A composition for preventing and treating thrombosis diseases containing 7,8,3',4'- tetrahydroxyflavone as an active ingredient

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of thrombosis containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient, a health functional food for the prevention or improvement of thrombosis, and a method for manufacturing the same. More specifically, the invention relates to a composition for the prevention or treatment of thrombosis containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient, isolated and extracted from Bidens pilosa, a member of the Asteraceae family that is widely distributed worldwide, a health functional food for the prevention or improvement of thrombosis, and a method for manufacturing the same. The composition according to the present invention can effectively inhibit platelet aggregation and thrombus formation caused by platelet activation, thereby preventing and treating thrombosis caused by thrombosis.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of thrombosis containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient, a health functional food for the prevention or improvement of thrombosis, and a method for manufacturing the same. More specifically, the invention relates to a composition for the prevention or treatment of thrombosis containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient, isolated and extracted from Bidens pilosa, a member of the Asteraceae family that is widely distributed worldwide, a health functional food for the prevention or improvement of thrombosis, and a method for manufacturing the same. The composition according to the present invention can effectively inhibit platelet aggregation and thrombus formation caused by platelet activation, thereby preventing and treating thrombosis caused by thrombosis. Background Technology

[0002] With the rapid advancement of industrial and medical technologies leading to changes in dietary habits and an increase in life expectancy, the number of people suffering from cardiovascular diseases, such as arterial, venous, heart, and cerebrovascular diseases, is rapidly increasing. Most of these cardiovascular diseases are thrombosis, which occurs when blood clots abnormally form within blood vessels and obstruct the flow of blood.

[0003] Generally, when a blood clot is formed through the body's hemostatic and coagulation systems when a blood vessel is injured due to various factors, it blocks the injury to the vessel to prevent further bleeding; thus, the formation of blood clots is one of the very important physiological phenomena of the human body. Typically, when a blood vessel is injured, platelets are activated to form the clot, and a blood clot is formed through an aggregation process in which multiple platelets clump together.

[0004] A blood clot is formed when multiple platelets become activated and adhere to each other at a site of injury within a blood vessel, and it serves to stop bleeding through the wound site, thereby preventing the spread of damage caused by the injury. In other words, the human body generates blood clots through the normal functioning of the hemostasis and coagulation system, and dissolves the generated clots to prevent bleeding through wounds within blood vessels and to allow blood to move normally within the blood vessels.

[0005] However, when platelets become abnormally activated, there is a problem in that thrombosis can occur as blood clots rapidly form in blood vessels even without damage to the vessels, blocking the movement of blood within the vessels. In particular, if platelets become abnormally activated and form blood clots in human arteries, veins, peripheral blood vessels, capillaries, cardiovascular vessels, or cerebrovascular vessels, it can cause fatal problems for humans.

[0006] To address these problems, antithrombotic agents designed to treat thrombosis by inhibiting platelet aggregation caused by platelet activation have recently been developed and commercialized. Generally, representative antithrombotic agents include those using heparin or aspirin.

[0007] Meanwhile, in the case of antithrombotic agents using heparin, heparin itself does not have the effect of inhibiting platelet aggregation, but by combining with antithrombin to inhibit the production of thrombin that acts on platelet aggregation, it enhances the function of antithrombin, thereby enabling it to inhibit platelet aggregation.

[0008] However, conventional antithrombotic agents using heparin cannot inhibit thrombin that has already attached to a blood clot, so they only inhibit the formation of new blood clots rather than preventing the spread of existing blood clots, and they have problems such as inducing further bleeding at the site of blood vessel injury or causing systemic bleeding, which can lead to various side effects.

[0009] In addition, antithrombotic agents using aspirin can inhibit platelet aggregation by irreversibly inhibiting cyclooxygenase (COX), which causes inflammation in the body, thereby inhibiting the production of receptors that induce platelet aggregation. However, antithrombotic agents using aspirin have the problem of potentially causing gastric ulcers or gastrointestinal bleeding, as well as various side effects such as headaches or vitamin C deficiency. Prior art literature

[0010] Korean Registered Patent No. 10-820575 (Published April 10, 2008) Korean Registered Patent No. 10-139060 (Published May 19, 2014) US Registered Patent No. US 11802138 (Published October 31, 2023) Korean Registered Patent No. 10-1757674 (Published July 14, 2017) Korean Published Patent No. 10-2021-0130595 (Published November 1, 2021) The problem to be solved

[0011] The present invention, devised to solve the aforementioned problems, aims to provide a composition for antithrombotic prevention and treatment that is excellent in treating thrombosis without having the side effects of existing antithrombotic agents or preventive agents.

[0012] Another objective of the present invention is to provide a composition that can effectively prevent and treat various thrombosis caused by platelet aggregation and thrombus formation due to platelet activation by inhibiting platelet activation and the formation of thrombi caused by platelet activation.

[0013] Another objective of the present invention is to provide a composition for preventing or treating thrombosis containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient and a method for preparing the same.

[0014] Another objective of the present invention is to provide a health functional food for preventing or improving thrombosis containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient and a method for manufacturing the same.

[0015] Another objective of the present invention is to provide a composition for preventing or treating thrombosis containing a Bidens pilosa plant extract as an active ingredient, a health functional food for preventing or improving thrombosis, and a method for manufacturing the same.

[0016] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0017] The present invention, devised to achieve the above-mentioned purpose, preferably comprises a pharmaceutical composition for the prevention or treatment of thrombosis containing 7,8,3',4'-tetrahydroxyflavone having the following chemical formula as an active ingredient.

[0018]

[0019] In addition to the features described above, the present invention is also preferably a pharmaceutical composition for the prevention or treatment of thrombosis characterized in that the composition contains the 7,8,3',4'-tetrahydroxyflavone at a concentration of 2 mM to 20 mM in a solvent mixed with DMSO (dimethyl sulfoxide) and water in a volume ratio of 1:9, or additionally, the 7,8,3',4'-tetrahydroxyflavone is extracted from Bidens bipinnata L.

[0020] In addition, the present invention is also preferably a pharmaceutical composition for the prevention or treatment of thrombosis containing a plant extract of Bidens bipinnata L. as an active ingredient.

[0021] In addition, the present invention is also desirable as a health functional food for the prevention or improvement of thrombosis containing 7,8,3',4'-tetrahydroxyflavone having the following chemical formula as an active ingredient.

