KR101945733B1 - Pharmaceutical composition comprising the ethanol extracts of yunnongchamssal as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an ethanol extract of Yunnongchamssal as an active ingredient and more specifically to a pharmaceutical composition for preventing or treating thrombosis comprising ethanol extract of Yunonongchamssal, The present invention relates to a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis through inhibition of strong platelet aggregation inhibition and blood coagulation, which comprises a water residue after fractionation as an active ingredient. The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the ethanol extract of Yunnong rice as an active ingredient of the health functional food inhibit the coagulation inhibition effect of platelets which inhibits the thrombogenesis- And exhibits excellent hemostatic activity against human erythrocytes and exhibits excellent thermal stability and exhibits a blood coagulase inhibitory effect, a blood coagulation factor inhibitory effect and a thrombogenic effect even in an acidic condition of pH 2 and in plasma It is expected that it can be used as a pharmaceutical composition and health functional food for prevention and treatment / improvement of thrombosis such as ischemic stroke and haemorrhagic stroke through improvement of blood circulation because the loss of the related enzyme inhibitory effect does not appear, , Powder, ring, and tablet. Since the excellent effects that can be easily prepared into a form is very useful inventions the pharmaceutical industry and the food industry.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, which comprises ethanol extract of Yongin rice,

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing an ethanol extract of Yunnongchamssal as an active ingredient and more specifically to a pharmaceutical composition for preventing or treating thrombosis comprising ethanol extract of Yunonongchamssal, The present invention relates to a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis through inhibition of strong platelet aggregation inhibition and blood coagulation, which comprises a water residue after fractionation as an active ingredient.

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

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

Rice is one of the world's three largest crops, with more than half of the world's population using stocks, and is a key resource in the country that can not be confronted with other crops in Asia. In Korea, which uses rice as a stock, rice consumption per capita in 2016 is steadily decreasing to 61.9 kilograms (as a result of the National Statistical Office's 2016 grain consumption survey), which is 1.0 kilogram less than in 2015. However, recently, nutritional superiority and various functionalities of coloring matter such as black rice and red rice have become known, so that the consumption of coloring rice is increasing.

The functional components of rice, which are known to date, include dietary fiber, phytic acid, ferulic acid, isovitectin, γ-aminobutyric acid and various phenolic compounds. Antioxidant activity and thrombin inhibitory activity of these functional ingredients , 2005. Korean Journal of Food Science and Nutrition 34: 593-598), blood glucose lowering effect and carcinogenesis inhibitory effect (Mi-Sook, 2004, Food and Nutrition Industry, 9: 19-25). In particular, regarding the antithrombotic activity of rice, it has been reported that thrombin inhibitory effect of colored rice and commercial rice is partly reported (Son Ho Yong et al., 2005. Korean Journal of Food Science and Nutrition 34: 593-598) Thrombin, prothrombin and blood coagulation factor inhibitory effects are rarely studied, and the efficacy of rice in relation to platelet aggregation, which determines the initial response to thrombus formation, Recently, improved rice for the purpose of enhancing specific nutrients and enhancing functionality has been spreading, but there has been no development of functional rice that can inhibit blood clot formation and prevent cardiovascular diseases.

The patent relating to the antithrombogenic effect of rice is disclosed in Korean Patent No. 10-1121087, in which rice coated with a black garlic liquid and its preparation method, in No. 10-0683233, a composition containing a red rice fermented product, And monacukus strains for these have been disclosed, but they are known to be anti-thrombogenic activity by the black garlic liquid and the Monacukus strain, not by the rice itself. Therefore, there is no patent for a rice composition which is excellent in thrombogenesis inhibition and platelet aggregation inhibiting activity so far.

KR 10-1121087 B

 Son Ho Yong et al., 2005. Antithrombotic and Antioxidant Activity of Methanol Extract of Commercial Rice, Korean Journal of Food Science and Nutrition 34: 593-598

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems of the prior art, and it is an object of the present invention to provide a pharmaceutical composition for preventing or treating thrombosis, which comprises ethanol extract of sunflower rice and active fractions thereof, Health functional foods.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which comprises ethanol extract of sunflower seed rice as an active ingredient.

