Plant polypeptide and application thereof in lowering blood pressure

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of plant polypeptide. The schisandra chinensis active polypeptide is prepared by carrying out enzymolysis reaction on schisandra chinensis protein. The schisandra chinensis active polypeptide can effectively inhibit the activity of angiotensin converting enzyme (ACE), so that the schisandra chinensis active polypeptide can be used as an ACE inhibitor to reduce blood pressure. The plant extract disclosed by the invention not only has the functions of high-efficiency antihypertensive effect, no toxicity, no side effect and the like, but also has the advantages of simplicity in preparation, easiness in absorption and the like, and avoids adverse effects of side effects of traditional chemical medicines on human bodies.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a plant-derived active polypeptide, application thereof to treatment of hypertension, and a preparation method thereof. BACKGROUND

[0002] Hypertension is a chronic disease characterized by systolic pressure ≥ 140 mmHg and diastolic pressure ≥ 90 mmHg, which can cause a series of cardiovascular diseases such as stroke, coronary heart disease, and atherosclerosis. According to statistics, in 2000, there were about 972 million adults worldwide with hypertension, and it is estimated that the number of patients will increase to 156 million, an increase of 60%, by 2025. However, by 2015, the number of adult hypertensive patients had increased to 113 million, of which 52.8% were men and 47.2% were women. It can be seen that hypertension has become a global health problem.

[0003] The causes of hypertension include the following aspects: (1) family history of hypertension; (2) high stress in modern society, mental tension, anxiety, etc., which may cause hypertension; (3) high intake of salt and its products; (4) lack of exercise, obesity, and some bad habits such as smoking. Obesity can cause compression of blood vessels, and the pressure in blood vessels will gradually increase. Nicotine in tobacco stimulates the heart and kidneys to release catecholamines, which increases heart rate and causes blood vessels to contract, leading to increased blood pressure. In medicine, hypertension is divided into primary and secondary types. The primary hypertension patients have unknown causes, and therefore need to rely on medication to control blood pressure for life. Secondary hypertension is caused by some other physical diseases, such as kidney disease, endocrine disorders, or external damage, and these patients can be cured by symptomatic treatment.

[0004] Currently, the drugs for treating hypertension mainly include chemically synthesized western medicines, such as angiotensin-converting enzyme (ACE) enzyme inhibitors, beta-receptor blockers, calcium channel blockers, diuretics, etc. Although these drugs have significant therapeutic effects, they can cause adverse reactions such as dermatitis, slow heartbeat, dry cough, and vascular edema.

[0005] Bioactive peptides are low molecular weight polymers that are beneficial to the biological body and easily absorbed by the body, which are protein fragments composed of 2-20 amino acids released from proteins, and have a molecular weight of less than 10 kDa, with the advantages of no side effects and easy absorption by the human body. Natural plant source bioactive peptide ingredients are rich in sources, low in cost, and simple in raw material preparation, such as grain straw (wheat, oat, corn) seeds, which are commonly used to extract bioactive peptides. Among them, the bioactive peptides with antihypertensive function obtained from plants can be used as an ideal substitute for commercially available antihypertensive drugs. The biological mechanism is mainly to inhibit the activity of angiotensin converting enzyme (ACE), and then affect the renin-angiotensin system to regulate systolic and diastolic blood pressure. The safety, mildness and no side effects of natural plant source ACE inhibitory peptides are outstanding, so it is necessary to screen new active peptides of Schisandra to meet the demand for antihypertensive drugs in clinical practice. SUMMARY

[0006] In view of the prior art, the purpose of the present application is to provide a plant polypeptide, in particular, a Schisandra active polypeptide, which can effectively inhibit the activity of angiotensin converting enzyme (ACE), and thus can be used as an ACE inhibitor to reduce blood pressure. The plant polypeptide of the present application not only has the functions of significant antihypertensive effect, non-toxicity, no side effects, etc., but also has the advantages of simple preparation, easy absorption, etc., avoiding the adverse effects of the side effects of traditional chemical drugs on human body.

[0007] The present application first provides a plant polypeptide, characterized in that the plant polypeptide is a Schisandra active polypeptide, and the amino acid sequence thereof is shown as SEQ ID NO: 1.

[0008] In some embodiments, the plant polypeptide is an ACE inhibitor.

[0009] Another aspect of the present application also provides a nucleic acid molecule encoding the plant polypeptide of the present application.

[0010] Another aspect of the present application also provides a vector comprising the nucleic acid molecule of the present application.

