KR20190009929A - Pharmaceutical composition comprising the extraction of leaf of nelumbo nucifera 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 functional health food for preventing or treating thrombosis, comprising extracts of Nelumbo nucifera leaf, and more specifically, to a pharmaceutical composition and a functional health food for preventing or treating/alleviating thrombosis through potent platelet coagulation suppression and blood coagulation suppression, which comprise, as active ingredients, hot water extracts or ethanol extracts of Nelumbo nucifera leaf, hexene fractions thereof, ethyl acetate fractions thereof, and butanol fractions thereof. The extracts of Nelumbo nucifera leaf, as the active ingredients of the pharmaceutical composition and the functional health food for preventing or treating thrombosis of the present invention, show potent anti-thrombotic activities due to inhibitory activities on platelet coagulation, which serves to initiate thrombus formation, as well as inhibitory activities on thrombus formation-associated enzymes and blood-clotting factors, while showing no hemolytic activity to human erythrocytes; have excellent thermal stability; and show no decrease in inhibitory activities on blood-clotting factors and inhibitory activities on thrombus formation-associated enzyme, even in an acidic environment at pH 2 and in blood plasma, and thus, may be beneficially used in a pharmaceutical composition and a functional health food that prevent or treat thrombosis, such as ischemic stroke and hemorrhagic stroke, through improving blood circulation. Further, the active ingredients of the present invention may be processed in various forms and ingestible at any time, such as extracts, powder, pills, tablets, and the like, and thus may be extremely useful in the drug and food industries.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, which comprises an extract of a lotus leaf extract as an active ingredient, and a health functional food. [0002] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis,

The invention lotus leaf (Leaf of Nelumbo The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis which contains an extract of Nucifera as an active ingredient and more specifically to a composition comprising a hot-water extract or an ethanol extract of a lotus leaf and a hexane fraction thereof, an ethyl acetate fraction And a butanol fraction as active ingredients, and to a pharmaceutical composition and health functional food for preventing or treating / improving thrombosis through inhibition of blood coagulation.

As a constituent of human body, blood has various important functions such as oxygen and nutrients, the function and buffering function of waste products, maintenance of body temperature, control of osmotic pressure and maintenance of ion balance, maintenance of moisture, regulation of fluidity, maintenance and regulation of blood pressure, have. Normal blood circulation facilitates blood circulation by complementary regulation of the blood coagulation system and thrombolysis system in the body. Among them, the mechanism of the blood coagulation system is that the platelets adhere to the blood vessel walls and coagulate to form platelet thrombus , It is reported that the blood coagulation system is activated and fibrin thrombus is formed centering on the platelet aggregation mass.

On the other hand, the production of fibrin thrombus is activated by thrombin involved in fibrin clotting through several steps of many blood coagulation factors to finally produce fibrin monomers from fibrinogen. Fibrin monomers are polymerized by calcium, Cells to form a cross-linked fibrin polymer by factor XIII and produce a permanent thrombus. In addition, thrombin plays a pivotal role in thrombus formation by activating platelet, V factor, and Factor VII to promote blood coagulation. Therefore, the activity inhibitor of thrombin can be used as a prophylactic and therapeutic agent very useful for various thrombotic diseases caused by excessive blood coagulation. On the other hand, prothrombin activation after sequential activation of factor XII, factor XI, factor IX and factor X is known to activate thrombin in the endogenous thrombogenesis pathway, so that specific inhibition of blood coagulation factors is also important. It is becoming a target. To date, various anticoagulants such as heparin, coumarin, aspirin and europine have been used for the prevention and treatment of thrombotic diseases. However, they are not only very expensive but also have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.

Meanwhile, Nelumbo nucifera ) is a perennial aquatic plant with dicotyledonous plants, and is a good substitute for rice, which is a good crop to plant in rivers, marshes and wetlands and to purify water with the decrease of carbon dioxide. . The undergrowth of the undergrowth in the mud has nodes, is white and thin, and grows in the autumn to become a lotus root. In this lotus root, there are stalks and blooming stalks, flowers bloom in summer, and fruit blooms in autumn. The fruit is in the ovary called the honeycomb, which is called honeycomb, and the seed is called the softener. Kite is used for medicinal and edible purposes in all areas without discarding it. It is used for medicinal and edible purposes. It can be used for various purposes such as rootstocks, seeds (seeds), seeds (embryos), ovaries It is also used as a medicinal part.

