Pharmaceutical composition comprising the extract of barbarea orthoceras ledeb 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 *Barbarea orthoceras Ledeb*, 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 the whole plant of *Barbarea orthoceras Ledeb* having potent thrombin inhibitory and blood coagulation factor inhibitory activities. The extract of *Barbarea orthoceras* (Ledeb) 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 wild mustard ( Barbarea orthoceras The invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an extract of *Ledeb*, and more specifically, to *Ledeb* having potent thrombin inhibitory and blood coagulation factor inhibitory activity. Barbarea orthoceras This 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 the whole plant of Ledeb 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, Nado-naengi ( Barbarea orthocerasLedeb is a biennial plant belonging to the Brassicaceae family of the Papaverales order of dicotyledonous plants. It grows mainly along streams or in wetlands in Korea, Japan, China, Eastern Siberia, and North America. The stems stand upright and branch out, reaching a height of about 1 meter. Leaves emerging from the roots grow in clusters, have long petioles, are inverted egg-shaped, and are deeply pinnately divided. The seeds are oval-shaped and surrounded by narrow wings. Among the shepherd's purse species, *Lespedeza bicolor* is relatively tall and slender, with multiple stems growing upwards. The leaves are edible, either eaten raw in salads or blanched. In folk medicine, similar to the common shepherd's purse, it is used for hemostatic, diuretic, constipation relief, detoxification, anti-inflammatory, and antibacterial effects, and is known to be effective for menstrual irregularities and symptoms of uterine bleeding.

[0005] Studies related to *Barbarea* include the distribution characteristics of naturalized plants in Dadohae Marine National Park (Kim Ha-song et al., 2010, Journal of the Korean Society of Plant Resources 23: 187-196) and *Glucosinolates, flea beetle resistance, and leaf pubescence as taxonomic characters in the genus Barbarea (Agerbirk N et al., 2003. Phytochemistry 63: 69-80); there has been almost no research on this, and in particular, strong antithrombotic activity of *Barbarea* has not been reported to date.

[0006] Regarding patents related to *Nadogaegi* (a type of edible edible plant), while no domestic patents are known, relevant documents include U.S. Published Patent No. 2003-0165557 "Food, medicine and method relating to Bifidobacterium proliferation promoting action, allergy preventive action and human cholesterol lowering action"; U.S. Registered Patent No. 7838045 "Anti-inflammatory activity of phenethylisothiocyanate (PEITC) and the Barbarea verna seed preparation containing this compound"; U.S. Published Patent No. 2024-0389603 "USE OF AN EXTRACT OF PART OF A PLANT FOR STIMULATING THE DEFENCES OF PLANTS AGAINST PATHOGENS, ASSOCIATED COMPOSITION AND METHODS"; and Japanese Published Patent No. 2013-241467 "FOOD HAVING HUMAN SERUM HYPOCHOLESTEROLEMIC ACTIVITY, AND PROPHYLACTIC OR THERAPEUTIC AGENT FOR Only "HUMAN HYPERCHOLESTEROLEMIA OR HUMAN ARTERIOSCLEROSIS", International Patent WO1999-047006 "FOODS, MEDICAL TREATMENTS AND METHOD RELATING TO EFFECTS OF PROMOTING THE GROWTH OF LACTOBACILLUS BIFIDUS, PREVENTING ALLERGY AND LOWERING HUMAN CHOLESTEROL LEVEL" is known, and to date, no potent antithrombotic activity of *Nasturtium vitis-idaea* has been reported. Prior art literature

[0007] (Patent Document 0001) JP 2013-241467 A 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 a type of cress ( Barbarea orthoceras 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 an extract of Ledeb as an active ingredient. means of solving the problem

[0009] In order to solve the above problems, the present invention relates to a type of cress ( Barbarea orthoceras A pharmaceutical composition for the prevention or treatment of thrombosis containing an extract of Ledeb as an active ingredient is provided.

[0010] The above-mentioned wild mustard ( Barbarea orthoceras Ledeb) extract is a type of cress ( Barbarea orthoceras It is preferable that it be a hot water extract of the whole plant (Ledeb).

[0011] In addition, the present invention relates to a type of wild mustard ( Barbarea orthoceras A blood coagulation inhibitor containing an extract of Ledeb as an active ingredient is provided.

