KR20200004460A - 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 for preventing or treating thrombosis by containing Nelumbo nucifera seedpod ethanol extract as an active ingredient; and health functional food including the same and, more specifically, to a pharmaceutical composition for preventing or treating and alleviating thrombosis through strong platelet aggregation inhibition and blood coagulation inhibition by containing Nelumbo nucifera seedpod ethanol extract, an organic solvent fraction of the extract or water residue after fractionation as an active ingredient, and health functional food including the same. The Nelumbo nucifera seedpod ethanol extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis and the health functional food: exhibits strong antithrombotic activity by the coagulation inhibitory effect of platelets performing an initiating role of thrombosis generation along with the inhibition of blood coagulation factors and thrombus generation-related enzymes; shows no hemolytic activity against human erythrocytes; has excellent thermal stability; does not show loss in blood coagulation factor inhibitory effects and enzyme generation-related enzyme inhibitory effects even in plasma and acidic conditions of pH 2; and thus, can be expected to be used as the pharmaceutical composition for preventing or treating and alleviating thrombosis such as ischemic stroke and hemorrhagic stroke by improving blood circulation and as the health functional food. The active ingredient is a very useful invention for pharmaceutical industries and food industries by having an excellent effect which can be processed into various forms such as extract, powder, pills, tablets, etc. to be taken at all times.

Description

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 SAME}

The present invention relates to a pharmaceutical composition and health functional food for preventing or treating thrombosis (Seedpod of Nelumbo nucifera ) containing ethanol extract as an active ingredient, more specifically, the ethanol extract yeonjabbang, The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating / improving thrombosis through strong platelet aggregation inhibition and blood coagulation inhibition containing organic solvent fraction or water residue after fractionation as an active ingredient.

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

On the other hand, the generation of fibrin thrombus is a multi-step reaction of numerous blood coagulation factors that activates thrombin, which is involved in fibrin coagulation, and finally generates fibrin monomers from fibrinogen, and the fibrin monomers are polymerized by calcium, thereby forming platelets and endothelial cells. It binds to cells and creates permanent thrombi, forming fibrin polymers cross-linked by factor XIII. In addition, thrombin plays a pivotal role in thrombus formation by activating platelets, factor V and factor VII to promote blood coagulation. Therefore, thrombin activity inhibitors can be used as a prophylactic and therapeutic agent that is very useful for various thrombotic diseases that occur due to excessive blood clotting. Meanwhile, in the endogenous thrombus generation pathway, sequential activation of XII, XI, IX and X factors is followed by the activation of prothrombin, which is known to activate thrombin. It is targeted. To date, various anticoagulants such as heparin, coumarin, aspirin, urokinase, antiplatelets, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases. Its use is limited to such circumstances.

The lotus ( Nelumbo nucifera ) is a perennial plant of the dicotyledon, the genus Amphitaceae, and the trunks crossing the mud have nodes, white and thin, and grow in lotus root in autumn. This lotus root produces leafy stems and flowering stems. Flowers bloom in summer and fruits bear in autumn. Fruits are produced in a seed room called honeycomb shaped like honeycomb, and seeds are called softwood. The kite is used for medicinal and edible parts without throwing away the ground stem (lotus root), seeds (lotus root, lotus root, lotus root), embryos in the seed (lotus root), ovary (lotus leaf), lotus leaf, lotus flower, ground stem (Word) is also used for medicinal purposes. In particular, the soft room is also called the federal, and after drying some of it is drinking tea, most of them are harvested and discarded or used only for the ornamental use of unusual shapes.

