KR101997260B1 - Pharmaceutical composition comprising the extraction of nodulus of nelumbinus rhizoma 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 the use of Nodulus of Nelumbinis The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis which comprises an extract of rheumatoid arthritis and rhizoma as an active ingredient and more specifically to a pharmaceutical composition and a health functional food for preventing or treating thrombosis comprising an extract of rheumatic ethanol and its butanol fraction or a water residue after sequential organic solvent fraction And a pharmaceutical composition for preventing or treating / improving thrombosis through inhibition of blood clotting and a health functional food. The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the rheumatoid extract as an active ingredient of the health functional food are effective for inhibiting thrombogenesis-related enzymes and blood coagulation factors and 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.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, which contains an enteral 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 present invention relates to the use of Nodulus of Nelumbinis The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis which comprises an extract of rheumatoid arthritis and rhizoma as an active ingredient and more specifically to a pharmaceutical composition and a health functional food for preventing or treating thrombosis comprising an extract of rheumatic ethanol and its butanol fraction or a water residue after sequential organic solvent fraction And a pharmaceutical composition for preventing or treating / improving thrombosis through inhibition of blood clotting and a health functional food.

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 a number of blood coagulation factors to finally produce a fibrin monomer from fibrinogen. The fibrin monomers are polymerized by calcium, Cells, which produce permanent blood clots while forming cross-linked fibrin polymers by factor XIII. 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 europaine have been used for the prevention and treatment of thrombotic diseases. However, these anticoagulants are not only very high in price, but also have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.

On the other hand, Lotus Root (Nelumbo nucifera) is a perennial aquatic plant with dicotyledonous plants. It has a node in the mud and has a node. It is white, thin, and becomes broad in 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 "lecture room" which is shaped like a honeycomb, and the seed is called "lecturer". Kite is used for medicinal and edible purposes in all parts without discarding it. It is used for medicinal and edible purposes. It is used for the seedling (lotus root), seed (rice, lemon juice, "Is also used for medicinal purposes. In particular, it is a node of the root of the root of the root of the ground. It is called a rustic wave, a giant rustic temple, etc. It is used as an important medicinal herb to treat blood, blood, nosebleeds, hemorrhage, hemorrhoids and uterine bleeding. In fact, in some parts of Korea, a root canal is used in combination with the root of the root of the lotus root, but the nodulus of Nelumbinis Rhizoma is a square cylinder, unlike a cylindrical lotus root, Has 2 ~ 4cm, 7 ~ 9 round holes, and the surface is yellowish brown to grayish brown.

In the present study, we investigated the phenolic compounds catechin, (+) - gallocatechin, (+) - gallocatechin- (4α-8) -catchin and scopoletin, 603), and alkaloids such as nuciferin, nornuciferin, pronuciferin, dehydronuciferine, liensine, isoliensine, and neferine and flavonoids such as quercetin and kaempferol and amino acids such as tryptophan, asparagine and tyrosine Paper). The diuretic effect (Mukherjee PK et al., 1997, J. Ethnopharmacol. 58, 207-213; Kim Okkyung, 2014, Journal of the Korean Chemical Society, 31, 509-516) 1996, Phytotherapy Res. 10, 424-425), antipyretic effect (Sinha S et al., 2000, Phytotherapy Research, 14, 272-274), psychological stress relief effect 158), hyperlipidemia and hypercholesterolemia (Lee Jae Jun et al., 2006, Korean Journal of Food Preservation and Distribution, 13, 634-642) and the effect of renal failure therapy (Hong Kwang Hae, 2009, Dongshin University doctoral dissertation). In particular, it is well known that hemostatic function and blood circulation promotion effect in the private circulation in relation to blood circulation, and hydrothermal extract in the right hemisphere is helpful in the treatment of ischemia due to local cerebral blood flow increasing activity in brain tissue damage in the rat (Kim, Young-Gyun et al., 2005. Korean Journal of Inorganic Chemistry, 20, 75-81, Lee, Geumsoo, 2005. Journal of Physiology and Pathology, 19, 1546-1551). However, in the report on the promotion of blood circulation, it is confirmed that the natural product used in the experiment is a lotus root.

Meanwhile, patents related to the rupture are disclosed in Korean Patent Laid-Open No. 10-2005- 0028272, which is effective for dieting, anti-diabetic, hyperlipidemia, and antioxidation including bacon, mackerel, livestock, The present invention relates to a food composition, a food composition, a cosmetic composition, a cosmetic composition, and a pharmaceutical composition using the lotus root. There is no known patent for an active extract of folium that shows activity but is not toxic. In addition, reports of anti-thrombotic activity related to pandemic to date show that neferine isolated from seed embryo of zhou seeds reported by Zhou et al. In 2013 inhibits platelet aggregation and exhibits antithrombotic activity (Zhou YJ et al. 2013. Thrombosis Res. 132, 202-210) is the only situation.

