Pharmaceutical composition comprising the extract of acer pseudosieboldianum as an effective component for prevention or treatment of thrombosis and health functional food comprising the same

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an extract of Acer pseudosieboldianum, and more specifically, to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through inhibition of blood coagulation containing, as an active ingredient, a hot water extract or ethanol extract of Acer pseudosieboldianum leaves having potent thrombin inhibitory and blood coagulation factor inhibitory activity. The extract of *Acer pseudosieboldianum*, used as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis according to the present invention, exhibits excellent antithrombotic activity through strong inhibition of thrombus-related enzymes such as thrombin and blood coagulation factors. At the same time, it does not exhibit any hemolytic activity against human red blood cells, has excellent thermal stability, and does not show loss of blood coagulation factor inhibitory effects or thrombus-related enzyme inhibitory effects even under acidic conditions of pH 2 and in plasma. Therefore, it is expected to be usable for the prevention and treatment of thrombosis, such as ischemic stroke and hemorrhagic stroke, through the improvement of blood circulation. Furthermore, since the active ingredient has the excellent effect of being processed into various forms such as extract, powder, pills, and tablets, allowing for formulation into a form suitable for regular consumption, this invention is highly useful for the pharmaceutical and food industries.

Description

Technology Field

[0001] The present invention relates to a Japanese maple tree ( Acer pseudosieboldianum The invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an extract of ) more specifically, a Japanese maple tree having potent thrombin inhibitory and blood coagulation factor inhibitory activity ( Acer pseudosieboldianum The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through inhibition of blood coagulation, containing a hot water extract or ethanol extract of the leaves as an active ingredient. Background Technology

[0002] As a component of the human body, blood possesses various important functions, including the transport of oxygen, nutrients, and waste products, buffering, maintaining body temperature, regulating osmotic pressure and ion balance, maintaining constant fluid levels, regulating humor, maintaining and regulating blood pressure, and providing biological defense. Normal blood circulation is facilitated by the complementary regulation of the blood coagulation and thrombolytic systems within the body. Among these, the mechanism of the blood coagulation system is reported to involve platelets adhering to and aggregating on the blood vessel walls to form a platelet thrombus, after which the blood coagulation system is activated to form a fibrin thrombus centered around the platelet aggregate.

[0003] The formation of fibrin clots involves a multi-step reaction of numerous blood coagulation factors, through which thrombin, which is involved in fibrin coagulation, is activated. This ultimately leads to the generation of fibrin monomers from fibrinogen. These fibrin monomers are polymerized by calcium and bind to platelets and endothelial cells, forming a permanent blood clot by creating a fibrin polymer cross-linked by Factor XIII. Furthermore, thrombin plays a pivotal role in clot formation by activating platelets, Factor V, and Factor VII to promote blood coagulation reactions. Therefore, thrombin inhibitors can be used as highly useful preventive and therapeutic agents for various thrombotic diseases resulting from excessive blood coagulation abnormalities. Meanwhile, the endogenous thrombosis pathway is known to involve the sequential activation of Factor XII, Factor XI, Factor IX, and Factor X, followed by the activation of prothrombin, which ultimately leads to the activation of thrombin; thus, the specific inhibition of blood coagulation factors is also an important target for the development of treatments for thrombotic diseases. To date, various anticoagulants, antiplatelet agents, and thrombolytics such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases; however, their use is limited due to their very high cost, as well as hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions.

[0004] Meanwhile, the Japanese maple ( Acer pseudosieboldianum Acer palmatum is a deciduous broad-leaved tree belonging to the genus Acer in the family Aceraceae, and its origin is known to be Korea, China, and Russia. In Korea, it is the most commonly seen tree among maple species and is also called the mountain maple. It is highly resistant to cold and shows good growth in areas with moderate moisture rather than dry places, and is widely distributed in mountainous areas between 100 and 1,700 meters above sea level.

