A multifunctional bioactive peptide based on millet protein and its preparation method

Abstract

This invention belongs to the field of biotechnology, specifically relating to a multifunctional bioactive peptide based on millet protein and its preparation method. The amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1. The bioactive peptide of this invention can activate alcohol dehydrogenase activity and inhibit xanthine oxidase and angiotensin-converting enzyme activity. It has the characteristics of small molecular weight and easy absorption. Furthermore, bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. It can be used to develop drugs with therapeutic effects on diseases such as hangover relief, uric acid reduction, and blood pressure reduction, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a multifunctional bioactive peptide based on millet protein and its preparation method. Background Technology

[0002] Millet is a grain with a long history. As a high-quality plant protein source, it accounts for about 9.7% of the dry weight of protein and is often used to supplement people's daily protein needs. In addition, millet is rich in carbohydrates, protein, dietary fiber, B vitamins, various minerals, and antioxidants such as flavonoids. Therefore, millet has many health benefits, such as anti-oxidation, blood sugar regulation, digestion promotion, sleep improvement, and cardiovascular protection. Millet protein can be processed by enzymatic hydrolysis and other methods to obtain small molecule peptides. Some of these peptides have specific biological activities, such as anti-oxidation and blood pressure lowering. However, there are still problems such as the unclear amino acid sequence and efficacy of characteristic peptides in millet.

[0003] Therefore, the screening of millet peptides with multiple biological activities and clearly defined effects is of great significance for the application of millet protein products in the development of pharmaceuticals, health products or functional foods. Summary of the Invention

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

[0005] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0006] In the research process of millet bioactive peptides, the inventors screened a large number of virtual screening operations and experimental verifications to obtain a bioactive peptide KDFPF that simultaneously activates alcohol dehydrogenase activity and inhibits xanthine oxidase and angiotensin-converting enzyme activity. 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 this bioactive peptide using the Fmoc solid-phase synthesis method and verified through experiments that it has the expected alcohol dehydrogenase activating activity and inhibits xanthine oxidase and angiotensin-converting enzyme biological activities.

[0007] In a first aspect, the present invention provides a bioactive peptide. According to embodiments 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 and inhibit xanthine oxidase and angiotensin-converting enzyme activity. It has the characteristics of small molecular weight and easy absorption, and bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. It can be used to develop drugs for the treatment of diseases such as hangovers, uric acid reduction, and blood pressure reduction, and has broad application prospects.

[0008] In a second aspect, the present invention provides 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 first aspect bioactive peptide encoded by the nucleic acid molecule can activate alcohol dehydrogenase activity, inhibit xanthine oxidase and angiotensin-converting enzyme activity, and has the characteristics of small molecular weight and easy absorption. Furthermore, bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The nucleic acid molecule enables the efficient expression of this bioactive peptide, facilitates its large-scale industrial production, and provides convenience for the subsequent development and application of related drugs for hangover relief, uric acid reduction, and blood pressure reduction.

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

[0010] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, it comprises the nucleic acid molecule described in the second aspect or the construct described in the third aspect. According to embodiments of the invention, the recombinant cell can efficiently express the aforementioned bioactive peptide under suitable conditions. This bioactive peptide can activate alcohol dehydrogenase activity, inhibit xanthine oxidase and angiotensin-converting enzyme activity, and has the characteristics of small molecular weight and easy absorption. Furthermore, bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The recombinant cell method enables the efficient expression of this bioactive peptide, facilitating its large-scale industrial production.

[0011] In a fifth aspect, the invention proposes the use of the bioactive peptides described in the first aspect, the nucleic acid molecules described in the second aspect, the constructs described in the third aspect, or the recombinant cells described in the fourth aspect in the preparation of a medicament. According to embodiments of the invention, the medicament has at least one of the following uses: relieving hangovers; 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 understand that the features and advantages described above for bioactive peptides also apply to this use, and will not be repeated here.

[0013] In a sixth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, it comprises one or more of the following: 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 understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells also apply to this use, and will not be repeated here.

[0015] According to embodiments of the present invention, the above-described pharmaceutical composition may further have the following additional technical features:

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

[0017] In a seventh aspect of the invention, a method is provided for increasing the activity of alcohol dehydrogenase and inhibiting the activity of xanthine oxidase and / or angiotensin-converting enzyme in a sample. According to embodiments of the invention, the method includes contacting the sample with the bioactive peptide described in the first aspect. The method according to embodiments of the invention involves co-culturing the sample with the bioactive peptide of the invention to increase the activity of alcohol dehydrogenase and inhibit the activity of xanthine oxidase and / or angiotensin-converting enzyme in the sample.

