2 mL / min, and more preferably 1.0 mL / min, the detection wavelength is 200-240 nm, preferably 210-230 nm, and more preferably 220 nm;

[0086] The specific steps of the chromatography are as follows: the filtrate is dried to obtain the freeze-dried powder of hairtail polypeptide, the freeze-dried powder of hairtail polypeptide and ultrapure water are mixed to prepare a sample loading solution with a concentration of 5 mg / mL, and Sephadex G-15 Sephadex gel is selected as the material for Sephadex gel column chromatography.

[0087] In the present invention, in step (4), the elution is linear gradient elution;

[0088] The specific steps of the elution are as follows: 10 μL of the sample is loaded onto a C18-reversed phase column (0.15×150 mm, 5 μm); the column is eluted with a mobile phase composed of 0.1% formic acid aqueous solution (buffer A) and a mixed solution (0.1% formic acid and 84% acetonitrile solution mixed, buffer B) by linear gradient elution: the linear gradient of B solution is from 4% to 50% (0-50 min), the linear gradient of B solution is from 50% to 100% (50-54 min), and B solution is maintained at 100% (54-60 min); ESI-MS is set to positive ion mode, the scanning range is 300-1800 m / z, the resolution of the first-stage mass spectrometry is 70000, and the maximum dwell time is 40 ms; the resolution of the second-stage mass spectrometry is 17500, and the dwell time is 60 ms.

[0089] In the present invention, in step (4), the bioinformatics database is the PeptideRanker database.

[0090] The present invention also provides the application of the hairtail peptide for reducing uric acid or the hairtail peptide for reducing uric acid prepared by the preparation method in the preparation of drugs or health products for reducing uric acid.

[0091] The present invention also provides the application of the hairtail uric acid-lowering peptide or the hairtail uric acid-lowering peptide prepared by the preparation method in the preparation of a drug or health product for inhibiting the activity of xanthine oxidase.

[0092] The present invention also provides the application of the hairtail uric acid-lowering peptide or the hairtail uric acid-lowering peptide prepared by the preparation method in the preparation of a drug or health product for treating liver and kidney injuries.

[0093] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0094] Example 1

[0095] (1) Wash the hairtail, remove the head, tail, internal organs and skin, and use a chopper to break it into fish paste. First, pre-cool the fish paste in a refrigerator at -80°C for 6 h, then perform preliminary drying at 40°C and 10 Pa for 12 h, and then heat it to 30°C for secondary drying for 24 h to obtain freeze-dried hairtail powder;

[0096] (2) Mix the freeze-dried hairtail powder and water at a mass-to-volume ratio of 1 g:80 mL to obtain a suspension, and then add 12000 U / g of papain, and enzymatically hydrolyze it at 55°C and pH 7 for 5 h to obtain an enzymatic hydrolysate;

[0097] (3) Heat the enzymatic hydrolysate in a water bath at 95°C for 15 min to inactivate the enzyme, and then add absolute ethanol to obtain a mixture, where the addition amount of absolute ethanol is 80% of the volume of the mixture; Centrifuge the mixture at 12000 rpm for 10 min and take the supernatant; Vacuum concentrate the supernatant at -0.1 MPa and 55°C to a total solid content of 25%, dry it, then pre-cool it in a refrigerator at -80°C for 6 h, then perform preliminary drying at 40°C and 10 Pa for 12 h, and then heat it to 30°C for secondary drying for 24 h to obtain freeze-dried hairtail protein hydrolysate powder;

[0098] (4) Take the freeze-dried hairtail protein hydrolysate powder and mix it with ultrapure water to prepare a sample with a concentration of 1 mg / mL, filter it using an ultrafiltration membrane, collect the filtrates in different molecular weight ranges, and name them UH1 to UH4: UH1 (<1 KDa), UH2 (1 - 3 KDa), UH3 (3 - 5 KDa), UH4 (>5 KDa);