[0012] The above-mentioned wild mustard ( Barbarea orthoceras Ledeb) extract is a type of cress ( Barbarea orthoceras It is preferable that it be a hot water extract of the whole plant (Ledeb).

[0013] In addition, the present invention relates to a type of wild mustard ( Barbarea orthoceras A health functional food for the prevention or improvement of thrombosis is provided, containing an extract of Ledeb as an active ingredient.

[0014] The above-mentioned wild mustard ( Barbarea orthoceras Ledeb) extract is a type of cress ( Barbarea orthoceras It is preferable that it be a hot water extract of the whole plant (Ledeb). Effects of the invention

[0015] The pharmaceutical composition for the prevention or treatment of thrombosis and the active ingredient of a health functional food according to the present invention, *Nasturtium officinale* ( Barbarea orthocerasLedeb extract exhibits excellent antithrombotic activity through strong inhibition of thrombus-related enzymes thrombin and blood coagulation factors, while simultaneously showing no hemolytic activity against human red blood cells, having excellent thermal stability, and showing no loss of inhibitory effects on blood coagulation factors or thrombus-related enzymes even under acidic conditions of pH 2 and in plasma; therefore, it is expected to be used for the prevention and treatment of thrombosis, such as ischemic stroke and hemorrhagic stroke, by improving 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, it is a very 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 certain method and evaluated their antithrombotic and antioxidant activities, and confirmed strong anticoagulant activity in the extract of *Nasturtium vitis-idaea*. They recovered the hot water extract of *Nasturtium vitis-idaea* 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. Therefore, 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 *Nasturtium officinale* to develop pharmaceutical compositions and health functional foods for the prevention, treatment, or improvement of thrombosis using *Nasturtium officinale*, 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 extract of the whole plant of *Nasturtium officinale* exhibits 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 the hawk's-eye ( Barbarea orthoceras A pharmaceutical composition for the prevention or treatment of thrombosis containing an extract of Ledeb as an active ingredient is provided.

[0020] The above-mentioned wild mustard ( Barbarea orthoceras Ledeb) extract is a type of cress ( Barbarea orthoceras It is preferable that it be a hot water extract of the whole plant (Ledeb).

[0021] In addition, the present invention relates to a type of wild mustard ( Barbarea orthoceras A blood coagulation inhibitor containing an extract of Ledeb as an active ingredient is provided.

[0022] The above-mentioned wild mustard ( Barbarea orthoceras Ledeb) extract is a type of cress ( Barbarea orthoceras It is preferable that it be a hot water extract of the whole plant (Ledeb).

[0023] In addition, the present invention relates to a type of wild mustard ( Barbarea orthoceras A health functional food for the prevention or improvement of thrombosis is provided, containing an extract of Ledeb as an active ingredient.

[0024] The above-mentioned wild mustard ( Barbarea orthoceras Ledeb) extract is a type of cress ( Barbarea orthocerasIt is preferable that it be a hot water extract of the whole plant (Ledeb).

[0025] In the following, the Japanese radish of the present invention ( Barbarea orthoceras The manufacturing method and efficacy experiments of the Ledeb extract are explained in more detail.

[0026] The present invention comprises the steps of: recovering the whole plant of *Nasturtium vitis-idaea*; extracting the recovered whole plant with hot water and 70% ethanol to prepare an extract; evaluating the antithrombotic activity of the extract and evaluating the acute toxicity of the extract.

[0027] "Nado-naengi (included in the composition of the present invention Barbarea orthoceras The “Ledeb) extract” can be obtained by the steps of extracting the mature whole plant of the Japanese knotweed 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 Japanese daisy ( Barbarea orthocerasExtracts of *Ledeb* were prepared, and their 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 *Ledeb* inhibited thrombin formation by exhibiting stronger thrombin inhibition and stronger inhibition of blood coagulation factors than aspirin. Furthermore, the *Ledeb* extract showed no hemolytic activity against human red blood cells, suggesting that it can replace existing antithrombotic agents such as aspirin, which have a high risk of side effects.

