KR20180138480A - Pharmaceutical composition comprising ethanol extracts of seedpod 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 food for preventing or treating thrombosis, comprising an ethanol extract of seedpods of Nelumbo nucifera as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition and a functional food for preventing or treating thrombosis through potent inhibition of platelet coagulation and inhibition of blood coagulation, comprising an ethanol extract of seedpods of Nelumbo nucifera, an organic solvent fraction of the extract, or residual water remaining after fractionation, as an active ingredient. The active ingredient in a pharmaceutical composition and a functional food for preventing or treating thrombosis according to the present invention, the ethanol extract of seedpods of Nelumbo nucifera, shows potent anti-thrombotic activities due to inhibitory activities on coagulation of platelets, which serve to initiate thrombus formation, as well as inhibitory activities on enzymes associated with thrombus formation and blood-clotting factors, while showing no hemolytic activity to human red blood cells; has excellent thermal stability; and shows no decrease in inhibitory activities on blood-clotting factors and inhibitory activities on enzyme related to thrombus formation, even in an acid environment of pH 2 and inside blood plasma, and thus, may be beneficially used in a pharmaceutical composition and a functional health food that prevent or treat/alleviate thrombosis such as ischemic stroke and hemorrhagic stroke, through improving blood circulation. Further, the active ingredient of the present invention may be processed in various forms that can be ingested 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 The present invention relates to a pharmaceutical composition for the prevention or treatment of thrombosis, which contains ethanol extract as an active ingredient, and a pharmaceutical composition for treating or preventing thrombosis. SAME}

The invention ligatures room (Seedpod of Nelumbo The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis which contains an ethanol extract of nucifera as an active ingredient and more specifically, The present invention relates to a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis through inhibition of strong platelet aggregation inhibition and blood coagulation inhibition containing the residue as an active ingredient.

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, these anticoagulants and thrombolytic agents are not only very expensive but also have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.

Nelumbo nucifera ) is a perennial aquatic plant with dicotyledonous plants and buttercups. The subterranean stalks that pass through the mud are nodules, white and thin, and become broaden in the autumn. 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. It is used for medicinal and edible purposes in all areas without discarding kite. It is used for seedling (lotus root), seed (soft liquor, rice, livestock), embryo in seed, ovary, (Ridiculous) are also used for medicinal purposes. Particularly, the lecture room is also called the federation, and some of the lecture rooms are drunk after being dried, but most of them are only used for ornamental use by disposing of the lecture after harvesting.

The antioxidant activity of procyanidins (Ling ZQ et al., 2005, J Agric Food Chem. 53, 2441-2445) and the autophagy-inducing activity in human liver cancer cells are known Y, et al., 2016, Biomed Pharmacother. 79, 135-152), and the ability to protect against radioactivity by acetone-water extract in a stool chamber (Duan Y et al., 2010, Food Chem Toxicol. 48, 3374-3384). In addition, the stator chamber can be used for oxidative stress relaxation (Luo X et al., 2016, Biomed Pharmacother. 82, 640-648), neuronal cell protection (Yin C et al., 2016, Biomed Pharmacother. 82, 628- 639), prevention of impaired immune function (Zhang H et al., 2016, Biomed Pharmacother. 82, 364-372). On the other hand, Xiao JS et al. Reported separation of polymerized procyanidins and quercetin glucuronide from a continuous chamber. In Korea, the study on lecture room is at an early stage, and the addition of lectin powder (10%) during the high fat diet method has been reported to lower blood triglyceride levels (Kim, Yong-hwan, 684-691). However, the inhibition of platelet aggregation and the activity of inhibiting blood clotting have not been reported up to now.

