Bioactive peptide and application thereof in treating cardiovascular and cerebrovascular diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a bioactive peptide and application thereof in treating cardiovascular and cerebrovascular diseases. The oat active polypeptide is prepared by carrying out enzymolysis on oat protein. The active polypeptide effectively reduces secretion of Renin, ANGII and LDL-C, promotes synthesis and secretion of BK and HDL-C, reduces an atherosclerosis index, and / or reduces systolic pressure and / or diastolic pressure, improves cardiovascular and cerebrovascular diseases, has certain clinical application value, also has the advantages of simple preparation, easy absorption and the like, and is easy to popularize and apply.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a bioactive peptide and application thereof in treating cardiovascular and cerebrovascular diseases. BACKGROUND

[0002] Cardiovascular and cerebrovascular diseases (CVD) are a general term for cardiovascular diseases and cerebrovascular diseases, mainly including hypertension, coronary heart disease and stroke, and the pathological basis of the occurrence of which is atherosclerosis, which is mainly characterized by thickening and hardening of the arterial wall, loss of elasticity and narrowing of the lumen. The "endothelial injury response theory" considers that the formation of atherosclerosis is the result of the inflammatory-fibroplastic response of the artery to the injury of the intima. Under the condition of long-term hyperlipidemia, the increased lipoprotein can cause functional damage to the arterial intima, change the surface characteristics of endothelial cells and white blood cells, increase the expression of adhesion factors, and increase the number of mononuclear cells adhering to the endothelial cells and migrating into the subendothelial space to become macrophages. Macrophages transform into foam cells after phagocytosis of oxidized low-density lipoprotein and then form atherosclerotic lipid stripes, which will develop into fibrofatty lesions under the action of cytokines, and eventually develop into fibrous plaques leading to atherosclerosis. Cardiovascular and cerebrovascular diseases have the characteristics of "four high and one multiple", i.e. high incidence, high disability rate, high mortality, high recurrence rate and multiple complications, which seriously threaten the health of human beings, especially the middle-aged and elderly population. However, with the development of social economy and the improvement of people's living standards in China, the prevalence rate of cardiovascular and cerebrovascular diseases has shown a significant upward trend. After years of research, it has been confirmed that hypertension, diabetes, dyslipidemia, overweight / obesity, metabolic syndrome, waist circumference, smoking, alcohol consumption and the like have been identified as risk factors for traditional cardiovascular and cerebrovascular diseases. Hypertension is a chronic disease characterized by elevated systolic or diastolic blood pressure, which can cause changes and damage to the structure and function of the heart, brain, kidney, blood vessels and ocular structures, and cause various complications, and has become a major factor in the occurrence of cardiovascular diseases, myocardial infarction, stroke and the like, seriously endangering people's lives and health. Hyperlipidemia is a common clinical disease and an important risk factor for stroke, coronary heart disease and sudden cardiac death. High-fat diet usually leads to hyperlipidemia, which is characterized by high levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), and low levels of high-density lipoprotein cholesterol (HDL-C). Epidemiological and clinical studies have also shown that high blood lipid levels in the blood are positively correlated with cardiovascular diseases, and hyperlipidemia induced by high-fat and high-cholesterol diet can cause non-alcoholic fatty liver disease.

[0003] Polypeptide is a new bioactive effective component, whose relative molecular weight is between macromolecular protein / antibody component and small molecule chemical component. It is widely used in disease treatment field because of its characteristics of strong bioactivity, target specificity, chemical biology diversity and low toxicity. With the rapid development of genomics, proteomics and bioinformatics research and the progress of polypeptide screening technology, more and more polypeptide drugs are discovered and used in disease diagnosis, treatment and prevention. Polypeptide drugs show various effects and potential commercial value in many system diseases. Among them, the application of polypeptide in the field of cardiovascular and cerebrovascular diseases is gradually becoming a hot spot. A variety of polypeptide drugs have been found, such as heat shock protein, galectin-3, meso leaflet, angiotensin 1-7 and hematin hexapeptide, which have ideal effects on preventing and treating ischemic cardiovascular and cerebrovascular diseases.