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

[0019] In a ninth aspect of the invention, an inhibitor of xanthine oxidase and / or angiotensin-converting enzyme is provided. According to an embodiment of the invention, it comprises: the bioactive peptide described in the first aspect. The activator according to an embodiment of the 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 invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

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

[0022] Figure 1 This is a chromatogram of the HPLC detection results of the pure KDFPF bioactive peptide sample in Example 1 of the present invention;

[0023] Figure 2 This is a graph showing the MS detection results of the pure KDFPF bioactive peptide sample in Example 1 of this invention.

[0024] Figure 3 This is a schematic diagram illustrating the docking of the bioactive polypeptide KDFPF with alcohol dehydrogenase in Example 2 of the present invention;

[0025] Figure 4 This is a schematic diagram illustrating the docking of the bioactive polypeptide KDFPF with xanthine oxidase in Example 2 of the present invention;

[0026] Figure 5 This is a schematic diagram of the docking of the bioactive polypeptide KDFPF with angiotensin-converting enzyme in Example 2 of the present invention;

[0027] Figure 6 This is the evaluation result of the alcohol-relieving, uric acid-lowering, and blood pressure-lowering functions of the bioactive polypeptide KDFPF in Example 3 of the present invention. Detailed Implementation

[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 construed 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 construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0030] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0032] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0033] Terms and Definitions

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

[0035] In this article, the term "xanthine oxidase" refers to a key enzyme in purine catabolism, which catalyzes the formation of xanthine from hypoxanthine, which then produces uric acid. Excessively high levels of xanthine oxidase can also lead to the deposition of uric acid in the body. Xanthine oxidase inhibitors can block the biosynthesis of uric acid, thus xanthine oxidase is an important therapeutic target for the treatment of gout.

[0036] In this article, the term "angiotensin-converting enzyme" is used to describe its role in regulating blood pressure in the human body. It plays a role in promoting blood pressure elevation in the renin-angiotensin system and the kinin-kinin-generating enzyme system, which control blood pressure in the human body.

[0037] Bioactive peptides

[0038] This invention proposes a bioactive peptide. According to embodiments of the invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1. The bioactive peptide of this invention can activate alcohol dehydrogenase activity and inhibit xanthine oxidase and angiotensin-converting enzyme activity. It has the characteristics of small molecular weight and easy absorption, and bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. It can be used to develop drugs with therapeutic effects on diseases such as hangover relief, uric acid reduction, and blood pressure reduction, 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 this invention are all shown in the order from N-terminus to C-terminus.

[0042] Exemplary examples include: the bioactive peptides of the present invention having alcohol dehydrogenase activating activity, thereby enabling further development of drugs with hangover relief effects, for the prevention and / or treatment of alcohol poisoning, and for the prevention and / or treatment of alcohol-induced liver damage; the bioactive peptides of the present invention having xanthine oxidase inhibitory activity, thereby enabling further development of drugs for the prevention and / or treatment of gout; and the bioactive peptides of the present invention having angiotensin-converting enzyme inhibitory activity, thereby enabling further development of drugs for the prevention and / or treatment of hypertension.

[0043] Nucleic acid molecules

[0044] This invention proposes a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned bioactive peptide. According to an embodiment of the invention, the aforementioned bioactive peptide encoded by the nucleic acid molecule can activate alcohol dehydrogenase activity, inhibit xanthine oxidase and angiotensin-converting enzyme activity, and has the characteristics of small molecular weight and easy absorption. Furthermore, bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The nucleic acid molecule enables the efficient expression of this bioactive peptide, facilitates its large-scale industrial production, and provides convenience for the subsequent development and application of related drugs for hangover relief, uric acid reduction, and blood pressure reduction.

[0045] It should be noted that those skilled in the art should understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands; although in most cases only one strand is given, the other complementary strand is actually disclosed as well; in addition, the nucleic acid molecule sequences in this invention include DNA or RNA forms, and disclosing one of them means that the other is also disclosed.

[0046] Construct

[0047] This invention proposes a construct. According to embodiments of the invention, it includes the aforementioned nucleic acid molecule. According to embodiments of the invention, the construct enables the nucleic acid molecule to be efficiently expressed in host cells, thereby achieving stable and efficient production of the aforementioned bioactive peptide.

