Plant polypeptides and their use for lowering blood pressure

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a preparation method of plant polypeptide and application thereof. The present application prepares active polypeptide of Schisandra chinensis (Turcz.) Baill. by enzyme hydrolysis of Schisandra chinensis (Turcz.) Baill. protein. The active polypeptide of Schisandra chinensis (Turcz.) Baill. 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 extract of the present application not only has the functions of high blood pressure reducing effect, non-toxicity, no side effect, etc., but also has the advantages of simple preparation, easy absorption, etc., and can avoid the adverse effects of side effects of traditional chemical drugs on human body.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a plant-derived active polypeptide, its application in the treatment of hypertension, and its preparation method. Background Technology

[0002] Hypertension is a chronic disease characterized by systolic blood pressure ≥140 mmHg and diastolic blood pressure ≥90 mmHg. It can cause a series of cardiovascular diseases such as stroke, coronary heart disease, and atherosclerosis. Statistics show that in 2000, approximately 972 million adults worldwide suffered from hypertension, and this number is projected to increase to 1.56 billion by 2025, an increase of 60%. However, by 2015, the number of adult hypertension patients had already increased to 1.13 billion, with men accounting for 52.8% and women for 47.2%. This demonstrates that hypertension has become a global health problem.

[0003] The causes of hypertension include the following: (1) a family history of hypertension; (2) high stress in modern life, which can lead to mental tension and anxiety, and may cause hypertension; (3) excessive intake of salt and its products; (4) lack of exercise, obesity, and some bad habits, such as smoking. Excessive obesity can compress blood vessels, causing blood pressure to gradually increase. Tobacco contains nicotine, which stimulates the heart and kidneys to release catecholamines, thereby increasing heart rate and causing blood vessels to constrict, leading to increased blood pressure. In medicine, hypertension is divided into two types: primary and secondary. Patients with primary hypertension need to rely on medication to control their blood pressure for life because the cause is still unclear. Secondary hypertension is caused by other physical diseases, such as kidney disease, endocrine disorders, or external injuries. These patients can be cured through symptomatic treatment.

[0004] Currently, the main medications for treating hypertension are chemically synthesized Western medicines, such as angiotensin-converting enzyme (ACE) inhibitors, beta-blockers, calcium channel blockers, and diuretics. Although these drugs are highly effective, they can cause adverse reactions such as dermatitis, bradycardia, dry cough, and angioedema.

[0005] Bioactive peptides are low-molecular-weight polymers that are beneficial to the body and easily absorbed. They are protein fragments composed of 2-20 amino acids released from proteins, with a molecular weight of less than 10 kDa. They have the advantages of being free of side effects and easily absorbed by the human body. Natural plant-derived bioactive peptides are abundant, inexpensive, and easy to prepare from raw materials. For example, seeds from cereal straws (wheat, oats, corn) are commonly used to extract bioactive peptides. Among them, bioactive peptides with antihypertensive functions obtained from plants can serve as ideal alternatives to commercially available antihypertensive drugs. Their biological mechanism mainly involves inhibiting the activity of angiotensin-converting enzyme (ACE), thereby affecting the renin-angiotensin system to regulate systemic systolic and diastolic blood pressure. Natural plant-derived ACE-inhibiting peptides have significant advantages in terms of safety, mildness, and lack of side effects. Therefore, it is necessary to screen for new Schisandra chinensis bioactive peptides to meet the clinical demand for antihypertensive drugs. Summary of the Invention

[0006] In view of the current state of the technology, the purpose of this invention is to provide a plant polypeptide, specifically a Schisandra chinensis active polypeptide, which can effectively inhibit the activity of angiotensin-converting enzyme (ACE), and therefore can be used as an ACE inhibitor to lower blood pressure. The plant polypeptide of this invention not only has significant antihypertensive effects, is non-toxic, and has no side effects, but also has the advantages of simple preparation and easy absorption, avoiding the adverse effects of traditional chemical drugs on the human body.

[0007] The present invention first provides 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.

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

[0009] Another aspect of the present invention provides a nucleic acid molecule encoding the plant polypeptide described herein.

[0010] Another aspect of the present invention provides a carrier comprising the nucleic acid molecules described herein.

[0011] Another aspect of the present invention provides an isolated host cell comprising the plant polypeptides, nucleic acid molecules, or vectors described in the present invention.