[0004] Oat (Avena satival) has high nutritional value. With the in-depth scientific research, the functional characteristics of oat have been gradually discovered, and oat has become a globally recognized health food. Oat contains a large amount of fat, amino acids, proteins, and trace amounts of vitamin E, calcium, phosphorus, etc. Compared with other grains, fat is the most advantageous nutrient component. In addition, due to the high content of fatty acids in oat, it can stimulate the synthesis of bile acid and cholesterol, so it also has obvious effect on reducing blood lipids. In addition, oat contains stachyose and raffinose, which can stimulate the proliferation of bifidobacterium and enhance human immunity and a series of effects. Among the grains consumed by humans, the content of high-quality grain protein in oat is much higher than that in other grains. Research results show that the protein components of naked oat are mainly composed of globulin, albumin, prolamin and glutelin. The content of globulin is 50.0%-80.0%, 1.0%-12.0%, 4.0%-15.0% and 19.0%-22.0%, respectively. Moreover, due to the high content and proportion of globulin, the amino acid composition of oat protein is better than that of other grains. Studies have shown that oat polypeptide has the effects of reducing blood lipids and controlling blood pressure. In addition, studies have shown that oat protein has obvious effect on reducing low-density lipoprotein cholesterol (LDL-C). Experiments have proved that the mechanism of oat in reducing cholesterol is to promote the synthesis of cholesterol 7α-hydroxylase to promote the transformation of cholesterol into bile acid. Therefore, oat-derived polypeptide may have good therapeutic effect on cardiovascular diseases, which is of great significance to improve the nutrition and health level of Chinese people. SUMMARY

[0005] In view of the prior art, the present application aims to provide an oat polypeptide for treating cardiovascular and cerebrovascular diseases and application thereof. Specifically, the oat polypeptide provided by the present application can reduce the secretion of Renin and ANGII, promote the synthesis and secretion of BK and HDL-C, reduce the atherosclerosis index, reduce systolic and / or diastolic blood pressure, and improve cardiovascular and cerebrovascular diseases. In addition, the oat polypeptide provided by the present application is non-toxic and has no side effects, and has the advantages of simple preparation, easy absorption, etc., and can be used for preparing health products or drugs.

[0006] The present application first provides an oat polypeptide, characterized in that the amino acid sequence thereof is shown as SEQ ID NO: 1.

[0007] In some embodiments, the oat polypeptide can reduce the secretion of Renin and ANGII.

[0008] In some embodiments, the oat polypeptide can promote the secretion of BK.

[0009] In some embodiments, the oat polypeptide can promote the increase of HDL-C content.

[0010] In some embodiments, the oat polypeptide can promote the decrease of LDL-C content.

[0011] In some embodiments, the oat polypeptide can reduce the atherosclerosis index.

[0012] In some embodiments, the oat polypeptide can treat cardiovascular and cerebrovascular diseases.

[0013] Another aspect of the present application also provides a nucleic acid molecule encoding the oat polypeptide of the present application.

[0014] Another aspect of the present application also provides a vector comprising the nucleic acid molecule of the present application.

[0015] Another aspect of the present application also provides an isolated host cell comprising the oat polypeptide of the present application, the nucleic acid molecule of the present application, or the vector of the present application.

[0016] Another aspect of the present application also provides the use of the oat polypeptide for preparing health products or drugs for treating cardiovascular and cerebrovascular diseases.

[0017] Another aspect of the present application provides a pharmaceutical composition comprising an effective amount of the oat polypeptide of the present application, and the amino acid sequence of the oat polypeptide is shown as SEQ ID NO: 1.

[0018] The oat polypeptide of the present application or the pharmaceutical composition comprising the oat polypeptide of the present application is administered in the form of a freeze-dried powder.

[0019] In some embodiments, the oat polypeptide or the pharmaceutical composition comprising the oat polypeptide of the present application can be further used in combination with other drugs for treating cardiovascular and cerebrovascular diseases.

[0020] In some embodiments, the drug for treating cardiovascular and cerebrovascular diseases can be selected from antihypertensive drugs such as captopril, enalapril, losartan, valsartan, amlodipine, nifedipine, metoprolol, bisoprolol, hydrochlorothiazide, furosemide, nitroglycerin, isosorbide dinitrate, amiodarone, propafenone, metoprolol, verapamil, digoxin, spironolactone, dapagliflozin, aspirin, clopidogrel, warfarin, rivaroxaban, simvastatin, atorvastatin, rosuvastatin, PCSK9 inhibitor, sodium nitroprusside, alteplase, and the like.

