KR102128622B1 - 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 for preventing or treating thrombosis and a health functional food containing a sesame leaf (Leaf of Perilla) extract as an active ingredient, and more specifically, a powerful blood using the 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. The sesame leaf extract as an active ingredient in the pharmaceutical composition and health functional food for prevention or treatment/improvement of thrombosis of the present invention exhibits strong antithrombotic activity through inhibition of blood clotting factors and enzymes related to thrombosis, and also against human red blood cells. Since it does not show any hemolytic activity, has excellent thermal stability, and does not exhibit loss of blood clotting factor inhibitory effect and enzyme inhibitory effect related to thrombus production even in acidic conditions of pH 2 and plasma, 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, pill, tablet, etc., so it has an excellent effect that can be prepared in a form that can be taken at all times. It is a very useful invention in the industry.

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 for preventing or treating thrombosis and a health functional food containing a sesame leaf (Leaf of Perilla) extract as an active ingredient, and more specifically, a powerful blood using the 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 component of the human body has various important functions such as transport function and buffer function of oxygen, nutrients, wastes, maintenance of body temperature, regulation of osmotic pressure and maintenance of ion balance, maintenance of moisture, fluid control, maintenance and regulation of blood pressure, and defense of the body. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolysis reaction system in the body are complementarily regulated, and among these, the mechanism of the blood coagulation reaction system is that platelets adhere to and adhere to blood vessel walls to form platelet thrombus. , It has been reported that the blood coagulation system activates to form a fibrin thrombus around a platelet aggregate.

On the other hand, the production of fibrin thrombus is activated by thrombin, which is involved in fibrin coagulation through several step reactions of numerous blood coagulation factors, to finally produce fibrin monomer from fibrinogen, and the fibrin monomers are polymerized by calcium, causing platelets and endothelial It binds to cells and forms a permanent thrombus, forming a fibrin polymer cross-linked by factor XIII. In addition, thrombin plays a pivotal role in thrombus production, such as promoting blood clotting reactions by activating platelets, factor V and factor VII. Therefore, an active inhibitor of thrombin can be used as a very useful preventive and therapeutic agent for various thrombotic diseases caused by excessive blood clotting abnormalities. On the other hand, it is known that the activation of prothrombin following sequential activation of factor XII, factor XI, factor IX, factor X in the endogenous thrombus production pathway finally activates thrombin. It is targeted. To date, various anticoagulants such as heparin, coumarin, aspirin, and eurokinase, antiplatelet agents, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases, but they are not only very expensive, but also have bleeding side effects, gastrointestinal disorders, and hypersensitivity reactions. The use thereof is limited.

Perilla frutescens var japonica ( Perilla frutescens var japonica) is a perennial herbaceous plant that is widely cultivated in Southeast Asia such as Korea, China, and India. Perilla is a crop that is usually cultivated to squeeze oil, but it is the sesame leaf that is harvested and edible during growth.

Korea is the only country that eats sesame leaves for food, and since it contains aromatic essential oil ingredients such as perillaaldehyde, limonene, and perylketone, its unique aroma enhances the taste, and is mainly used as a wrapped vegetable. It can be eaten as a side dish of kimchi or used as a spice for seasoning or hot water. Recently, noodle using sesame leaves (Hyo-Hyun Hyun, 2011, Korean Journal of Food and Nutrition 40: 1688-1693), fish cake added with sesame leaves, and medicinal liquor using sesame leaves (sesame leaf liquor) are known. Sesame leaves contain more than twice as much iron as spinach, and have high nutritional value such as calcium, minerals, and vitamin A and vitamin C. It is effective for gastrointestinal disorders such as anorexia, diarrhea, and constipation. It is also known to help prevent various adult diseases, and it is also rich in vitamin K, which acts to promote blood clotting.