[0022]

[0023] In addition, in addition to the features described above, it is also desirable to use a health functional food for the prevention or improvement of thrombosis characterized by the 7,8,3',4'-tetrahydroxyflavone being extracted from Bidens bipinnata L.

[0024] The present invention is also preferably a health functional food for the prevention or improvement of thrombosis containing a plant extract of Bidens bipinnata L. as an active ingredient, or additionally, the plant extract is characterized by being prepared in the form of a powder, granule, tablet, capsule, or beverage from an extract obtained from the leaves and stems of Bidens bipinnata.

[0025] The present invention also preferably comprises a method for extracting a pharmaceutical composition for the prevention or treatment of thrombosis from Bidens bipinnata, characterized by the following steps: washing the leaves and stems of Bidens bipinnata in water, drying them, and then grinding them to make a powder; placing the powder in a solvent and stirring, filtering, and concentrating it to obtain a concentrate; resuspending the concentrate in water and removing lipid components; separating and extracting flavonoids from the concentrate from which lipid components have been removed; separating and extracting 7,8,3',4'-tetrahydroxyflavone from the flavonoids through chromatography; and preparing a composition by mixing the 7,8,3',4'-tetrahydroxyflavone at a concentration of 2 mM to 20 mM in a solvent in which DMSO (dimethyl sulfoxide) and water are mixed in a volume ratio of 1:9, respectively.

[0026] In addition to the features described above, the present invention also preferably provides a method for extracting a pharmaceutical composition for the prevention or treatment of thrombosis from Bidens pilosa, characterized in that the powder is dried at 40°C for 48 hours to reduce the moisture content to 5% or less and then ground to a size of 0.5 to 1 mm, the solvent is a 70% aqueous ethanol solution, and the concentrate is obtained by adding 500 mL of the solvent per 100 g of the powder and circulating extracting it with an extractor at a temperature of 70°C for 7 hours, filtering it through a 0.45 μm filter paper, and then concentrating it with an evaporator at a temperature of 50°C and a vacuum of 250 mbar.

[0027] The present invention also preferably comprises a method for manufacturing a health functional food for the prevention or improvement of thrombosis using an extract of Bidens bipinnata, characterized by the following steps: washing the leaves and stems of Bidens bipinnata L. in water, drying them, and then grinding them to make a powder; placing the powder in a solvent and stirring to obtain an extract; filtering and concentrating the extract to obtain a concentrate; removing the solvent from the concentrate and drying it to obtain a dried product; grinding the dried product to make a fine powder; and adding an excipient to the fine powder, mixing it, and then molding it. Effects of the invention

[0028] As seen above, the “composition for preventing or treating thrombosis and health functional food containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient” according to the present invention is harmless to the human body and effectively inhibits platelet aggregation and thrombus formation caused by platelet activation, thereby having the effect of preventing, treating, and improving various thrombotic diseases caused by platelet aggregation and thrombus formation.

[0029] In addition, the “composition for preventing or treating thrombosis and health functional food containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient” according to the present invention has the effect of preventing, treating, and improving various thrombotic diseases including thrombosis because it inhibits platelet aggregation and thrombus formation by increasing the production of cAMP in platelets.

[0030] In addition, the “composition for preventing or treating thrombosis and health functional food containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient” according to the present invention has the effect of preventing, treating, and improving various thrombotic diseases including thrombosis by increasing the production of cGMP in platelets and inhibiting platelet aggregation and thrombus formation.

[0031] In addition, the “composition for preventing or treating thrombosis and health functional food containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient” according to the present invention inhibits platelet aggregation and thrombus formation by inhibiting calcium mobilization for platelet activation, thereby having the effect of preventing, treating, and improving various thrombotic diseases including thrombosis.

[0032] In addition, the “composition for preventing or treating thrombosis and health functional food containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient” according to the present invention inhibits the release of ATP released from alpha granules in the initial stage of platelet activation, thereby inhibiting platelet aggregation and thrombus formation, and thus has the effect of preventing, treating, and improving various thrombotic diseases including thrombosis.

[0033] In addition, the “composition for preventing or treating thrombosis and health functional food containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient” according to the present invention has the effect of preventing, treating, and improving various thrombotic diseases including thrombosis because it inhibits platelet aggregation and thrombus formation by inhibiting the release of serotonin released from alpha granules in the initial stage of platelet activation.

[0034] In addition, the “composition for preventing or treating thrombosis and health functional food containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient” according to the present invention has the effect of preventing, treating, and improving various thrombotic diseases including thrombosis because it inhibits platelet aggregation and thrombus formation by inactivating integrin αIIbβ3, a fibrinogen receptor in platelets, thereby inhibiting fibrinogen, which amplifies platelet aggregation, from binding to integrin αIIbβ3. Brief explanation of the drawing

[0035] FIG. 1 is a schematic diagram showing the mechanism of action of a composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient according to the present invention and the inhibitory pathway for platelet activation in the signal transduction process within platelets. Figure 2 is a diagram showing the platelet aggregation inhibitory effect when a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention is applied to human platelets stimulated with collagen. Figure 3 is a diagram showing the platelet aggregation inhibitory effect when a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention is applied to human platelets stimulated with U46619. Figure 4 is a diagram showing the effect of increasing the production of cAMP in human platelets by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient to collagen-stimulated platelets. Figure 5 is a diagram showing the effect of increasing the production of cAMP in human platelets stimulated with U46619 by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient. FIG. 6 is a diagram showing the effect of increasing the production of cGMP in human platelets by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient to collagen-stimulated platelets. Figure 7 is a diagram showing the effect of increasing the production of cGMP in human platelets stimulated with U46619 by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient. FIG. 8 is a diagram showing the effect of reducing calcium mobilization when a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention is applied to human platelets stimulated with collagen. FIG. 9 is a diagram showing the effect of reducing calcium mobilization when a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention is applied to human platelets stimulated with U46619. FIG. 10 is a diagram showing the degree of inhibition of ATP release from dense granules of platelets by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient to human platelets stimulated with collagen. FIG. 11 is a diagram showing the degree of inhibition of ATP release from dense granules of platelets by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient to human platelets stimulated with U46619. FIG. 12 is a diagram showing the degree of inhibition of serotonin release from dense granules of platelets by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient to human platelets stimulated with collagen. FIG. 13 is a diagram showing the degree of inhibition of serotonin release from dense granules of platelets by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient to human platelets stimulated with U46619. FIG. 14 is a diagram showing the degree of inhibition of fibrinogen release from dense granules of platelets by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient to human platelets stimulated with collagen. FIG. 15 is a diagram showing the degree of inhibition of fibrinogen release from dense granules of platelets by applying a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient to human platelets stimulated with U46619. FIG. 16 is a diagram evaluating whether a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient exhibits toxicity in human platelet cells after application. Specific details for implementing the invention

[0036] The present invention will be described in detail below so that the aforementioned objectives and features may be clearly understood, and accordingly, a person skilled in the art to which the present invention pertains will be able to easily implement the technical concept of the present invention. Furthermore, in describing the present invention, regarding known technologies related to the present invention that are already well known in the technical field, if it is determined that a detailed description of such known technology might unnecessarily obscure the essence of the present invention, such detailed description will be omitted.