Especially, the lotus leaf is dried by the leaf of the kite and tastes, and the nature is good. It has been called as bureaucratic in the civilian and oriental medicine since ancient times and is called hemorrhagic gastric ulcer, gastritis, hemorrhage, diarrhea, headache and dizziness, , Enuresis, and detoxification. As the edible product, the lotus leaves are limited to the lotus leaf tea and the lotus leaf tea, and they are used for some alcoholic liquor production (Yuhanna et al., 2011, The Korean Society of Culinary Science 27, 577-587). In recent years, studies on the production of various processed foods using lotus leaf and lotus leaf powder have been carried out. For example, The Korean Journal of Food Preservation 21, 328-333), the bread with the addition of lotus leaf powder (Park, Sang-Ha et al., 2009, Korean Journal of Food Preservation 16, 47-52) ) Is known.

Studies on the useful components of Korean lotus leaf have been reported to have alkaloid components such as analgesic, sedative roemerine, nuciferin, armepavine, N-nor-nuciferine and pronuciferine and tartaric acid, citric acid, malic acid, succinic acid and tannin (2008), Phloroglucinol and pyrocatechol, which have excellent antioxidant and antimicrobial activity, have been isolated from the leaves of Yeonhak (Korean Journal of Food Science and Nutrition, 2008, Hoseo University). In addition, the antioxidative effect of Lee et al. (Lee, KS et al., 2006. J. Korean Soc Food Sci Nutr 35: 182-186) and the antibacterial effect (Lee KS et al., 2006. J. Korean Soc Food Sci Nutr 35: 219-223), the inhibitory effect of starcholytic enzymes on Ko (et al., 2006, Korean J. Food Sci. Technol. 38: 114-120), Lee et al. (Shin MK and Han SH 2006. Korean J. Food Culture 21: 202-208) have been known to suppress blood cholesterol and neutral lipid metabolism by Shin et al. (Park KM et al., 2016, Journal of Photochemistry and Photobiology. B, Biology 161, 1011-1344), whitening effect (Yudansae et al., 2015, Journal of Life Science 25, 1115-1123) (Rajendra K, 2008; Majo University, Rajendra K, 2012, Evidence-based Complementary and Alternative Medicine, 153568) Libin D, 2000, Chinese Traditional Herbal drugs 31, 526-527), anti-diabetic effect (Sharma BR et al., Chinese Traditional Herbal drugs 36, 71-77), anti-obesity effect (Onoa Y, 2006. J. Ethnopharmacol. : 238-244; Huna D et al., 2010, Journal of Biomedical Science 17, suppl. 1, 42). However, no strong antithrombotic activity has been reported to date due to inhibition of blood clotting activity and inhibition of platelet aggregation. Recently, there has been a report on the effect of improving the blood circulation of functional natural fermented food using lotus, lotus leaf, and lotus root (Hongjong, 2016. Research Report), but it shows antithrombotic activity through fermentation. Respectively.

Patent No. 10-1578595 of Korean Patent No. 10-1578595 discloses a hand disinfectant containing a lotus leaf extract and a method for producing the same. Cholesterolemia or obesity], 10-1558530 [Method for producing an anti-obesity functional tea containing fermented ginseng leaf, lotus leaf and green tea as an active ingredient, and an anti-obesity functional tea prepared by the above method] , 10-1658618 [Diet food composition using a lotus leaf and its preparation method], 10-1625366 [Soybean oil containing lotus leaf and its preparation method] and 10-0583088 [A lotus leaf tea containing lotus leaf tea [Production method], 10-0863817 [Lotus leaf tea and its preparation method], 10-0395872 [Bleached leaf tea production method], 10-1121980 [Method of manufacturing lotus leaf extract and the extract of lotus leaf and its Containing composition], 10-09788 49] discloses a [chlorella suspension for fish feed containing lotus leaf extract]. Korean Patent Application No. 10-2008-0019675 discloses a pharmaceutical composition and health food having diabetic prophylactic and therapeutic effect containing extract extracted from a lotus leaf, in Patent Application No. 10-2010-0031301, A composition for reducing blood sugar containing a lotus leaf extract extracted as a solvent], and 10-2003-0003828 discloses a process for producing a hangover-resolved beverage containing a lotus leaf extract and a method for producing the same.

On the other hand, as a patent relating to the anti-thrombotic activity of yeast, 2 ~ 20 parts by weight of a lotus root was added to soybean fermented chungkukjang powder to Korean patent No. 10-0722361 [ The present inventors have found that the prothrombin time is not changed and the aprotic time is shortened and the thrombogenic activity is promoted rather than the prothrombin time. However, because of the error in interpretation of results, the antithrombotic activity There is a problem that is described.