[0021] Beneficial effects

[0022] The active polypeptide extracted and prepared from the oat of the present application can effectively reduce the secretion of Renin, ANGII and LDL-C, promote the synthesis and secretion of BK and HDL-C, reduce the atherosclerosis index, and / or reduce systolic and / or diastolic blood pressure, improve cardiovascular and cerebrovascular diseases, and has certain clinical application value. In addition, the polypeptide 7G5 provided by the present application is easy to synthesize, low in cost, and easy to popularize and apply. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1A The results shown are the changes in systolic blood pressure of spontaneous hypertensive rats (SHR) after treatment with 7G5 polypeptide.

[0024] Figure 1B The results shown are the changes in diastolic blood pressure of SHR after treatment with 7G5 polypeptide.

[0025] Figure 2A The results shown are the decrease in the content of Renin in the serum of SHR after treatment with 7G5 polypeptide.

[0026] Figure 2B The results shown are the decrease in the content of Angiotensin II (ANGII) in the serum of SHR after treatment with 7G5 polypeptide.

[0027] Figure 2C The results shown are the increase in the content of Bradykinin (BK) in the serum of SHR after treatment with 7G5 polypeptide.

[0028] Figure 3A The results shown are the decrease in the content of LDL-C in the serum of hyperlipidemic rats after treatment with 7G5 polypeptide.

[0029] Figure 3B The results shown are the increase in the content of HDL-C in the serum of hyperlipidemic rats after treatment with 7G5 polypeptide.

[0030] Figure 4 The results shown are the changes in the AI ​​index of hyperlipidemic rats after treatment with 7G5 peptide. Detailed Implementation

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0032] Except as provided in the operational examples or otherwise indicated, all figures for the amount of expressed components or reaction conditions used herein should be understood to be modified by the term "about" in all cases. When used in conjunction with percentages, the term "about" may mean ±1%.

[0033] In some embodiments, the pharmaceutical compositions provided herein comprise about 1 pg to about 2000 mg of the active polypeptide described herein (e.g., a polypeptide component, which may be a single polypeptide in some embodiments), optionally wherein the pharmaceutical composition comprises about 1 pg to about 1000 mg, about 1 pg to about 500 mg, about 1 pg to about 400 mg, about 1 pg to about 300 mg, about 1 pg to about 200 mg, about 1 pg to about 100 mg, about 1 pg to about 50 mg, about 1 pg to about 25 mg, about 1 pg to about 20 mg, about 1 pg to about 15 mg, about 1 pg to about 10 mg, about 1 pg to about The active polypeptides described herein (e.g., polypeptide components, which in some embodiments may be a single polypeptide) of 5 mg, about 1 pg to about 1 mg, about 1 pg to about 500 pg, about 1 pg to about 250 pg, about 1 pg to about 200 pg, about 1 pg to about 150 pg, about 1 pg to about 100 pg, about 1 pg to about 50 pg, about 1 mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg to about 400 mg, about 1 mg to about 300 mg, about 1 mg to about 200 mg, about 1 mg to about 100 mg, about 1 mg to about 50 mg, about 1 mg to about 25 mg.

[0034] As used herein, the terms “treatment” or “improvement” are used interchangeably. These terms refer to the means by which a beneficial or desired outcome is achieved, including but not limited to therapeutic and / or preventative benefits.

[0035] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include", "includes" and "included" is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising a single unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Further, the use of the term "part" can include a part of a part or an entire part. Throughout this specification, unless the context requires otherwise the word "comprise", or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0036] The term "therapeutically effective amount" refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition, in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of a disease, disorder, and / or condition. Those of ordinary skill in the art will understand that a therapeutically effective amount does not necessarily result in successful treatment in every particular treated individual. Rather, a therapeutically effective amount can be an amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount can be a reference to an amount as measured in one or more particular tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will understand that, in some embodiments, a particular agent or therapy can be formulated and / or administered in a single dose to achieve a therapeutically effective amount. In some embodiments, a therapeutically effective agent can be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0037] The pharmaceutical composition of the present application further comprises a pharmaceutically acceptable carrier.

[0038] Further, the pharmaceutical compositions of the present application can be in oral dosage forms. Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active polypeptide is mixed with at least one conventional inert diluent such as sodium citrate or dicalcium phosphate or with such other ingredients as binders, (a) fillers or solubilizers, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) humectants, such as hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) moisturizing agents, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate; (e) solution retarders, such as paraffin; (f) absoφtion accelerators, such as quaternary ammonium compounds; (g) moistening agents, such as lecithin and

[0039] The provided formulations can include a lyoprotectant, for example, which can be selected from the group consisting of sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, maltose, lactulose, maltitol, glucitol, lactitol, isomaltulose, and mannitol; an amino acid, for example, arginine or histidine or proline or glycine; a kosmotropic salt, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethylcellulose, and combinations thereof.

