Active peptide capable of reducing uric acid as well as preparation method and application of active peptide

Abstract

The invention belongs to the technical field of biological small molecule active peptides, and more specifically relates to a uric acid reducing active peptide, a preparation method and an application thereof. The uric acid reducing activity LQKW has remarkable XOD inhibitory activity, IC50 is 2.70 mg / mL, and the IC50 is superior to that of many known natural source XOD inhibitory peptides; good gastrointestinal digestion stability is shown, and the activity retention rate after in-vitro simulated gastrointestinal digestion is (61.84 + / -0.82)%. According to the invention, a specific binding mechanism of uric acid reduction activity and XOD active sites (Ile648, Phe649 and the like) is clarified through molecular docking, and a basis is provided for rational design based on a structure. According to the invention, the method for efficiently preparing the active peptide fragment from the white spirit vinasse is established, and the high-valued utilization of the wine brewing byproducts is realized. The uric acid reducing activity LQKW provided by the invention can be prepared through solid-phase synthesis, the process is mature, the quality is controllable, and a foundation is laid for industrialization. In addition, as the XOD inhibitor of a natural source, the uric acid reducing activity LQKW has good safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological small molecule active peptides, and more specifically relates to a uric acid-lowering active peptide with XOD inhibitory activity, a preparation method and an application thereof. Background Art

[0002] Hyperuricemia is a metabolic disease characterized by elevated uric acid concentrations in the blood due to excessive uric acid production or decreased excretion. Long-term hyperuricemia can lead to serious complications such as gout, kidney stones, and chronic kidney disease. Xanthine oxidase (XOD) is a key enzyme in uric acid production, and inhibiting XOD activity is an important therapeutic strategy for hyperuricemia.

[0003] Currently, commonly used XOD inhibitors in clinical practice, such as allopurinol and febuxostat, are effective but have serious adverse reactions, including allergic reactions, hepatotoxicity, and cardiovascular events. Therefore, the development of safe and effective XOD inhibitors from natural sources is of great significance.

[0004] Food-derived bioactive peptides have become a research hotspot for new XOD inhibitors due to their high safety and minimal side effects.

[0005] Liquor lees, a byproduct of the brewing industry, are rich in protein but have low utilization rates. While studies have examined the extraction of bioactive peptides from lees, no specific peptides with significant XOD inhibitory activity have been reported, particularly short peptides that can bind to the XOD active site through a specific interaction mechanism. Therefore, there is an urgent need to develop a uric acid-lowering peptide with high XOD inhibitory activity, good gastrointestinal stability, and a clear mechanism of action, as well as to establish an efficient preparation method for it, to provide new options for the treatment of hyperuricemia. Summary of the Invention

[0006] In view of the above technical problems, the present invention provides a uric acid-lowering active peptide, a preparation method and its application, in order to obtain a small molecule peptide with high XOD inhibitory activity, which has high oral stability, a clear mechanism of action and improves the utilization efficiency of white wine lees.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a uric acid-lowering active peptide, wherein the amino acid sequence of the uric acid-lowering active peptide is Leu-Gln-Lys-Trp.

[0008] In some preferred embodiments, the half inhibitory concentration IC 50 =2.70mg / mL.

[0009] More preferably, after simulated gastrointestinal digestion in vitro, the XOD inhibitory activity retention rate of the uric acid-lowering active peptide is (61.84±0.82)%.

[0010] As a preferred uric acid-lowering active peptide of the present invention, the uric acid-lowering active peptide binds to the active site of XOD through hydrophobic interaction and hydrogen bonds. The uric acid-lowering active peptide forms 7 hydrophobic interactions with the active sites Ile648, Phe649, Phe914, Phe1009, Val1011, Phe1013, and Leu1014 in XOD, and forms 5 hydrogen bonds with the active sites Leu648, Glu802, Ser876, Thr1010, and Val1011 in XOD.

[0011] In a second aspect, the present invention provides a pharmaceutical composition comprising the uric acid-lowering peptide according to the first aspect of the present invention and a pharmaceutically acceptable excipient.

[0012] In a third aspect, the present invention provides a method for preparing the uric acid-lowering peptide according to the first aspect, comprising the following steps:

[0013] S1: Using liquor lees as raw material, the enzymatic hydrolysate was obtained by alkaline protease enzymolysis;

[0014] S2: ultrafiltration separation of the enzymatic hydrolysate to collect the active peptide component of the lees with a molecular weight of less than 1 kDa;

[0015] S3: Identify the peptide sequences of the active peptide components of the lees with a molecular weight of less than 1 kDa by LC-MS, and screen the peptides with a bioactivity score of ≥0.5 and a docking energy of <-7 kcal / mol;

[0016] S4: Solid phase chemical synthesis of the uric acid-lowering active peptide LQKW.