Studies on ovarian ovaries include the antioxidant activity of procyanidins (Ling ZQ et al., 2005, J Agric Food Chem. 53, 2441-2445) and autophagy-inducing activity in human liver cancer cells (Duan). Y et al., 2016, Biomed Pharmacother. 79, 135-152), and the protection against radiation exposure by acetone-water extracts of lotus root (Duan Y et al., 2010, Food Chem Toxicol. 48, 3374-3384) is known. In addition, soft-bangs can be used to mitigate oxidative stress caused by electromagnetic exposure and low-frequency electric fields (Luo X et al., 2016, Biomed Pharmacother. 82, 640-648), neuronal cell protection (Yin C et al., 2016, Biomed Pharmacother. 82, 628-). 639), which is known to be effective in preventing immune impairment (Zhang H et al., 2016, Biomed Pharmacother. 82, 364-372). Meanwhile, Xiao JS et al. Reported the separation of polymerized procyanidins and quercetin glucuronide from the ovary. In Korea, research on the ovaries is in the beginning stage, and the addition of yeonjab powder (10%) has been reported to lower blood triglyceride levels when high fat diet is administered (Kim Yong-hwan, 2016, Korean Journal of Food and Nutrition, 29, 684-691). However, no strong platelet aggregation and blood coagulation inhibitory activity has been reported.

In addition, as a patent related to the soft room, Korean Patent No. 10-1529227 [Yunwoobangbang and federal whitening cosmetic composition for improving and herbal cosmetics containing the same], Patent No. 10-1529227 [ A composition for the prevention or treatment of immune thyroid disease] is disclosed, and [Patent No. 10-2016-0090098] [Cosmetic composition for anti-aging and whitening using extracts containing a large amount of polyphenols] 10-2016-0148824 discloses [compositions for the prevention and treatment of allergic diseases or inflammatory diseases comprising a lotus leaf and a soft surgical extract as an active ingredient]. However, until now, there is no known patent on the active extract of the ovarian ovary, which shows potent antithrombotic activity through inhibition of blood coagulation and inhibition of platelet aggregation.

To date, anti-thrombotic activity related to kite has been reported to indicate that neferine isolated from seed embryos of kite seeds reported by Zhou et al. 2013 showed antithrombotic activity by inhibiting platelet aggregation (Zhou YJ et al., 2013). Thrombosis Res. 132, 202-210).

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.

The present invention has been made to solve the problems of the prior art as described above, the problem to be solved in the present invention is a pharmaceutical composition for the prevention or treatment of thrombosis containing yeonjabang ethanol extract which is a part of the kite as an active ingredient and health It is to provide a nutraceutical.

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

The soft ethanol extract ethanol extract is preferably a hexene fraction, an ethyl acetate fraction, butanol fraction or water residue obtained by sequentially fractionating the soft ethanol extract of hexene, ethyl acetate and butanol.

The present invention also provides a dietary supplement for the prevention or improvement of thrombosis containing Seedpod of Nelumbo nucifera ethanol extract as an active ingredient.

The soft ethanol extract ethanol extract is preferably a hexene fraction, an ethyl acetate fraction, butanol fraction or water residue obtained by sequentially fractionating the soft ethanol extract of hexene, ethyl acetate and butanol.

Inhibitory effect of platelet aggregation to inhibit the thrombosis-producing enzyme and blood coagulation factor in combination with the inhibition of thrombosis-related enzymes and blood coagulation factors It exhibits strong antithrombotic activity and no hemolytic activity against human erythrocytes, excellent thermal stability, loss of blood coagulation factor inhibitory effect and clotting production-related enzyme inhibition effect even in acidic conditions and plasma of pH 2 Since it does not appear, it is expected to be used as a pharmaceutical composition and health functional food for the prevention and treatment / improvement of thrombosis such as ischemic stroke and hemorrhagic stroke through the improvement of blood circulation, and the active ingredient is extract, powder, pill, tablet It can be processed into various forms such as It is a very useful invention in the pharmaceutical and food industry because it has an excellent effect.