 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 for preventing or treating a thrombotic disease, Health 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 Nodulus of Nelumbinis rhizoma as an active ingredient.

The rheumatoid extract is preferably a rheumatoid ethanol extract.

It is preferable that the rheumatic ethanol extract is the butanol fraction or the water residue obtained by successively fractionating the rheumatic ethanol extract with hexane, ethyl acetate and butanol.

In addition, the present invention provides a health functional food for preventing or ameliorating thrombosis, which comprises the flesh extract of the present invention.

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the rheumatoid extract as an active ingredient of the health functional food are effective for inhibiting thrombogenesis-related enzymes and blood coagulation factors and 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.

Fig. 1 is a photograph of a lotus leaf, a lecture room, a lecture room, a lotus root,
FIG. 2 is a graph showing thrombin inhibition according to the concentrations of the extracts of the sweet potatoes (A), soft potatoes (B) and fruiting (C)
FIG. 3 is a graph showing the prothrombin inhibition by prothrombin time according to the concentration of the extracts of the potato (A), the potato (B) and the fruity (C)
FIG. 4 is a graph showing the degree of inhibition of blood coagulation factors according to the concentrations of the extracts of the soft pot (A), soft pot (B) and feces (C)
FIG. 5 shows the inhibitory activity of human platelet aggregation of the ethanol extract and the sequential organic solvent fractions thereof. Herein, a mixture of 1: solvent control (DMSO), 2: aspirin (0.5 mg / ml), 3: aspirin (0.25 mg / ml) (0.25 mg / ml), 6: ethylacetate fraction (0.25 mg / ml) of the ethanol extract of rumen, 7: butanol fraction (0.25 mg / ml) of the rumen ethanol extract, 8: water after the organic solvent fraction of the rumen ethanol extract The residue (0.25 mg / ml)
FIG. 6 shows human platelet aggregation inhibitory activity of the ethanol extract, the butanol fraction and the water residue thereof. Herein, the butanol fraction (0.25 mg / ml) of the ethanol extract of 4: flesh extract, the 5: fructose ethanol (5 mg / ml) of the extract of 1: solvent control (DMSO), 2: aspirin (0.5 mg / 7: water residue (0.25 mg / ml), 8: water residue of ethanol extract of fruiting body (0.125 mg / ml), 6: ethanol fraction of butanol fraction (0.062 mg / Water (0.125 mg / ml), and 9: water residue of ethanol extract (0.062 mg / ml).

Hereinafter, the present invention will be described in detail.

In order to test the anti-thrombotic efficacy of kites, the inventors of the present invention collected various parts of kites to prepare extracts of each part by a certain method, and evaluated the activity to determine the specific part of the kite showing strong antithrombotic activity . The extracts and fractions of the extracts and fractions showed no hemolytic activity on human erythrocytes, but the extracts and fractions showed no hemolytic activity on human erythrocytes, And the extracts and fractions were used as pharmaceutical composition and health functional food for preventing or treating / improving 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 fleshy extracts showing strong activity were fractionated with hexane, ethyl acetate and butanol, respectively, and finally, a water residue was 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 the evaluation of the inhibitory activity, strong anti - coagulant activity and platelet aggregation inhibitory activity were confirmed in the ethanol extract of rumen and its butanol fraction and water residue. This anti-thrombotic activity was strong antithrombotic activity exceeding that of aspirin which is used clinically as antithrombotic agent. Furthermore, it was confirmed that the above-described rheumatoid extract and active fractions 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 Nodulus of Nelumbinis rhizoma as an active ingredient.

Preferably, the rheumatoid extract is a rheumatic ethanol extract, and the rheumatic ethanol extract is preferably a butanol fraction or a water residue obtained after successive fractionation of rheumatic ethanol extract with hexane, ethyl acetate and butanol.

In addition, the present invention relates to a nodulus of Nelumbinis rhizoma ) extract of the present invention for preventing or ameliorating thrombosis.

Preferably, the rheumatoid extract is a rheumatic ethanol extract, and the rheumatic ethanol extract is preferably a butanol fraction or a water residue obtained after successive fractionation of rheumatic ethanol extract with hexane, ethyl acetate and butanol.

Hereinafter, the feces extract of the present invention, the method for producing each active fraction and the experiment of efficacy will be described in more detail.

