Bioactive peptides from highland barley and their applications

Abstract

The present invention discloses a bioactive peptide and its application, wherein the bioactive peptide has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence of a conservatively modified form thereof. The bioactive peptide of the present invention has alcohol dehydrogenase activation activity and xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme inhibition activity, has the advantages of low toxicity, low carcinogenicity, high hydrophilicity, long half-life, and can be absorbed through the intestine, and can be used to develop polypeptide drugs or functional foods with disease treatment effects, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to highland barley-derived bioactive peptides and applications thereof. Background Art

[0002] Bioactive peptides can be separated from proteins by enzymatic hydrolysis or fermentation, and are widely discussed and accepted because of their potential functions, including anti-oxidation, alcohol sobering, blood pressure lowering, and uric acid lowering. However, peptides that have multiple biological activities, such as alcohol sobering, uric acid lowering, and metabolic syndrome relief, are relatively rare.

[0003] Highland barley, also known as naked barley, belongs to the genus Hordeum of the Poaceae family. It is a plateau specialty grain with rich protein content and a balanced amino acid ratio. It can be used as a source of high-quality plant protein peptides. At present, how to use highland barley to develop new peptide products and provide new ways to deeply utilize highland barley resources is an urgent problem that the industry needs to solve. 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 bioactive peptide derived from highland barley and its application. The bioactive peptide of the present invention has alcohol dehydrogenase activation activity and xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme inhibition activity, has the advantages of low toxicity, low carcinogenicity, high hydrophilicity, long half-life, and can be absorbed through the intestine, etc., and can be used to develop polypeptide drugs with disease treatment effects, and has good application prospects.

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

[0006] During the research process of highland barley polypeptides, the inventors accidentally screened out a bioactive peptide PGRPLP that can effectively activate alcohol dehydrogenase activity and inhibit the activity of xanthine oxidase, pancreatic lipase, and angiotensin-converting enzyme through a large amount of virtual screening work and experimental verification. The amino acid sequence of this bioactive peptide is: Pro-Gly-Arg-Pro-Leu-Pro (PGRPLP), with a molecular weight of 635.84Da.

[0007] The bioactive peptide was further prepared using the Fmoc solid phase synthesis method, and its expected biological activity was verified by experiments, thereby further clarifying the mechanism by which the bioactive peptide exerts its biological activity.

[0008] Therefore, in one aspect of the present invention, the present invention provides a bioactive peptide. According to an embodiment of the present invention, the bioactive peptide has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence of a conservatively modified form thereof.

[0009] According to an embodiment of the present invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO:1.

[0010] PGRPLP (SEQ ID NO: 1).

[0011] It should be noted that the amino acid sequences mentioned in the present invention are all shown from N-terminus to C-terminus.

[0012] The bioactive peptide PGRPLP according to the embodiment of the present invention has alcohol dehydrogenase activating activity and xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme inhibitory activity, has the advantages of low toxicity, low carcinogenicity, high hydrophilicity, long half-life, and can be absorbed through the intestine, and can be used to develop polypeptide drugs with disease treatment effects, and has good application prospects.

[0013] Alcohol dehydrogenase is the rate-limiting enzyme for normal human metabolism of alcohol. It can catalyze the dehydrogenation of alcohol to produce acetaldehyde, which is further catalyzed by acetaldehyde dehydrogenase to produce acetic acid that 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 accelerate alcohol metabolism, alcohol and metabolic intermediates excretion from the body, but also reduce the adverse effects of alcohol on the liver. The bioactive peptide of the present invention has alcohol dehydrogenase activation activity, and thus, can be further used to develop drugs with the efficacy of sobering up, for preventing and / or treating alcohol poisoning, and preventing and / or treating alcohol-induced liver damage.

[0014] Xanthine oxidase is a key enzyme in purine catabolism. It catalyzes hypoxanthine to generate xanthine and then uric acid. At the same time, excessive xanthine oxidase levels can also cause uric acid to deposit 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. The bioactive peptide of the present invention has xanthine oxidase inhibitory activity and can be further used to develop drugs for the prevention and / or treatment of gout.