Patent No. 10-1529227 of Korea Patent No. 10-1529227 discloses a cosmetic composition for whitening improvement containing a cosmetics and a cosmetics and a cosmetic composition containing the same, A composition for preventing or treating immune thyroid disease], and Japanese Patent Laid-open Publication No. 10-2016-0090098 discloses a composition for anti-aging and whitening using an extract of a sweet potato containing a large amount of polyphenol component, 10-2016-0148824 discloses [a composition for the prevention and treatment of allergic diseases or inflammatory diseases comprising an extract of soft and dry leaves as an active ingredient]. However, there is no known patent for an active extract of a sweet potato which exhibits a potent antithrombotic activity through inhibition of blood clotting and inhibition of platelet aggregation and is not toxic to date.

To date, reports of antithrombotic activity related to lead have been reported by Zhou et al. (2013) that neferine isolated from the seed embryo of seeds inhibits platelet aggregation and thus exhibits antithrombotic activity (Zhou YJ et al., 2013 Thrombosis Res. 132, 202-210) is the only situation.

KR 10-2016-0090098 A

 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 keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a pharmaceutical composition and a pharmaceutical composition for preventing or treating thrombosis, Functional foods.

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

Preferably, the soft spinning ethanol extract is a hexane fraction, an ethyl acetate fraction, a butanol fraction or a water residue obtained by successively fractionating the soft spinning ethanol extract with hexene, ethyl acetate and butanol.

In addition, the present invention relates to a method and apparatus for detecting the presence or absence of a seedpod of Nelumbo The present invention provides a health functional food for preventing or ameliorating thrombosis comprising an ethanol extract of Nucifera as an active ingredient.

Preferably, the soft spinning ethanol extract is a hexane fraction, an ethyl acetate fraction, a butanol fraction or a water residue obtained by successively fractionating the soft spinning ethanol extract with hexene, ethyl acetate and butanol.

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the extract of liquorice ethanol as an active ingredient of the health functional food inhibit the coagulation inhibition effect of platelets which inhibits thrombogenesis-related enzymes and blood coagulation factors, And exhibits excellent hemostatic activity against human erythrocytes, exhibits excellent thermal stability, exhibits an acidic condition of pH 2 and a loss of blood coagulation factor inhibitory effect and thrombogenesis-related enzyme inhibitory effect even in plasma It is expected that it can be used as a pharmaceutical composition and a health functional food for prevention and treatment / improvement of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredient is expected to be used as an extract, powder, Etc., so that it can be prepared at any time. It is a very useful invention in the pharmaceutical industry and the food industry because of its excellent effect.

Fig. 1 is a photograph of a lotus leaf, a lecture room, a lecture room, a lotus root,
FIG. 2 is a graph showing the thrombin inhibition according to the concentration of the ethanol extract of the sweet potato as thrombin time,
FIG. 3 is a graph showing the prothrombin inhibition by the concentration of the ethanol extract of the soft potato as prothrombin time,
FIG. 4 is a graph showing the degree of inhibition of blood coagulation factor according to the concentration of the extract of ethanol in soft boiled water,
(DMSO), 2: aspirin (0.25 mg / ml), and 3: aspirin (0.5 mg / ml) in the sequential organic solvent fraction. 6: extract of hexane fraction (0.125 mg / ml) of ethanol extract of soft potato, 7: extract of ethanol extract of soft potato (0.25 mg / ml) (0.25 mg / ml) of ethanol extract, 8: ethyl acetate fraction (0.125 mg / ml) of ethanol extract of soft spinach, 9: butanol fraction (0.25 mg / ml) of ethanol extract of soft spinach, 10: (0.125 mg / ml) of ethanol extract, 11: water residue (0.25 mg / ml) after sequential organic solvent fractionation of the ethanol extract of the spinach, 12: water residue after sequential organic solvent fractionation of ethanol extract 0.125 mg / ml), respectively.

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 anti-thrombocyte active component from the extract of the sweet potato, and the extract and the fraction had hemolytic activity , The extracts and fractions were used as pharmaceutical compositions and health functional foods for the prevention, treatment, and improvement of thrombosis.