[0099] The inhibitory activity of the filtrates in different molecular weight ranges against XOD was determined according to the method of He et al. (He W, Su G, Sun-Waterhouse D, et al. In vivo anti-hyperuricemic and xanthine oxidase inhibitory properties of tuna protein hydrolysates and its isolated fractions [J]. Food Chemistry, 2019, 272: 453-461.): 0.5 mL of the filtrates in different molecular weight ranges (sample groups, UH1, UH2, UH3, UH4), the freeze-dried powder of lizardfish protein hydrolysate (HNPH), and the XOD solution (0.5 mL, 0.1 U / mL) were mixed respectively, incubated at 37 °C for 10 min. Subsequently, the xanthine solution (1.5 mL, 0.40 mmol / L) was added to the mixture to initiate the enzymatic reaction, and the change in the absorbance value at 290 nm within 2 min was recorded using an ultraviolet-visible spectrophotometer. The results are as Figure 1 shown.

[0100] The calculation formula for the XOD inhibitory activity is as follows:

[0101]

[0102] Note: V S and V O respectively represent the initial rates of the enzymatic reaction in the presence and absence of the polypeptide.

[0103] It can be seen from Figure 1 that at a certain concentration (5 mg / mL), the XOD inhibition rates of the separated components UH1-UH4 showed a gradually decreasing trend, and the XOD inhibitory activity of UH1 was the highest and significantly higher than that of the component HNPH before separation. Therefore, the filtrate of the UH1 group was selected for subsequent separation.

[0104] Example 2

[0105] The filtrate of the UH1 group described in Example 1 was pre-cooled in a refrigerator at -80 °C for 6 h, then preliminarily dried at 40 °C and 10 Pa for 12 h, and then heated to 30 °C for the second drying for 24 h to obtain the freeze-dried powder of lizardfish polypeptide. The freeze-dried powder of lizardfish polypeptide and ultrapure water were mixed to prepare a sample solution with a concentration of 5 mg / mL. Sephadex G-15 dextran gel was selected for dextran gel column chromatography. The specific conditions were as follows: the sample loading volume was 5 mL, the flow rate was 1 mL / min, and the detection wavelength was 220 nm. According to the ultraviolet spectrum of the gel chromatography ( Figure 2 ) Each component of the dextran gel column chromatography was collected and named H1, H2, H3, H4, H5;

[0106] The inhibitory activities of the components of Sephadex column chromatography against XOD were determined according to the determination method described in Example 1, and the results are as Figure 3 shown.

[0107] It can be seen from Figure 3 that at a certain concentration (5 mg / mL), the XOD inhibition rates of the separated components H1 - H5 showed a gradually increasing trend, and the XOD inhibitory activity of H5 was the highest, with the XOD inhibition of component H5 reaching more than 80%. Therefore, H5 was selected for subsequent polypeptide sequence identification.

[0108] Example 3

[0109] 10 μL of H5 described in Example 2 was loaded onto a C18-reverse phase column (0.15×150 nm, 5 μm); the column was eluted with a mobile phase consisting of 0.1% formic acid aqueous solution (buffer A) and a mixed solution (a mixture of 0.1% formic acid and 84% acetonitrile solution, buffer B) by linear gradient elution; the elution process was a linear gradient: the linear gradient of solution B was from 4% to 50% (0 - 50 min), the linear gradient of solution B was from 50% to 100% (50 - 54 min), and solution B was maintained at 100% (54 - 60 min); ESI-MS was set to positive ion mode, the scanning range was 300 - 1800 m / z, the resolution of the first-order mass spectrometry was 70000, and the maximum dwell time was 40 ms; the resolution of the second-order mass spectrometry was 17500, and the dwell time was adjusted to 60 ms; 107 peptide sequences (i.e., Harpadon nehereus oligopeptides) were eluted and separated;

[0110] Using the PeptideRanker database screening and molecular docking prediction simulation methods, Harpadon nehereus hypouricemic peptide sequences with a functional score > 0.8, mass spectrometry identification > 20, and non-toxicity were screened out from 107 peptide sequences: Leu-Arg-Phe (LRF, LF3), Phe-Leu-His (FLH, FH3), Phe-Gly-Lys-Phe (FGKF, FF4), Leu-His-Leu-Phe (LHLF, LF4), Ile-Trp-His-His-Thr-Phe (IWHHTF, IF6), Trp-His-His-Thr-Phe-Tyr (WHHTFY, WY6), Asn-Pro-Tyr-Asp-Phe-Pro-Met (NPYDFPM, NM7). Then, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize Harpadon nehereus hypouricemic peptides: LF3, FH3, FF4, LF4, IF6, WY6, NM7.