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

The present invention also provides a health functional food for preventing or ameliorating thrombosis, which comprises an ethanol extract of Yunnan rice and rice as an active ingredient.

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

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the ethanol extract of Yunnong rice as an active ingredient of the health functional food inhibit the coagulation inhibition effect of platelets which inhibits the thrombogenesis- And exhibits excellent hemostatic activity against human erythrocytes and exhibits excellent thermal stability and exhibits a blood coagulase inhibitory effect, a blood coagulation factor inhibitory effect and a thrombogenic effect even in an acidic condition of pH 2 and in plasma It is expected that it can be used as a pharmaceutical composition and health functional food for prevention and treatment / improvement of thrombosis such as ischemic stroke and haemorrhagic stroke through improvement of blood circulation because the loss of the related enzyme inhibitory effect does not appear, , Powder, ring, and tablet. Since the excellent effects that can be easily prepared into a form is very useful inventions the pharmaceutical industry and the food industry.

Fig. 1 shows rice, fresh glutinous rice, and lupine rice used in the examples of the present invention, and rice, fresh glutinous rice, and lupine rice are shown from the left side.
Fig. 2 shows rice powder, fresh glutinous rice, and lupine rice powder used in the examples of the present invention, and rice, fresh glutinous rice and lupine rice powder are shown from the left side.
(DMSO), 2: aspirin (0.5 mg / ml), 3: aspirin (0.25 mg / ml), and aspirin 4: Ethanol extract of rice (0.25 mg / ml), 5: Fresh glutinous rice ethanol extract (0.25 mg / ml) and 6: Ethanol extract of wheat rice (0.25 mg / ml).
(DMSO), 2: aspirin (0.5 mg / ml), 3: aspirin (0.25 mg / ml), and 4: 6: ethyl acetate fraction (0.25mg / ml) of ethanol extract of yunon rice, 7: ethanol extract of yunnung rice (rice extract) (0.25mg / ml) (0.25 mg / ml), and 8: water residue (0.25 mg / ml) after the sequential organic solvent fractionation of the ethanol extract of Yongon rice.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared an extract of commercially available rice, glutinous rice (freshly glutinous rice) and rice paddy rice to evaluate the anti-thrombotic efficacy against rice, evaluate human platelet aggregation inhibitory activity and blood coagulation inhibitory activity, After extracting the hexane fraction, the ethyl acetate fraction, the butanol fraction and the water residue after the fractionation of the organic solvent from the yunon rice extract, the antithrombotic active ingredient was identified. And fractions have excellent heat stability and acid stability, while showing no hemolytic activity against human erythrocytes. Thus, the extract and fractions can be used as pharmaceutical compositions and health functional foods for preventing or treating / improving thrombosis .

More specifically, the inventors of the present invention prepared a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis of rayon rice, evaluated the antithrombotic activity by preparing an ethanol extract of yunon rice, Ethanol extracts of rice paddy rice, which showed better activity than glutinous rice, were prepared by sequential organic solvent fractions with hexene, ethyl acetate and butanol. Finally, water residues were recovered after fractionation. The antithrombotic activity of the extract and of each of the fractions was compared with that of thrombin time, prothrombin time and activated partial thromboplastin time (aPTT) and platelet aggregation for human plasma and human thrombin As a result of evaluation of the inhibitory activity, both the ethanol extract of Yongon rice and its fractions and the water residue showed excellent blood coagulation inhibitory activity, and the water residue showed a strong inhibitory activity against platelet aggregation. These antithrombotic activities were comparable to those of aspirin, which is used clinically as an antithrombotic agent. In addition, it was confirmed that the ethanol extracts, active fractions and water residues of the above-mentioned leavened rice extract did not show hemolytic activity on human erythrocytes.

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

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

The present invention also provides a health functional food for preventing or ameliorating thrombosis, which comprises an ethanol extract of Yunnan rice and rice as an active ingredient.

