Pharmaceutical composition comprising the extract of lindera glauca blume 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 Lindera glauca Blume, 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 of Lindera glauca Blume leaves having potent thrombin inhibitory and blood coagulation factor inhibitory activity. The extract of *Lindera glaucaBlume*, 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 the Camellia japonica tree ( Lindera glauca The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an extract of *Blume* (Blume), and more specifically, to *Gamtaenamu* (which has potent thrombin inhibitory and blood coagulation factor inhibitory activities). Lindera glauca 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 of Blume 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 spindle tree is a deciduous shrub belonging to the genus Lindera in the family Lauraceae, typically growing up to 7 meters tall. Its leaves are stiff and turn grayish when dry. The flowers are dioecious and bloom in April in umbel inflorescences. The flowers are yellow, and the fruit ripens in September and is bean-shaped. It thrives in sunny forests. The fruit of the Japanese spindle tree is called Yeonsumok or Sanhocho and is used as a medicine in traditional Korean medicine, while the leaves and fruit are consumed as tea. In folk medicine, the fruit is used to improve blood circulation, stabilize blood pressure, promote cardiovascular health, reduce cholesterol, treat stroke, detoxify, and provide hemostatic effects; it is also used for the prevention and treatment of diabetes.

[0005] Studies related to Lindera glauca include: anti-inflammatory and analgesic effects of the ethyl acetate fraction of Lindera glauca fruit (Kim Jong-soo et al., 2022 Journal of the Korean Society of Pharmacognosy 53: 49-56), evaluation of the physiological activity of Lindera glauca extracts as cosmetic materials from leaves and stems (Park Ina, 2023, Master's thesis, Chonnam National University), anti-inflammatory activity of Lindera glauca (Kim Jong-soo, 2013, Master's thesis, Woosuk University), study on anti-inflammatory components of Lindera glauca using LC-QTOF MS / MS-based cell extraction method (Kim Ye-bin, 2023, Master's thesis, Korea University), antioxidant and anti-inflammatory activities of leaves and branches (Antioxidant and Anti-Inflammatory Activities of Lindera glauca Extracts, Park YN et al., 2024, Microb Physiol. 34: 78-87), and a study on the antioxidant and inhibitory effects of human colon cancer cell proliferation by ethanol extracts of Lindera glauca (fruit) (Kim Ye-eon et al., 2015. Journal of the Korean Society of Food Science and Nutrition 44: 635-640), Effects of Lindera glauca on Amyloid β 1-42-induced memory decline in mice (Kim Yun-na, 2016. Master's thesis, Kyung Hee University), Growth inhibition and apoptosis induction of colon cancer cells by Lindera glauca root extract (Kim Ye-eon, 2021. J. Korean Society of Food Culture 36: 235-245), Antioxidant activity of Lindera glauca fruit (Flavonoids from the Heartwood of Lindera glauca Blume and Their LDL-antioxidative Activities, Heo Gyu-won, 2012. Master's thesis, Kyung Hee University; Lindera glauca (Siebold et Zucc.) Blume Stem Extracts Protect Against tert-Butyl Hydroperoxide-Induced Oxidative Stress. Kim YS et al., 2019. J Med Food.Although anti-obesity activity (Park, 2016, Ph.D. dissertation, Yonsei University) and other effects are known, no strong antithrombotic activity of *Gamtae* has been reported to date.

[0006] Patents related to the Japanese knotweed include Korean registered patent No. 10-2175732, which discloses an antiviral composition containing a compound derived from the Japanese knotweed as an active ingredient; Korean registered patent No. 10-0909768, which discloses a cosmetic composition for preventing skin aging containing a Japanese knotweed extract as an active ingredient; Korean registered patent No. 10-2351363, which discloses an anti-allergic composition using water-soluble mastic gum or a Japanese knotweed extract; Korean registered patent No. 10-2328824, which discloses a composition for promoting hair growth or inhibiting hair loss using water-soluble mastic gum and a Japanese knotweed extract; and Korean registered patent No. 10-2305520, which discloses a red chili paste containing *Sophora triphylla*, *Japanese knotweed*, and *Euryale ferox*. Additionally, Korean published patent No. 10-2021-0103676 discloses a composition for the prevention, treatment, or improvement of inflammatory diseases containing an ethyl acetate-soluble fraction of the Japanese knotweed as an active ingredient. However, to date, no strong antithrombotic activity of the Japanese knotweed has been reported. Prior art literature

[0007] Evaluation of the physiological activity of Lindera glauca extracts. Park, Ina, 2023. Master's thesis, Chonnam National University. Antioxidant and Anti-Inflammatory Activities of Lindera glauca Extracts, Park, YN et al., 2024. Microb Physiol. 34: 78-87 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 sea tangle tree ( Lindera glauca The 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 Blume extract as an active ingredient. means of solving the problem

[0009] In order to solve the above-mentioned problems, the present invention relates to a sea tangle tree ( Lindera glauca A pharmaceutical composition for the prevention or treatment of thrombosis containing Blume extract as an active ingredient is provided.

