A ginseng polypeptide and use thereof in treating cardiovascular and cerebrovascular diseases

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a preparation method of ginseng polypeptide and application thereof. The ginseng active polypeptide is prepared by enzyme hydrolysis of ginseng protein. The ginseng active polypeptide can effectively inhibit the activity of thrombin, and thus can be used as a thrombus formation inhibitor to treat cardiovascular and cerebrovascular diseases such as myocardial infarction and ischemic stroke. The plant extract of the present application not only has an anti-thrombus effect, but also has no toxicity and no side effects, and has the advantages of simple preparation, easy absorption and the like, thereby avoiding the adverse effects of side effects of traditional chemical drugs such as bivalirudin on human body.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a ginseng polypeptide and its use in the treatment of cardiovascular and cerebrovascular diseases. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are a group of diseases caused by multiple factors, mainly including coronary heart disease (ischemic heart disease), cerebrovascular disease, rheumatic heart disease, congenital heart disease, aortic aneurysm, and other peripheral vascular diseases. Coronary heart disease and cerebrovascular disease have the highest mortality rates and are the leading causes of death worldwide, accounting for 80% of the burden in low- and middle-income countries. Primary prevention is crucial for improving cardiovascular health and preventing cardiovascular disease. The American Heart Association defines ideal cardiovascular health as the absence of cardiovascular disease and meeting all seven indicators of cardiovascular health, including four healthy behaviors (smoking, physical activity, weight, and diet) and three health risk factors (total cholesterol, blood pressure, and fasting blood glucose). Considering the global burden of cardiovascular disease, a cost-effective peptide drug that can improve cardiovascular health and prevent cardiovascular disease is highly attractive.

[0003] Ginseng holds a unique position in the history of Chinese medicine, hailed as the "King of Herbs," and is a nationally recognized iconic medicinal herb. Modern medical research has proven that ginseng has excellent protective effects on the central nervous system, cardiovascular system, respiratory system, blood and hematopoietic system, endocrine system, and reproductive system. Furthermore, research has shown that ginseng also possesses significant anti-tumor, anti-radiation, anti-aging, and detoxification functions. Thanks to advancements in chemical analysis methods, significant progress has been made in the isolation, purification, and structural identification of ginseng components over the past thirty years. To date, ginseng roots are known to contain various effective components, including ginsenosides, polysaccharides, volatile oils, fatty acids, sterols, vitamins, proteins, and polypeptides. Further research has revealed that many ginseng polypeptides have anti-lipid decomposition and blood sugar and liver glycogen-lowering effects. However, current research on ginseng polypeptides mainly focuses on metabolic diseases, with limited research on their role in cardiovascular diseases, particularly in the important area of ​​anti-thrombosis. The pathophysiological mechanisms of thrombosis are complex, involving multiple processes such as cardiomyocyte hypertrophy, fibrosis, energy metabolism disorders, and apoptosis. Existing therapeutic drugs still have limitations and cannot fully meet clinical needs. Therefore, it is necessary to screen and identify novel bioactive peptides from ginseng, systematically evaluate their protective effects on the cardiovascular system and their molecular mechanisms, and provide scientific evidence and candidate molecules for the development of innovative drugs for the treatment of cardiovascular and cerebrovascular diseases, especially those that inhibit thrombus formation. Summary of the Invention

[0004] In view of the current state of the technology, the purpose of this invention is to provide a ginseng polypeptide, specifically a ginseng active polypeptide, which can effectively inhibit thrombin activity and therefore can be used as a thrombosis inhibitor to treat cardiovascular and cerebrovascular diseases such as myocardial infarction and ischemic stroke. The plant extract of this invention not only has antithrombotic effects but is also non-toxic and has no side effects. It also has the advantages of simple preparation and easy absorption, avoiding the adverse effects on the human body caused by the side effects of traditional chemical drugs such as bivalirudin and other thrombosis inhibitors. The present invention first provides a ginseng polypeptide, characterized in that the polypeptide is a ginseng active polypeptide, and its amino acid sequence is shown in SEQ ID NO: 1.

[0005] In some embodiments, the ginseng polypeptide is a thrombin inhibitor.

[0006] Another aspect of the present invention provides a nucleic acid molecule encoding the ginseng polypeptide described herein.

[0007] Another aspect of the present invention provides a carrier comprising the nucleic acid molecule described herein.