In addition, sesame leaves contain luteolin, a type of plant pigment flavonoids, to relieve 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 sneezing symptoms, has strong antioxidant activity (Kim Min-Jung, 2011. Master's thesis in Kyungnam University), is excellent in preventing hyperlipidemia (Hyun Kwang-Ok et al., 2003. Korean Journal of Food Science and Technology 35: 975-979), anti-cancer 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. Korean J. Food Nutr. 28: 1107-1112). In addition, carcinogenesis inhibitory effect (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 restoration ability (Lee Hyun-ah, 2017. Hannam University master's thesis), melanin pigment synthesis inhibitory effect (Hwang JH, 2007. J Toxicol Environ Health A. 70: 393-407) have been reported, and it is reported that it can be used as a functional cosmetic material. , Recently, sesame leaf hot water extract has been reported to shorten prothrombin time and has excellent hemostatic effect (Ohkura N et al., 2015. J Intercult Ethnopharmacol. 4: 19-23, Screening for hemostatic activities of popular Chinese medicinal herbs in vitro).

As a patent related to sesame leaf, Korean Patent Registration No. 10-0644762 [Pharmaceutical composition and functional food for prevention and treatment of obesity containing sesame leaf extract], [10-1152168] contains sesame leaf extract having antibacterial activity The composition is known, and in relation to the cosmetics industry, [No. 10-1808091 [Moisturizing cosmetic composition for skin moisturizing using sesame leaf extract], No. 10-1814090 [No skin trouble containing sesame leaf chewable tablet as an active ingredient] Palliative composition] is known, and in 10-1298148, [Noodles containing sesame leaf extract having antioxidant activity and a method for manufacturing the same] and 10-1414260 [Sesame leaf rice wine and a method for manufacturing the same], 10-1734992 No. [Medium composition for inducing growth of sesame leaf callus and increasing antioxidant ability and method for cultivating sesame leaf callus using the same] is registered. However, there are no known patents or research results on the active extract of sesame leaves that exhibits strong antithrombotic activity through the blood coagulation inhibitory activity of sesame leaves.

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 was devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to prevent or treat/improve pharmaceutical compositions and health functional foods for thrombosis containing sesame leaf extract as an active ingredient. Is to provide.

In order to solve the above problems, 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 hot water extract or sesame leaf ethanol extract.

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

The sesame leaf ethanol extract is preferably an ethyl acetate fraction, a 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.

In addition, the present invention 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 hot water extract or sesame leaf ethanol extract.

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

The sesame leaf ethanol extract is preferably an ethyl acetate fraction, a 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.

In addition, the present invention provides a platelet aggregation inhibitor containing sesame leaf hot water extract as an active ingredient.

The sesame leaf extract as an active ingredient in the pharmaceutical composition and health functional food for prevention or treatment/improvement of thrombosis of the present invention exhibits strong antithrombotic activity through inhibition of blood clotting factors and enzymes related to thrombosis, and also against human red blood cells. Since it does not show any hemolytic activity, has excellent thermal stability, and does not exhibit loss of blood clotting factor inhibitory effect and enzyme inhibitory effect related to thrombus production even in acidic conditions of pH 2 and plasma, 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, pill, tablet, etc., so it has an excellent effect that can be prepared in a form that can be taken at all times. It is a very useful invention in the industry.

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy against sesame leaves, the inventors of the present invention hot-dried sesame leaves, prepared the extracts in a certain way, and evaluated the activity to confirm the sesame leaf extracts exhibiting antithrombotic activity, and then sesame leaf extract From the hexene fraction, ethyl acetate fraction, butanol fraction and water residue after the organic solvent fraction were prepared to recover the organic solvent fraction showing antithrombotic activity, the active extract and fraction showing no hemolytic activity against human red blood cells at all , It was intended to utilize the antithrombotic active extract and fraction as a pharmaceutical composition for preventing or treating/improving thrombosis and a health functional food by confirming that it has excellent characteristics of thermal stability and acid stability.

Specifically, the present inventors prepared a dry powder of sesame leaves after preparing a dry powder of sesame seeds to develop a pharmaceutical composition and a health functional food for preventing or treating/improving thrombosis using sesame leaves used in various purposes in the private sector, followed by ethanol extract and hot water extract Was prepared respectively, and their antithrombotic activity was evaluated. Subsequently, sesame leaf extracts were subjected to sequential organic solvent fractionation with hexene, ethyl acetate, and butanol, respectively. Finally, water residues were prepared after the organic solvent fractionation. The antithrombotic activity of each extract and fraction was evaluated by direct thrombin inhibition, prothrombin time, and activated partial thromboplastin time (aptt) for human plasma and human thrombin. As a result, strong blood clotting inhibitory activity was confirmed in sesame leaf hot water extract and ethanol extract and their fractions, and they confirmed that hemolytic activity against human red blood cells was not shown.