[0010] The above Camellia tree ( Lindera glauca Blume) extract is a type of Camellia japonica ( Lindera glauca It is preferable that it be a hot water extract of Blume leaves.

[0011] In addition, the present invention relates to a sea tangle tree ( Lindera glauca A blood coagulation inhibitor containing Blume extract as an active ingredient is provided.

[0012] The above Camellia tree ( Lindera glauca Blume) extract is a type of Camellia japonica ( Lindera glauca It is preferable that it be a hot water extract of Blume leaves.

[0013] In addition, the present invention relates to a sea tangle tree ( Lindera glauca A health functional food for preventing or improving thrombosis is provided, containing Blume extract as an active ingredient.

[0014] The above Camellia tree ( Lindera glauca Blume) extract is a type of Camellia japonica ( Lindera glauca It is preferable that it be a hot water extract of Blume leaves. Effects of the invention

[0015] Camellia japonica as an active ingredient in the pharmaceutical composition for the prevention or treatment of thrombosis and health functional food of the present invention ( Lindera glaucaThe extract of Blume 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 in plasma. Therefore, it is expected to be useful 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, it is a highly useful invention 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 *Gamtae* (a type of tree), and recovered the hot water extract of *Gamtae* leaves as an antithrombotic active component. By confirming that the extract exhibits excellent thermal and acid stability while showing no hemolytic activity against human red blood cells, they intended to utilize the extract as a pharmaceutical composition and health functional food for the prevention or treatment / improvement of thrombosis.

[0018] Specifically, in order to develop pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of thrombosis using *Gamtae* (a type of tree) which is known in folk medicine to be effective for various diseases of the vascular and circulatory systems, digestive systems, and metabolic systems, the inventors prepared various solvent extracts from the leaves and branches of *Gamtae* and evaluated the antithrombotic activity of these extracts by assessing direct thrombin inhibition (Thrombin Time), prothrombin inhibition (Prothrombin Time), and activated partial thromboplastin time (aPTT) against human thrombin, *Gamtae* ( Lindera glauca It was confirmed that the hot water extract of Blume leaves possesses very potent anticoagulant activity. Furthermore, it was confirmed that the extract does not exhibit hemolytic activity against human red blood cells, thereby verifying its feasibility for practical use.

[0019] Therefore, the present invention ( Lindera glauca A pharmaceutical composition for the prevention or treatment of thrombosis containing Blume extract as an active ingredient is provided.

[0020] The above Camellia tree ( Lindera glauca Blume) extract is a type of Camellia japonica ( Lindera glauca It is preferable that it be a hot water extract of Blume leaves.

[0021] In addition, the present invention relates to a sea tangle tree ( Lindera glauca A blood coagulation inhibitor containing Blume extract as an active ingredient is provided.

[0022] The above Camellia tree ( Lindera glauca Blume) extract is a type of Camellia japonica ( Lindera glauca It is preferable that it be a hot water extract of Blume leaves.

[0023] In addition, the present invention relates to a sea tangle tree ( Lindera glauca A health functional food for preventing or improving thrombosis is provided, containing Blume extract as an active ingredient.

[0024] The above Camellia tree ( Lindera glaucaBlume) extract is a type of Camellia japonica ( Lindera glauca It is preferable that it be a hot water extract of Blume leaves.

[0025] In the following, the sea tangle tree of the present invention ( Lindera glauca The manufacturing method and efficacy experiments of Blume extract are explained in more detail.

[0026] The present invention relates to the Camellia japonica tree ( Lindera glauca The method includes the steps of: recovering leaves and branches of Blume; preparing an extract by extracting the recovered leaves and branches with hot water and 70% ethanol; evaluating the antithrombotic activity of the extract and evaluating the acute toxicity of the extract.

[0027] "Gamtae tree (included in the composition of the present invention Lindera glauca The “Blume) extract” can be obtained by the steps of extracting the leaves of a mature Camellia japonica with hot water, 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 Camellia japonica tree ( Lindera glaucaExtracts of *Blume* were prepared and adjusted to a concentration of 2 mg / ml to measure thrombin time, prothrombin time, and APT time. As a result, it was confirmed that the hot water extract of *Blume* leaves inhibited thrombus formation by exhibiting thrombin inhibition similar to aspirin and strong inhibition of blood coagulation factors. Furthermore, since the *Blume* extract showed no hemolytic activity against human red blood cells, it is suggested that it can replace existing antithrombotic agents such as aspirin, which have high concerns regarding side effects.

