Multifunctional bioactive peptide based on millet protein and preparation method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a multifunctional bioactive peptide based on millet protein and a preparation method thereof, and the amino acid sequence of the bioactive peptide is shown as SEQ ID NO: 1. The bioactive peptide can activate the activity of alcohol dehydrogenase and inhibit the activity of xanthine oxidase and angiotensin converting enzyme, has the characteristics of small molecular weight, easiness in absorption and the like, and has the advantages of no toxicity, no carcinogenicity, high hydrophilicity, longer half-life period and the like through biosignal analysis and prediction; the compound can be used for developing medicines with the disease treatment effects of dispelling the effects of alcohol, reducing uric acid, reducing blood pressure and the like, and has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a multifunctional bioactive peptide based on millet protein and a preparation method thereof. Background Art

[0002] Millet is a cereal with a long history. As a high-quality plant protein source, its protein content accounts for about 9.7% of its dry weight, and it is often used to supplement people's protein needs in their daily diet. In addition, millet is rich in carbohydrates, protein, dietary fiber, B vitamins, various minerals and antioxidant ingredients such as flavonoids. Therefore, millet has many health benefits such as antioxidant, blood sugar regulation, digestion promotion, sleep improvement and cardiovascular protection. Small molecule peptides can be obtained by treating millet protein through enzymatic hydrolysis and other methods. Among them, some peptides have specific biological activities, such as antioxidant, blood pressure lowering, etc. However, there are still problems such as unclear amino acid sequence and efficacy of millet characteristic peptides.

[0003] Therefore, screening out millet peptides with multiple biological activities and clear efficacy is of great significance for the application of millet protein products in the development of medicines, health products or functional foods. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a multifunctional bioactive peptide based on millet protein and a preparation method thereof. The bioactive peptide of the present invention can activate the activity of alcohol dehydrogenase, inhibit the activity of xanthine oxidase and angiotensin converting enzyme, has the characteristics of small molecular weight and easy absorption, and is predicted by bioinformatics analysis to have the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. It can be used to develop drugs for the treatment of diseases such as hangover, lowering uric acid and lowering blood pressure, and has broad application prospects.

[0005] The present invention is based on the inventor's discovery and understanding of the following facts and problems:

[0006] In the process of researching millet bioactive peptides, the inventors screened out a bioactive peptide KDFPF that has the activity of activating alcohol dehydrogenase and inhibiting xanthine oxidase and angiotensin converting enzyme through a large amount of virtual screening and experimental verification. The amino acid sequence of this bioactive peptide is: lysine-aspartic acid-phenylalanine-proline-phenylalanine (Lys-Asp-Phe-Pro-Phe, KDFPF); the inventors further prepared the bioactive peptide by the Fmoc solid phase synthesis method, and verified through experiments that it has the expected biological activity of activating alcohol dehydrogenase and inhibiting xanthine oxidase and angiotensin converting enzyme.

[0007] In the first aspect of the present invention, the present invention proposes a bioactive peptide. According to an embodiment of the present invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1. The bioactive peptide of the present invention can activate the activity of alcohol dehydrogenase, inhibit the activity of xanthine oxidase and angiotensin converting enzyme, has the characteristics of small molecular weight and easy absorption, and is predicted by bioinformatics analysis to have the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life, and can be used to develop drugs for the treatment of diseases such as alcohol sobering, lowering uric acid and lowering blood pressure, and has broad application prospects.

[0008] In the second aspect of the present invention, the present invention proposes a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the bioactive peptide described in the first aspect. According to an embodiment of the present invention, the nucleic acid molecule encodes the bioactive peptide of the first aspect, which can activate alcohol dehydrogenase activity, inhibit xanthine oxidase and angiotensin converting enzyme activity, has the characteristics of small molecular weight and easy absorption, and is predicted by bioinformatics analysis to have the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The nucleic acid molecule can further achieve efficient expression of the bioactive peptide, help to achieve large-scale industrial production of the bioactive peptide, and provide convenience for the subsequent development and application of related drugs such as alcohol sobering, lowering uric acid and lowering blood pressure.

[0009] In the third aspect of the present invention, the present invention provides a construct. According to an embodiment of the present invention, it includes the nucleic acid molecule described in the second aspect. According to an embodiment of the present invention, the construct enables the nucleic acid molecule to be efficiently expressed in a host cell, thereby achieving stable and efficient production of the aforementioned bioactive peptide.

[0010] In the fourth aspect of the present invention, the present invention proposes a recombinant cell. According to an embodiment of the present invention, it includes the nucleic acid molecule described in the second aspect or the construct described in the third aspect. According to an embodiment of the present invention, the recombinant cell can efficiently express the aforementioned bioactive peptide under appropriate conditions, and the bioactive peptide can activate the activity of alcohol dehydrogenase, inhibit the activity of xanthine oxidase and angiotensin converting enzyme, has the characteristics of small molecular weight and easy absorption, and is predicted by bioinformatics analysis to have the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The recombinant cell can achieve efficient expression of the bioactive peptide, which is conducive to its large-scale industrial production.