[0015] Pancreatic lipase is a key rate-limiting enzyme for treating and alleviating obesity. It is a key enzyme that catalyzes the hydrolysis of fat into glycerol, fatty acids, etc. By inhibiting its activity, the hydrolysis of fat in the diet can be delayed, and the absorption of fatty acids, etc. can be reduced, thereby achieving the purpose of treating or preventing obesity. The bioactive peptide of the present invention has pancreatic lipase inhibitory activity and can be further used to develop drugs for preventing and / or treating obesity.

[0016] Angiotensin converting enzyme can participate in the regulation of human blood pressure. It plays a role in promoting blood pressure increase in the renin-angiotensin system and kinin-kinase system that control blood pressure in the human body. The bioactive peptide of the present invention has angiotensin converting enzyme inhibitory activity and can be further used to develop drugs for preventing and / or treating hypertension.

[0017] It should be noted that, without substantially affecting the tyrosinase inhibitory activity of the polypeptide of the present invention (retaining at least 90% of the activity), one or more amino acid residues of the polypeptide of the present invention may be replaced by other amino acid residues from the same side chain family, and the functional assay method described herein may be used to test the retained function of the altered polypeptide. Preferably, the number of conservative modifications does not exceed 1 or 2.

[0018] According to an embodiment of the present invention, the bioactive peptide is derived from highland barley. Therefore, the bioactive peptide of the present invention can be prepared from highland barley, and the source of the peptide is healthy, which provides a new way to deeply utilize highland barley resources.

[0019] In a second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned bioactive peptide.

[0020] According to an embodiment of the present invention, the aforementioned bioactive peptide encoded by the nucleic acid has alcohol dehydrogenase activating activity and xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme inhibitory activity, has the advantages of low toxicity, low carcinogenicity, high hydrophilicity, long half-life, and can be absorbed through the intestine, and can be used to develop polypeptide drugs with disease treatment effects.

[0021] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0022] In the third aspect of the present invention, the present invention provides a construct. According to an embodiment of the present invention, the construct contains the aforementioned nucleic acid molecule. Thus, the constructed construct, such as a vector or a transformant, can effectively express the aforementioned bioactive peptide.

[0023] According to an embodiment of the present invention, the construct may be a vector or a transformant.

[0024] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the aforementioned functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0025] According to an embodiment of the present invention, the construct (such as a vector or a transformant) may include an optional control sequence, which is operably connected to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The construct (such as a vector or a transformant) thus constructed can effectively express the aforementioned polypeptide.

[0026] When the aforementioned nucleic acid molecule is connected to the construct (such as a vector or a transformant), such as an expression vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. The nucleic acid molecule can be operably connected to the control element.

[0027] In some embodiments of the present invention, the vector may refer to a cloning vector or an expression vector, which can be obtained by operably connecting the nucleic acid to a commercially available vector (such as a plasmid or a viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0028] In this article, the term "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors may be, for example, viral vectors, plasmids, bacteriophages, etc.

[0029] After the expression vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, it can effectively express the aforementioned nucleic acid molecule under the mediation of the regulatory system, thereby achieving large-scale in vitro production of the polypeptide encoded by the nucleic acid molecule.

[0030] In some embodiments of the present invention, the vector is a eukaryotic vector or a prokaryotic vector.

[0031] In the fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell contains the aforementioned nucleic acid molecule or the aforementioned construct. According to an embodiment of the present invention, the cell can efficiently express the aforementioned bioactive peptide under appropriate conditions, and further obtain a bioactive peptide having alcohol dehydrogenase activation activity and xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme inhibition activity, low toxicity, low carcinogenicity, high hydrophilicity, long half-life, and can be absorbed through the intestine.

[0032] In the fifth aspect of the present invention, the present invention proposes the use of the aforementioned 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 alcoholism; preventing and / or treating alcohol-induced liver damage; preventing and / or treating obesity; preventing and / or treating hypertension; preventing and / or treating gout.

[0033] 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.

[0034] In a sixth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned peptide.

[0035] Those skilled in the art will appreciate that the features and advantages described above for the bioactive peptides are also applicable to the pharmaceutical composition and will not be described in detail herein.