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

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

In a preferred embodiment, the present invention provides a method for preparing a fermented soybean oil, Evaluating the antithrombotic activity of the extract; Preparing sequential organic solvent fractions of hexane, ethyl acetate, and butanol from an ethanol extract of rayon seed rice showing excellent antithrombotic activity and preparing a water residue obtained thereafter; Evaluating the antithrombotic activity of the extract and the fractions and examining the stability of the active substance.

The "Yunnongchamssal" referred to in the present invention is a new variety in which the variety name is registered in the National Seeds Institute of Korea as the variety name registration No. 01-0001-242 by the applicant Kim Yoon Soo on Oct. 20, 2008 to be. Yongnong rice was selected for the production of Dongjin rice in 2001 and hybridized with the rice in the following year. After that, hybrid seeds were sown again, (National Seed Source: www.seed.go.kr, March 13, 2012) and registered with the Korean Intellectual Property Office as Trademark Registration No. 40-0757976 (2008 8 21, Applicant: Kim, Yoon-soo), and current breeder rights holders are Baek Myeong Ok and Kim Young Won. The botanical characteristics of the Yongnong rice registered in the National Seed Source are as follows:

- Early leaves, leaves, leafy leaves No anthocyanin pigment, leaf green is normal.

- There is no or little amenorrhea in the leaves, and there is no anthocyanin pigment in the leaves, leaflets, and leaves are colorless.

- Leaf length and width are 50cm and 17mm, respectively.

- The shape of the stem is semi-upright, and the heading is August 10th.

- There is no or very soft anthocyanin pigment on the outside, and the main color is white.

- The length of the stem is 96cm, the length of the ears is about 27cm, and maturity is 9/25.

- The exterior color is brown, and there is no exterior pattern.

- Base length is medium, color is straw color, there is phenol reaction of outer plant.

- The brown color is white and the ending oil is pure.

"Ethanol Extract of Lactic Acid Ethanol Extract of the Present Invention" Extracting the ground powder with ethanol; And filtering the extract using a filter net of 0.06 mm or less and concentrating it under reduced pressure.

In addition, the ethanol extract of rayon seed rice contained in the pharmaceutical composition and the health functional food of the present invention may be a butanol fraction or a water residue obtained by sequentially or separately fractionating the ethanol extract with an organic solvent of hexene, ethyl acetate and butanol.

In the present invention, when the concentration of ethanol extract of rayon seed rice was adjusted to 5 mg / ml, the thrombin time, the prothrombin time and the apathy time were measured. As a result, the blood coagulation time was 1.2 times longer than that of the no- Respectively. It is considered that this activity is superior to commercially available rice and glutinous rice, and it will have a great effect on Asians who use rice as a stock. As a result of measurement of human platelet aggregation inhibitory activity at a concentration of 0.25 mg / ml of ethanol extract of yunnung rice, the platelet aggregation was weakly promoted, but the effect of promoting aggregation was promoted by the effect of platelet aggregation promoting effect of ethanol extract of commercial rice and glutinous rice And relatively small compared to the others.

As a result of evaluation of blood coagulation inhibition activity of organic solvent fractions and water residues of ethanol extract of yunnung rice, concentration-dependent inhibition of thrombogenesis was observed in all fractions. In particular, in the butanol fraction, excellent blood coagulation inhibitory activity Respectively. In the case of thrombin time measurement, butanol fractions> water residues> ethanol fractions> hexane fractions> ethylacetate fractions were superior in order of activity; in the case of prothrombin time measurement, butanol fraction> hexene fraction> ethyl acetate fraction> ethanol extract> Water, respectively. In the case of aprotic time for measuring blood coagulation factor inhibition, the activity was excellent in the order of ethyl acetate fraction, hexane fraction, butanol fraction, ethanol fraction, and water residue. This means that the extracts and fractions (5 mg / ml) of Yongin rice paddy are superior or similar to the aspirin (1.5 mg / ml) activity used in clinical practice. As a result of evaluating the inhibition activity of platelet aggregation in organic solvent fractions and water residues of ethanol extract of yunnung rice, it was confirmed that the water residue had a concentration - dependent inhibitory activity against platelet aggregation.