1 is a photograph of lotus leaf, lotus, lotus root, lotus root and verse from the left,
Figure 2 is a graph showing the thrombin inhibition degree by thrombin time of the ethanol extract of Yeonbangbang,
Figure 3 is a graph showing the prothrombin inhibition rate by the concentration of the ethanol extract of Yeonbangbang as a prothrombin time,
Figure 4 is a graph showing the blood coagulation factor inhibitory rate by the time of the ethanol extract of Yonjabang ethanol,
Figure 5 shows the human platelet aggregation inhibitory activity of the ethanol extract ethanol extract and its sequential organic solvent fraction, 1: solvent control (DMSO), 2: aspirin (0.25mg / ml), 3: aspirin (0.5mg / ml) , 4: ovarian ethanol extract (0.25 mg / ml), 5: hexene fraction of ethanol extract ethanol extract (0.25 mg / ml), 6: hexene fraction of ethanol extract ethanol extract (0.125 mg / ml), 7: yeonjabang Ethyl acetate fraction of ethanol extract (0.25mg / ml), 8: ethyl acetate fraction of ethanol extract (0.125mg / ml), 9: butanol fraction of ethanol extract of ethanol extract (0.25mg / ml), 10: yeonbangbang Butanol fraction of ethanol extract (0.125 mg / ml), 11: Water residue after sequential organic solvent fraction of soft ethanol extract (0.25 mg / ml), 12: Water residue after sequential organic solvent fraction of soft ethanol extract ( 0.125 mg / ml).

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

The inventors of the present invention, in order to assay the antithrombotic efficacy against the kite, by extracting the various parts of the kite to prepare extracts for each part in a certain way, and to evaluate the human platelet aggregation inhibitory activity and blood coagulation inhibitory activity potent antithrombotic activity After the identification of the ovaries, the hexene fractions, ethyl acetate fractions, butanol fractions and water residues from the organic solvent fractions were recovered as antithrombotic active ingredients, and the extracts and fractions were hemolytic activity against human erythrocytes. While not exhibiting at all, the thermal stability and acid stability by confirming that the excellent characteristics of the extract and fractions were intended to utilize as a pharmaceutical composition and health functional food for the prevention or treatment / improvement of thrombosis.

Specifically, the present inventors prepared ethanol extracts in various regions of the kite respectively in order to develop pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of thrombosis using kites known to be excellent in blood circulation improvement in the private sector. Then, antithrombotic activity was evaluated, and then sequentially extracted organic solvent fractions of hexene, ethyl acetate and butanol were extracted from ethanol extracts showing strong activity. Finally, water residue was recovered after the fractionation. The antithrombotic activity of each extract and fractions was determined by direct thrombin time, prothrombin time, and activated partial thromboplastin time (aPTT) and platelet aggregation on human plasma and human thrombin. As a result of evaluating the inhibitory activity, it was confirmed that the ethanol extract of Y. bark, each fraction and water residue thereof showed potent blood coagulation inhibitory activity and platelet aggregation inhibitory activity. This antithrombotic activity was a potent antithrombotic activity that surpassed aspirin, which is used in the clinic as an antithrombotic agent. In addition, it was confirmed that the ethanol extract and the active fraction and water residues of the ovary did not show hemolytic activity against human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing Seedpod of Nelumbo nucifera ethanol extract as an active ingredient.

The soft ethanol extract ethanol extract is preferably a hexene fraction, an ethyl acetate fraction, butanol fraction or water residue obtained by sequentially fractionating the soft ethanol extract of hexene, ethyl acetate and butanol.

The present invention also provides a dietary supplement for the prevention or improvement of thrombosis containing Seedpod of Nelumbo nucifera ethanol extract as an active ingredient.

The soft ethanol extract ethanol extract is preferably a hexene fraction, an ethyl acetate fraction, butanol fraction or water residue obtained by sequentially fractionating the soft ethanol extract of hexene, ethyl acetate and butanol.

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

In a preferred embodiment, the present invention comprises the steps of preparing an extract from various lead sites; Evaluating antithrombotic activity of various lotus extracts; Preparation of a sequential organic solvent fraction of hexene, ethyl acetate, butanol from the ethanol extract ethanol extract exhibiting strong antithrombotic activity, and subsequent preparation of water residues; Evaluation of the antithrombotic activity of the extracts and fractions and the investigation of the stability of the active substance.

