KR20140044692A - Pharmaceutical composition comprising the lees extract of korean rice wine 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 health functional food comprising an extract of Korean rice wine lees as an active ingredient for prevention or treatment of thrombosis and, more specifically, to a pharmaceutical composition and a health functional food comprising, as an active ingredient, an ethanol extract or a hot water extract of Korean rice wine lees, or a mixture thereof, particularly, an ethyl acetate fraction, a butanol fraction, or a water residue of the extract of Korean rice wine lees, or a mixture thereof, for prevention or treatment of thrombosis. The extracts of Korean rice wine lees as an active ingredient of the pharmaceutical composition and the health functional food of the present invention are prepared by performing extraction on Korean rice wine lees using ethanol or the like to prepare an extract and then performing sequential fraction on the extract using hexene, ethyl acetate, and butanol. These extracts have an effect of efficiently inhibiting antithrombotic activity, antiplatelet activity, and thrombosis, due to the direct inhibition of human thrombin, and has an excellent effect of preventing or treating thrombosis, such as ischemic stroke and hemorrhagic stroke, by improving blood circulation. In particular, the extract of Korean rice wine lees of the present invention does not show acute oral toxicity, has excellent thermal stability, and does not show losses due to the direct inhibition of human thrombin, inhibition of prothrombin, and inhibition of blood clotting factors, even in acidic conditions of pH 2 and in the plasma. Therefore, the extract of Korean rice wine lees of the present invention is processed into various forms such as an extract liquid, a powder, a pill, and a tablet, and thus can be taken at all times, so that the present invention is very useful in the pharmaceutical industry and food industry.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE LEES EXTRACT OF KOREAN RICE WINE AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis containing sake extract as an active ingredient, and more particularly, an ethanol extract of sake extract, hot water extract or a mixture thereof. In particular, the present invention relates to a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing ethyl acetate fraction, butanol fraction, water residue or mixtures thereof 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 as the blood coagulation reaction system and the thrombolytic reaction system in the body are complementarily regulated. Among them, the mechanism of the blood coagulation reaction system forms platelet thrombus due to the adhesion of platelets to the blood vessel walls and aggregation. 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 activated by thrombin, which is involved in fibrin coagulation through several stage reactions of numerous coagulation factors, and finally, fibrin monomers are generated from fibrinogen, and 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 caused by excessive blood clotting. In the endogenous thrombosis pathway, sequential activation of factor XII, XI, IX, and X is followed by activation of prothrombin, which is known to activate thrombin. It is becoming. Various anticoagulants such as heparin, coumarin, aspirin, urokinase, antiplatelets, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases.However, they are very expensive and have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity reactions. As a result, its use is limited.

On the other hand, Jukbu refers to the residue left after filtering fermented liquor such as Takju, and is called sake ji-gee, janggang, or argyu. Generally, in the production of Takju, it is made up of the raw rice → semi-semitransparent → salting → increase and cooling → immigration → manufacture → primer → primary fermentation → secondary fermentation → aging → fermentation, The process is completed through various physical, chemical and biological reactions of the yeast, water, and rice starch. After the fermentation, the Takju product is completed through the above-described sifting process (a process of filtering the takju with water using a sieve etc.) , And lees of Korean rice wine. Therefore, it is well known that apricot has a large amount of nutrients and useful physiologically active ingredients. Actually, in the case of Takju, which uses rice as its main ingredient, it has been used as a substitute for food, and recently, it has been attracting attention as a functional cosmetic material. However, the use of sake lees in Korea is mainly used to make vinegar using alcohol in sake lees, or to make pickles using flavor ingredients, and is used as some eco-friendly fertilizer and animal feed, but most of them are disposed of. It is true.

The research related to sake lees mainly focused on sake lees in Japan, and during the breakthrough of the sake industry in Japan, which was stagnated in the 1990s, while the useful physiological activity was verified in sake lees, researchers by Saito et al. Activated as isolated, reported (Saito et al., 1994. Biosci. Biotechnol. Biochem. 58: 1767-1177). In the present study, the effect of the extract on the removal of environmental pollutants (Adachi et al., 2005, Chemosphere 58: 817 ~ 822), whitening effect (Jeon et al., 2006. J. Agric. Food Chem. 54: 9827-9833) , 2011, Biosci. Biotechnol. Biochem. 75: 140-144) have been known, and various studies on apricot are underway.

