Pharmaceutical composition comprising the extract of ammammia multiflora 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 Ammannia multiflora, a domestic freshwater plant, as an active ingredient. More specifically, the 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 70% ethanol extract of the above-ground parts of Ammannia multiflora as an active ingredient, which has strong thrombin inhibitory activity and prothrombin and blood coagulation factor inhibitory activity. The extract of *Lysimachia japonica*, 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 strong antithrombotic activity by inhibiting thrombus-forming enzymes such as thrombin, prothrombin, 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-forming 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 domestic freshwater plant, *Lysimachia japonica* ( Ammannia multiflora The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an extract as an active ingredient, 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 a 70% ethanol extract of the above-ground parts of *Lysimachia japonica* as an active ingredient, which has strong thrombin inhibitory activity and prothrombin and blood coagulation factor inhibitory activity. 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, small lotus ( Ammannia multiflora ) is an annual plant belonging to the family Lythraceae in the order Myrtales of dicotyledonous plants. It is native to Korea and is mainly found in sunny wetlands in the central region. Lythraceae ( Lythrum anceps Although similar in name and form to *Mulberry*, *Mulberry* differs in that it is an annual plant, whereas *Mulberry* is a perennial. *Mulberry* branches out in a cross shape from the stem, and its leaves are opposite, spreading horizontally with the base clasping the main stem. Flowers bloom from September to October, and it bears capsules. In particular, it is characterized by a square cross-section of the stem. In other countries, it is also called Many-flowered ammannia.

[0005] Small-flowered ( Ammannia multiflora There has been almost no research on ), and studies on the antimicrobial activity (Upadhyay, Harish C et al., 2012. Bioenhancing and antimycobacterial agents from Ammannia multiflora. Planta medica 78, 79-81), hyperglycemic inhibitory effect (Upadhyay, Harish C et al., 2012. Antihyperglycemic agents from Ammannia multiflora. Natural product communications 7, 1934578X1200700), and antioxidant activity (Jayasingha, Jayasingha Arachchige Chathuranga Chanaka et al., 2023. Antioxidant activities of plant extracts (Ammannia multiflora, Ammannia coccinea, and Salix gracilistyla activate the Nrf2 / HO-1 signaling pathway. Applied sciences 13, 6701; Jayasingha, Jayasingha Arachchige Chathuranga Chanaka et al., Only the following are known: 2023. Free radical scavenging and antioxidant activities of water extracts from Amannia multiflora, Amannia coccinea, Salix gracilistyla inhabiting along the Nakdong river. 2022 Autumn Conference of the Korean Society of Plant Resources 31)

[0006] On the other hand, the alien weed American borer ( Ammannia coccineaResearch on ) is relatively well known, and studies on vegetation (Hwang Sun-min et al., 2014. Spreading and distribution of exotic weed Ammannia coccinea in Korea, Weed & Turfgrass Science 3, 292-298), and the effects of competition by American buttercup on rice growth and yield (Shin Hyang-il et al., 2008. Weed & Turfgrass Science 25-31) are known.

[0007] However, to date, no research reports or patents regarding the potent antithrombotic activity of *Lysimachia japonica* are known. Prior art literature

[0008] Antimicrobial activity of *Ammannia multiflora* (Upadhyay, Harish C et al., 2012. Bioenhancing and antimycobacterial agents from Ammannia multiflora. Planta medica 78, 79-81) Antihyperglycemic effect (Upadhyay, Harish C et al., 2012. Antihyperglycemic agents from Ammannia multiflora. Natural product communications 7, 1934578X1200700) Antioxidant activity (Jayasingha, Jayasingha Arachchige Chathuranga Chanaka et al., 2023. Antioxidant activities of plant extracts (Ammannia multiflora, Ammannia coccinea, and Salix gracilistyla activate the Nrf2 / HO-1 signaling pathway. Applied sciences 13, 6701; Jayasingha, Jayasingha Arachchige Chathuranga Chanaka et al., 2023. Free radical scavenging and antioxidant Activities of water extracts from Amannia multiflora, Amannia coccinea, and Salix gracilistyla inhabiting along the Nakdong River. 2022 Autumn Conference of the Korean Society of Plant Resources 31) The problem to be solved

[0009] 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 small lotus ( Ammannia multiflora The purpose is to provide a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through inhibition of blood coagulation using an extract as an active ingredient. means of solving the problem

[0010] In order to solve the above problems, the present invention relates to a small lotus ( Ammannia multiflora Provides a pharmaceutical composition for the prevention or treatment of thrombosis containing an extract as an active ingredient.

