Application of plant extracts in the treatment of hypertension

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a plant extract and its application. This invention prepares tartary buckwheat active polypeptides by enzymatically hydrolyzing tartary buckwheat protein. The tartary buckwheat active polypeptides can effectively inhibit the activity of angiotensin-converting enzyme (ACE), and therefore can be used as ACE inhibitors to lower blood pressure. The plant extract of this invention not only has highly effective 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.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a plant extract and its application in the treatment of hypertension, as well as its preparation method. Background Art

[0002] Hypertension is a prevalent chronic disease. Statistics show that approximately 70% of strokes and 50% of myocardial infarctions are closely linked to hypertension, making it a significant cause of cardiovascular death. China has a large population of people with hypertension. With my country's aging population and urbanization, the number of hypertension patients is increasing annually. Therefore, how to prevent and treat hypertension has become a challenge for the medical community, and research into hypertension drugs and treatment methods is constantly deepening. Currently, there are various types of antihypertensive drugs commonly used in clinical practice, but all have certain side effects. Some are unsuitable for certain special populations, limiting their long-term use. For example, calcium channel blockers may cause rapid heartbeat, facial flushing, and ankle edema in hypertensive patients with long-term use. Diuretics promote potassium excretion, and high doses may cause hypokalemia in patients with this condition. Long-term use of beta-blockers may cause side effects such as fatigue and slowed heart rate. Common side effects of angiotensin-converting enzyme inhibitors include dry cough; some patients may also experience hypotension, rash, taste disturbance, and angioedema. Therefore, researchers are paying increasing attention to developing safe, effective, and less toxic natural antihypertensive active substances from food that can replace Western medicines, and antihypertensive peptides derived from food proteins are particularly suitable for making various health foods.

[0003] Tartary buckwheat (Fagopyrum tataricum), also known as Tatar buckwheat, belongs to the Polygonaceae family and the Fagopyrum genus. Tartary buckwheat is relatively rich in amino acids, fatty acids, and vitamins, possessing high and unique nutritional value. It plays a role in preventing and treating diabetes, fighting cancer, anti-tumor activity, and combating fatigue, offering various health benefits. Tartary buckwheat protein has a complete amino acid composition, making it a full-value protein. Tartary buckwheat protein peptides are hydrolyzed products of tartary buckwheat protein. Studies have shown that tartary buckwheat protein peptides possess various physiological functions, with currently the most widely studied being antioxidant, hypoglycemic, hypotensive, and antibacterial effects. Li et al. reported that buckwheat flavonoids do not have a blood pressure lowering effect, while buckwheat protein, after being treated with pepsin, with the addition of trypsin and chymotrypsin, significantly enhanced ACE inhibitory activity, and extracted two ACE inhibitory peptides, Tyr-Gln-Tyr and Pro-Ser-Tyr (Li CH, Matsui T, Matsui K, et al. Latent production of angiotensin I-converting enzyme inhibitors from Tartary buckwheat protein[J]. Journal of Peptide Science, 2002, 8(6): 267-274.), indicating that buckwheat protein peptides can be used as functional food ingredients with antihypertensive properties.

[0004] Angiotensin-converting enzyme (ACE) is an enzyme found on vascular endothelial cells. Its main function is to convert angiotensin I (Ang I) into angiotensin II (Ang II). Ang II is a potent vasoconstrictor, causing vasoconstriction and increased vascular resistance, thus leading to elevated blood pressure. Furthermore, Ang II stimulates adrenaline secretion, increases sympathetic nerve excitability, and promotes sodium and water reabsorption, increasing blood volume and further raising blood pressure. Therefore, ACE is an important therapeutic target for regulating blood pressure. ACE inhibitors are commonly used antihypertensive drugs that inhibit ACE activity, reduce Ang II synthesis, and decrease the vasoconstrictive effect of Ang II, thus causing vasodilation. In addition, ACE inhibitors can increase angiotensin levels and inhibit the degradation of angiotensin by ACE, thereby achieving the purpose of vasodilation and lowering blood pressure.