[0048] Exemplarily, the construct may be a vector; it should be noted that, for the purposes of this document, a vector refers to a nucleic acid molecule capable of self-replication within a suitable host, which transfers the inserted nucleic acid molecule to host cells and / or between host cells; the vector may include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for replicating DNA or RNA, and expression vectors primarily used for transcription and / or translation of DNA or RNA; the vector may be a polynucleotide capable of being 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 bioactive peptides, the vector including viral vectors, plasmids, bacteriophages, etc.

[0049] Recombinant cells

[0050] This invention proposes a recombinant cell. According to embodiments of the invention, it includes the aforementioned nucleic acid molecule or the aforementioned construct. According to embodiments of the invention, the recombinant cell can efficiently express the aforementioned bioactive peptide under suitable conditions. This bioactive peptide can activate alcohol dehydrogenase activity, inhibit xanthine oxidase and angiotensin-converting enzyme activity, and has the characteristics of small molecular weight and easy absorption. Furthermore, bioinformatics analysis predicts that it has advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and long half-life. The recombinant cell method enables the efficient expression of this bioactive peptide, facilitating its large-scale industrial production.

[0051] application

[0052] This invention proposes the application of the aforementioned bioactive peptides, nucleic acid molecules, constructs, or recombinant cells in the preparation of pharmaceuticals. According to embodiments of the invention, the pharmaceuticals have at least one of the following uses: relieving hangovers; 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 understand that the features and advantages described above for bioactive peptides also apply to this use, and will not be repeated here.

[0054] Pharmaceutical Composition

[0055] This invention provides a pharmaceutical composition. According to embodiments of the invention, it comprises one or more of the aforementioned bioactive peptides, nucleic acid molecules, constructs, and recombinant cells.

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

[0057] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. Thus, the addition of excipients allows the pharmaceutical composition to better exert the efficacy of the bioactive peptides.

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

[0059] method

[0060] This invention provides a method for increasing the activity of alcohol dehydrogenase and inhibiting the activity of xanthine oxidase and / or angiotensin-converting enzyme in a sample. According to embodiments of the invention, the method includes contacting the sample with the aforementioned bioactive peptide. The method according to embodiments of the invention further enhances the activity of alcohol dehydrogenase and inhibits the activity of xanthine oxidase and / or angiotensin-converting enzyme by co-culturing the sample with the bioactive peptide of the invention.

[0061] It should be noted that the "sample" in this invention refers to a sample containing or possibly containing alcohol dehydrogenase, xanthine oxidase, or angiotensin-converting enzyme; exemplaryly, the sample may be derived from a healthy individual or a patient or subject, including but not limited to blood, urine, saliva, feces, tissue samples, cell samples, cerebrospinal fluid, semen, amniotic fluid, or other bodily fluids such as synovial fluid, pleural effusion, etc.; it may also be derived from other biological materials, including but not limited to cells, microorganisms, etc. obtained through biotechnology.

[0062] Activator

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

[0064] Inhibitors

[0065] This invention provides an inhibitor of xanthine oxidase and / or angiotensin-converting enzyme. According to embodiments of the invention, it comprises the aforementioned bioactive peptide. The activator according to embodiments of the 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 this invention are detailed in Table 1.

[0067] Table 1

[0068] SEQ ID NO: sequence 1 KDFPF 2 WLLPH 3 LLNPF 4 LLFRL 5 FLPLH 6 FNPTLP 7 APHWNLN 8 YLTPF 9 FDPVTWR 10 FGLPGPK

[0069] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

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

[0071] 1. Preparation of millet protein

[0072] Sufficient millet (purchased from Shanxi Dongfangliang Life Science and Technology Co., Ltd.) was soaked in water at a ratio of 1:2 (w / v) for 2 hours at room temperature. The soaked millet was then ground into a paste, with an equal amount of water added during the grinding process. The paste was then filtered through a 100-mesh sieve to obtain millet paste. The pH of the millet paste was adjusted to 10 using a 4% sodium hydroxide solution, and then stirred at 20-30℃ for 3 hours to obtain alkali-soluble millet paste. The alkali-soluble millet paste was then centrifuged at 3500 r / min for 5 minutes to separate it into a liquid phase, a protein solid phase, and a starch solid phase. The liquid phase and the protein solid phase were collected to obtain millet protein paste. The pH of the millet protein paste was then adjusted to 7 using a 4% hydrochloric acid solution. The millet protein paste was then freeze-dried to obtain millet protein, which was stored at -20℃ for later use.