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. Afterwards, the extracts of the sweet potatoes, which showed strong activity, were prepared with sequential organic solvent fractions with hexene, ethyl acetate and butanol. Finally, the water residues were recovered 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 the evaluation of the inhibitory activity, it was confirmed that the extract of the soft - room ethanol and its fractions and the water fractions exhibited a strong coagulation inhibitory activity and a platelet aggregation inhibitory activity. 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 extracts of the soft-boiled ethanol, the active fractions and the water residues do not show hemolytic activity on human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis comprising an extract of Seedpod of Nelumbo nucifera as an active ingredient.

Preferably, the soft spinning ethanol extract is a hexane fraction, an ethyl acetate fraction, a butanol fraction or a water residue obtained by successively fractionating the soft spinning ethanol extract with hexene, ethyl acetate and butanol.

In addition, the present invention relates to a method and apparatus for detecting the presence or absence of a seedpod of Nelumbo The present invention provides a health functional food for preventing or ameliorating thrombosis comprising an ethanol extract of Nucifera as an active ingredient.

Preferably, the soft spinning ethanol extract is a hexane fraction, an ethyl acetate fraction, a butanol fraction or a water residue obtained by successively fractionating the soft spinning ethanol extract with hexene, ethyl acetate and butanol.

Hereinafter, the extract of the soft-boiled ethanol 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.

As a preferred embodiment, the present invention provides a method for preparing an extract, comprising: Evaluation of antitumor activity of various kite extracts; Preparing sequential organic solvent fractions of hexane, ethyl acetate, and butanol from a water softener extract that exhibits potent antithrombotic activity, and then preparing a water residue obtained therefrom; Evaluating the antithrombotic activity of the extract and the fractions and examining the stability of the active substance.

The "soft drink ethanol extract " included in the pharmaceutical composition and health functional food of the present invention comprises a step of harvesting the matured quartz chamber; Removing the string in the string room; Suspending the softened room at room temperature, or drying the softened room at 60 to 70 DEG C for 2 to 3 days; Crushing the dry soft room; Extracting the ground powder with ethanol; And filtering the extract using a filter net of 0.06 mm or less and concentrating it under reduced pressure.

The "soft pot extract of ethanol " contained in the pharmaceutical composition and the health functional food of the present invention is a fraction of hexane, ethyl acetate, and ethyl acetate obtained by sequentially or fractionally fractionating the ethanol extract with an organic solvent of hexane, ethyl acetate and butanol, Butanol fraction or water residue.

In the present invention, the thrombin time, the prothrombin time and the apathy time were measured at a concentration of 5 mg / ml of the ethanol extract of the soft-boiled water, and the blood coagulation time was extended more than 15 times as compared with the non- Respectively. In addition, the human platelet aggregation inhibitory activity was measured at a concentration of 0.25 mg / ml of the ethanol extract of the soft potato, and the platelet aggregation inhibitory activity was more potent than the aspirin 0.5 mg / ml concentration treatment.

As a result of evaluating the blood coagulation inhibitory activity of the organic solvent fraction and the water residue of the ethanol extract of the sweet potato, concentration-dependent inhibition of thrombogenesis was observed in all the fractions. Especially, in the butanol fraction and water residue, 5 ~ 10 times more potent thrombin, prothrombin and blood coagulation factor inhibition. As a result of evaluating platelet aggregation inhibition activity of the organic solvent fraction and the water residue of the ethanol extract of the soft potato, the concentration dependent platelet aggregation inhibitory activity was confirmed in all the fractions.

Therefore, it has been confirmed that the ethanol extract and active fractions thereof in the sweet potato can be developed as anticoagulants and antiplatelet agents capable of replacing aspirin, which is highly likely to cause side effects such as gastrointestinal disorders.

The soft spinning ethanol extract of the present invention and its active fractions can be made into powders through conventional powdering processes such as vacuum drying and freeze drying, spray drying and the like. They are not degraded by various degrading enzymes in the plasma and remain active even at 100 ° C heat treatment and pH 2 pH conditions on the human body.