[0036] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0037] According to an embodiment of the present invention, the auxiliary material includes at least one of an excipient, a diluent, a stabilizer and a carrier.

[0038] According to an embodiment of the present invention, the medicine is an oral preparation. The bioactive peptide of the present invention has high hydrophilicity and can be absorbed through the intestine, and can be further developed into an oral preparation.

[0039] In addition to having good solubility in water, hydrophilic peptides usually have enhanced biocompatibility and good cell permeability. Hydrophilic peptides usually have low immunogenicity. This makes them have good biocompatibility in biomedical applications such as drug delivery and tissue engineering. Hydrophilic peptides can also pass through cell membranes, which helps peptide molecules enter the cell and play a role.

[0040] Peptides with long half-lives have significant advantages in the pharmaceutical field, such as reducing dosing frequency, improving treatment outcomes, reducing the risk of side effects, enhancing patient convenience, economy, and predictability of pharmacokinetics. A longer half-life means that the effective concentration of the drug in the body can be maintained for a longer time, so patients do not need to take the drug frequently, which improves patient compliance; stable drug concentrations help achieve better therapeutic effects, especially in chronic diseases that require long-term management; due to the reduced frequency of dosing, the risk of side effects and toxic accumulation caused by frequent dosing may be reduced; long-acting peptide drugs can reduce the number of visits to medical institutions by patients, which is a significant advantage for patients who require long-term treatment; although the initial development cost of long-acting drugs may be higher, they may have a better cost-effectiveness ratio due to the reduced number of dosing; peptide drugs with long half-lives usually have more predictable pharmacokinetic properties, which helps to simplify dosage design and treatment monitoring.

[0041] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

[0042] As used herein, the term "pharmaceutically acceptable ingredients" refers to substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), ie, substances with a reasonable benefit / risk ratio.

[0043] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipient incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition interacting in a harmful manner, their use is also within the scope of the present invention.

[0044] In a seventh aspect of the present invention, the present invention provides an in vitro method for increasing the activity of alcohol dehydrogenase in a sample and inhibiting the activity of xanthine oxidase and / or pancreatic lipase and / or angiotensin converting enzyme in the sample. According to an embodiment of the present invention, the method comprises: contacting the sample with the aforementioned peptide.

[0045] It should be noted that the "sample" of the present invention refers to a sample that contains or may contain alcohol dehydrogenase, xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme. Exemplarily, the sample can be from a healthy individual or a patient or a subject, and can include but is 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 from other biological materials, such as, but not limited to: cells and microorganisms obtained by biotechnology.

[0046] 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

[0047] The foregoing 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:

[0048] Figure 1 This is the HPLC chart of the crystalline sample of polypeptide PGRPLP in Example 1;

[0049] Figure 2 This is the MS graph of the crystalline sample of polypeptide PGRPLP in Example 1;

[0050] Figure 3 This is a visualization result diagram of the peptide PGRPLP molecule docked with alcohol dehydrogenase in Example 2;

[0051] Figure 4 This is a visualization result diagram of the docking of the polypeptide PGRPLP molecule to xanthine oxidase in Example 2;

[0052] Figure 5 This is a visualization result diagram of the docking of the polypeptide PGRPLP molecule with pancreatic lipase in Example 2;

[0053] Figure 6 This is a visualization result diagram of the docking of the polypeptide PGRPLP molecule with angiotensin converting enzyme in Example 2;

[0054] Figure 7 The graphs are the results of the investigation of the interaction between the polypeptide PGRPLP molecule and alcohol dehydrogenase, xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme in Example 2, wherein (A) is the result of the investigation of the alcohol dehydrogenase activation rate; (B) is the result of the investigation of the xanthine oxidase inhibition rate; (C) is the result of the investigation of the pancreatic lipase inhibition rate; and (D) is the result of the investigation of the angiotensin converting enzyme inhibition rate. DETAILED DESCRIPTION

[0055] 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.

[0056] 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.

[0057] The quality inspection index data in the embodiments are all average values.

[0058] Terms and Definitions

[0059] In the text, the term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the biological activity of the polypeptide comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the biologically active peptides of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0060] 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.