The present invention relates to a method for preparing an extract, Evaluating the antithrombotic activity of various extracts of Yan; Preparing sequential organic solvent fractions of hexane, ethyl acetate, and butanol from a flesh extract that exhibits potent antithrombotic activity, and then preparing a water residue obtained therefrom; Evaluating the antithrombotic activity of the extract and fraction and examining the stability of the active substance of the butanol fraction and the water residue.

"Rectal extract" included in the composition of the present invention is prepared by cutting a mature portion of a mature Lotus root, collecting it, and washing it; Drying the ridicule at 60 to 70 DEG C for 1 to 2 days; Crushing the dry fade; Extracting the ground powder with an organic solvent; And filtering the extract using a filter net of 0.06 mm or less and concentrating it under reduced pressure.

The organic solvent used in the present invention may be any organic solvent such as water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol (methanol, ethanol, alcohol, propanol, butanol etc.) having 1 to 4 carbon atoms, a mixed solvent of the lower alcohol and water , And hot water or 95% ethanol extraction is most preferred.

In a preferred embodiment, the rugae extract of the present invention can be used by extracting rugae 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, the thrombin time, prothrombin time, and apathy time were measured at a concentration of 5 mg / ml of the ryo myrmid extract. As a result, the blood coagulation time was extended by at least 15 times, Respectively. In addition, the human platelet aggregation inhibitory activity was measured at a concentration of 0.25 mg / ml of the rheumatoid arthritis extract. As a result, it showed similar activity of aspirin-like aggregation inhibition.

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 right side, concentration-dependent thrombogenesis inhibitory activity was confirmed in all the fractions. Especially, in the butanol fraction and water residue, 10 times more potent thrombin, prothrombin and blood coagulation factor inhibitory activity. Therefore, it has been confirmed that the ethanol extract and the active fractions thereof can be developed as an anticoagulant and antiplatelet agent capable of replacing aspirin, which is highly likely to cause side effects such as gastrointestinal disorders. On the other hand, there was no significant antithrombotic activity in the same root, but in the lotus root, which is mainly used for edible purposes.

The extract of the present invention and the active fraction thereof may be powdered by a conventional powdering process such as vacuum drying and freeze drying or spray drying. 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 can include 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  1: Preparation of extracts from various parts of yeast and analysis of their useful components

Dried longevity, dried liquor, liquor, lotus root and fruity were cultivated and harvested in Mungyeong, Kyungbuk Province in 2016, and their ethanol extracts were prepared (Fig. 1). In the case of rootstocks and ridges, it was dried at 60 ~ 70 ℃ for 2 days, and then ground for 20 ~ 30 mesh using a food grinder. In preparing the ethanol extracts, 10 times of ethanol was added to each sample, and the extracts were repeatedly extracted three times at room temperature. The extracts were collected by filtration and concentrated under reduced pressure to prepare ethanol extracts. The results of each extraction efficiency and component analysis of the extract are shown in Table 1. Total polyphenols, total flavonoids, total sugars and reducing sugar content were determined by component 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, 400 μl of 90% diethylene glycol was added to 400 μl of the filtered extract, and 40 μl of 1 N NaOH was added thereto. After reacting for 1 hour at 37 ° C., absorbance was measured at 420 nm . 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] Extraction efficiency and useful components of ethanol extracts of various soft tissues

As shown in Table 1, the extraction efficiency was in the order of Yeonpyeong> Yeonja> Lotus root> Yeonjae> Yeonjeol, and the fruition showed the lowest extraction yield of 1.31%. The total polyphenol content of each ethanol extract was found to be 203 mg / g, which is the highest in the stigma, and 103 mg / g, which is higher in the stigma. After that, the contents of 74mg / g in the lotus root and 10.5mg / g in the root lot were shown. On the other hand, total flavonoid content was highest in leaves of Yeonpyeon, followed by fruition, lecture room, lecture, and lotus root. Total sugars and reducing sugars also showed higher contents than those of the leaves, leaves, and lotus root in the smoker. Therefore, it was judged to exhibit various physiological activities due to high content of useful components in the case of the stigma, stigma, and fruition.

Example  2: Coagulase inhibitory activity of various extracts

The blood coagulation inhibitory activity of the ethanol extracts of various soft tissues of Example 1 was evaluated, and the results are shown in Table 2. In this case, the evaluation method of blood clotting inhibition activity was evaluated according to the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time and apathy time were measured. Plasma was obtained from a 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, and the coagulation factor inhibitory activity was expressed as aPTT time divided by the aPTT time of the solvent control.