On the other hand, in Korea, due to the recent well-being fever, various makgeolli are developed and manufactured as the social demand for Takju increases.Takju added powder and chitooligosaccharides (Park Sang-sun et al., 2011. 169) has been reported, and Takju, which contains functional plants such as blueberries, cornus, palm cactus, deodeok, licorice and bokbunja, has also been developed. Studies on the production of rice koji related to the production of takju in Korea have mainly been carried out on the study of fungi, yeast and yeast related to starch saccharification and fermentation, and the study of extension of storage period of takju (Myomin et al., 1999, Journal of the Korean Society of Food Science and Nutrition, 12: 427 ~ 432; Wang Sangjin et al., 2012, Korea Food Research Journal, 19: 116 ~ 122). However, up to now, studies on physiological activity of Takju and their extracts have been at a basic level. The main researches on the physiological activities of Takju and sake lees in Korea are the antibacterial and antioxidant activities of sake lees prepared using barley, wheat, and situational mushrooms (Kim Tae-young et al., 2010, Korean Food). Korean Journal of Nutrition Science, 23: 299 ~ 305), antioxidative effect of gourd extract, promotion of procollagen synthesis, and anti-wrinkle effect through inhibition of expression of MMP-1 (Yu Jeong-min et al., 2010, Journal of the Korean Life Science, 20: 1838 ~ 1843), Efficacy of Ethylacetate Extracts of Zucchini on the IgE-mediated Allergy (Kang, Yeo-Jin et al., 2011. Korean Society of Life Science, 21: 1364 ~ 1369), Diabetes improvement by Takju intake 21: 638-643). However, there are few reports on the blood circulation related useful physiological activity of gourds. In particular, the effects of thrombin inhibition and platelet aggregation on human blood coagulation are not known.

The research on the development of the food using the Chinese cabbage has been reported as the functional food added with the Chinese cabbage, the functional noodle, the pork additive, etc. (Jo, Eunja et al., 2007, East Asian Journal of Food and Nutrition, 17: 94 ~ 102; , 2006. Korean Journal of Food Culture, 21: 565 ~ 570, Kim Sun-Mi et al., 2007. Korean Journal of Food Culture, 22: 359 ~ 364) : 1562 ~ 1570), and the production of yeast spores from the seeds (Invitrogen et al., 2004, Korean Society of Microbial Biotechnology, 32: 184 ~ 189).

On the other hand, as patent documents related to sake lees, the outer shellfish using the Republic of Korea Patent No. 10-115263 Bokbunja sake and its manufacturing method (the date of registration 29 May 2012), using the Republic of Korea Patent No. 10-1101313 brewing waste Method of manufacturing ethanol (Registration date December 26, 2011), Republic of Korea Patent No. 10-0736327 Functional natural cosmetics for moisturizing, whitening, wrinkle improvement using sake lees and its manufacturing method (Registration date June 29, 2007 ), Korea Registered Patent No. 10-0737862, functional organic fertilizer containing alcohol and by-product strain of Bacillus genus (Registration date July 4, 2007), Republic of Korea Patent No. 10-11194147 It discloses a composition for enhancing immune activity (January 26, 2012), etc. comprising as an active ingredient.

However, there has been no report on the effects of thrombocytopenia through direct inhibition of human thrombin enzyme, prothrombin enzyme, blood coagulation-related factors and platelet aggregation inhibition.

Therefore, the present inventors aimed at developing a functional food material exhibiting anti-thrombotic and blood flow improving activity. After preparing saccharin extract, the present inventors evaluated human blood plasma, human thrombin, prothrombin, and blood coagulation factors to evaluate thrombogenic inhibitory activity. The platelet aggregation inhibitory activity of the gourd was measured using the gourd extract, and the potent antithrombotic activity was confirmed in the gourd extract and its sequential fraction.

Republic of Korea Patent No. 10-115263

The present invention has been made to recover the useful physiologically active substance from the discarded gourds, increase the utility of the gourds as biological resources, and to overcome the risks associated with the side effects of the existing antithrombotic agents. The task is to include a pharmaceutical composition for the prevention or treatment of thrombotic diseases caused by direct inhibition of human thrombin, inhibition of prothrombin, inhibition of endogenous coagulation factor and inhibition of platelet aggregation, containing the extract of sake lees as an active ingredient, and the extract To provide a dietary supplement that is.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which contains sake extract (酒 粕) as an active ingredient.

The gourd extract is preferably gourd ethanol extract, gourd hot water extract or a mixture thereof.

The gourd extract is preferably an ethyl acetate fraction, a butanol fraction, a water residue, or a mixture thereof, of a gourd ethanol extract or a gourd hydrothermal extract.