[0005] Studies related to the Japanese maple include the activity in improving benign prostatic hyperplasia (Son Se-yeon, 2024, Master's thesis, Chung-Ang University), the cancer cell growth inhibitory activity of ellagitannins derived from the Japanese maple (Yin, Jun, 2020, Ph.D. thesis, Chung-Ang University), and antibacterial activity by inhibition of quorum sensing (Niu K et al., 2017, Indian J Microbiol. 57: 329-338), but to date, no potent antithrombotic activity of the Japanese maple has been reported.

[0006] Regarding patents related to the Japanese maple, Korean registered patent No. 10-2479869 discloses a composition for the prevention, treatment, or improvement of prostate cancer containing a Japanese maple extract or a fraction thereof; No. 10-2697085 discloses an anti-obesity composition using a Japanese maple extract; and No. 10-1656875 discloses a quorum detection inhibitor for inhibiting Gram-negative bacteria using maple extract, Japanese maple extract, and Japanese holly extract. Additionally, Korean published patent No. 10-2009-0089951 discloses a method for manufacturing a lactic acid bacteria fermented beverage using the sap of the Japanese maple and the lactic acid bacteria fermented beverage produced therefrom, and No. 10-2017-0091238 discloses an antioxidant composition containing a complex extract of the sugar maple, the Japanese maple, and the Chinese maple; however, to date, no potent antithrombotic activity of the Japanese maple has been reported. Prior art literature

[0007] Benign prostate hypertrophy improvement effects of phenolic compounds isolated from Acer pseudosieboldianum (Pax) Komarov leaves. Son, Se-yeon, 2024. Master's thesis, Chung-Ang University. The problem to be solved

[0008] The present invention has been devised to solve the problems of the prior art as described above, and the problem to be solved by the present invention is the Japanese maple ( Acer pseudosieboldianumThe aim is to provide a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through inhibition of blood coagulation, comprising an extract as an active ingredient. means of solving the problem

[0009] In order to solve the above problems, the present invention relates to a Japanese maple tree ( Acer pseudosieboldianum Provides a pharmaceutical composition for the prevention or treatment of thrombosis containing an extract as an active ingredient.

[0010] The above Japanese maple tree ( Acer pseudosieboldianum ) extract is Japanese maple ( Acer pseudosieboldianum It is preferable that the extract be a hot water extract or an ethanol extract of the leaf.

[0011] In addition, the present invention relates to a Japanese maple tree ( Acer pseudosieboldianum Provides a blood coagulation inhibitor containing ) extract as an active ingredient.

[0012] The above Japanese maple tree ( Acer pseudosieboldianum ) extract is Japanese maple ( Acer pseudosieboldianum It is preferable that the extract be a hot water extract or an ethanol extract of the leaf.

[0013] In addition, the present invention relates to a Japanese maple tree ( Acer pseudosieboldianum Provides a health functional food for preventing or improving thrombosis containing an extract as an active ingredient.

[0014] The above Japanese maple tree ( Acer pseudosieboldianum ) extract is Japanese maple ( Acer pseudosieboldianum It is preferable that the extract be a hot water extract or an ethanol extract of the leaf. Effects of the invention

[0015] A maple tree as an active ingredient in a pharmaceutical composition for the prevention or treatment of thrombosis and a health functional food according to the present invention ( Acer pseudosieboldianumThe extract exhibits excellent antithrombotic activity through strong inhibition of thrombus-related enzymes, such as thrombin and blood coagulation factors, while simultaneously showing no hemolytic activity against human red blood cells. It also exhibits excellent thermal stability and does not lose its inhibitory effects on blood coagulation factors or thrombus-related enzymes even under acidic conditions of pH 2 or within plasma. Therefore, it is expected to be usable for the prevention and treatment of thrombosis, such as ischemic stroke and hemorrhagic stroke, by improving blood circulation. Furthermore, since the active ingredient can be processed into various forms such as extract, powder, pills, and tablets, and prepared in a form suitable for regular consumption, this invention is highly useful for the pharmaceutical and food industries. Specific details for implementing the invention

[0016] The present invention will be described in detail below.