[0008] Another aspect of the present invention provides an isolated host cell comprising the ginseng polypeptide, the nucleic acid molecule, or the carrier described in the present invention.

[0009] Another aspect of the present invention provides the use of the ginseng polypeptide in the preparation of a medicament for treating cardiovascular and cerebrovascular diseases.

[0010] In some embodiments, the cardiovascular and cerebrovascular diseases are myocardial infarction or ischemic stroke.

[0011] Another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the ginseng polypeptide of the present invention, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0012] The ginseng polypeptide of the present invention, or a pharmaceutical composition containing the ginseng polypeptide of the present invention, is applied in the form of lyophilized powder.

[0013] Beneficial effects This invention extracts and prepares an active polypeptide from ginseng. This active polypeptide effectively inhibits thrombin activity and protects endothelial cells, inhibiting thrombus formation. Therefore, the polypeptide of this invention has excellent therapeutic effects on cardiovascular and cerebrovascular diseases and has certain clinical application value. The polypeptide S3-136 provided by this invention is easy to synthesize, low in cost, and easy to promote and apply. Furthermore, the S3-136 polypeptide of this invention does not excessively inhibit coagulation function and does not exhibit the serious side effect of uncontrollable bleeding that may occur with bivalirudin. Attached Figure Description

[0014] Figure 1 The results show that the S3-136 peptide inhibits thrombin-induced S1PR2 expression.

[0015] Figure 2 The results shown represent the survival rates of mice in the acute thrombosis model after different treatment groups.

[0016] Figure 3 The results shown are the PT indexes of mice in the acute thrombosis model after different treatments.

[0017] Figure 4 The results shown are the aPTT index of mice in the acute thrombosis model after different treatments. Detailed Implementation

[0018] 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.

[0019] 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%.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The compositions of the present invention can be administered daily or intermittently, with a frequency of once daily 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 cardiovascular disease treatments. 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.

[0035] 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.

[0036] Example 1: Screening of Ginseng Bioactive Polypeptides 1.1 Ginseng protein extraction Take a quantity of raw ginseng, pulverize it using a high-speed tissue homogenizer, weigh 500g, add 10 times the volume of 10mM pH7.4 Tris-HCl (0.15M NaCl) buffer, stir well, and extract at 4℃ for 12h. Centrifuge at 5000r / min for 30min, add 10 times the volume of 10mM pH7.4 Tris-HCl (0.15M NaCl) to the precipitate for a second extraction, centrifuge, and combine the supernatants. Add solid (NH4)2SO4 to the supernatant to a final concentration of 80%, stir well, let stand overnight at 4℃, centrifuge at 5000r / min for 30min, dissolve the precipitate in distilled water, dialyze thoroughly with water, and freeze-dry to obtain total ginseng protein.

[0037] 1.2 Ginseng protein hydrolysis A 3% ginseng protein solution was prepared using deionized water. Papain was selected for enzymatic hydrolysis of the ginseng protein. The hydrolysis conditions were: enzyme-to-substrate ratio of 1%, hydrolysis time of 3 hours, pH of 7, and hydrolysis temperature of 50℃. After hydrolysis, the enzyme was inactivated by boiling in a 100℃ water bath for 15 minutes, cooled to room temperature, centrifuged at 10000 rpm for 10 minutes, and then freeze-dried to obtain the ginseng protein hydrolysate.

[0038] 1.3 Isolation and purification of ginseng polypeptides Anhydrous ethanol was added to the ginseng protein hydrolysate at a volume ratio of 1:3 and stirred until homogeneous. The mixture was then allowed to stand at 4°C for 12 hours. The neutral enzyme hydrolysate after ethanol precipitation was then centrifuged (8000 r / min, 20 min) to remove the anhydrous ethanol, and the supernatant was collected to obtain the ginseng protein ethanol precipitate. The concentration of the ginseng protein ethanol precipitate was adjusted, and the precipitate was filtered using an aqueous filter membrane. Ultrafiltration was then performed using a 3 kDa ultrafiltration membrane. The resulting retentate (S1) and ultrafiltrate (S2) were freeze-dried under vacuum. Furthermore, S2 was fractionated using a 1 kDa ultrafiltration membrane, and the resulting retentate (S3) and ultrafiltrate (S4) were used to prepare and collect freeze-dried powders, which were stored together with the other components at -20°C.