[0017] In some preferred embodiments, the enzymatic hydrolysis conditions in step S1 are: the amount of alkaline protease added is 6000 U / g of vinasse protein, the pH value is 9.5, the temperature is 50° C., and the time is 5 h.

[0018] In a fourth aspect, the present invention provides the use of the uric acid-lowering active peptide according to the first aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention in the preparation of a medicament for treating hyperuricemia. Preferably, the medicament is used to lower serum uric acid levels, improve renal index or alleviate renal pathological damage.

[0019] In a fifth aspect, the present invention provides use of the uric acid-lowering peptide according to the first aspect of the present invention in the preparation of an XOD inhibitor.

[0020] In a sixth aspect, the present invention provides a food additive comprising the uric acid-lowering active peptide as described in the first aspect of the present invention, wherein the food additive is used to prepare functional food or dietary supplement.

[0021] Different from the existing technology, the uric acid-lowering active peptide LQKW provided by the above technical solution has significant XOD inhibitory activity, IC 50 The uric acid-lowering active peptide LQKW can be prepared by solid-phase synthesis, with a mature process and controllable quality, which lays the foundation for industrialization. Moreover, as a natural food-derived XOD inhibitor, the uric acid-lowering active peptide LQKW has high safety.

[0022] The above-mentioned records related to the content of the invention are only an overview of the technical solution of this application. In order to enable ordinary technicians in this field to understand the technical solution of this application more clearly, and then implement it according to the text of the specification and the contents recorded in the drawings, and to make the above-mentioned purposes and other purposes, features and advantages of this application easier to understand, the following is an explanation in combination with the specific implementation methods and drawings of this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings are only used to illustrate the principles, implementation methods, applications, characteristics and effects of the specific embodiments of this application and other related contents, and are not to be considered as limiting this application.

[0024] In the drawings of the specification:

[0025] Figure 1 In the specific embodiment, the XOD inhibitory activity IC 50 Results of the effects of in vitro simulated digestion of four peptides with a concentration less than 4 mg / mL on the XOD inhibitory activity of the peptides;

[0026] Figure 2 This is a schematic diagram of the molecular docking results of the 1FIQ drug in a specific embodiment and the uric acid-lowering active peptide LQKW provided by the present invention;

[0027] Figure 3 The effect of the uric acid-lowering active peptide LQKW provided by the present invention on the renal index of hyperuricemia HUA mice, wherein, compared with the Control group, *. indicates a significant difference (P < 0.05), **. indicates an extremely significant difference (P < 0.01); compared with the Model group, #. indicates a significant difference (P < 0.05), ##. indicates an extremely significant difference (P < 0.01);

[0028] Figure 4 The results show the effect of the uric acid-lowering active peptide LQKW provided by the present invention on the serum uric acid UA(A) level in hyperuricemia HUA mice;

[0029] Figure 5 The results show the effect of the uric acid-lowering active peptide LQKW provided by the present invention on the serum creatinine CRE content (B) level in hyperuricemia HUA mice;

[0030] Figure 6 The results show that the uric acid-lowering active peptide LQKW provided by the present invention affects the urea nitrogen (BUN) content (C) level in hyperuricemia HUA mice;

[0031] Figure 7 The results of the effect of the uric acid-lowering active peptide LQKW provided by the present invention on the renal pathological changes in hyperuricemia HUA mice (400×). DETAILED DESCRIPTION

[0032] In order to explain in detail the possible application scenarios, technical principles, specific solutions that can be implemented, and the purpose and effects of this application, the following is a detailed description of the specific embodiments listed in conjunction with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of this application and are therefore only examples and are not intended to limit the scope of protection of this application.

[0033] References to "embodiments" herein mean that the specific features, structures, or characteristics described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the word "embodiment" in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or relevance to other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the various technical features mentioned in the embodiments can be combined in any manner to form a corresponding implementable technical solution.

[0034] Unless otherwise defined, the technical terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms herein is only for describing specific embodiments and is not intended to limit this application.

[0035] In the description of this application, the term "and / or" is used to describe a logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and both A and B exist. In addition, the character " / " in this document generally indicates that the objects before and after are in a logical "or" relationship.

[0036] In this application, terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, priority or sequence relationship between these entities or operations.