[0037] Furthermore, while the terms used in this invention have been selected to be as widely used as possible, there are also terms arbitrarily chosen by the applicant in specific cases; since the meanings of such terms are described in detail in the relevant description of the invention, it should be noted that the invention should be understood based on the meaning of the terms rather than their mere names. The terms used in the description of the embodiments are used merely to explain specific embodiments and are not intended to limit the embodiments. Unless the context clearly indicates otherwise, singular expressions include plural expressions.

[0038] Embodiments may be modified in various forms and may have various additional embodiments, and specific embodiments are shown in the drawings and described in detail. However, this is not intended to limit the embodiments to specific forms, and it should be understood that they include all modifications, equivalents, or substitutions that fall within the spirit and scope of the embodiments.

[0039] In the description of various embodiments, expressions such as “first,” “second,” “first,” or “second” may modify various components of the embodiments, but do not limit said components. For example, said expressions do not limit the order and / or importance of said components. Such expressions may be used to distinguish one component from another.

[0040] The present invention will be described below with reference to the attached drawings. The present invention relates to a composition for preventing and treating thrombotic diseases containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient, characterized by containing 7,8,3',4'-tetrahydroxyflavone, which is isolated and extracted from Bidens pilosa, a species of Asteraceae that is widely distributed worldwide, as an active ingredient, thereby inhibiting platelet aggregation and thrombus formation caused by platelet activation within blood vessels, and enabling the prevention and treatment of thrombotic diseases.

[0041] Bidens bipinnata L., also known as Devil's Needle, is an annual plant belonging to the Asteraceae family and is widely distributed globally in Korea, Japan, China, Australia, India, and Africa. Its leaves are deeply divided, bipinnately compound, and feather-shaped with serrated margins. The flowers are yellow capitula, and the stems are erect, reaching a height of 50 to 150 cm, and are densely hairy. The fruit consists of black seeds covered with densely packed hook-shaped spines that attach to animals or human clothing, thereby dispersing the seeds.

[0042] Bidens pilosa has traditionally been known for its antipyretic, anti-inflammatory, liver function-improving, and diuretic effects. In folk medicine, fresh leaves were crushed to disinfect wounds, or a decoction was used to treat gastritis or the common cold. The main active ingredients of Bidens pilosa include 7,8,3',4'-tetrahydroxyflavone, luteolin, and quercetin derivatives.

[0043] The present invention aims to provide a composition for preventing or treating thrombosis containing 7,8,3',4'-tetrahydroxyflavone extracted from Bidens bipinnata L. as an active ingredient, and to provide a health functional food for preventing or improving thrombosis containing 7,8,3',4'-tetrahydroxyflavone extracted from Bidens bipinnata L. as an active ingredient.

[0044] FIG. 1 shows a schematic diagram illustrating the mechanism of action of a composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient according to the present invention and the inhibition pathway for platelet activation in the signal transduction process within platelets.

[0045] As illustrated in Fig. 1, the signaling process of platelet aggregation following platelet activation is such that when stimulating substances such as collagen or U46619 stimulate platelets, Phospholipase C (PLase C) is activated and hydrolyzes the platelet membrane phospholipid (phosphatidylinositol 4,5-bisphosphate, PIP2) into IP3 (inositol 1,4,5-trisphosphate) and DG (diacylglycerol).

[0046] IP3 generated by hydrolysis binds to IP3 receptors located on the membrane of the endoplasmic reticulum (ER), a calcium storage site within the cytoplasm of platelets, opening calcium channels to release calcium (Ca 2+ It performs the function of causing mobilization.

[0047] Calcium, which is increased by calcium mobilization, binds to calmodulin and activates myosin light chain kinase (C-kinase), causing platelets to aggregate by phosphorylating the myosin light chain and pleckstrin proteins responsible for cell contraction.

[0048] At this time, the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient can significantly inhibit platelet aggregation caused by calcium mobilization by increasing the production of cAMP and cGMP, which are known as calcium antagonists, thereby significantly reducing thrombus formation.

[0049] In addition, a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient can significantly reduce thrombus formation by inhibiting the secretion of substances within platelet cell granules that promote platelet activation, such as ATP, and by inhibiting the binding of fibrinogen, which amplifies platelet aggregation, to integrin αIIbβ3, thereby significantly inhibiting platelet aggregation.

[0051] The present invention will be described in more detail below through examples and experimental examples. The examples and experimental examples described below are disclosed to aid in understanding the present invention, and it should be understood that the present invention may be implemented with various modifications different from the examples or experimental examples described herein. It is obvious to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, and it is also natural that such modifications and changes fall within the scope of the appended claims.

[0053] <Example 1>: Extraction of 7,8,3',4'-tetrahydroxyflavone from Bidens pilosa

[0054] (a) Preparation of raw materials

[0055] The leaves and stems of the devil's needle plant were harvested, washed in running water, and then dried in the shade.

[0056] - It was dried in a dryer at 40℃ for 48 hours to reduce the moisture content to 5% or less.

[0057] - It was ground using a grinder and processed into a powder form of 0.5 to 1 mm. (Improved extraction efficiency due to increased surface area)

[0058] (2) Extraction using a solvent

[0059] - Since flavonoids are well soluble in polar solvents, a 70% aqueous ethanol solution was used as the solvent.

[0060] 500 mL of solvent was added to 100 g of Bidens pilosa powder, and circulating extraction was performed at 70°C for 7 hours using a Soxhlet extractor.

[0061] - The extract was filtered through a 0.45 μm filter paper.

[0062] (3) Extract concentration

[0063] - 10 mL of concentrate was obtained by concentrating with a rotary evaporator at a temperature of 50°C and a vacuum of 250 mbar.