[0040] Alternatively or additionally, in some embodiments, the provided formulation can comprise a penetration enhancer, for example, it can be selected from the group consisting of bile salts, such as trihydroxy salt cholic acid sodium, glycocholic acid sodium, taurocholic acid sodium and dihydroxy salts, deoxycholic acid sodium, glycodeoxycholic acid sodium, taurodeoxycholic acid sodium; fatty acids, their salts and esters, such as, for example, oleic acid, lauric acid, cod liver extract, sodium laurate, sodium caprate, glycerol monostearate, diethylene glycol monoethyl ether and various sucrose fatty acid esters, medium chain fatty acid glycerides, polycaprolactone eoma-3 fatty acids, lecithin (phosphatidylcholine), lysophosphatidylcholine; surfactants, such as sodium dodecyl (lauryl) sulfate, polysorbate (polysorbate 80), lauryl ether, brijs and benzalkonium chloride; complexing agents, such as cyclodextrins, dextran sulfate, dextran sulfate, sodium ethylenediaminetetraacetate; complexing agents, such as cyclodextrins, dextran sulfate, dextran sulfate, sodium ethylenediaminetetraacetate, cosolvents, such as ethanol and propylene glycol, a combination of 1% oleic acid and 5% / 10% polyethylene glycol 200, a combination of 2% glyceryl monolaurate and 40% alcohol, sodium caprate and alcohol or propylene glycol, a combination of 10% lauric acid in propylene glycol, polyoxyethylene, 2,3-lauryl ether, menthol, sodium caprate, sodium caprylate, sodium glycodeoxycholate, ethylene glycol; polysaccharides, such as chitosan and chitosan glutamate; and others, such as aprotinin, benzalkonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, sodium salicylate, lysophosphatidylcholine, methoxy salicylate, hydroxymethyl salicylate, sodium edta, sulfoxides, various alkyl glycosides, ethylenediaminetetraacetic acid (edta), tartaric acid; lyotropic salts, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethyl cellulose and combinations thereof.

[0041] Optionally or additionally, in some embodiments, the provided formulation can include an absorption enhancer, for example, which can be selected from a surfactant, a cholate, a glyceride, a salicylate, a bile salt, a chelating agent, sodium caprate, a salt of capric acid, and others including N-(5-chlorosalicyloyl)-8- aminooctanoic acid (5-CNAC), 4-((4-chloro-2-hydroxybenzoyl))-amino)butyric acid (4- CNAB), and N-(8-(2-hydroxybenzoyl))-amino)octanoic acid, also known as sodium nicosulfate (SNAC, octanoic acid, C8, castor oil, medium-chain, acylcarnitine, EDTA, glycerol monolaurate, bovine P- casein, tocopherol succinate ethylene glycol chitosan conjugate, lecithin, glycerol monostearate (GMS), chitosan and alginate, PLGA, silicon dioxide, stearic acid, oleic acid, hydrogenated castor oil, and trimyristin, etoposide phosphate, sulindac, enalapril maleate, ramipril, olmesartan medoxomil, valacyclovir, midodrine, gabapentin enacarbil, sulfasalazine, or alternatively or additionally, in some embodiments, the provided formulation can include a mucosal bioadhesive agent, for example, which can be selected from sucrose, lactose, trehalose, dextran, erythritol, arabinose, xylitol, sorbitol, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, and mannitol; an amino acid, for example, arginine or histidine or proline or glycine; a lyotropic salt, such as magnesium sulfate; propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol);Gelatin, dextrin, modified starch, carboxymethyl cellulose and combinations thereof, mucoadhesive systems, such as mucoadhesive systems from nature, such as gelatin, agarose, chitosan, hyaluronic acid and synthetic polymers, such as polyvinylpyrrolidone (PVP), polyacrylate, polyvinyl alcohol, sodium carboxymethyl cellulose (SCMC) and pectin, all anionic polymers, chitosan (cationic) and hydroxypropyl methylcellulose (HPMC) as non-ionic polymer, polyacrylic acid (PAA) derivatives (CP 934, CP 940, PCP), 15% CMC and 35% CP, copolymer of acrylic acid and poly(ethylene glycol) monomethyl ether monomethacrylate (PEG MM), eudragit l NE 40D is a neutral poly(ethyl acrylate methacrylate), hydrophilic polymers such as, methocel K4M, methocel K15M, SCMC 400, Cekol 700, Cekol 10000, CP 934P, CP 971P and CP 974P, carboxyvinyl polymers and triethanolamine, HPC (hydroxypropyl cellulose), CP (carbopol 934P), carbopol (CP) Ex-55 CMC (sodium carboxymethyl cellulose), HPMC (hydroxypropyl methylcellulose), HEC (hydroxyethyl cellulose), PIP [poly(isoprene)], PIB [poly(isobutylene)], xanthan gum, locust bean gum, pectin, polycarbophil, benzyl ester, hydroxyethyl cellulose, poly(acrylic acid), poly(acrylic acid-co-acrylamide), poly(acrylic acid-co-methyl methacrylate), poly(acrylic acid-co-butyl acrylate), (bioadhesive polymer blend of CP and PIB), formulations consisting of PVP, cetylpyridinium chloride (as stabilizer), chitosan chloride, polyethylene oxide, polymethylvinyl ether / maleic anhydride (PME / MA) and tragacanth gum, polyethylene glycol monomethyl ether monomethacrylate, drum-dried waxy maize starch (DDWM), carbopol 974P and sodium stearyl fumarate and cellulose derivatives; hydrogels - acrylic acid (polar) and butyl acrylate (non-polar) and combinations thereof.