[0011] Another aspect of the present application also provides an isolated host cell comprising the plant polypeptide of the present application, the nucleic acid molecule of the present application, or the vector of the present application.

[0012] Another aspect of the present application also provides the use of the plant polypeptide for preparing a medicament for treating hypertension.

[0013] Another aspect of the present application provides a pharmaceutical composition comprising an effective amount of the plant polypeptide of the present application, and the amino acid sequence of the plant polypeptide is shown as SEQ ID NO: 1.

[0014] The plant polypeptide or the pharmaceutical composition comprising the plant polypeptide of the present application is administered in the form of a lyophilized powder.

[0015] In some embodiments, the plant polypeptide or the pharmaceutical composition comprising the plant polypeptide of the present application can be further used in combination with other anti-hypertensive drugs.

[0016] In some embodiments, the anti-hypertensive drug can be selected from diuretic antihypertensive drugs, beta receptor blockers, alpha receptor blockers, calcium ion antagonists, angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists. In some embodiments, the anti-hypertensive drug can be reserpine, guanethidine, prazosin, spironolactone, propranolol, furosemide, hydralazine, nifedipine, amlodipine, hydrochlorothiazide, aminophylline, losartan, candesartan, amiloride or captopril.

[0017] Beneficial effects The present application extracts and prepares an active polypeptide from Schisandra chinensis, which effectively inhibits the activity of angiotensin converting enzyme (ACE) and can promote the release of NO in human umbilical vein endothelial cells (HUVECs) while inhibiting the production of ET-1. Therefore, the polypeptide of the present application has excellent therapeutic effect on hypertension and has certain clinical application value. In addition, the polypeptide 7A81 provided by the present application is easy to synthesize, low in cost and easy to popularize and apply. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The results shown are the ACE inhibition activity retention rate of the active polypeptide 7A81 under different pH conditions.

[0019] Figure 2 The results shown are the ACE inhibition activity retention rate of the active polypeptide 7A81 under different temperatures.

[0020] Figure 3 The results shown are the effects of the active polypeptide 7A81 on the release of NO in HUVECs.

[0021] Figure 4 The results shown are the effects of the active polypeptide 7A81 on the level of ET-1 in HUVECs. DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be understood by one of ordinary skill in the art to which this application belongs.

[0023] Except in the Examples, or where otherwise indicated, all numbers used herein are to be understood as being modified in all instances by the term "about." The term "about" used in connection with a percentage when used in connection with a percentage can mean ± 1%.

[0024] In some embodiments, the pharmaceutical compositions provided herein comprise from about 1 pg to about 2000 mg of an active polypeptide described herein (e.g., a polypeptide component, which in some embodiments can be a single polypeptide), optionally wherein the pharmaceutical composition comprises from about 1 pg to about 1000 mg, from about 1 pg to about 500 mg, from about 1 pg to about 400 mg, from about 1 pg to about 300 mg, from about 1 pg to about 200 mg, from about 1 pg to about 100 mg, from about 1 pg to about 50 mg, from about 1 pg to about 25 mg, from about 1 pg to about 20 mg, from about 1 pg to about 15 mg, from about 1 pg to about 10 mg, from about 1 pg to about 5 mg, from about 1 pg to about 1 mg, from about 1 pg to about 500 pg, from about 1 pg to about 250 pg, from about 1 pg to about 200 pg, from about 1 pg to about 150 pg, from about 1 pg to about 100 pg, from about 1 pg to about 50 pg, from about 1 mg to about 1000 mg, from about 1 mg to about 500 mg, from about 1 mg to about 400 mg, from about 1 mg to about 300 mg, from about 1 mg to about 200 mg, from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, from about 1 mg to about 25 mg of an active polypeptide described herein (e.g., a polypeptide component, which in some embodiments can be a single polypeptide).

[0025] As used herein, "treatment" or "treating," or "amelioration" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and / or prophylactic benefit.

[0026] 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 "includes" and "included," is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Additionally, the use of the term "part" can include a part or all of a 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.

[0027] 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 of 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.

[0028] The pharmaceutical composition of the present application further comprises a pharmaceutically acceptable carrier.

[0029] 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

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 vehicles include water, ethanol, polyols and suitable mixtures thereof.

[0036] Dosage forms of the polypeptides of the application for topical administration include ointments, powders, patches, sprays and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or necessary propellants as can be required.

[0037] The pharmaceutical compositions of the application are formulated to 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 and sodium bicarbonate, and sodium phosphate dibasic and monobasic.