[0064] (4) Step 1 Purification: Liquid-liquid separation

[0065] 10 mL of concentrate was mixed with 200 mL of distilled water, and then washed with 200 mL of hexane to remove lipid components from the upper layer.

[0066] - The concentrate from which the lipid layer had been removed was divided into 200 mL of ethyl acetate to separate and extract the flavonoid components.

[0067] (5) Step 2 Purification: Chromatography

[0068] - The hexane-ethyl acetate mixture was flowed through a silica gel column.

[0069] - The silica gel column used silica gel as a packing material in a glass column with a diameter of 5 cm × a length of 40 cm.

[0070] - The location of the target compound was confirmed using TLC (Thin Layer Chromatography).

[0071] (6) Final separation: HPLC (High Performance Liquid Chromatography)

[0072] - HPLC: A C18 column was used, and acetonitrile-water was applied as the mobile phase.

[0073] - The acidic conditions of acetonitrile-water were set to 0.1% TFA.

[0074] - The target was fractionated and obtained after confirming the peak with a UV detector (254-280 nm).

[0075] (7) Analysis and verification

[0076] - Molecular weight and separation efficiency were confirmed using a mass spectrometer (MS).

[0077] - To confirm the structure, the specific signal of 7,8,3',4'-tetrahydroxyflavone was confirmed by Nuclear Magnetic Resonance (NMR) analysis.

[0079] <Example 2> : Preparation of a composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient

[0080] A composition was prepared by adding 5.72 mg to 57.20 mg of the 7,8,3',4'-tetrahydroxyflavone prepared in Example 1 to 10 mL of a solvent composed of 10% DMSO (dimethyl sulfoxide) and 90% water, such that the 7,8,3',4'-tetrahydroxyflavone has a concentration of 2 mM to 20 mM in the solvent.

[0082] <Example 3> : Preparation of samples for the experiment

[0083] 390 μL of platelet suspension (i.e., Platelet Rich Plasma (PRP)) was prepared as a sample to evaluate the effect of the "composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient" according to the present invention, and the "composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient" prepared above was added to the prepared platelet suspension (sample) so that the concentrations of 7,8,3',4'-tetrahydroxyflavone in the 99.75% aqueous solution and 0.25% DMSO were finally 16 μM, 32 μM, 48 μM, 56 μM, 64 μM, 96 μM, and 128 μM, respectively, thereby preparing seven types of experimental samples for evaluation.

[0085] <Example 4> : Preparation of a health functional food prototype

[0086] (1) Powder processing

[0087] The leaves and stems of the devil's needle plant were harvested and washed in water to remove impurities.

[0088] - Soaked in a 70% ethanol solution for 15 minutes to remove microorganisms, then rinsed with running water.

[0089] - It was dried in a dryer at 40℃ for 48 hours to reduce the moisture content to 5% or less.

[0090] - It was ground using a grinder and processed into a powder form of 0.5 to 1 mm. (Improved extraction efficiency due to increased surface area)

[0091] (2) Flavonoid extraction

[0092] - The solvent was a 70% ethanol solution, and it was added at a ratio of 10L of solvent per 1Kg of powdered raw material.

[0093] Heat reflux extraction was performed while stirring for 3 hours at a temperature of -75℃.

[0094] After the first extraction, a new solvent was added to the filtered residue, and a second extraction was performed for 3 hours.

[0095] (3) Filtration, concentration

[0096] The extracts obtained in two stages were combined, and impurities were removed using a filter.

[0097] - The solvent was removed from the extract with impurities removed using a rotary evaporator at 40~50℃.

[0098] (4) Drying and formulation

[0099] - For freeze-drying, it was rapidly frozen at -40℃ and dried under a low-temperature vacuum. (High preservation rate of active ingredients)

[0100] The dried extract was ground and processed into a 100 mesh fine powder.

[0101] - Maltodextrin was added as an excipient to improve fluidity.

[0102] - Molded into various forms such as capsules, tablets, and liquids.

[0103] (5) Analysis of active ingredients

[0104] - The 7,8,3',4'-tetrahydroxyflavone content was confirmed through HPLC (High Performance Liquid Chromatography) analysis.

[0105] - If the content is above the standard (0.57 mg / g to 5.7 mg / g or higher), it was decided to use it as the final product.

[0107] <Experimental Example 1>

[0108] To investigate the inhibitory effect on platelet aggregation in human platelets, an experiment was conducted to test the inhibitory effect on platelet aggregation. For the aggregation effect experiment, aggregation of platelets in the sample was induced using collagen, which is known to induce platelet aggregation in the human body, as an agonist, and aggregation of platelets in the sample was induced using U46619, an analog of TXA2 (thrimboxane A2), which is another substance known to induce platelet aggregation in the human body, as an agonist.

[0109] (a) Five samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to achieve concentrations of 16 μM, 32 μM, 48 μM, 56 μM, and 64 μM, respectively.

[0110] A platelet suspension stimulated with 2.5 μg / mL of collagen was used as a control group, and five experimental groups were prepared in which each sample was added to the platelet suspension stimulated with 2.5 μg / mL of collagen. The aggregation rate was measured by light transmittance for 5 minutes. The platelet aggregation rate in each sample was determined based on the measured light transmittance.

[0111] As shown in Figure 2, the experimental results indicate that the platelet aggregation rate was 72.0% for the control group stimulated only with collagen in the platelet suspension, and the platelet aggregation rates for each sample at concentrations of 7,8,3',4'-tetrahydroxyflavone of 16μM, 32μM, 48μM, 56μM, and 64μM were 60.5%, 57.4%, 26.3%, 21.0%, and 1.0%, respectively.

[0112] (b) Six samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to achieve concentrations of 32 μM, 48 μM, 64 μM, 96 μM, 128 μM, and 144 μM, respectively.

[0113] A platelet suspension stimulated with 0.25 μM U46619 was used as a control group, and six experimental groups were prepared in which each sample was added to the platelet suspension stimulated with 0.25 μM U46619. The aggregation rate was measured by light transmittance for 5 minutes. The platelet aggregation rate in each sample was determined based on the measured light transmittance.

[0114] As shown in Figure 3, the experimental results indicate that the platelet aggregation rate was 81.5% for the control group stimulated only with U46619 in the platelet suspension, and the platelet aggregation rates for each sample at concentrations of 7,8,3',4'-tetrahydroxyflavone of 32μM, 48μM, 64μM, 96μM, 128μM, and 144μM were 71.3%, 68.5%, 48.7%, 38.0%, 19.3%, and 12.5%, respectively.

