KR20200015326A - Pharmaceutical composition comprising the extract of perilla leaf as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis, comprising a perilla leaf extract as an effective component, and more specifically, to a pharmaceutical composition and a health functional food comprising a perilla leaf extract as an effective component for preventing/alleviating thrombosis via potent blood coagulation inhibition activities. The perilla leaf extract as the effective component of the pharmaceutical composition and the health functional food for preventing or treating/alleviating thrombosis, while exhibiting a potent anti-thrombotic activity via inhibition of thrombus formation-associated enzymes and blood coagulation factors, does not show hemolytic activity with respect to human red blood cells; has excellent thermostability; and even in an acidic condition of pH 2 and in plasma, does not exhibit a loss of the inhibitory effect on blood coagulation factors or the inhibitory effect on thrombus formation-associated enzymes, and thus is expected to be usable for preventing and treating thrombosis, such as ischemic stroke and hemorrhagic stroke, by improving blood circulation. Furthermore, the effective component can be processed in various forms such as extracts, powder, pills, tablets, etc., to be prepared in a form that can be conveniently taken at any time, and thus is extremely useful in the food and pharmaceutical industries.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF PERILLA LEAF AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis containing sesame leaf (Leaf of Perilla) extract as an active ingredient, and more particularly, powerful blood containing sesame leaf extract as an active ingredient. The present invention relates to a pharmaceutical composition for preventing or treating / improving thrombosis through coagulation inhibitory activity and a dietary supplement.

Blood as a human component has various important functions such as transporting and buffering oxygen, nutrients and wastes, maintaining body temperature, controlling osmotic pressure and ion balance, maintaining moisture, controlling fluid, maintaining and controlling blood pressure, and defending the body. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolytic reaction system in the body complement each other, and among them, the mechanism of the blood coagulation reaction system forms platelet thrombus by adhering and agglomerating platelets to blood vessel walls. In addition, it has been reported that the blood coagulation system is activated to form fibrin clots around platelet aggregates.

On the other hand, the generation of fibrin thrombus is a multi-step reaction of numerous blood coagulation factors to activate the thrombin involved in fibrin coagulation, finally to produce fibrin monomer from fibrinogen, fibrin monomers are polymerized by calcium, platelets and endothelial They bind to cells and form permanent thrombus, forming fibrin polymers cross-linked by factor XIII. In addition, thrombin plays a pivotal role in thrombus generation by activating platelets, factor V and factor VII to promote blood coagulation. Therefore, thrombin's activity inhibitor can be used as a prophylactic and therapeutic agent which is very useful for various thrombotic diseases caused by excessive blood clotting. Meanwhile, in the endogenous thrombus generation pathway, sequential activation of XII, XI, IX and X factors is followed by the activation of prothrombin, which is known to activate thrombin. It is targeted. Until now, various anticoagulants such as heparin, coumarin, aspirin, urokinase, antiplatelet, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases, but they are not only expensive but also have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity reactions. Its use is limited to such circumstances.

Perilla frutescens var japonica, a perennial herbaceous plant belonging to the family Asteraceae, is widely grown in Southeast Asia, including Korea, China, and India. Perilla is a crop that is usually grown to squeeze oil. It is sesame leaves that harvest and edible leaves during growth.

Korea is the only country that eats sesame leaves for food, and it contains fragrant essential oils such as perillaaldehyde, limonene, and peryl ketone. It can be eaten as a side dish such as kimchi, or used as a spice in radish or hot water. Recently, noodles using sesame leaves (Hyun-eun, 2011, Korean Journal of Food and Nutrition Science 40: 1688-1693), fish cakes with sesame leaves, and medicinal sake (seeds) using sesame leaves are known. Sesame leaves contain more than twice as much iron as spinach, and are rich in minerals such as calcium, vitamin A and vitamin C. It is effective for gastrointestinal disorders such as anorexia, diarrhea and constipation, and is known to help prevent various adult diseases, and it is particularly rich in vitamin K, which promotes blood clotting.