The ethyl acetate fraction, butanol fraction and water residues are preferably obtained by sequentially distilling the sake ethanol extract or sake hot water extract after the hexene fraction, and then sequentially distilling with ethyl acetate and butanol.

In addition, the present invention provides a dietary supplement comprising the sake extract of the present invention.

The extract of sake lees as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis and the health functional food of the present invention, as shown in the examples of the present specification, extracts sake lees with ethanol or the like, and then extracts hexene, It is prepared by fractionation using ethyl acetate and butanol sequentially. They have the effect of effectively inhibiting antithrombotic activity, antiplatelet activity and thrombus formation by the direct inhibitory effect of human thrombin, and ischemic stroke through blood circulation improvement. And excellent effects that can be used for the prevention and treatment of thrombosis, such as hemorrhagic stroke. In particular, the gourd extract of the present invention does not show acute oral toxicity, excellent thermal stability, direct inhibition of human thrombin, prothrombin inhibition, blood coagulation factor inhibitory effect in acidic conditions and plasma of pH 2, extract, It is a very useful invention for the pharmaceutical industry and the food industry because it has an excellent effect that can be processed into various forms such as powder, pills, tablets, etc., so that it can be always taken.

Figure 1 is a graph showing the results of human platelet aggregation inhibitory activity of the ethanol extract of sorghum and sequential organic solvent fractions. From left 1: solvent control (DMSO), 2: aspirin (0.50 mg / ml), 3: aspirin (0.25 mg / ml), 4: gourd ethanol extract (0.25 mg / ml), 5: gourd hexene fraction (0.25 mg / ml), 6: sake ethyl acetate fraction (0.25 mg / ml), 7: sake butanol fraction (0.25 mg / ml) and 8: sake residue (0.25 mg / ml), respectively.

The present invention relates to pharmaceutical compositions and health functional foods for preventing or treating thrombosis having human thrombin direct inhibition, prothrombin inhibition, endogenous blood coagulation factor inhibition and platelet aggregation inhibitory activity. More specifically, the inventors of the present invention recovered the antithrombotic active ingredient from the gourd extract obtained by a certain method, by confirming that these components have excellent thermal stability and acid stability without showing oral acute toxicity. The extract was intended to be used as a pharmaceutical composition and health functional food for the prevention or treatment of thrombosis.

Specifically, the present inventors prepared ethanol extract or hot water extract of sake lees in order to develop a pharmaceutical composition for preventing or treating thrombosis and health functional food using sake lees, and sequential organic solvent fractionation, hexene fraction, ethyl acetate fraction , Butanol fractions and water residues, and the like, thrombin Time, Prothrombin Time and Activated Partial Thromboplastin Time (aPTT) for human plasma and human thrombin, respectively ), Platelet aggregation inhibitory ability was evaluated. As a result, thrombin thyme, prothrombin thyme and active part thromboplastin thyme showed prolonged activity in the ethanol extract or hot water extract of ethanol extract, and platelet aggregation inhibitory activity was 0.5 mg / ml at 0.25 mg / ml concentration. Efficacy corresponding to the. This means that gourd extracts exhibit stronger platelet aggregation inhibitory activity than aspirin. On the other hand, among the organic solvent fractions of ethanol extract of saccharin, ethyl acetate fraction showed thrombinogenesis inhibitory activity that was extended by 15 times or more in the thrombin time, the prothrombin time, and the active part thromboplastin time, compared to the non-added, 2.5 At the mg / ml concentration, thrombin time, prothrombin time and active partial thromboplastin time were extended 2.4-, 2.2- and 2.0-fold, respectively, compared with no addition. In addition, butanol fractions and water residues have completed the present invention by identifying potent platelet aggregation inhibitory effects comparable to aspirin (commercial antithrombotic agents).

In order to confirm the effect of the sake lees, the inventors of the present invention to prepare a sake lees extract and organic solvent fractions, analyze the useful components such as total polyphenols, flavonoids, etc., and directly inhibit the human thrombin of the extracts and fractions, Prothrombin inhibitory effect, active part thromboplastin time extension effect by inhibiting blood coagulation factors (VIII factor, IX factor, XI factor and XII factor), and platelet aggregation inhibitory effect using concentrated human platelets, were evaluated. After confirming that the extract has antithrombotic activity similar to that of the commercially available antithrombotic aspirin (trade name: Protect), human erythrocyte hemolytic activity does not appear, and the thermal and acid stability of the substance were investigated, and then the substance Was administered orally to rats, and acute toxicity test was conducted to confirm that the extract had little human toxicity. It was.