[0115] (c) As shown in FIGS. 2 and 3, it was found that the composition for the prevention and treatment of thrombosis containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient can significantly inhibit platelet aggregation in a dose-dependent, i.e., concentration-dependent manner in platelets stimulated with collagen or U46619.

[0116] Through this, it can be seen that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient can effectively inhibit platelet aggregation by inhibiting the activity of collagen and TXA2 when acting in the human body.

[0118] <Experimental Example 2>

[0119] An experiment was conducted to evaluate the effect on cAMP (cyclic adenosine monophosphate), which is known to inhibit platelet aggregation by lowering calcium (Ca2+), a factor closely related to platelet aggregation; specifically, to assess the amount of cAMP produced. In this experiment, the unit of cAMP production was pmoL / 10 9 It is Platelets.

[0120] (a) Two samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to increase the production of cAMP in human platelets stimulated with collagen, so that the concentrations of 7,8,3',4'-tetrahydroxyflavone were 32 μM and 64 μM, respectively.

[0121] Platelet suspensions not stimulated with collagen and platelet suspensions stimulated with 2.5 μg / mL of collagen were prepared, respectively. In addition, two experimental groups were created by adding each sample to the platelet suspension stimulated with 2.5 μg / mL of collagen, and the effect of increasing cAMP production was measured.

[0122] As shown in Figure 4, it can be seen that when platelet suspensions are not stimulated with collagen and when stimulated with collagen, the cAMP generated in the platelets is 6.6782 and 3.7662, respectively. When the two experimental group samples were stimulated with collagen, the cAMP was 6.625 at 32 μM and 12.67 at 64 μM, indicating that the production of cAMP significantly increased as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0123] (b) To examine the effect of increasing cAMP production in human platelets stimulated with U46619, 7,8,3',4'-tetrahydroxyflavone was added to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to obtain concentrations of 64 μM, 96 μM, and 128 μM of 7,8,3',4'-tetrahydroxyflavone, respectively, and three samples were prepared for evaluation.

[0124] Platelet suspensions not stimulated with U46619 and platelet suspensions stimulated with 0.25 μM of U46619 were prepared, respectively, and three experimental groups were created by adding each sample to the platelet suspension stimulated with 0.25 μM of U46619 to measure the effect of increasing cAMP production.

[0125] As shown in Figure 5, it can be seen that when platelet suspensions are not stimulated with U46619 and when stimulated with U46619, the cAMP produced in the platelets is 6.6782 and 3.7662, respectively. When the three experimental group samples were stimulated with U46619, the concentration of 7,8,3',4'-tetrahydroxyflavone was 6.27 at 64 μM, 8.45 at 96 μM, and 16.80 at 128 μM, indicating that the production of cAMP significantly increased as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0126] (c) As shown in FIGS. 4 and 5, it was found that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient significantly increased the production of cAMP in platelets stimulated with collagen or U46619 in a dose-dependent, i.e., concentration-dependent manner.

[0127] Through this, it can be seen that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient, when acting in the human body, significantly enhances the production of cAMP when platelets are activated due to various factors, thereby effectively inhibiting platelet aggregation caused by platelet activation.

[0129] <Experimental Example 3>

[0130] An experiment was conducted to evaluate the effect on cGMP (cyclic guanosine monophosphate), which is known to regulate platelet activation and prevent platelet aggregation; specifically, the amount of cGMP produced. In this experiment, the unit of cGMP production was pmoL / 10 9 It is Platelets.

[0131] (a) Two samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to increase the production of cAMP in human platelets stimulated with collagen, so that the concentrations of 7,8,3',4'-tetrahydroxyflavone were 32 μM and 64 μM, respectively.

[0132] Platelet suspensions not stimulated with collagen and platelet suspensions stimulated with 2.5 μg / mL of collagen were prepared, respectively, and two experimental groups were created by adding each sample to the platelet suspension stimulated with 2.5 μg / mL of collagen to measure the effect of increasing cGMP production.

[0133] As shown in Figure 6, it can be seen that when platelet suspensions are not stimulated with collagen and when stimulated with collagen, the cGMP generated in the platelets is 7.60 and 2.78, respectively. When the two experimental group samples were stimulated with collagen, the concentration of 7,8,3',4'-tetrahydroxyflavone was 6.38 at 32 μM and 8.08 at 64 μM, indicating that the production of cGMP significantly increased as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0134] (b) To examine the effect of increasing cGMP production in human platelets stimulated with U46619, 7,8,3',4'-tetrahydroxyflavone was added to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to obtain concentrations of 64 μM, 96 μM, and 128 μM, respectively, to prepare three samples for evaluation.

[0135] Platelet suspensions not stimulated with U46619 and platelet suspensions stimulated with 0.25 μM of U46619 were prepared, respectively, and three experimental groups were created by adding each sample to the platelet suspension stimulated with 0.25 μM of U46619 to measure the effect of increasing cGMP production.

[0136] As shown in Figure 7, it can be seen that the cGMP produced in platelets in the case of platelet suspension not stimulated with U46619 and when stimulated with U46619 was 3.85 and 2.03, respectively. When the three experimental group samples were stimulated with U46619, the concentration of 7,8,3',4'-tetrahydroxyflavone was 3.02 at 64 μM, 4.86 at 96 μM, and 5.73 at 128 μM, indicating that the production of cGMP increased significantly as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0137] (c) As shown in FIGS. 6 and 7, it was found that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient significantly increased the production of cGMP in platelets stimulated with collagen or U46619 in a dose-dependent, i.e., concentration-dependent manner.

[0138] Through this, it can be seen that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient, when acting in the human body, significantly enhances the production of cGMP when platelets are activated due to various factors, thereby effectively inhibiting platelet aggregation caused by platelet activation.

[0140] <Experimental Example 4>

[0141] Calcium ([Ca 2+ ] i Since mobilization is an important step required for platelet aggregation, as platelets are activated and degranulated, the effect on calcium mobilization was evaluated for a composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient.

[0142] (a) Four samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to examine the effect of reducing calcium mobilization in human platelets stimulated with collagen, so that the concentrations of 7,8,3',4'-tetrahydroxyflavone were 32 μM, 48 μM, 64 μM, and 96 μM, respectively.