It is noteworthy that the antithrombotic activity reported to date in relation to kinship is due to neferine, a lipid soluble bis-benzylisoquinoline alkaloid isolated from seed embryo, (Zhou YJ et al., 2013. Thrombosis Res. 132, 202-210), but neferine is limited to chloroform, dichloromethane, ethylacetate, acetone and rarely detected in other parts of the vertebrate . In addition, the content of neferine is below 0.25% at the maximum in the simulated seawater. Therefore, it is not possible to predict the antithrombotic activity of the plant itself, which is not pure neferine. Therefore, no strong antithrombotic activity has been reported to date due to the inhibition of blood coagulation and the inhibition of platelet aggregation, especially in the leaves.

KR 10-0722361 B

 Zhou YJ et al., 2013. Thrombosis Res. 132, 202-210 [Neferine exerts its antithrombotic effect by inhibiting platelet aggregation and promoting dissociation of platelet aggregates]

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and an object of the present invention is to provide a pharmaceutical composition for preventing or treating a thrombotic disease, .

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis comprising Leaf of Nelumbo nucifera extract as an active ingredient.

The lotus leaf extract is preferably a lotus leaf hot water extract or a lotus leaf ethanol extract.

It is preferable that the long-leaf ethanol extract is selected from the group consisting of a hexane fraction, an ethyl acetate fraction and a butanol fraction of a lotus leaf extract.

The invention also lotus leaf (Leaf of Nelumbo The present invention provides a health functional food for preventing or ameliorating thrombosis comprising an extract of Nucifera as an active ingredient.

The pharmaceutical compositions for the prevention or treatment of thrombosis of the present invention and the leaf extract of leaves as an active ingredient of the health functional food are effective for inhibiting thrombogenesis-related enzymes and blood coagulation factors as well as for inhibiting the aggregation of platelets, Exhibit strong antithrombotic activity, exhibit no hemolytic activity on human erythrocytes, exhibit excellent thermal stability, exhibit an acidic condition of pH 2 and a loss of blood coagulation factor inhibitory effect and thrombogenic enzyme-inhibiting effect even in plasma Therefore, it is expected that it can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. The active ingredient can be processed into various forms such as extract, powder, The pharmaceutical industry and the pharmaceutical industry, Industrially very useful inventions.

(DMSO), 2: aspirin (0.25 mg / ml), and 3: aspirin (0.5 mg / ml) in the human platelet aggregation inhibitory activity of the ethanol extract of the lotus leaf and the sequential organic solvent fraction thereof. ml), 4: Ethyl acetate fraction (0.25 mg / ml), 4: Yeast leaf ethanol extract (0.25 mg / ml), 5: Hexane fraction (0.25 mg / ml) of the extract, and 8: water residue (0.25 mg / ml) of the ethanol extract of the lotus leaf after the organic solvent fractionation.

Hereinafter, the present invention will be described in detail.

In order to test the anti-thrombotic efficacy against kites, the inventors of the present invention collected various parts of the yeast to prepare extracts of each part by a certain method, evaluated human platelet aggregation inhibitory activity and blood coagulation inhibitory activity, , And then the hexane fraction, the ethyl acetate fraction, the butanol fraction and the water residue after the organic solvent fraction were recovered as an antithrombotic active ingredient from the extract of the lotus leaf ethanol, and the extract and the fraction had hemolytic activity against human erythrocytes The extracts and fractions were used as pharmaceutical composition and health functional food for prevention or treatment / improvement of thrombosis by confirming that they have excellent heat stability and acid stability.

In order to develop a pharmaceutical composition and a health functional food for preventing or treating / improving thrombosis using a star which is known to have excellent blood circulation improvement effect in a private sector, The antithrombotic activity was evaluated. After that, the extracts of the extracts of long-leafed leaves, which showed strong activity, were successively fractionated with hexene, ethyl acetate and butanol. Finally, water residues were prepared after the fractionation. The antithrombotic activity of each of the extracts and fractions was compared with that of thrombin time, prothrombin time and activated partial thromboplastin time (aPTT) and platelet aggregation for human plasma and human thrombin As a result of evaluation through inhibition activity, strong coagulation inhibitory activity and platelet aggregation inhibitory activity were confirmed in ethanol extract of lotus leaf and its hexane fraction, ethylacetate fraction and butanol fraction. This anti-thrombotic activity was strong antithrombotic activity exceeding that of aspirin which is used clinically as antithrombotic agent. In addition, it was confirmed that the lotus leaf extract and the active fractions did not show hemolytic activity on human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, which comprises a lotus leaf extract as an active ingredient.

