Application of hyperoside and metabolite thereof in preparation of antiplatelet drugs

Abstract

The invention belongs to the field of medicine, and relates to application of hyperoside and metabolite thereof in preparation of antiplatelet drugs, hyperoside or 3 '-O-methylated hyperoside in the application has inhibitory activity on PDI reductase, and can also directly inhibit platelet adhesion, platelet activity, blood clot contraction and the like.

Description

Technical Field

[0001] The present application belongs to the field of medicine, and specifically relates to the application of hyperoside and its metabolites in the preparation of antiplatelet drugs. Background Art

[0002] Currently, many antiplatelet drugs have problems such as bleeding risk, drug resistance, and individual differences leading to different efficacy. The development of new, safe and effective antiplatelet drugs has always been the focus and difficulty of cardiovascular research.

[0003] However, the existing drugs still need further exploration in terms of both performance and mechanism. Summary of the invention

[0004] The purpose of the present application is to provide a new drug use of hyperoside or a pharmaceutically acceptable salt thereof, 3'-O-methylated hyperoside or a pharmaceutically acceptable salt thereof.

[0005] The first aspect of the present application is to provide a use of hyperoside or a pharmaceutically acceptable salt thereof in the preparation of an antiplatelet drug; wherein the hyperoside has a structure as shown in the following formula I:

[0006]

[0007] The second aspect of the present application provides a use of 3'-O-methylated hyperoside or a pharmaceutically acceptable salt thereof in the preparation of an antiplatelet drug; wherein the 3'-O-methylated hyperoside has a structure as shown in the following formula II:

[0008]

[0009] In some embodiments of the present application, the application described in the present application, wherein the above-mentioned hyperoside, pharmaceutically acceptable salt of hyperoside, 3'-O-methylated hyperoside or pharmaceutically acceptable salt of 3'-O-methylated hyperoside has the effect of inhibiting the activity of protein disulfide isomerase.

[0010] In some embodiments of the present application, the use described in the present application, wherein the above-mentioned hyperoside, hyperoside pharmaceutically acceptable salt, 3'-O-methylated hyperoside or 3'-O-methylated hyperoside pharmaceutically acceptable salt has the function of inhibiting the upregulation of platelet ROS, inhibiting thrombin-induced platelet Ca 2+ Mobilization, inhibition of platelet adhesion, and inhibition of blood clot retraction.

[0011] In some embodiments of the present application, the above-mentioned drug comprises any one or more of hyperoside, a pharmaceutically acceptable salt of hyperoside, 3'-O-methylated hyperoside or a pharmaceutically acceptable salt of 3'-O-methylated hyperoside.

[0012] In some embodiments of the present application, the drug comprises hyperoside and 3'-O-methylated hyperoside, and the molar ratio (molar ratio) between the hyperoside and 3'-O-methylated hyperoside is 1:100 to 100:1;

[0013] In some embodiments of the present application, the ratio of the amount of substance (molar ratio) is 1:50 to 50:1;

[0014] In some embodiments of the present application, the ratio of the amounts of substances (molar ratio) is 1:1.

[0015] In these embodiments, the present application discloses that the amount ratio (molar ratio) of hyperoside to 3'-O-methylated hyperoside is any one of 1:100, 10:100, 20:100, 30:100, 40:100, 50:100, 60:100, 70:100, 80:100, 90:100, 100:100, 100:90, 100:80, 100:70, 100:60, 100:50, 100:40, 100:30, 100:20, 100:10, 100:5, and 100:1.

[0016] The third aspect of the present application is to provide a pharmaceutical composition, which comprises hyperoside or a pharmaceutically acceptable salt thereof and 3'-O-methylated hyperoside or a pharmaceutically acceptable salt thereof, wherein the hyperoside has a structure shown in the following formula I, and the 3'-O-methylated hyperoside has a structure shown in the following formula II:

[0017]

[0018] In some embodiments of the present application, the pharmaceutical composition comprises hyperoside and 3'-O-methylated hyperoside, and the molar ratio (molar ratio) between the hyperoside and 3'-O-methylated hyperoside is 1:100 to 100:1;

[0019] In some embodiments of the present application, the ratio of the amount of substance (molar ratio) is 1:50 to 50:1;

[0020] In some embodiments of the present application, the ratio of the amounts of substances (molar ratio) is 1:1.

[0021] In these embodiments, the present application discloses that the amount ratio (molar ratio) of hyperoside to 3'-O-methylated hyperoside is any one of 1:100, 10:100, 20:100, 30:100, 40:100, 50:100, 60:100, 70:100, 80:100, 90:100, 100:100, 100:90, 100:80, 100:70, 100:60, 100:50, 100:40, 100:30, 100:20, 100:10, 100:5, and 100:1.

[0022] In some embodiments of the present application, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier;

[0023] In some embodiments of the present application, the above-mentioned pharmaceutical composition is prepared into any pharmaceutically acceptable dosage form;

[0024] In some embodiments of the present application, the pharmaceutically acceptable dosage form is selected from any one of tablets, capsules, solutions, granules, powders, ointments, pills, suspensions, suppositories, ointments, emulsions, smears, patches and sprays.

[0025] In some embodiments of the present application, the above dosage form is in the form of unit dosage, and each unit dosage contains the compound hyperoside or the compound 3'-O-methylated hyperoside: 1 mg to 10 mg, or 10 mg to 100 mg, or 100 mg to 1000 mg, or 1000 mg to 10000 mg.

[0026] The following is an explanation and description of the terms used in this application:

[0027] The English name of hyperoside is Hyperoside, the English abbreviation is Hyp, and the molecular formula is C 21 H 20 O 12 , the structural formula is as follows, CAS number: 482-36-0.

[0028]

[0029] The English name of 3'-O-methylated hyperoside (Isorhamnetin-3-O-galactoside) is Isorhamnetin-3-O-galactoside, the English abbreviation is I3G, and the molecular formula is C 22 H 22 O 12 , the structural formula is as follows, CAS number: 6743-92-6.