[0011] In addition, the present invention relates to the small lotus ( Ammannia multiflora Provides a health functional food for the prevention or improvement of thrombosis containing an extract as an active ingredient. Effects of the invention

[0012] The extract of *Lysimachia japonica*, 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 strong antithrombotic activity by inhibiting thrombus-forming enzymes such as thrombin, prothrombin, 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-forming 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. Specific details for implementing the invention

[0013] The present invention will be described in detail below.

[0014] The inventors of the present invention prepared various solvent extracts using the above-ground parts of *Buddleja davurica*, a native Korean freshwater plant, to test the antithrombotic efficacy of the plant. They evaluated the antithrombotic and antioxidant activities of these extracts and confirmed strong anticoagulant activity in the 70% ethanol extract of the above-ground parts of *Buddleja davurica*, recovering it 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.

[0015] Specifically, in order to develop pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of thrombosis using *Buddleja davurica*, which is known in folk medicine to be effective for various diseases such as hemostasis, the digestive system, and the metabolic system, the inventors prepared various solvent extracts from the above-ground parts of *Buddleja davurica* and 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 70% ethanol extract of the above-ground parts of *Buddleja davurica* has very strong anticoagulant activity.

[0016] Therefore, the present invention relates to the small lotus (Ammannia multiflora Provides a pharmaceutical composition for the prevention or treatment of thrombosis containing an extract as an active ingredient.

[0017] The above small lotus (Ammannia multiflora ) extract is small lotus (Ammannia multiflora It is preferable that the above-ground part be a 70% ethanol extract.

[0018] In addition, the present invention relates to the small lotus (Ammannia multiflora Provides a health functional food for the prevention or improvement of thrombotic diseases containing an extract as an active ingredient.

[0019] The above small lotus (Ammannia multiflora ) extract is small lotus (Ammannia multiflora It is preferable that the above-ground part be a 70% ethanol extract.

[0020] In the following, the small lotus of the present invention (Ammannia multiflora The manufacturing method and efficacy experiments of the extract are explained in more detail.

[0021] The present invention relates to the small lotus (Ammannia multiflora The method includes the steps of: recovering the above-ground parts of the plant; preparing an extract by extracting with 70% ethanol; and evaluating antithrombotic activity.

[0022] The organic solvent used for extraction 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 70% ethanol extraction is most preferable.

[0023] The above 70% ethanol 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.

[0024] As a preferred embodiment, the small lotus included in the composition of the present invention (Ammannia multiflora The extract can be obtained by the steps of extracting the above-ground parts of mature Lysimachia japonica with 70% ethanol, filtering the extract using a filter mesh of 0.06 mm or less, and concentrating it under reduced pressure.

[0025] In the present invention, the small lotus (Ammannia multiflora Extracts 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 70% ethanol extract inhibited thrombin formation by exhibiting stronger thrombin inhibition, prothrombin inhibition, and blood coagulation factor inhibition than aspirin.

[0026] The small lotus of the present invention (Ammannia multiflora 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The present invention will be explained in more detail below through examples. The following examples are merely preferred embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[0044] [Example]

[0045] Example 1: Preparation of extract of Lysimachia japonica

[0046] The hot water and ethanol extracts of the above-ground parts of *Lysimachia japonica* 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 above-ground parts of *Lysimachia japonica* were harvested, foreign substances were removed, and they were dried. The hot water extracts were prepared by adding 20 times the weight of hot water to the dried powder, extracting the mixture, filtering it through a Whatman No. 2 filter, concentrating the filtrate using a rotary vacuum evaporator (EYELA, Japan), and freeze-drying it. Meanwhile, the ethanol extracts were prepared by adding 10 times the weight of 70% ethanol to the dried powder of *Lysimachia japonica*, extracting the mixture twice using a Whatman No. 2 filter, concentrating the filtrate using a rotary vacuum evaporator (EYELA, Japan), and freeze-drying it. Each extract was prepared by dissolving it in DMSO to a concentration of 20 mg / ml. The samples for use are announced on the Freshwater Biological Resources Bank website.

[0047] [Table 1] Information on the freshwater plant Lysimachia japonica

[0048]

[0049] Example 2: Analysis of Total Polyphenol Content of Small-flowered Buddleja Extract

[0050] The total polyphenol content of the hot water and ethanol extracts of *Buddleja davidii* prepared in Example 1 was measured, and the results are shown in Table 2. The extracts of the above-ground parts of *Buddleja davidii* showed a very high content of 66.7–68.9 mg / g regardless of the extraction solvent. Since polyphenol components are associated with various beneficial physiological activities such as antioxidant, antithrombotic, and anti-inflammatory effects, it was expected that the hot water and 70% ethanol extracts of *Buddleja davidii* would exhibit various beneficial physiological activities. At this time, 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 sample, leaving it at room temperature for 1 hour, and measuring the absorbance at 725 nm. Tannic acid was used as the standard reagent.