[0017] The inventors of the present invention, in order to verify the antithrombotic efficacy of native Korean plants, prepared various plant extracts by a specific method and evaluated their antithrombotic and antioxidant activities, thereby confirming strong anticoagulant activity in the extract of the Japanese maple tree. They recovered the hot water extract or ethanol extract of the Japanese maple tree leaves as an antithrombotic active component, and confirmed that the extract exhibited excellent thermal and acid stability while showing no hemolytic activity against human red blood cells. Consequently, they intended to utilize the extract as a pharmaceutical composition and health functional food for the prevention or treatment / improvement of thrombosis.

[0018] Specifically, the inventors prepared various solvent extracts of Acer palmatum leaves to develop pharmaceutical compositions and health functional foods for the prevention, treatment, or improvement of thrombosis using Acer palmatum, which is known in folk medicine to be effective against various diseases of the vascular and circulatory systems, digestive systems, and metabolic systems. They evaluated the antithrombotic activity of these extracts by measuring direct thrombin inhibition (Thrombin Time), prothrombin inhibition (Prothrombin Time), and activated partial thromboplastin time (aPTT) against human thrombin, and confirmed that the hot water or ethanol extracts of Acer palmatum leaves exhibit very strong anticoagulant activity. Furthermore, they confirmed that the extracts do not exhibit hemolytic activity against human red blood cells, thereby confirming their practical applicability.

[0019] Therefore, the present invention relates to a Japanese maple tree ( Acer pseudosieboldianum Provides a pharmaceutical composition for the prevention or treatment of thrombosis containing an extract as an active ingredient.

[0020] The above Japanese maple tree ( Acer pseudosieboldianum ) extract is Japanese maple ( Acer pseudosieboldianum It is preferable that the extract be a hot water extract or an ethanol extract of the leaf.

[0021] In addition, the present invention relates to a Japanese maple tree ( Acer pseudosieboldianum Provides a blood coagulation inhibitor containing ) extract as an active ingredient.

[0022] The above Japanese maple tree ( Acer pseudosieboldianum ) extract is Japanese maple ( Acer pseudosieboldianum It is preferable that the extract be a hot water extract or an ethanol extract of the leaf.

[0023] In addition, the present invention relates to a Japanese maple tree ( Acer pseudosieboldianum Provides a health functional food for preventing or improving thrombosis containing an extract as an active ingredient.

[0024] The above Japanese maple tree ( Acer pseudosieboldianum ) extract is Japanese maple ( Acer pseudosieboldianum It is preferable that the extract be a hot water extract or an ethanol extract of the leaf.

[0025] In the following, the Japanese maple of the present invention ( Acer pseudosieboldianum The manufacturing method and efficacy experiments of the extract are explained in more detail.

[0026] The present invention comprises the steps of: recovering leaves of a Japanese maple tree; preparing an extract by extracting the recovered leaves with hot water and 70% ethanol; evaluating the antithrombotic activity of the extract and evaluating the acute toxicity of the extract.

[0027] "Japanese maple tree (included in the composition of the present invention Acer pseudosieboldianum The “) extract” can be obtained by the steps of extracting the leaves of a mature Japanese maple tree with hot water or ethanol, filtering the extract using a filter mesh of 0.06 mm or less, and concentrating it under reduced pressure.

[0028] The organic solvent used in the present invention may be water (cold water, hot water), hexene, methylene chloride, acetone, ethanol, anhydrous or hydrated lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, ethanol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc., and hot water extraction at 100°C is most preferable.

[0029] The above hot water extract can be further obtained by sequentially or separately fractionating it with organic solvents of hexene, ethyl acetate, and butanol to obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

[0030] In the present invention, the Japanese maple ( Acer pseudosieboldianumExtracts were prepared, and thrombin time, prothrombin time, and APT time were measured after adjusting the concentration to 2 mg / ml. As a result, it was confirmed that the hot water extract of Japanese maple leaves exhibited stronger thrombin inhibition, prothrombin inhibition, and blood coagulation factor inhibition than aspirin, thereby inhibiting thrombus formation. Furthermore, the Japanese maple extract showed no hemolytic activity against human red blood cells, suggesting that it can replace existing antithrombotic agents such as aspirin, which have high concerns regarding side effects.