[0037] Without further limitations, in this application, the words "include", "comprise", "have" or other similar expressions used in the sentences are intended to cover non-exclusive inclusion. These expressions do not exclude the presence of additional elements in the process, method or product including the elements, so that the process, method or product including a series of elements may include not only those defined elements, but also other elements not explicitly listed, or elements inherent to such process, method or product.

[0038] Consistent with the understanding in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceed" are understood to exclude the number itself; expressions such as "above," "below," and "within" are understood to include the number itself. Furthermore, in the description of the embodiments of this application, "multiple" means more than two (including two), and similar expressions related to "multiple" are also understood in this manner, such as "multiple groups," "multiple times," etc., unless otherwise specifically defined.

[0039] In the description of the embodiments of the present application, the space-related expressions used, such as "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "vertical", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicate the orientation or position relationship based on the orientation or position relationship shown in the specific embodiments or drawings, and are only for the convenience of describing the specific embodiments of the present application or facilitating the reader's understanding, and do not indicate or imply that the device or component referred to must have a specific position, a specific orientation, or be constructed or operated in a specific orientation. Therefore, it should not be understood as a limitation on the embodiments of the present application.

[0040] Unless otherwise expressly specified or limited, in the description of the embodiments of the present application, the terms "installed", "connected", "connected", "fixed", "set", etc. used should be understood in a broad sense. For example, the "connection" can be a fixed connection, a detachable connection, or an integrated setting; it can be a mechanical connection, an electrical connection, or a communication connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be the internal connection of two elements or the interaction relationship between two elements. For those skilled in the art of the present application, the specific meanings of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0041] Chronic diseases have become a major threat to the health of Chinese residents. The incidence of hyperuricemia (HUA) has been rising year by year, becoming the second largest metabolic disease after diabetes. The number of people suffering from hyperuricemia in my country has reached 190 million, and the trend is towards younger age. An unreasonable diet high in calories, fat, protein, and purine is the primary factor leading to the chronic disease HUA. Xanthine oxidase (XOD) is a key enzyme that leads to the formation of uric acid. The principle of existing drug treatments is mostly to reduce the production of uric acid by inhibiting XOD, such as allopurinol (AP) and febuxostat. Food-derived active peptides have the advantages of being mild, easily absorbed, and having no side effects. Research on uric acid-lowering active peptides has mostly focused on extracts from by-products such as marine fish, while there is less research on the extraction of uric acid-lowering active peptides from plants and their waste.

[0042] my country is a leading producer of baijiu (white spirits), with an annual production of over 24 million tons of distiller's grains (liquor grains) and up to 8 million tons of dry grains. Distiller's grains are highly nutritious, consisting of a variety of components, including proteins, polysaccharides, lipids, and minerals. In recent years, they have often been underutilized, with large quantities used as a source of feed protein in animal husbandry. Distiller's grains (liquor grains) are the primary by-product of winemaking. They are a solid residue formed after rice, sorghum, wheat, and other grains are steamed, fermented, and distilled to produce alcohol. Most of the starch in these raw materials is liquefied, saccharified, and fermented into alcohol, while protein conversion and utilization is extremely low. Currently, research on distiller's grains focuses on the isolation of specific microorganisms, the development of condiments, and protein extraction. High-value utilization of processing by-products rich in grain protein sources requires in-depth technological development. The present invention uses ultrafiltration to separate the enzymatic hydrolysate of liquor lees, and verifies the uric acid-lowering function of the component with the highest XOD inhibitory activity in HUA mice. Sequence analysis is performed by liquid chromatography-mass spectrometry (LC-MS). Potential highly active XOD inhibitory peptides are screened based on computer analysis, and in vitro functional verification and in vitro simulated gastrointestinal digestion tolerance are examined. Molecular docking is used to reveal the interaction mechanism between the peptide segment and the XOD target, and the uric acid-lowering and kidney-protective effects of the peptide segment in HUA mice are clarified, in order to provide technical support for the high-value utilization of winemaking by-products and the development of active uric acid-lowering peptides derived from liquor lees.

[0043] The following is a description of the reagents, raw materials, instruments, equipment, testing methods, etc. used in the specific embodiments of the present invention.