[0011] In the fifth aspect of the present invention, the present invention proposes the use of the bioactive peptide described in the first aspect, the nucleic acid molecule described in the second aspect, the construct described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of a drug. According to an embodiment of the present invention, the drug has at least one of the following uses: sobering up; preventing and / or treating alcohol poisoning; preventing and / or treating alcohol-induced liver damage; lowering uric acid; preventing and / or treating gout; lowering blood pressure; preventing and / or treating hypertension.

[0012] Those skilled in the art will appreciate that the features and advantages described above for the bioactive peptides are also applicable to this use and will not be described in detail here.

[0013] In the sixth aspect of the present invention, the present invention provides a pharmaceutical composition, which, according to an embodiment of the present invention, comprises: one or more of the bioactive peptide described in the first aspect, the nucleic acid molecule described in the second aspect, the construct described in the third aspect, and the recombinant cell described in the fourth aspect.

[0014] Those skilled in the art will appreciate that the features and advantages described above for the bioactive peptides, nucleic acid molecules, constructs or recombinant cells are also applicable to this use and will not be described in detail here.

[0015] According to an embodiment of the present invention, the above-mentioned pharmaceutical composition may also have the following additional technical features:

[0016] According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

[0017] In the seventh aspect of the present invention, the present invention provides a method for increasing the activity of alcohol dehydrogenase in a sample and inhibiting the activity of xanthine oxidase and / or angiotensin converting enzyme in the sample. According to an embodiment of the present invention, the method comprises: contacting the sample with the bioactive peptide described in the first aspect. According to the method of an embodiment of the present invention, the activity of alcohol dehydrogenase in the sample is increased and the activity of xanthine oxidase and / or angiotensin converting enzyme in the sample is inhibited by co-culturing the sample with the bioactive peptide of the present invention.

[0018] In an eighth aspect of the present invention, the present invention provides an alcohol dehydrogenase activator. According to an embodiment of the present invention, the activator comprises: the bioactive peptide described in the first aspect. The activator according to an embodiment of the present invention can specifically activate the activity of alcohol dehydrogenase through the bioactive peptide described in the first aspect.

[0019] In the ninth aspect of the present invention, the present invention provides a xanthine oxidase and / or angiotensin converting enzyme inhibitor. According to an embodiment of the present invention, it includes: the bioactive peptide described in the first aspect. The activator according to an embodiment of the present invention can specifically inhibit the activity of xanthine oxidase and / or angiotensin converting enzyme through the bioactive peptide described in the first aspect.

[0020] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0022] Figure 1 This is a graph showing the HPLC test results of the pure sample of the biologically active peptide KDFPF in Example 1 of the present invention;

[0023] Figure 2 This is a graph showing the MS detection results of the pure sample of the biologically active peptide KDFPF in Example 1 of the present invention;

[0024] Figure 3 This is a diagram showing the docking of the biologically active polypeptide KDFPF and the alcohol dehydrogenase molecule in Example 2 of the present invention;

[0025] Figure 4 This is a diagram showing the docking of the biologically active polypeptide KDFPF and the xanthine oxidase molecule in Example 2 of the present invention;

[0026] Figure 5 This is a schematic diagram of the docking of the biologically active polypeptide KDFPF and the angiotensin converting enzyme molecule in Example 2 of the present invention;

[0027] Figure 6 These are the evaluation results of the alcohol-relieving, uric acid-lowering and blood pressure-lowering functions of the biologically active polypeptide KDFPF in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0030] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0031] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0032] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0033] Terms and Definitions

[0034] In this article, the term "alcohol dehydrogenase" is the rate-limiting enzyme in the normal human body's metabolism of alcohol. It can catalyze the dehydrogenation of alcohol to produce acetaldehyde, which is further catalyzed by acetaldehyde dehydrogenase to produce acetic acid, which is harmless to humans, and finally decomposes into water and carbon dioxide and is excreted from the body. Activating the activity of acetaldehyde dehydrogenase can not only speed up alcohol metabolism and the excretion of alcohol and metabolic intermediates from the body, but also reduce the adverse effects of alcohol on the liver.

[0035] In this article, the term "xanthine oxidase" is a key enzyme in purine catabolism, which catalyzes hypoxanthine to produce xanthine and then uric acid. At the same time, excessive xanthine oxidase levels can also lead to uric acid deposition in the body. Xanthine oxidase inhibitors can block the biosynthesis of uric acid. Therefore, xanthine oxidase is an important therapeutic target for the treatment of gout.

[0036] In this article, the term "angiotensin converting enzyme" can be involved in the regulation of blood pressure in the human body, and plays a role in promoting blood pressure increase in the renin-angiotensin system and kinin-kinin generating enzyme system that control blood pressure in the human body.

[0037] Bioactive Peptides

[0038] The present invention proposes a bioactive peptide. According to an embodiment of the present invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1. The bioactive peptide of the present invention can activate alcohol dehydrogenase activity, inhibit xanthine oxidase and angiotensin converting enzyme activity, has the characteristics of small molecular weight and easy absorption, and is predicted by bioinformatics analysis to have the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. It can be used to develop drugs with the effects of treating diseases such as sobering up, lowering uric acid and lowering blood pressure, and has broad application prospects.

[0039] According to an embodiment of the present invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO:1.