[0061] Example 1: Screening of polypeptide PGRPLP and prediction of biological activity

[0062] 1. Preparation of highland barley protein

[0063] The highland barley is crushed into powder, and the highland barley powder is defatted with n-hexane, wherein the solid-liquid ratio is 1:5 (w / v, g / mL), and the n-hexane is discarded after standing overnight to obtain defatted highland barley powder. The defatted highland barley powder and distilled water are mixed in a ratio of 1:25 (w / v, g / mL), and the pH value of the solution is adjusted to 11 with 1mol / L NaOH, and after continuous stirring at 60°C for 30min, the solution is centrifuged at 4°C and 7000×g for 10min, and the supernatant is collected. The pH value of the supernatant is adjusted to 7 with 1mol / L HCl, and the solution is allowed to stand for 1h at room temperature to facilitate protein precipitation. The protein precipitate is collected, and the precipitate is washed three times with distilled water, and then the pH value of the protein precipitate is adjusted to 7.0 with 1mol / L NaOH to obtain highland barley protein, which is freeze-dried to obtain a freeze-dried product of highland barley protein and stored at -20°C for standby use.

[0064] 2. Enzymatic hydrolysis of highland barley protein

[0065] The freeze-dried product of highland barley protein was mixed evenly with 35mM sodium phosphate buffer at a concentration of 5% (w / v, g / mL), the pH value of the solution was adjusted to 7.0 with NaOH and HCl, alkaline protease was added at a mass ratio of 1:20 (w:w) of enzyme substrate, and the resulting solution was incubated at 50°C for 120min for enzymolysis. After the enzymolysis was completed, the enzyme was inactivated in a boiling water bath for 10min to terminate the reaction. After the solution was cooled to room temperature, it was centrifuged at 4°C and 10000×g for 15min, and the supernatant was collected to obtain highland barley protein hydrolyzate.

[0066] 3. Isolation and purification of protein hydrolysates and peptide sequence identification

[0067] 12 mL of highland barley protein hydrolysate solution was transferred to a 3 kDa centrifugal ultrafiltration tube and centrifuged at 4 °C, 5000 × g for 30 min to obtain a fraction sample of highland barley protein hydrolysate with a molecular weight of less than 3 kDa. The fraction sample was desalted with a C18 desalting column and analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was a Q-ExactivePlus mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200.

[0068] The LC-MS / MS analysis method is as follows: 1 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm×25 cm), and the sample was separated by a gradient of 60 min, column flow rate: 0.4 uL / min, column temperature: 40°C, and electrospray voltage: 2 kV. Mobile phase A: 0.1% formic acid in water, 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.

[0069] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 200-2000; resolution: 70000; AGC target: 3e6; maximum injection time: 60 ms; (2) HCD-MS / MS: resolution: 17500; AGC target: 5e4; maximum injection time: 80 ms; collision energy: 27; dynamic exclusion time: 20 s.

[0070] The tandem mass spectra were 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 was set for enzymatic digestion. 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, candidate peptides were obtained by virtual screening by setting ALC (%) ≥ 80 (see Table 1 for typical screening results).

[0071] 3. Prediction of Biological Activity of Candidate Peptides

[0072] The PeptideRanker online system (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.Php) was used to predict the biological activity of the aforementioned candidate polypeptides, that is, the ability of polypeptide molecules to produce biological effects or activities on organisms; ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to predict water solubility and toxicity; Expasy (https: / / web.expasy.org / protparam / ) was used for isoelectric point and half-life analysis; admetSAR (http: / / lmmd.ecust.edu.cn / admetsar1 / predict / ) was used to predict human intestinal absorption performance and carcinogenicity.

[0073] Preliminary screening resulted in 12 peptides with high biological activity and good biological performance. The prediction results of peptideRanker prediction value, toxicity, carcinogenicity, hydrophilicity, half-life, isoelectric point, and intestinal absorption activity are shown in Table 1.