[0031] The sea tangle of the present invention ( Lindera glauca Blume 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 extracts from leaves and stems of *Gamtae* tree

[0051] The hot water extract and 70% ethanol extract of the leaves and stems of *Gamtaenamu* 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 and stems of *Gamtaenamu* 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 filter (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 and stems, and the extract was extracted at 100°C for 1 hour. The filtrate filtered through a filter (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 Ecklonia cava extract used in the present invention

[0053]

[0054] Example 2: Analysis of Total Polyphenol Content in Leaf and Branch Extracts of *Gamtaenia cava*

[0055] The total polyphenol content of the *Gamtae* extract prepared in Example 1 was measured, and the results are shown in Table 2. The extracts from the leaves and branches of *Gamtae* exhibited high total polyphenol content ranging from 53.7 to 56.2 mg / g, regardless of the extraction solvent. Since polyphenol components in plants are associated with various beneficial physiological activities such as antioxidant, antithrombotic, and anti-inflammatory effects, the high total polyphenol content of these leaves and branches suggests the beneficial physiological activities of *Gamtae*. 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 Ecklonia cava extract used in the present invention

[0057]

[0058] Example 3: Evaluation of the antithrombotic activity of various Ecklonia cava extracts

[0059] The blood coagulation inhibitory activity of the *Ecklonia cava* extract prepared in Example 1 was evaluated, and the results are shown in Table 3. The blood coagulation inhibitory activity was evaluated in accordance with 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 Ecklonia cava 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, 1.33, and 1.42 times, respectively, confirming that it exhibited excellent antithrombotic activity. Meanwhile, regarding the extracts of *Gamtae*, strong anticoagulant activity was observed only in the hot water extract of the leaves; when thrombin time and APT time were measured at a concentration of 2 mg / ml, the effect of extending thrombus formation by more than 15 times was observed. In the case of prothrombin time, which is associated with exogenous thrombus formation, a 1.27-fold extension effect was observed. Therefore, it was determined that the hot water extract of *Gamtae* leaves can effectively inhibit endogenous thrombus formation through the inhibition of blood coagulation factors and thrombin, and can replace aspirin, which is known to cause side effects such as gastrointestinal disorders.

[0069] Example 4: Evaluation of the antioxidant activity of Ecklonia cava 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 Ecklonia cava 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 Ecklonia cava extract of the present invention

[0076]

[0077] As shown in Table 4, vitamin C used as a control exhibited very strong antioxidant activity, 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 at a concentration of 0.05 mg / ml. The prepared Ecklonia cava extract also showed excellent antioxidant activity, with the ethanol extract of the branches showing the most excellent active radical scavenging activity and reducing power. However, in the case of the hot water extract of Ecklonia cava leaves of the present invention, excellent antioxidant activity was confirmed by showing 58.7% DPPH anion scavenging activity at a concentration of 0.125 mg / ml, 71.9% ABTS cation scavenging activity at a concentration of 0.063 mg / ml, a reducing power of 1.471 at a concentration of 0.5 mg / ml, and 72.4% nitrite scavenging activity at a concentration of 0.05 mg / ml. Therefore, it is believed that the potent antioxidant activity of the hot water extract of *Gamtaenamu* leaves contributes to antithrombotic activity.

[0078] Example 5: Human erythrocyte hemolytic activity of Ecklonia cava extract

[0079] To evaluate the potential for acute toxicity of the Camellia japonica 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 *Gamtae* 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. However, the hot water extract of *Gamtae* branches showed very strong hemolytic activity, indicating that further research is required regarding the continuous consumption of *Gamtae* branches. These results suggest that the hot water extract of *Gamtae* leaves of the present invention exhibits antithrombotic activity without erythrocyte hemolytic activity, indicating that it can safely replace antithrombotic agents with side effects, such as aspirin, in the future.

[0083] [Table 5] Human erythrocyte hemolytic activity of the *Gamtae* extract of the present invention

[0084]

[0085] Example 6: Evaluation of Plasma, Acid, and Thermal Stability of Camellia Extract

[0086] Plasma stability, heat stability, and acid stability regarding antithrombotic activity were confirmed for the hot water extract of *Gamtae* leaves obtained in Example 1 above. The hot water extract of *Gamtae* 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 *Gamtae* 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 Camellia tree Lindera glauca A pharmaceutical composition for the prevention or treatment of thrombosis containing Blume extract as an active ingredient. Claim 2 In Article 1, the above-mentioned Camellia tree ( Lindera glauca Blume) extract is a type of Camellia japonica ( Lindera glauca A pharmaceutical composition characterized by being a hot water extract of Blume leaves. 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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