[Table 2] Blood coagulation inhibitory activity of various ethanol extracts

As a result, the extracts of lotus root and rootstock used for edible purposes showed very little blood coagulation inhibitory activity at the concentration of 5 mg / ml. Prothrombin time, and apathy time by 15 times or more, indicating strong blood coagulation inhibitory activity. At the 5 mg / ml concentration of aspirin used as a control, thrombin time, prothrombin time, and apathy time were extended by 15 times or more, respectively. At 1.5 mg / ml, thrombin time, prothrombin time, , 1.29 times, and 1.49 times, respectively. Thus, the blood coagulation inhibitory activity was evaluated according to the concentrations of the extracts of the sweet potato, the sweet potato, and the fruity extract, and the results are shown in FIG. 2, FIG. 3 and FIG. As a result, the extracts of soft tissues, soft tissues, and rheumatoid arthritis showed more potent inhibitory activity than aspirin.

Example  3: Platelet Aggregation Inhibitory Activity of Extracts of Various Pods

The activity of inhibiting human platelet aggregation of the extract of various soft tissues of Example 1 was evaluated and the results are shown in Table 3. [ 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] Platelet Aggregation Inhibitory Activity of Various Yeast Extracts

As shown in Table 3, first, aspirin showed strong inhibition of platelet aggregation in a concentration-dependent manner at a concentration of 0.25-0.5 mg / ml, and thus it was found to be used as an antithrombotic agent in clinical practice. On the other hand, lotus root extract showed a hemostatic effect by strongly promoting human platelet aggregation at a concentration of 0.25 mg / ml. Therefore, it was confirmed that the underground stems of the stems showed both hemostatic effect and thrombogenesis inhibitory effect at the site, and it was found that the ground of the stomach was used for the purpose of hemostasis and blood circulation at the same time.

 On the other hand, the zygosporin and lectin showed platelet aggregation inhibitory activity several times stronger than aspirin, and the lotus leaf showed coagulation inhibitory activity better than aspirin.

Example  4: Human erythrocyte hemolytic activity of various soft tissue extracts

Yeast has been used for a long time as an edible and medicinal product, and lotus root and lotus leaf are registered as food raw materials and safety is secured. In addition, lecture rooms, lectures, and festivals have been used as cars. In the present invention, human erythropoietic activity was evaluated in order to evaluate the acute toxicity of various soft tissue extracts, and the results are shown in Table 4. In this case, hemolytic activity was assessed in accordance with the existing report (Jung In-chang, Son Ho Yong, 2014 ㆍ Korean J. Microbiol. Biotechnol. 42: 285-292), and 100 μl of human erythrocytes, 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 hemoglobin efflux 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.

( % ) Hemolysis = [( Abs .S- Abs .C) / ( Abs .T- Abs . C)] x 100

Abs . S: Sample Additive  Absorbance,

Abs . C: DMSO Additive  Absorbance,

Abs . T: Triton X-100 Additive  Absorbance.

[Table 4] Hemolytic activity of human erythrocytes in various soft tissue extracts

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. On the other hand, hemolytic activity did not appear at all at 1 mg / ml. Amphoteracin B, an anticancer drug known to have haemolytic activity, has a haemolytic activity of 55% at a concentration of 0.00625 mg / ml and 93.7% at a concentration of 0.05 mg / ml.

Example  5: Fear  Sequential organic solvent of ethanol extract Fraction  Preparation and analysis of their components

The rumen was finally selected through the evaluation of the extracts of various kite parts, and a large amount of the rumen ethanol extract was prepared in the same manner as described in Example 1, and then fractionated with hexene, ethyl acetate and butanol sequentially, To recover the water residue. The extraction efficiency of ethanol extract, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction are shown in Table 5.

[Table 5] Fear  Ethanol extract and its sequential Fraction  Comparison of yield and component analysis

As shown in Table 5, the rumen ethanol extracts were similarly performed with 29.7%, 27.9%, and 26.5% of hexane fraction, ethyl acetate fraction, and butanol fraction, respectively, and water residue was the least 14.4%. This result implies that the extracts of various kinds contain various kinds of polar substances, and it is possible to recover about 0.35 g of the butanol fraction and about 0.19 g of the water residue from 100 g dried feces.

As a result of total polyphenol content analysis, high content of 172.1 ~ 139.9 was high in butanol fractions and water residues, showing the highest content among fractions, which was similar in total flavonoid content analysis. In addition, high butanol fraction and water residue showed high total sugar and reducing sugar content. Therefore, various physiological activities of the ethanol extracts of the feces were expected to appear in butanol fractions and water residues, and the fractions showed strong antioxidative activity and nitrite scavenging ability.