[0005] Because different proteases have different optimal conditions and cleavage sites, the types and effects of ACE-inhibiting peptides obtained after enzymatic digestion of tartary buckwheat protein also differ. Therefore, it is necessary to screen for new bioactive tartary buckwheat 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 extract, specifically a tartary buckwheat 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 extract of this invention not only has highly effective 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 extract, characterized in that the plant extract is an active polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

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

[0009] Another aspect of the present invention provides a nucleic acid molecule that encodes the plant extract described herein.

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

[0011] Another aspect of the present invention provides an isolated host cell comprising the plant extract described in this invention, the nucleic acid molecule described in this invention, or the carrier described in this invention.

[0012] Another aspect of the invention provides the use of the plant extract 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 extract described herein, the amino acid sequence of which is shown in SEQ ID NO:1.

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

[0015] In some embodiments, the plant extracts of the present invention or pharmaceutical compositions comprising the plant extracts 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, propranolol, hydralazine, nifedipine, amlodipine, hydrochlorothiazide, or captopril.

[0018] Beneficial effects

[0019] This invention extracts and prepares an active polypeptide from tartary buckwheat plants. This active polypeptide effectively inhibits the activity of angiotensin-converting enzyme (ACE) and plays a protective role in a human umbilical vein endothelial cell (HUVECs) oxidative stress injury model. The polypeptide of this invention has excellent therapeutic effects on hypertension and has certain clinical application value. Furthermore, the polypeptide 3G7 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 illustrate the effect of the active peptide 3G7 on the survival rate of HUVECs in the H2O2 model.

[0021] Figure 2 The results shown illustrate the effect of the active peptide 3G7 on NO release from HUVECs in the H2O2 model.

[0022] Figure 3 The results shown indicate the effect of active peptide 3G7 on SOD enzyme activity in HUVECs in the H2O2 model.

[0023] Figure 4 The results shown indicate the effect of the active peptide 3G7 on the MDA content in HUVECs in the H2O2 model. 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 of achieving a beneficial or desired outcome (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 references 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 may 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 active polypeptides from tartary buckwheat

[0044] 1.1 Extraction of buckwheat protein

[0045] Fresh buckwheat was ground into powder, passed through a 60-mesh sieve, defatted with petroleum ether, and air-dried to obtain defatted buckwheat powder. The powder was mixed with water at a ratio of 1:12 (m / v), and the pH was adjusted to 10.00 using NaOH (1 mol / L). The mixture was heated to 55℃ and stirred for 50 min to extract crude protein. The supernatant was centrifuged at 5000 rpm for 15 min to obtain the supernatant. The supernatant was adjusted to pH 3.00 using HCl (1 mol / L), and allowed to precipitate for 1 h. The precipitate was then centrifuged at 5000 rpm for 15 min, dialyzed for 48 h, and freeze-dried to obtain crude buckwheat protein.

[0046] 1.2 Tartary buckwheat protein hydrolysis

[0047] A tartary buckwheat protein dispersion was prepared using PBS buffer (pH 7.00), and the pH was adjusted to 2.00. Pepsin with a protein content of 4% was added, and hydrolysis was carried out at 37°C for 2 hours. Then, the pH was adjusted to 7.00, a certain amount of trypsin was added, and hydrolysis was carried out at 37°C for 2 hours. After boiling to inactivate the enzyme, the mixture was centrifuged at 10000 rpm for 15 minutes, and the supernatant was the tartary buckwheat protein hydrolysate.

[0048] 1.3 Assay for ACE inhibitory activity

[0049] 100 μL of 1 mol / L furanopropyl tripeptide FAPPGG (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 added sequentially to a 96-well plate, and the reaction was carried out at 37 °C. The decrease in absorbance at 340 nm was recorded, lasting for 30 min. A blank experiment was performed using the buffer solution instead of the sample solution.

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

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

[0052] ΔA blank represents the decrease in absorbance within 30 minutes under blank conditions, while ΔA sample represents the decrease in absorbance within 30 minutes for the sample group.

[0053] 1.4 Isolation and purification of ACE inhibitory peptides

[0054] Gel filtration chromatography (GFC)

[0055] The tartary buckwheat protein hydrolysate exhibiting the best ACE inhibitory effect was purified. The hydrolysate was first filtered through a 0.22 μm microfiltration membrane, then separated using an AKTA Avant 25 system Superdux peptide 10 / 300GL gel column (10 mm × 300 mm) with water as the mobile phase at a flow rate of 0.4 mL / min. Peaks were collected at 280 nm, and the collected peak components were diluted to the same concentration (0.1 mg / mL). The inhibition rates of different peak components on ACE were compared. The results showed that GFC separated three chromatographic peaks, with the last peak (24 min), A3, exhibiting the highest ACE inhibition rate of 72.39%.