Accordingly, the present invention provides a pharmaceutical composition and health functional food for preventing or treating thrombosis, which contains sake extract as an active ingredient.

In a preferred embodiment, the sake lees extract is preferably sake ethanol extract, hot water extract or a mixture thereof.

In a preferred embodiment, the sake extract is preferably an ethyl acetate fraction, a butanol fraction, a water residue or a mixture thereof of sake ethanol extract or sake hot water extract.

The ethyl acetate fraction, butanol fraction and water residue may be obtained by hexene fractionation of the saccharin ethanol extract or saccharin hot water extract, and then sequentially fractionated into ethyl acetate and butanol, and water fraction may be obtained as a final residue after fractionation. have.

Hereinafter, the method of preparing a sake extract of the present invention and an organic solvent fraction thereof, and an efficacy test will be described in more detail.

The present invention comprises the steps of preparing an active extract from the commercial sake of Takju; Crude extract from crude extract with organic solvent fractions such as hexene, ethyl acetate, butanol and water residues; Investigation of the antithrombotic efficacy and stability of the extracts and fractions; And an acute toxicity test step of the fraction.

The "scavenger extract" included in the composition of the present invention comprises the steps of reducing the water content by compressing the sake lees obtained after the preparation process of the takju, extracting the obtained sake lees with an organic solvent, and using the filter network of 0.06 mm or less. It can be obtained by filtration and concentration under reduced pressure. The organic solvent used in the present invention may be water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, and the like. And 80 to 95% ethanol is most preferred.

In the present invention, the thrombin time, the prothrombin time and the epitaxial time of the ethanol extract or hot water extract of the sake extract showed increased antithrombotic activity compared to the non-added. In particular, the ethyl acetate fraction of the sequential organic solvent fraction of saccharin ethanol extract showed a prolonged antithrombotic activity at the concentration of 5 mg / ml, in which thrombin time, prothrombin time and apitime were extended by 15 times or more than the non-added group. , 2.5 mg / ml showed antithrombotic activity at 1.5 mg / ml of aspirin. The butanol fraction showed a 1.9-fold extension of only thrombin time at a concentration of 5 mg / ml. On the other hand, aspirin used as an antithrombotic agent extended thrombin time, prothrombin time, and epitaxial time by 8.6, 1.8, and 1.9 times, respectively, at 2.5 mg / ml concentration, and 2.7 and 1.55 at 1.5 mg / ml, respectively. Fold and 1.37 fold prolonged effect. These results indicate that the ethyl acetate fraction of saccharin extract, especially saccharin ethanol extract, acts on thrombin, prothrombin and blood coagulation factors to inhibit thrombogenesis, and anticoagulants such as aspirin have been reported to have side effects such as gastrointestinal disorders. It suggests that it can be replaced.

Squash extract of the present invention and its ethyl acetate fraction, butanol fraction and water residue can be prepared into a powder through a conventional powdering process such as distillation under reduced pressure and lyophilization or spray drying. They maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The gourd extract 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 extract of the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, 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 pharmaceutical composition 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.

In a preferred embodiment of the present invention, the effective amount of the extract of the present invention in the pharmaceutical composition of the present invention may vary depending on the age, sex and body weight of the patient. Generally, 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight, 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 used in a variety of foods and drinks effective for preventing or improving thrombosis. The food of the present invention containing the extract of the present invention may be in the form of powder, granule, tablet, capsule or beverage, for example, various foods, beverages, gums, tea, vitamin complexes and health supplement foods. .

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

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

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

Examples of the flavoring agents include natural flavoring agents such as tautatin, rebaudioside A and stiglycerin such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame. The ratio of the natural carbohydrate is generally 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 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 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the specific method of the present invention will be described in more detail by way of examples. The following examples are only one preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[ Example ]

Example  One: The  Extracts and organic solvents Fraction  pharmacy

We recovered the spruce produced at the Takju factory in Hoengseong, Gangwon Province in 2012. Ethanol extract was added twice a volume of ethanol (95%, Deoksan, Korea) with respect to the gourd and extracted twice twice at room temperature. Hot water extract was added 5 times the volume of water to the gourd and extracted by heating at 100 ℃ for 1 hour. Each extract was recovered after removing the foreign matter by using a filter, concentrated under reduced pressure to prepare a powder and stored at low temperature until use. Then, each extract was suspended in distilled water, and then partitioned sequentially with hexene, ethyl acetate, butanol, and finally a water residue was obtained.