[0012] Another aspect of the invention provides the use of the plant polypeptide in the preparation of a medicament for treating hypertension.

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

[0014] The plant polypeptides of the present invention, or pharmaceutical compositions containing the plant polypeptides of the present invention, are applied in the form of lyophilized powder.

[0015] In some embodiments, the plant polypeptides of the present invention or pharmaceutical compositions containing the plant polypeptides of the present invention may be used in combination with other antihypertensive drugs.

[0016] In some embodiments, the antihypertensive drug may be selected from diuretics, beta-blockers, alpha-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, or angiotensin II receptor antagonists.

[0017] In some embodiments, the antihypertensive drug may be reserpine, guanethidine, prazosin, spironolactone, propranolol, furosemide, hydralazine, nifedipine, amlodipine, hydrochlorothiazide, triamterene, losartan, candesartan, amiloride, or captopril.

[0018] Beneficial effects

[0019] This invention extracts and prepares an active polypeptide from Schisandra chinensis. This active polypeptide effectively inhibits the activity of angiotensin-converting enzyme (ACE) and promotes the release of NO from human umbilical vein endothelial cells (HUVECs), while simultaneously inhibiting the production of ET-1. Therefore, the polypeptide of this invention has excellent therapeutic effects on hypertension and has certain clinical application value. Furthermore, the polypeptide 7A81 provided by this invention is easy to synthesize, low in cost, and easy to promote and apply. Attached Figure Description

[0020] Figure 1 The results shown are the retention rates of ACE inhibitory activity of the active peptide 7A81 under different pH conditions.

[0021] Figure 2 The results shown are the retention rates of ACE inhibitory activity of active peptide 7A81 at different temperatures.

[0022] Figure 3 The results shown illustrate the effect of active peptide 7A81 on NO release in HUVECs.

[0023] Figure 4 The results shown illustrate the effect of active peptide 7A81 on ET-1 levels in HUVECs. Detailed Implementation

[0024] 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.

[0025] 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%.

[0026] 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.

[0027] 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.

[0028] In this application, unless otherwise specifically stated, the use of the singular includes the plural. In this application, unless otherwise stated, the use of “or” means “and / or”. Furthermore, the use of the term “including” and other forms such as “includes” and “included” is not restrictive. Additionally, unless otherwise specifically stated, terms such as “element” or “component” cover elements and components that include one unit as well as elements and components that include more than one subunit. Additionally, the use of the term “part” can include a portion of a part or an entire portion. Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply inclusion of the specified integer or group of integers, but not to exclude any other integer or group of integers.

[0029] The term "therapeuticly effective amount" refers to the amount that produces the desired effect of its administration. In some embodiments, the term refers to an amount sufficient to treat a disease, condition, and / or ailment when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to such a disease, condition, and / or ailment. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, condition, and / or ailment, and / or delays its onset. Those skilled in the art will understand that a therapeutically effective amount does not necessarily achieve successful treatment in every particular individual. Rather, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a large number of subjects when administered to patients who require such treatment. In some embodiments, references to a therapeutically effective amount can be to an amount measured, such as in one or more specific tissues (e.g., tissues affected by a disease, condition, or ailment) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, a specific agent or therapy can be formulated and / or administered in a single dose. In some implementations, the therapeutic agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0030] The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier.

[0031] Furthermore, the pharmaceutical compositions of the present invention can be 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 excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opaque agents, and the release of the active peptide or peptide in such compositions may be delayed at a site in the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active peptide may also be formed into microcapsules with one or more of the excipients described above. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active peptide, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0032] The provided formulation may include lyophilization protectants, such as those selected from sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucol, maltitol, lactitol, isomaltulose, and mannitol; amino acids, such as arginine, histidine, proline, or glycine; 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.