[0073] 2. Enzymatic hydrolysis of millet protein

[0074] The millet protein obtained in step 1 was mixed evenly in 35 mM sodium phosphate buffer at a ratio of 5% (w / v); the pH was adjusted to 7.0 using NaOH and HCl, and then alkaline protease (purchased from Sigma; Sigma-Aldrich, St. Louis, MO, USA) was added at an enzyme-substrate ratio of 1:20 (w:w) to obtain millet protein hydrolysate; the millet protein hydrolysate was incubated at 50°C for 120 min, and then placed in a boiling water bath for 10 min to terminate the enzymatic hydrolysis reaction; after the millet protein hydrolysate cooled to room temperature, it was centrifuged at 4°C and 10000×g for 15 min, and the supernatant was collected to obtain the millet protein hydrolysate solution for later use.

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

[0076] The 12 mL millet protein hydrolysate solution obtained in step 1 was transferred to a 3 kDa ultrafiltration tube and centrifuged at 4°C and 5000 × g for 30 min to obtain millet protein hydrolysate samples with a molecular weight <3 kDa. The obtained millet protein hydrolysate samples with a molecular weight <3 kDa were then desalted using a C18 desalting column to obtain desalted millet protein hydrolysate samples with a molecular weight <3 kDa. LC-MS / MS analysis was then performed on these samples using an online nanojet ion source. The system used for this analysis was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) with a tandem EASY-nanoLC 1200. The specific LC-MS / MS analysis steps are as follows:

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

[0078] The Q-Exactive Plus mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters are 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 spectra were then analyzed using PEAKS Studio version 10.6 (BioinformaticsSolutions Inc., Waterloo, Canada). The database used was uniprot-Hordeum_vulgare_subsp_vulgare (version 2023, 34528 entries), with the enzymatic digestion set to none. The search parameters were: fragment ion mass tolerance: 0.02 Da; parent ion mass tolerance: 10 ppm; variable modifications: Oxidation (M) 15.99; peptide card value: -10lgP ≥ 20. For peptides not found 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 Partial Target Peptide Sequences

[0086] Target peptide sequence Serial Number KDFPF SEQ ID NO: 1 WLLPH SEQ ID NO: 2 LLNPF SEQ ID NO: 3 LLFRL SEQ ID NO: 4 FLPLH SEQ ID NO: 5 FNPTLP SEQ ID NO: 6 APHWNLN SEQ ID NO: 7 YLTPF SEQ ID NO: 8 FDPVTWR SEQ ID NO: 9 FGLPGPK SEQ ID NO: 10

[0087] 4. Virtual screening of functional peptides from millet

[0088] First, the PeptideRanker online system (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.Php) was used to predict the bioactivity 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. Next, 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 / admetSAR1 / predict / ) was used to predict the human intestinal absorption and carcinogenicity of the target peptide sequence obtained in step 3.

[0089] Table 2 shows the superior results of in vitro functional prediction of millet peptides through virtual screening.

[0090] Table 2. Optimal results of in vitro functional prediction for millet peptide virtual screening.

[0091] Xiaomi polypeptide sequence peptideranker molar weight toxicity Carcinogenicity hydrophilic half life isoelectric point KDFPF 0.952122 653.36 Non-toxic none 0.2 1.3 hours 5.84 WLLPH 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 APHWNLN 0.833448 851.03 Non-toxic none -0.83 4.4 hours 6.79 YLTPF 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 a bioactivity greater than 0.8 were obtained through screening. Among them, KDFPF had good bioactivity, human intestinal absorption and low toxicity, and also had 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. 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 from the solvents.

[0097] (2) Resin swelling

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

[0099] (3) Introduce the first amino acid

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

[0101] (4) Removal of Fmoc protecting groups

[0102] Remove the solvent from step 3 by core filtration. Add 10 mL of 20% piperidine DMF solution to the above resin, stir for 10 min under N2 protection, and filter out the solution. Add another 10 mL of 20% piperidine DMF solution, stir under N2 protection for 5 min, and filter out the solution again. Repeat the above operation twice. Then wash with DMF 4 times and methanol 2 times, 6 mL each time.

[0103] (5) Detection of Fmoc protecting group removal effect by ninhydrin

[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 minutes. If it turns dark blue, it is a positive reaction, indicating that the Fmoc protecting group has been completely removed and the next reaction operation can be carried out. If it is colorless, it means that the protecting group has not been completely removed and the above step (4) Fmoc protecting group removal operation needs to be repeated.