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 hot water extract or sesame leaf ethanol extract.

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

The sesame leaf ethanol extract is preferably an ethyl acetate fraction, a 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.

In addition, the present invention 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 hot water extract or sesame leaf ethanol extract.

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

The sesame leaf ethanol extract is preferably an ethyl acetate fraction, a 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 blood coagulation inhibitory activity described above, the hot water extract showed excellent platelet aggregation inhibitory activity.

Therefore, the present invention provides a platelet aggregation inhibitor containing sesame leaf hot water extract as an active ingredient.

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

The present invention is a step of drying sesame leaves; Preparing an extract from the dry powder; Evaluation of antithrombotic activity of sesame leaf extract; Sequential organic solvent fraction preparation of hexene, ethyl acetate, butanol from sesame leaf extract showing antithrombotic activity and preparation of water residues obtained thereafter; It includes the step of evaluating the antithrombotic activity of the extract and fractions and the step of investigating the stability of the active substance.

"Sesame leaf extract" contained in the composition of the present invention is to collect the mature sesame leaves, and washing them; Drying the sesame leaves at 70-80° C. for 1 day; Grinding the dried sesame leaves; It can be obtained by the step of 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 alcohols having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, ethyl acetate, etc.), a mixed solvent of the lower alcohol and water Etc. can be used, and hot water or 95% ethanol extraction is most preferred.

In the present invention, the sesame leaf powder was extracted with hot water or ethanol to obtain a hot water extract or an ethanol extract, wherein the hot water or ethanol extract was sequentially or separately fractionated with an organic solvent of hexene, ethyl acetate and butanol, respectively to hexene fraction, ethyl acetate fraction, butanol Fractions and water residues can additionally be obtained.

In the present invention, as a result of measuring thrombin time, prothrombin time and apity time with sesame leaf hot water extract at a concentration of 5 mg/ml, it was increased by 1.62, 1.05, and 1.61 times, respectively, compared to no additives, thereby showing good thrombin inhibition and coagulation factor. Thrombin time, prothrombin time, and apity time were measured with a sesame leaf ethanol extract at a concentration of 5 mg/ml, and increased by 1.29, 1.60, and 1.36 times, respectively, compared to the no-additives, indicating good blood coagulase inhibition and Blood coagulation factor inhibition was shown. Subsequently, as a result of evaluating the blood coagulation inhibitory activity on the organic solvent fraction and water residue of the sesame leaf extract, it was confirmed that the concentration-dependent thrombus production inhibitory activity was observed in all the fractions, especially in the ethyl acetate fraction, strong thrombin comparable to aspirin , Prothrombin and blood coagulation factor inhibition. Therefore, it was confirmed that the sesame leaf extract, especially the ethyl acetate fraction of the sesame leaf extract, can be developed as an antithrombotic agent that can replace aspirin with high side effects such as gastrointestinal disorders.

On the other hand, it was confirmed that the sesame leaf extract from the sesame leaf extract of the present invention exhibits thrombin inhibition, blood coagulation enzyme inhibition and blood coagulation factor inhibitory activity as well as concentration-dependent platelet aggregation inhibitory activity as described above. That is, it exhibits a platelet aggregation ability of 45.7% at a concentration of 0.50 mg/ml of hot water extract, which is expected to be used as a use (antiplatelet agent) of platelet aggregation inhibitors.

The sesame leaf extract and the active fraction materials thereof of the present invention can be prepared as 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 they maintain activity even at a heat treatment at 100°C and a 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 related to thrombosis. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, motor abnormalities, sensory abnormalities, personality changes, decreased vision, epilepsy seizures, pulmonary thrombosis , Deep vein thrombosis, swelling of the lower extremities, pain and acute peripheral arterial occlusion, and intravenous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, brain venous sinus thrombosis and central retinal vein occlusion.