[0038] The compositions of the 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, their frequency of administration can be the same or different from each other. In addition, the compositions of the application can be used alone or in combination with other antihypertensive drugs. In view of all the above factors, it is important to administer the lowest dose possible to achieve the best efficacy without side effects, which can be easily determined by one skilled in the art. In some embodiments, the dosing regimen is repeated, e.g., once, twice, three times or more; for example, for the remainder of the life of the individual in need.

[0039] The main antihypertensive drugs are diuretic antihypertensive drugs, beta receptor blockers, alpha receptor blockers, calcium antagonists, angiotensin converting enzyme inhibitors, and angiotensin II receptor antagonists. The existing antihypertensive drugs all affect blood pressure regulation directly or indirectly, and according to the main action site and mechanism of various drugs in blood pressure regulation, they are roughly divided into: drugs acting on the central nervous system, such as clonidine, moxonidine, etc.; ganglionic blockers, such as mecamylamine, mioxedil, etc. This class of drugs blocks both sympathetic and parasympathetic ganglia, which can cause widespread and serious adverse reactions, so they are usually used for severe hypertension or rapid reduction of blood pressure in "hypertensive crisis" when other drugs are ineffective; adrenergic neurotransmitter drugs, such as reserpine, guanethidine, etc.; adrenergic receptor blockers, such as prazosin, propranolol, etc.; vasodilators, such as hydralazine; calcium antagonists, such as nifedipine, amlodipine, etc.; the basic action of calcium antagonists is to inhibit the influx of extracellular calcium ions, which relaxes smooth muscle cells and dilates blood vessels to lower blood pressure; diuretics, such as hydrochlorothiazide; drugs affecting the renin-angiotensin system, such as captopril.

[0040] The present application will be further described in detail below with reference to the accompanying drawings and examples, but in no way limits the present application, any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0041] Example 1 Screening of active polypeptide of Schisandra chinensis 1.1 Extraction of Schisandra chinensis protein The fruit of Schisandra chinensis was removed, crushed, and dried after defatting with petroleum ether to obtain defatted Schisandra chinensis powder. A certain amount of defatted Schisandra chinensis powder was weighed, homogenized with deionized water at a solid-liquid ratio of 1:35, and the pH of the original solution was adjusted to 9.5 with 1 mol / L NaOH solution. After stirring in a 35°C water bath for 3 h, centrifugation was performed (3500 r / min, 15 min), and the precipitate was discarded. The pH of the supernatant was adjusted to 3.4, and it was placed at 4°C for 2 h before centrifugation. The supernatant was discarded, and the precipitate was dissolved in a small amount of deionized water. After adjusting the pH to neutral, it was loaded into a dialysis bag (MW: 8000-14000) and dialyzed at 4°C for 48 h, with deionized water replaced every 2 h. The liquid in the dialysis bag was freeze-dried, and the freeze-dried material was Schisandra chinensis protein, which was stored at -20°C for use.

[0042] 1.2 Enzymatic hydrolysis of Schisandra chinensis protein A 3% Schisandra chinensis protein solution was prepared with deionized water, and alkaline protease was selected for enzymatic hydrolysis of the Schisandra chinensis protein. The enzyme-to-substrate ratio was 1%, the hydrolysis time was 3 h, the pH was 9, and the hydrolysis temperature was 55°C. After hydrolysis, the enzyme was inactivated by boiling in a 100°C water bath for 15 min, cooled to room temperature, adjusted to neutral pH, and centrifuged at 10000 r / min for 10 min. The freeze-dried material was Schisandra chinensis protein hydrolysis peptide.

[0043] 1.3 Determination of ACE inhibitory activity Furanpropionic acid FAPGG was used as a mimic substrate of angiotensin I (Ang I), and the product after reaction with ACE would reduce the absorbance of the solution at 340 nm. Specifically, 100 μL of FAPGG (1 mM), 50 μL of sample, and 30 μL of ACE (0.1 U / mL) were sequentially added to a 96-well plate and reacted at 37°C. The blank control group used HEPES buffer (1 mM) instead of the sample solution. The absorbance values before and after 30 min of reaction at 37°C were measured using a microplate reader at a wavelength of 340 nm.

[0044] The calculation method of ACE inhibition rate was calculated according to the following formula: ACE inhibition rate (%) = (ΔAblank-ΔAsample) / ΔAblank x 100% Where ΔA is the difference in absorbance of the blank group within 30 min, and ΔAsample is the difference in absorbance of the sample group within 30 min.