[0040] KDFPF (SEQ ID NO: 1).

[0041] It should be noted that the amino acid sequences mentioned in the present invention are all shown in the form of N-terminus to C-terminus.

[0042] Exemplarily, the bioactive peptides of the present invention have alcohol dehydrogenase activating activity, and thus can be further used to develop drugs with sobering efficacy, for preventing and / or treating alcoholism, and preventing and / or treating alcohol-induced liver damage; the bioactive peptides of the present invention have xanthine oxidase inhibitory activity, and thus can be further used to develop drugs for preventing and / or treating wind; the bioactive peptides of the present invention have angiotensin converting enzyme inhibitory activity, and thus can be further used to develop drugs for preventing and / or treating hypertension.

[0043] Nucleic acid molecules

[0044] The present invention proposes a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned bioactive peptide. According to an embodiment of the present invention, the aforementioned bioactive peptide encoded by the nucleic acid molecule can activate alcohol dehydrogenase activity, inhibit xanthine oxidase and angiotensin converting enzyme activity, has the characteristics of small molecular weight and easy absorption, and is predicted by bioinformatics analysis to have the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The nucleic acid molecule can further realize the efficient expression of the bioactive peptide, which is helpful to realize the large-scale industrial production of the bioactive peptide, and provides convenience for the subsequent development and application of related drugs such as alcohol sobering, lowering uric acid and lowering blood pressure.

[0045] It should be noted that, for the nucleic acid molecules mentioned in this article, those skilled in the art should understand that they actually include any one or both of the complementary double strands; although in most cases only one strand is given, the other strand complementary to it is actually also disclosed; in addition, the nucleic acid molecule sequence in the present invention includes DNA form or RNA form, and disclosing one of them means that the other is also disclosed.

[0046] Construct

[0047] The present invention provides a construct. According to an embodiment of the present invention, it includes the aforementioned nucleic acid molecule. According to an embodiment of the present invention, the construct enables the nucleic acid molecule to be efficiently expressed in a host cell, thereby achieving stable and efficient production of the aforementioned bioactive peptide.

[0048] Exemplarily, the construct may be a vector; it should be noted that the vector mentioned in this article refers to a nucleic acid molecule that can be inserted into a suitable host and self-replicate, which transfers the inserted nucleic acid molecule into the host cell and / or between host cells; the vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for the transcription and / or translation of DNA or RNA; the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell; by culturing a suitable host cell containing the vector, the vector can produce the aforementioned biologically active peptide, and the vector includes a viral vector, a plasmid, a bacteriophage, etc.

[0049] Recombinant cells

[0050] The present invention proposes a recombinant cell. According to an embodiment of the present invention, it includes the aforementioned nucleic acid molecule or the aforementioned construct. According to an embodiment of the present invention, the recombinant cell can efficiently express the aforementioned bioactive peptide under appropriate conditions, and the bioactive peptide can activate alcohol dehydrogenase activity, inhibit xanthine oxidase and angiotensin converting enzyme activity, has the characteristics of small molecular weight and easy absorption, and is predicted by bioinformatics analysis to have the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The recombinant cell can achieve efficient expression of the bioactive peptide, which is conducive to its large-scale industrial production.

[0051] application

[0052] The present invention proposes the use of the aforementioned bioactive peptide, the aforementioned nucleic acid molecule, the aforementioned construct or the aforementioned recombinant cell in the preparation of a drug. According to an embodiment of the present invention, the drug has at least one of the following uses: sobering up; preventing and / or treating alcohol poisoning; preventing and / or treating alcohol-induced liver damage; lowering uric acid; preventing and / or treating gout; lowering blood pressure; preventing and / or treating hypertension.

[0053] Those skilled in the art will appreciate that the features and advantages described above for the bioactive peptides are also applicable to this use and will not be described in detail here.

[0054] Pharmaceutical composition

[0055] The present invention provides a pharmaceutical composition, which, according to an embodiment of the present invention, comprises: one or more of the aforementioned bioactive peptide, the aforementioned nucleic acid molecule, the aforementioned construct, and the aforementioned recombinant cell.

[0056] Those skilled in the art will appreciate that the features and advantages described above for the bioactive peptides, nucleic acid molecules, constructs or recombinant cells are also applicable to this use and will not be described in detail here.

[0057] According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable excipients. Thus, by adding the excipients, the pharmaceutical composition can better exert the efficacy of the bioactive peptide.

[0058] Exemplarily, the excipients include excipients, diluents, stabilizers, etc.; it should be noted that pharmaceutically acceptable excipients may include any solvents, solid excipients, diluents or other liquid excipients, etc., suitable for specific target dosage forms, except for any conventional excipients that are incompatible with the functional ingredients in the pharmaceutical composition of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of consideration of the present invention.

[0059] method

[0060] The present invention provides a method for increasing the activity of alcohol dehydrogenase in a sample and inhibiting the activity of xanthine oxidase and / or angiotensin converting enzyme in the sample. According to an embodiment of the present invention, the method includes: contacting the sample with the aforementioned bioactive peptide. According to the method of an embodiment of the present invention, the activity of alcohol dehydrogenase in the sample is increased and the activity of xanthine oxidase and / or angiotensin converting enzyme in the sample is inhibited by co-culturing the sample with the bioactive peptide of the present invention.