The pharmaceutical composition comprising the active ingredient of the present invention is an oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, injection of sterile injectable solution according to a conventional method according to each purpose of use It can be formulated and used in various forms, such as oral administration or intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

The pharmaceutical composition may further include a carrier, excipient or diluent, 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 And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., such solid preparations comprising at least one excipient in the pharmaceutical composition, for example starch, calcium carbonate, It is formulated by mixing sucrose, lactose and gelatin. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

As a preferred embodiment, a liquid preparation for oral use may be exemplified by suspending agents, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, Fragrances, preservatives, and the like may be included.

As a preferred embodiment, the preparation for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injections can 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, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, drug activity, Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs and other factors well known in the medical arts. 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 a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those 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 patient's age, sex and body weight, and generally 1 to 5,000 mg per kg body weight, preferably 100 to 3,000 mg daily Or it can be administered every other day or divided into 1 to 3 times a day. However, since the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, and age, the dosage is not limited to the scope of the present invention in any way.

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

In the present invention, "administration" means providing a predetermined substance to a patient in any suitable method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to target cells.

“Subject” in the present invention includes, but is not particularly limited to, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, mice, rabbits, or guinea pigs, for example. And, preferably, a mammal, more preferably a human.

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

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

The dietary supplement of the present invention may contain, as an essential ingredient in the indicated proportions, the above compound as an essential ingredient, and may contain, as an additional ingredient, a food additive that is food-acceptable, such as natural carbohydrates and various flavoring agents.

Examples of the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and sugars such as xylitol, sorbitol, and erythritol, and common sugars such as dextrin and cyclodextrin.

The flavoring agent includes taumatin; Natural flavors such as stevia such as rebaudioside A or glycyrrhizine, and synthetic flavors such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally used in about 1 to 20 g, preferably about 5 to 12 g per 100 ml of 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 flavors such as natural flavors, colorants and neutralizers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective colloids It may contain a thickening agent, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The ratio of these 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 through 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: Sesame leaf extract preparation and component analysis of the extract

In 2017, we purchased sesame leaves grown and harvested in Andong, North Gyeongsang Province, removed foreign substances, washed them, and dried them in hot air at 80°C for 1 day to prepare powder (50~100 mesh). Thereafter, when preparing the ethanol extract, 10 times ethanol was added to the sesame leaf powder, and the extract was collected three times at room temperature, filtered, collected, filtered, and concentrated under reduced pressure to prepare an ethanol extract, and the hot water extract was added to the sesame leaf powder. About 10 times, distilled water was added, and the extract was collected three times at 100° C. for 1 hour, collected, filtered, and concentrated under reduced pressure to prepare a powder to prepare a hot water extract. Table 1 shows the results of each extraction efficiency and component analysis of the extract. Total polyphenols, total flavonoids, total sugars, and reducing sugars were determined by component analysis. The total polyphenol content was 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ saturated solution in 400 μl of the extracted sample solution, and allowed to stand at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. The total flavonoid content was extracted by stirring each sample with methanol for 18 hours, adding 4 ml of 90% diethylene glycol to 400 μl of the filtered extraction sample, adding 40 μl of 1 N NaOH again, and reacting at 37° C. for 1 hour to 420 nm. Absorbance was measured at. Rutin was used as a standard reagent. The reducing sugar was quantified using the DNS method and the total sugar was phenol-sulfuric acid method.

[Table 1] Analysis of extraction efficiency and useful components 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 hot water extract. It was confirmed that sesame leaves contain a higher total polyphenol content than ordinary vegetables.

Example 2: Blood coagulation inhibitory activity of sesame leaf extract

The blood coagulation inhibitory activity of the sesame leaf extracts of Example 1 was evaluated, and the results are shown in Table 2. At this time, the blood clotting 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 affinity time were measured. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China). Thrombin time, prothrombin time and apity time measurements were performed in the following procedure.

Thrombin Time

After mixing 50 μl of 0.5 U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ and 10 μl of the sample extract at various concentrations at 37° C. in a tube of Amelung coagulometer KC-1A (Japan), reacted for 2 minutes, After adding 100 μl of plasma, the time until plasma clot was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of a sample as a solvent control. In the case of DMSO, the clotting time was 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 value of the solidification time when the sample was added divided by the solidification time of the solvent control.