[0031] The Japanese maple of the present invention ( Acer pseudosieboldianum The extracts can be prepared into powder through conventional pulverization processes such as vacuum drying, freeze-drying, or spray drying. They are not degraded by various enzymes in plasma and maintain their activity even under heat treatment at 100°C and at a pH of 2 in the human stomach.

[0032] The active ingredient of the present invention can be used for the prevention or treatment of various diseases related to thrombosis. The diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, motor abnormalities, sensory abnormalities, personality changes, visual impairment, epileptic seizures, pulmonary thrombosis, deep vein thrombosis, lower extremity edema, pain, and acute peripheral arterial occlusion, and venous thrombosis such as deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral sinus thrombosis, and central retinal vein occlusion.

[0033] A pharmaceutical composition containing the active ingredient of the present invention can be formulated into various forms according to conventional methods to suit each intended use, such as oral formulations like powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injectable formulations like sterile injectable solutions, and can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

[0034] These pharmaceutical compositions may additionally include carriers, excipients, or diluents, and examples of suitable carriers, excipients, or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical compositions of the present invention may additionally include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0035] As a preferred embodiment, solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid formulations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above pharmaceutical composition. In addition, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

[0036] As a preferred embodiment, oral liquid formulations may be exemplified as suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients in addition to commonly used simple diluents such as water and liquid paraffin, such as humectants, sweeteners, flavorings, preservatives, etc.

[0037] As a preferred embodiment, formulations for parenteral administration may be exemplified as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

[0038] The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above-mentioned factors into consideration, and this can be easily determined by a person skilled in the art.

[0039] As a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and body weight, and generally, 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0040] The pharmaceutical composition of the present invention may be administered to a subject via various routes. Any mode of administration may be anticipated, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intradural, or intracerebroventricular injection.

[0041] In the present invention, "administration" means providing a specific substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention may be oral or parenteral through any general route capable of reaching the target tissue. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

[0042] In the present invention, "object" includes, but is not specifically limited to, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, and preferably means mammals, more preferably humans.

[0043] In addition, the health functional food of the present invention can be used in various ways in foods and beverages, etc., that are effective in preventing or improving thrombosis. Foods containing the active ingredients of the present invention include, for example, various types of food, beverages, chewing gum, tea, vitamin complexes, health supplements, etc., and can be used in the form of powder, granules, tablets, capsules, or beverages.

[0044] The active ingredient of the present invention can generally be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition can be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

[0045] In addition to containing the above compound as an essential component in the indicated proportions, the health functional food of the present invention may contain food-grade acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional components.

[0046] Examples of the above natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, as well as common sugars and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0047] As the above flavoring agents, natural flavoring agents such as stevia, thaumatin, rebaudioside A, or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of the above natural carbohydrates is generally about 1 to 20g, preferably about 5 to 12g, per 100ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various nutritional supplements, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the health functional food of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, vegetable beverages, etc. These ingredients may be used independently or in combination. The proportion of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

[0048] The present invention will be explained in more detail below through specific embodiments. The details described in the following embodiments describe a preferred embodiment of the present invention, and the scope of the present invention is not to be interpreted as being limited by the details described in the following embodiments.

[0049] [Example]

[0050] Example 1: Preparation of extract of Japanese maple leaves

[0051] The hot water extract and 70% ethanol extract of the leaves of the Japanese maple used in the present invention were obtained from the Freshwater Biological Resources Bank (FBCC) of the Resource Banking Department at the National Nakdong River Biological Resources Center, and sample information is shown in Table 1. To prepare the extracts, the leaves of the Japanese maple were collected, foreign substances were removed, and they were dried. The dried powder was extracted twice with 10 times its weight in 70% by weight ethanol at room temperature for 24 hours, and the filtrate filtered through a Whatman No. 2 was concentrated using a rotary vacuum evaporator (EYELA, Japan) and freeze-dried to produce the ethanol extract. Meanwhile, 20 times its weight in distilled water was added to the dried powder of the leaves, extracted at 100°C for 1 hour, and the filtrate filtered through a Whatman No. 2 was concentrated using a rotary vacuum evaporator (EYELA, Japan) and freeze-dried to produce the hot water extract. The prepared extract was dissolved in DMSO to a concentration of 20 mg / ml. The sample used is disclosed in the Freshwater Biological Resources Bank.