[0044] Xanthine oxidase was purchased from Beijing Solebow Technology Co., Ltd., 0.5 U / mg;

[0045] Alkaline protease was purchased from Jiangsu Ruiyang Biotechnology Co., Ltd. with an enzyme activity of 200,000 U / g;

[0046] The urea nitrogen test kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., catalog number C013-1-1;

[0047] Creatinine kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., catalog number C011-2;

[0048] Potassium oxonate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number P137112-5g;

[0049] Adenine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number A108804-5g;

[0050] The X-ray diffraction three-dimensional structure data of the XOD receptor (PDB code: 1FIQ) of the drug 1FIQ were downloaded from the Protein Data Bank (www.rcsb.org). Unrelated small molecules were deleted using Pymol 2.1 software. After constructing the mutant pdb using pymol, it was imported into AutoDock Tools-1.5.6 software, water molecules were deleted, hydrogen atoms were added, and the atom types were set, and the data were saved as a pdbqt file.

[0051] The Maotai-flavor liquor lees in the embodiment of the present invention were provided by Fujian Zhenghe Dongping Laojiao Distillery Co., Ltd., dried at 60° C. and ultrafinely ground for later use, with a protein content of 16.3%.

[0052] Ethics statement: The animal experiments in the specific embodiments of the present invention were approved by the Experimental Animal Ethics Committee of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences (approval number [MYLLSC2024-006]).

[0053] The optical microscope, tissue slicer, staining equipment, microscopic imaging system, etc. involved in obtaining the tissue pathology test results in the present invention all adopt instruments, equipment and methods well known and commonly used by those skilled in the art.

[0054] The computer programs, software, and systems used in the present invention are all well-known and commonly used instruments, equipment, and methods by those skilled in the art.

[0055] Example 1

[0056] Preparation, activity determination and structural characterization of the uric acid-lowering peptide LQKW

[0057] (1) Preparation of enzymatic hydrolysate of liquor lees

[0058] Take 15g of Maotai-flavor baijiu lees and mix thoroughly at a material-to-water ratio of 1:10. Enzymatic hydrolysis conditions: add 6000U of alkaline protease per gram of lees protein, pH 9.5, incubate for 5 hours, and incubate at 50°C. After the hydrolysis is complete, inactivate the enzyme in a 95°C water bath for 10 minutes. After the hydrolysis product cools, centrifuge at 2°C and 10,000 rpm for 15 minutes. Collect the supernatant, which is the baijiu lees hydrolyzate.

[0059] (2) Ultrafiltration separation

[0060] Ultrafiltration membranes with molecular weight cutoffs of 5kDa and 1kDa were selected to ultrafilter the liquor lees hydrolysate to obtain liquor lees hydrolysates with different molecular weight ranges (>5kDa, 5kDa~1kDa and <1kDa). The filtrates were collected for XOD inhibitory activity determination, and the components with the best inhibitory activity were evaluated for uric acid-lowering efficacy in mice and analyzed by mass spectrometry.

[0061] (3) Determination of XOD inhibitory activity

[0062] Add 50 μL of 0.1 U / mL xanthine oxidase and 50 μL of sample to a 96-well plate, incubate at 37°C for 10 min, then add 150 μL of 0.3 mmol / L xanthine solution, continue incubation for 15 min, and measure the absorbance at 292 nm.

[0063] XOD inhibition rate I (%) = [(D3-D4)-(D1-D2)] / (D3-D4)×100%

[0064] Where I represents the inhibition rate of XOD; D1 is the absorbance value of the sample group with xanthine as the substrate; D2 is the absorbance value of the sample group with buffer replacing the xanthine solution; D3 is the absorbance value of the blank group with buffer replacing the sample; and D4 is the absorbance value of the blank group with buffer replacing the sample and xanthine solution. A linear equation is obtained using the natural logarithm of the sample protein concentration as the horizontal axis and the XOD inhibition rate as the vertical axis. The sample concentration at which the inhibition rate is 50% is calculated, i.e., the half-inhibitory concentration (IC). 50 .

[0065] (4) Sequence identification

[0066] The amino acid sequence of the XOD inhibitory peptide was identified using Q Exactive LC-MS. The sample was first subjected to reductive alkylation and desalting, then dissolved in 0.1% formic acid for mass spectrometry analysis. The chromatographic conditions were: Acclaim PepMap C 18The chromatographic column was 150 μm × 150 mm; the flow rate was 600 nL / min; the mobile phase A consisted of 0.1% formic acid in water, and the mobile phase B consisted of 80% acetonitrile and 0.1% formic acid. The gradient elution program was as follows: 0 min, 4% B; 3 min, 8% B; 89 min, 28% B; 109 min, 40% B; 110 min, 95% B; 120 min, 95% B. The mass spectrometry raw files were searched using Byonic software, using the Uniprot-Glutelin database.