[0030]

[0031] Hyperoside or 3'-O-methylated hyperoside can be purchased from the market, or can be synthesized according to the literature in the prior art.

[0032] The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or formed salt is generally chemically or physically compatible with the other ingredients that constitute a pharmaceutical dosage form and physiologically compatible with the receptor.

[0033] The pharmaceutical composition described in the present application can be any reusable pharmaceutical preparation form, such as oral, injection, external use, etc. The oral dosage form includes but is not limited to tablets, capsules, oral liquids, granules, pills, suspensions, injections selected from water injections and powder injections, and external preparations selected from patches and ointments. All preparations can be prepared according to conventional pharmaceutics techniques, such as using the compound of the present application, or any one of its stereoisomers, or its pharmaceutically acceptable salt as a pharmaceutically active ingredient, adding a pharmaceutically acceptable carrier when necessary, and preparing the above-mentioned pharmaceutical dosage form suitable for taking, wherein the pharmaceutically active ingredient of a unit dose can be 0.1 mg to 1000 mg, such as each tablet containing 0.1 mg to 1000 mg, preferably 5 mg to 500 mg, of the pharmaceutically active ingredient.

[0034] The terms "salt" and "pharmaceutically acceptable salt" refer to acidic and / or basic salts of the above-mentioned compounds or their stereoisomers, formed with inorganic and / or organic acids and bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final separation and purification of the compound. It can also be obtained by mixing the above-mentioned compound, or its stereoisomer, with a certain amount of acid or base appropriately (e.g., equivalent). These salts may form a precipitate in the solution and be collected by filtering, or be recovered after solvent evaporation, or be obtained by freeze drying after reaction in an aqueous medium. The salt described in the present application can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

[0035] In certain embodiments, one or more compounds of the present application can be used in combination with each other. It is also possible to select the compounds of the present application to be used in combination with any other active agent to prepare a drug or pharmaceutical composition for regulating cell function or treating a disease. If a group of compounds is used, these compounds can be administered to the subject simultaneously, separately or in order.

[0036] Obviously, based on the above content of this application, in accordance with common technical knowledge and customary means in this field, without departing from the above basic technical ideas of this application, other various forms of modifications, replacements or changes can be made.

[0037] Beneficial technical effects of this application:

[0038] 1. The compounds Hyp and I3G of the present application have been confirmed to have inhibitory activity against PDI reductase, and they may directly inhibit the activity of PDI.

[0039] 2. The compounds Hyp and I3G of the present application also have the effects of directly inhibiting platelet adhesion, platelet activity, and blood clot contraction. This comprehensive intervention strategy may provide a more comprehensive and more potent antiplatelet effect, thereby reducing the possible limitations of single-target therapy. Therefore, the compounds Hyp and I3G of the present application may have good application prospects in the preparation and treatment of peripheral arterial disease, diabetic microangiopathy, hypertension-related cardiovascular and cerebrovascular diseases, thromboembolism caused by atrial fibrillation (AF), chronic obstructive pulmonary disease (COPD), cardiovascular complications related to chronic kidney disease, and hypertension complications such as stroke and myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present application. Moreover, the same reference numerals are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0041] Figure 1 This is a test diagram of the inhibitory activity of the compounds Hyp and I3G of some embodiments of this application on PDI reductase, wherein: Figure 1 A is the fluorescence curve of EGSH produced by recombinant PDI cleaving Di-E-GSSG after treatment with Hyp and control solvent. Figure 1 C is Figure 1 A Maximum EGSH fluorescence bar graph (n = 3 per group, Veh: control solvent); *P < 0.05;

[0042] Similarly, Figure 1 B is the fluorescence curve of EGSH produced by recombinant PDI cleaving Di-E-GSSG after I3G and control solvent treatment. Figure 1 D is Figure 1 B is the maximum EGSH fluorescence bar graph (n=3 per group, Veh: control solvent); *P<0.05.

[0043] Figure 2 Molecular docking simulation diagram of compounds Hyp and I3G according to some examples of this application.

[0044] Figure 3The DCF fluorescence intensity detected by flow cytometry when the compounds Hyp and I3G of some examples of the present application have different concentrations, wherein Veh is the control solvent group.

[0045] Figure 4 for Figure 3 The vertical axis is the fluorescence intensity.

[0046] Figure 5 This is a diagram showing the change of calcium signal in platelets over time detected by flow cytometry when the compounds Hyp and I3G of some embodiments of the present application have different concentrations.

[0047] Figure 6 for Figure 5 The vertical axis is the area under the maximum fluorescence curve (AUC), which can also represent the total intracellular Ca 2+ concentration.

[0048] Figure 7 This is a representative diagram of platelet adhesion on the collagen surface when the compounds Hyp and I3G of some examples of the present application have different concentrations.

[0049] Figure 8 for Figure 7 (n=3 for each group; Veh: control solvent).

[0050] Fig. 9 This is a representative diagram showing that the compounds Hyp and I3G according to some embodiments of the present application inhibit blood clot contraction at different concentrations.

[0051] Fig.10 for Fig. 9 The bar graph is shown in Figure 1 (n=3 for each group, Veh: control solvent, Thr: thrombin, Fib: fibrinogen), where the ordinate is the percentage of blood clot in the total volume.

[0052] Fig.11 This is a schematic diagram of the effect of compound Hyp on four coagulation parameters in some embodiments of this application, wherein: Fig.11 A is prothrombin time (PT); Fig.11 B is prothrombin time activity (PT%); Fig.11 C is the international normalized ratio (PTINR); Fig.11 D is the partial prothrombin time (APTT); Fig.11 E is fibrinogen quantification (FBGC); Fig.11 F is the thrombin time (TT).