[0074] Table 1 Peptides with good in vitro function prediction results in virtual screening

[0075] Peptide amino acid sequence peptideranker toxicity Carcinogenicity Hydrophilicity half life Isoelectric point Intestinal absorption PGRPLP 0.863611 none none 0.2 >20hours 10.18 + FNPTLP 0.833778 none none -0.75 1.1 hours 5.52 + APHWNL 0.833448 none none -0.83 4.4 hours 6.79 + YLTP 0.833399 none none -1.4 2.8 hours 5.52 + FDPVTWR 0.833127 none none -0.26 1.1 hours 5.84 + FGLPGPK 0.832603 none none -0.19 1.1 hours 8.75 + GFPRPQ 0.83244 none none 0.12 30 hours 9.75 + SDPAKLF 0.831792 none none 0.21 1.9 hours 5.55 + LFLRL 0.830943 none none -0.98 5.5 hours 9.75 + APGLL 0.830508 none none -0.82 4.4 hours 5.57 + MPAGL 0.801151 none none -0.72 >10hours 5.28 + CSPVPMPQ 0.80046 none none -0.41 >10hours 5.52 -

[0076] Note: PeptideRanker is an algorithm based on machine learning to predict and rank peptide activity. The higher the predicted value of PeptideRanker, the greater the possibility that the corresponding peptide has biological activity; "+" means it can be absorbed through the intestine, and "-" means it cannot be absorbed through the intestine.

[0077] The results showed that the polypeptide PGRPLP has good biological activity, can be absorbed through the human intestine, has low toxicity and low carcinogenicity, and has good hydrophilicity and a long half-life.

[0078] Therefore, the inventors used the Fmoc solid phase synthesis method to prepare a highland barley hexapeptide polypeptide PGRPLP sample with a purity greater than 95%, which was further used to identify the activity of biological enzymes such as alcohol dehydrogenase, xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme.

[0079] The specific experimental method of Fmoc solid phase synthesis is as follows:

[0080] (1) Solvent treatment: N,N-dimethylformamide (DMF) and methanol were soaked in G3 molecular sieves overnight before use to remove impurities and water.

[0081] (2) Fully swell the resin: 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.

[0082] (3) Receiving the first amino acid: At room temperature, filter out the solvent from the previous step through a sand core, add 1 mmol of a 5-fold molar excess of the first amino acid at the C-terminus, a 5-fold molar excess of DMAP, a 5-fold molar excess of N,N-diisopropylcarbodiimide, and DMF as solvent, and react at room temperature for 3 hours. After the reaction is completed, wash with DMF 4 to 6 times, 5-6 mL each time. Then add pyridine and acetic anhydride in a volume ratio of 1:1 and react for 30 minutes. After the reaction is completed, wash with DMF 4 to 6 times, 5-6 mL each time.

[0083] (4) Removal of Fmoc protecting group: Remove the solvent of step (3) by suction filtration, add 10 mL of 20% piperidine DMF solution to the resin, stir under N2 for 10 min, filter out the solution, add 10 mL of 20% piperidine DMF solution, stir under N2 for 5 min, and filter out the solution. Repeat this operation twice, then wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0084] (5) Ninhydrin test for removal effect: Take out a small amount of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105℃-110℃ for 5 min. A dark blue color indicates a positive reaction, indicating that the removal is complete and the next step can be carried out. If it is colorless, it means that the protecting group is not completely removed and the above deprotection operation needs to be repeated.

[0085] (6) Removal of the second amino acid and Fmoc protecting group: Weigh a 3-fold molar excess of the second amino acid at the C-terminus, a 3-fold molar excess of HBTU and a 3-fold molar excess of 1-hydroxybenzotriazole in a reaction tube, add an appropriate amount of DMF solution to completely dissolve it, then add a 10-fold molar excess of N,N-diisopropylethylamine, react at room temperature for 40 minutes, and wash with DMF 4 to 6 times, 5-6 mL each time. Take a small amount of resin and detect it with ninhydrin detection reagent, which is colorless, then add 10 mL of 20% piperidine DMF solution to remove Fmoc, and perform it twice, 10 minutes and 5 minutes respectively, then wash it with DMF 4 times and methanol 2 times, 5-6 mL each time. Take out a small amount of resin and detect it with ninhydrin detection reagent, and it is blue, then proceed to the next step of reaction.

[0086] (7) Repeat step (6) until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then drain.