[0067] (5) Virtual screening

[0068] The peptides identified by mass spectrometry were virtually screened to screen peptides with less than 10 peptides. The peptides were analyzed for biological activity using the Peptide Ranker program (http: / / distttilldeep.ucd.ie / PeptideRanker / ), and peptides with a biological activity score ≥ 0.5 were selected. Autodock software was used for simulated flexible docking to select peptides with an amino acid sequence binding energy score less than -7 Kcal / mol. Peptides with XOD inhibitory activity characterized by hydrophobic amino acids and aromatic amino acids (alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F), methionine (M), proline (P), tryptophan (W), and tyrosine (Y)) accounting for more than 50% were screened.

[0069] (6) Peptide synthesis

[0070] The peptides were synthesized by entrusting Sangon Biotech (Shanghai) Co., Ltd. with solid-phase chemical synthesis, with a purity of 99%, and the XOD inhibition rate was determined.

[0071] (7) Evaluation of in vitro simulated gastrointestinal digestion tolerance

[0072] The uric acid-lowering peptide was dissolved in simulated gastric fluid and digested in a water bath at 200 rpm and 37°C for 2 hours. The pH was adjusted to 6.8 ± 0.1 with 1 mol / L NaOH. The supernatant was collected as the gastric digestion fluid and the XOD inhibition rate was measured before and after digestion.

[0073] Equal proportions of gastric digestive fluid and simulated intestinal fluid were thoroughly mixed and then placed in a constant-temperature shaking water bath at 2000 rpm and 37°C for 4 hours to simulate intestinal digestion. Following completion, the enzymes were inactivated in a boiling water bath at 100°C for 10 minutes. The mixture was cooled to room temperature and centrifuged at 4°C and 8000 rpm for 10 minutes. The supernatant was collected as the gastrointestinal digestive fluid and assayed for XOD inhibition.

[0074] (8) Molecular docking

[0075] The molecular docking model of XOD substrate and peptide was established by Discover studio 2019 software. The X-ray diffraction three-dimensional structure data of XOD receptor (PDB code: 1FIQ) were downloaded from the Protein Data Bank (www.rcsb.org). Pymol2.1 software was used to delete irrelevant small molecules. After constructing the mutant pdb using pymol, it was imported into AutoDockTools-1.5.6 software, water molecules were deleted, hydrogen atoms were added, and the atom types were set, and saved as pdbqt files. The ligand-active peptide was energy minimized using the MMFF94 force field in Chem 3D, converted into pdb format, and imported into AutoDock Tools-1.5.6 software. While adding atomic charges and assigning atom types, all flexible bonds were rotatable by default, and finally saved as pdbqt files.

[0076] The receptor protein and the active peptide were semi-flexibly docked using the autodock vina software, and the docking results were analyzed using PLIP and visualized using pymol.

[0077] Example 2

[0078] Verification of the uric acid-lowering efficacy of the uric acid-lowering active peptide LQKW

[0079] ICR male mice (SPF grade, 20-25 g, 5 weeks old) were raised in the SPF grade animal room of the Experimental Animal Center under the following breeding conditions: room temperature (23±2)℃, relative humidity (45-55)%, free access to water and food, and 12-h light and dark cycles.

[0080] (1) Construction, grouping, and drug administration of the hyperuricemia HUA mouse model

[0081] After 7 days of adaptive feeding, the mice were randomly divided into 7 groups (n=6 per group): blank control group (Control), model group (Model), allopurinol group (10 mg / kg, AP), <1 kDa distillers grains active peptide group (1600 mg / kg), LQKW low-dose (200 mg / kg, LQKW-L), medium-dose (400 mg / kg, LQKW-M), and high-dose (800 mg / kg, LQKW-H) groups. The modeling drug was prepared by suspending potassium oxonate and adenine in 0.5% sodium carboxymethylcellulose (CMC-Na). Except for the blank control group, mice in each group were gavaged daily with the modeling drug (potassium oxonate 300 mg / kg, adenine 200 mg / kg) to establish a hyperuricemia HUA mouse model. Two hours after gavage with the modeling drug, mice in each group were gavaged with the corresponding drug. The blank control group and model group were given the same dose of 0.5% CMC-Na once a day for 7 consecutive days. After 7 days, oral administration of modeling drugs was stopped and oral administration of corresponding drugs was continued for 7 days. The blank control group and model group were treated in the same way. 2 hours after the last administration on day 14, mice were anesthetized with 2% sodium pentobarbital, blood was collected from the eyeballs, and kidneys were removed for testing.

