Peptides and their applications

Abstract

This application discloses a polypeptide and its application, wherein the polypeptide is selected from any one of SEQ ID Nos:1-42, and the polypeptide can bind to XOD enzyme, thereby inhibiting the activity of XOD enzyme to reduce uric acid and improving hyperuricemia.

Description

Technical Field

[0001] This application relates to the field of polypeptide preparation technology, and in particular to a polypeptide and its applications. Background Technology

[0002] The Chinese giant salamander (scientific name: *Andrias davidianus*) is the world's largest living amphibian, possessing extremely high edible and medicinal value, and showing great potential for development and application in the field of life and health. Wild giant salamanders are a Class II protected aquatic animal in China, but in the past two decades, artificial breeding techniques have gradually matured, leading to the rapid development of the giant salamander industry. Large-scale giant salamander farming industries and markets have formed in several provinces and regions, including Shaanxi, Hunan, Henan, and Sichuan. This provides a sustainable biological resource for extracting life and health products with medicinal and health-promoting value from giant salamander bones. Summary of the Invention

[0003] This application provides a polypeptide that can inhibit XOD activity in vitro and in vivo to reduce uric acid and improve hyperuricemia.

[0004] The specific technical solution of this application is as follows:

[0005] 1. A polypeptide selected from any one of SEQ ID Nos:1-42.

[0006] 2. A nucleic acid used to encode the polypeptide described in item 1.

[0007] 3. A composition comprising the polypeptide described in item 1.

[0008] 4. The composition according to claim 3, wherein it further comprises a pharmaceutically acceptable carrier, excipients, and active ingredient.

[0009] 5. The use of the polypeptide described in item 1 or the composition described in item 3 or 4 in the preparation of a medicament for lowering uric acid or treating hyperuricemia.

[0010] 6. A medicament for lowering uric acid or treating hyperuricemia, comprising the polypeptide described in claim 1 or the composition described in claim 3 or 4.

[0011] The effects of the invention

[0012] The polypeptide described in this application can bind to XOD enzyme, thereby inhibiting XOD enzyme activity and lowering uric acid. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the molecular weight distribution of giant salamander bone peptide extract (ADBP).

[0014] Figure 2 This diagram illustrates the primary and secondary separation processes of the giant salamander bone peptide extract (ADBP), as well as its XOD inhibition rate. Figure 2 In the diagram, A is a schematic diagram of the initial separation of ADBP. Figure 2 In the diagram, B represents the inhibition rate of XOD by the product of the initial separation of ADBP. Figure 2 C in the diagram represents a secondary separation of the product with a high XOD inhibition rate from the initial separation. Figure 2 D in the diagram represents the inhibition rate of XOD by the product of secondary separation.

[0015] Figure 3 This is a schematic diagram illustrating the uric acid-lowering effect of peptides on mice with hyperuricemia. Figure 3 In the diagram, A represents the experimental data. Figure 3 B in the diagram represents the activity of xanthine oxidase in the liver. Figure 3 The 'C' in the diagram represents the blood uric acid level. Figure 3 D in the diagram represents the serum creatinine level. Figure 3 E in the diagram represents the blood urea nitrogen level. Detailed Implementation

[0016] The present application will now be described in detail with reference to the accompanying drawings, wherein the same numerals in all the drawings denote the same features. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0017] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0018] This application provides a polypeptide selected from any one of SEQ ID Nos:1-42.

[0019] Among them, the sequence of SEQ ID NO:1 is TEAPLNPK;

[0020] The sequence of SEQ ID NO:2 is HDEPVTIPNKPN;

[0021] The sequence of SEQ ID NO:3 is GPRGPQGPS;

[0022] The sequence of SEQ ID NO:4 is RGPPGPS;

[0023] The sequence of SEQ ID NO:5 is LDFE;

[0024] The sequence of SEQ ID NO:6 is PAW;

[0025] The sequence of SEQ ID NO:7 is GPAGPSGPR;

[0026] The sequence of SEQ ID NO:8 is AVLVFR;

[0027] The sequence of SEQ ID NO:9 is VLIPVK;

[0028] The sequence of SEQ ID NO:10 is VMVLEIR;

[0029] The sequence of SEQ ID NO:11 is ALSLPLIK;

[0030] The sequence of SEQ ID NO:12 is IIPNIVK;

[0031] The sequence of SEQ ID NO:13 is ALTLDIY;

[0032] The sequence of SEQ ID NO:14 is IIWEFIK;

[0033] The sequence of SEQ ID NO:15 is DVLNLVY;

[0034] The sequence of SEQ ID NO:16 is YLAFLR;