[0042] In addition to these inert diluents, the compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0043] In addition to the active polypeptide, the suspension can contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide gel, and agar-agar, or mixtures of these substances.

[0044] Compositions for parenteral injection can include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0045] Dosage forms of the polypeptides of the present application for topical administration include powders, patches, sprays, and inhalers. The active ingredients are mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as can be required. Ophthalmic formulations, ear drops, eye drops, ear drops, eye drops, and the like, are also contemplated as being within the scope of the present application.

[0046] The pharmaceutical compositions of the present application are formulated to have a pH of 5.5 to 7.5. In one embodiment, the pH of the aqueous medium can be adjusted by low concentrations of suitable biocompatible buffer ingredients, non-limiting examples of which are tromethamine, sodium carbonate and sodium bicarbonate, and sodium phosphate monobasic and dibasic.

[0047] The compositions of the present application can be administered daily or intermittently, and the frequency of administration can be once daily or 2 to 3 times daily. If each of the two active ingredients is a single formulation, the frequency of administration thereof can be the same as or different from each other. In addition, the compositions of the present application can be used alone or in combination with other drugs for treating cardiovascular and cerebrovascular diseases. In view of all the above factors, it is important to administer at the lowest dose to achieve the best efficacy without side effects, which can be easily determined by those skilled in the art. In some embodiments, the dosing regimen is repeated, for example, once, twice, three times or more; for example, for the remainder of the life of the individual in need thereof.

[0048] The present application is further described in detail by the following drawings and examples, but in no way limited by the present application, any transformation or improvement based on the teaching of the present application, falls within the scope of the present application.

[0049] Example 1. Preparation and screening of oat active polypeptides

[0050] Oat powder was obtained by mechanically crushing oat through a 40-mesh sieve and placing it in a drying dish for subsequent use. The oat powder was defatted using petroleum ether, mixed with petroleum ether at a ratio of 1:5 (g / mL), and stirred magnetically at room temperature for 3 h, centrifuged at 4000 rpm for 30 min, and repeated the extraction 3 times. The precipitate was collected and dried at 60°C to obtain oat defatted powder, which was finally stored at 4°C for later use.

[0051] The oat powder was mixed with distilled water at a ratio of 1:10 g / mL, and the pH was adjusted to 10 with 1 mol / L NaOH solution. After magnetic stirring at 50°C for 1 hour, centrifugation was performed at 5000 r / min for 15 min. The liquid was adjusted in pH by adding hydrochloric acid, and then centrifuged again. The precipitate was further adjusted in pH until neutral. The obtained liquid was poured into a dialysis bag for 24 hours of dialysis, and the dialyzed liquid was finally freeze-dried.