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

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

[0107] (7) Introducing amino acids again

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

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

[0110] Cutting: Add cutting solution (97.5% trifluoroacetic acid) in 6 times the volume of resin, shake on a shaker for 2 hours, filter out the resin, precipitate the filtrate with anhydrous diethyl ether, wash the precipitate 3 times with anhydrous diethyl ether, and finally place the precipitate in a vacuum drying oven and dry at room temperature for 24 hours. Then, desalt and purify the crude peptide by HPLC, freeze-dry to precipitate crystals, which is the pure bioactive peptide KDFPF, and store at -20℃ for later use.

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

[0112] Take a small amount of the pure bioactive peptide KDFPF sample obtained in step (8), dissolve it in trifluoroacetic acid solution by ultrasound to obtain the test solution; place the test solution in a high performance liquid chromatograph for detection to perform the quality test of the pure bioactive peptide KDFPF.

[0113] 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] The MS parameter settings are as follows:

[0116] Ion source: Electrospray ionization source (ESI source); Atomizing 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 results of the pure bioactive peptide KDFPF sample are shown below. Figure 1 MS detection results of pure KDFPF bioactive peptide sample are shown in [the table below]. Figure 2 .

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

[0119] Example 2: Molecular docking and interaction force analysis of bioactive peptide KDFPF

[0120] Using the bioactive peptide KDFPF screened in Example 1 as a ligand and alcohol dehydrogenase, xanthine oxidase, and angiotensin-converting enzyme as receptors, the inventors used molecular docking technology to clarify the interaction sites and interactions between peptide KDFPF and the enzymes, thereby realizing the theoretical verification of its functions of detoxification, uric acid reduction, and blood pressure reduction.

[0121] (1) Ethanol dehydrogenase

[0122] The three-dimensional structure of the bioactive peptide KDFPF was created using Chimera 1.16. An alcohol dehydrogenase (PDB ID: 5ENV) was obtained from the PDB database (http: / / www.rcsb.org / ) and water molecules and other ligands were removed for molecular docking. Then, ZDOCK was used to perform molecular docking between the bioactive peptide KDFPF and the enzyme. After screening for the optimal binding conformation, 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+. Finally, the binding between the ligand (bioactive peptide KDFPF) and the receptor (alcohol dehydrogenase) was visualized using PyMOL software.

[0123] Opinion on molecular docking of bioactive peptide KDFPF with 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 mainly depended on hydrophobic interactions and hydrogen bonds. Specifically, the Phe3(C), Pro4(C), and Phe5(C) amino acid residues of the bioactive peptide KDFPF formed significant hydrophobic interactions with the Pro24(A), Pro26(A), Thr130(A), Val25(A), Ile10(A), and His214(A) amino acid residues of alcohol dehydrogenase, while Asp2(C) and Lys59(A) formed hydrogen bonds (hydrogen bond distance was 0.2 kcal / mol). Therefore, it is inferred that the bioactive polypeptide KDFPF binds tightly to alcohol dehydrogenase through the synergistic effect of hydrophobic interactions and hydrogen bonds, thereby activating enzyme activity.

[0125] The above results indicate that the bioactive polypeptide KDFPF has a good affinity for alcohol dehydrogenase, suggesting that it has a good function in relieving hangovers.

[0126] (2) Xanthine oxidase

[0127] Xanthine oxidase (3NRZ) was obtained from the PDB database (http: / / www.rcsb.org / ), and water molecules, metal atoms, and co-crystallization ligands were removed using PyMOL. Then, the three-dimensional structure of the bioactive peptide KDFPF was created using Chimera 1.16. The docking process was performed using ZDOCK to molecularly dock the bioactive peptide KDFPF with the enzyme. After screening for the optimal binding conformation, the binding force between the bioactive peptide KDFPF and the enzyme was analyzed using PDBePISA, and the interaction forces were further analyzed in detail using LigPlus+. Finally, the binding between the ligand (bioactive peptide KDFPF) and the receptor (xanthine oxidase) was visualized using PyMOL software.

[0128] Opinion on molecular docking of bioactive peptide KDFPF with 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 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 Leu744(A), Gln585(A), Gly796(A), Val1200(A), Phe798(A), and Ile1229(A) amino acid residues of the enzyme, while the amino acid residues Asp2(C), Lys1(C), and Ile1235(A) formed two hydrogen bonds. Therefore, it is speculated that the bioactive peptide KDFPF binds tightly 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 for xanthine oxidase, suggesting that it has a good uric acid-lowering function.