[0111] Example 4 In vitro activity determination of polypeptides

[0112] The in vitro digestion simulation of the polypeptide refers to the method of AO et al. (Ao J, Li B. Stability and antioxidative activities of casein peptide fractions during simulated gastrointestinal digestion in vitro: Charge properties of peptides affect digestive stability[J]. Food Research International, 2013, 52(1): 334-341.): Accurately weigh 40 mg of the polypeptide and add it to the gastric digestive juice (2 mL, pH = 2). Incubate it in a shaker (37 °C, 2 h) to simulate the gastric digestion process. After the end, adjust the pH to 7 with sodium hydroxide to terminate the gastric digestion. Then add 2 mL of intestinal digestive juice and culture it in a shaker incubator at 37 °C for 4 h. Place the mixture in a water bath at 100 °C and heat it for 5 min to terminate the digestion, and then centrifuge to obtain the supernatant, which is the digestion product.

[0113] The inhibitory activities of the uric acid-lowering peptide of Harpadon nehereus synthesized in Example 3 (before digestion) and the digestion product (after digestion) against XOD were measured respectively by the measurement method described in Example 2. The measurement results are as Figure 4 shown.

[0114] As can be seen from Figure 4 it, the inhibitory activity of the uric acid-lowering peptide FF4 of Harpadon nehereus against XOD increased significantly after in vitro simulated digestion, indicating that the uric acid-lowering peptide FF4 of Harpadon nehereus was stable in activity after gastrointestinal digestion. Therefore, the uric acid-lowering peptide FF4 of Harpadon nehereus was selected for the verification of the in vivo uric acid-lowering activity in subsequent animal experiments.

[0115] Example 5 Determination of the in vivo uric acid-lowering activity

[0116] Randomly select 60 C57BL / 6 mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd., production license number: SCXK (Jiangsu) 2023-0009), and adaptively raise them for 7 days before the experiment; randomly divide the mice into groups, with 12 mice in each group, a total of 5 groups: blank group, low-dose polypeptide group of Harpadon nehereus, high-dose polypeptide group of Harpadon nehereus, model group, and allopurinol group; except for the blank group, mice in other groups were intraperitoneally injected with potassium oxonate (PO) daily for 7 consecutive days to establish a hyperuricemia model, with an injection volume of 280 mg / (kg·d), and the blank group was given the same volume of normal saline; starting from the 8th day, 1 hour after intraperitoneal injection of PO to the mice, each group was intragastrically administered the corresponding dose of the drug: the drug intragastrically administered to the low-dose polypeptide group of Harpadon nehereus was 10 mg / kg of Harpadon nehereus uric acid-lowering peptide FF4 (FF4-L), the drug intragastrically administered to the high-dose polypeptide group of Harpadon nehereus was 50 mg / kg of Harpadon nehereus uric acid-lowering peptide FF4 (FF4-H), the drug intragastrically administered to the allopurinol group was 25 mg / kg of allopurinol, and the model group (MC) and the blank group (NC) were intragastrically administered the same volume of normal saline. The intragastric administration volume of each group was 1 mL / 100 g, and continuous intragastric administration was carried out for 28 days.

[0117] During the experiment, the body weights of the mice were recorded on the 0th, 7th, 14th, 21st, and 28th days respectively, and the results are as Figure 5 shown;

[0118] From Figure 5 it can be seen that during the experiment, the body weights of the mice in the NC group continued to increase, while the body weights of the mice in other groups increased slowly, indicating that PO has side effects on the growth of mice; taking xanthinol will aggravate the side effects, while Harpadon nehereus uric acid-lowering peptide FF4 can relieve the side effects of the drug.