In addition, sesame leaves contain luteolin, a type of vegetable pigment flavonoid, which helps to reduce inflammation in the body (Uemura et al., 2018, J Agric Food Chem. 66: 3443-3448), anti-allergic effect, cough or runny nose, It has the effect of alleviating the symptoms of sneezing, has strong antioxidant activity (Kim Min-jeong, 2011. Kyungnam University Master's thesis), and is effective in preventing hyperlipidemia (Hwang, Gwang-ok et al., 2003. Korean Journal of Food Science and Technology 35: 975-979), anticancer The substance Phytol is known to enhance immune function by removing cancer cells and pathogenic bacteria (Kwak Young-eun et al., 2013, Korean J. Food Cookery Sci 29: 241-248; Kim KH et al., 1993. Food Nutr. 28: 1107-1112). In addition, carcinogenic efficacy (Park KY et al., 1992. J. Korean Soc. Food Nutr. 21: 149-153; Lee KI, 1992. Korean J. Food Nutr. 21: 302-307) and DNA in skin cells Damage repair ability (Hyeon-A Lee, 2017. Hannam University Master's thesis), melanin synthesis inhibitory effect (Hwang JH, 2007. J Toxicol Environ Health A. 70: 393-407) has been reported to be available as a functional cosmetic material. Recently, sesame leaf hot water extract has been reported to shorten prothrombin time, resulting in excellent hemostatic action (Ohkura N et al., 2015. J Intercult Ethnopharmacol. 4: 19-23, Screening for hemostatic activities of popular Chinese medicinal herbs in vitro).

Patents related to sesame leaves include Korean Registered Patent No. 10-0644762 in [Pharmaceutical Prevention and Treatment Composition and Functional Foods Containing Sesame Leaf Extract], and No. 10-1152168 in [Per sesame Leaf Extract with Antibacterial Activity] Composition] is known, and in connection with the cosmetic industry, No. 10-1808091 [Cosmetic moisturizing composition using sesame leaf extract] is disclosed in No. 10-1814090 [Skin trouble containing sesame leaf chewable tablet as an active ingredient]. Relaxing compositions] are known, and 10-1298148 discloses [noodles containing sesame leaf extract having antioxidant activity and its preparation method] and 10-1414260 [sesame leaf rice wine and its manufacturing method], 10-1734992. No. [Medium composition for inducing growth of sesame leaf callus and increasing antioxidant capacity and a method of cultivating sesame leaf callus using the same] is registered. However, there is no known patent or research result on the active extract of sesame leaf, which shows no potent antithrombotic activity through the anticoagulant activity of sesame leaf.

 Ohkura N et al., 2015. J Intercult Ethnopharmacol. 4: 19-23, Screening for hemostatic activities of popular Chinese medicinal herbs in vitro

The present invention has been made to solve the problems of the prior art as described above, the problem to be solved in the present invention is to prevent or treat / improve the pharmaceutical composition and health functional food of thrombosis containing sesame leaf extract as an active ingredient It is to provide.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing sesame leaf extract as an active ingredient.

The sesame leaf extract is preferably sesame leaf hydrothermal extract or sesame leaf ethanol extract.

The sesame leaf hydrothermal extract is preferably an ethyl acetate fraction obtained by sequentially distilling the sesame leaf hydrothermal extract with an organic solvent of hexene, ethyl acetate and butanol, water residue or a mixture thereof.

The sesame leaf ethanol extract is preferably an ethyl acetate fraction, butanol fraction or a mixture thereof obtained by sequentially fractionating the sesame leaf ethanol extract with an organic solvent of hexene, ethyl acetate and butanol.

The present invention also provides a health functional food for preventing or improving thrombosis containing sesame leaf extract as an active ingredient.

The sesame leaf extract is preferably sesame leaf hydrothermal extract or sesame leaf ethanol extract.

The sesame leaf hydrothermal extract is preferably an ethyl acetate fraction obtained by sequentially distilling the sesame leaf hydrothermal extract with an organic solvent of hexene, ethyl acetate and butanol, water residue or a mixture thereof.

The sesame leaf ethanol extract is preferably an ethyl acetate fraction, butanol fraction or a mixture thereof obtained by sequentially fractionating the sesame leaf ethanol extract with an organic solvent of hexene, ethyl acetate and butanol.

The present invention also provides a platelet aggregation inhibitor containing sesame leaf hydrothermal extract as an active ingredient.