"Yangjabang ethanol extract" included in the pharmaceutical composition and the health functional food of the present invention comprises the steps of harvesting the mature lotus bark; Removing the soft (seed) from the soft room; Hanging or hanging the softened room with the softening at room temperature, or drying for 2-3 days at 60 ~ 70 ℃; Pulverizing the dried soft-bang; Extracting the ground powder with ethanol; And the extract is filtered using a filter net of 0.06 mm or less, it can be obtained by the step of concentration under reduced pressure.

In addition, the "ethanol ethanol extract" included in the pharmaceutical composition and health functional food of the present invention is a hexene fraction, ethyl acetate fraction obtained by sequentially or respectively fractionating the ethanol extract with an organic solvent of hexene, ethyl acetate and butanol, Butanol fraction or water residue.

In the present invention, by measuring the thrombin time, prothrombin time and epitaxial ethanol extract concentration of 5mg / ml, all coagulation time is extended by 15 times or more compared to the non-added furniture, very strong blood coagulation inhibitory effect It was confirmed. In addition, as a result of measuring human platelet aggregation inhibitory activity at a concentration of 0.25 mg / ml of the ethanol extract of Yeonbangbang, it showed more potent platelet aggregation inhibitory activity than 0.5 mg / ml concentration of aspirin.

On the other hand, as a result of evaluating the blood coagulation inhibitory activity of the organic solvent fraction and water residues of the ethanol extract of Yonjabang, the concentration-dependent clotting inhibitory activity was confirmed in all fractions, in particular, butanol fraction and water residues aspirin 5-10 times more potent thrombin, prothrombin and blood coagulation factor inhibition. In addition, as a result of evaluating platelet aggregation inhibitory activity of the organic solvent fraction and water residues of the ethanol extract of Yeonjabang, concentration-dependent platelet aggregation inhibitory activity was confirmed in all fractions.

Therefore, it was confirmed that the ethanol extract and active fractions of the ovarian ovary could be developed as anticoagulants and antiplatelets to replace aspirin, which has high side effects such as gastrointestinal disorders.

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

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. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis , Deep vein thrombosis, lower extremity edema, pain, and acute peripheral arterial obstruction. Examples of venous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous thrombosis, and central retinal vein occlusion.

The pharmaceutical composition comprising the active ingredient of the present invention may be orally formulated as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It may be formulated and used in various forms, and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further include carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include a filler, an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

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

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

As a preferred embodiment, the preparation for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories and the like. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

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

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

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

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

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

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

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

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

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

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

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

EXAMPLE

Example 1 Preparation of Yeonjabang Ethanol Extract and Sequential Organic Solvent Fractions and Their Component Analysis

In 2016, the Yeonjabang grown (cultivated and harvested) in Mungyeong, Gyeongbuk was purchased, and its ethanol extract was prepared. The dried softwood was used after grinding to 20 mesh without any treatment. When preparing the ethanol extract, 10 times ethanol was added to the sample, and extracted three times at room temperature, the extract was collected and filtered, and then concentrated under reduced pressure to prepare a ethanol extract. In addition, after preparing a large amount of the ethanol extract of soft-banged room, the organic solvent fractions were sequentially subjected to hexene, ethyl acetate, butanol, and finally the water residue was recovered. Extraction efficiency, organic solvent fractionation efficiency, and extract / fraction component analysis results of ethanol extraction are shown in Table 1. Component analysis determined total polyphenols, total flavonoids, total sugars and reducing sugars content. The total polyphenol content was added to 400μl of the extracted sample solution, 50μl of Folin-ciocalteau, 100μl of Na ₂ CO ₃ saturated solution, and left at room temperature for 1 hour and absorbance at 725nm. Tannic acid was used as the standard reagent. For the total flavonoid content, each sample was extracted by stirring for 18 hours with methanol, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extract sample, 40 μl of 1N NaOH was added thereto, and the absorbance was measured at 420 nm after reaction for 1 hour at 37 ° C. Rutin was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was determined by phenol-sulfuric acid method.