The lotus leaf extract is preferably a lotus leaf hot water extract or a lotus leaf ethanol extract.

It is preferable that the long-leaf ethanol extract is selected from the group consisting of a hexane fraction, an ethyl acetate fraction and a butanol fraction of a lotus leaf extract.

The present invention also provides a health functional food for preventing or ameliorating thrombosis, which comprises a lotus leaf extract as an active ingredient.

Hereinafter, the lotus leaf extract of the present invention, the method for producing the respective active fractions, the experiment of efficacy and the like will be described in more detail.

The present invention relates to a method for preparing an extract, comprising the steps of: preparing an extract from a lotus leaf; Evaluating the antithrombotic activity of the maple leaf extract; Preparing a sequential organic solvent fraction of hexane, ethyl acetate, and butanol from a lotus leaf ethanol extract showing strong antithrombotic activity, and preparing a water residue obtained thereafter; And the step of evaluating the antithrombotic activity and the step of examining the stability of the extract and the fraction.

The "lotus leaf extract" contained in the composition of the present invention comprises the steps of harvesting the matured lotus leaves; Drying the long leaves at 60 to 70 ° C for 2 to 3 days; Milling the dried lotus leaves; Extracting the ground powder with a solvent, filtering the extract using a filter net of 0.06 mm or less, and concentrating it under reduced pressure.

The solvent to be used in the present invention may be selected from the group consisting of water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol), a mixed solvent of the lower alcohol and water Most preferred is hot water, or 95% ethanol extraction.

As a preferred embodiment, the lotus leaf extract of the present invention can be used by extracting the lotus leaf with hot water or ethanol. Herein, the hydrothermal or ethanol extract may be sequentially or separately fractionated with an organic solvent of hexene, ethyl acetate and butanol to obtain a hexane fraction, an ethyl acetate fraction, a butanol fraction and a water residue.

In the present invention, thrombin time, prothrombin time, and apathy time were measured at a concentration of 5 mg / ml of hot water extract of longevity, and blood clotting times were extended by 15 times, 0.98 times, and 1.51 times Strong thrombin inhibition and inhibition of blood coagulation by inhibition of coagulation factors were confirmed. However, hardy leaf extract showed little inhibitory activity on human platelet aggregation at a concentration of 0.25 mg / ml.

On the other hand, when thrombin time, prothrombin time, and apathy time were measured at a concentration of 5 mg / ml of ethanol extract of oranges, the blood coagulation time was prolonged by 2.97 times, 0.88 times, and 1.17 times, respectively, Inhibition of blood coagulation by inhibition of coagulation factors was confirmed. The anticoagulant activity was increased in a dose - dependent manner. At 7 mg / ml, thrombin time was extended by 8.75 times as compared with no - added. In addition, the human platelet aggregation inhibitory activity was measured at a concentration of 0.25 mg / ml of ethanol extract of the lotus leaf and the activity was similar to the platelet aggregation inhibitory activity at the concentration of 0.25 mg / ml of aspirin. Therefore, it was confirmed that the ethanol extract of the lotus leaf can exhibit more potent antithrombotic activity than aspirin through its excellent anticoagulant activity and strong platelet aggregation inhibitory activity.

Based on the above results, the organic solvent fractions and the water residues of the ethanol extract of oranges were prepared, and their blood coagulation inhibitory activities were evaluated. As a result, the concentration-dependent inhibition of thrombogenesis in all the fractions was confirmed, Acetate and butanol fractions showed very strong antithrombotic activity. In particular, in the ethyl acetate fraction, it was confirmed that thrombin time, prothrombin time, and apytime time were 5 to 15 mg / ml. Among the fractions of ethanol extract of the lotus leaf, the fraction of hexane showed about 50% inhibition activity of platelet aggregation at the concentration of 0.25 mg / ml, and each fraction showed antithrombotic activity by different mechanism. Therefore, it has been confirmed that the ethanol extract of the lotus leaf and its active fractions can be developed as an anticoagulant and an antiplatelet agent capable of replacing aspirin, which is highly likely to cause side effects such as gastrointestinal disorders.