The sesame leaf extract as an active ingredient in the preventive or therapeutic / improvement of thrombosis of the present invention and a health functional food exhibits potent antithrombotic activity through inhibition of enzymes and blood coagulation factors related to thrombus formation, It shows no hemolytic activity, excellent thermal stability, no loss of blood coagulation factor inhibitory effect and thrombus formation related enzyme inhibitory effect even in acidic condition and plasma of pH 2, thus improving blood circulation, such as ischemic stroke and hemorrhagic stroke. It is expected to be used for the prevention and treatment of thrombosis, and the active ingredient is processed into various forms such as extract, powder, pills, tablets, etc. It is an industrially useful invention.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The inventors of the present invention, in order to assay the antithrombotic efficacy of sesame leaves, hot air dried sesame leaves, prepared extracts by a certain method, and evaluated the activity to identify sesame leaf extract showing antithrombotic activity, sesame leaf extract Hexene fraction, ethyl acetate fraction, butanol fraction and water residue after organic solvent fraction were prepared to recover the organic solvent fraction showing antithrombotic activity, and the active extract and fraction showed no hemolytic activity against human erythrocytes. In addition, the anti-thrombotic active extracts and fractions were used as pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of thrombosis by confirming that they have excellent thermal stability and acid stability.

Specifically, the present inventors prepared a dry powder of sesame leaves, ethanol extract and hot water extract in order to develop a pharmaceutical composition and health functional food for the prevention or treatment / improvement of thrombosis by using sesame leaves, which are used for various purposes in the private sector. Were prepared, and their antithrombotic activity was evaluated. Then, sesame leaf extracts were subjected to sequential organic solvent fractionation with hexene, ethyl acetate and butanol, respectively, and finally water residue after organic solvent fractionation was prepared. The antithrombotic activity of each extract and fractions was evaluated for Thrombin Time, Prothrombin Time and Activated Partial Thromboplastin Time (aPTT) against human plasma and human thrombin. As a result, it was confirmed that potent anticoagulant activity in sesame leaf hydrothermal extract and ethanol extract and their fractions, they do not show hemolytic activity against human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing sesame leaf extract as an active ingredient.

The sesame leaf extract is preferably sesame leaf hydrothermal extract or sesame leaf ethanol extract.

The sesame leaf hydrothermal extract is preferably an ethyl acetate fraction obtained by sequentially distilling the sesame leaf hydrothermal extract with an organic solvent of hexene, ethyl acetate and butanol, water residue or a mixture thereof.

The sesame leaf ethanol extract is preferably an ethyl acetate fraction, butanol fraction or a mixture thereof obtained by sequentially fractionating the sesame leaf ethanol extract with an organic solvent of hexene, ethyl acetate and butanol.

The present invention also provides a health functional food for preventing or improving thrombosis containing sesame leaf extract as an active ingredient.

The sesame leaf extract is preferably sesame leaf hydrothermal extract or sesame leaf ethanol extract.

The sesame leaf hydrothermal extract is preferably an ethyl acetate fraction obtained by sequentially distilling the sesame leaf hydrothermal extract with an organic solvent of hexene, ethyl acetate and butanol, water residue or a mixture thereof.

The sesame leaf ethanol extract is preferably an ethyl acetate fraction, butanol fraction or a mixture thereof obtained by sequentially fractionating the sesame leaf ethanol extract with an organic solvent of hexene, ethyl acetate and butanol.

Among the sesame leaf extracts exhibiting antithrombotic activity by the anticoagulant inhibitory activity described above, the hydrothermal extract showed excellent platelet aggregation inhibitory activity.

Accordingly, the present invention provides a platelet aggregation inhibitor containing sesame leaf hydrothermal extract as an active ingredient.

Hereinafter, the preparation method and efficacy experiment of sesame leaf extract and each active fraction of the present invention will be described in more detail.

The present invention comprises the steps of drying the sesame leaves; Preparing an extract from the dry powder; Evaluation of antithrombotic activity of sesame leaf extract; Preparing a sequential organic solvent fraction of hexene, ethyl acetate, butanol from sesame leaf extract exhibiting antithrombotic activity, and then preparing a water residue; Evaluation of the antithrombotic activity of the extracts and fractions and investigation of the stability of the active material.

"Perilla leaf extract" included in the composition of the present invention is to harvest the mature perilla leaves, and washing them; Drying the sesame leaves at 70-80 ° C. for 1 day; Grinding the dried sesame leaves; Extracting the ground powder with an organic solvent and filtering the extract using a filter network of 0.06 mm or less, and concentrating it under reduced pressure.