Prothrombin time

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations to the tube of Amelung coagulometer KC-1A (Japan), warm at 37°C for 3 minutes, and then add 130 μl of PT reagent The time until plasma clot was expressed as the average value of the experiment repeated 3 times. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of a sample as a solvent control. In the case of DMSO, a solidification time of 16.8 seconds was shown. The prothrombin inhibitory activity was expressed as a value obtained by dividing the coagulation time when the sample was added by the coagulation time of the solvent control.

aPTT (activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of sample extracts of various concentrations were added to a tube of Amelung coagulometer KC-1A (Japan), heated at 37° C. for 3 minutes, then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added and again 37 Incubated for 3 minutes at ℃. After 50 μl CaCl ₂ (35 mM) was added, the time until the plasma was clot was measured. DMSO was used as a solvent control instead of a sample, and in this case, a clotting time of 42.2 seconds was exhibited. The result of aPTT was expressed as the average value of the experiment repeated 3 times, and the blood coagulation factor inhibitory activity was expressed by dividing the aPTT at the time of adding the sample by the aPTT of the solvent control.

[Table 2] Blood coagulation inhibitory activity of various sesame leaf extract

As a result, the hot water extract of sesame leaf increased thrombin time, prothrombin time, and apity time by 1.62, 1.05, and 1.61 times at 5 mg/ml concentration, showing good thrombin inhibition and blood coagulation factor inhibition, and ethanol extract 5 mg At a concentration of /ml, thrombin time, prothrombin time, and ap time were increased by 1.29, 1.60, and 1.36 times, respectively, to show anticoagulant activity.

Example 3: Platelet aggregation inhibitory activity of sesame leaf hot water extract

Table 1 shows the results of evaluation of human platelet aggregation inhibitory activity of the sesame leaf hot water extract of Example 1. Platelets are discoid-shaped small cells that circulate blood vessels with various blood cells, and instead of having nuclei, they have cytoplasmic granules containing high concentrations of various substances related to vascular damage protection and platelet aggregation, and aggregation when damage to the vascular lining appears It is an important cell that secretes factors and combines with collagen exposed due to damage to endothelial cells to form a primary hemostatic plug to initiate thrombosis. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombus production. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Human platelets were used as 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, after resuspending 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), at 3,000 rpm After centrifugation for 10 minutes, the suspension was suspended again in a suspending buffer, and the platelet number was adjusted to be 4x10 ⁹ /ml. Thereafter, 2.5 μl collagen was added to the 1 ml suspension and reacted 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 hot water extract

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

Example 4: Preparation of sequential organic solvent fractions of sesame leaf extract and their component analysis

The various sesame leaf extracts of Example 1 were subjected to sequential organic solvent fractionation with hexene, ethyl acetate, and butanol, and finally water residues were recovered. Table 4 shows the yield of the fractions and the analysis results of the fractions.

Table 4 Comparison of sequential fraction yield and composition analysis of sesame leaf extract

As shown in Table 4, the sesame leaf hot water extract was carried out 0.1% and 3.9%, respectively, to the hexene fraction and ethyl acetate fraction, and 45.9% and 55.5%, respectively, to the butanol fraction and water residue. On the other hand, the sesame leaf ethanol extract was carried out by ethyl acetate fraction and butanol fraction by 7.4% and 8.6%, respectively, and the hexene fraction and water residue accounted for 41.0% and 46.5%. These results indicate that the sesame leaf hot water extract contains mostly water-soluble components, and the sesame leaf ethanol extract contains fat-soluble components and water-soluble components in similar amounts. In addition, it means that about 0.865 g of the ethyl acetate fraction of the hot water extract and about 0.72 g of the ethyl acetate fraction of the ethanol extract can be recovered from 100 g dried sesame leaves.