[0061] It should be noted that the "sample" of the present invention refers to a sample that contains or may contain alcohol dehydrogenase, xanthine oxidase or angiotensin-converting enzyme; illustratively, the sample can be from a healthy individual or a patient or a subject, including but not limited to blood, urine, saliva, feces, tissue samples, cell samples, cerebrospinal fluid, semen, amniotic fluid or other body fluids such as joint cavity fluid, pleural effusion, etc.; it can also be derived from other biological materials, including but not limited to cells and microorganisms obtained by biotechnology means.

[0062] Activator

[0063] The present invention provides an alcohol dehydrogenase activator. According to an embodiment of the present invention, the activator comprises: the aforementioned bioactive peptide. The activator according to the embodiment of the present invention can specifically activate the activity of alcohol dehydrogenase through the aforementioned bioactive peptide.

[0064] Inhibitors

[0065] The present invention provides a xanthine oxidase and / or angiotensin converting enzyme inhibitor. According to an embodiment of the present invention, it includes: the aforementioned bioactive peptide. The activator according to an embodiment of the present invention can specifically inhibit the activity of xanthine oxidase and / or angiotensin converting enzyme through the aforementioned bioactive peptide.

[0066] The amino acid sequences involved in the present invention are shown in Table 1.

[0067] Table 1

[0068] SEQ ID NO: sequence 1 KDF 2 WLn 3 LLNPF 4 LLFRL 5 FLPLH 6 FNPTLP 7 APHWNL 8 YLTP 9 FDPVTWR 10 FGLPGPK

[0069] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0070] Example 1: Isolation and preparation of biologically active polypeptide KDFPF

[0071] 1. Preparation of millet protein

[0072] A sufficient amount of millet (purchased from Shanxi Dongfangliang Life Science Co., Ltd.) is soaked in water at a ratio of 1:2 (w / v), and after soaking for 2 hours at room temperature, the soaked millet is ground into pulp, and water equal to the mass of the millet is added during the grinding process, and the millet is filtered with a 100-mesh sieve after grinding to obtain millet pulp; the millet pulp is adjusted to pH = 10 with a 4% sodium hydroxide solution, and alkali-dissolved for 3 hours under the condition of 20-30°C to obtain alkali-soluble millet pulp; the alkali-soluble millet pulp is then centrifuged at a speed of 3500r / min for 5min to separate it into a liquid phase layer, a protein solid phase layer and a starch solid phase layer, and the liquid phase layer and the protein solid phase layer are collected to obtain millet protein pulp; then the millet protein pulp is adjusted to pH = 7 with a 4% hydrochloric acid solution; the millet protein pulp is freeze-dried to obtain millet protein, and stored at -20°C for standby use.

[0073] 2. Enzymatic hydrolysis of millet protein

[0074] The millet protein obtained in step 1 is mixed evenly in a 35 mM sodium phosphate buffer at a ratio of 5% (w / v); the pH is adjusted to 7.0 using NaOH and HCL, and then alkaline protease (purchased from Sigma; Sigma-Aldrich, St. Louis, MO, USA) is added at an enzyme-substrate ratio of 1:20 (w:w) to obtain a millet protein hydrolysate; the millet protein hydrolysate is incubated at 50°C for 120 minutes, and then placed in a boiling water bath for 10 minutes to terminate the enzymatic reaction; after the millet protein hydrolysate is cooled to room temperature, it is centrifuged at 4°C and 10,000×g for 15 minutes, and the supernatant is collected to obtain a millet protein hydrolysate solution for later use.

[0075] 3. Isolation, purification and sequence identification of peptides

[0076] The 12 mL millet protein hydrolysate solution obtained in step 1 was transferred to a 3 kDa centrifugal ultrafiltration tube, and centrifuged at 4°C and 5000×g for 30 min to obtain a millet protein hydrolysate sample with a molecular weight of less than 3 kDa; the millet protein hydrolysate sample with a molecular weight of less than 3 kDa was desalted using a C18 desalting column to obtain a desalted millet protein hydrolysate sample with a molecular weight of less than 3 kDa; and the sample was subjected to LC-MS / MS analysis equipped with an online nanospray ion source. The system used for the analysis was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200. The specific LC-MS / MS analysis steps were as follows:

[0077] 1 μL of desalted millet protein hydrolysate sample of the molecular weight <3 kDa fraction was loaded (analytical column: Acclaim PepMap C18, 75 μm×25 cm), and the loaded sample was separated by a gradient of 60 min, column flow rate: 400 nL / min, column temperature: 40°C, electrospray voltage: 2 kV; mobile phase A: 0.1% formic acid aqueous solution, phase B: 80% ACN solution containing 0.1% formic acid, the gradient started from 0% phase B, increased to 60% in 46 min with a nonlinear gradient, increased to 100% within 4 min, and maintained for 10 min.

[0078] The Q-Exactive Plus mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition, and the mass spectrometry parameters were set as follows:

[0079] (1)MS

[0080] Scan range (m / z): 200-2000; resolution: 70000; AGC target: 3e6; maximum injection time: 60ms;

[0081] (2) HCD-MS / MS

[0082] Resolution: 17500; AGC target: 5e4; Maximum injection time: 80ms; Collision energy: 27; Dynamic exclusion time: 20s.