The organic solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, ethyl acetate, etc.), the mixed solvent of the lower alcohol and water Or the like, hot water or 95% ethanol extraction is most preferred.

In the present invention, sesame leaf powder was extracted with hot water or ethanol to obtain a hot water extract or ethanol extract. Fractions and water residues can additionally be obtained.

In the present invention, when the sesame leaf hot water extract was measured at a concentration of 5 mg / ml, the thrombin time, prothrombin time, and epitaxial time were measured, and were increased by 1.62, 1.05, and 1.61 times as compared to the non-added groups, respectively, indicating good thrombin inhibition and coagulation factor. Thrombin time, prothrombin time, and epitaxial time were measured using sesame leaf ethanol extract at a concentration of 5 mg / ml, which was 1.29, 1.60, and 1.36 times higher than those of the non-added groups, respectively. Blood coagulation factor inhibition was shown. Subsequently, as a result of evaluating blood coagulation inhibitory activity in the organic solvent fraction and water residues of sesame leaf extract, all the fractions were confirmed to have a concentration-dependent clotting inhibitory activity, especially in ethyl acetate fraction, a strong thrombin comparable to aspirin. , Prothrombin and coagulation factor inhibition. Therefore, it was confirmed that the sesame leaf extract, especially the ethyl acetate fraction of the sesame leaf extract, could be developed as an antithrombotic agent that can replace aspirin, which has high side effects such as gastrointestinal disorders.

On the other hand, the sesame leaf hydrothermal extract of the sesame leaf extract of the present invention was found to exhibit a concentration-dependent platelet aggregation inhibitory activity, as well as thrombin inhibitory, blood coagulation enzyme inhibition and blood coagulation factor inhibitory activity described above. In other words, it exhibits 45.7% platelet aggregation ability at a concentration of 0.50 mg / ml of hydrothermal extract, which is expected to be used as a platelet aggregation inhibitor (antiplatelet agent).

The sesame leaf extract of the present invention and the active fractions thereof may be prepared into a powder through a conventional powdering process such as vacuum drying and freeze drying or spray drying. They are not degraded by various degrading enzymes in plasma and remain active even at a heat treatment of 100 ° C. and pH in the human stomach at pH 2.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases associated with thrombosis. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis , Deep vein thrombosis, lower extremity edema, pain, and acute peripheral arterial obstruction. Examples of venous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous thrombosis, and central retinal vein occlusion.

The pharmaceutical composition comprising the active ingredient of the present invention may be prepared in accordance with conventional methods for the purpose of use, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and injectables of sterile injectable solutions. It may be formulated and used in various forms, and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further 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 rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include a filler, an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like may also be used.

As a preferred embodiment, oral liquid preparations may be exemplified by suspending agents, solvents, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, Fragrances, preservatives and the like.

As a preferred embodiment, the preparation for parenteral administration may be sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized, suppository and the like. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, activity of the drug, Sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg body weight daily. Or every other day or divided into 1 to 3 times daily. However, since the dose may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

As used herein, "administration" means providing a patient with any substance by any suitable method, wherein the route of administration of the pharmaceutical composition of the present invention is oral or parenteral via all common routes as long as the target tissue can be reached. Oral administration. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

"Subject" in the present invention is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And preferably mammals, and more preferably humans.

In addition, the health functional food of the present invention can be used in a variety of foods and beverages and the like effective in preventing or improving thrombosis. Examples of the food containing the active ingredient of the present invention include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and can be used in the form of powders, granules, tablets, capsules, or beverages. .

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

In addition to containing the compound as an essential ingredient in the indicated proportions, the health functional food of the present invention may contain as food additives food additives such as natural carbohydrates and various flavoring agents.