[0051] [Table 2] Analysis of Total Polyphenol Content in Freshwater Plant Buddleja Extract

[0052]

[0053] Example 3: Evaluation of the antithrombotic activity of *Buddleja davidii* extract

[0054] The blood coagulation inhibitory activity of the *Lysimachia japonica* 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.

[0055] Thrombin Time

[0056] 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 30.1 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.

[0057] Prothrombin time

[0058] 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 15.5 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.

[0059] activated Partial Thromboplastin Time (aPTT)

[0060] 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 25.1 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 at the time of sample addition by the aPTT of the solvent control.

[0061] [Table 3] Inhibitory activity of freshwater plant *Buddleja davidii* extract on blood coagulation

[0062]

[0063] As shown in Table 3, when the thrombin time, prothrombin time, and APT time were measured at a concentration of 1.5 mg / ml for aspirin (antithrombotic agent trade name: Protect), which was used as a control, it was confirmed that it exhibited excellent antithrombotic activity by extending them by 1.54 times, 1.33 times, and 1.42 times, respectively. In the case of the hot water extract of *Lysimachia japonica*, when the thrombin time, prothrombin time, and APT time were measured at a concentration of 2 mg / ml, it was confirmed that it exhibited excellent antithrombotic activity by extending them by 1.30 times, 1.27 times, and 1.57 times, respectively.

[0064] On the other hand, when the thrombin time was measured at a concentration of 2 mg / ml using the 70% ethanol extract of *Lysimachia japonica*, it was extended by more than 15 times compared to the control group, confirming very strong thrombin inhibitory activity. In addition, the prothrombin time and APT time measurements were extended by 1.38 times and 4.63 times, respectively, compared to the control group, demonstrating superior anticoagulant activity compared to aspirin. Therefore, it was determined that the 70% ethanol extract of the above-ground parts of *Lysimachia japonica* can be used as an antithrombotic agent, and in particular, due to its strong thrombin inhibition and inhibition of blood coagulation factors, it can effectively suppress the formation of endogenous thrombi and can replace aspirin, which is known to cause side effects such as gastrointestinal disorders.

[0065] Example 4: Evaluation of the antioxidant activity of *Lysimachia japonica* extract

[0066] 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 *Buddleja davurica* extract was evaluated and the results are shown in Table 4.

[0067] At this time, the extract was dissolved in DMSO (dimethylsulfoxide) and diluted to an appropriate concentration 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 nitrites dissolved in 99.5% ethanol were added to 20 μl of samples diluted to various concentrations. -4380 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.

[0068]

[0069] 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.

[0070]

[0071] 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.

[0072] [Table 4] Antioxidant activity of freshwater plant *Buddleja davidii* extract

[0073]

[0074] As shown in Table 4, vitamin C used as a control exhibited very strong antioxidant activity, showing 87.5% DPPH anion scavenging activity, 89.7% ABTS cation scavenging activity, 79.3% nitrite scavenging activity, and a reducing power of 1.805 at a concentration of 0.5 mg / ml. The extract of *Buddleja davurica* of the present invention also exhibited excellent antioxidant activity; in particular, the 70% ethanol extract showed 61.3% DPPH anion scavenging activity, 87.2% ABTS cation scavenging activity, and a reducing power of 1.386 at a concentration of 0.5 mg / ml, and 77.9% nitrite scavenging activity at a concentration of 0.2 mg / ml, confirming antioxidant activity similar to that of vitamin C. Therefore, it is judged that the strong antioxidant activity of the 70% ethanol extract of the above-ground parts of *Buddleja davurica* contributes to antithrombotic activity.

[0075] Example 5: Evaluation of plasma, acid, and thermal stability of *Lysimachia japonica* extract

[0076] Plasma stability, heat stability, and acid stability regarding antithrombotic activity were confirmed for the 70% ethanol extract of the above-ground parts of *Buddleja davidii* obtained in Example 1 above. The 70% ethanol extract of *Buddleja davidii* 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 70% ethanol extract of *Buddleja davidii* 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 Small-flowered ( Ammannia multiflora A pharmaceutical composition for the prevention or treatment of thrombosis containing an extract as an active ingredient. Claim 2 In Article 1, the above small lotus ( Ammannia multiflora ) extract is small buttercup ( Ammannia multiflora A pharmaceutical composition characterized by being a 70% ethanol extract of the above-ground parts. 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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