The extract of the lotus leaf extract of the present invention and its active fractions can be made into powders through a conventional powdering process such as vacuum drying, freeze drying, spray drying and the like. They are not degraded by various degradation enzymes in the plasma, and maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases associated with thrombosis. Such diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, , Deep vein thrombosis, lower limb edema, pain, and acute peripheral artery occlusion. Vein thrombosis includes deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition containing the active ingredient of the present invention may be formulated into tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, injections of sterilized injection solutions And may be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, 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 flavoring agent, an emulsifying agent, an antiseptic, and the like.

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

Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Perfumes, preservatives, and the like.

As a preferable specific example, the preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester 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 the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered 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, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

As a preferable example, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight per day Or every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell.

In the present invention, the term "object" includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , Preferably a mammal, more preferably a human.

In addition, the health functional food of the present invention can be variously used for foods and beverages effective for prevention or improvement of thrombosis. Examples of foods containing the active ingredient of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they can be used in the form of powder, granule, tablet, capsule or beverage .

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

The health functional food of the present invention may contain, as an additional ingredient, a food-acceptable food-aid additive such as natural carbohydrates and various flavors, in addition to containing the above-mentioned compound as an essential ingredient in the indicated ratio.

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

Examples of the flavoring agents include natural flavoring agents such as tautatin, rebaudioside A and stiglycerin such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably 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 may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio 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.

Hereinafter, the present invention will be described in more detail by way of examples. The following examples are only exemplary embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[ Example ]

Example  1: lotus leaf Heat number  Preparation of extracts and ethanol extracts and analysis of their components

In 2016, a lotus leaf cultivated and harvested in Mungyeong, Kyungbuk Province was purchased, and its hot water and ethanol extracts were prepared, and their composition and antithrombotic activity were evaluated. Dried leaves were ground with 20 mesh without further treatment. To prepare the hot-water extract, 20 times of distilled water was added to the sample, and the mixture was repeatedly extracted twice at 100 ° C for 1 hour. The extract was collected and filtered, and then concentrated under reduced pressure to obtain a powder. In preparing the ethanol extract, 10 times of ethanol was added to the sample, and the mixture was repeatedly extracted three times at room temperature. Then, the extract was collected by filtration in the same manner as above, and concentrated under reduced pressure to prepare an ethanol extract. Total polyphenols, total flavonoids, total sugars and reducing sugar contents were measured by composition analysis. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau and 100 μl of saturated Na ₂ CO ₃ solution to 400 μl of the extract solution, leaving it at room temperature for 1 hour and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. The total flavonoid content of each sample was measured by stirring for 18 hours in methanol, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extract, 40 μl of 1N NaOH was added, and the absorbance at 420 nm was measured at 37 ° C. for 1 hour. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Heat number  Extraction efficiency and useful components of extracts and ethanol extracts

As shown in Table 1, the efficacy of extracting the leaves was 13.1%, which was 1.4 times higher than the ethanol extraction efficiency. Total polyphenols and total flavonoids were also 179.7 mg / g and 68.5 mg / g in the hot water extract, Of total polyphenols and total flavonoids were 2.42 times and 1.98 times higher than those of total polyphenols and total flavonoids, respectively. On the other hand, total sugar was 1.2 times higher than ethanol extract and reducing sugar content was 1.68 times higher than ethanol extract. Therefore, it can be seen that the lotus leaf can be expected to have excellent efficacy even when it is drunk with a hot water extraction type tea or developed with various processed products after organic solvent extraction.

Example  2: Heat number  Anticoagulant activity of extracts and ethanol extracts

The blood coagulation inhibitory activity of the hot water extract and ethanol extract prepared from Example 1 was evaluated, and the results are shown in Table 2. At this time, evaluation methods of blood clotting inhibition activity were evaluated according to the previously reported methods (Sohn et al., 2004. Kor J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14 J., < / RTI > Life Science, 20: 922-928), thrombin time, prothrombin time and apathy time were measured. Plasma was obtained from commercial control plasma (MD Pacific Technology Co., Ltd., Huayuan Industrial Area, China), and thrombin time, prothrombin time and apitime time were measured by the following procedure.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA) and 50 μl of 20 mM CaCl ₂ and 10 μl of various concentrations of sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes at 37 ° C., and 100 μl of plasma was added And the time until the plasma coagulated was measured. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 24.2 seconds. The thrombin inhibitory effect was expressed as the average value of the experiments repeated three or more times. The thrombin inhibitory activity was expressed by the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

Prothrombin time ( prothrombin  time)