The yields of ethanol extract and hot water extract were 10.02 ± 1.6% and 17.05 ± 3.3%, which showed higher extraction efficiency than ethanol extract. The yields of hexene fraction, ethyl acetate fraction, butanol fraction and water residues of the saccharin ethanol extract were 17.31%, 2.31%, 27.52% and 49.37%, respectively, showing a very small amount of ethyl acetate fraction. This result means that about 0.23 g of ethyl acetate fraction is obtained for 100 g of sake (water content 90 ± 5.3%), and the ethyl acetate fraction is considered to be considerably purified. On the other hand, the yields of hexene fraction, ethyl acetate fraction, butanol fraction and water residues of saccharine hot water extract were 0.27%, 0.54%, 5.37% and 90.09%, respectively, indicating that most of the hydrothermal extracts were transferred to water residues.

Example  2: The  Component analysis of extracts and Anti-thrombosis  Activity evaluation

PH of sake lees used in this example was 4.06, Brix was 13.8, alcohol content was 13.8%, solid content was 8.7-10%, and water content was 90-91.2%. The protein content was 3.06 ± 0.10%, carbohydrate 34%, crude fat 0.6%, crude fiber 2.38%.

Total flavonoids, total polyphenols, total sugars and reducing sugars of the sake extracts prepared in Example 1 were measured. The total flavonoid content was measured by stirring and extracting each sample with methanol for 18 hours, 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 1 hour reaction at 37 ° C. . As a standard reagent, rutin was used. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ Saturated solution was added, and allowed to stand at room temperature for 1 hour, and then absorbance was measured at 725 nm. Tannic acid was used as a standard reagent. Reducing sugar was measured by DNS method, and total sugar was quantified by phenol-sulfuric acid method. The results are shown in Table 1 below.

Yields and Analysis of Components of Squash Ethanol Extract and Hot Water Extract extract Extraction efficiency (%) Content (mg / g) Total polyphenol Total flavonoid Per total Reducing sugar Ethanol extract 10.02 ± 1.6 21.6 ± 0.7 2.52 ± 0.1 133.6 ± 10.2 114.1 ± 3.3 Hot water extract 17.05 ± 3.3 112.7 ± 3.5 40.2 ± 2.1 366.9 ± 15.4 137.8 ± 4.1

The extraction efficiency of saponium ethanol extract was 58% of hydrothermal extract, total polyphenol and flavonoid contents were 6.2% and 19%, total sugar content was 36% and reducing sugar content was 83%. Therefore, it is judged that hot water extraction of sake lees is suitable for extraction of a large amount of bioactive substances.

Next, the antithrombotic activity of the gourd extracts was evaluated. Antithrombotic activity was assessed in accordance with previously reported methods (Sohn et < RTI ID = 0.0 > 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 prepared from the whole blood of the applicant, and immediately after collection, the plasma was separated by centrifugation at 5,000g for 5 minutes at 4 ° C and stored frozen (fresh frozen plasma) and thawed at room temperature if necessary. The thrombin time, prothrombin time and apathy measurement were performed as follows.

Thrombin  time( Thrombin Time )

50 μl of 0.5 U thrombin (Sigma Co., USA) at 20 ° C. and 20 mM CaCl ₂ 50 μl, and 10 μl of various concentrations of sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) and allowed to react for 2 minutes. Then, 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 solidification time of 32.1 seconds. In the case of thermal stability measurement, sample solutions at various concentrations were heat-treated at 100 ° C for 30 minutes, allowed to stand at room temperature for 1 hour, and residual activity was measured. The thrombin inhibitory effect was expressed as the average value of the experiments repeated three or more times. The value obtained by dividing the solidification time at the time of adding the sample by the solidification time of the solvent control was multiplied by 100 and expressed as%.

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), incubate 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. 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 18.1 seconds.

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. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 55.1 seconds. The results of aPTT were expressed as the average of three replicate experiments. The value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control was multiplied by 100 and expressed in%.

The antithrombotic measurement results according to the above-described thrombin time, prothrombin time and episode time measurement are shown in Table 2 below.