[0035] The sequence of SEQ ID NO:17 is FFAAFASR;

[0036] The sequence of SEQ ID NO:18 is YALVFFYR;

[0037] The sequence of SEQ ID NO:19 is LNLNVLY;

[0038] The sequence of SEQ ID NO:20 is DLMLW;

[0039] The sequence of SEQ ID NO:21 is VGW;

[0040] The sequence of SEQ ID NO:22 is NENIIIL;

[0041] The sequence of SEQ ID NO:23 is VTIETVTY;

[0042] The sequence of SEQ ID NO:24 is EVLMLR;

[0043] The sequence of SEQ ID NO:25 is VNWL;

[0044] The sequence of SEQ ID NO:26 is LMISMLDR;

[0045] The sequence of SEQ ID NO:27 is FLDCAFR;

[0046] The sequence of SEQ ID NO:28 is MWLP;

[0047] The sequence of SEQ ID NO:29 is FWFP;

[0048] The sequence of SEQ ID NO:30 is TWL;

[0049] The sequence of SEQ ID NO:31 is AHF;

[0050] The sequence of SEQ ID NO:32 is FTLR;

[0051] The sequence of SEQ ID NO:33 is DMLLR;

[0052] The sequence of SEQ ID NO:34 is SHIADF;

[0053] The sequence of SEQ ID NO:35 is IPYW;

[0054] The sequence of SEQ ID NO:36 is WPF;

[0055] The sequence of SEQ ID NO:37 is FWYY;

[0056] The sequence of SEQ ID NO:38 is DWF;

[0057] The sequence of SEQ ID NO:39 is FWQ;

[0058] The sequence of SEQ ID NO:40 is PHF;

[0059] The sequence of SEQ ID NO:41 is FDEW;

[0060] The sequence of SEQ ID NO:42 is FWER.

[0061] Among them, SEQ ID Nos:1-7 is obtained by a "top-down" method, which involves enzymatically hydrolyzing giant salamander bone powder, followed by alcohol precipitation to obtain giant salamander bone peptide extract (ADBP), and then separating the giant salamander bone peptide extract and measuring the XOD inhibition rate to obtain the polypeptide as described in SEQ ID Nos:1-7.

[0062] In some embodiments, the preparation method of giant salamander bone peptide extract (ADBP) is as follows:

[0063] The supernatant obtained by enzymatic hydrolysis and centrifugation of giant salamander bone powder is the enzymatic hydrolysis extract of giant salamander bone powder.

[0064] The enzymatic hydrolysis extract of giant salamander bone powder was subjected to alcohol precipitation and centrifugation to obtain giant salamander bone peptide extract (ADBP).

[0065] In some embodiments, the protease is an alkaline protease, preferably 2709 alkaline protease; preferably, the protease hydrolysis is carried out at 30-40°C; preferably, the protease hydrolysis is carried out at pH 9-11; preferably, the hydrolysis time is 3-6 hours; preferably, the mass ratio of protease to giant salamander bone powder is 1:6-15. In some embodiments, ethanol is used for precipitation; preferably, the mass concentration of the ethanol is 55-85%; preferably, the precipitation time is 3-12 hours.

[0066] In some embodiments, the obtained giant salamander bone peptide extract is separated using conventional high-performance liquid chromatography (HPLC) separation methods, and the inhibition rate of xanthine oxidase (XOD) of each separated product is evaluated using chemical methods. The product with a higher XOD inhibition rate is selected for secondary separation to obtain a secondary separated product. The XOD inhibition rate of each secondary separated product is evaluated, and the secondary separated product with a higher XOD inhibition rate is identified by mass spectrometry to obtain the polypeptide as described in SEQ ID NOs:1-7.

[0067] In some embodiments, the chromatographic conditions for the second separation are the same as those for the separation described above.

[0068] In some embodiments, the conditions for the high-performance liquid chromatography are as follows:

[0069] The chromatographic column is an Agilent 5 prep-C18 column; and / or

[0070] Mobile phase A: an aqueous solution containing 0.05-0.1 wt% TFA; mobile phase B is an acetonitrile solution containing 0.05-0.1 wt% TFA, preferably, the volume ratio of mobile phase A to mobile phase B is 5-25:95-75; and / or

[0071] The flow rate is 5-15 mL / min; and / or

[0072] The injection volume is 0.5-1.5 mL; and / or

[0073] The detection wavelength is 210-220nm or 280nm.

[0074] In this application, no restrictions are placed on the determination of protein content in the giant salamander bone peptide extract. It can be determined according to conventional methods in the art, such as the method for determining protein in the first method of national standard GB 5009.5-2016.