The active ingredient of the pharmaceutical composition 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 agent include tau martin; Natural flavoring agents such as stevia such as rebaudioside A or 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  One: Lecture room  Ethanol extract and sequential organic solvent Fraction  Preparation and analysis of their components

The ethanol extracts were prepared by purchasing the cultured and harvested sweet potatoes (Fig. 1) from Mungyeong, Kyungbuk Province in 2016. The dried softener was ground to 20 mesh without any treatment. In preparing the ethanol extract, 10 times of ethanol was added to the sample, and the extract was repeatedly extracted three times at room temperature. The extracts were collected by filtration and concentrated under reduced pressure to prepare an ethanol extract. In addition, after preparing a large amount of the extract of ethanol as a softener, the fraction was fractionated with hexane, ethyl acetate and butanol sequentially, and finally the water residue was recovered. The extraction efficiency of ethanol extract, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction are shown in Table 1. Total polyphenols, total flavonoids, total sugars and reducing sugar contents were measured by compositional 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 determined by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Lecture room  Ethanol extract and its sequential Organic solvent Fraction  Comparison of yield and component analysis

As shown in Table 1, most of the ethanol extracts of the soft potatoes were 70.0% and 24.4%, respectively, of the butanol fraction and the hexane fraction, while the ethyl acetate fraction and water residues accounted for 3.5% and 3.3% of the extract, respectively. These results indicate that the ethanol extract of the soft pot contains a variety of various polar substances, and it means that about 1.61 g of butanol fraction and about 0.076 g of water residue can be recovered from 100 g of the dried soft pot. As a result of total polyphenol content analysis, the content of butanol fraction was 261.4 mg / g, which was the highest among the fractions. In the analysis of total flavonoid content, 14.5 mg / g of butanol fraction was followed by ethyl acetate fraction High content was confirmed. In addition, it exhibited a high total sugar and reducing sugar content in the butanol fraction and the water residue, and was significantly different from the characteristics of the fraction of general natural products. Therefore, various physiological activities of the ethanol extract of the soft potato were expected to appear in butanol fractions and water residues. As a result of the actual analysis, the fractions showed strong antioxidative activity and nitrite scavenging ability.

Example  2: Lecture room  Ethanol extract and sequential Organic solvent Fraction  Evaluation of blood coagulation inhibitory activity

The coagulation inhibitory activity of the extract of the soft-boiled ethanol obtained in Example 1 and its fractions was evaluated. The results are shown in Table 2 and FIG. 2 to FIG. 4. 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 the thrombin time, prothrombin time and api assay were performed as follows.

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. Then, 50 μl CaCl ₂ (35 mM) was added and 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] Lecture room  Ethanol extract and its sequential Organic solvent Fraction  Anticoagulant activity

As shown in Table 2, at 1.5 mg / ml concentration of aspirin, prolonged thrombin time, prothrombin time, and apathy time were 1.32 times, 1.37 times, and 1.43 times, respectively, showing excellent blood coagulation inhibitory activity. On the other hand, the ethanol extract of the sweet potato extended the thrombin time, prothrombin time, and apathy time at a concentration of 1.25-5 mg / ml by 15 times or more than that of the no-added solution, and the prothrombin time and apathy time were 15 More than twice and thrombin time was extended 4.43 times. The hexane fraction and the ethyl acetate fraction of the fractions of the extracts of the sweet potato also showed good concentration-dependent blood coagulation inhibitory activity, but significant potent activities were found in the butanol fractions and water residues. In the butanol fraction, prothrombin time and apathy time were extended 15 times and thrombin time was extended 3.25 times at 0.62 mg / ml concentration. For water residues, thrombin time, prothrombin time, All of the extracts were lengthened more than 15 times compared to the non - added extracts. At the concentration of 1.25 mg / ml, the thrombin time was increased by 5.25 times and the apathy time by 5.12 times. These results suggest that the ethanol extracts of the soft potato contain blood coagulation inhibitory substances with various solubilities and that the most active fraction is the butanol fraction. In addition, the present results indicate that the ethanol extract of the sweet potato and its butanol fraction and the water residue can be developed as a commercial anti-thrombotic agent, and it is possible to replace aspirin, which is a conventional antithrombotic agent showing side effects such as gastrointestinal disorder .