[0087] (8) Resin removal and pure product separation and detection: Finally, use trifluoroacetic acid cutting solution (95% trifluoroacetic acid: 2% triisopropylsilane: 2% ethanedithiol: 1% H2O) to cut for 2 hours, filter the reaction solution to obtain trifluoroacetic acid solution of oligopeptide, blow dry the cleavage solution with nitrogen, precipitate with ether, centrifuge, and then wash with ether 3 to 5 times to obtain a white solid. After dissolving in pure water, desalt and purify by HPLC, and lyophilize to precipitate polypeptide crystals.

[0088] (9) Peptide quality detection: Take a small amount of sample and dissolve it in ultrasound, then place it in a high performance liquid chromatograph for detection. HPLC parameters are: chromatographic column: ZORBAX SB-C18, 4.6×250 mm, 5 μm; aqueous phase: 100% water plus 0.1% trifluoroacetic acid; organic phase: 100% acetonitrile plus 0.1% trifluoroacetic acid; flow rate: 1 mL / min; injection volume: 10 μL, detection wavelength: 220 nm.

[0089] Mass spectrometry parameters: the ion source is an electrospray ionization source (ESI source), the 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.

[0090] Through HPLC high performance liquid chromatography and MS mass spectrometry analysis, it was determined that a polypeptide with an amino acid sequence of PGRPLP was obtained, and the purity of the crystal sample containing the polypeptide was greater than 95% ( Figure 1 , Figure 2 ).

[0091] Example 2: Prediction of affinity between peptide PGRPLP and enzyme

[0092] The peptide PGRPLP with the best in vitro function prediction effect was used as a ligand, and alcohol dehydrogenase, xanthine oxidase, pancreatic lipase, and angiotensin-converting enzyme were used as receptors. The action site and interaction force between the peptide PGRPLP and the enzyme were determined by molecular docking technology, thereby predicting whether the peptide PGRPLP has the aforementioned enzyme activity. The specific method is as follows:

[0093] 1. Alcohol dehydrogenase

[0094] The peptide PGRPLP with the best in vitro function prediction effect was used to create a three-dimensional structure of the peptide using Chimera 1.16, and molecular docking was performed with the 3D structure of alcohol dehydrogenase (PDB number: 5ENV) obtained from the PDB database (http: / / www.rcsb.org / ) and removing water molecules and other ligands. The molecular docking process used AutoDock Vina, and the docking center coordinates were set to (-36.916, 63.248, -8.711) (x, y, z), and the docking box size was 26×26×26.

[0095] The peptide ligands were docked individually onto the alcohol dehydrogenase crystal structure in PDBQT format. The binding of the ligand and receptor was visualized using PyMOL software.

[0096] The visualization results are as follows Figure 3 shown.

[0097] The results showed that the binding energy of the peptide PGRPLP molecule was -5.5 kcal / mol.

[0098] The above results show that the polypeptide PGRPLP has a good affinity with alcohol dehydrogenase.

[0099] 2. Xanthine oxidase

[0100] The peptide PGRPLP with the best in vitro function prediction effect was used to create the three-dimensional structure of the peptide using Chimera 1.16. 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. The three-dimensional structure of the peptide PGRPLP ligand was molecularly docked with the 3D structure of the xanthine oxidase (3NRZ) receptor after removing water molecules, metal atoms and co-crystallization. The molecular docking process used AutoDockVina, and the docking center coordinates were set to (44.052, -8.088, 40.183) (x, y, z), and the docking box size was 17.33×20.33×19.12. The peptide ligand was docked separately to the xanthine oxidase crystal structure in PDBQT format. The binding of the ligand and the receptor was visualized using PyMOL software.

[0101] The visualization results are as follows Figure 4 shown.

[0102] The results showed that the binding energy of the peptide PGRPLP molecule was -6.1 kcal / mol.

[0103] The above results indicate that the polypeptide PGRPLP has a good affinity with xanthine oxidase.