[0143] Platelet suspensions not stimulated with collagen and platelet suspensions stimulated with 2.5 μg / mL of collagen were prepared, respectively, and four experimental groups were created by adding each sample to the platelet suspension stimulated with 2.5 μg / mL of collagen to measure the effect of reducing calcium mobilization.

[0144] As shown in Figure 8, when the platelet suspension was not stimulated with collagen, the platelets were found to mobilize calcium equivalent to 50.77 (nM), and when only collagen was applied to the platelet suspension, the calcium mobilized by the corresponding platelets was significantly increased to 395.34 (nM), indicating that the platelets were activated.

[0145] And when the four experimental group samples were stimulated with collagen, the concentration of 7,8,3',4'-tetrahydroxyflavone was 270.47 at 32 μM, 151.54 at 48 μM, 97.00 at 64 μM, and 68.47 at 96 μM, indicating that calcium mobilization in platelets decreased significantly as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0146] (b) To examine the effect of reducing calcium mobilization in human platelets stimulated with U46619, 7,8,3',4'-tetrahydroxyflavone was added to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to obtain concentrations of 16 μM, 32 μM, 48 μM, and 64 μM, respectively, to prepare four samples for evaluation.

[0147] Platelet suspensions not stimulated with U46619 and platelet suspensions stimulated with 0.25 μM of U46619 were prepared, respectively, and four experimental groups were created by adding each sample to the platelet suspension stimulated with 0.25 μM of U46619 to measure the effect of reducing calcium mobilization.

[0148] As shown in Figure 9, when the platelet suspension was not stimulated with U46619, it was found that the platelets mobilized calcium equivalent to 28.37 nM, and when only U46619 was applied to the platelet suspension, the calcium mobilized by the corresponding platelets was significantly increased to 686.69 nM, indicating that the platelets were activated.

[0149] And when four experimental group samples were stimulated with U46619, the concentration of 7,8,3',4'-tetrahydroxyflavone was 192.26 nM at 16 μM, 119.26 nM at 32 μM, 56.96 nM at 48 μM, and 41.76 nM at 64 μM, indicating that calcium mobilization in platelets decreased significantly as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0150] (c) As shown in FIGS. 8 and 9, it was found that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient significantly lowered calcium mobilization in platelets stimulated with collagen or U46619 in a dose-dependent, i.e., concentration-dependent manner.

[0151] Through this, it can be seen that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient, when acting in the human body, significantly lowers calcium mobilization when platelets are activated due to various factors, thereby effectively inhibiting platelet aggregation caused by platelet activation.

[0153] <Experimental Example 5>

[0154] ATP (adenosine triphosphate) is an energy transfer substance produced by platelets and is involved in platelet activation and coagulation. In the initial stage of platelet activation, ATP is rapidly released from the dense granules of the corresponding platelet and stimulates other platelets, thereby performing the function of causing multiple platelets to rapidly attach and aggregate. Accordingly, the effect of a composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention on inhibiting ATP release was evaluated.

[0155] (a) Four samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to examine the degree of inhibition of ATP release from dense granules of platelets when applied to human platelets stimulated with collagen, so that the concentrations of 7,8,3',4'-tetrahydroxyflavone were 32 μM, 48 μM, 64 μM, and 96 μM, respectively.

[0156] Platelet suspensions not stimulated with collagen and platelet suspensions stimulated with 2.5 μg / mL of collagen were prepared, respectively, and four experimental groups were created by adding each sample to the platelet suspension stimulated with 2.5 μg / mL of collagen, and the degree of ATP release inhibition was measured.

[0157] As shown in Figure 10, it was found that when the platelet suspension was not stimulated with collagen, 240.06 μM of ATP was released from the platelets, and when the platelet suspension was treated only with collagen, the corresponding platelets released a significantly increased amount of ATP, up to 2186.01 μM.

[0158] And when the four experimental group samples were stimulated with collagen, the concentration of 7,8,3',4'-tetrahydroxyflavone was 1413.99 μM at 32 μM, 996.92 μM at 48 μM, 694.75 μM at 64 μM, and 682.57 μM at 96 μM, showing that ATP release from platelets decreased as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0159] (b) To examine the degree of inhibition of ATP release in human platelets stimulated with U46619, 7,8,3',4'-tetrahydroxyflavone was added to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to obtain concentrations of 7,8,3',4'-tetrahydroxyflavone of 16 μM, 48 μM, 64 μM, and 96 μM, respectively, to prepare four samples for evaluation.

[0160] Platelet suspensions not stimulated with U46619 and platelet suspensions stimulated with 0.25 μM of U46619 were prepared, respectively, and four experimental groups were created by adding each sample to the platelet suspension stimulated with 0.25 μM of U46619 to measure the ATP release inhibition effect.

[0161] As shown in Figure 11, it was found that when the platelet suspension was not stimulated with U46619, it released ATP equivalent to 240.06 μM, and when the platelet suspension was treated only with U46619, the ATP released from the corresponding platelets increased significantly to 1328.69 μM.

[0162] And when four experimental group samples were stimulated with U46619, ATP was 1211.33 μM at a concentration of 7,8,3',4'-tetrahydroxyflavone of 32 μM, 780.09 μM at 48 μM, 522.92 μM at 64 μM, and 595.78 μM at 96 μM, indicating that ATP release from platelets was inhibited as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0163] (c) As shown in FIGS. 10 and 11, the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient can be seen to have the effect of effectively inhibiting the aggregation of platelets by significantly inhibiting the release of ATP from platelets stimulated with collagen or U46619.

[0164] Through this, it can be seen that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient, when acting in the human body, significantly inhibits the release of ATP when platelets are activated due to various factors, thereby effectively inhibiting platelet aggregation caused by platelet activation.

[0166] <Experimental Example 6>

[0167] Excessive release of serotonin can increase the risk of thrombosis. Therefore, the effect of the “composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient” according to the present invention on inhibiting the release of serotonin was evaluated.

[0168] (a) Four samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to examine the degree of inhibition of serotonin release from the dense granules of the platelets when applied to human platelets stimulated with collagen, so that the concentrations of 7,8,3',4'-tetrahydroxyflavone were 32 μM, 48 μM, 64 μM, and 96 μM, respectively.

[0169] Platelet suspensions not stimulated with collagen and platelet suspensions stimulated with 2.5 μg / mL of collagen were prepared, respectively, and four experimental groups were created by adding each sample to the platelet suspension stimulated with 2.5 μg / mL of collagen, and the degree of inhibition of serotonin release was measured.