On the other hand, as a result of analyzing the total polyphenol and total flavonoid content, the highest content in the ethyl acetate fraction (318~377 mg/g polyphenol and 146~273 mg/g flavonoid), various physiological activities of sesame leaf extract are ethyl acetate fraction It was expected to appear in

Example 5: Evaluation of blood coagulation inhibitory activity of sequential organic solvent fraction of sesame leaf extract

The blood coagulation inhibitory activity of the organic solvent fraction 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 activity of organic solvent fractions obtained from sesame leaf extract

As shown in Table 5, aspirin showed excellent blood coagulation inhibitory activity by extending the thrombin time, prothrombin time, and apity 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 the sesame leaf hot water extract, strong anticoagulant activity was confirmed in the ethyl acetate fraction, and the thrombin time, prothrombin time, and apity time were extended 1.53 times, 2.36 times, and 2.59 times compared to the additive-free group at a concentration of 5 mg/ml. Prothrombin time was extended by 4.14 times at a concentration of 7 mg/ml, but both thrombin time and apity time were extended by 15 times or more to show strong antithrombotic activity. In addition, the thrombin time was specifically extended in the case of the water residue of the hot water extract to exhibit thrombin specific inhibitory activity. On the other hand, the fraction of sesame leaf ethanol extract showed concentration-dependent strong anticoagulant activity in both the hexene fraction, ethyl acetate fraction, and butanol fraction, especially in the case of ethyl acetate fraction, thrombin time, prothrombin time, and apity at 5 mg/ml concentration. The time was extended 15 times, 6.21 times, and 9.1 times compared to the additive-free group, and at 7 mg/ml, all were extended 15 times or more to show the strongest antithrombotic activity. This strong activity was found to be comparable to aspirin used in clinical practice. Therefore, it is judged that the ethyl acetate fraction of sesame leaf extract can be developed as a commercial antithrombotic agent, and it can be replaced with aspirin, an existing antithrombotic agent that exhibits side effects such as gastrointestinal disorders.

Example 6: Human red blood cell hemolytic activity of sesame leaf extract and its fractions

Sesame leaves have been edible as ssam vegetables and spices for a long time, and are currently registered as food raw materials in Korea to ensure safety. In the present invention, to evaluate the acute toxicity of sesame leaf extract and its fractions, human red blood cell hemolytic activity was evaluated, and the results are shown in Table 6. At this time, the hemolytic activity was evaluated according to the previous report (Kim Mi-sun et al., 2014. J. Life Sci. 24: 515-521), and simply added 100 μl of human red blood cells washed three times with PBS to a 96-well microplate. After adding 100 μl of various concentrations of the sample solution and reacting for 30 minutes at 37° C., the reaction solution was centrifuged for 10 minutes (1,500 rpm) to transfer 100 μl of the supernatant to a new microtiter plate and the degree of hemoglobin outflow due to hemolysis was determined. Measured 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 red blood cell hemolytic activity of sesame leaf extract and its fractions

As a result, as shown in Table 6, first, DMSO and water used as controls did not have hemolytic activity, and triton X-100 confirmed that hemolysis of 100% of red blood cells was performed at a concentration of 1 mg/ml. On the other hand, the sesame leaf extract had no hemolytic activity in both the hot water extract and the ethanol extract. However, the hexene fraction of the ethanol extract showed a hemolytic activity of 47.9% at a concentration of 1 mg/ml, and no hemolytic activity was observed in other fractions and water residues. Therefore, it is judged that sesame leaf hot water extract, ethanol extract, their ethyl acetate fraction, butanol fraction, and water residue will not exhibit a problem of inducing separate acute toxicity.

Example 7: Evaluation of plasma, acid and thermal stability of sesame leaf extract and its fractions

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

Claims (6)

A blood clotting inhibitor that contains Leaf of Perilla extract as an active ingredient. According to claim 1, wherein the sesame leaf extract is a blood coagulation inhibitor characterized in that the sesame leaf hot water extract or sesame leaf ethanol extract. The blood coagulation inhibitor according to claim 2, wherein the sesame leaf hot water extract is an ethyl acetate fraction obtained by sequentially fractionating a sesame leaf hot water extract with an organic solvent of hexene, ethyl acetate, and butanol, a water residue, or a mixture thereof. The blood coagulation inhibitor according to claim 2, wherein the sesame leaf ethanol extract is an ethyl acetate fraction, a 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. delete delete