[0052] [Table 1] Information on the Japanese maple extract used in the present invention

[0053]

[0054] Example 2: Analysis of Total Polyphenol Content in Japanese Maple Leaf Extract

[0055] The total polyphenol content of the Acer palmatum extract prepared in Example 1 was measured, and the results are shown in Table 2. The hot water and ethanol extracts of Acer palmatum leaves exhibited very high total polyphenol content ranging from 59.8 to 60.2 mg / g. Since polyphenol components in plants are associated with various beneficial physiological activities such as antioxidant, antithrombotic, and anti-inflammatory effects, this high total polyphenol content of Acer palmatum leaves suggests the beneficial physiological activities of Acer palmatum. The total polyphenol content was analyzed by adding 50 μl of Folin-Ciocalteau and 100 μl of saturated Na2CO3 solution to 400 μl of the extract solution, leaving it at room temperature for 1 hour, and measuring the absorbance at 725 nm. Tannic acid was used as the standard reagent.

[0056] [Table 2] Analysis of total polyphenol content of the Japanese maple extract used in the present invention

[0057]

[0058] Example 3: Evaluation of the antithrombotic activity of various Japanese maple extracts

[0059] The blood coagulation inhibitory activity of the Acer palmatum extract prepared in Example 1 was evaluated, and the results are shown in Table 3. The blood coagulation inhibitory activity was evaluated according to previously reported methods (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al., 2010. J. Life Science, 20. 922-928), and thrombin time, prothrombin time, and APT time were measured. Commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and the thrombin time, prothrombin time, and APT time measurements were performed according to the following procedure.

[0060] Thrombin Time

[0061] At 37°C, 50 μl of 0.5 U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl2, and 10 μl of sample extracts of various concentrations were mixed in the tube of an Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes. Afterward, 100 μl of plasma was added, and the time until the plasma coagulated was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used as the solvent control instead of the sample. In the case of DMSO, a coagulation time of 25.0 seconds was observed. Thrombin inhibition was expressed as the average value of experiments repeated at least three times, and thrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time upon sample addition by the coagulation time of the solvent control.

[0062] Prothrombin time

[0063] 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solutions of various concentrations were added to the tubes of an Amelung coagulometer KC-1A (Japan). After heating at 37°C for 3 minutes, 130 μl of PT reagent was added, and the time until the plasma coagulated was recorded as the average of three repeated experiments. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used as the solvent control instead of the sample. In the case of DMSO, a coagulation time of 14.5 seconds was observed. Prothrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time upon sample addition by the coagulation time of the solvent control.

[0064] activated Partial Thromboplastin Time (aPTT)

[0065] 100 μl of plasma and 10 μl of sample extracts of various concentrations were added to the tube of an Amelung coagulometer KC-1A (Japan) and heated at 37°C for 3 minutes, after which 50 μl of aPTT reagent (Sigma, ALEXIN TM) was added and incubated again at 37°C for 3 minutes. Afterwards, 50 μl of CaCl2 (35 mM) was added, and the time until plasma coagulation was measured. DMSO was used as the solvent control instead of the sample, and in this case, a coagulation time of 32.5 seconds was observed. The aPTT result was expressed as the average of three repeated experiments, and blood coagulation factor inhibitory activity was expressed as the value obtained by dividing the aPTT time at the time of sample addition by the aPTT time of the solvent control.