[0170] As shown in Fig. 12, in the case where the platelet suspension was not stimulated with collagen 10 8 It was found that 97.65 ng of serotonin is released per platelet, and when collagen stimulation is applied to the platelet suspension, the corresponding platelets release 267.65 ng of serotonin, which is a significant increase.

[0171] And when the four experimental group samples were stimulated with collagen, it was found that 235.61 ng of 7,8,3',4'-tetrahydroxyflavone was released at a concentration of 32 μM, 101.64 ng at 48 μM, 90.04 ng at 64 μM, and 63.35 ng at 96 μM. Based on this, it was found that the release of serotonin from platelets decreased as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0172] (b) To examine the degree of inhibition of serotonin release in human platelets stimulated with U46619, 7,8,3',4'-tetrahydroxyflavone was added to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to obtain concentrations of 7,8,3',4'-tetrahydroxyflavone of 16 μM, 48 μM, 64 μM, and 96 μM, respectively, to prepare four samples for evaluation.

[0173] Platelet suspensions not stimulated with U46619 and platelet suspensions stimulated only with 0.25 μM U46619 were prepared, respectively. In addition, four experimental groups were created by adding each sample to the platelet suspension stimulated with 0.25 μM U46619, and the inhibitory effect on serotonin release was measured.

[0174] As shown in Fig. 13, when the platelet suspension was not stimulated with U46619, 10 8 It was found that 97.65 ng of serotonin is released per platelet, and when only U46619 was treated to the platelet suspension, the serotonin released from the platelets increased significantly to 261.38 ng.

[0175] And when four experimental group samples were stimulated with U46619, serotonin was 177.96 ng at 32 μM, 109.62 ng at 48 μM, 82.56 ng at 64 μM, and 97.22 ng at 96 μM, indicating that serotonin release from platelets was inhibited as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0176] (c) As shown in FIGS. 12 and 13, the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient can be seen to have the effect of effectively inhibiting platelet aggregation by significantly inhibiting the release of serotonin from platelets stimulated with collagen or U46619.

[0177] Through this, it can be seen that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient, when acting in the human body, significantly inhibits the release of serotonin when platelets are activated due to various factors, thereby effectively inhibiting platelet aggregation caused by platelet activation.

[0179] <Experimental Example 7>

[0180] Fibrinogen is closely related to thrombosis because it is a key protein in the blood coagulation process. The binding of fibrinogen to integrin αIIbβ3 is one of the important substances that causes platelet activation by inducing structural changes in platelets, and is an essential element for platelet aggregation. Therefore, the effect of a composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient on the number of platelets aggregated by fibrinogen in activated platelets and on fibrinogen binding to integrin αIIbβ3 was evaluated. In this experiment, the number of platelets aggregated by fibrinogen was measured using induced cytometry with FITC (fluorescein isothiocyanate)-A, a derivative of fluorescein.

[0181] (a) Four samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to obtain concentrations of 7,8,3',4'-tetrahydroxyflavone of 32 μM, 48 μM, 64 μM, and 96 μM, respectively, to examine the degree to which fibrinogen binds to integrin αIIbβ3 from the dense granules of the platelets when applied to human platelets stimulated with collagen.

[0182] Platelet suspensions not stimulated with collagen and platelet suspensions stimulated with 2.5 μg / mL of collagen were prepared, respectively. In addition, four experimental groups were created by adding each sample to the platelet suspension stimulated with 2.5 μg / mL of collagen, and the degree of fibrinogen binding was measured.

[0183] As shown in Figure 14, when the platelet suspension was not stimulated with collagen, the degree of fibrinogen binding to integrin αIIbβ3 was 12.75%, and when only collagen was applied to the platelet suspension, it increased to 79.95%.

[0184] And when the four experimental group samples were stimulated with collagen, the concentration of 7,8,3',4'-tetrahydroxyflavone decreased to 69.43% at 32 μM, 47.90% at 48 μM, 27.53% at 64 μM, and 17.77% at 96 μM. In other words, it was found that the binding degree of fibrinogen to platelets decreased as the concentration of 7,8,3',4'-tetrahydroxyflavone increased. In addition, it was found that the number of platelets aggregated by fibrinogen also approached the state of not being stimulated with collagen as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0185] (b) To examine the extent to which fibrinogen binds to integrin αIIbβ3 in human platelets stimulated with U46619, 7,8,3',4'-tetrahydroxyflavone was added to a solvent mixed with 10% DMSO (dimethyl sulfoxide) and 90% water to achieve concentrations of 16 μM, 48 μM, 64 μM, and 96 μM, respectively, to prepare four samples for evaluation.

[0186] Platelet suspensions not stimulated with U46619 and platelet suspensions stimulated with 0.25 μM of U46619 were prepared, respectively. In addition, four experimental groups were created by adding each sample to the platelet suspension stimulated with 0.25 μM of U46619, and the degree of fibrinogen binding to integrin αIIbβ3 was measured.

[0187] As shown in Figure 15, the binding degree of fibrinogen in the platelet suspension was 12.75% when not stimulated with U46619, and it increased significantly to 82.95% when only U46619 was treated in the platelet suspension.

[0188] When four experimental group samples were stimulated with U46619, the fibrinogen binding rate decreased from 69.33% at 32 μM, to 64.90% at 48 μM, to 29.00% at 64 μM, and to 23.33% at 96 μM at 7,8,3',4'-tetrahydroxyflavone concentration, indicating that the binding of fibrinogen to integrin αIIbβ3 in platelets was inhibited as the concentration of 7,8,3',4'-tetrahydroxyflavone increased. Additionally, it was found that the number of platelets aggregated by fibrinogen also approached the state not stimulated by U46619 as the concentration of 7,8,3',4'-tetrahydroxyflavone increased.

[0189] (c) As shown in FIGS. 14 and 15, the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient can be seen to have the effect of effectively inhibiting platelet aggregation by reducing the number of platelets aggregated by fibrinogen in platelets stimulated with collagen or U46619 and inhibiting the degree to which fibrinogen binds to integrin αIIbβ3.

[0190] Through this, it can be seen that the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient, when acting in the human body, reduces the number of platelets aggregated by fibrinogen when platelets are activated due to various factors and significantly inhibits the binding of fibrinogen and integrin αIIbβ3, thereby effectively inhibiting platelet aggregation caused by platelet activation.

[0192] <Experimental Example 8>

[0193] The composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient according to the present invention was evaluated to see if it exhibits toxicity in platelet cells.