[0075] In some embodiments, based on the giant salamander bone peptide extract, glycine is 4-11 mg / kg; and / or alanine is 2-9 mg / kg; and / or valine is 0.5-7 mg / kg; and / or leucine is 2-9 mg / kg; and / or isoleucine is 2-7 mg / kg; and / or proline is 2-9 mg / kg; and / or phenylalanine is 1-7 mg / kg; and / or methionine is 0-4 mg / kg; and / or tyrosine is 0-6 mg / kg; and / or serine is 1-8 mg / kg; and / or threonine is 0-7 mg / kg; and / or aspartic acid is 3-10 mg / kg; and / or glutamic acid is 9-15 mg / kg; and / or lysine is 2-9 mg / kg; and / or arginine is 2-9 mg / kg; and / or histidine is 0-5 mg / kg.

[0076] For example, based on the giant salamander bone peptide extract, glycine can be 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, etc.;

[0077] Alanine can be in doses of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, etc.

[0078] Valine can be in doses of 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, etc.

[0079] Leucine can be in doses of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, etc.

[0080] Isoleucine can be in doses of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, etc.

[0081] Proline can be in the form of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, etc.

[0082] Phenylalanine can be in doses of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, etc.

[0083] Methionine can be available in concentrations of 0, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, etc.

[0084] Tyrosine can be in the form of 0, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, etc.

[0085] Serine can be in doses of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, etc.

[0086] Threonine can be in the form of 0, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, etc.

[0087] Aspartic acid can be in the form of 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, etc.

[0088] Glutamic acid can be in doses of 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, etc.

[0089] Lysine can be in doses of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, etc.

[0090] Arginine can be in the form of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, etc.

[0091] Histidine can be in doses of 0, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, etc.

[0092] In this application, no restrictions are placed on the method for determining the amino acid content in the giant salamander bone peptide extract. It can be determined according to conventional methods in the field, such as the method in national standard GB 5009.124-2016.

[0093] In this application, no restrictions are placed on the method for determining the amino acid sequence of the polypeptide. It can be performed according to conventional methods in the art, such as UPLC-Orbitrap Fusion MS. For example, gradient elution can be performed using an aqueous solution containing 0.1% TFA as mobile phase A and an acetonitrile solution containing 0.15% TFA as mobile phase B. Preferably, the gradient elution method is as follows:

[0094] 0-2 min, 3-7 vol% mobile phase B;

[0095] 2-52 min, 7-22 vol% mobile phase B;

[0096] 52-62 min, 22-35% (v / v) mobile phase B;

[0097] 62-64 min, 35-90% (v / v) of mobile phase B;

[0098] 64-84 min, 90% by volume mobile phase B

[0099] In some embodiments, the flow rate is 250 nL / min. In some embodiments, the mass spectrometry conditions are: m / z scan range 300–2000, resolution 120,000, and target ion concentration 2 × 10⁻⁶. 5 In this application, no restrictions are placed on the method for determining the abundance; it can be determined according to conventional methods in the art, such as calculation using ProteinDiscorvery software.

[0100] In some embodiments, the weight-average molecular weight of the giant salamander bone peptide extract is 490-600 g / mol, preferably 490-550 g / mol, and more preferably 490-530 g / mol;

[0101] Preferably, the number-average molecular weight of the giant salamander bone peptide extract is 300-600 g / mol, and more preferably 350-550 g / mol.

[0102] For example, the weight-average molecular weight of the giant salamander bone peptide extract can be 490 g / mol, 500 g / mol, 510 g / mol, 520 g / mol, 530 g / mol, 540 g / mol, 550 g / mol, 560 g / mol, 570 g / mol, 580 g / mol, 590 g / mol, 600 g / mol, etc.; the number-average molecular weight of the giant salamander bone peptide extract can be 300 g / mol, 310 g / mol, 320 g / mol, 330 g / mol, 340 g / mol, 350 g / mol, 360 g / mol, 370 g / mol, 380 g / mol, 390 g / mol, 400 g / mol, 410 g / mol, 420 g / mol, 430 g / mol, 440 g / mol, 450 g / mol, 460 g / mol, 470 g / mol, 480 g / mol, etc. g / mol, 490 g / mol, 500 g / mol, 510 g / mol, 520 g / mol, 530 g / mol, 540 g / mol, 550g / mol, 560 g / mol, 570 g / mol, 580 g / mol, 590 g / mol, 600 g / mol, etc.

[0103] In some embodiments, the weight-average molecular weight of the giant salamander bone peptide extract is 513±17.04 g / mol, and the number-average molecular weight is 454.67±97.35 g / mol.