[0039] 1.4 In vitro thrombin inhibitory effect of ultrafiltration components Prepare solutions S1, S2, S3, and S4 at the same concentration. Set the microplate reader to 405 nm wavelength and 37°C. Dissolve fibrinogen at a concentration of 0.1% in Tris-HCl buffer (pH 7.2), and prepare thrombin at a concentration of 10 U / ml. Add 140 μL of fibrinogen solution and 40 μL of sample solution to the wells of the plate, mix, and incubate at 37°C for 5 min, then measure the absorbance. Add 10 μL of thrombin to start the reaction, incubate for 5 min, and measure the absorbance again. Calculate the thrombin inhibition rate of the peptide using the following formula.

[0040] Thrombin inhibition rate % = ASB−AS / ACB−AC × 100%, where AS and AC represent the absorbance values ​​of the sample group and the blank group before thrombin addition, respectively; ASB and ACB represent the absorbance values ​​of the sample group and the blank group after thrombin incubation, respectively.

[0041] The results showed that at a concentration of 1 mg / ml, the S3 component had the highest thrombin inhibition rate of 36.26%, while the thrombin inhibition rates of S1, S2 and S4 were 13.16%, 25.37% and 7.21%, respectively.

[0042] 1.5 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) for Peptide Omics Detection S3 fraction was ultrafiltered and desalted before mass spectrometry detection. Solution B was a 0.1% formic acid aqueous solution and a 0.1% formic acid solution in acetonitrile (84% acetonitrile). The LC column (0.15 mm × 150 mm, rp-c18) was equilibrated with 95% liquid A, and the sample was loaded onto a Zorbax 300sb-c18 peptide trap via an autosampler. After separation on another LC column, the relevant LC gradient settings were as follows: 0–50 min, linear gradient of liquid B from 4% to 50%; 50–54 min, linear gradient of liquid B from 50% to 100%; 54–60 min, where liquid B was maintained at 100%. Digested products were separated by capillary HPLC and analyzed by precision mass spectrometry over 60 min. Detection mode: positive ion. Peptides and peptide fragment mass-to-charge ratios were collected according to the following method: 10 fragments were obtained after each full scan (MS2 scan).

[0043] The raw mass spectrometry data were retrieved using Max quant 1.5.5.1 software to search the UniProtAspidochirotida 31557 20221021 database. Charcot parameters were as follows: enzyme (absent), maximum deletion cleavage (2), fixed modification (-), variable modification (oxidation (M)), Decoy database mode (reverse), peptide tolerance (20 ppm), MS / MS tolerance (0.1 Da), and FDR (≤0.01). Finally, protein identification and quantification results, as well as etymological analysis of all peptide sequences, were obtained.

[0044] PeptidRanker (https: / / distilldeep.ucd.ie / PeptideRanker / ) predicts the probability of biological activity in each peptide sequence within the enzymatic digest, setting a threshold of 0.6 to further evaluate peptide sequences and predict activity scores. The proportion of peptide sequences with scores higher than 0.6 in each sample group was calculated. Finally, the physicochemical properties were analyzed using the online tool Expasy ProtoParam (www.expasy.org): molecular weight (WM); theoretical isoelectric point (pI); and average number of hydrophilicities (GRAVY).

[0045] The top 20 ranked peptides were selected. Shanghai Qiangyao Biotechnology Co., Ltd. was commissioned to synthesize the pre-selected thrombin-inhibiting peptides using a solid-phase synthesis method, with a purity greater than 90%. The thrombin-inhibiting activity of the synthesized peptides was verified, and the peptide with the best thrombin-inhibiting activity was S3-136, whose amino acid sequence is shown in SEQ ID NO: 1.

[0046] Example 2. Effect of peptide S3-136 on thrombin-induced endothelial cell injury Excessive thrombin production can damage vascular endothelial cells. When blood vessels are damaged, the normal physiological functions of the vascular endothelial cells are disrupted, leading to the aggregation and activation of fibrinogen and platelets at the site of damage, ultimately forming a thrombus. Therefore, the homeostasis of vascular endothelial cells has a significant impact on whether or not a thrombus forms.