[0083] The obtained tandem mass spectrum was then analyzed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada), the database was uniprot-Hordeum_vulgare_subsp_vulgare (version 2023, 34528 entries), and none enzymatic digestion was set; the search parameters were fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm; variable modification: Oxidation (M) 15.99, peptide card value -10lgP ≥ 20; for peptides not retrieved in the database, ALC (%) ≥ 80 was set to obtain the target peptide sequence.

[0084] Some target peptide sequences are shown in Table 2.

[0085] Table 2 Some target peptide sequences

[0086] Target peptide sequence Sequence number KDF SEQ ID NO: 1 WLn SEQ ID NO: 2 LLNPF SEQ ID NO: 3 LLFRL SEQ ID NO: 4 FLPLH SEQ ID NO: 5 FNPTLP SEQ ID NO: 6 APHWNL SEQ ID NO: 7 YLTP SEQ ID NO: 8 FDPVTWR SEQ ID NO: 9 FGLPGPK SEQ ID NO: 10

[0087] 4. Virtual screening of millet functional peptides

[0088] First, the PeptideRanker online system (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.Php) was used to predict the biological activity of the target peptide sequence obtained in step 3; then, ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to predict the water solubility and toxicity of the target peptide sequence obtained in step 3; then, Expasy (https: / / web.expasy.org / protparam / ) was used to analyze the isoelectric point and half-life of the target peptide sequence obtained in step 3; finally, admetSAR (http: / / lmmd.ecust.edu.cn / adm etsar1 / predict / ) was used to predict the human intestinal absorption and carcinogenicity of the target peptide sequence obtained in step 3.

[0089] The better results of in vitro function prediction of millet peptide virtual screening are shown in Table 2.

[0090] Table 2 The better results of in vitro function prediction of millet peptide virtual screening

[0091] Millet peptide sequence peptideranker Molar weight toxicity Carcinogenicity Hydrophilicity half life Isoelectric point KDF 0.952122 653.36 Non-toxic none 0.2 1.3 hours 5.84 WLn 0.834327 664.88 Non-toxic none -1.5 2.8 hours 6.74 LLNPF 0.834233 602.8 Non-toxic none -1.18 5.5 hours 5.52 LLFRL 0.833998 660.93 Non-toxic none -0.98 5.5 hours 9.75 FLPLH 0.833814 625.84 Non-toxic none -1.32 1.1 hours 6.74 FNPTLP 0.833778 687.87 Non-toxic none -0.75 1.1 hours 5.52 APHWNL 0.833448 851.03 Non-toxic none -0.83 4.4 hours 6.79 YLTP 0.833399 639.81 Non-toxic none -1.4 2.8 hours 5.52 FDPVTWR 0.833127 920.12 Non-toxic none -0.26 1.1 hours 5.84 FGLPGPK 0.832603 714.96 Non-toxic none -0.19 1.1 hours 8.75

[0092] The results showed that 10 millet peptides with biological activities greater than 0.8 were screened, among which KDFPF had good biological activity, human intestinal absorption and low toxicity, and a long half-life. KDFPF was selected for subsequent experiments.

[0093] 5. Artificial synthesis of bioactive peptide KDFPF

[0094] The inventors prepared the bioactive peptide KDFPF using the Fmoc solid phase synthesis method, and the specific steps are as follows:

[0095] (1) Solvent pretreatment

[0096] Before use, N,N-dimethylformamide (DMF) and methanol were soaked overnight using G3 pore molecular sieves to remove impurities and moisture in the solvent.

[0097] (2) Resin swelling

[0098] Weigh 2.0 g of blank Wang resin into a clean and dry reaction tube, add 15 mL of DMF, and activate at room temperature for 30 min.

[0099] (3) Insertion of the first amino acid

[0100] At room temperature, the DMF in step 2 was removed by sand core filtration, and 1 mmol of a 5-fold molar excess of the first amino acid (phenylalanine) at the C-terminus of the target sequence, as well as a 5-fold molar excess of 4-dimethylaminopyridine (DMAP), a 5-fold molar excess of N,N-diisopropylcarbodiimide (DIC), and 60 ml of DMF were added as solvent, and the reaction was carried out at room temperature for 3 hours; after the reaction was completed, the mixture was washed with DMF 5 times, 6 ml each time; then 6 ml of pyridine and acetic anhydride in a volume ratio of 1:1 were added, and the reaction was carried out for 30 minutes; after the reaction was completed, the mixture was washed with DMF 5 times, 6 ml each time.

[0101] (4) Removal of Fmoc protecting group

[0102] The solvent in step 3 was removed by sand core filtration, 10 mL of 20% piperidine DMF solution was added to the above resin, and the solution was filtered out after stirring for 10 min under N2 protection; 10 mL of 20% piperidine DMF solution was added again, and the solution was filtered out again after blowing and stirring for 5 min under N2 protection. After repeating the above operation twice, the resin was washed 4 times with DMF and 2 times with methanol, each time with 6 mL.