Therefore, it was confirmed that the ethanol extract and its active fractions of Yongin rice paddy rice could be developed as a blood coagulation inhibitor and a platelet aggregation inhibitor having indications for thrombosis that can replace aspirin, which is highly likely to cause side effects such as gastrointestinal disorders.

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

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

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

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

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

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

As a preferable specific example, the preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

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

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

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

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

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

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

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

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

Examples of the flavoring agents include natural flavoring agents such as tautatin, rebaudioside A and stiglycerin such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

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

[ Example ]

Example  One: Yongon rice , Nutritional Components and Physicochemical Properties of Commercial Rice and Glutinous Rice

In 2016, Yongon rice, commercial rice, and commercial glutinous rice (fresh glutinous rice) cultivated and harvested in Andong, Gyeongbuk Province were purchased (Fig. 1), and their moisture content and nutrient content were analyzed. First, moisture content was 9.6 ~ 13.7%, respectively. On the other hand, crude protein content was 12.6% in Yoon - nong rice, which was 1.7 ~ 2 times higher than commercial rice and glutinous rice. In addition, crude lipid content was 2.6 times higher than commercial rice, and ash content was 1.34% higher than that of commercial rice, 3.35 times higher than commercial rice, 1.9 times higher than glutinous rice (Table 1).

[Table 1] Yongon rice , Nutritional analysis of commercial rice and glutinous rice

On the other hand, Yoonong rice was different from the conventional rice and glutinous rice, and each rice was pulverized (Fig. 2). As a result, Yoonong rice was lower in brightness than conventional rice and glutinous rice, Redness and yellowness were high (Table 2). This is considered to be the result of the inclusion of various colors of rice grains in the yunon rice (Figs. 1 and 2).

[Table 2] Color analysis of commercially available rice, glutinous rice,

Example  2: Yongon rice , Ethanol extracts of commercial rice and glutinous rice

Ethanol extracts were prepared from each of the rice powders prepared in Example 1. In preparing the ethanol extract, 10 times of ethanol was added to the sample, and the extract was repeatedly extracted three times at room temperature. The extracts were collected by filtration and concentrated under reduced pressure to prepare an ethanol extract. Table 3 shows the ethanol extraction efficiency and the component analysis of the extract of rice, rice, commercially available rice and glutinous rice. 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, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extract, 40 μl of 1N NaOH was added, and the absorbance at 420 nm was measured at 37 ° C. for 1 hour. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 3] Yongon rice . Comparison of yield and composition of ethanol extracts of commercial rice and glutinous rice

As shown in Table 3, the ethanol extracting efficiency of yunnung rice was 9.4 times higher than that of rice and 3.1 times higher than that of fresh glutinous rice. The content of total polyphenol and total flavonoid were similar to those of fresh glutinous rice. However, total sugar and reducing sugar contents were similar to rice. Therefore, the content of useful components of ethanol extract per 100 g of rice considering extraction efficiency was calculated, and the results are shown in Table 4.

[Table 4] Yongon rice . Content of active ingredients in ethanol extract prepared per 100 g of commercial rice and glutinous rice

As shown in Table 4, the lean rice contains 12.13 mg and 12.41 mg of total polyphenols and total flavonoids per 100 g, which is 12.6 times and 11.4 times higher than that of rice, and 2.7 times and 2.9 times Respectively. Therefore, it is anticipated that the leavened rice will exhibit various useful physiological activities with high total polyphenol and total flavonoid contents.

Example  3: Yongon rice , Evaluation of anticoagulant activity of ethanol extracts of commercial rice and glutinous rice

The blood coagulation inhibitory activity of the ethanol extract of Yunon rice, commercial rice, and glutinous rice obtained in Example 2 was evaluated, and the results are shown in Table 5. At this time, evaluation methods of blood clotting inhibition activity were evaluated according to the previously reported methods (Sohn et al., 2004. Kor J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14 J., < / RTI > Life Science, 20: 922-928), thrombin time, prothrombin time and apathy time were measured. Plasma was obtained from commercial control plasma (MD Pacific Technology Co., Ltd., Huayuan Industrial Area, China), and thrombin time, prothrombin time and apitime time were measured by the following procedure.