[0033] Alternatively or additionally, in some embodiments, the provided formulation may comprise a penetration enhancer, such as one selected from bile salts, such as sodium trihydroxycholate, sodium glycocholate, sodium taurocholate and dihydroxycholate, sodium deoxycholate, sodium glycodeoxycholate, sodium taurodeoxycholate; fatty acids, their salts and esters, such as oleic acid, lauric acid, cod liver oil extract, sodium lauryl laurate, sodium decanoate, glyceryl 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 lauryl sulfate, polysorbate (polysorbate 80), lauryl ether, Brijs and benzalkonium chloride; complexing agents, such as cyclodextrin, dextran sulfate, ethylenediaminetetraacetic acid sodium salt; complexing agents, such as cyclodextrin, dextran sulfate, ethylenediaminetetraacetic acid sodium salt; Dextran, sodium EDTA, 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 decanoate and alcohol or propylene glycol, a combination of 10% lauric acid in propylene glycol, polyoxyethylene, 2,3-lauryl ether, menthol, sodium decanoate, sodium octanoate, sodium glycinate, ethylene glycol; polysaccharides such as chitosan and chitosan glutamate; and others It includes, for example, aprotinin, benzalkonium chloride, hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, sodium salicylate, lysophosphatidylcholine, methoxysalicylate, hydroxymethyl oleate, sodium EDTA, sulfoxide, 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.

[0034] Optionally or additionally, in some embodiments, the provided formulation may include an absorption enhancer, such as one selected from surfactants, cholesterol, glycerides, salicylates, bile salts, chelating agents, sodium decanoate, salts of decanoic acid, and others including N-(5-chlorosalicylic acid)-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 salicylate (SNAC, octanoic acid, C8, castor oil, medium chain, acylcarnitine, EDTA, glyceryl monolaurate, bovine P-casein, tocopheryl succinate glycol chitosan conjugate, lecithin, glyceryl monostearate (GMS), chitosan, and alginate. PLGA, silica, stearic acid, oleic acid, hydrogenated castor oil and trimyridine glyceryl, etoposide phosphate, enalapril maleate, ramipril, olmesartan medoxomil, valacyclovir, midodrine, gabapentin enalacarbide, sulfasalazine, or alternatively or additionally, in some embodiments, the provided formulation may contain a mucosal bioadhesive, such as selected from sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucosyl alcohol, maltitol, lactitol, isomaltulose and mannitol; amino acids, such as arginine or histidine or proline or glycine; 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; mucosal adhesion systems, such as those derived from natural sources, such as gelatin, agarose, chitosan, hyaluronic acid, and synthetic polymers, such as polyvinylpyrrolidone (PVP), polyacrylates, polyvinyl alcohol, sodium carboxymethyl cellulose (SCMC), and pectin; all anionic polymers, chitosan (cationic), and hydroxypropyl methyl cellulose (HPMC) as a nonionic polymer; polyacrylic acid (PAA) derivatives (CP934, CP940, PCP), 15% CMC and 35% CP; copolymers of acrylic acid and poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMM); eudragitlNE40D is a neutral poly(ethyl acrylate methacrylate); hydrophilic polymers, such as methocel K4M, methocel K15M, SCMC 400, Cekol 700, Cekol 10000, CP934P, CP971P and CP974P, carboxyvinyl polymers and triethanolamine, HPC (hydroxypropyl cellulose), CP (Carbopol 934P), Carbopol (CP) Ex-55CMC (sodium carboxymethyl cellulose), HPMC (hydroxypropyl methyl cellulose), HEC (hydroxyethyl cellulose), PIP [poly(isoprene)], PIB [poly(isobutylene)], xanthan gum, locust bean gum, pectin, polycarbofil, benzyl ester, hydroxyethyl cellulose Formulations comprising: poly(acrylic acid), poly(acrylic acid-co-acrylamide), poly(acrylic acid-co-methyl methacrylate), poly(acrylic acid-co-butyl acrylate), (bioadhesive polymer blends of CP and PIB), composed of PVP, hexadecylpyridinium chloride (as stabilizer), chlorinated chitosan, polyethylene oxide, polymethyl vinyl ether / maleic anhydride (PME / MA) and tragacanth gum, polyethylene glycol monomethyl ether monomethyl acrylate, drum-dried waxy corn starch (DDWM), carbopol 974P and stearyl fumarate sodium, and cellulose derivatives; hydrogels of acrylic acid (polar) and butyl acrylate (non-polar) and combinations thereof.

[0035] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0036] In addition to active peptides, suspensions may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0037] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0038] Dosage forms of the polypeptides of the present invention 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, if necessary, propellants.