[0066] [Table 3] Blood coagulation inhibitory activity of the Japanese maple extract of the present invention

[0067]

[0068] As shown in Table 3, when aspirin (antithrombotic agent brand name: Protect), used as a control, was measured at a concentration of 1.5 mg / ml, the thrombin time, prothrombin time, and APT time were extended by 1.54 times, 1.33 times, and 1.42 times, respectively, confirming that it exhibited excellent antithrombotic activity. Meanwhile, in the case of the Japanese maple extract, the hot water extract of the leaves showed strong anticoagulant activity, and when the thrombin time, prothrombin time, and APT time were measured at a concentration of 2 mg / ml, all showed an effect of extending more than 15 times. When the hot water extract of the Japanese maple leaves was adjusted to a concentration of 1.5 mg / ml, both the thrombin time and APT time were extended by more than 15 times, and the prothrombin time showed an effect of extending 4.52 times. Meanwhile, when the anticoagulant activity of the 70% ethanol extract of Acer palmatum leaves was evaluated at a concentration of 2.0 mg / ml, the thrombin time was extended 7.8 times, and both the prothrombin time and APT time were extended more than 15 times. In both the hot water extract and the ethanol extract of Acer palmatum leaves, the APT time was extended more than 15 times even at a concentration of 1 mg / ml. Therefore, it was determined that the hot water extract of Acer palmatum leaves can effectively inhibit endogenous thrombus formation through potent inhibition of blood coagulation factors and inhibition of thrombin and prothrombin enzymes involved in thrombus formation, and can replace aspirin, which is known to cause side effects such as gastrointestinal disorders.

[0069] Example 4: Evaluation of the antioxidant activity of Japanese maple extract

[0070] Since polyphenol components of natural products are closely related to antioxidant activity and oxidative stress is also closely related to thrombus formation (Wang, L. et al., Sci. Rep. 2017, 7, 12429; Martinez, M. et al., Free Radic. Biol. Med. 2013, 65, 411-418; Bijak, M. et al., Thromb. Res. 2012, 130, e123-e128), the antioxidant activity of Acer palmatum extracts was evaluated and the results are shown in Table 4. At this time, the extracts were dissolved in DMSO (dimethylsulfoxide) and diluted to appropriate concentrations for use in measuring DPPH (1,1-diphenyl-2-picryl hydrazyl) anion radical scavenging activity, ABTS [2,2-azobis(3-ethylbenzo thiazoline-6-sulfonate)] cation radical scavenging activity, nitrite scavenging activity, and reducing power. First, for DPPH scavenging activity, 2 x 10⁻⁶ compounds dissolved in 99.5% ethanol were added to 20 μl of samples diluted to various concentrations. -4 380 μl of M DPPH solution was added and mixed, and the mixture was reacted at 37°C for 30 minutes. Subsequently, the absorbance was measured at 516 nm using a microplate reader (Asys Hitech, Expert96, Asys Co., Austria). DPPH radical scavenging activity was expressed as a percentage of the sample-added group versus the sample-free group. For ABTS scavenging activity, 5 ml of 7 mM ABTS (Sigma Co., USA) and 88 ml of 140 mM potassium persulfate were mixed and exposed to light at room temperature for 16 hours to form ABTS cations. The solution was then diluted with ethanol until the absorbance value at 414 nm was 1.5. 190 ml of the prepared diluted solution was mixed with 10 ml of samples prepared at various concentrations, reacted at room temperature for 6 minutes, and the absorbance was measured at 734 nm. The ABTS radical scavenging activity was then calculated using the following formula.

[0071]

[0072] Meanwhile, for the measurement of nitrite scavenging activity, the sample solution was added to a nitrite solution (1 mM), and 0.1 N HCl was added to adjust the pH to 1.2. After reacting at 37°C for 1 hour, Griess reagent (Sigma Co., USA) was added and mixed. Subsequently, the amount of residual nitrite was determined by measuring the absorbance at 520 nm after leaving the solution at room temperature for 15 minutes. The nitrite scavenging activity (%) was calculated using the following formula.

[0073]

[0074] For the evaluation of reducing power, a modified method of Oyaizu et al. (Ahn et al., 2011. J. Life Sci. 21: 576-583) was used. To 2.5 ml of the sample dissolved in ethanol, 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 10% potassium ferricyanide were added and reacted at 50°C for 20 minutes. Afterward, 2.5 ml of 10% trichloroacetic acid was added to terminate the reaction, and the supernatant was collected by centrifuging at 4000 rpm for 10 minutes. The collected supernatant was diluted twofold with distilled water, mixed with a freshly prepared 0.1% ferric chloride solution at a ratio of 5:1 (v / v), and evaluated by measuring the absorbance at 700 nm.