[0053] Fig.12 This is a schematic diagram of the effect of compound I3G on four coagulation items in some embodiments of the present application, wherein: Fig.12A is prothrombin time (PT); Fig.12 B is prothrombin time activity (PT%); Fig.12 C is the international normalized ratio (PTINR); Fig.12 D is the partial prothrombin time (APTT); Fig.12 E is fibrinogen quantification (FBGC); Fig.12 F is the thrombin time (TT). DETAILED DESCRIPTION

[0054] Hyperoside, also known as quercetin-3-O-β-D-galactopyranoside, is an organic compound with a chemical formula of C 21 H 20 O 12 . It is easily soluble in ethanol, methanol, acetone and pyridine, and is stable under normal conditions. The aglycone is quercetin, and the glycosyl is galactopyranose, with the 3-O atom of quercetin linked to the glycosyl by a β-glycosidic bond. Hyperoside is widely distributed and has multiple physiological activities such as anti-inflammatory, antispasmodic, diuretic, antitussive, antihypertensive, cholesterol-lowering, protein assimilation, local and central analgesic, and protective effects on the heart and brain blood vessels. It is an important natural product. 3'-O-methylated hyperoside (isorhamnetin-3-O-galactoside) is a metabolite of hyperoside, which also has anti-inflammatory effects and is referred to as I3G.

[0055] PDI (Protein Disulfide Isomerase) is an important class of enzymes responsible for catalyzing the formation, cleavage and rearrangement of disulfide bonds in proteins. PDI belongs to the thioredoxin family, and its structure usually contains two or more active regions, each of which contains a region that catalyzes disulfide bonds (Cys-Gly-His-Cys). The mechanism of action of these catalytic disulfide bonds is similar to redox reactions, catalyzing the formation of disulfide bonds (-SS-) between thiol (-SH) groups of proteins. PDI inhibitors work by interfering with the catalytic activity of PDI, usually by binding to the active site of PDI or its key functional region, thereby blocking its catalytic effect on protein disulfide bonds.

[0056] At present, PDI inhibitors have shown potential application value as drug targets in cancer treatment and antiviral therapy. In cancer treatment, many cancer cells rely on the endoplasmic reticulum stress response to deal with the large amount of unfolded or misfolded proteins produced during rapid proliferation. As a key folding enzyme, the inhibition of PDI may lead to further aggravation of endoplasmic reticulum stress and induce cell apoptosis. Therefore, PDI inhibitors are considered as a potential anticancer drug, especially for tumor cells that are resistant to conventional therapies. In antiviral effects: PDI also plays an important role in the process of virus invasion of host cells. Some viruses (such as HIV, dengue virus, etc.) use the host's PDI to help the correct folding and assembly of viral proteins. Inhibiting the function of PDI may prevent viral replication and thus have antiviral potential. In neurodegenerative diseases, due to the role of PDI in protein folding, its dysregulation may be related to protein misfolding and accumulation in some neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease). Therefore, regulating PDI activity may be helpful in the treatment of these diseases.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0058] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0059] Conventional techniques of inorganic chemistry within the art can be used in this application. In the following examples, efforts are made to ensure the accuracy of the numbers used (including amounts, temperatures, reaction times, etc.), but some experimental errors and deviations should be considered. The temperatures (in degrees Celsius) used in the following examples are expressed in ° C, and the pressure is atmospheric pressure or near atmospheric pressure. All pharmaceutical reagents are purchased at AR grade, and all reactions are carried out under argon protection. Unless otherwise noted, all reagents are obtained from commercial channels.

[0060] The present application is further described below by way of examples, but is not intended to limit the present application.

[0061] Example 1: Inhibitory activity of hyperoside (Hyp) and 3'-O-methylated hyperoside (I3G) on protein disulfide isomerase;

[0062] The inhibitory activity of Hyp and its I3G on PDI reductase was verified by the Di-E-GSSG (diosmin-oxidized glutathione) method. Specifically, the experiment was carried out using a 96-well plate, and human recombinant PDI was incubated with I3G, Hyp and control solvent at 37°C for 20 minutes. Di-E-GSSG and DTT (dithiothreitol) were dissolved in a reaction buffer solution (0.1M KH2PO4, 2mM EDTA, pH7.0), and the resulting mixture was added to the above system to make Di-E-GSSG, DTT, and PDI 150nM, 5μM, and 750nM, respectively. The concentration gradients of I3G and Hyperoside were 100μM, 20μM, 4μM, 0.8μM, and 0.16μM, and the total volume of each well was 300μL.

[0063] Figure 1 The different concentration gradients of I3G and Hyp (0, 160nM, 800nM, 4μM, 20μM, 100μM) are obtained by diluting the 1mM stock solution with a reaction buffer solution (0.1M KH2PO4, 2mM EDTA, pH7.0). After mixing, the fluorescence intensity change is immediately detected on an ELISA reader, and the excitation wavelength (Excitation Wavelength) and emission wavelength (Emission Wavelength) are 525nm and 545nm, respectively. The total measurement time is 45min, and the signal reading time interval is 1min.

[0064] Considering that PDI participates in thrombosis mainly in the form of reductase in vivo, PDI was first incubated with Hyp and I3G separately. After incubation, DTT was added to induce PDI to convert into a reduced state, and then Di-E-GSSE was added for detection by an enzyme marker.

[0065] The basic principle of Example 1 of the present application is that in the presence of DTT, PDI exerts reductase activity, catalyzes the opening of the disulfide bond of Di-E-GSSE, increases the distance between the two dye (eosin) molecules, and eliminates fluorescence quenching. By exciting at a wavelength of 525 nm, a fluorescence signal can be detected at 545 nm. Therefore, by observing the fluorescence signal, the lower the fluorescence signal, the lower the PDI activity and the stronger the inhibitory ability of the small molecule.

[0066] As the instruction manual Figure 1 As shown in Figures A and C, the fluorescence signal in the Hyp experimental group decreased in a dose-dependent manner compared with the control group.