[0039] The pharmaceutical compositions of the present invention 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 buffering agents, non-limiting examples of which are glycerol, sodium carbonate and sodium bicarbonate, and sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0040] The compositions of the present invention can be administered daily or intermittently, with a frequency of once or two to three times daily. If each of the two active ingredients is a single formulation, their administration frequencies can be the same or different. Furthermore, the compositions of the present invention can be used alone or in combination with other antihypertensive drugs. Considering all the foregoing factors, it is important to administer the lowest possible dose to achieve optimal efficacy without side effects, which can be readily 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, repeated over the remaining lifespan of the individual in need.

[0041] Antihypertensive drugs are mainly classified into six categories: diuretics, beta-blockers, alpha-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, and angiotensin II receptor antagonists. Existing antihypertensive drugs affect blood pressure regulation directly or indirectly. Based on their primary sites of action and mechanisms of action in blood pressure regulation, they can be broadly categorized as: drugs acting on the central nervous system, such as clonidine and moxonidine; and ganglion blocking drugs, such as mecamidine and mithifene. These types of drugs block both sympathetic and parasympathetic ganglia, which can cause widespread and severe adverse reactions. Therefore, they are usually only used for rapid blood pressure reduction in severe hypertension or "hypertensive crisis" when other drugs are ineffective. Other drugs include those that affect adrenergic neurotransmitters, such as reserpine and guanethidine; adrenergic receptor blockers, such as prazosin and propranolol; vasodilators, such as hydralazine; calcium channel blockers, such as nifedipine and amlodipine; the basic function of calcium channel blockers is to inhibit the influx of extracellular calcium ions, which, for blood vessels, relaxes smooth muscle cells, dilates blood vessels, and lowers blood pressure; diuretics, such as hydrochlorothiazide; and drugs that affect the renin-angiotensin system, such as captopril.

[0042] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0043] Example 1: Screening of Schisandra chinensis active peptides

[0044] 1.1 Schisandra chinensis protein extraction

[0045] Remove the pulp from the Schisandra chinensis fruit, pulverize it, defatted it with petroleum ether, and then dry it to obtain defatted Schisandra chinensis powder. Weigh a certain amount of the dried defatted Schisandra chinensis powder, add deionized water at a material-to-liquid ratio of 1:35 to homogenize it, and adjust the pH of the original solution to 9.5 with 1 mol / L NaOH solution. Extract by stirring in a water bath at 35℃ for 3 hours, then centrifuge (3500 r / min, 15 min), discard the precipitate, adjust the pH of the supernatant to 3.4, let it stand at 4℃ for 2 hours, centrifuge again, discard the supernatant, dissolve the precipitate with a small amount of deionized water, adjust the pH to neutral, and then put it into a dialysis bag (MW: 8000~14000). Dialyze at 4℃ for 48 hours, changing the deionized water every 2 hours. Freeze-dry the liquid in the dialysis bag. This freeze-dried product is Schisandra chinensis protein, which is dried and stored at -20℃ for later use.

[0046] 1.2 Schisandra chinensis protein hydrolysis

[0047] A 3% substrate concentration of Schisandra chinensis protein solution was prepared using deionized water. Alkaline protease was selected to hydrolyze the Schisandra chinensis protein under the following conditions: enzyme-to-substrate ratio of 1%, hydrolysis time of 3 hours, pH of 9, and hydrolysis temperature of 55℃. After hydrolysis, the enzyme was inactivated by boiling in a 100℃ water bath for 15 minutes. The solution was then cooled to room temperature, the pH was adjusted to neutral, centrifuged at 10000 rpm for 10 minutes, and freeze-dried to obtain the Schisandra chinensis protein hydrolysate.

[0048] 1.3 Assay for ACE inhibitory activity

[0049] Using furanylpropionyl FAPPGG as a mimic substrate for angiotensin I (Ang I), the product of its reaction with ACE decreased the absorbance of the solution at 340 nm. Specifically, 100 μL of FAPPGG (1 mM), 50 μL of sample, and 30 μL of ACE (0.1 U / mL) were added sequentially to a 96-well plate, and the reaction was carried out at 37 °C. The blank experimental group used HEPES buffer (1 mM) instead of the sample solution. The absorbance was measured at 340 nm before and after 30 min of reaction at 37 °C using a microplate reader.

[0050] The ACE inhibition rate is calculated using the following formula:

[0051] ACE inhibition rate (%) = (ΔA blank - ΔA sample) / ΔA blank × 100%

[0052] Where ΔA is the difference in absorbance of the blank group within 30 minutes, and ΔAsample is the difference in absorbance of the sample group within 30 minutes.