[0119] During the experiment, orbital venous blood was collected on the 0th, 7th, 14th, 21st, and 28th days respectively, and then the serum uric acid (UA) level of the mice was measured using a kit (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.), and the results are as Figure 6 shown;

[0120] From Figure 6 it can be seen that on the 28th day of continuous drug administration, compared with the MC group, the serum UA levels of the mice in the PC and FF4 groups were significantly decreased, and the high-dose polypeptide group could achieve a similar effect to the positive drug group, indicating that Harpadon nehereus uric acid-lowering peptide FF4 can effectively reduce the serum UA level in HUA mice.

[0121] One hour after the last administration, mice were anesthetized with 0.3% sodium pentobarbital anesthetic (30 mg / kg BW), blood was collected from the orbital vein, and tissue samples were taken from the mice. For histopathological examination, fresh liver and kidney tissues were placed in 4% paraformaldehyde solution for 2 days for tissue fixation, and then the tissues were taken out. The tissues were dehydrated using different concentrations of ethanol gradients (100%, 90%, 80%, 70% and 50%), washed with xylene in sequence, and embedded in paraffin. Finally, the tissues were sectioned routinely, dewaxed and stained with HE, and observed under an optical microscope at a magnification of ×400. The results are as Figures 7 - 8 shown;

[0122] As can be seen from Figure 7 , the morphological structure of the kidney tissue of the mice in the NC group was clear, the glomeruli were intact, and no obvious pathological phenomena such as inflammatory cell infiltration were observed. Compared with the NC group, the glomeruli in the MC group were atrophied, the renal tubules were significantly dilated, and inflammatory cell infiltration was visible. The PC group also had glomerular atrophy and renal tubule dilation. The glomerular atrophy and renal tubule dilation in the FF4-L group were alleviated, and the kidney structure in the FF4-H group was clear and the glomeruli were intact. It shows that PO modeling can induce kidney injury in mice, and intragastric administration of the uric acid-lowering peptide FF4 from Harpadon nehereus can significantly improve kidney injury in mice;

[0123] As can be seen from Figure 8 , the organizational structure of the liver of the mice in the NC group was clear, the hepatocytes were of normal size and arranged neatly, and no obvious pathological changes were observed. Compared with the NC group, there was a small amount of fatty degeneration around the central vein of the liver structure in the MC group, with round vacuoles of different sizes in the cytoplasm, and a little lymphocyte infiltration was visible. The hepatocytes in the PC group were mildly edematous, and the fatty degeneration in the PC and FF4 groups was improved. It shows that PO modeling can induce mild liver injury in mice, and intragastric administration of the uric acid-lowering peptide FF4 from Harpadon nehereus can relieve liver injury to a certain extent.

[0124] As can be seen from the above examples, the present invention provides a uric acid-lowering peptide from Harpadon nehereus, its preparation method and application. The invention uses the marine low-value fish Harpadon nehereus as a raw material to prepare a uric acid-lowering peptide from Harpadon nehereus, which can achieve the effect of lowering uric acid by inhibiting the activity of xanthine dehydrogenase, and at the same time has an obvious improvement effect on liver oxidative damage and kidney immune inflammatory response.

[0125] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A hairtail fish-derived uric acid-lowering peptide, characterized in that, The lizardfish uric acid-lowering peptide comprises one or several of the following peptide segments: LF3, FH3, FF4, LF4, IF6, WY6, and NM7; The amino acid sequence of LF3 is Leu-Arg-Phe; The amino acid sequence of FH3 is Phe-Leu-His; The amino acid sequence of FF4 is Phe-Gly-Lys-Phe; The amino acid sequence of LF4 is Leu-His-Leu-Phe; The amino acid sequence of IF6 is Ile-Trp-His-His-Thr-Phe; The amino acid sequence of WY6 is Trp-His-His-Thr-Phe-Tyr; The amino acid sequence of NM7 is Asn-Pro-Tyr-Asp-Phe-Pro-Met.