Table 1 Comparison of Yield and Ethanol Extracts of Yeonbangbang and Its Sequential Organic Solvent Fractions

As shown in Table 1, the ethanol extract of lotus leaf ethanol extract was mostly transferred to butanol fraction and hexene fraction, respectively 70.0% and 24.4%, and ethyl acetate fraction and water residue accounted for 3.5% and 3.3% of the extract, respectively. These results indicate that the ethanol extract of soft ovary contains various substances with different polarities, and that about 1.61 g of butanol fraction and about 0.076 g of water residue can be recovered from 100 g of dried soft yam. On the other hand, as a result of analysis of total polyphenol content, the butanol fraction showed the highest content of 261.4 mg / g, showing the highest content among the fractions, and in the total flavonoid content analysis, 14.5 mg / g in the butanol fraction after the ethyl acetate fraction High content was confirmed. In addition, the high total sugar and reducing sugar contents in the butanol fraction and water residues were significantly different from those of the natural fraction. Therefore, it was expected that various physiological activities of the ethanol extracts of the ovary ovary were shown in the butanol fraction and the water residue, and in actual analysis, the fractions showed strong antioxidant activity and nitrite scavenging ability.

Example 2: Evaluation of blood coagulation inhibitory activity of ethanol extract of yeonjabang and sequential organic solvent fractions

The coagulation inhibitory activity of the ethanol extract and fractions thereof obtained in Example 1 was evaluated, and the results are shown in Table 2 and FIGS. 2 to 4. At this time, the blood coagulation inhibitory activity evaluation method was evaluated according to the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time and episode time. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and thrombin time, prothrombin time, and APT measurement were performed as follows.

Thrombin Time

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

Prothrombin time

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

aPTT (activated Partial Thromboplastin Time)

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

[Table 2] Anticoagulant Activity of Ethanol Extracts of Yeonbangbang and Its Sequential Organic Solvent Fractions

As shown in Table 2, aspirin showed excellent blood coagulation inhibitory activity by extending thrombin time, prothrombin time, and epitaxial time by 1.32 times, 1.37 times, and 1.43 times, respectively, at a concentration of 1.5 mg / ml. On the other hand, the ethanol extract of lotus ovary extended the thrombin time, prothrombin time, and epitaxial time by 15 times more than the non-added group at the concentration of 1.25 ~ 5mg / ml, and prothrombin time and epitaxial time was 15 even at 0.62mg / ml. More than twice the thrombin time was 4.43 times longer. The hexene fraction and ethyl acetate fraction in the fractions of the ethanol extracts of lotus root extract also showed good concentration-dependent blood coagulation inhibitory activity, but significant potent activity was found in the butanol fraction and water residue. Butanol fractions increased prothrombin time and epitaxial time by 15 times and thrombin time by 3.25 times, even at 0.62 mg / ml concentration, and water residues showed thrombin time, prothrombin time and epitaxial time at 2.5 mg / ml concentration. All were prolonged more than 15 times compared to the non-added, and the thrombin time was 5.25 times and the apitime time was 5.12 times even at 1.25mg / ml. These results indicate that the ethanol extracts of the ovary plant contain blood coagulation inhibitory substances with various solubility, and the most powerful active fraction is butanol fraction. In addition, through this result, it is determined that the ethanol extract of ethanol extract, butanol fraction and water residue thereof may be developed as a commercial antithrombotic agent, and may replace aspirin, which is an existing antithrombotic agent that shows side effects such as gastrointestinal disorders. Judging.

Example 3 Evaluation of Human Platelet Aggregation Inhibitory Activity of Ethanol Extracts of Strains and Sequential Organic Solvent Fractions

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

Platelet aggregation inhibition activity

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

TABLE 3 Human Platelet Aggregation Inhibitory Activity of Ethanol Extract and Sequential Organic Solvent Fractions