[0053] 1.4 Isolation and purification of ACE inhibitory peptides

[0054] Ultrafiltration

[0055] 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).

[0056] Sample desalination

[0057] 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.

[0058] LC-MS / MS detection of peptides

[0059] 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.

[0060] PEAKS software was used to search databases for raw data, combined with manual de novo sequencing to identify different peptides. Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the bioactivity of all identified peptides; peptides with an activity score >0.5 were considered potentially bioactive. The AHTpin website (https: / / webs.iiitd.edu.in / raghava / ahtpin / index.php) was used to calculate and predict potential ACE-inhibiting peptides. Computer analysis was used to screen and rank the identified peptides, selecting the top 20. The pre-selected ACE-inhibiting peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. using a solid-phase synthesis method, achieving a purity greater than 90%. The ACE-inhibiting activity of the synthesized peptides was verified, and their IC50 values ​​were calculated. 50 Value. Among them, the peptide with the best ACE inhibitory activity was 7A81 (IC). 50 =0.96±0.03mg / mL), and its amino acid sequence is shown in SEQ ID NO: 1.

[0061] Example 2. Stability study of peptide 7A81

[0062] 2.1 pH stability

[0063] The pH of the solutions was 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 each solution at different pH values, and the reactions were allowed to proceed for 2 hours. The ACE inhibition rate was then measured. The ACE inhibition rate of the peptide solution at pH 7 was used as a control group.

[0064] ACE inhibition activity retention rate (%) = (100 / ACE inhibition rate of control group) × ACE inhibition rate of experimental group.

[0065] The results are as follows Figure 1 As shown, 7A81 exhibited higher inhibitory activity at pH 5 than at pH 7, with slightly reduced inhibitory activity at other pH values. The lowest level was observed at pH 11 (91.22 ± 0.52%), indicating that 7A81 has some resistance to pH changes.

[0066] 2.2 Thermal stability

[0067] A 0.5 mg / mL peptide solution was prepared, and 500 μL of each solution was heated in a water bath at 20, 40, 60, 80, and 100 °C for 2 h. The samples were then rapidly cooled to room temperature in an ice-water bath to determine ACE inhibitory activity. An untreated 0.5 mg / mL peptide solution was used as a control group, and the retention rate of its ACE inhibitory activity was calculated.

[0068] The results are as follows Figure 2 As shown, the inhibitory activity of 7A81 is enhanced from 25-45℃, and the retention rate of inhibitory activity gradually decreases above 45℃. However, the retention rate of ACE inhibitory activity remains above 85% at 80℃, indicating that 7A81 has good thermal stability.

[0069] Example 3. Effects of peptide 7A81 on the function of human umbilical vein endothelial cells

[0070] 3.1 Determination of Nitric Oxide (NO) Content

[0071] NO can antagonize the angiotensinogenic function of Ang II by downregulating the synthesis of Ang II receptors and ACE, thereby participating in the regulation of peripheral and central cardiovascular function and exerting an angioprotective effect. Therefore, by detecting changes in the release levels of NO regulatory factors, the effect of ACE inhibitory peptides on HUVECs cell function can be assessed.

[0072] Specifically, the density is 1×10 5HUVECs were seeded at 200 μL / mL in 96-well plates. 200 μL of 500 μg / mL peptide 7A81 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 culturing for 24 h, the cells were centrifuged at 12,000 rpm for 15 min at 4°C, and the pellet was discarded. NO levels were measured using a NO assay kit (Nanjing Jiancheng Biotechnology Institute). A blank control group was also included, with 200 μL of serum-free culture medium added for 24 h.

[0073] The results are as follows Figure 3 As shown, NO release increased significantly after treatment with the active peptide 7A81; and the effect was comparable to that of the ACE inhibitor captopril.

[0074] 3.2 Determination of human endothelin-1 (ET-1) content

[0075] ET-1 is a peptide composed of 21 amino acids. It is an endogenous mediator of cardiovascular disease, with a strong vasoconstrictive effect and a pressor effect. Increased ET-1 may lead to endothelial cell abnormalities. It can exert its blood pressure-lowering function by inhibiting the secretion of ET-1 in HUVECs.

[0076] Specifically, the density is 1×10 5 HUVECs 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.

[0077] 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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