Examples of the natural carbohydrate include conventional sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

As the flavoring agent, taumartin; Natural flavors such as stevia such as rebaudioside A or glycyrzin and synthetic flavors such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally used from about 1 to 20 g, preferably from about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals, synthetic flavors and natural flavoring agents, colorants and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids Thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for preparing natural fruit juice, fruit juice beverage, vegetable beverage and the like. These components can be used independently or in combination. The proportion of such additives is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

EXAMPLE

Example 1: Preparation of Sesame Leaf Extract and Component Analysis of the Extract

Purchasing sesame leaves cultivated and harvested in Andong, Gyeongsangbuk-do, 2017, removed foreign substances, washed and dried them for 1 day at 80 ℃ to prepare a powder (50 ~ 100 mesh). Thereafter, when preparing ethanol extract, 10 times ethanol was added to sesame leaf powder, and extracted three times at room temperature, and the extracts were collected and filtered, and then concentrated under reduced pressure to prepare an ethanol extract. 10 times distilled water was added, and the extract was repeatedly extracted three times at 100 ° C. for 1 hour, and the extracts were collected and filtered, and concentrated under reduced pressure to prepare a hot water extract. Each extraction efficiency and the results of the component analysis of the extract are shown in Table 1. Component analysis determined total polyphenols, total flavonoids, total sugars and reducing sugars content. The total polyphenol content was added to 400 μl of the extracted sample solution, 50 μl of Folin-ciocalteau and 100 μl of saturated Na ₂ CO ₃ solution were left at room temperature for 1 hour, and then absorbance was measured at 725 nm. Tannic acid was used as the standard reagent. For the total flavonoid content, each sample was extracted by stirring for 18 hours with methanol, 4 ml of 90% diethylene glycol was added to 400 µl of the filtered extract sample, and 40 µl of 1 N NaOH was added again. Absorbance was measured at. Rutin was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was determined by phenol-sulfuric acid method.

[Table 1] Extraction efficiency and useful ingredient analysis of hot water extract and ethanol extract of sesame leaf powder

As shown in Table 1, the hot water extraction efficiency was about 2.3 times higher than the ethanol extraction efficiency, and the total flavonoid, total sugar and reducing sugar contents of the extract were similar. However, the total polyphenol content of the ethanol extract was about 1.5 times higher than that of the hydrothermal extract. Sesame leaves were found to contain higher total polyphenol content than normal vegetables.

Example 2: Blood Coagulation Inhibitory Activity of Sesame Leaf Extract

The coagulation inhibitory activity of sesame leaf extracts of Example 1 was evaluated, and the results are shown in Table 2. At this time, blood coagulation inhibitory activity evaluation method was evaluated according to the previously reported method (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), thrombin time, prothrombin time and episode time. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and thrombin time, prothrombin time and apititime measurements were performed as follows.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ , and 10 μl of various sample extracts were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes. After the addition of 100 μl of plasma, the time until the plasma coagulated was measured. Aspirin (Sigma Co., USA) was used as a control and DMSO was used instead of the sample as a solvent control. DMSO showed a solidification time of 24.2 seconds. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the thrombin inhibitory activity was expressed as the coagulation time when the sample was added divided by the coagulation time of the solvent control.

Prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations were added to a tube of Amelung coagulometer KC-1A (Japan), warmed at 37 ° C. for 3 minutes, and then 130 μl of PT reagent was added. The time to plasma coagulation is expressed as the average of three repeated experiments. Aspirin (Sigma Co., USA) was used as a control and DMSO was used instead of the sample as a solvent control. DMSO had a solidification time of 16.8 seconds. The prothrombin inhibitory activity was expressed by the solidification time of sample addition divided by the solidification time of the solvent control.

aPTT (activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of various concentrations of the sample extract were added to a tube of Amelung coagulometer KC-1A (Japan), warmed at 37 ° C. for 3 min, and then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added again. Incubated for 3 minutes at ℃. Since 50 μl CaCl ₂ (35 mM) was added to measure the time until the plasma coagulate. DMSO was used as a solvent control instead of the sample, in which case it showed a solidification time of 42.2 seconds. The results of aPTT were shown as the average value of three repeated experiments, and the coagulation factor inhibitory activity was expressed as aPTT at the time of sample addition divided by aPTT of the solvent control.

[Table 2] Hemocoagulation inhibitory activity of various sesame leaf extracts

As a result, the hot water extract of sesame leaf increased the thrombin time, prothrombin time, and epitaxial time by 1.62, 1.05, and 1.61 times at 5 mg / ml concentration, respectively, and showed good thrombin inhibition and coagulation factor inhibition. The anticoagulant activity was increased by 1.29, 1.60, and 1.36-fold increase in thrombin time, prothrombin time, and apitime at / ml concentration, respectively.