[0067] Similarly, as shown in the instruction manual Figure 1 As shown in Figures B and D, the fluorescence signal of the I3G experimental group also showed a dose-dependent decrease compared with the control group.

[0068] Instructions attached Figure 2 Molecular docking showed that Hyp and I3G may mainly bind to the x domain of PDI (compared with other domains, the x binding domain has the highest binding energy), and may affect the conformation of PDI reductase and inhibit its binding to the substrate to exert reductase activity. Figure 2 The possible mechanism of action of Hyp, I3G and PDI is provided. Figure 2 The hydrophobic interaction in the formula refers to the hydrophobic interaction, the π-cation interaction refers to the π-cation interaction, the salt bridge refers to the salt bridge, the hydrogen bond refers to the hydrogen bond, and the π-stacking refers to the π-stacking. In addition, PHE-305, HIS-354, ARG-316, LEU-355, LYS-375, GLU-433, PRO-353, ILE-351, PHE-346, TYR-327, THR-325, MET-324, and THR-319 all refer to PDI-related amino acid residues.

[0069] Example 2: Effects of Hyp and I3G on Rosup-induced platelet ROS formation;

[0070] 1. Preparation of washed platelets from SD rats:

[0071] a. Whole blood was collected from SD rats through the retro-orbital venous plexus, and the blood was passed through a capillary tube (inner diameter 0.9 mm to 1.1 mm) rinsed with heparin (1 mg / mL) and dried, and then flowed into an Eppendorf (EP) tube pre-filled with 100 μL 3.2% sodium citrate to receive 900 μL of rat whole blood (volume ratio of sodium citrate: whole blood = 1:9).

[0072] b. Immediately centrifuge at 900 rpm for 20 min at room temperature and take the upper layer to obtain platelet-rich plasma (PRP).

[0073] c. The obtained PRP was allowed to stand at 37°C for 20 minutes, and then 180 μL of ACD solution (20% of the volume of PRP) and PGE1 solution (0.2 μM) were added in sequence, and the mixture was gently inverted upside down to mix well. The ACD solution was mainly composed of three components: sodium citrate (85 mM), citric acid (71 mM) and glucose (122 mM). PGE1 solution refers to prostaglandin E1 solution, and the models of these two solutions are conventional in the art.

[0074] d. Centrifuge at 2000 rpm for 10 min at room temperature, discard the supernatant, add 675 μL HTG solution preheated at 37°C to resuspend the platelets (75% of the original PRP volume), and let stand at 37°C for 20 min.

[0075] e. Take the resuspended platelets, first add 135 μL of ACD solution (HTG volume 20%), then add PGE1 (0.2 μM), and gently invert upside down to mix.

[0076] f. Centrifuge at 2000 rpm for 10 min at room temperature, discard the supernatant, add 675 μL of preheated HTG (75% of the original PRP volume) to resuspend the platelets, and let stand at 37°C for 20 min.

[0077] g. After counting the platelet suspension, adjust it to the required platelet concentration.

[0078] 2. Testing process:

[0079] a. preparing washed rat platelets using a platelet preparation kit to obtain washed platelets;

[0080] b. Add 300 μL of suspended platelets to each EP tube, add 30 μL of I3G, Hyp or control solvent DMSO, and incubate at 37°C for 15 min;

[0081] c. Dilute DCFH-DA with platelet suspension, add 40 μL (20 μM) to each EP tube, and add 30 μL Rosup (450 μg / mL), and incubate at 37°C for 20-30 min;

[0082] d. Centrifuge at 5000 rpm for 3 min, wash with PBS three times, and resuspend the platelets in 300 μL PBS solution;

[0083] e. The DCF fluorescence intensity was detected by flow cytometry (FL1 channel, 488 nm excitation, 530 nm emission). Washed rat platelets were incubated with Hyp, I3G or control solvent (DMSO) at 37°C for 10 min, and the stimulator Rosup (50 μg / mL) was added. DCFDA was also added, and the DCF fluorescence intensity on the platelet surface was detected by flow cytometry.

[0084] As the instruction manual Figure 3 As shown, Hyp and I3G inhibit the upregulation of platelet ROS. Washed rat platelets were incubated with Hyp, I3G or control solvent (DMSO) at 37°C for 10 min, and the stimulator Rosup and DCFDA were added at the same time. The DCF fluorescence intensity on the platelet surface was detected by flow cytometry.

[0085] Figure 3 Figure A is the control group, Figures B, C and D are all experimental groups, and Figure B shows the fluorescence intensity of the Hyp group with different concentrations, Figure C shows the fluorescence intensity of the I3G group with different concentrations, and Figure D shows the fluorescence intensity of the Hyp and I3G mixture with a molar ratio (molar ratio) of 1:1 at different concentrations. Figure 3 A to Figure 3 D, and Figure 4 It can be seen that compared with resting platelets, the calcium ions in platelets increased significantly after thrombin stimulation. After Hyp and I3G intervention alone, the calcium ion content can be effectively downregulated. It was also found that after the combined administration of Hyp and I3G (1:1, 4μM+4μM), strong inhibition can be achieved at a low dose (8μM). Hyp, I3G, and the combined administration of Hyp and I3G (1:1) all inhibited ROS upregulation in a dose-dependent manner. Therefore, the combined intervention of Hyp and I3G can achieve enhanced inhibition at the same dose compared with the intervention of Hyp and I3G alone, and there is a potential synergistic effect between the two.

[0086] Example 3: Effects of Hyp and I3G on thrombin-induced platelet calcium mobilization;

[0087] a. Wash platelets from rats and dilute them with the platelet suspension in the platelet washing kit;

[0088] b. Add calcium ion fluorescent probe Fluo 3-AM (4 μM) in dark and incubate at 37°C in dark for 30 min;

[0089] c. Add different concentrations of I3G and Hyp (4 μM, 20 μM, 100 μM) and incubate at 37°C in the dark for 30 min;

[0090] d. After incubation, the platelet suspension of each group was transferred into a flow tube. After the baseline was stable, CaCl2 (final concentration 1 nM) and α-thrombin (final concentration 0.08 U / mL) were added and the detection was continued for 5 min.