[0104] 3. Pancreatic lipase

[0105] The peptide PGRPLP with the best in vitro function prediction effect was used to create the three-dimensional structure of the peptide using Chimera 1.16. The 3D structure of pancreatic lipase was retrieved from the Protein Database (1LPB) (https: / / www.rcsb.org), and PyMOL was used to remove water molecules and other impurities. The three-dimensional structure of the peptide PGRPLP ligand was molecularly docked with the 3D structure of the pancreatic lipase receptor after removing water molecules and other impurities. The molecular docking process used AutoDock Vina, and the docking center coordinates were set to (4.395, 24.584, 34.542) (x, y, z), and the docking box size was 18×18×18. The peptide ligand was docked separately to the xanthine oxidase structure in PDBQT format. The binding of the ligand and the receptor was visualized using PyMOL software.

[0106] The visualization results are as follows Figure 5 shown.

[0107] The results showed that the binding energy of the peptide PGRPLP molecule was 8.5 kcal / mol.

[0108] The above results indicate that the polypeptide PGRPLP has a good affinity with pancreatic lipase.

[0109] 4. Angiotensin-converting enzyme

[0110] The peptide PGRPLP with the best in vitro function prediction effect was used to create the three-dimensional structure of the peptide using Chimera 1.16, and molecular docking was performed with the 3D structure of angiotensin-converting enzyme (PDB number: 1O86) obtained from the PDB database (http: / / www.rcsb.org / ) and water molecules and other impurities removed. The molecular docking process used AutoDock Vina, binding site parameters (x: 39.556, y: 37.112, z: 48.915) and docking box size (26×26×26). The binding of ligands and receptors was visualized using PyMOL software.

[0111] The visualization results are as follows Figure 6 shown.

[0112] The results showed that the binding energy of the peptide PGRPLP molecule was -8.4 kcal / mol.

[0113] The above results indicate that the polypeptide PGRPLP has a good affinity with angiotensin converting enzyme.

[0114] Example 3: Investigation of the interaction between the polypeptide PGRPLP and alcohol dehydrogenase, xanthine oxidase, pancreatic lipase, and angiotensin converting enzyme

[0115] Furthermore, the inventors investigated the activation activity of the polypeptide PGRPLP on alcohol dehydrogenase, and the inhibitory activity on xanthine oxidase, pancreatic lipase and angiotensin converting enzyme. The specific methods are as follows:

[0116] 1. Alcohol dehydrogenase activation rate experiment

[0117] Incubate 1.5 mL of pH 8.8 sodium pyrophosphate buffer, 0.5 mL of 8 mg / mL PGRPLP peptide solution (distilled water as the control group), 1 mL of coenzyme solution, and 0.1 mL of distilled water 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 at 340 nm every 1 min for 10 min until the absorbance reaches a stable value.

[0118] The calculation formula of alcohol dehydrogenase activity is:

[0119] In formula 1:

[0120] E 340 It is the increase of absorbance value per minute at 340nm;

[0121] E w is the enzyme content per milliliter of enzyme solution (mg / mL);

[0122] 6.22 is the molar absorption coefficient of NADH;

[0123] 3.2 is the total volume of the reaction solution (mL).

[0124] The calculation formula of alcohol dehydrogenase activity activation rate is:

[0125] In formula 2:

[0126] H is the enzyme activity activation rate (%);

[0127] A is the enzyme activity of the blank reaction solution (U / mg);

[0128] B is the enzyme activity of the sample reaction solution (U / mg).

[0129] 2. Xanthine oxidase inhibition test

[0130] After incubating 50 μL of 10 mg / mL peptide PGRPLP solution and 50 μL of xanthine oxidase solution at 37°C for 5 minutes, 150 μL of xanthine solution was added. After oscillation for 30 seconds, the mixture was incubated at 25°C for 25 minutes, and the absorbance was recorded at 290 nm by a microplate reader. The calculation formula for the inhibition rate of xanthine oxidase activity is as follows:

[0131]

[0132] In formula 3: A is the absorbance of the blank tube; B is the absorbance of the sample tube.

[0133] 3. Pancreatic lipase inhibition rate experiment

[0134] A 2.5 mg / mL pancreatic lipase solution was prepared using a pH 7.3 phosphate buffer, and then centrifuged at 5500 rpm for 5 min to obtain the supernatant. p-Nitrophenylbutyrate was diluted to 10 mM using a pH 7.3 phosphate buffer.