[0104] In this application, no restrictions are placed on the method for determining the weight-average molecular weight of the giant salamander bone peptide extract. It can be determined according to conventional methods in the art, such as GPC analysis.

[0105] In some embodiments, the giant salamander bone peptide extract contains 600.00-900.00 mg / kg of phosphorus, preferably 628.43-888.90 mg / kg; 7000.00-9000.00 mg / kg of potassium, preferably 7177.97-8308.69 mg / kg; 5000.00-6000.00 mg / kg of sodium, preferably 5571.64-5901.69 mg / kg; 6.00-7.00 mg / kg of iron, preferably 6.36-6.67 mg / kg; 3.00-4.00 mg / kg of zinc, preferably 3.37-3.63 mg / kg; and 0.50-1.00 mg / kg of selenium, preferably 0.64-0.73 mg / kg.

[0106] For example, the phosphorus content in the giant salamander bone peptide extract can be 600.00 mg / kg, 650.00 mg / kg, 700.00 mg / kg, 750.00 mg / kg, 800.00 mg / kg, 850.00 mg / kg, 900.00 mg / kg, etc.

[0107] Potassium can be 7000.00 mg / kg, 7100.00 mg / kg, 7200.00 mg / kg, 7300.00 mg / kg, 7400.00 mg / kg, 7500.00 mg / kg, 7600.00 mg / kg, 7700.00 mg / kg, 7800.00 mg / kg, 7900.00mg / kg, 8000.00 mg / kg, 8100.00 mg / kg, 8200.00 mg / kg, 8300.00 mg / kg, 8400.00 mg / kg, 8500.00 mg / kg, 8600.00 mg / kg, 8700.00 mg / kg, 8800.00 mg / kg, 8900.00 mg / kg, 9000.00mg / kg, etc.;

[0108] Sodium can be in the following concentrations: 5000.00 mg / kg, 5100.00 mg / kg, 5200.00 mg / kg, 5300.00 mg / kg, 5400.00 mg / kg, 5500.00 mg / kg, 5500.00 mg / kg, 5600.00 mg / kg, 5700.00 mg / kg, 5800.00 mg / kg, 5900.00 mg / kg, 6000.00 mg / kg, etc.

[0109] Iron concentrations can be 6.00 mg / kg, 6.10 mg / kg, 6.20 mg / kg, 6.30 mg / kg, 6.40 mg / kg, 6.50 mg / kg, 6.60 mg / kg, 6.70 mg / kg, 6.80 mg / kg, 6.90 mg / kg, 7.00 mg / kg, etc.

[0110] Zinc concentrations can be 3.00 mg / kg, 3.10 mg / kg, 3.20 mg / kg, 3.30 mg / kg, 3.40 mg / kg, 3.50 mg / kg, 3.60 mg / kg, 3.70 mg / kg, 3.80 mg / kg, 3.90 mg / kg, 4.00 mg / kg, etc.

[0111] Selenium can be present in concentrations of 0.50 mg / kg, 0.64 mg / kg, 0.65 mg / kg, 0.66 mg / kg, 0.67 mg / kg, 0.68 mg / kg, 0.69 mg / kg, 0.70 mg / kg, 0.71 mg / kg, 0.72 mg / kg, 0.73 mg / kg, 0.80 mg / kg, 0.90 mg / kg, and 1.00 mg / kg.

[0112] In this application, no restrictions are placed on the method for determining the content of macro- and micro-elements in the giant salamander bone peptide extract. The determination can be carried out in accordance with conventional methods in the field, such as the method in the first method of national standard GB5009.268-2016.

[0113] In some embodiments, SEQ ID Nos:8-42 is obtained through a bottom-up approach. This involves obtaining the transcriptomic information of *Andrias davidianus* cartilage using conventional transcriptomics methods, simulating translation to obtain proteins, and establishing a *Andrias davidianus* cartilage protein library. This library serves as a protein sequence comparison database for protein identification. Proteomic sequencing of *Andrias davidianus* cartilage yields polypeptide sequences, which are then used as a reference to obtain proteins. These proteins are then subjected to single-enzyme and multi-enzyme simulating digestion to establish a *Andrias davidianus* cartilage polypeptide library. Using known polypeptide sequences reported in the literature as a reference, machine learning is employed to screen the *Andrias davidianus* polypeptide library. The obtained polypeptides are then molecularly docked with XOD to obtain polypeptides that bind well to XOD, and these polypeptides are sorted according to hydrophobicity to obtain the polypeptides of SEQ ID Nos:8-42.