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample solution to tubes of Amelung coagulometer KC-1A (Japan), heat at 37 ° C for 3 minutes, add 130 μl of PT reagent, The time from the start of the experiment to the start of the experiment was expressed as the average value of the experiments repeated three times. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 16.8 seconds. The prothrombin inhibitory activity was expressed as the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

aPTT  (activated Partial Thromboplastin  Time)

Add 50 μl of aPTT reagent (Sigma, ALEXIN ^™ ) to the tubes of Amelung coagulometer KC-1A (Japan) and incubate for 3 minutes at 37 ° C. Add 100 μl of plasma and 10 μl of various concentrations of sample extract Min. After the addition of 50 μl CaCl ₂ (35 mM), the time until the plasma coagulated was measured. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 42.2 seconds. The results of aPTT were expressed as the mean value of three repeated experiments. The activity of inhibiting blood coagulation factor was represented by aPTT divided by the aPTT of the solvent control when the sample was added.

[Table 2] Heat number  Coagulase inhibitory activity of extracts and ethanol extracts

As shown in Table 2, aspirin (commercial antithrombotic agent, trade name: Protect) used as a control showed a strong concentration-dependent inhibition of blood coagulation, and at a concentration of 5 mg / ml, thrombin time, prothrombin time, The effect was extended by 15 times or more in comparison with the non-additive. In addition, the concentration of thrombin, prothrombin time, and apathy time was extended by 1.32 times, 1.37 times, and 1.43 times, respectively, at the concentration of 1.5 mg / ml, which is the normal use concentration.

On the other hand, the hot-water extract of the lotus leaf showed a strong thrombin inhibitory activity by prolonging thrombin time, prothrombin time, and apathy time at a concentration of 5 mg / ml by 15 times, 0.98 times, and 1.51 times, respectively, At the concentration, the thrombin time and apathy time were extended more than 15 times compared to the no - added period. In the case of ethanol extract of the lotus leaf, the antioxidant activity was slightly weaker than that of the hot-water extract, but the prothrombin time, prothrombin time, and apathy time were 2.97 times, 0.88 times, and 1.17 times longer than the no- ml, respectively, which were 8.75 times, 4.61 times, and 1.48 times longer than those of the non - supplemented group, respectively, indicating strong thrombin and prothrombin inhibition. These results suggest that the extract of the lotus leaves, especially the hot - water extract and the ethanol extract, can replace the commercially available anti - thrombotic agent aspirin.

Example  3: Heat number  Inhibitory Activity of Platelet Aggregation of Extracts and Ethanol Extracts

The human platelet aggregation inhibitory activity of the hot-water extract and ethanol extract prepared from Example 1 was evaluated, and the results are shown in Table 3. < tb > < TABLE > Platelets are cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentration instead of nucleus, and circulating blood vessels together with various blood cells. It is an important cell that secretes factors and binds to collagen exposed by damage of endothelial cells to form a primary hemostatic plug to initiate thrombogenesis. Therefore, inhibition of platelet aggregation is a very important activity to prevent thrombogenesis. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity (Platelet aggregation inhibition activity)

Human platelets were used as platelets and 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). Thereafter, the cells were resuspended in a 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 incubated at 3,000 rpm for 10 minutes After centrifugation, the cells were resuspended in a suspending buffer and the platelet count was adjusted to 4 × ^{10 9} / ml. Then, 2.5 μl of collagen was added to 1 ml of the suspension, and the mixture was reacted for 5 minutes. Platelet aggregation was measured at 37 ° C using a whole-blood aggregator (Chrono-log, USA).

[Table 3] Heat number  Inhibitory Activity of Platelet Aggregation of Extracts and Ethanol Extracts

As shown in Table 3, aspirin used as an active control showed strong inhibition of platelet aggregation in a concentration-dependent manner at a concentration of 0.25-0.5 mg / ml, and it was found that the aspirin used as an antithrombotic agent in clinical practice. On the other hand, the hot water extract of the lotus leaf weakly promoted the aggregation of human platelets at the concentration of 0.25 mg / ml, so that the coagulation inhibition effect was not recognized. On the other hand, ethanol extract of lotus leaf showed an aggregation degree of 41.7% at the concentration of 0.25 mg / ml and showed similar inhibition activity of platelet aggregation similar to aspirin which is currently used as an antithrombotic agent. These results suggest that the ethanol extract of the lotus leaf can replace aspirin, a commercially used antithrombotic agent.