[0091] Washed platelets from rats were incubated with different concentrations of Hyp, I3G or control solvent, and the platelet calcium ions were labeled with Fluo 3-AM and stimulated with thrombin (0.08 U / mL). The changes of platelet calcium signals over time were detected by flow cytometry. Figure 5 ,right Figure 5 The image was further processed and the maximum fluorescence area under the curve (AUC) was used as the ordinate to plot Figure 6 , where the area under the maximum fluorescence curve (AUC) also represents the total intracellular Ca 2+ concentration.

[0092] like Figure 5 , Figure 6As shown, washed platelets of rats were incubated with different concentrations of Hyp, I3G or control solvent, and the calcium ions in platelets were labeled with Fluo3-AM and stimulated with thrombin (0.08U / mL): in the control group, after the application of the agonist thrombin, the calcium ion signal in platelets increased significantly, but in the experimental group, it was found that Hyp (50μM) significantly inhibited the enhancement of calcium ion signals. Similarly, I3G (50μM) also had a significant inhibitory effect. And when Hyp and I3G were co-administered (1:1, 4μM+4μM), strong inhibition could be achieved at a low dose (8μM). Hyp, I3G, and the combined administration of Hyp and I3G all inhibited the enhancement of calcium ion signals in a dose-dependent manner. Therefore, after the combined intervention of Hyp and I3G, strong inhibition can be achieved at a low dose (8μM).

[0093] Example 4: Effects of Hyp and I3G on platelet adhesion;

[0094] a. Take a clean round glass cover slip with a diameter of 13 mm and place it in a 24-well plate. Add 400 μL of 5 mg / mL collagen solution to the well plate and coat it in a 4°C refrigerator overnight.

[0095] b. Discard the solution in the wells and add 1× PBS solution to wash the plate three times.

[0096] c. Add 300 μL of 1% BSA solution (prepared using 1× PBS solution) to each well and block at room temperature for 1 hour.

[0097] d. The platelet suspension was mixed with low, medium and high concentrations of I3G and Hyp solutions (dissolved in DMSO), a mixed solution of I3G and Hyp, and a control solvent DMSO, and incubated at 37°C for 10 min.

[0098] e. After the incubation, 300 μL of the platelet mixture solution from each group was added to the corresponding 24-well plate and incubated at 37°C for 30 min.

[0099] f. After the incubation, discard the platelet suspension in the wells and wash three times with 1× PBS solution to remove unattached platelets.

[0100] g. Add 500 μL of 4% paraformaldehyde fixative to each well and fix at room temperature for 30 minutes.

[0101] h. After fixation, discard the fixative solution in the wells and wash three times with 1× PBS solution.

[0102] i. Dissolve TRITC-phalloidin in 1×PBS solution, then add Triton X-100 (0.1%) to prepare a mixed solution. Add 200 μL of the mixed solution to each well, wrap the well plate with tin foil and incubate for 2 h in the dark.

[0103] j. After staining, discard the solution in the wells and wash the wells 5 times with PBS, shaking for 2 minutes each time.

[0104] k. Take out the round glass slide from the well plate under light-proof conditions, place the side with expanded platelets close to the slide, and lightly apply a layer of glycerol around the round glass slide to fix it.

[0105] l. Observe the platelet morphology under a fluorescence microscope. Randomly select 10 fields of view in each group, use Image J software to measure the expansion area of ​​each platelet, and classify and count the platelet expansion morphology.

[0106] As shown in the attached figure of the instruction manual Figure 7 , 8 As shown in the figure, washed rat platelets were incubated with different concentrations of Hyp, I3G or control solvent for 10 minutes, added to the surface of a slide pre-coated with collagen, and incubated at 37°C for 30 minutes. The platelet skeleton was stained with TRITC-Phalloidin, and images were collected using a fluorescence microscope. Figure 7 Representative images of platelet adhesion on collagen surfaces. Figure 8 It is a bar graph of platelet adhesion (n=3 in each group, Veh: control solvent).

[0107] In the present application, before thrombus formation, there is often damage to the vascular endothelium. The damaged part will expose the collagen in the lower layer, and then the platelets will specifically bind to the collagen to achieve platelet adhesion. In order to simulate the adhesion environment of platelets in vivo, a round glass slide is first placed in a 24-well plate, and then collagen is added for coating. A layer of collagen is attached to the glass slide, and BSA is further added for sealing to prevent non-specific binding between the platelets and the area on the glass slide not occupied by collagen. After the substrate is prepared, platelets incubated with a solvent (DMSO) and platelets incubated with I3G, Hyp, and I3G and Hyp are added respectively, and the platelets are allowed to adhere to the collagen substrate through incubation. After the platelets adhere, the non-adherent platelets are washed away, and the adhered platelets are fixed by adding PFA paraformaldehyde, and then the platelet skeleton is labeled, and the adhesion of the platelets is observed under a fluorescence microscope. As Figure 7 As shown, in the solvent control group, platelets were fully spread on collagen. Compared with the experimental group, both I3G and Hyp could significantly reduce the platelet coverage. For the I3G and Hyp combined administration group (1:1, 4μM+4μM), even at a low dose (8μM), the platelet coverage can be significantly reduced. Figure 8 As shown, the platelet coverage rate decreased in a dose-dependent manner after Hyp, I3G, and the combined administration of Hyp and I3G.