[0056] RP-HPLC

[0057] After GFC separation, the peak with the highest ACE inhibition rate was concentrated and injected into an X Bridge Prep C18RP-HPLC column (10 mm × 100 mm). Gradient elution was performed using water (phase A) containing 0.1% TFA and acetonitrile (phase B) containing 0.1% TFA as the mobile phase, with a flow rate controlled at 2 mL / min. Eluent was collected over time at 220 nm, one tube every 6 min. The collected fractions were diluted to the same concentration (0.1 mg / mL), and the inhibition rates of different peak fractions on ACE were compared. Based on hydrophobicity, fraction A3 was divided into 10 subfractions (B1, B2, B3, B4, B5, B6, B7, B8, B9, and B10). B1 exhibited the highest hydrophilicity, while B9 showed the highest hydrophobicity. B5 exhibited the highest ACE inhibition rate, at 60.09%.

[0058] Identification of ACE inhibitory peptides

[0059] After purification by GFC and RP-HPLC, the fraction with the highest ACE inhibition rate was desalted using a Pierce C18 Spin Tips column and lyophilized. The peptide sample was redissolved in 0.1% formic acid-water (solvent A), and its concentration was analyzed using a Q-Exactive Plus and a Thermo Fisher Scientific EASY-nanoLC 1200 system. In a 60-min gradient, 3 μL of peptide sample (1.5 mg / mL) was loaded onto a PepMap C18 column (75 μm × 250 mm), starting with 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, maintaining this level for 12 min. The flow rate was maintained at 300 nL / min, and the column temperature at 40 °C. The electrospray voltage was set to 2 kV. Full-scan MS spectra (m / z 200–1800) were obtained in Orbitrap at a resolution of 70,000.

[0060] A total of 1648 peptides were detected by HPLC-MS / MS. They were screened and ranked using computer analysis, and 32 ACE inhibitory peptides were selected. Finally, peptide 3G7 with the best ACE inhibitory activity was selected, and its amino acid sequence is shown in SEQ ID NO:1.

[0061] Example 2. Effects of peptide 3G7 on the function of human umbilical vein endothelial cells

[0062] 2.1 Cell viability assay

[0063] A cell model of oxidative stress injury in human umbilical vein endothelial cells (HUVECs) was constructed using H2O2 as an inducer. Specifically, cells with a density of 1×10⁻⁶ cells were used. 5 HUVECs were seeded at 200 μL / mL in 96-well plates. For the experimental groups, 100 μL of either 1.2 mg / mL peptide 3G7 or the ACE inhibitor captopril was added to the HUVECs. After 24 h of culture, 200 μL of 1000 μmol / L H2O2 was added to the HUVECs, and the cells were incubated for 4 h. Cell viability was assessed using the MTT assay. Control groups were also included: a blank control group (normal growth) and a model control group (treated with only H2O2).

[0064] After H2O2 treatment and removal of the culture medium, cell viability was determined using an MTT assay kit (Nanjing Jiancheng Bioengineering Institute). The calculation formula is as follows:

[0065] Cell viability (%) = (OD570nm of experimental group / OD570nm of blank group) × 100%

[0066] The results are as follows Figure 1 As shown, in the H2O2 model, the active peptide 3G7 can effectively promote the survival of HUVECs, indicating that it can play a protective role in oxidative stress damage.

[0067] 2.2 Determination of Nitric Oxide (NO) Content

[0068] When endothelial cells are damaged by H2O2, the production and release of the vasoactive substance NO are inhibited. Therefore, by detecting changes in the release level of NO regulatory factors, the protective effect of ACE inhibitory peptides against oxidative damage in HUVECs cells can be evaluated.