[0114] The polypeptide described in this application has a good inhibition rate against XOD, with an IC50 of 0.31-0.35 mg / mL.

[0115] This application provides a nucleic acid for encoding a polypeptide of any one of SEQ ID Nos:1-42.

[0116] This application provides a composition comprising a polypeptide of any one of SEQ ID Nos: 1-42. In some embodiments, it further comprises a pharmaceutically acceptable carrier, excipients, and an active ingredient.

[0117] In this application, no restrictions are placed on pharmaceutically acceptable carriers, excipients and active ingredients, which may be carriers, excipients or active ingredients commonly used in the art.

[0118] This application provides the use of the above-described polypeptide or composition in the preparation of a medicament for lowering uric acid or treating hyperuricemia.

[0119] This application provides a drug for lowering uric acid or treating hyperuricemia, comprising the polypeptide or the composition described above.

[0120] Example

[0121] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0122] Example 1: Obtaining peptides using a top-down method

[0123] Giant salamander bone powder was added to deionized water at a material-to-liquid ratio of 1:12.5. The pH was adjusted to 10.0±0.5 using 1M sodium hydroxide. Then, 2709 alkaline protease was added at a concentration of 0.6% (i.e., the volume ratio of protease to solution). The mixture was stirred and hydrolyzed in a water bath at 40℃, pH 9, and time for 4 hours. The protease was then inactivated (100℃, 10 min). After cooling the hydrolysate to room temperature, it was centrifuged (5,000×g, 10 min) to obtain the supernatant, which is the giant salamander bone powder enzymatic extract. 75% ethanol was added to the giant salamander bone powder enzymatic extract for alcohol precipitation for 6 hours. The mixture was then allowed to stand overnight and centrifuged (5,000×g, 20 min) to obtain the supernatant. The supernatant was then obtained by rotary evaporation at 50℃ to obtain the giant salamander bone peptide extract (ADBP).

[0124] (1) ADBP quality assessment

[0125] According to national standards, the nutritional indicators (moisture, total protein, ash), mineral elements (calcium, phosphorus, potassium, sodium, magnesium, iron, zinc, selenium), amino acid composition, and heavy metal indicators (mercury, lead, arsenic, cadmium) of the giant salamander bone peptide extract were evaluated. The results are shown in Tables 1-4. The nutritional indicators were determined using the protein determination method in Method 1 of the national standard GB 5009.5-2016.

[0126] The moisture content was determined according to the first method of the national standard GB 5009.3-2016;

[0127] Crude fat content was determined according to the second method of national standard GB 5009.6-2016;

[0128] Ash content was determined according to the first method of national standard GB 5009.4-2016;

[0129] The determination of mineral elements was carried out according to the method in Method I of the national standard GB 5009.268-2016;

[0130] The amino acid composition was determined according to the method in the national standard GB 5009.124-2016.

[0131] The methods for determining heavy metal indicators (mercury, lead, arsenic, cadmium) are as follows:

[0132] Total mercury was determined using the first method of GB 5009.17-2014, lead content was determined using the second method of GB 5009.12-2017, total arsenic was determined using the first method of GB 5009.11-2014, and cadmium content was determined using the first method of GB 5009.15-2014.

[0133] Table 1 Basic physicochemical properties of ADBP

[0134]

[0135] Table 2. Constant and trace elements of ADBP

[0136]

[0137] Table 3 Heavy Metal Index of ADBP

[0138]

[0139] Note: ND indicates not detected.

[0140] Table 4. Amino acid composition and content of ADBP

[0141]

[0142] Table 1-4 shows that ADBP is rich in crude protein, reaching 83.75±1.91%; ADBP is rich in macroelements such as P, K, and Na, and is also rich in Se (0.69±0.05 mg / kg); the actual content of various heavy metal indicators in ADBP is very low or below the detection limit; the hydrophobic amino acid content in ADBP is 28.01±1.04 mg / kg, accounting for 36.1% of the total amino acids.

[0143] (2) Molecular weight distribution of ADBP, as well as weight-average and number-average molecular weight