[0045] 1.4 Isolation and purification of ACE inhibitory peptides Ultrafiltration The enzymatic hydrolysate was sequentially separated by ultrafiltration using different MWCO ultrafiltration membranes (10, 5, and 3 kDa) into three fractions: fraction I (<10 kDa), fraction II (<5 kDa), and fraction III (<3 kDa). The obtained ultrafiltrates were lyophilized to obtain Schisandra chinensis polypeptide powders of corresponding molecular weights. After reconstitution at a concentration of 1 mg / mL, the ACE inhibitory activity of each fraction was determined. The results showed that fraction III had the lowest IC50 value (1.35 ± 0.03 mg / mL), exhibiting the strongest ACE inhibitory activity. This was followed by fraction II (1.77 ± 0.05 mg / mL) and fraction I (2.14 ± 0.02 mg / mL).

[0046] Sample desalination Take the ultrafiltration lyophilized sample with the strongest ACE inhibitory activity (<3kDa), dissolve it thoroughly in 0.1% TFA, rinse the C18 column 10 times with 50μL 60% ACN / 0.1% TFA, then wash the C18 column 10 times with 10μL 0.1% TFA. Aspirate and expel the sample from the C18 column 20 times, and drain the liquid. Wash the C18 column 5 times with 10μL 0.1% TFA. Finally, elute the peptide with 10μL 60% ACN and 0.1% TFA, transfer the peptide eluent to a new EP tube, and vacuum dry.

[0047] LC-MS / MS detection of peptides The peptide fragments were dissolved in 30 μL of dissolving buffer (0.1% formic acid, 5% acetonitrile), vortexed thoroughly, and centrifuged at 13500 rpm at 4℃ for 20 min. The supernatant was transferred to a sample tube, and 8 μL was taken for mass spectrometry identification. Liquid chromatography settings: Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% ACN; Elution method: 0–6.00 min 95.0% A, 5.0% B; 6.00–90.00 min 60.0% A, 40.0% B; 90–110.00 min 40.0% A, 60.0% B; 110.00–115.00 min 5.0% A, 95.0% B; 115.00–120.00 min 95.0% A, 5.0% B; Flow rate: 400 μL / min. Mass spectrometry settings: Primary and secondary mass spectrometry scan range: 50 - 1700 m / z; Mass spectrometry information: AGC target: 1e5; Maximum IT: 100 ms; TopN: 12; NCE / stepped NCE: 27.

[0048] The original data were subjected to database searching using PEAKS software, combined with manual de novo sequencing to identify different peptides. Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the possibility of biological activity of all identified peptides, and peptides with an activity score >0.5 were considered to be potentially bioactive peptides. The AHTpin website (https: / / webs.iiitd.edu.in / raghava / ahtpin / index.php) was used for the computational prediction of potential ACE-inhibitory peptides. The identified peptides were screened and ranked using computer analysis, and the top 20 peptides were selected. The ACE-inhibitory peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. using solid-phase synthesis, with a purity of more than 90%. The ACE-inhibitory activity of the synthesized peptides was verified, and their IC 50 values were calculated. The polypeptide with the optimal ACE-inhibitory activity was 7A81 (IC 50 = 0.96 ± 0.03 mg / mL), and its amino acid sequence is shown in SEQ ID NO: 1.

[0049] Example 2. Stability study of polypeptide 7A81 2.1 pH stability The pH values of the solutions were adjusted to 3, 5, 7, 9, and 11 using 1 mol / L HCl and NaOH solutions, respectively. An equal volume of 1 mg / mL peptide solution was added to the solutions at different pH values, and the reaction was allowed to proceed for 2 h. The ACE-inhibitory rate was determined. The ACE-inhibitory rate of the peptide solution at pH 7 was used as the control group.

[0050] ACE-inhibitory activity retention rate (%) = (100 / control group ACE-inhibitory rate) x test group ACE-inhibitory rate.

[0051] The results are shown in Table 1. Figure 1 As shown in Table 1, 7A81 exhibited higher inhibitory activity at pH 5 than at pH 7, and the inhibitory activity was slightly reduced at other pH values, with the lowest level at pH 11 (91.22 ± 0.52%), indicating that 7A81 has certain resistance to pH changes.

[0052] 2.2 Thermal stability A 0.5 mg / ml peptide solution was prepared, and 500 μL was taken and placed in a water bath at 20, 40, 60, 80, and 100°C for 2 h, respectively. The samples were then quickly cooled to room temperature in an ice water bath to determine the ACE-inhibitory activity. The untreated 0.5 mg / mL peptide solution was used as the control group, and the ACE-inhibitory activity retention rate was calculated.