[0103] (5) Ninhydrin to detect the removal effect of Fmoc protecting group

[0104] Take out a small amount of resin treated in step 4, wash it three times with methanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 110°C for 5 min, and turn dark blue for a positive reaction, indicating that the Fmoc protecting group is completely removed, and the next step of reaction can be performed; if it is colorless, it means that the protecting group is not completely removed, and it is necessary to repeat the above step (4) to remove the Fmoc protecting group.

[0105] (6) Insertion of the second amino acid and removal of the Fmoc protecting group

[0106] Weigh a 3-fold molar excess of the second amino acid at the C-terminus of the target sequence, a 3-fold molar excess of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and a 3-fold molar excess of 1-hydroxybenzotriazole (HOBT) into the above reaction tube, add an appropriate amount of DMF to completely dissolve it; then add a 10-fold molar excess of N,N-diisopropylethylamine (DIPEA), react at room temperature for 40 minutes, and after the reaction is completed, wash with DMF 5 times, 6 mL each time.

[0107] (7) Reintroduction of amino acids

[0108] Repeat the operation described in step (6) until the last amino acid at the N-terminus of the target sequence is synthesized, remove the Fmoc protecting group and then drain the solution to obtain a resin containing the target bioactive peptide KDFPF.

[0109] (8) Resin shedding and separation and detection of pure bioactive peptide KDFPF

[0110] Cutting: Add 6 times the volume of resin cutting solution (97.5% trifluoroacetic acid), shake on a shaker for 2 hours, filter out the resin, precipitate the filtrate with anhydrous ether, and wash the precipitate with anhydrous ether 3 times. Finally, put the precipitate in a vacuum drying kettle and dry it at room temperature for 24 hours. Then desalt and purify the crude polypeptide by HPLC, freeze-dry and precipitate crystals, which is the pure product of the biologically active peptide KDFPF, and store it at -20°C for future use.

[0111] (9) Quality testing of pure bioactive peptide KDFPF

[0112] A small amount of the pure sample of the bioactive peptide KDFPF obtained in step (8) is taken and dissolved in a trifluoroacetic acid solution by means of ultrasound to obtain a test solution; the test solution is placed in a high performance liquid chromatograph for testing to perform a quality test on the pure sample of the bioactive peptide KDFPF.

[0113] Among them, the HPLC parameters are set as follows:

[0114] Chromatographic column: ZORBAX SB-C18, 4.6×250mm, 5μm; aqueous phase: 100% water plus 0.1% trifluoroacetic acid; organic phase: 100% acetonitrile plus 0.1% trifluoroacetic acid; flow rate: 1mL / min; injection volume: 10μL; detection wavelength: 220nm;

[0115] Among them, the MS parameters are set as follows:

[0116] Ion source: electrospray ionization source (ESI source); nebulizer gas flow rate: 1.5 L / min; CDL: -20.0 V; CDL temperature: 250°C; heating block temperature: 200°C; ion source voltage: +4.5 kV; detector voltage: 1.5 kV; mobile phase flow rate: 0.2 mL / min; mobile phase ratio: 50% H2O / 50% ACN.

[0117] The HPLC test results of the pure sample of the biologically active peptide KDFPF are shown in Figure 1 The MS test results of the pure sample of the biologically active peptide KDFPF are shown in Figure 2 .

[0118] The results showed that: the purity of the pure product of the bioactive peptide KDFPF obtained in step (8) was greater than 95% as determined by HPLC; and the polypeptide sequence of the pure product of the bioactive peptide KDFPF obtained in step (8) was the target sequence KDFPF as determined by MS.

[0119] Example 2: Molecular docking and interaction force analysis of the biologically active peptide KDFPF

[0120] The inventors used the bioactive peptide KDFPF screened out in Example 1 as a ligand, and alcohol dehydrogenase, xanthine oxidase, and angiotensin-converting enzyme as receptors, and used molecular docking technology to clarify the action site and interaction force between the peptide KDFPF and the acting enzyme, thereby achieving theoretical verification of its functions of sobering up, lowering uric acid, and lowering blood pressure.

[0121] (1) Alcohol dehydrogenase

[0122] The three-dimensional structure of the bioactive peptide KDFPF was created using Chimera 1.16; and the alcohol dehydrogenase (PDB number: 5ENV) with water molecules and other ligands removed was obtained from the PDB database (http: / / www.rcsb.org / ) for molecular docking; then the bioactive peptide KDFPF was molecularly docked with the enzyme using ZDOCK, and after the best binding conformation was screened, the binding force between the bioactive peptide KDFPF and the enzyme was analyzed by PDBePISA, and the interaction force was further analyzed in detail using LigPlus+; then the binding of the ligand (bioactive peptide KDFPF) and the receptor (alcohol dehydrogenase) was visualized using PyMOL software.

[0123] Molecular docking of biologically active peptide KDFPF and alcohol dehydrogenase Figure 3 .