Thrombin Time

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

Prothrombin time ( prothrombin  time)

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

aPTT (activated Partial Thromboplastin  Time)

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

[Table 5] Rice, glutinous rice, Yongon rice  Coagulase inhibitory activity of ethanol extracts

As a result, the thrombin time, prothrombin time, and apathy time were increased by 1.36 times, 1.42 times, and 1.52 times, respectively, at the 1.5 mg / ml concentration of aspirin used as an antithrombotic agent. Or more to confirm strong antithrombotic activity. On the other hand, unlike other rice, Yoonong rice showed relatively good thrombin time, prothrombin time and apathy time extension effect.

Example  4: Yongon rice , Human platelet aggregation inhibitory activity of ethanol extracts of commercially available rice and glutinous rice

The human platelet aggregation inhibitory activity of the ethanol extracts of the leavened rice, commercial rice, and glutinous rice obtained in Example 2 was evaluated, and the results are shown in Table 6 and FIG. Platelets are cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentration instead of nucleus, and circulating blood vessels together with various blood cells. It is an important cell that secretes factors and binds to collagen exposed by damage of endothelial cells to form a primary hemostatic plug to initiate thrombogenesis. Therefore, inhibition of platelet aggregation is a very important activity to prevent thrombogenesis. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity (Platelet aggregation inhibition activity)

Human platelets were used as platelets and washed once with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, the cells were resuspended in a suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 0.49 mM MgCl ₂ , 0.25% gelatin, pH 7.4) and incubated at 3,000 rpm for 10 minutes After centrifugation, the cells were resuspended in a suspending buffer and the platelet count was adjusted to 4 × ^{10 9} / ml. Then, 2.5 μl of collagen was added to 1 ml of the suspension, and the mixture was reacted for 5 minutes. Platelet aggregation was measured at 37 ° C using a whole-blood aggregator (Chrono-log, USA).

[Table 6] Rice, glutinous rice, Yongon rice  Platelet Aggregation Inhibitory Activity of Ethanol Extract

As a result, as shown in Table 6 and FIG. 3, in the case of DMSO as a solvent control, human platelets rapidly and strongly aggregated by the addition of collagen, and aspirin as a platelet aggregation inhibitor strongly inhibited platelet aggregation in a concentration-dependent manner . At this time, the aspirin showed an aggregation degree of 50.6% at the concentration of 0.25 mg / ml. On the other hand, the ethanol extracts of the used rice showed no inhibitory effect on platelet aggregation. Especially, commercial rice and glutinous rice showed strong agglutination promoting activity. On the contrary, platelet aggregation of yunnung rice showed 131% coagulation compared to no platelets, and the coagulation promoting activity was relatively weak.

Example  5: Yongon rice  Ethanol extract, its sequential organic solvent Fraction  Preparation and analysis of their components

On the basis of the above results, a large amount of ethanol extract of yunnungun rice was prepared, and then fractionated with hexene, ethyl acetate and butanol in order of organic solvent. Finally, the water residue was recovered. The extraction efficiency of ethanol extract, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction are shown in Table 7.

[Table 7] Yongon rice  Ethanol extract and its sequential Fraction  Yield and component analysis

As shown in Table 7, the ethyl acetate fraction and the water residue of 7.1% and 3.0% of the extract, respectively, were replaced with 71.6% and 20.3% of the hexane fraction and the water residue, respectively. These results indicate that the ethanol extract of Yongin rice paddy contains a variety of polar substances, and it means that about 0.085g of butanol fraction and 0.57g of water residue can be recovered from 100g of dried paddy rice. As a result of total polyphenol content analysis, the highest content was found in fractions having the highest content of 19.3 mg / g, which is the highest in water residue, and in the total flavonoid content analysis, 4.6 mg / g. < / RTI > The contents of total sugar and reducing sugar showed the highest contents of 205mg / g and 14.2mg / g in water residue. Therefore, various physiological activities of ethanol extracts of Yongin rice paddy were expected to appear in butanol fractions and water residues.