[0108] Example 5: Effects of Hyp and I3G on blood clot contraction;

[0109] Wash platelets from SD rats, add I3G, Hyp, or control solvent, and pretreat at 37°C for 10 minutes. Add thrombin, fibrinogen, and CaCl2 solutions dissolved in ultrapure water at concentrations of 1U / mL, 2mg / mL, and 1mM, respectively, and incubate at 37°C for 15 minutes. Take photos after 15 minutes to record the retraction of blood clots. Different concentration gradients of I3G and Hyp were obtained by taking 1mM stock solutions and diluting them with ultrapure water. Hyp and I3G intervention alone effectively inhibited blood clot retraction. When the two were intervened together, they showed similar inhibitory effects as I3G intervention alone.

[0110] As the instruction manual Fig. 9 , 10 As shown, washed platelets from rats were incubated with different concentrations of Hyp, I3G or control solvent for 10 min, and thrombin (1 U / mL), fibrinogen (2 mg / mL), and calcium ions (1 mM) were added and incubated at 37°C for 60 min. Fig. 9 Representative images of clot retraction in different groups. Fig.10 The figure is a bar graph showing the percentage of blood clot in the total volume (n=3 in each group, Veh: control solvent; Thr: thrombin; Fib: fibrinogen).

[0111] The blood clot contraction in this application mainly occurs in the later stage of thrombosis, and the platelet contraction makes the thrombosis tighter and improves the stability of the thrombosis. Fig. 9 As shown in the figure, compared with the resting state, the blood clot retracted significantly after agonist stimulation. After Hyp and I3G were given alone, blood clot retraction was effectively inhibited. When Hyp and I3G were combined (1:1), the inhibition was enhanced. Fig.10 This indicates that the volume of blood clots increases in a dose-dependent manner when Hyp, I3G, or the combined administration of Hyp and I3G, and blood clot contraction can be inhibited in a dose-dependent manner.

[0112] Example 6: Effects of Hyp and I3G on four coagulation parameters;

[0113] a. Take 1 mL of healthy human plasma and add the compounds I3G and Hyp of the present application at concentrations of 0.16 μM, 0.8 μM, 4 μM, 20 μM, and 100 μM, respectively.

[0114] b. After adding, incubate at room temperature for 5 minutes and use an automatic hematology analyzer to detect four coagulation parameters.

[0115] The effects of the compounds Hyp and I3G of the present application on four coagulation parameters, including prothrombin time, partial prothrombin time, fibrinogen content, and thrombin time. Fig.11 The results showed that Hyp did not significantly affect the four coagulation parameters. Fig.12 The results showed that I3G did not significantly affect the four coagulation tests. The experimental subjects were human plasma. Figure 11 to Figure 12 The range between the two dotted lines is the normal numerical range of the corresponding indicators in human plasma.

[0116] It can be seen from the above examples that the Hyp and / or I3G provided in the present application have inhibitory activity against PDI reductase, and also have the performance of inhibiting platelet adhesion, platelet activation and blood clot contraction.

[0117] In addition, the Hyp and / or I3G provided in the present application may also be used in the preparation of a pharmaceutical composition for treating any one or more of peripheral arterial disease, diabetic microangiopathy, hypertension-related cardiovascular and cerebrovascular diseases, thromboembolism caused by atrial fibrillation (AF), chronic obstructive pulmonary disease (COPD), cardiovascular complications associated with chronic kidney disease, and hypertension complications of stroke and myocardial infarction.

[0118] In some embodiments, peripheral artery disease (PAD) is a vascular disease caused by arteriosclerosis, which mainly affects blood flow to the limbs, especially the legs. The core pathological characteristics of PAD are the formation of intravascular plaques and stenosis of the vascular lumen, leading to insufficient blood supply and hypoxia in the tissues. Symptoms of this disease include intermittent claudication, limb pain, ulcers and even gangrene. In the course of PAD, abnormal activation and functional changes of platelets play a key role. By regulating platelet function, the symptoms of PAD can be effectively alleviated and the prognosis can be improved. The following details strategies for treating peripheral artery disease by affecting platelet function:

[0119] 1) The role of platelets in peripheral arterial disease:

[0120] Platelet activation and aggregation: PAD patients often have increased platelet activation, and platelets tend to aggregate on damaged blood vessel walls, further promoting thrombosis and vascular stenosis.

[0121] Inflammation and oxidative stress: Platelet activation releases inflammatory factors and reactive oxidative species (ROS), which aggravate arterial inflammation and damage and lead to disease progression.

[0122] Platelets and atherosclerotic plaques: Platelets are involved in the instability of plaques, and the rapid aggregation of platelets after plaque rupture can lead to acute thrombotic events.

[0123] 2) Therapeutic strategies by affecting platelet function:

[0124] Treatments that affect platelet function mainly focus on inhibiting platelet activation, aggregation, and the release of inflammatory mediators, thereby reducing thrombosis, improving blood flow, and alleviating symptoms. By regulating platelet function, especially inhibiting platelet activation, adhesion, and aggregation, peripheral arterial disease can be effectively prevented and treated.

[0125] In some embodiments, diabetic microangiopathy is a common complication of diabetes, which is mainly manifested by changes in microvascular structure and function, leading to microcirculatory disorders. This type of lesion mainly affects the retina (diabetic retinopathy), glomerulus (diabetic nephropathy) and nerves (diabetic peripheral neuropathy). Platelets play an important role in diabetic microangiopathy, and abnormal platelet function aggravates vascular damage and microcirculatory disorders. By regulating platelet function, diabetic microangiopathy can be effectively intervened and treated. The following details how to treat diabetic microangiopathy by affecting platelet function:

[0126] 1) The role of platelets in diabetic microangiopathy:

[0127] Platelet activation: The platelets of diabetic patients are in a highly activated state and easily adhere to the damaged vascular endothelium, leading to thrombosis and microvascular obstruction.

[0128] Inflammation and oxidative stress: Activated platelets release inflammatory factors, thromboxane A2 (TXA2), and reactive oxidative substances (ROS), which promote endothelial cell damage and aggravate microvascular lesions.