[0053] The results are shown in Table 2. Figure 2As shown in the table, the inhibitory activity of 7A81 was improved when the temperature was from 25-45°C, and the retention rate of the inhibitory activity gradually decreased when the temperature was higher than 45°C. However, the retention rate of the ACE inhibitory activity was still maintained at more than 85% at 80°C, indicating that 7A81 has good thermal stability.

[0054] Example 3. Effect of polypeptide 7A81 on the function of human umbilical vein endothelial cells 3.1 Determination of the content of nitric oxide (NO) NO can antagonize the vasoconstrictive function of Ang II by down-regulating the synthesis of Ang II type receptors and ACE, and thus participate in the regulation of the peripheral and central functions of the cardiovascular system, and play a vascular protective role. Therefore, by detecting the changes in the release level of NO regulatory factors, the effect of ACE inhibitory peptides on the function of HUVECs can be evaluated.

[0055] Specifically, 1 x 10 5 / mL HUVECs were inoculated into a 96-well culture plate at 200 μL per well. 200 μL of polypeptide 7A81 with a concentration of 500 μg / mL was added to the experimental group, 1 μg / mL of ACE inhibitor Captopril (Cap) was added to the positive control group, and 0.5 μg / mL of norepinephrine (NE) was added to the negative control group. After 24 h of culture, the cells were centrifuged at 4°C and 12000 rpm for 15 min, and the precipitate was discarded. The NO level was determined using a NO kit (Nanjing Jiancheng Biological Engineering Institute). At the same time, a blank control group was set up, in which 200 μL of serum-free culture medium was added for 24 h of culture.

[0056] The results are shown in the table. Figure 3 As shown in the table, the release of NO was significantly increased after treatment with active polypeptide 7A81; and the effect was comparable to that of ACE inhibitor Captopril.

[0057] 3.2 Determination of the content of human endothelin-1 (ET-1) ET-1 is a peptide composed of 21 amino acids, which is an endogenous mediator of cardiovascular diseases, has a strong vasoconstrictive effect, and has a blood pressure raising effect. The increase of ET-1 can lead to endothelial cell abnormalities, and its function of lowering blood pressure can be exerted by inhibiting the secretion of ET-1 in HUVECs.

[0058] Specifically, 1 x 10 5HUVECs were seeded at 200 μL / mL in 96-well plates. 200 μL of peptide 7A81 (500 μg / mL) was added to each well in the experimental group, 1 μg / mL of the ACE inhibitor captopril (Cap) was added to the positive control group, and 0.5 μg / mL of norepinephrine (NE) was added to the negative control group. After 24 h of culture, ET-1 levels were measured using an ET-1 ELISA kit (Sangon Biotech (Shanghai) Co., Ltd.). A blank control group was also included, with 200 μL of serum-free culture medium added and cultured for 24 h.

[0059] The results are as follows Figure 4 As shown, ET-1 levels decreased significantly after treatment with the active peptide 7A81; and the effect was comparable to that of the ACE inhibitor captopril.

Claims

1. A plant polypeptide, characterized in that, The plant polypeptide is a Schisandra chinensis active polypeptide, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The plant polypeptide according to claim 1, wherein the active polypeptide is an ACE inhibitor.

3. A nucleic acid molecule encoding a plant polypeptide as described in claim 1 or 2.

4. A vector comprising the nucleic acid molecule as described in claim 3.

5. An isolated host cell comprising a plant polypeptide as claimed in claim 1 or 2, a nucleic acid molecule as claimed in claim 3, or a carrier as claimed in claim 4.

6. Use of the plant polypeptide according to claim 1 or 2 in the preparation of a medicament for treating hypertension.

7. A pharmaceutical composition comprising an effective amount of the plant polypeptide as described in claim 1 or 2.

8. The pharmaceutical composition according to claim 7, wherein it is administered in the form of a lyophilized powder.

9. The pharmaceutical composition according to claim 7 or 8 may further be used in combination with other antihypertensive drugs.

10. The pharmaceutical composition according to claim 9, wherein the antihypertensive drug may be reserpine, guanethidine, prazosin, spironolactone, propranolol, furosemide, hydralazine, nifedipine, amlodipine, hydrochlorothiazide, triamterene, losartan, candesartan, amiloride, or captopril.

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