[0144] The method of Yu et al. (YU Y, HU Q, LIU J, et al. Isolation, purification and identification of immunologically active peptides from Hericium erinaceus[J]. Food and Chemical Toxicology, 2021, 151: 112111.) was referenced and modified. An Agilent 1260 high-performance liquid chromatograph (equipped with a differential detector) was used to analyze the molecular weight distribution of ADBP. The chromatographic column was a TSK-GelG2000SWXL (300 mm × 7.8 mm), the mobile phase was acetonitrile-water (45:55, containing 0.1% trifluoroacetic acid), the detection wavelength was 220 nm, the flow rate was 0.5 mL / min, the column oven temperature was 30℃, and the injection volume was 10 μL. The standards used for the standard curve were cytochrome C (12,384 Da, purchased from Beijing Solarbio), aprotinin (6,511 Da, purchased from Beijing Solarbio), the synthetic peptide VPSGPLGPEGPR (SEQ ID NO:21, 1,161.6 Da, purchased from Hefei Guotai Biotechnology Co., Ltd.), glutathione (307 Da, purchased from Beijing Solarbio), and Glutamate (147 Da, purchased from Beijing Solarbio). A standard curve was plotted as y = -0.3705x + 10.4769 (y = Log MW, R² = 0.997). The molecular weight distribution of the giant salamander bone peptide extract was determined as follows: Figure 1 As shown.

[0145] from Figure 1It can be seen that in ADBP, the proportion of peptides with a molecular weight of "less than 1000 Da" is 77.78±1.15%, the proportion of peptides with a molecular weight of "1000-3000 Da" is 21.22±1.15%, the proportion of peptides with a molecular weight of "3000-5000 Da" is 0.81±0.27%, and the proportion of peptides with a molecular weight of "greater than 5000 Da" is 0.17±0.11%.

[0146] The bone peptide extract of the giant salamander was analyzed by GPC (gel permeation chromatography), and the weight-average molecular weight of the extract was 513±17.04 g / mol, and the number-average molecular weight was 454.67±97.35 g / mol.

[0147] 1.3 Obtaining Polypeptides

[0148] The ADBP prepared by the above method was initially separated using an Agilent 1260 Infinity II preparative high-performance liquid chromatography (HPLC). Chromatographic conditions: Agilent 5 prep-C18 column (50 × 21.2 mm); mobile phase: pump A was aqueous phase (containing 0.1% TFA), pump B was acetonitrile (containing 0.1% TFA); flow rate: 10 mL / min; injection volume: 0.9 mL; detection wavelength: 214 nm, 280 nm; elution conditions: isocratic elution in 10% acetonitrile system for 30 min. A 150 mg / mL ADBP stock solution (2.97 g ADBP) was prepared and filtered through a 0.22 μm filter membrane. The ADBP chromatogram was separated into three components: ADBP-1 (1.73 g), ADBP-2 (0.38 g), and ADBP-3 (0.43 g). Figure 2 A), after rotary concentration and lyophilization at 60℃, the total recovery rate of the sample was 85.52%. The XOD inhibition rate of the ADBP-separated components was evaluated using chemical methods. The XOD inhibition rate of the 20 mg / mL ADBP-2 component was 67.82±6.96%, significantly higher than that of ADBP-1 (57.76±11.74%) and ADBP-3 (40.80±6.54%) (p<0.05). Figure 2 B). Therefore, the ADBP-2 component was selected for the second separation.

[0149] The chemical method is operated as follows:

[0150] Take 50 μL of the test sample and 50 μL of XOD (0.05 U / mL) and incubate at 37℃ for 10 min in a 96-well plate. Then add 150 μL of 0.1 mmol / L xanthine solution and immediately place the plate in a microplate reader. Record the kinetic change of absorbance at 290 nm over 2 min (every 20 s). PBS and allopurinol were used instead of the sample solution for the blank control and positive control, respectively. The XOD inhibition rate was calculated using the following formula:

[0151]

[0152] Where V0 is the initial reaction rate of the enzyme-catalyzed reaction system in the control group, and V1 is the initial reaction rate of the enzyme-catalyzed reaction system in the sample group.

[0153] ADBP-2 was separated a second time using an Agilent 1260 Infinity II preparative high-performance liquid chromatography (HPLC). Chromatographic conditions: Agilent 5 prep-C18 column (50 × 21.2 mm); mobile phase: pump A was aqueous phase (containing 0.1% TFA), pump B was acetonitrile (containing 0.1% TFA); flow rate: 5 mL / min; injection volume: 0.9 mL; detection wavelength: 214 nm, 280 nm; elution conditions: isocratic elution in a 10% acetonitrile system for 25 min. A 150 mg / mL ADBP-2 stock solution (1.05 g) was prepared and filtered through a 0.22 μm filter membrane. The ADBP chromatogram was then separated into three fractions: ADBP-21 (0.27 g), ADBP-22 (0.23 g), and ADBP-23 (0.39 g). Figure 2 C), after rotary concentration and lyophilization at 60℃, the overall recovery rate was 84.76%. The XOD inhibition rates of the ADBP-2 separated components were evaluated using chemical methods. The XOD inhibition rates of ADBP-21 and ADBP-22 at 20 mg / mL were 73.91±4.10% and 74.20±4.02%, respectively, significantly higher than those of ADBP-23 (55.07±8.00%). Figure 2 (D) The chemical methods are the same as above. Therefore, ADBP-22 was selected as the component for subsequent mass spectrometry identification.