[0129] Platelet-leukocyte interactions: Activated platelets interact with leukocytes, further initiating local inflammation and vascular damage.

[0130] Microthrombosis: Platelet activation and aggregation can easily lead to the formation of microthrombi in microvessels, aggravating microcirculation disorders.

[0131] 2) Therapeutic strategies by regulating platelet function:

[0132] By affecting platelet activation, aggregation, release of inflammatory mediators and other processes, it can reduce microvascular damage, improve microcirculation, and delay the progression of diabetic microangiopathy.

[0133] In some embodiments, hypertension-related cardiovascular and cerebrovascular diseases (such as hypertensive heart disease, stroke, myocardial infarction, etc.) are one of the main health threats to the cardiovascular system. Hypertension can lead to changes in the structure and function of the vascular wall, increase the risk of vascular sclerosis and thrombosis, and platelets play a key role in these pathological processes. Abnormal platelet function can aggravate cardiovascular and cerebrovascular damage in patients with hypertension. Therefore, hypertension-related cardiovascular and cerebrovascular diseases can be effectively treated and prevented by regulating platelet function. The following details strategies for treating hypertension-related cardiovascular and cerebrovascular diseases by affecting platelet function:

[0134] 1) The role of platelets in hypertension-related cardiovascular and cerebrovascular diseases:

[0135] Platelet activation and aggregation: Hypertensive patients often have platelet activation and aggregation, which can lead to vascular endothelial damage and microthrombosis, thereby increasing the risk of cardiovascular and cerebrovascular diseases.

[0136] Promoting inflammatory response: Activated platelets release a variety of pro-inflammatory factors, such as thromboxane A2 (TXA2), platelet factor 4 (PF4), 5-hydroxytryptamine (5-HT), etc. These factors can induce local inflammation and accelerate the progression of atherosclerosis.

[0137] Oxidative stress and vascular damage: Activated platelets produce reactive oxygen species (ROS), which further damage the vascular endothelium, leading to increased vascular stiffness and further worsening hypertension.

[0138] 2) Therapeutic strategies by regulating platelet function:

[0139] By affecting platelet function, especially inhibiting its activation, aggregation, adhesion and the release of proinflammatory factors, the risk of hypertension-related cardiovascular and cerebrovascular events can be effectively reduced.

[0140] In some embodiments, atrial fibrillation (AF) is a common arrhythmia that can easily lead to blood turbulence and stagnation in the atria, thereby forming blood clots and increasing the risk of stroke and other thromboembolic events. Platelets play an important role in the formation of AF-related blood clots. By regulating platelet function, the risk of thromboembolism in patients with AF can be effectively reduced. The following details strategies for treating thromboembolism caused by AF by affecting platelet function:

[0141] 1) The role of platelets in atrial fibrillation-related thromboembolism:

[0142] Platelet activation and aggregation: During atrial fibrillation, blood flow in the atria is disordered and slow, and platelets are easily activated, causing platelets to aggregate in areas of blood flow stagnation such as the left atrial appendage, forming a thrombus.

[0143] Interaction between platelets and coagulation system: Activated platelets release procoagulant substances (such as thromboxane A2, P-selectin, etc.), activate the coagulation cascade reaction, and aggravate thrombosis.

[0144] Proinflammatory response: After platelet activation, inflammatory mediators (such as IL-1β, TNF-α) are released, which can promote endothelial damage and vascular inflammatory response, further promoting the formation of thrombus.

[0145] 2) Therapeutic strategies by regulating platelet function:

[0146] By affecting platelet function, especially inhibiting platelet activation, aggregation and procoagulant effects, the risk of thromboembolism caused by atrial fibrillation can be effectively reduced.

[0147] In some embodiments, patients with chronic obstructive pulmonary disease (COPD) often have increased platelet activation and oxidative stress. Inhibiting platelet calcium and ROS levels may be beneficial for reducing COPD-related inflammatory responses and vascular lesions. \COPD\ is a chronic inflammatory lung disease characterized by airflow limitation, accompanied by respiratory and systemic inflammatory responses. In recent years, studies have found that COPD patients often have abnormal platelet function, including increased platelet activation, release of pro-inflammatory molecules, etc. These changes are closely related to COPD-related cardiovascular complications, chronic inflammation and deterioration of lung function. Therefore, treating COPD by affecting platelet function has become a new research direction. The following details strategies for treating COPD by affecting platelet function:

[0148] 1) The role of platelets in COPD

[0149] Platelet activation and systemic inflammation: COPD patients usually show a systemic inflammatory state. After platelet activation, they will release a large number of proinflammatory factors, such as thromboxane A2 (TXA2), 5-hydroxytryptamine (5-HT), P-selectin, etc. These factors not only aggravate lung inflammation, but also lead to systemic vascular inflammation and cardiovascular complications.

[0150] Oxidative stress and ROS production: COPD patients have increased platelet activity, accompanied by increased production of reactive oxygen species (ROS), which can lead to oxidative stress in blood vessels and airways, further damaging lung tissue and aggravating the condition.

[0151] Interaction between platelets and leukocytes: After activation, platelets in COPD can interact with leukocytes such as neutrophils to form platelet-leukocyte complexes, further aggravating the inflammatory response and tissue damage in the lungs.

[0152] Platelets and pulmonary vascular disease: In COPD patients, platelet activation and aggregation can easily lead to pulmonary vascular inflammation and thrombosis, thereby increasing the risk of pulmonary hypertension and cor pulmonale.

[0153] 2) Therapeutic strategies by regulating platelet function:

[0154] By inhibiting platelet activation and reducing platelet-mediated inflammatory response and oxidative stress, the pulmonary and systemic inflammation of COPD can be alleviated, thereby improving symptoms and prognosis.