[0069] Specifically, the density is 1×10 5 HUVECs were seeded at 200 μL / mL in 96-well plates, with 200 μL of 1000 μmol / L H2O2 added to each well, and cultured for 24 h. In the experimental group, 200 μL of 1.2 mg / mL peptide 3G7 or the ACE inhibitor captopril was added to HUVECs, and after 24 h of culture, NO levels were measured using a NO assay kit (Nanjing Jiancheng Bioengineering Institute). Control groups were also set up: a blank control group was cultured in 200 μL of serum-free medium for 48 h, and a model control group was cultured in 200 μL of 1000 μmol / L H2O2 for 48 h.

[0070] The results are as follows Figure 2 As shown, in the H2O2 model, NO release increased significantly after treatment with the active peptide 3G7; and the effect was comparable to that of the ACE inhibitor captopril.

[0071] 2.3 Superoxide dismutase (SOD) activity assay

[0072] Endothelial cells are damaged by H2O2, which leads to a decrease in SOD activity. Therefore, by detecting SOD activity, the protective effect of ACE inhibitory peptides against oxidative damage in HUVECs cells can be evaluated.

[0073] Specifically, the density is 1×10 5HUVECs cells were seeded at a concentration of 2 mL / well in 6-well plates. 200 μL of 1000 μmol / L H₂O₂ was added to each well, and the cells were cultured for 24 h. In the experimental group, 100 μL of either 1.2 mg / mL peptide 3G7 or the ACE inhibitor captopril was added to the HUVECs cells. After 24 h of culture, the culture medium was discarded, cells were scraped off with a cell scraper, sonicated, allowed to stand for 2 min, and centrifuged to collect the supernatant. The SOD activity in the supernatant was measured using a SOD detection kit (Nanjing Jiancheng Biotechnology Institute). Control groups were also set up: a blank control group was cultured in 200 μL of serum-free culture medium for 48 h, and a model control group was cultured in 200 μL of 1000 μmol / L H₂O₂ for 48 h.

[0074] The results are as follows Figure 3 As shown, in the H2O2 model, SOD activity was significantly increased after treatment with the active peptide 3G7; and the effect was comparable to that of the ACE inhibitor captopril.

[0075] 2.4 Determination of malondialdehyde (MDA) content

[0076] Lipid peroxidation is an important indicator of the degree of cell damage in an organism, and intracellular MDA content is often used as an indirect indicator of lipid peroxidation. After endothelial cells are damaged by H2O2, the intracellular MDA content increases significantly. Therefore, by detecting MDA content, the protective effect of ACE inhibitory peptides against oxidative damage in HUVECs cells can be assessed.

[0077] Specifically, the density is 1×10 5 HUVECs cells were seeded at a concentration of 2 mL / well in 6-well plates. 200 μL of 1000 μmol / L H₂O₂ was added to each well, and the cells were cultured for 24 h. In the experimental group, 100 μL of either 1.2 mg / mL peptide 3G7 or the ACE inhibitor captopril was added to the HUVECs cells. After 24 h of culture, the culture medium was discarded, cells were scraped off with a cell scraper, sonicated, allowed to stand for 2 min, and centrifuged to collect the supernatant. The MDA content in the supernatant was measured using an MDA detection kit (Nanjing Jiancheng Bioengineering Institute). Control groups were also set up: the blank control group was cultured in 200 μL of serum-free culture medium for 48 h, and the model control group was cultured in 200 μL of 1000 μmol / L H₂O₂ for 48 h.

[0078] The results are as follows Figure 4 As shown, in the H2O2 model, the MDA content decreased significantly after treatment with the active peptide 3G7; and the effect was comparable to that of the ACE inhibitor captopril.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the polypeptide as described in claim 1.

3. A vector comprising the nucleic acid molecule as described in claim 2.

4. An isolated host cell comprising the nucleic acid molecule as described in claim 2, or the vector as described in claim 3.

5. Use of the polypeptide according to claim 1 in the preparation of a medicament for treating hypertension.

6. A pharmaceutical composition comprising an effective amount of the polypeptide as claimed in claim 1.

7. The pharmaceutical composition according to claim 6, wherein it is administered in the form of a lyophilized powder.

8. The pharmaceutical composition according to claim 6 or 7, further used in combination with other antihypertensive drugs.

9. The pharmaceutical composition according to claim 8, wherein the antihypertensive drug is reserpine, guanethidine, prazosin, propranolol, hydralazine, nifedipine, amlodipine, hydrochlorothiazide, or captopril.

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