Antithrombotic Activity of Lime Ethanol Extract and Hot Water Extract Final concentration
(mg / mL) Thrombin time (seconds) Prothrombin Time (sec) Age time (seconds) ethanol
extract Hydrothermal
extract aspirin ethanol
extract Hydrothermal
extract aspirin ethanol
extract Hydrothermal
extract aspirin 0 32.1
± 0.5 32.1
± 0.5 32.1
± 0.5 18.1
± 0.3 18.1
± 0.3 18.1
± 0.3 55.1
± 1.4 55.1
± 1.4 55.1
± 1.4 1.5 - - 86.3
± 6.6 - - 28.1
± 2.4 - - 75.5
± 5.1 2.5 33.0
± 0.9 39.5
± 2.7 277.1
± 11.9 20.0
± 0.7 22.9
± 1.1 32.8
± 3.8 53.5
± 6.5 72.0
± 6.7 104.3
± 7.2 5.0 38.5
± 1.5 57.8
± 1.2 > 480 20.1
± 0.3 26.5
± 0.5 > 270 61.0
± 4.2 88.1
± 0.9 > 800

Aspirin used as a control showed excellent antithrombotic effect at 5 mg / ml concentration. And 1.1-fold extension. However, in the case of hot water extracts, thrombin times, prothrombin times, and aptitimes extended 1.8 times, 1.4 times, and 1.6 times or more at 5 mg / ml concentrations, indicating that hot water extracts exhibited stronger antithrombotic activity than ethanol extracts. It was.

Example  3: The  The sequential organic solvent of the extract Fraction  Component analysis and Anti-thrombosis  Activity evaluation

The components and antithrombotic activity of the sequential organic solvent fractions obtained from the ethanol extract and hot water extract prepared in Example 1 were investigated. The results of measuring the total flavonoids, total polyphenols, total sugars and reducing sugars content of the fractions are shown in Tables 3 and 4, and the antithrombotic activity is shown in Tables 5 and 6.

Component Analysis of Organic Solvent Fraction from Sour Ethanol Extract extract Fractionation efficiency
(%) Content (mg / g) Total polyphenol Total flavonoid Per total Reducing sugar Hexene fraction 17.31 7.61 ± 0.26 4.28 ± 0.14 31.38 ± 0.78 23.0 ± 0.47 Ethyl acetate fraction 2.31 151.13 ± 4.65 19.77 ± 2.19 236.20 ± 8.55 61.64 ± 4.13 Butanol fraction 27.52 92.12 ± 5.47 16.57 ± 0.66 429.87 ± 14.30 224.48 ± 14.26 Water residue 49.37 97.64 ± 6.88 16.57 ± 5.07 345.67 ± 10.26 333.23 ± 14.51

Component Analysis of Organic Solvent Fraction from Zhuk Hot Water Extract extract Fractionation efficiency
(%) Content (mg / g) Total polyphenol Total flavonoid Per total Reducing sugar Hexene fraction 0.27 222.14 ± 9.88 29.27 ± 2.04 332.08 ± 12.90 124.57 ± 11.74 Ethyl acetate fraction 0.54 109.31 ± 8.15 38.84 ± 2.89 90.12 ± 6.58 29.90 ± 1.41 Butanol fraction 5.37 97.55 ± 2.34 16.17 ± 0.78 334.15 ± 18.23 161.55 ± 11.58 Water residue 90.09 98.14 ± 3.85 21.33 ± 1.50 357.78 ± 11.44 280.79 ± 10.12

Water residue was the most abundant fraction in the organic solvent fraction of saccharin ethanol extract, and ethyl acetate fraction was only 2.31%. However, the ethyl acetate fraction showed a very high content of polyphenols and flavonoids (151 mg / mg), and the butanol fraction and water residues were 35-43% saccharides. The total polyphenol and flavonoid contents are shown in the order of ethyl acetate fraction> water residue, butanol fraction> hexene fraction. It was confirmed that it contained.

On the other hand, sake hot water extract is more than 90% water-soluble component, hexene fraction and ethyl acetate fraction was very low fractionation efficiency of less than 1%. However, the ethyl acetate fraction and the hexene fraction showed very high polyphenol contents of 109.31 and 222.14 mg / ml and high flavonoid contents of 38.84 and 29.27 mg / ml.

Antithrombotic Activity of Organic Solvent Fractions and Water Residues of Squash Ethanol Extract sample Concentration (mg / ml) Thrombus generation time (seconds) Thrombin time Prothrombin time Apathy time DMSO (solvent) - 32.1 ± 0.5 18.1 ± 0.3 55.1 ± 1.4 aspirin 1.5 86.3 ± 6.6 28.1 ± 2.4 75.5 ± 5.1 Hexene fraction 5.0 32.1 ± 0.6 19.9 ± 1.2 60.6 ± 3.4 2.5 32.0 ± 0.4 18.4 ± 0.7 55.1 ± 0.9 Ethyl acetate fraction 5.0 > 480 > 280 > 800 2.5 77.0 ± 2.5 39.8 ± 3.5 110.2 ± 8.4 1.25 38.5 ± 1.4 21.7 ± 0.9 66.1 ± 4.0 Butanol fraction 5.0 60.9 ± 2.2 21.7 ± 2.6 60.6 ± 4.9 2.5 41.1 ± 3.5 19.8 ± 1.1 55.2 ± 0.6 Water residue 5.0 41.7 ± 1.7 19.9 ± 0.9 60.6 ± 1.6 2.5 33.5 ± 0.6 18.5 ± 1.4 58.5 ± 3.6