[0194] (a) Five samples for evaluation were prepared by adding 7,8,3',4'-tetrahydroxyflavone to a solvent mixture of 10% DMSO (dimethyl sulfoxide) and 90% water to achieve concentrations of 32 μM, 48 μM, 64 μM, 96 μM, and 128 μM, respectively. Platelet suspensions without samples were used as a control group, and platelet suspensions containing each sample were prepared as five experimental groups.

[0195] (b) As a result of the evaluation, as shown in Figure 16, it can be seen that more than 98% of almost all platelets survive regardless of the concentration of 7,8,3',4'-tetrahydroxyflavone included in each sample. Through this, it can be seen that the present invention is harmless to the human body, and at the same time, a composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient can effectively inhibit platelet aggregation and thrombus formation caused by platelet activation.

[0197] <Summary of Experimental Results>

[0198] As observed in Fig. 1, when stimulating substances such as collagen or U46619 stimulate platelets, Phospholipase C (PLase C) is activated, hydrolyzing platelet membrane phospholipids (phosphatidylinositol 4,5-bisphosphate, PIP2) into IP3 (inositol 1,4,5-trisphosphate) and DG (diacylglycerol). The resulting IP3 binds to IP3 receptors located on the endoplasmic reticulum (ER) membrane, which serves as a calcium reservoir within the platelet cytoplasm, thereby opening calcium channels and allowing calcium (Ca 2+It performs the function of inducing mobilization. In addition, calcium increased by calcium mobilization binds to calmodulin and activates myosin light chain kinase (C-kinase), causing platelets to aggregate by phosphorylating the myosin light chain and pleckstrin proteins responsible for cell contraction.

[0199] At this time, the composition containing 7,8,3',4'-tetrahydroxyflavone of the present invention as an active ingredient can significantly inhibit platelet aggregation caused by calcium mobilization by increasing the production of cAMP and cGMP, which are known as calcium antagonists, thereby significantly reducing thrombus formation.

[0200] In addition, a composition containing 7,8,3',4'-tetrahydroxyflavone according to the present invention as an active ingredient can significantly reduce thrombus formation by inhibiting the secretion of substances within platelet cell granules that promote platelet activation, such as ATP, and by inhibiting the binding of fibrinogen, which amplifies platelet aggregation, to integrin αIIbβ3, thereby significantly inhibiting platelet aggregation.

[0201] Therefore, a composition containing 7,8,3',4'-tetrahydroxyflavone as an active ingredient inhibits platelet aggregation and thrombus formation caused by platelet activation, thereby enabling the prevention and treatment of thrombosis that may occur in human arteries, veins, cardiovascular vessels, peripheral vessels, microvessels, cerebrovascular vessels, etc., due to thrombosis.

[0203] Although the present invention has been described with various examples as stated above, the present invention is not necessarily limited to these examples and may be implemented with various modifications within the scope of the technical concept of the present invention. Accordingly, the examples disclosed in the present invention are intended to explain, not limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by these examples. The scope of protection of the present invention shall be interpreted by the claims below, and all technical concepts within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of thrombosis containing 7,8,3',4'-tetrahydroxyflavone having the following chemical formula as an active ingredient Claim 2 A pharmaceutical composition for the prevention or treatment of thrombosis according to claim 1, wherein the composition comprises containing the 7,8,3',4'-tetrahydroxyflavone at a concentration of 2 mM to 20 mM in a solvent prepared by mixing DMSO (dimethyl sulfoxide) and water in a volume ratio of 1:9, respectively. Claim 3 A pharmaceutical composition for the prevention or treatment of thrombosis according to claim 1, characterized in that the 7,8,3',4'-tetrahydroxyflavone is extracted from Bidens bipinnata L. Claim 4 Pharmaceutical composition for the prevention or treatment of thrombosis containing a plant extract of Bidens bipinnata L. as an active ingredient Claim 5 A health functional food for the prevention or improvement of thrombosis containing 7,8,3',4'-tetrahydroxyflavone having the following chemical formula as an active ingredient Claim 6 A health functional food for the prevention or improvement of thrombosis according to claim 5, characterized in that the 7,8,3',4'-tetrahydroxyflavone is extracted from Bidens bipinnata L. Claim 7 A health functional food for the prevention or improvement of thrombosis containing Bidens bipinnata L. plant extract as an active ingredient Claim 8 A health functional food for the prevention or improvement of thrombosis according to claim 7, characterized in that the plant extract is an extract obtained from the leaves and stems of Bidens pilosa prepared in the form of powder, granules, tablets, capsules, or a beverage. Claim 9 A method for extracting a pharmaceutical composition for the prevention or treatment of thrombosis from Bidens bipinnata, characterized by comprising: a process of washing the leaves and stems of Bidens bipinnata in water, drying them, and then grinding them to make a powder; a process of placing the powder in a solvent, stirring, filtering, and concentrating it to obtain a concentrate; a process of resuspending the concentrate in water and removing lipid components; a process of separating and extracting flavonoids from the concentrate from which lipid components have been removed; a process of separating and extracting 7,8,3',4'-tetrahydroxyflavone from the flavonoids through chromatography; and a process of preparing a composition by mixing the 7,8,3',4'-tetrahydroxyflavone at a concentration of 2 mM to 20 mM in a solvent in which DMSO (dimethyl sulfoxide) and water are mixed in a volume ratio of 1:9, respectively. Claim 10 A method for extracting a pharmaceutical composition for the prevention or treatment of thrombosis from Bidens pilosa, characterized in that, in claim 9, the powder is dried at 40°C for 48 hours to reduce the moisture content to 5% or less and then ground to a size of 0.5 to 1 mm; the solvent is a 70% aqueous ethanol solution; and the concentrate is obtained by adding 500 mL of the solvent per 100 g of the powder, circulating extracting with an extractor at 70°C for 7 hours, filtering through a 0.45 μm filter paper, and then concentrating using an evaporator at a temperature of 50°C and a vacuum of 250 mbar. Claim 11 A method for manufacturing a health functional food for the prevention or improvement of thrombosis using an extract of Bidens bipinnata, characterized by comprising the following steps: washing the leaves and stems of Bidens bipinnata in water, drying them, and then grinding them to form a powder; placing the powder in a solvent and stirring to obtain an extract; filtering and concentrating the extract to obtain a concentrate; removing the solvent from the concentrate and drying it to obtain a dried product; grinding the dried product to form a fine powder; and adding an excipient to the fine powder, mixing it, and then molding it.

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