As shown in Table 3, in the case of the solvent control DMSO, human platelets were rapidly and strongly aggregated by collagen addition, and aspirin, a platelet aggregation inhibitor, strongly inhibited platelet aggregation in a concentration-dependent manner. At this time, the aspirin showed 38.6% aggregation at 0.25mg / ml concentration. On the other hand, ethanol extract and hexene fraction thereof showed 39.8% and 38% aggregation inhibition similar to aspirin at 0.25 mg / ml concentration, respectively. In addition, the ethyl acetate fraction and butanol fraction showed weaker activity than the aspirin at the same concentration, but it was confirmed that it can be sufficiently used as a platelet aggregation inhibitor by showing aggregation of 51.8% and 49.3% at 0.25 mg / ml concentration, respectively. The strongest inhibition of aggregation appeared in water residues, with a degree of 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 extracts and its fractions of ovarian ovary has been suggested to be possible to manufacture anti-thrombotic and blood circulation-enhancing health functional foods using yeonjabang, and yeonjabang contains various antithrombotic components with various solubilities. Suggests that.

Example 4 Human Erythrocyte Hemolysis Activity of Ethanol Extracts and Sequential Organic Solvent Fractions

Yeonjabang has been ingested in tea for a long time, and in the present invention, human erythrocyte hemolytic activity was evaluated to evaluate acute toxicity of the ethanol extract of ethanol and its sequential organic solvent fractions, and the results are shown in Table 4. The hemolytic activity was evaluated according to the previous report (Korean J. Microbiol. Biotechnol. 2014, 42: 285-292) .Simply, 100 μl of human red blood cells washed three times with PBS was added to a 96-well microplate, 100 μl of the sample solution was added and then reacted at 37 ° C. for 30 minutes. After that, the reaction solution was centrifuged for 10 minutes (1,500 rpm) to transfer 100 μl of the supernatant to a new microtiter plate, and the degree of hemoglobin leakage according to hemolysis was measured at 414 nm. DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1 mg / ml) was used as an experimental control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

[Table 4] Human erythrocyte hemolytic activity of ethanol extract and sequential organic solvent fractions

First, DMSO and water used as a control did not have hemolytic activity, triton X-100 was confirmed that 100% hemolysis of red blood cells at 1mg / ml concentration. In addition, empoterasin B, an anticancer agent known to have hemolytic activity, exhibited hemolytic activity of 55.5% at a concentration of 0.00625 mg / ml and 93.7% at a concentration of 0.05 mg / ml. In the case of the ethanol extract of lotus ovary, there was no hemolytic activity at the concentration of 1.0 mg / ml, and all fractions of the ethanol extract of the lotus ovary did not show any hemolytic activity. Therefore, ethanol extract, sequential organic solvent fractions and water residues of lotus leaf are not considered to show the problem of causing acute toxicity.

Example 5 Evaluation of Plasma, Acid and Thermal Stability of Ethanol Extracts, Sequential Organic Solvent Fractions and Water Residues thereof

Plasma stability, thermal stability, and acid stability against antithrombotic activity of the ethanol extract ethanol extract obtained in Example 1, sequential organic solvent fractions, and water residue thereof were confirmed. The samples showed high stability due to no significant decrease in blood coagulation inhibition and platelet aggregation inhibitory activity even when treated at 100 ° C. for 1 hour, 1 hour at pH 2 (0.01 M HCl), and 1 hour at plasma. . Therefore, the extracts and active fractions are expected to maintain excellent antithrombotic activity during digestion absorption and food manufacturing.

Claims (4)

A pharmaceutical composition for preventing or treating thrombosis, comprising an ethanol extract of Seedpod of Nelumbo nucifera as an active ingredient. The pharmaceutical composition according to claim 1, wherein the lotus leaf ethanol extract is hexene fraction, ethyl acetate fraction, butanol fraction or water residue obtained by sequentially fractionating the lotus leaf ethanol extract with hexene, ethyl acetate and butanol. Seedpod of Nelumbo nucifera Health functional food for preventing or improving thrombosis containing ethanol extract as an active ingredient. 4. The dietary supplement of claim 3, wherein the ethanol extract of Hyeonjabang is hexene fraction, ethyl acetate fraction, butanol fraction or water residue obtained by sequentially fractionating the ethanol extract ethanol extract of hexene, ethyl acetate and butanol.

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