Example  4: Ethanol extract of lotus leaf and sequential organic solvent Fraction  Preparation and analysis of their components

In the above results, the platelet aggregation inhibitory activity and the blood coagulation inhibitory activity associated with thrombogenesis were all strong ethanol extract of the lotus leaf. Thus, a large amount of ethanol extract of the lotus leaf was prepared in the same manner as described in Example 1, followed by sequential organic solvent fractionation with hexene, ethyl acetate and butanol. Finally, the water residue was recovered. The fractional efficiency of the various fractions and the component analysis results of the fractions are shown in Table 4.

[Table 4] Ethanol extract of lotus leaf and its sequential Organic solvent Fraction  Comparison of yield and component analysis

As shown in Table 4, 50.4% of the ethanol extract of the lotus leaf was converted to the hexane fraction and 31.3% was the water residue. Ethyl acetate fraction and butanol fraction accounted for 7.8% and 15.0%, respectively, of the lotus leaf ethanol extract. These results indicate that the ethanol extract of the lotus leaf contains a variety of substances ranging from water soluble to lipid soluble. From 100g of the leaves, 4.69g of the hexane fraction, 0.73g of the ethyl acetate fraction, about 1.4g of the butanol fraction, Means that about 2.91 g can be recovered.

As a result of total polyphenol content analysis, ethyl acetate fraction showed a very high content (386.3 mg / g), followed by butanol fraction, hexene fraction and water residue. In the total flavonoid content analysis, ethyl acetate fraction showed the highest content of 149.6 mg / g, followed by hexane fraction, butanol fraction and water residue. Total sugar content was relatively high in the order of ethyl acetate fraction, butanol fraction and water residue in the order of 194.7 ~ 227.6 mg / g. The reducing sugar content was also 88.3 ~ 103.0 mg / g in the order of ethyl acetate fraction, butanol fraction, , And the hexene fraction had a total sugar content of 71.6 mg / g of reducing sugar and 74.1 mg / g of total sugar. Considering that polyphenols and flavonoids exhibit various physiological activities, the ethyl acetate fraction and the butanol fraction of the ethanol extract of the lotus leaf extract are expected to show various physiological activities.

Example  5: sequential organic solvent of ethanol extract of lotus leaf Fraction  Evaluation of blood coagulation inhibitory activity

The blood coagulation inhibitory activity of the extract of the lotus leaf ethanol obtained in Example 4 and its fractions was evaluated in the same manner as in Example 2, and the results are shown in Table 5.

[Table 5] Ethanol extract of lotus leaf and its Organic solvent Fraction  Anticoagulant activity

First, the aspirin used as the active control showed an excellent blood coagulation inhibitory activity by prolonging thrombin time, prothrombin time, and apathy time at a concentration of 1.5 mg / ml by 1.32 times, 1.37 times, and 1.43 times, respectively, / ml, prolonged thrombin time, prothrombin time, and apathy time by 15 times or more to confirm strong anticoagulant activity. In addition, the concentration of 5mg / ml of lanolin ethanol extract was 2.97, 0.88, and 1.17 times longer than that of the no-added solution at the concentration of 5mg / ml, and 8.75 times, 4.61 times, 1.48 The boat was extended. On the other hand, the fraction of ethanol extract of the lotus leaf showed a strong anticoagulant activity in a concentration dependent manner. First, the ethyl acetate fraction exhibited strong anticoagulant activity by prolonging thrombin time, prothrombin time, and apathy time at a concentration of 5 mg / ml by 15 times or more. The thrombin time and prolonged thrombin time at a concentration of 2.5 mg / Times longer than apitate time. Butanol fraction showed prothrombin time which was prolonged more than 15 times at the concentration of 6mg / ml, confirming strong prothrombin inhibition and showing stronger anticoagulant activity than aspirin 1.5mg / ml concentration. In particular, the hexane fractions exhibited an aprotic time of 15 times or more at a concentration of 6 mg / ml, thus confirming the activity of inhibiting the coagulation factor of the hexane fraction.

Therefore, the ethyl acetate fraction of the fractions showed strong inhibitory activity against thrombin, prothrombin, and blood coagulation factors, while the butanol fraction was found to be effective against prothrombin and the hexene fraction had strong inhibitory activity against blood coagulation factors. These results suggest that the ethanol extract of the lotus leaf and the organic solvent fractions thereof can be developed as a commercial antithrombotic agent and that it is possible to replace aspirin and the like which is a conventional antithrombotic agent exhibiting side effects such as gastrointestinal disorder, It is concluded that it can be used specifically for the study of inhibition of blood coagulation.