[0124] The results showed that the molecular binding energy between the bioactive peptide KDFPF and the amino acid residues of alcohol dehydrogenase was -3.2 kcal / mol, and the binding between the two mainly depended on hydrophobic interactions and hydrogen bonds. Specifically, the amino acid residues Phe3 (C), Pro4 (C), and Phe5 (C) of the bioactive peptide KDFPF formed significant hydrophobic interactions with the amino acid residues Pro24 (A), Pro26 (A), Thr130 (A), Val25 (A), Ile10 (A), and His214 (A) of alcohol dehydrogenase, while Asp2 (C) and Lys59 (A) formed hydrogen bonds (hydrogen bond distance of ); Therefore, it is speculated that the biologically active polypeptide KDFPF tightly binds to alcohol dehydrogenase through the synergistic effect of hydrophobic interaction and hydrogen bonding, thereby activating the enzyme activity.

[0125] The above results indicate that the bioactive polypeptide KDFPF has a good affinity with alcohol dehydrogenase, and it is speculated that it has a good alcohol sobering function.

[0126] (2) Xanthine oxidase

[0127] Xanthine oxidase (3NRZ) was obtained from the PDB database (http: / / www.rcsb.org / ), and PyMOL was used to remove water molecules, metal atoms and co-crystallized ligands; then Chimera 1.16 was used to create the three-dimensional structure of the bioactive peptide KDFPF, and ZDOCK was used to dock the bioactive peptide KDFPF with the enzyme. After the best binding conformation was screened, the binding force between the bioactive peptide KDFPF and the enzyme was analyzed by PDBePISA, and LigPlus+ was used to further analyze the interaction force in detail; then PyMOL software was used to visualize the binding of the ligand (bioactive peptide KDFPF) and the receptor (xanthine oxidase).

[0128] Molecular docking of biologically active peptide KDFPF and xanthine oxidase Figure 4 .

[0129] The results showed that the molecular binding energy between the bioactive peptide KDFPF and the amino acid residues of xanthine oxidase was -7.4 kcal / mol, and the binding between the two mainly depended on hydrophobic interactions and hydrogen bonds. Specifically, the Phe3(C), Pro4(C), and Phe5(C) amino acid residues of the peptide formed significant hydrophobic interactions with the enzyme's Leu744(A), Gln585(A), Gly796(A), Val1200(A), Phe798(A), and Ile1229(A) amino acid residues, while the amino acid residues Asp2(C) and Lys1(C) formed two hydrogen bonds with Ile1235(A). Therefore, it is speculated that the bioactive peptide KDFPF tightly binds to xanthine oxidase through the synergistic effect of hydrophobic interactions and hydrogen bonds, thereby inhibiting the activity of xanthine oxidase.

[0130] The above results indicate that the bioactive polypeptide KDFPF has a good affinity with xanthine oxidase, and it is speculated that it has a good uric acid-lowering function.

[0131] (3) Angiotensin-converting enzyme

[0132] Angiotensin-converting enzyme (PDB ID: 1O86) was purified by PyMOL to remove water molecules and other impurities. ZDOCK was used to dock the bioactive peptide KDFPF with the enzyme. After the optimal binding conformation was screened, the binding force between the bioactive peptide KDFPF and the enzyme was analyzed by PDBePISA, and LigPlus+ was used to further analyze the interaction force in detail. PyMOL software was then used to visualize the binding of the ligand and receptor.

[0133] Molecular docking of biologically active peptide KDFPF and angiotensin converting enzyme Figure 5.

[0134] The results showed that the molecular binding energy between the bioactive peptide KDFPF and the amino acid residues of angiotensin converting enzyme was -6.6 kcal / mol, and the binding between the two mainly depended on hydrophobic interactions and hydrogen bonds. Specifically, the Phe3(B), Pro4(B), and Phe5(B) amino acid residues of the bioactive peptide KDFPF formed significant hydrophobic interactions with the Tyr520(A), Tyr523(A), Phe512(A), Phe457(A), Glu411(A), His513(A), His353(A), Thr282(A), His383(A), and His387(A) amino acid residues of the enzyme, while the amino acid residues Asp2(B) and Lys1(B) formed hydrogen bonds with Lys511(A), Glu162(A). In summary, it was speculated that the bioactive peptide KDFPF tightly bound to angiotensin converting enzyme through the synergistic effect of hydrophobic interactions and hydrogen bonds, thereby inhibiting the activity of angiotensin converting enzyme.

[0135] The above results indicate that the bioactive polypeptide KDFPF has a good affinity with angiotensin converting enzyme, and it is speculated that it has a good blood pressure lowering function.

[0136] Example 3: Experimental verification of the biologically active peptide KDFPF's ability to sober up, lower uric acid and lower blood pressure

[0137] 1. Determination of alcohol dehydrogenase activation rate of biologically active peptide KDFPF

[0138] 1.5 mL of sodium pyrophosphate buffer solution with pH=8.8, 0.5 mL of 8 mg / mL pure solution of the bioactive peptide KDFPF obtained in Example 1 (blank control group: replaced with 0.5 mL of distilled water), 1 mL of coenzyme solution and 0.1 mL of distilled water were mixed and incubated in a 25°C water bath for 5 min, then 0.1 mL of 0.25 U / mL alcohol dehydrogenase solution was added and timing was started; the absorbance value at 340 nm was read every 1 min for 10 min until the absorbance value reached stability; wherein, the alcohol dehydrogenase activity was calculated according to Formula 1, and the enzyme activity activation rate was calculated according to Formula 2:

[0139]

[0140] In formula 1: E340 is the increase in absorbance at 340 nm per minute; Ew is the amount of enzyme in each milliliter of ethanol dehydrogenase solution (mg / mL); 6.22 is the molar absorbance coefficient of NADH (coenzyme); 3.2 is the total volume of the reaction solution (mL);

[0141]

[0142] In formula 2: H: enzyme activity activation rate (%); A: enzyme activity of blank reaction solution (control group) (U / mg); B: enzyme activity of bioactive peptide KDFPF (experimental group) (U / mg).