[0075] [Table 4] Antioxidant activity of the Japanese maple extract of the present invention

[0076]

[0077] As shown in Table 4, vitamin C used as a control exhibited very strong antioxidant activity at a concentration of 0.05 mg / ml, showing 88.4% DPPH anion scavenging activity, 89.7% ABTS cation scavenging activity, 88.9% nitrite scavenging activity, and a reducing power of 2.207. The prepared Japanese maple extracts also showed excellent antioxidant activity, and in particular, the hot water extracts showed excellent active radical scavenging activity and reducing power. Therefore, it is judged that the strong antioxidant activity of the hot water and ethanol extracts of Japanese maple leaves contributes to antithrombotic activity.

[0078] Example 5: Human erythrocyte hemolytic activity of Japanese maple extract

[0079] To evaluate the potential for acute toxicity of the Acer palmatum extract, human erythrocyte hemolytic activity was assessed. Hemolytic activity was evaluated according to previous reports (Son Ho-yong, 2014 · Korean J. Microbiol. Biotechnol. 42: 285-292). Simply put, 100 μl of human erythrocytes washed three times with PBS were placed in a 96-well microplate, 100 μl of sample solutions of various concentrations were added, and the mixture was reacted at 37°C for 30 minutes. Afterward, the reaction mixture was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate. The degree of hemoglobin leakage due to hemolysis was then measured at 414 nm. DMSO (2%) was used as the solvent control for the samples, and Triton X-100 (1 mg / ml) was used as the experimental control for erythrocyte hemolysis. Hemolytic activity was calculated using the following formula.

[0080]

[0081] As shown in Table 5, DMSO and water used as controls showed no hemolytic activity, and it was confirmed that Triton X-100 lysed 100% of red blood cells at a concentration of 1 mg / ml. In addition, it was confirmed that amphotericin B, which is used as an anticancer and antifungal agent, lysed more than 50% of red blood cells at a concentration of 0.025 mg / ml.

[0082] Meanwhile, the hot water extract of the Japanese maple leaves did not exhibit erythrocyte hemolysis up to a concentration of 1 mg / ml, confirming that it has no acute toxicity or erythrocyte hemolytic activity. These results suggest that the hot water extract of the Japanese maple leaves of the present invention exhibits antithrombotic activity without erythrocyte hemolytic activity, and thus can safely replace antithrombotic agents that cause side effects, such as aspirin, in the future.

[0083] [Table 5] Human erythrocyte hemolytic activity of the Japanese maple extract of the present invention

[0084]

[0085] Example 6: Evaluation of Plasma, Acid, and Thermal Stability of Japanese Maple Leaf Extract

[0086] Plasma stability, heat stability, and acid stability regarding antithrombotic activity were confirmed for the hot water extract of the Japanese maple leaves obtained in Example 1 above. The hot water extract of the Japanese maple leaves did not show a decrease in blood coagulation inhibitory activity even when heat-treated at 100°C for 1 hour, treated at pH 2 (0.01M HCl) for 1 hour, or treated in plasma for 1 hour. Therefore, it was confirmed that the hot water extract of the Japanese maple leaves contains antithrombotic active substances with acid resistance and heat resistance, and thus confirmed that there is a high possibility of practical use as an antithrombotic food or pharmaceutical material in the future.

Claims

Claim 1 Japanese maple ( Acer pseudosieboldianum A pharmaceutical composition for the prevention or treatment of thrombosis containing an extract as an active ingredient. Claim 2 In Article 1, the above-mentioned Japanese maple ( Acer pseudosieboldianum ) extract is Japanese maple ( Acer pseudosieboldianum A pharmaceutical composition characterized by being a hot water extract or ethanol extract of a leaf. Claim 3 A health functional food for the prevention or improvement of thrombosis containing the active ingredient described in Paragraph 1 or 2.

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