Example  3: Lecture room  Ethanol extract and sequential Organic solvent Fraction  Evaluation of human platelet aggregation inhibitory activity

The human platelet aggregation inhibitory activity of the extract of the sweet potato extract and its fractions obtained in Example 1 was evaluated, and the results are shown in Table 3 and FIG. 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] Lecture room  Ethanol extract and sequential Organic solvent Fraction  Human platelet aggregation inhibitory activity

As shown in Table 3, in the case of DMSO as the solvent control, human platelets rapidly and strongly aggregated by collagen addition, and aspirin, which is a platelet aggregation inhibitor, strongly inhibited platelet aggregation in a concentration-dependent manner. At this time, aspirin exhibited 38.6% aggregation at 0.25 mg / ml concentration. On the other hand, the extracts of the stigma ethanol and the hexane fractions thereof showed 39.8% and 38% aggregation inhibition similar to aspirin at the concentration of 0.25 mg / ml, respectively. In addition, the ethyl acetate fraction and the butanol fraction showed weaker activity than aspirin at the same concentration, but 51.8% and 49.3% were aggregated at a concentration of 0.25 mg / ml, respectively, and thus it was confirmed that the fraction could be sufficiently used as a platelet aggregation inhibitor. The most potent inhibition of aggregation, as seen in water residues, showed an aggregation comparable to 0.25 mg / ml of aspirin at a concentration of 0.125 mg / ml. The potent platelet aggregation inhibitory activity of the ethanol extract and its fractions of the sweet potatoes suggests that it is possible to produce an anti-thrombotic agent and a blood circulation improving health functional food by using a sweet potato. The sweet potato contains various antithrombotic ingredients having various solubilities And that the

Example  4: Lecture room  Ethanol extract and sequential Organic solvent Fraction  Human erythrocyte hemolytic activity

In the present invention, human erythrocyte hemolytic activity was evaluated in order to evaluate the acute toxicity of the ethanol extract of the sweet potato and the sequential organic solvent fraction thereof, and the results are shown in Table 4. 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, 100 μl of the sample solution was added and reacted at 37 ° C for 30 minutes. Then, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate and 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 4] Lecture room  Ethanol extract and sequential 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 haemolytic activity, showed hemolytic activity of 55.5% at a concentration of 0.00625 mg / ml and 93.7% at a concentration of 0.05 mg / ml. There was no hemolytic activity at the concentration of 1.0 mg / ml for the ethanol extract of the sweet potato, and the fraction of the ethanol extract of the soft pot did not show hemolytic activity either. Therefore, the ethanol extracts of the sweet potatoes, the sequential organic solvent fractions thereof and the water residues do not show any problem of causing acute toxicity.

Example  5: Lecture room  Ethanol extract, its sequential Organic solvent Fraction  ≪ / RTI > and plasma residue, acid and thermal stability of water residues

The plasma stability, thermal stability and acid stability of anti-thrombotic activity of the elongated ethanol extract, the sequential organic solvent fractions and water residues obtained in Example 1 were confirmed. 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)

Seedpod of Nelumbo The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, comprising an ethanol extract of nucifera as an active ingredient. The pharmaceutical composition according to claim 1, wherein the soft spinning ethanol extract is a hexane fraction, an ethyl acetate fraction, a butanol fraction or a water residue obtained by successively fractionating a soft spinning ethanol extract with hexene, ethyl acetate and butanol. Seedpod of Nelumbo nucifera ) is an effective ingredient for preventing or ameliorating thrombosis. [Claim 5] The health functional food according to claim 3, wherein the soft spinning ethanol extract is a hexane fraction, an ethyl acetate fraction, a butanol fraction or a water residue obtained by successively fractionating the soft spinning ethanol extract with hexene, ethyl acetate and butanol.

Images