Example 3: Platelet aggregation inhibitory activity of sesame leaf hydrothermal extract

Human platelet aggregation inhibitory activity of the sesame leaf hydrothermal extract of Example 1 was evaluated and the results are shown in Table 3. Platelets are discoid small cells that circulate blood vessels with various blood cells. They have a cytoplasmic granule that contains high concentrations of various substances related to vascular damage protection and platelet aggregation, instead of the nucleus. It is an important cell that secretes factors and binds collagen and the like exposed to damage of endothelial cells to form a primary hemostatic plug to initiate thrombus formation. Thus, platelet aggregation inhibition is a very important activity for preventing thrombus formation. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Platelets were human concentrated platelets, which were washed once with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Then, resuspend in suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 0.49 mM MgCl ₂ , 0.25% gelatin, pH 7.4) and then at 3,000 rpm. After centrifugation for 10 minutes, the suspension was resuspended in suspending buffer, and the platelet count was adjusted to 4x10 ⁹ / ml. Thereafter, 2.5 μl collagen was added to the 1 ml suspension for 5 minutes, and platelet aggregation was measured at 37 ° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 3] Platelet aggregation inhibitory activity of sesame leaf hydrothermal extract

As shown in Table 3, first, aspirin showed a strong concentration of platelet aggregation (aggregation of 39.5-63.7%) at a concentration of 0.125-0.25 mg / ml, indicating the basis for use as an antithrombotic agent in the clinic. On the other hand, hot water extract of sesame leaves did not affect human platelet aggregation at 0.25 mg / ml concentration, but increased concentration of 84% at 0.35 mg / ml concentration and 45.7% at 0.5 mg / ml concentration. It was found to inhibit aggregation.

Example 4 Preparation of Sequential Organic Solvent Fractions of Sesame Leaf Extract and Their Component Analysis

The organic solvent fractions were sequentially sequential to hexene, ethyl acetate, and butanol with respect to various sesame leaf extracts of Example 1, and finally water residue was recovered. The yield of the fractions and the results of component analysis of the fractions are shown in Table 4.

[Table 4] Yield and component analysis of sequential fractions of sesame leaf extract

As shown in Table 4, the sesame leaf hydrothermal extract was transferred to the hexene fraction and the ethyl acetate fraction by 0.1% and 3.9%, respectively, and to the butanol fraction and the water residue by 45.9% and 55.5%, respectively. In contrast, sesame leaf ethanol extracts were 7.4% and 8.6%, respectively, with ethyl acetate fraction and butanol fraction, and the hexene fraction and water residue accounted for 41.0% and 46.5%, respectively. These results indicate that sesame leaf hydrothermal extract contains most water-soluble components, and sesame leaf ethanol extract contains similar amounts of fat-soluble components and water-soluble components. In addition, the ethyl acetate fraction of the hydrothermal extract from 100 g dried sesame leaves is about 0.865 g, the ethyl acetate fraction of the ethanol extract means that it can recover about 0.72 g.

On the other hand, total polyphenol and flavonoid content analysis showed the highest content in the ethyl acetate fraction (318 ~ 377 mg / g polyphenol and 146 ~ 273 mg / g flavonoids), the various physiological activity of sesame leaf extract was ethyl acetate fraction Was expected to appear.

Example 5 Evaluation of Blood Coagulation Inhibitory Activity of the Fraction of Organic Solvents from Perilla Leaf Extract

The coagulation inhibitory activity of the organic solvent fractions obtained from the sesame leaf extract obtained in Example 4 was evaluated in the same manner as in Example 2, and the results are shown in Table 5.

[Table 5] Blood Coagulation Inhibitory Activities of Organic Solvent Fractions Obtained from Sesame Leaf Extract