Antithrombotic Activity of Fractions of Organic Solvents and Water Residues of Squash Extracts sample density
(mg / ml) Thrombus generation time (seconds) Thrombin time Prothrombin time Apathy time DMSO (solvent) - 32.1 ± 0.5 18.1 ± 0.3 55.1 ± 1.4 aspirin 1.5 86.3 ± 6.6 28.1 ± 2.4 75.5 ± 5.1 Hexene fraction 5.0 67.41 ± 4.3 23.5 ± 1.6 82.6 ± 7.6 2.5 37.2 ± 1.4 18.0 ± 1.3 57.1 ± 4.5 Ethyl acetate fraction 5.0 > 480 > 280 > 800 2.5 93.1 ± 6.5 45.3 ± 6.3 110.2 ± 9.8 1.25 53.2 ± 3.6 38.7 ± 7.2 81.1 ± 10.6 Butanol fraction 5.0 83.5 ± 6.3 30.0 ± 2.5 104.7 ± 11.2 2.5 51.4 ± 2.2 21.7 ± 2.6 60.6 ± 4.9 Water residue 5.0 54.6 ± 7.1 21.7 ± 3.1 66.2 ± 3.4 2.5 50.2 ± 4.5 20.2 ± 1.2 58.2 ± 5.3

As a result of evaluating the antithrombotic activity of the organic solvent fraction from the ethanol extract of sake, the ethyl acetate fraction showed the best antithrombotic activity as shown in Table 5 above. The ethyl acetate fraction was more potent than the antithrombotic effect of aspirin 1.5 mg / ml at 5 mg / ml, and the thrombin time was similar at 2.5 mg / ml, but the prothrombin time and epitaxial time were further prolonged, resulting in stronger antithrombotic activity than aspirin. Indicated.

On the other hand, as a result of evaluating the antithrombotic activity of the fraction of saccharine hot water extract, as shown in Table 6 above, the antihistogenic activity of the hexene fraction, ethyl acetate fraction and butanol fraction at 5 mg / ml concentration was comparable to that of aspirin (1.5 mg / ml concentration). It showed thrombus activity. However, at 2.5 mg / ml concentration, only ethyl acetate fraction showed better antithrombotic activity than aspirin. Therefore, it was confirmed that ethyl acetate fraction of saccharin extract can be developed into an antithrombotic composition better than aspirin. In addition, the present results suggest that the ethyl acetate sequential solvent fraction has a low fraction yield but shows strong antithrombotic activity, which is very efficient as a method for purifying anti-thrombotic active substance of gourd.

Example  4: The  Extract, sequential organic solvent Fraction  Human platelet aggregation inhibitory activity

As a part of the antithrombotic activity of the ethanol extract and fractions of the gourd, the aggregation inhibitory activity against human platelets was evaluated, and the results are shown in FIGS. 1 and 7. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibitory activity ( Platelet aggregation inhibition activity )

Platelets are mixed with 3.2% sodium citrate solution in a ratio of 1: 9 from healthy human whole blood, followed by centrifugation at 1,100 rpm for 10 minutes to obtain PRP (platelet rich plasma) in the upper layer, which is washed with 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), centrifuged at 3,000 rpm for 10 minutes. After separation, the cells were resuspended in suspending buffer, and the platelet count was adjusted to 4 × ^{10 9} / ml. Then, 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).