[0052] The oat protein was mixed with 20 times distilled water to prepare an oat protein solution, and neutral protease was added for enzymolysis for 3 hours. After enzymolysis, the oat protein solution was immersed in boiling hot water for 10 minutes for enzyme inactivation. After cooling to room temperature, centrifugation was performed at 10000 r / min for 10 minutes. The supernatant was collected, and the yield of oat polypeptide was determined before freeze-drying.

[0053] The oat protein enzymolysis solution was filtered through a water phase 0.45 μm microporous filter membrane, and then fractionated by ultrafiltration membranes with molecular weights of 10 KDa, 5 KDa and 3 KDa. Four kinds of oat protein enzymolysis solutions with different molecular weights (<3 kDa, 3-5 kDa, 5-10 kDa and >10 kDa) were obtained by ultrafiltration fractionation, and each was configured to a concentration of 5 mg / mL. The inhibition rates of each molecular weight on angiotensin converting enzyme (ACE) were 75.37±0.15%, 60.95±0.13%, 53.11±0.08% and 38.55±0.07%, respectively.

[0054] The ACE inhibition rate was detected by the following method: 100 μL of 1 mol / L furanpropyl tripeptide FAPGG (dissolved in 50 mmol / L Tris-HCl buffer containing 0.3 mmol / L NaCl, pH 7.50), 50 μL of sample and 50 μL of ACE (dissolved in borate buffer, pH 8.30) were sequentially added in a 96-well plate, and the reaction was performed at 37°C. The decrease in absorbance at 340 nm was recorded for 30 min. The blank experiment was performed by replacing the sample solution with the buffer. The calculation method of ACE inhibition rate was calculated according to the following formula: ACE inhibition rate (%) = (ΔA blank-ΔA sample) / ΔA blank x 100%, wherein ΔA blank is the decrease in absorbance in 30 min under blank conditions, and ΔA sample is the decrease in absorbance in 30 min in the sample group.

[0055] The polypeptide component with a molecular weight of <3 kDa was purified by Sephadex G-25 gel chromatography. The concentration of the polypeptide solution was 10 mg / mL, 2 mL was loaded, the flow rate was adjusted to 0.8 mL / min, detection was performed at 220 nm, and 1 tube was collected every 5 min. The components belonging to the same elution peak were combined. The collected peak components were diluted to the same concentration, and the ACE inhibition rates of different peak components were compared. The results showed that two chromatographic peaks F1 (38 min) and F2 (52 min) were separated, and the ACE inhibition rates were 54.35% and 78.62%, respectively.

[0056] The F2 component with the highest ACE inhibition rate was concentrated and injected into an Agilent C18 chromatographic column. Pure water containing 1% TFA was used as mobile phase A, and a solution of 1% TFA in acetonitrile was used as mobile phase B; the injection volume was 10 μl; the speed was 1 mL / min; and the detection wavelength was 220 nm. One tube of eluent was collected every 6 min. The collected components were diluted to the same concentration (0.1 mg / mL), and the ACE inhibition rates of different peak components were compared. According to the hydrophobicity, the F2 component was divided into 10 sub-components (A1-A10). Among them, A1 has the highest hydrophilicity, and A10 has the highest hydrophobicity. A5 has the highest ACE inhibition rate, which is 65.09%.

[0057] After gel chromatography and RP-HPLC purification, the part with the highest ACE inhibition rate was desalted by Pierce C18 Spin Tips column and freeze-dried. The polypeptide sample was redissolved in 0.1% formic acid water (solvent A), and its concentration was analyzed using Q-ExactivePlus and Thermo Fisher Scientific EASY-nanoLC 1200 system. In a gradient of 60 min, 3 μL of polypeptide sample (1.5 mg / mL) was loaded onto a PepMap C18 column (75 μm x 250 mm), starting from 2% buffer B (80% acetonitrile plus 0.1% formic acid), gradually increasing to 35% buffer B, and then increasing to 100% buffer B within 1 min, and maintaining for 12 min. The flow rate was kept at 300 nL / min, and the column temperature was kept at 40°C. The electrospray voltage was set to 2 kV. Full scan MS spectra (m / z 200-1800) were obtained in the Orbitrap with a resolution of 70000.

[0058] A total of 1324 polypeptides were detected by HPLC-MS / MS, which were screened and ranked by computer analysis. 24 ACE inhibitory peptides were screened, and the polypeptide 7G5 with the optimal ACE inhibition activity was finally selected, and its amino acid sequence is shown as SEQ ID NO: 1.