[0082] (2) Analysis of biochemical indicators related to uric acid metabolism in mouse serum

[0083] Serum uric acid (UA) levels were determined by liquid chromatography. Approximately 100 μL of mouse blood was collected into a blood collection tube and rapidly centrifuged at 15,000 rpm for 10 min at 4°C. Serum was then aspirated and stored at 4°C until further use. Serum UA levels were determined by HPLC using a Waters XBridge HILIC column (4.6 mm × 150 mm, 3.5 μm). Isocratic elution was used with a methanol:ammonium acetate ratio of 80:20 (v:v), an ammonium acetate concentration of 20 mmol / L, a flow rate of 1 mL / min, an injection volume of 10 μL, a column temperature of 35°C, and a detection wavelength of 280 nm.

[0084] The levels of urea nitrogen (BUN) and creatinine (CRE) in serum were determined using a urea nitrogen kit and a creatinine kit according to the kit instructions.

[0085] (3) Mouse kidney index analysis and pathological observation

[0086] Kidney index analysis: Mouse kidneys were removed and weighed, and the weight was calculated according to the following formula: Kidney index (%) = kidney weight (g) / mouse body weight (g) × 100.

[0087] Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned into 4 μm sections. The sections were stained with hematoxylin-eosin, and the histopathological changes were observed under an optical microscope.

[0088] (4) Preparation of the uric acid-lowering active peptide LQKW and verification of its uric acid-lowering efficacy in HUA mice

[0089] The enzymatic hydrolysate of liquor lees was prepared from the lees of Maotai-flavor liquor. When the mass concentration was 6.5 mg / kg, the XOD inhibition rate was 61.15%. The ultrafiltration fractions (>5kDa, 5kDa~1kDa and <1kDa) of the liquor lees enzymatic hydrolysate were collected and the XOD inhibitory activity was compared. The results showed that the XOD inhibitory activity IC 50 The values ​​were (11.74±0.02) mg / mL, (10.47±0.02) mg / mL and (5.70±0.11) mg / mL, respectively. The <1 kDa vinasse active peptide fraction had higher XOD inhibitory activity.

[0090] Uric acid is the end product of purine base metabolism. Potassium oxonate, with its chemical structure similar to the purine ring of uric acid, acts as a uricase inhibitor, competitively inhibiting uricase activity and increasing uric acid production. Potassium oxonate was used to establish a HUA mouse model, and a <1 kDa vinasse active peptide fraction was collected to validate its uric acid-lowering efficacy in HUA mice. Serum uric acid levels in the control, model, and <1 kDa vinasse active peptide fractions were (152.09 ± 12.81), (248.76 ± 25.97), and (175.34 ± 46.97) μmol / L, respectively. Compared with the control group, serum uric acid levels in the model group increased significantly by 63.56% (P < 0.01), indicating successful HUA mouse modeling. Compared with the model group, serum uric acid levels in the <1 kDa vinasse active peptide fraction decreased by 29.51%. These results indicate that the <1 kDa vinasse active peptide fraction has a significant uric acid-lowering effect in chronic HUA mice.

[0091] Example 3

[0092] Sequence analysis and virtual screening of uric acid-lowering peptides

[0093] Mass spectrometry analysis of uric acid-lowering peptides identified a total of 3775 peptides, of which 1988 were less than 10 nucleotides in length, accounting for 52.66% of the total peptides. Peptides with less than 10 nucleotides were screened and bioactivity scores were predicted using the Peptide Ranker program. A total of 1251 peptides with activity scores ≥0.5 were identified, accounting for 33.25% of the total peptides. High-throughput virtual molecular docking using Autodock-Vina software revealed 860 amino acid sequences with docking energies <-7 kcal / mol. Based on these results and screening for potential highly active peptides using amino acids characteristic of inhibitory activity, 12 peptides were identified (Table 1).

[0094] Twelve peptides were synthesized by solid phase chemical synthesis, and the XOD inhibition rate and XOD inhibitory activity IC were determined. 50 There are four peptides with a concentration of less than 4 mg / mL. Among them, the peptide with the best XOD inhibitory activity is LQKW, IC 50 was 2.70 mg / mL, followed by WDLPF, WPQ, WFPE, and IC 50 They are 2.88, 3.51 and 3.71 mg / mL respectively.

[0095] Table 1 Sequences and predicted activities of 12 peptides

[0096]

[0097] Note: Red indicates hydrophobic amino acids or aromatic amino acids.