[0047] Specifically, the density is 1×10 6 HUVECs cells were seeded at 2 mL / well in 6-well plates and cultured for 24 h. 2 U / mL thrombin (Prolytix, HCT-0020) was added to each well. Additionally, 50 μM of peptide S3-136 was added to the experimental group, and 30 μg / mL bivalirudin (Selleck, P1004) was added to the positive control group. After 2 h of culture, the cells were centrifuged at 12000 rpm for 15 min at 4°C, and the pellet was discarded. An appropriate amount of RNA extraction buffer was added to extract RNA from each group of cells. RT-PCR was then performed to obtain the corresponding DNA products. Q-PCR was subsequently performed to measure the expression levels of thrombosis-related proteins. Results are as follows: Figure 1 As shown, the antithrombotic peptide S3-136 effectively inhibits thrombin-induced S1PR2 protein expression. The results indicate that the antithrombotic peptide S3-136 can inhibit thrombin-induced endothelial damage, thereby effectively inhibiting thrombus formation.

[0048] Example 3. In vivo antithrombotic activity of peptide S3-136 An acute thrombosis model induced by thrombin was established. First, animals were randomly divided into four groups (Group A, Group B, Group C, and Group D), with seven male and seven female mice in each group. Group A received PBS buffer; Group B received 750 U / kg thrombin solution; Group C received 1 mg / kg bivalirudin solution and 750 U / kg thrombin solution; and Group D received 10 mg / kg S3-136 peptide and 750 U / kg thrombin solution. All reagents were dissolved in PBS (0.01M, pH 7.4) buffer. The number of mice with hemiplegia and deaths within five minutes was recorded, and the survival rate of thrombin-induced acute thrombosis in each group was calculated. Immediately afterwards, blood was collected from each group of mice, anticoagulated with 3.2% sodium citrate, and centrifuged at 3000 rpm for 10 min. The supernatant was collected, plasma was obtained, and the activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured.

[0049] Before the experiment, 25 mM calcium chloride solution was transferred to a 5 mL centrifuge tube and preheated at 37°C in the sample preheating chamber of a coagulation analyzer (MC4, Merlin, Germany). The activated partial thromboplastin time (aPTT) kit (Sunbiote, 6011) was used. Magnetic beads, 100 μL of plasma, and 100 μL of aPTT reagent were added to the coagulation vessel and incubated at 37°C for 180 s. Then, 100 μL of calcium chloride solution was added to the coagulation vessel, and the aPTT time of the plasma was measured.

[0050] The PT reagent powder was dissolved and transferred to a 5 mL centrifuge tube, which was then placed in the sample preheating chamber of a coagulation analyzer (MC4, Merlin, Germany) and preheated at 37°C. The prothrombin time kit (Sunbiote, 4001) was used for the experiment. First, magnetic beads and 100 μL of plasma were added to the coagulation vessel and incubated at 37°C for 60 s. Then, 200 μL of PT reagent solution was added to the coagulation vessel, and the PT time of the plasma was measured.

[0051] The results of S3-136 polypeptide on thrombin-induced acute thrombosis in mice are as follows: Figure 2 As shown in the figure. The results indicate that the S3-136 peptide can effectively inhibit thrombin-induced acute thrombosis in mice. All mice treated with the S3-136 peptide survived, while the mortality rate of mice in the experimental group treated with thrombin alone exceeded 90%.

[0052] The results of PT and aPTT indices are as follows: Figure 3-4 As shown in the figure. The results showed that the blood parameters of mice in group A were normal. Group B mice, due to the large consumption of clotting factors, exhibited different blood clotting characteristics compared to group A, but their blood still clotted within a relatively short time. In contrast, group C showed a PT delay of more than 3 times and an aPTT delay of more than 30 times compared to group A, indicating coagulation abnormalities in group C, which could potentially lead to serious side effects such as uncontrollable bleeding. In comparison, while group D also showed prolonged clotting compared to group A, its PT and aPTT values ​​were less than twice that of group A and showed no significant difference from group B, indicating that its blood could still clot normally. These results demonstrate that S3-136 can significantly reduce the mortality rate caused by thrombin-induced acute thrombosis without causing adverse conditions such as uncontrollable bleeding.

Claims

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

1.

2. A nucleic acid molecule encoding the ginseng 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 ginseng polypeptide as described in claim 1, the nucleic acid molecule as described in claim 2, or the carrier as described in claim 3.

5. The use of the ginseng polypeptide according to claim 1 in the preparation of a drug for treating cardiovascular and cerebrovascular diseases, wherein the cardiovascular and cerebrovascular diseases are myocardial infarction or ischemic stroke.

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

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

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