[0059] Example 2.7 Effects of G5 peptide on spontaneously hypertensive rats

[0060] Blood pressure lowering effect measurement:

[0061] A 42-day antihypertensive effect study was conducted using spontaneously hypertensive rats (SHR) with a systolic blood pressure (SBP) above 170 mmHg. Specifically, 24 male SHR rats aged 14-17 weeks (body weight 280±20g) (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into a low-dose group, a high-dose group, a positive control group, and a blank control group, with 6 rats in each group. The 7G5 peptide groups were administered 0.25 g / kg and 0.5 g / kg via gavage, respectively, while the positive control group was administered captopril (Changzhou Pharmaceutical Co., Ltd.) at a gavage dose of 30 mg / kg. All treatments were administered using purified water. A blank control group was also included, administered once daily for 42 consecutive days. SBP and diastolic blood pressure (DBP) were measured weekly to observe the effects of each test substance on the blood pressure of the SHR rats.

[0062] The results are as follows Figure 1A and 1B As shown, long-term oral administration of oat bioactive peptide 7G5 can reduce systolic blood pressure in rats. Figure 1A ) and diastolic blood pressure ( Figure 1B The effect was obvious, and the high-dose group had a better antihypertensive effect than the low-dose group, with an earlier onset of action.

[0063] Serum markers measurement

[0064] After the last gavage, the animals in each group were fasted for 12 hours but allowed free water. They were anesthetized intraperitoneally with 10% chloral hydrate at 400 mg / kg body weight. About 5 mL of whole blood was collected from the abdominal aorta. The blood was allowed to clot naturally at room temperature and then centrifuged (3500 rpm, 10 min, 4℃). The yellow, transparent supernatant was collected as serum and aliquoted into multiple 1.5 mL Eppendorf (EP) tubes. The serum was stored at -20℃ for later use.

[0065] The levels of renin, angiotensin II (ANGII), and bradykinin (BK) in serum were determined using a microplate spectrophotometer and an ELISA kit (purchased from Nanjing Senbega Biotechnology Co., Ltd.).

[0066] The results for Renin, ANGII, and BK are as follows: Figures 2A-2CAs shown, the results showed that long-term gavage of oat active polypeptide 7G5 could reduce the levels of Renin and ANGII, while increasing the content of BK. The antihypertensive effect of the high-dose group of 7G5 was comparable to that of captopril (30 mg / kg).

[0067] Body weight and organ index

[0068] During the 42-day antihypertensive test, the body weight of SHR was measured once a week. The results showed that there was no significant difference in body weight between the low-dose group, the high-dose group, the captopril group and the blank control group (P>0.05), indicating that oat active polypeptide 7G5 and captopril had no significant effect on the health status of SHR.

[0069] The body weight of rats was weighed before anesthesia, and the rats were dissected after anesthesia and exsanguination. The heart, liver, spleen, lung and kidney were taken, the surface excess blood was washed with physiological saline, and the surface residual liquid was absorbed with filter paper. The organs were weighed and the organ index was calculated. The organ index calculation formula is:

[0070] Organ index (%) = organ weight (g) / body weight (g) x 100%.

[0071] The results are shown in the following table. There was no significant difference in organ index of liver, spleen, lung and kidney between the blank control group, the low-dose group and the high-dose group of 7G5 polypeptide (P>0.05), indicating that oat active polypeptide 7G5 had no significant effect on the above organs of SHR. The heart organ index of the high-dose group of 7G5 polypeptide and the captopril group was significantly lower than that of the blank control group (P<0.05), indicating that high-dose 7G5 polypeptide and captopril could effectively reduce blood pressure, improve myocardial remodeling, and reduce the damage of long-term high-level blood pressure to the heart. In addition, the kidney organ index of the captopril group was significantly increased, indicating that long-term gavage of captopril might have an impact on the kidney.