[0154] The composition of ADBP-22 was analyzed using an Orbitrap Fusion mass spectrometer. Gradient elution was performed using a mobile phase of 0.1% trifluoroacetic acid aqueous solution (A) and 0.1% trifluoroacetic acid acetonitrile solution (B) at a flow rate of 250 nL / min. The gradient program was: 0–2 min, 3–7% B; 2–52 min, 7–22% B; 52–62 min, 22–35% B; 62–64 min, 35–90% B; 64–84 min, 90–90% B. The m / z scan range was 300–2000, and the target 2e 5 Ions were collected at a resolution of 120,000. The mass spectrometer was set to the highest-rate high-energy collisional dissociation mode for data-dependent acquisition. Based on the collected MS / MS spectral data, peptide sequences were identified using DeNovoGUI software (Version 1.16.2, Max Planck Institute, Munich, Germany). A total of 329 peptide sequences were identified in this experiment. Based on the abundance and amino acid characteristics of the peptides, 7 peptides were selected, as shown in Table 5.

[0155]

[0156] Example 2: Obtaining peptides using a bottom-up approach

[0157] This experiment used the cartilage of farmed giant salamanders in Luoyang, Henan Province as the research subject. First, transcriptomics methods were used to obtain the transcriptome information of the giant salamander cartilage. A total of 15,183 proteins were annotated using simulated translation, and a giant salamander cartilage protein library was established as a protein sequence information comparison database required for protein identification in proteomic data processing. Then, proteomic sequencing was performed on the giant salamander cartilage, identifying a total of 6,385 polypeptide sequences. Using the established giant salamander cartilage protein database as a reference, a total of 1,271 proteins were annotated. The 1271 proteins mentioned above were imported into existing computer-aided protein hydrolysis servers (PeptideCutter, PeptideMass, BIOPEP, etc.). Single-enzyme and complex-enzyme simulated hydrolysis was performed using trypsin, chymotrypsin, trypsin / chymotrypsin, pepsin, proteinase K, and thermolysin, respectively, yielding 21504, 26767, 27049, 30651, 16248, and 26647 giant salamander cartilage polypeptides, totaling 148866 (molecular weight 250-3000 Da). The inventors of this application compiled 60 identified uric acid-lowering peptide sequences from databases such as Web of Science and CNKI over the past decade (2013-2023). Using these databases, machine learning was applied to 148,866 peptides in this experiment, resulting in the selection of 3,638 peptides with a similarity higher than 85%. The 3D structure of xanthine oxidase (XOD) was obtained from the protein database PDB (http: / / www.rcsb.org / ), from which 3NVY—a bovine-derived XOD crystal structure—was retrieved. Using 3NVY as the receptor protein and the 3,638 selected peptides as ligands, molecular docking was performed using semi-flexible docking, yielding 900 peptides, of which 524 were 2-8 peptides. Studies showed that hydrophobic peptides bind better to XOD for inhibitory effects. These 524 peptides were then sorted by hydrophobicity, and the top 35 peptides were selected as potential active peptide sequences, as shown in Table 6.

[0158] Table 6

[0159]

[0160] Note: Overall average hydrophilicity (GRAVY): A larger positive value indicates greater hydrophobicity, and a larger negative value indicates greater hydrophilicity.

[0161] Experimental Example 1: In vitro xanthine oxidase inhibitory effect of artificially synthesized peptides

[0162] Forty-two peptides (purity >95%) were obtained through artificial synthesis. The xanthine oxidase inhibition rate (IC50) was used as the evaluation index, and conventional methods in the field were employed to determine the IC50. 50 The results are shown in Table 7.

[0163] Table 7. IC50 sequence of artificially synthesized uric acid-lowering giant salamander bone peptide active polypeptide. 50 value

[0164]

[0165] The above 42 uric acid-lowering peptides were found to have good in vitro inhibitory effects on xanthine oxidase activity, IC50. 50 The values ​​ranged from 0.31 to 3.35 mg / mL.

[0166] After sorting the above 42 peptides, the experiment further selected the first 8 uric acid-lowering peptides (IC50). 50 Intervention experiments were conducted on mice with hyperuricemia (with a value less than 1 mg / mL).