Example  6: Yongon rice  Ethanol extract and sequential Organic solvent Fraction  Evaluation of blood coagulation inhibitory activity

The blood coagulation inhibitory activity of the ethanol extract and the fractions thereof obtained in Example 5 was evaluated, and the results are shown in Table 8.

[Table 8] Yongon rice  Ethanol extract and its sequential Organic solvent Fraction  Anticoagulant activity

As a result, aspirin showed excellent anticoagulant activity in a concentration dependent manner, and fractions of ethanol extract of yunnung rice showed excellent anticoagulant activity. Concentration dependent inhibition of thrombogenesis was observed in all fractions. In particular, the butanol fraction showed the best blood coagulation inhibitory activity. In the case of the thrombin time measurement, the butanol fraction, the water residue, the ethanol fraction, the hexane fraction, and the ethyl acetate fraction were superior in order of activity. In the case of measuring the prothrombin time, the butanol fraction, the hexane fraction, the ethyl acetate fraction, Respectively. In the case of aprotic time, which measures inhibition of blood coagulation factors, the activity was in the order of ethyl acetate fraction> hexene fraction> butanol fraction> ethanol fraction> water residue. This means that the extracts and fractions (5mg / ml) of Yongin rice paddy rice are superior to or similar to the aspirin (1.5mg / ml) activity used in clinical practice. Especially, It can be used as a safe antithrombotic agent that can replace aspirin, in particular, as a blood coagulation inhibitor.

Example  7: Yongon rice  Ethanol extract and sequential Organic solvent Fraction  Evaluation of human platelet aggregation inhibitory activity

The human platelet aggregation inhibitory activity of the extract of the leavened rice and the fractions thereof obtained in Example 5 were evaluated, and the results are shown in Table 9 and FIG.

[Table 9] Yongon rice  Ethanol extract and sequential Organic solvent Fraction  Human platelet aggregation inhibitory activity

As a result, an excellent platelet aggregation inhibitory activity was confirmed in the water residue after the organic solvent fractionation of the ethanol extract of the yunnung rice. The water residue of the ethanol extract of the Yongin rice extract showed a 58.5% platelet aggregation at the concentration of 0.25 mg / ml for the water residue and an aggregation of 54.3% at the concentration of 0.25 mg / ml of the aspirin used as the antithrombotic agent. It may be possible to develop a safe antithrombotic agent, especially a platelet aggregation inhibitor, which can replace aspirin with high side effects such as gastrointestinal disorder.

Example  8: Yongon rice  Ethanol extract of butanol Fraction  ≪ / RTI > and plasma residue, acid and thermal stability of water residues

The plasma stability, thermal stability and acid stability of the anti-thrombotic activity of the butanol fraction and the water residue of the ethanol extract obtained from Example 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 clotting inhibition and platelet aggregation inhibition activity even for 1 hour treatment in plasma . Therefore, it is expected that the above extracts and active fractions will maintain excellent antithrombotic activity during digestive absorption process and food production process.

The butanol fractions and water residues of the ethanol extracts of the leavened turtle rice showed strong anticoagulant activity and platelet aggregation inhibitory activity, suggesting that it is possible to produce anti-thrombogenic agents and blood circulation improving health functional foods using the leavened egg rice, The present invention relates to a method for preventing or treating thrombosis which can replace conventional antithrombotic agents because it has both safety and stability as a stock which has been fed continuously in large quantities for a long period of time and has no side effects or problems, It can be developed as food.

Claims (4)

Korean Society for the Protection of Breeding Protection No. 3909 A pharmaceutical composition for the prevention or treatment of thrombosis containing an ethanol extract of Yunnongchamssal as an active ingredient. [Claim 2] The pharmaceutical composition according to claim 1, wherein the ethanol extract of lupine seed rice is a butanol fraction or a water residue obtained by successively fractionating an ethanol extract of lupine seed rice with hexene, ethyl acetate and butanol. A functional food for prevention or improvement of thrombosis containing ethanol extract as an active ingredient. [Claim 4] The health functional food according to claim 3, wherein the ethanol extract of the leavened rice extract is a butanol fraction or a water residue obtained by successively fractionating ethanol extract of leavened rice with leucine, ethyl acetate and butanol.

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