Human Platelet Aggregation Inhibitory Activity of Swallow Ethanol Extracts and Fractions Sample / Control burglar
(Amplitude: ohm) inclination
(Slope) Extension time
(Lag time: seconds) Descending area
(Area under) DMSO (solvent) 21 3 42 271.6 Aspirin (0.50mg / mL) 8 0 112 40.2 Aspirin (0.25 mg / mL) 13 One 50 117.6 Ethanol Extract (0.25mg / mL) 5 One 62 65.6 Hexene fraction (0.25 mg / mL) 9 9 4 147.8 Ethyl Acetate Fraction (0.25 mg / mL) 16 3 20 247.7 Butanol Fraction (0.25 mg / mL) 6 One 46 81.9 Water residue (0.25mg / mL) 7 2 41 108.3

* The smaller the descending area, the stronger the cohesion inhibition

First, aspirin exhibited excellent human platelet aggregation inhibitory activity, and confirmed concentration-dependent aggregation inhibitory activity. Sour ethanol extract showed a strong platelet aggregation inhibitory activity with a drop area of 65.6 at 0.25 mg / ml concentration, similar to that of aspirin 0.5 mg / ml, which is a commercial aggregation inhibitor (falling area 40.2). Among the fractions, flocculation inhibitory activity was observed in the order of butanol fraction> water residue> hexene fraction. Butanol fraction and water residue showed stronger platelet aggregation inhibitory activity than aspirin at the same concentration. This showed weak activity as 247.7. Thus, sake extract, fractions thereof, and water residues were found to be able to replace aspirin with antithrombotic agents.

Example  5: The  Ethyl acetate Fraction  And Butanol Fraction  Chemical Properties and Stability of Active Substances

Ethyl acetate and butanol fractions of the ethanol extract of saplings obtained in Example 1 were dried under pressure to prepare a powder, and then human erythrocyte hemolytic activity, plasma stability, thermal stability, and acid stability of the recovered active material were confirmed. The modulated actives showed high stability due to no inhibition of antithrombotic activity and a decrease in platelet aggregation inhibitory activity for 2 hours at 100 ° C., 2 hours at pH 2 (0.01 M HCl), and 2 hours at plasma. It was. In addition, hemolytic activity against human erythrocytes was not seen up to 5 mg / ml concentration (Table 8).

Erythrocyte Hemolytic Activity of Extracts and Fractions of Squash Sample (5 mg / ml) Human erythrocyte hemolytic activity (%) Ethanol extract 2.40 ± 1.10 Hexene fraction 0.10 ± 1.15 Ethyl acetate fraction 4.92 ± 3.81 Butanol fraction 2.01 ± 0.20 Water residue 3.05 ± 0.6 Triton X-100 0.1% 100.00 ± 0.00 Emhotericin B 125 μg / ml 75.30 ± 8.50

Anthrone reaction for qualitative detection of carbohydrates, ninhydrin reaction for amino acid colorimetric determination, diethylene glycol reaction for polyphenol detection and flavonoid detection reaction were carried out on ethyl acetate and butanol fractions. Silver has been identified as a substance containing polyphenols and flavonoid components, in part in the form of carbohydrate complexes that do not contain proteins or amino acids.

Example  6: The  Ethyl acetate and Butanol Fraction Single oral toxicity  exam

Four-week-old rats (Slc, ICR Mouse) were supplied from a central laboratory animal, and the temperature was 23 ± 3 ℃, relative humidity 50 ± 10%, and 12-hour intervals at 150-300Lux illuminance (8 am-8 pm). After 14 days of acclimatization in an animal laboratory controlled by), single oral toxicity of the ethyl acetate fraction and the butanol fraction of the ethanol extract obtained from Example 1 was evaluated. The animal experiment was conducted with the approval of the Animal Experiment Ethics Committee of Andong National University (Academic Research Support Division-2132, May 18, 2012). After dissolving the sample in DMSO and water, the concentration of 400, 800, 1500, 2000mg / kg After oral administration of 3 animals each, survival and pathologic symptoms were observed for 1 week. As a control, only DMSO was orally administered. As a result, the survival rate was 100% and there were no sign of pathological abnormality. Thus, the fraction of sake lees, which has been used for food and medicinal purposes, is classified as low or non-toxic, and it was determined that the use of low concentrations would not cause additional problems.

Claims (5)

A pharmaceutical composition for preventing or treating thrombosis, which contains sake extract as an active ingredient. The method according to claim 1,
The gourd extract is a pharmaceutical composition for preventing or treating thrombosis, characterized in that the gourd ethanol extract, gourd hot water extract or a mixture thereof. The method according to claim 1,
The edible extract is a pharmaceutical composition for preventing or treating thrombosis, characterized in that the ethyl acetate fraction, butanol fraction, water residue or a mixture thereof of ethanol extract or edible hydrothermal extract. The method of claim 3, wherein
The ethyl acetate fraction, butanol fraction and water residue is a hexene fraction of the saccharin ethanol extract or saccharin hot water extract, and then obtained by sequentially fractionating with ethyl acetate, butanol, thrombosis prevention or treatment pharmaceutical composition. A health functional food comprising the gourd extract of any one of claims 1 to 4.

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