[0155] In some embodiments, patients with chronic kidney disease (CKD) are often accompanied by high cardiovascular risk, the main reasons for which include inflammation, oxidative stress, increased platelet activation and endothelial dysfunction. Platelets play an important role in CKD-related cardiovascular complications. By regulating platelet function, the risk of cardiovascular events can be effectively reduced. The following details strategies for treating cardiovascular complications associated with chronic kidney disease by affecting platelet function:

[0156] 1) Role of platelets in CKD-related cardiovascular complications

[0157] Increased platelet activation: CKD patients often show increased platelet activation. The procoagulant substances released by platelets (such as thromboxane A2 and P-selectin) are closely related to vascular damage, atherosclerosis and thrombosis.

[0158] Oxidative stress and inflammatory response: Toxin retention in CKD patients (such as urea, urea toxins, etc.) can induce platelet activation, produce a large amount of oxidative stress substances (ROS), aggravate vascular inflammation, lead to endothelial dysfunction, and promote atherosclerosis and cardiovascular events.

[0159] Interaction between platelets and the coagulation system: Activated platelets activate the coagulation cascade by releasing procoagulant factors, which puts CKD patients in a hypercoagulable state, making them prone to thrombosis and increasing the risk of cardiovascular events.

[0160] 2) Therapeutic strategies by regulating platelet function:

[0161] By regulating platelet function, especially inhibiting platelet activation, reducing inflammation and oxidative stress, the risk of cardiovascular complications in CKD patients can be significantly reduced.

[0162] In some embodiments, hypertension is a major risk factor for cardiovascular and cerebrovascular complications (such as stroke and myocardial infarction). Abnormal platelet function in hypertensive patients, especially increased platelet activation and aggregation, is one of the key pathological mechanisms of events such as atherosclerosis, thrombosis, stroke and myocardial infarction. By regulating platelet function, the risk of these serious complications associated with hypertension can be reduced. The following details strategies for treating complications of hypertension (such as stroke and myocardial infarction) by affecting platelet function:

[0163] 1) The role of platelets in hypertension-related complications:

[0164] Increased platelet activation: Vascular endothelial damage, increased shear stress, and endothelial dysfunction caused by hypertension can lead to increased platelet activation. Activated platelets release thromboxane A2, P-selectin, and other procoagulant substances, accelerating atherosclerosis and thrombosis.

[0165] Platelet-leukocyte interaction: Hypertension promotes the interaction between platelets and leukocytes, forming platelet-leukocyte complexes, increasing the inflammatory response of the blood vessel wall, and aggravating arteriosclerosis and vascular stenosis.

[0166] Oxidative stress and ROS: Hypertension will increase oxidative stress in the body. The ROS generated after platelet activation will not only damage endothelial cells, but also promote further platelet aggregation, increasing the risk of thrombosis.

[0167] Platelet aggregation and cardiovascular and cerebrovascular events: Platelet aggregation is a key step in the occurrence of myocardial infarction and stroke. Platelet dysfunction caused by hypertension increases the instability of atherosclerotic plaques and leads to the occurrence of acute vascular events.

[0168] 2) Therapeutic strategies by regulating platelet function:

[0169] By regulating platelet function, especially inhibiting platelet activation and reducing inflammation and oxidative stress, the risk of serious complications such as stroke and myocardial infarction in patients with hypertension can be effectively reduced.

[0170] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. Use of hyperoside or a pharmaceutically acceptable salt thereof in the preparation of an antiplatelet drug, characterized in that: The hyperoside has a structure shown in the following formula I:

2. Use of 3'-O-methylated hyperoside or a pharmaceutically acceptable salt thereof in the preparation of an antiplatelet drug, characterized in that: The 3'-O-methylated hyperoside has a structure as shown in the following formula II:

3. The use according to claim 1 or 2, characterized in that: Any one or more of the hyperoside, the pharmaceutically acceptable salt of hyperoside, 3'-O-methylated hyperoside and the pharmaceutically acceptable salt of 3'-O-methylated hyperoside has the function of inhibiting the activity of protein disulfide isomerase.

4. The use according to claim 1 or 2, characterized in that: Any one or more of the hyperoside, the pharmaceutically acceptable salt of hyperoside, 3'-O-methylated hyperoside and the pharmaceutically acceptable salt of 3'-O-methylated hyperoside have the effects of inhibiting the upregulation of platelet ROS, inhibiting the platelet Ca2+ induced by thrombin, 2+ Mobilize, inhibit platelet adhesion or inhibit blood clot contraction.

5. The use according to claim 1 or 2, characterized in that: The drug comprises any one or more of hyperoside, a pharmaceutically acceptable salt of hyperoside, 3'-O-methylated hyperoside or a pharmaceutically acceptable salt of 3'-O-methylated hyperoside.

6. The use according to claim 1 or 2, characterized in that: The drug comprises hyperoside and 3'-O-methylated hyperoside, and the amount ratio of the hyperoside to the 3'-O-methylated hyperoside is 1:100 to 100:

1.

7. A pharmaceutical composition, characterized in that: The invention comprises hyperoside or a pharmaceutically acceptable salt thereof and 3'-O-methylated hyperoside or a pharmaceutically acceptable salt thereof, wherein the hyperoside has a structure shown in the following formula I, and the 3'-O-methylated hyperoside has a structure shown in the following formula II:

8. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition comprises hyperoside and 3'-O-methylated hyperoside, and the amount ratio of the hyperoside to the 3'-O-methylated hyperoside is 1:100 to 100:

1.

9. The pharmaceutical composition according to any one of claims 6 to 8, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier; The pharmaceutical composition is prepared into any pharmaceutically acceptable dosage form.

10. The pharmaceutical composition according to claim 9, characterized in that: The dosage form is a unit dosage form, and each unit dosage contains 1 mg to 10 mg, or 10 mg to 100 mg, or 100 mg to 1000 mg, or 1000 mg to 10000 mg of the compound hyperoside or the compound 3'-O-methylated hyperoside.

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