Example  6: sequential organic solvent of ethanol extract of lotus leaf Fraction  Evaluation of human platelet aggregation inhibitory activity

The human platelet aggregation inhibitory activity of the ethanol extract of the lotus leaf and its fractions obtained in Example 4 was evaluated in the same manner as in Example 3, and the results are shown in Table 6 and FIG.

[Table 6] Ethanol extract of lotus leaf and its Organic solvent Fraction  Human platelet aggregation inhibitory activity

As a result, in the case of DMSO as the solvent control, human platelets rapidly and strongly aggregated by the addition of collagen. Aspirin, which is a platelet aggregation inhibitor, strongly inhibited platelet aggregation in a concentration dependent manner, and 38.6% Respectively. On the other hand, the ethanol extract of the lotus leaf and its hexane fraction showed 41.7% and 50.2% aggregation similar to aspirin at 0.25 mg / ml concentration, respectively. However, ethyl acetate fraction and butanol fraction had little effect on platelet aggregation, and water fraction showed 152.2% aggregation at 0.25 mg / ml concentration, indicating rather than coagulation promoting effect. The result of inhibiting the platelet aggregation inhibition of ethanol extract of the lotus leaf and its hexane fraction suggests that it is possible to prepare an antithrombotic agent and a blood circulation improving health functional food using the lotus leaf and that the active ingredient of the platelet aggregation inhibitor of the lotus leaf ethanol extract is a fat soluble ingredient I am suggesting.

Example  7: Activity of leaf extract Fraction  Human erythrocyte hemolytic activity

The long leaves have been used for long time in a private house for the consumption of longevity tea and lotus leaf rice, and have been consumed in non-drinkable tea. Therefore, it was judged that acute toxicity such as erythrocyte hemolysis would not be exhibited in the extract of the lotus leaf extract and its active fraction. In this example, the acute toxicity of the active fraction of the lotus leaf extract to human hemolytic hemolysis was evaluated, and the results are shown in Table 7. The hemolytic activity was assessed in accordance with the existing report (Korean J. Microbiol. Biotechnol. 2014, 42: 285-292). In brief, 100 μl of human erythrocytes washed three times with PBS was added to a 96-well microplate, The reaction mixture was centrifuged at 1,500 rpm for 10 min. After transferring 100 μl of the supernatant to a new microtiter plate, the degree of hemoglobin leaching due to hemolysis was measured at 414 nm . Triton X-100 (1 mg / ml) was used as an experimental control for erythrocyte hemolysis. DMSO (2%) was used as a solvent control of the sample. Hemolytic activity was calculated using the following formula.

[Table 7] Ethanol extract of lotus leaf and its Organic solvent Fraction  Human erythrocyte hemolytic activity

First, DMSO and water used as controls did not have hemolytic activity, and triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. Amphoteracin B, an anticancer drug known to have hemolytic activity, showed hemolytic activity of 68.5% at a concentration of 0.0125 mg / ml and 92.6% at a concentration of 0.05 mg / ml. On the other hand, the ethanol extract of the lotus leaf and its fractions showed no hemolytic activity up to the concentration of 1 mg / ml. Therefore, the ethanol extract of the lotus leaf and its active fractions do not show any problem of acute toxicity induction.

Example  8: Ethanol extract of lotus leaf and its objection Organic solvent Fraction  Plasma, acid and thermal stability assessment

The ethanol extract of the lotus leaf and the organic solvent fraction thereof obtained in Example 4 were tested for plasma stability, thermal stability and acid stability against antithrombotic activity. The samples showed high stability due to no heat treatment at 100 ° C for 1 hour, 1 hour treatment at pH 2 (0.01M HCl), and no significant decrease in blood clotting inhibition and platelet aggregation inhibition activity even for 1 hour treatment in plasma . Therefore, it is expected that the above extracts and active fractions will maintain excellent antithrombotic activity during digestive absorption process and food production process.

Claims (4)

Leaf of Nelumbo The present invention relates to a pharmaceutical composition for preventing or treating thrombosis. [Claim 2] The pharmaceutical composition according to claim 1, wherein the leaf extract is a lotus leaf extract or a lotus leaf ethanol extract. [Claim 3] The pharmaceutical composition according to claim 2, wherein the ethanol extract of oranges is selected from the group consisting of hexane fraction, ethyl acetate fraction and butanol fraction. Leaf of Nelumbo nucifera ) as an active ingredient for the prevention or amelioration of thrombosis.

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