[0143] 2. Determination of xanthine oxidase inhibition rate of biologically active peptide KDFPF

[0144] After incubating 50 μL of a 10 mg / mL pure solution of the bioactive peptide KDFPF obtained in Example 1 (the blank control group was a 0.2 mol / L pH=7.5 phosphate buffer) and 50 μL of a xanthine oxidase solution at 37°C for 5 min, 150 μL of a xanthine solution was added; the absorbance value was recorded every 20 s at 290 nm using an ELISA reader, and the change in the absorbance value within 10 min was continuously recorded, wherein the xanthine oxidase activity inhibition rate was calculated according to Formula 3:

[0145]

[0146] In Formula 3: A: reaction rate of 0.2 mol / L pH=7.5 phosphate buffer (blank control group); B: reaction rate of pure reaction solution sample of bioactive peptide KDFPF obtained in Example 1 (experimental group).

[0147] 3. Determination of angiotensin converting enzyme inhibition rate of biologically active peptide KDFPF

[0148] 15 μL of the 5 mg / mL pure solution of the bioactive peptide KDFPF obtained in Example 1 was incubated with 10 μL of angiotensin converting enzyme (100 mU / mL) at 37°C for 10 min to obtain a mixed solution; then the mixed solution was mixed with 50 μL of a substrate solution containing 5 mM hippurylhistidylleucine and 100 mM sodium borate buffer (pH=8.3, 300 mM sodium chloride) and incubated at 37°C for 60 min, and then 75 μL of 1 M hydrochloric acid, 150 μL of pyridine and 75 μL of benzenesulfonyl chloride were added to obtain a mixed test solution; then the mixed test solution was vortexed for 1 min and immediately cooled in an ice bath; finally, the amount of hippuric acid released was measured at 410 nm using an ELISA instrument, wherein the angiotensin converting enzyme activity inhibition rate was calculated according to Formula 4:

[0149]

[0150] In formula 4: A is the absorbance of the control group (the reaction solution sample of the pure bioactive peptide KDFPF is replaced by 0.2 mol / L, pH=8.3 sodium borate buffer); B is the absorbance of the control blank group (the angiotensin converting enzyme solution is replaced by sodium borate buffer); C is the absorbance value of the reaction solution sample of the pure bioactive peptide KDFPF (experimental group).

[0151] The evaluation results of the alcohol-removing, uric acid-lowering and blood pressure-lowering functions of the bioactive peptide KDFPF are shown in Figure 6 .

[0152] The results showed that the alcohol dehydrogenase activation rate of the bioactive peptide KDFPF obtained in Example 1 was 124.14±2.97%, which could exert a good alcohol sobering effect; the xanthine oxidase inhibition rate of the bioactive peptide KDFPF obtained in Example 1 was 91.40±3.10%, which could exert a good uric acid lowering activity, and thus could be used to treat hyperuricemia or gout; the angiotensin converting enzyme inhibition rate of the bioactive peptide KDFPF obtained in Example 1 was 95.48±3.05%, which could exert a good blood pressure lowering effect, and thus could be used to treat diseases such as hypertension.

[0153] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0154] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A bioactive peptide, characterized in that: The amino acid sequence of the bioactive peptide is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the biologically active peptide according to claim 1.

3. A construct, characterized in that Comprising the nucleic acid molecule according to claim 2.

4. A recombinant cell, characterized in that It comprises the nucleic acid molecule according to claim 2 or the construct according to claim 3.

5. Use of the bioactive peptide according to claim 1, the nucleic acid molecule according to claim 2, the construct according to claim 3 or the recombinant cell according to claim 4 in the preparation of a drug, characterized in that: The drug has at least one of the following uses: hangover relief; prevention and / or treatment of alcoholism; Prevent and / or treat alcohol-induced liver damage; Lowers uric acid; Prevent and / or treat gout; Lowers blood pressure; Prevent and / or treat high blood pressure.

6. A pharmaceutical composition, characterized in that include: One or more of the bioactive peptide of claim 1, the nucleic acid molecule of claim 2, the construct of claim 3 and the recombinant cell of claim 4.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises: Pharmaceutically acceptable excipients.

8. A method for increasing the activity of alcohol dehydrogenase and inhibiting the activity of xanthine oxidase and / or angiotensin converting enzyme in a sample, characterized in that: include: The sample is contacted with the bioactive peptide according to claim 1.

9. An alcohol dehydrogenase activator, characterized in that include: The bioactive peptide according to claim 1.

10. A xanthine oxidase and / or angiotensin converting enzyme inhibitor, characterized in that: include: The bioactive peptide according to claim 1.

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