[0131] (3) Angiotensin-converting enzyme

[0132] Angiotensin-converting enzyme (PDB ID: 1O86) was processed using PyMOL to remove water molecules and other impurities. The docking process employed ZDOCK to perform molecular docking of the bioactive peptide KDFPF with the enzyme. After screening for the optimal binding conformation, the binding force between the bioactive peptide KDFPF and the enzyme was analyzed using PDBePISA, and further detailed analysis of the interaction forces was performed using LigPlus+. Finally, PyMOL software was used to visualize the binding of the ligand and receptor.

[0133] Opinion on molecular docking of bioactive peptide KDFPF with 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 (ACE) was -6.6 kcal / mol, and the binding 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. At the same time, the amino acid residues Asp2(B) and Lys1(B) formed hydrogen bonds with Lys511(A) and Glu162(A). In summary, it is speculated that the bioactive peptide KDFPF binds tightly to ACE through the synergistic effect of hydrophobic interactions and hydrogen bonds, thereby inhibiting the activity of ACE.

[0135] The above results indicate that the bioactive polypeptide KDFPF has a good affinity for angiotensin-converting enzyme, suggesting that it has a good antihypertensive function.

[0136] Example 3: Experimental verification of the functions of bioactive polypeptide KDFPF in detoxification, uric acid reduction, and blood pressure reduction.

[0137] 1. Determination of the activation rate of alcohol dehydrogenase in the bioactive polypeptide KDFPF

[0138] Mix 1.5 mL of pH 8.8 sodium pyrophosphate buffer, 0.5 mL of 8 mg / mL KDFPF pure solution obtained in Example 1 (blank control group: replaced with 0.5 mL distilled water), 1 mL of coenzyme solution, and 0.1 mL of distilled water, and incubate in a 25°C water bath for 5 min. Then add 0.1 mL of 0.25 U / mL alcohol dehydrogenase solution and start timing; read the absorbance value at 340 nm every 1 min for 10 min until the absorbance value stabilizes; the alcohol dehydrogenase activity is calculated according to Equation 1, and the enzyme activity activation rate is calculated according to Equation 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 bioactive polypeptide KDFPF

[0144] 50 μL of a 10 mg / mL pure solution of the bioactive peptide KDFPF obtained in Example 1 (the blank control group was 0.2 mol / L pH 7.5 phosphate buffer) and 50 μL of xanthine oxidase solution were incubated at 37°C for 5 min, followed by the addition of 150 μL of xanthine solution. The absorbance was recorded at 290 nm every 20 s using a microplate reader for 10 min. The xanthine oxidase activity inhibition rate was calculated according to Equation 3.

[0145]

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

[0147] 3. Determination of the angiotensin-converting enzyme inhibition rate of the bioactive polypeptide KDFPF

[0148] 15 μL of a 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 mixture. This mixture was then mixed with 50 μL of a substrate solution containing 5 mM hippuryl histidine leucine and 100 mM sodium borate buffer (pH = 8.3, 300 mM sodium chloride) and incubated at 37 °C for 60 min. 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. The test solution was then vortexed for 1 min and immediately cooled in an ice bath. Finally, the amount of hippuric acid released was measured at 410 nm using a microplate reader. The angiotensin-converting enzyme activity inhibition rate was calculated according to Equation 4.

[0149]

[0150] In Formula 4: A is the absorbance of the control group (the bioactive peptide KDFPF pure reaction solution sample is replaced with 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 with sodium borate buffer); C is the absorbance value of the bioactive peptide KDFPF pure reaction solution sample (experimental group).

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

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

[0153] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0154] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to 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 bioactive peptide of claim 1.

3. A construct, characterized in that, Includes the nucleic acid molecule described in claim 2.

4. A recombinant cell, characterized in that, Includes the nucleic acid molecule of claim 2 or the construct of claim 3.

5. The use of the bioactive peptide of claim 1, the nucleic acid molecule of claim 2, the construct of claim 3, or the recombinant cell of claim 4 in the preparation of a drug, characterized in that, The drug has at least one of the following uses: Sobering up; Prevention and / or treatment of alcohol poisoning; Prevention and / or treatment of alcohol-induced liver damage; Lowering uric acid; Prevention and / or treatment of gout; Lower blood pressure; Prevention and / or treatment of hypertension.

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 of claim 1; wherein the method is for non-diagnostic and non-therapeutic purposes.

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

10. An inhibitor of xanthine oxidase and / or angiotensin-converting enzyme, characterized in that, include: The bioactive peptide of claim 1.

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