As shown in Table 5, aspirin showed excellent blood coagulation inhibitory activity by extending thrombin time, prothrombin time, and epitaxial time by 1.60 times, 1.41 times, and 1.58 times, respectively, at a concentration of 1.5 mg / ml. On the other hand, among the fractions of sesame leaf hot water extract, strong anticoagulant activity was observed in the ethyl acetate fraction, and the thrombin time, prothrombin time, and apitime were extended by 1.53 times, 2.36 times, and 2.59 times at 5 mg / ml. At the 7 mg / ml concentration, prothrombin time was 4.14 times longer, but thrombin time and apity time were both extended at least 15 times, indicating strong antithrombotic activity. In addition, the water residue of the hydrothermal extract specifically exhibited thrombin-specific inhibitory activity by specifically extending the thrombin time. On the other hand, the fractions of the sesame leaf ethanol extract showed strong concentration-dependent anticoagulant activity in all hexene fraction, ethyl acetate fraction and butanol fraction. Especially, ethyl acetate fraction showed thrombin time, prothrombin time and apiti The time was extended by 15 times, 6.21 times, and 9.1 times compared to the non-added group, and at the 7 mg / ml concentration, all of them extended 15 times or more, showing the strongest antithrombotic activity. This potent activity was found to be comparable to that of aspirin used in the clinic. Therefore, the ethyl acetate fraction of sesame leaf extract may be developed as a commercial antithrombotic agent, and may be replaced with aspirin, which is an existing antithrombotic agent exhibiting side effects such as gastrointestinal disorders.

Example 6: Human Erythrocyte Hemolytic Activity of Sesame Leaf Extract and Fractions thereof

Sesame leaves have been eaten as a ssam vegetable and spices for a long time. Currently, it is registered as a food ingredient in Korea. In the present invention, human erythrocyte hemolytic activity was evaluated to evaluate the acute toxicity of sesame leaf extract and its fractions, and the results are shown in Table 6. At this time, the hemolytic activity was evaluated according to the existing report (Kim Mi-sun et al., 2014. J. Life Sci. 24: 515-521), and simply, 100 μl of human red blood cells washed three times with PBS was added to a 96-well microplate. 100 μl of various sample solutions were added and then reacted at 37 ° C. for 30 minutes. After that, the reaction solution was centrifuged (1,500 rpm) for 10 minutes to transfer 100 μl of the supernatant to a new microtiter plate. Measurement was made at 414 nm. DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1 mg / ml) was used as an experimental control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

TABLE 6 Human Erythrocyte Hemolytic Activity of Perilla Leaf Extract and Its Fractions

As a result, as shown in Table 6, first, DMSO and water used as a control had no hemolytic activity, triton X-100 was confirmed that 100% hemolysis of red blood cells at a concentration of 1 mg / ml. Meanwhile, sesame leaf extract had no hemolytic activity in both hot water extract and ethanol extract. However, the hexene fraction of the ethanol extract showed 47.9% hemolytic activity at a concentration of 1 mg / ml, and no other fraction and water residue showed hemolytic activity. Therefore, sesame leaf hydrothermal extract, ethanol extract, their ethyl acetate fraction, butanol fraction and water residues will not appear to exhibit a separate acute toxicity problem.

Example 7: Evaluation of Plasma, Acid and Thermal Stability of Perilla Leaf Extract and Its Fractions

Plasma stability, thermal stability and acid stability against antithrombotic activity of the sesame leaf extract, its fractions and water residues obtained in Examples 1 and 4 were confirmed. The samples showed high stability due to 1 hour heat treatment at 100 ° C., 1 hour treatment at pH 2 (0.01 M HCl), 1 hour treatment in plasma, and no significant decrease in blood coagulation inhibition and platelet aggregation inhibitory activity. . Therefore, the extracts and active fractions above are expected to maintain excellent antithrombotic activity during digestive absorption and food preparation.

Claims (6)

A pharmaceutical composition for preventing or treating thrombosis containing sesame leaf (Leaf of Perilla) extract as an active ingredient. The method of claim 1, wherein the sesame leaf extract is a pharmaceutical composition, characterized in that the sesame leaf hydrothermal extract or sesame leaf ethanol extract. 3. The pharmaceutical composition according to claim 2, wherein the sesame leaf hydrothermal extract is an ethyl acetate fraction obtained by sequentially distilling the sesame leaf hydrothermal extract with an organic solvent of hexene, ethyl acetate and butanol, water residue or a mixture thereof. 3. The pharmaceutical composition according to claim 2, wherein the sesame leaf ethanol extract is an ethyl acetate fraction, butanol fraction or mixture thereof obtained by sequentially fractionating the sesame leaf ethanol extract with an organic solvent of hexene, ethyl acetate and butanol. A health functional food for preventing or improving thrombosis, comprising the active ingredient according to any one of claims 1 to 4. Platelet aggregation inhibitor containing sesame leaves (Leaf of Perilla) as an active ingredient.