2. The preparation method of the lizardfish uric acid-lowering peptide according to claim 1, comprising the following steps: (1) Crush and dry the lizardfish to obtain lizardfish freeze-dried powder; (2) Mix the lizardfish freeze-dried powder and water to obtain a suspension; mix the suspension and papain, and enzymatically hydrolyze for 4 - 6 h to obtain an enzymatic hydrolysate; (3) Mix the enzymatic hydrolysate and absolute ethanol, perform alcohol precipitation for 12 - 18 h to obtain a mixture; centrifuge the mixture, take the supernatant; concentrate the supernatant to a total solid content of 20 - 30%, and dry to obtain lizardfish protein hydrolysate freeze-dried powder; (4) Ultrafilter the lizardfish protein hydrolysate freeze-dried powder, take the filtrate with a molecular weight < 1 kDa for chromatography, take the chromatographic fraction with a xanthine oxidase inhibition rate > 80% for elution to obtain lizardfish oligopeptides; screen the lizardfish oligopeptides with a functional score > 0.8 and a mass spectrometry identification > 20 using a bioinformatics database, which are the lizardfish uric acid-lowering peptides.

3. The preparation method according to claim 2, characterized in that, In step (1), the drying is freeze-drying; the drying includes precooling, primary drying, and secondary drying; The temperature of the precooling is -70 to -90 °C; the time of the precooling is 5 - 7 h; The temperature of the primary drying is 35 - 45 °C; the time of the primary drying is 10 - 14 h; the pressure of the primary drying is 8 - 12 Pa; The temperature of the secondary drying is 25 - 35 °C; the time of the secondary drying is 22 - 26 h; the pressure of the secondary drying is 8 - 12 Pa.

4. The preparation method according to claim 3, characterized in that, In step (2), the mass-volume ratio of the lizardfish freeze-dried powder to water for mixing is 1 g: 90 - 100 mL; The addition amount of the papain is 10000 - 14000 U / g; The temperature of the enzymatic hydrolysis is 50 - 60 °C.

5. The preparation method according to claim 4, characterized in that, In step (3), the volume ratio of the enzymatic hydrolysate to absolute ethanol for mixing is 18 - 25: 75 - 82; The rotation speed of the centrifugation is 10000 - 14000 rpm; the time of the centrifugation is 8 - 12 min; The concentration is vacuum concentration; the pressure of the concentration is -0.05 to -0.15 MPa; the temperature of the concentration is 50 - 60 °C; The drying is freeze-drying; the drying includes precooling, primary drying, and secondary drying; The temperature of the precooling is -70 to -90 °C; the time of the precooling is 5 - 7 h; The temperature of the preliminary drying is 35-45 °C; the time of the preliminary drying is 10-14 h; the pressure of the preliminary drying is 8-12 Pa; The temperature of the second drying is 25-35 °C; the time of the second drying is 22-26 h; the pressure of the second drying is 8-12 Pa.

6. The preparation method according to claim 5, characterized in that, In step (4), the chromatography is Sephadex gel column chromatography; The specific conditions of the chromatography are as follows: the sample loading volume is 4-6 mL, the flow rate is 0.5-1.5 mL / min, and the detection wavelength is 200-240 nm.

7. The preparation method according to claim 6, characterized in that, In step (4), the bioinformatics database is the PeptideRanker database.

8. Application of the uric acid-lowering peptide of Harpadon nehereus described in claim 1 or the uric acid-lowering peptide of Harpadon nehereus prepared by the preparation method described in any one of claims 2-7 in the preparation of a drug or health product for lowering uric acid.

9. Application of the uric acid-lowering peptide of Harpadon nehereus described in claim 1 or the uric acid-lowering peptide of Harpadon nehereus prepared by the preparation method described in any one of claims 2-7 in the preparation of a drug or health product for inhibiting the activity of xanthine oxidase.

10. Application of the uric acid-lowering peptide of Harpadon nehereus described in claim 1 or the uric acid-lowering peptide of Harpadon nehereus prepared by the preparation method described in any one of claims 2-7 in the preparation of a drug or health product for treating liver and kidney injuries.

Images