[0031] The wild mustard of the present invention ( Barbarea orthoceras Ledeb 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 Cardamine pratensis leaves

[0051] The hot water extract and 70% ethanol extract of *Nasturtium officinale* used in this 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 whole plant of *Nasturtium officinale* was harvested, impurities were removed, and it was dried. The dried powder was extracted twice with 10 times its weight in 70% by weight ethanol at room temperature for 24 hours. The filtrate was filtered through a Whatman No. 2 filter, concentrated using a rotary vacuum evaporator (EYELA, Japan), and freeze-dried to prepare the ethanol extract. Meanwhile, 20 times its weight in distilled water was added to the dried powder of the whole plant, extracted at 100°C for 1 hour, filtered through a Whatman No. 2 filter, concentrated using a rotary vacuum evaporator (EYELA, Japan), and freeze-dried to prepare the hot water extract. The prepared extracts were dissolved in DMSO to a concentration of 20 mg / ml. The samples for use are announced on the Freshwater Biological Resources Bank website.

[0052] [Table 1] Information on the *Nasturtium officinale* extract used in the present invention

[0053]

[0054] Example 2: Analysis of Total Polyphenol Content of Cardamine Extract

[0055] The total polyphenol content of the *Nasturtium officinale* extract prepared in Example 1 was measured, and the results are shown in Table 2. The hot water and ethanol extracts of *Nasturtium officinale* exhibited high total polyphenol content of 54.2 mg / ml and 39.8 mg / g, respectively. Unlike typical plant extracts, the hot water extract showed a higher polyphenol content than the 70% ethanol extract. 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 *Nasturtium officinale* extracts suggests the beneficial physiological activities of *Nasturtium officinale*. 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 *Nasturtium officinale* extract used in the present invention

[0057]

[0058] Example 3: Evaluation of Antithrombotic Activity of Cardamine Extract

[0059] The blood coagulation inhibitory activity of the *Nasturtium officinale* 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, ALEXINTM ) 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 *Nasturtium officinale* 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 *Nasturtium officinale* extract, the hot water extract showed strong anticoagulant activity; when the thrombin time and APT time were measured at a concentration of 2 mg / ml, both showed an effect of extending by more than 15 times, and the prothrombin time was extended by 1.36 times. On the other hand, the 70% ethanol extract showed relatively weak anticoagulant activity compared to the hot water extract. In particular, when the hot water extract of *Nadogae* was adjusted to a concentration of 1.5 mg / ml, the thrombin time, prothrombin time, and APT time were extended by 1.55 times, 1.12 times, and 1.55 times, respectively, exhibiting anticoagulant activity similar to that of aspirin. Therefore, it was determined that the hot water extract of *Nadogae* can effectively inhibit endogenous thrombus formation through potent inhibition of blood coagulation factors and inhibition of thrombin 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 Antioxidant Activity of Cardamine 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 Nasturtium officinale 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 *Nasturtium officinale* 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 *Nasturtium officinale* extract also showed excellent antioxidant activity, and in particular, the hot water extract showed significantly superior active radical scavenging activity and reducing power compared to the 70% ethanol extract. Therefore, it is judged that the strong antioxidant activity of the *Nasturtium officinale* hot water extract contributes to antithrombotic activity.

[0078] Example 5: Human erythrocyte hemolytic activity of *Nasturtium officinale* extract

[0079] To evaluate the potential for acute toxicity of the *Nasturtium officinale* extract, human erythrocyte hemolytic activity was evaluated. 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 and ethanol extracts of *Nasturtium officinale* did not exhibit erythrocyte hemolysis up to a concentration of 1 mg / ml, confirming that there was no acute toxicity or erythrocyte hemolytic activity. These results suggest that the hot water extract of *Nasturtium officinale* 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 *Nasturtium officinale* extract of the present invention

[0084]

[0085] Example 6: Evaluation of plasma, acid, and thermal stability of Cardamine lucidum leaf extract

[0086] Plasma stability, thermal stability, and acid stability regarding antithrombotic activity were confirmed for the hot water extract of *Nasturtium officinale* obtained in Example 1 above. The hot water extract of *Nasturtium officinale* 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 *Nasturtium officinale* contains antithrombotic active substances with acid resistance and heat resistance, confirming that it has high potential for practical use as an antithrombotic food and pharmaceutical material in the future.

Claims

Claim 1 Nado-naeng-i ( Barbarea orthoceras A pharmaceutical composition for the prevention or treatment of thrombosis containing an extract of Ledeb as an active ingredient. Claim 2 In claim 1, the above-mentioned Japanese daisy ( Barbarea orthoceras Ledeb) extract is a type of cress ( Barbarea orthoceras A pharmaceutical composition characterized by being a hot water extract of the whole plant (Ledeb). 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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