[0098] Example 4

[0099] Evaluation of uric acid-lowering peptides' tolerance to simulated gastrointestinal digestion in vitro

[0100] In vitro simulated gastrointestinal digestion tolerance of the four peptides Figure 1 The results showed that after gastrointestinal digestion, the XOD inhibitory activities of the four peptides decreased to varying degrees. After simulated gastric digestion, the XOD inhibition rate of LQKW was (74.9±0.88)%, and the activity retention rate was (76.36±0.45)%. After gastrointestinal digestion, the XOD inhibition rate was (76.36±0.45)%, and the activity retention rate was (61.84±0.82)%. The gastrointestinal digestion stability of LQKW was significantly higher than that of the other three peptides (P<0.01), indicating that it has better gastrointestinal digestion tolerance.

[0101] Example 5

[0102] Molecular docking of uric acid-lowering peptide LQKW and receptor XOD

[0103] XOD has a molecular weight of 300kDa and consists of two identical subunits, each of which is composed of three domains: a molybdenum cofactor (molybdopterin, Mo-pt), two iron-sulfur centers (2Fe-2S), and a FAD. The molybdopterin center of Mo-pt is the key site for XOD catalysis, and the surrounding amino acid residues are mainly: Phe649, Asn768, Glu802, Leu873, Arg880, Phe914, Phe1009, Thr1010, Leu1014, and Glu1261. Glu802, Arg880, and Glu1261 are key residues that promote the catalytic reaction. Uric acid-lowering peptides generally bind to the active site of the molybdopterin center, inhibiting XOD activity, reducing substrate hydroxylation, and lowering uric acid production. For semi-flexible docking of LQKW with the receptor XOD, please refer to [ 15 ]. Figure 2 The results showed that LQKW formed seven hydrophobic interactions with Ile648, Phe649, Phe914, Phe1009, Val1011, Phe1013, and Leu1014 in XOD, and formed five hydrogen bonds with Leu648, Glu802, Ser876, Thr1010, and Val1011. LQKW occupied the Mo-pt domain of the active cavity of the 1FIQ drug, involving interactions with six major amino acid residues in XOD. LQKW formed a hydrogen bond with the key residue Glu802. The hydrogen bonds prevent the substrate from entering the hydrophobic channel, thereby blocking the substrate from entering the active center.

[0104] Example 6

[0105] Evaluation of the uric acid-lowering efficacy of LQKW in HUA mice

[0106] (1) Effects of LQKW on renal index in HUA mice

[0107] The enlargement or atrophy of organs can reflect the damage of organs. A decrease in the renal index indicates that the kidneys may have a certain degree of atrophy, while an increase in the renal index indicates that the kidneys may have a certain degree of edema and hyperplasia. Figure 3As shown in the figure, compared with the control group, the renal coefficient and renal index of the model group were significantly increased, indicating that the modeling drug can cause renal enlargement in mice and the HUA mouse model was successfully established. Compared with the model group, the renal coefficients of the AP, LQKW-M, and LQKW-H groups were significantly reduced, with the renal coefficient of LQKW-H mice being the lowest. The effect of LQKW on reducing the renal coefficient of mice was dose-dependent. At the same time, the renal coefficients of the AP, LQKW-M, and LQKW-H groups were not significantly different from those of the control group, indicating that the effect of medium and high doses of LQKW on reducing the renal coefficient of mice is equivalent to that of 10 mg / kg allopurinol. In summary, it can be preliminarily concluded that LQKW can alleviate the renal damage induced by potassium oxonate and adenine in chronic HUA mice.

[0108] (2) Effects of LQKW on serum uric acid UA, serum creatinine CRE, and urea nitrogen BUN levels in HUA mice

[0109] Serum uric acid is the most direct and specific biochemical indicator for assessing the risk of HUA and gout. Figure 4 As shown, compared with the model group, the serum uric acid levels of mice in the AP group and the low-, medium-, and high-dose LQKW groups were significantly reduced (P < 0.01), decreasing by 25.13%, 32.59%, 35.96%, and 37.28%, respectively. There were no significant differences between the AP group and the LQKW group, indicating that both allopurinol and LQKW can reduce uric acid levels in mice with chronic HUA induced by potassium oxonate. The low-, medium-, and high-dose LQKW groups showed superior intervention effects on chronic HUA mice than the AP group, and the uric acid-lowering effect in mice was dose-dependent.