[0167] SPF-grade male C57BL / 6J mice, 6-8 weeks old and weighing 21 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in an SPF-grade animal laboratory at a temperature of 20-26℃ and a relative humidity of 40-70%, using a 12h:12h day / night intermittent lighting system. Mice underwent acclimatization feeding for 7 days, during which they had free access to food and water. From day 0 to 7, the control and model groups were administered 0.25% CMC-Na by gavage daily, while the positive control and experimental groups were administered 10 mg / kg allopurinol and 100 mg / kg different peptides by gavage, respectively. One hour after gavage, mice in each group were administered 0.25% CMC-Na via intraperitoneal injection. The control group received an intraperitoneal injection of 200 μL of 0.25% CMC-Na, while the other groups received an intraperitoneal injection of 200 μL of modeling agent (a 0.25% carboxymethyl cellulose sodium suspension containing 300 mg / kg potassium oxonate and 200 mg / kg hypoxanthine). One hour later, mice were anesthetized and blood was collected from the heart. Serum and liver samples were taken, and serum (uric acid, creatinine, blood urea nitrogen) and liver (XOD) levels were measured according to the kits used by Nanjing Jiancheng Bioengineering Institute. The results are as follows: Figure 3 As shown, where, Figure 3 In the diagram, A represents the experimental data. Figure 3 B in the diagram represents the activity of xanthine oxidase in the liver. Figure 3 The 'C' in the diagram represents the blood uric acid level. Figure 3 D in the diagram represents the serum creatinine level. Figure 3 E in the diagram represents the blood urea nitrogen level.

[0168] Depend on Figure 3According to the BC data, compared with the control group, the model group had significantly higher XOD activity and serum uric acid levels. p The value <0.05 indicates that the hyperuricemia mouse model was successfully established. Compared with the model group, the allopurinol group mice significantly inhibited XOD activity, thereby reducing the increase in serum uric acid levels. p <0.05, Figure 3 B-3C). By Figure 3 From B, it can be seen that, compared with the model group, the XOD activity of mice in the YLAFLR, HDEPVTIPNKPN, GPAGPSGPR, GPRGPQGPS, and AVLVFR groups was significantly decreased. p <0.05%, and there was no significant change in XOD activity in mice in the RGPPGPS, FTLR, and LDFE groups. p >0.05).

[0169] Figure 3 The results of the C-test showed that the serum uric acid level in the HDEPVTIPNKPN and AVLVFR groups was significantly lower than that in the model group. p <0.05%, and no significant changes were observed in other groups, but the serum levels of mice in the YLAFLR, RGPPGPS, FTLR, LDFE, and GPAGPSGPR groups showed a certain decreasing trend. p >0.05). Furthermore, the experiment further detected serum creatinine and urea nitrogen levels to evaluate the effects of eight peptides on renal function. Figure 3 According to DE, the levels of creatinine and urea nitrogen in the model group were significantly higher than those in the control group. p <0.05 indicates that acute hyperuricemia in mice is accompanied by kidney function impairment. There were no significant changes in serum kidney function impairment indicators between the allopurinol group and the model group. p >0.05). Compared with the model group, the serum creatinine of mice in the AVLVFR group was significantly decreased ( Figure 3 D in p <0.05, and the serum creatinine levels of mice in other polypeptide groups showed a certain degree of decreasing trend ( Figure 3 D in p >0.05, which has a protective effect on kidney function. In addition, the serum urea nitrogen levels in mice in the FTLR, LDFE, HDEPVTIPNKPN, GPAGPSGPR, GPRGPQGPS, and AVLVFR groups were slightly lower than those in the model group ( pThe concentration of these peptides (>0.05) suggests a potential trend towards reducing serum urea nitrogen. The timing and concentration of peptide intervention may be important factors in their protective effect on kidney function. In summary, HDEPVTIPNKPN and AVLVFR exhibit good inhibitory effects on XOD activity and uric acid reduction, making them promising candidate peptides for further research into their uric acid-lowering mechanisms. Furthermore, the other six peptides also demonstrated some XOD activity inhibition, uric acid-lowering effects, and renal protective effects.

[0170] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A polypeptide, the amino acid sequence of which is shown in SEQ ID No:

8.

2. A nucleic acid for encoding the polypeptide of claim 1.

3. A composition comprising the polypeptide of claim 1.

4. The composition according to claim 3, wherein, It also contains pharmaceutically acceptable carriers and / or excipients.

5. The use of the polypeptide of claim 1 or the composition of claim 3 or 4 in the preparation of a medicament for lowering uric acid or treating hyperuricemia.

6. A medicament for lowering uric acid or treating hyperuricemia, comprising the polypeptide of claim 1 or the composition of claim 3 or 4.

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