[0072] Table 1

[0073] Organ Control group 7G5 low dose group 7G5 high dose group Captopril group Heart % 0.409±0.011 0.383±0.012 0.372±0.009* 0.364±0.011* Lung % 2.653±0.122 2.575±0.103 2.586±0.091 2.534±0.109 Spleen % 0.153±0.011 0.144±0.012 0.151±0.011 0.156±0.012 Lung % 0.351±0.021 0.344±0.014 0.343±0.012 0.338±0.013 Kidney % 0.606±0.041 0.632±0.390 0.629±0.034 0.668±0.031*

[0074] "*" indicates P<0.05 compared with the blank control group

[0075] Example 3. Effect of 7G5 polypeptide on hyperlipidemic rats

[0076] The high-fat emulsion was used to gavage SD (Sprague-dawley, SD) rats to build a high blood fat rat model. After the model was successfully built, the oat active polypeptide 7G5 was gavaged to observe its effect on the lipid metabolism of rats. Specifically, male SD rats (body weight 120±20g) were randomly divided into 7G5 polypeptide low-dose group, high-dose group, positive control group, model control group and blank control group, 8 rats per group. The blank control group was gavaged with 1mL / 100g of normal saline, and the other groups were gavaged with high-fat emulsion at 1mL / 100g of body weight, once a day, for a total of 60 days, while normal drinking water and basic feed were provided. After the last gavage of each group of animals, the animals were fasted for 12h without water. The orbital venous plexus was used to collect blood, and the serum was separated to determine the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) to observe whether the model was successful.

[0077] After the model was successfully built, the 5 groups of mice were gavaged with different solutions, as follows: blank control group (distilled water), model control group (distilled water), positive control group (5mg / kg simvastatin (Tianfang Pharmaceutical Co., Ltd.)), 7G5 polypeptide low-dose group (0.25g / kg), and 7G5 polypeptide high-dose group (0.5g / kg). The gavage was performed at a fixed time every day, and the gavage dose for each mouse was 0.1mL / 10g, once a day, for 4 weeks.

[0078] After the last gavage of each group of animals, the animals were fasted for 12h without water, and the abdominal aorta was used to collect about 5mL of whole blood under abdominal cavity anesthesia with 10% chloral hydrate at 400mg / kg of body weight. The blood was left to naturally coagulate at room temperature, and the yellow transparent supernatant was obtained by centrifugation (3500rpm, 10min, 4℃). The content of TC, LDL-C and HDL-C in the serum was determined using a microplate spectrophotometer and an ELISA kit (purchased from Nanjing Jiancheng Bioengineering Institute), and the atherosclerotic index (AI) was calculated according to the following formula:

[0079] AI=(TC-HDL-C) / HDL-C

[0080] The results of LDL-C and HDL-C are shown in Figures 3A-3B Compared with the model control group, the oat polypeptide 7G5 can reduce the content of LDL-C in the serum of high blood fat rats and promote the increase of HDL-C, indicating that the oat polypeptide 7G5 has good efficacy in regulating lipid metabolism disorders.

[0081] The AI results are shown in Figure 4 Compared with the model control group, the atherosclerotic index (AI) of the oat polypeptide 7G5 group is reduced, indicating that the oat polypeptide 7G5 can improve the adverse conditions of atherosclerosis in rats and has a certain preventive effect on atherosclerosis, coronary heart disease, etc.

Claims

1. An active polypeptide, characterized in that, The active polypeptide amino acid sequence is shown as SEQ ID NO:

1.

2. The active polypeptide of claim 1, which is capable of reducing systolic and / or diastolic blood pressure, and / or reducing arterial stiffness index.

3. A nucleic acid molecule encoding the active polypeptide of claim 1 or 2.

4. A vector comprising the nucleic acid molecule of claim 3.

5. An isolated host cell comprising the active polypeptide of claim 1 or 2, the nucleic acid molecule of claim 3, or the vector of claim 4.

6. Use of the active polypeptide of claim 1 or 2 for the preparation of a health product or a medicament for treating cardiovascular and cerebrovascular diseases.

7. A pharmaceutical composition comprising an effective amount of the active polypeptide of claim 1 or 2.

8. The pharmaceutical composition of claim 7, which is administered in the form of a lyophilized powder.

9. The pharmaceutical composition of claim 7 or 8, which is further combined with other drugs for treating cardiovascular and cerebrovascular diseases.

10. The pharmaceutical composition of claim 9, wherein the drug for treating cardiovascular and cerebrovascular diseases can be captopril, enalapril, losartan, valsartan, amlodipine, nifedipine, metoprolol, bisoprolol, hydrochlorothiazide, furosemide, nitroglycerin, isosorbide dinitrate, amiodarone, propafenone, metoprolol, verapamil, digoxin, spironolactone, dapagliflozin, aspirin, clopidogrel, warfarin, rivaroxaban, simvastatin, atorvastatin, rosuvastatin, PCSK9 inhibitor, sodium nitroprusside, or alteplase.

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