[0135] After incubating 50 μL of 4 mg / mL peptide PGRPLP solution (an equal amount of distilled water as the control group), 40 μL of pancreatic lipase solution and 20 μL of p-nitrophenylbutyrate solution at 37°C for 30 min, the absorbance was recorded at 405 nm by a microplate reader. The calculation formula for the inhibition rate of pancreatic lipase activity is as follows:

[0136]

[0137] In formula 4: A is the control absorbance; B is the control blank absorbance; C is the sample absorbance; D is the sample blank absorbance.

[0138] 4. Angiotensin converting enzyme inhibition rate experiment

[0139] 15 μL of 5 mg / mL peptide PGRPLP solution was incubated with 10 μL of angiotensin converting enzyme (100 mU / mL) at 37 ° C for 10 min. The mixture was then incubated with 50 μL of a substrate solution containing 5 mM hippurylhistidylleucine, 100 mM sodium borate buffer (pH 8.3, 300 mM sodium chloride) 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. The solution was then vortexed for 1 min and immediately cooled in an ice bath. Finally, the amount of hippuric acid released was measured by an ELISA reader at 410 nm. The calculation formula for the angiotensin converting enzyme inhibition rate is as follows:

[0140]

[0141] In formula 5: A: control absorbance; B: control blank absorbance; C: sample absorbance

[0142] Test results such as Figure 7 shown.

[0143] The results showed that the activation rate of the polypeptide PGRPLP of the present invention on alcohol dehydrogenase was 118.51±5.04, and the inhibition rates on xanthine oxidase, pancreatic lipase and angiotensin converting enzyme activities were 96.76±2.13%, 41.30±1.54% and 97.96±0.53%, respectively.

[0144] The above results show that the polypeptide PGRPLP of the present invention can activate alcohol dehydrogenase, inhibit xanthine oxidase, pancreatic lipase and angiotensin converting enzyme, can effectively sober up, treat and prevent gout and metabolic syndrome, can be used as an effective ingredient to be widely used in sobering up, preventing or treating alcohol poisoning or alcohol-induced liver damage, preventing or treating obesity, preventing or treating hypertension, preventing or treating gout and other metabolism-related drugs, and can be used as a functional ingredient to be widely used in functional foods and health products for controlling body fat and maintaining healthy blood pressure levels, and has good application prospects.

[0145] The polypeptide of the present invention has the advantages of low toxicity, low carcinogenicity, high hydrophilicity, long half-life, and intestinal absorption, which is conducive to the development of long-acting, oral preparations and has good market development prospects.

[0146] Although the embodiments of the present invention have been shown and described above, it is to be understood that the aforementioned 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 aforementioned 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. The bioactive peptide according to claim 1, characterized in that The bioactive peptides are derived from highland barley.

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

4. A construct, characterized in that Containing the nucleic acid molecule according to claim 3.

5. A recombinant cell, characterized in that Containing the nucleic acid molecule according to claim 3 or the construct according to claim 4.

6. Use of the peptide according to claim 1 or 2, the nucleic acid molecule according to claim 3, the construct according to claim 4 or the recombinant cell according to claim 5 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; prevention and / or treatment of obesity; Prevent and / or treat high blood pressure; Prevent and / or treat gout.

7. A pharmaceutical composition, characterized in that include: The peptide according to claim 1 or 2.

8. The pharmaceutical composition according to claim 7, characterized in that Further including: Pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that The auxiliary materials include: one or more pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 8, characterized in that The auxiliary materials include: one or more pharmaceutically acceptable diluents.

11. The pharmaceutical composition according to claim 8, characterized in that The auxiliary materials include: one or more pharmaceutically acceptable stabilizers.

12. The pharmaceutical composition according to claim 8, characterized in that The auxiliary materials include: one or more pharmaceutically acceptable carriers.

13. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is an oral preparation.

14. A method for increasing the activity of alcohol dehydrogenase and inhibiting the activity of xanthine oxidase and / or pancreatic lipase and / or angiotensin converting enzyme in a sample in vitro, characterized in that: include: The sample is contacted with the peptide according to claim 1 or 2.

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