Example  6: Fear  Sequential organic solvent of ethanol extract Fraction  Evaluation of blood coagulation inhibitory activity

The blood coagulation inhibitory activity of the flesh extract and the fractions thereof obtained in Example 5 were evaluated in the same manner as in Example 2, and the results are shown in Table 6.

[Table 6] Fear  Ethanol extract and its Fraction  Anticoagulant activity

As shown in Table 6, the aspirin exhibited excellent blood coagulation inhibitory activity by prolonging thrombin time, prothrombin time, and apathy time by 1.38, 1.29 and 1.49 times, respectively, at a concentration of 1.5 mg / ml. On the other hand, in the 5 mg / ml concentration of the extract, the thrombin time, the prothrombin time and the apathy time were all extended 15 times or more than the no-added time, and the thrombin time and the apathy time were 15 times or more even at the 2.5 mg / Time was extended by 8.72 times. The hexane fraction and the ethyl acetate fraction in the fraction of the rheumatoid extract also showed good concentration-dependent blood coagulation inhibitory activity, but significant activity was confirmed in the butanol fraction and the water residue. In the case of butanol fraction, thrombin time, prothrombin time, and apathy time were extended by at least 15 times in the case of 2.5 mg / ml concentration. In the case of water residues, thrombin time, prothrombin time, The time was extended by more than 15 times compared to the no-added group, and the thrombin time, prothrombin time, and apathy time were extended by 5.19 times, 13.5 times, and 1.62 times, respectively, Fold more potent inhibitory activity. These results indicate that the rheumatoid arthritis extract and its butanol fractions and water residues can be developed as a commercial antithrombotic agent, and it is possible to replace aspirin, which is a conventional antithrombotic agent, exhibiting side effects such as gastrointestinal disorders. In addition, the fractions of the fruiting body exhibit excellent antioxidative and carcinostatic activities, and are expected to contribute to blood flow improvement and antithrombotic activity additionally.

Example  7: Fear  Sequential organic solvent of ethanol extract Fraction  Evaluation of human platelet aggregation inhibitory activity

The human platelet aggregation inhibitory activity of the flesh extract and the fractions thereof obtained in Example 5 were evaluated in the same manner as in Example 3, and the results are shown in Tables 7, 5 and 6.

[Table 7] Fear  Ethanol extracts and their Fraction  Human platelet aggregation inhibitory activity

As a result, aspirin strongly inhibited the platelet aggregation in a concentration dependent manner, and the ethanol extract of the right side showed inhibition similar to aspirin at the concentration of 0.25 mg / ml. Hexane fraction and ethylacetate fraction showed platelet aggregation promoting effect. On the other hand, the butanol fraction and the water residue exhibited very strong aggregation inhibition activity. In the butanol fraction, the same inhibition as 0.25 mg / ml of aspirin was obtained at a concentration of 0.125 mg / ml and 0.25 mg / ml. < / RTI > Thus, it was confirmed that the butanol residues were twice as effective as aspirin, and the water residues were more effective than aspirin four times.

Example  8: Fear  The activity of the extract Fraction  Human erythrocyte hemolytic activity

The follicle has been drinking for a long time as a blood sugar and the extract of the follicle did not show acute toxicity either. The acute toxicity of the active fractions of the enteral extracts according to the hemolysis of human erythrocytes was evaluated in the same manner as in Example 4.

[Table 8] Fear  Extract and its Fraction  Human erythrocyte hemolytic activity

As a result, as shown in Table 8, among the fractions of the fruiting extract, the hexene fraction showed a hemolysis activity of 43.8% at a concentration of 1 mg / ml, and no hemolytic activity was observed in other fractions and water residues. Therefore, the butanol fraction and the water residue of the rheumatoid arthritis extract do not show any problem of causing acute toxicity separately.

Example  9: Fear  Ethanol extract and its butanol Fraction , Plasma, acid and thermal stability assessment of water residues

Plasma stability, thermal stability and acid stability of anti-thrombotic activity of the feces extract and its butanol fraction and water residue obtained in Examples 1 and 5 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 coagulation 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)

delete delete A pharmaceutical composition for preventing or treating thrombosis comprising, as an active ingredient, a butanol fraction or a water residue obtained by successively fractionating an ethanol extract of Nodulus of Nelumbinis rhizoma with hexene, ethyl acetate and butanol. Nodulus of Nelumbinis rhizoma A health functional food for preventing or ameliorating thrombosis comprising, as an active ingredient, a butanol fraction or a water residue obtained by successively fractionating an ethanol extract with hexene, ethyl acetate and butanol.

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