[0110] Serum creatinine (CRE) and blood urea nitrogen (BUN) levels are often used to assess the degree of kidney damage. Figure 5 、 Figure 6 Compared with the control group, serum CRE and BUN levels in the model group were significantly increased (P ≤ 0.0001), indicating that renal function in HUA mice induced by potassium oxonate was impaired. However, these levels decreased to varying degrees in the AP group and the medium- and high-dose LQKW groups. There was no significant difference in CRE levels between the AP group and the LQKW-H group, which were 14.15 and 14.31 μmol / L, respectively. There was no significant difference in BUN levels between the AP group and the medium- and high-dose LQKW groups, which were 5.53, 7.81, and 6.60 mmol / L, respectively. Serum CRE and BUN levels in mice were dose-dependent and correlated with the administered dose.

[0111] (3) Renal pathology examination of hyperuricemia HUA mice treated with different regimens

[0112] See also Figure 7The results of renal tissue pathological changes in hyperuricemia HUA mice under different treatment schemes are shown. Figure 7 As can be seen, the renal corpuscles of the mice in the Control group were round or oval, with normal proximal and distal convoluted tubule sections around them. The renal capsule wrapped around the central glomerulus, and the intercellular space was normal. The glomerular structure of the Model group was disordered, the renal tubular epithelial cells were deformed, shed, and scattered irregularly, the renal tubules were significantly dilated, the tubular lumen was arranged in a disordered manner, and the width of the renal interstitium was significantly increased, and a large number of inflammatory cells were observed to be infiltrated. The pathological changes in the AP group and the LQKW dose groups were improved to varying degrees, the dilation of the renal tubules was reduced, and the glomerular enlargement was reduced. The glomerular morphology was close to normal. Mild inflammatory cell infiltration was observed in the LQKW-L group, and no inflammatory cell infiltration was observed in the AP group and the medium and high dose groups of LQKW. LQKW significantly alleviated the damage of hyperuricemia to the renal tissue of mice.

[0113] The uric acid-lowering peptide LQKW of the present invention can be produced through a mature solid-phase synthesis process with controlled quality. This uric acid-lowering peptide has significant XOD inhibitory activity and good stability, and can be used to develop drugs or functional foods for the treatment of hyperuricemia, with clear clinical applications and market prospects. Furthermore, the method provided by the present invention for screening active peptides from liquor lees provides a new approach for the high-value utilization of winemaking by-products.

[0114] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concepts of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. A uric acid-lowering active peptide, characterized in that: The amino acid sequence of the uric acid-lowering active peptide is Leu-Gln-Lys-Trp.

2. The uric acid-lowering active peptide according to claim 1, characterized in that Its half inhibitory concentration IC 50 It is 2.70mg / mL.

3. The uric acid-lowering active peptide according to claim 1, characterized in that After simulated gastrointestinal digestion in vitro, the XOD inhibitory activity retention rate was (61.84±0.82)%.

4. The uric acid-lowering active peptide according to any one of claims 1 to 3, characterized in that The uric acid-lowering active peptide binds to the active site of XOD through hydrophobic interaction and hydrogen bonds. The uric acid-lowering active peptide forms seven hydrophobic interactions with the active sites Ile648, Phe649, Phe914, Phe1009, Val1011, Phe1013, and Leu1014 in XOD, and forms five hydrogen bonds with the active sites Leu648, Glu802, Ser876, Thr1010, and Val1011 in XOD. The active site includes Ile648, Phe649, Glu802, Phe914, Phe1009, and Leu1014.

5. A pharmaceutical composition comprising the uric acid-lowering peptide according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

6. A method for preparing the uric acid-lowering active peptide according to claim 1, characterized in that: The following steps are involved: S1: Using liquor lees as raw material, the enzymatic hydrolysate was obtained by alkaline protease enzymolysis; S2: ultrafiltration separation of the enzymatic hydrolysate to collect the active peptide component of the lees with a molecular weight of less than 1 kDa; S3: Identify the peptide sequences of the components with a molecular weight of less than 1 kDa by LC-MS, and screen for peptides with a bioactivity score ≥ 0.5 and a docking energy < -7 kcal / mol; S4: Solid phase chemical synthesis of the uric acid-lowering active peptide LQKW.

7. The method according to claim 6, characterized in that The enzymatic hydrolysis conditions in step S1 are as follows: the amount of alkaline protease added is 6000 U / g of vinasse protein, the pH value is 9.5, the temperature is 50° C., and the time is 5 h.

8. Use of the uric acid-lowering peptide according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating hyperuricemia, preferably, the medicament is used to lower serum uric acid levels, improve renal index or alleviate renal pathological damage.

9. Use of the uric acid-lowering peptide according to any one of claims 1 to 4 in the preparation of an XOD inhibitor.

10. A food additive comprising the uric acid-lowering active peptide according to any one of claims 1 to 4, characterized in that: The food additive is used for preparing functional food or dietary supplement.

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