KR20160101887A - Pharmaceutical composition comprising the extract of phaseolus radiatus l. for prevention and control of thrombosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating thrombotic disease, containing a hot water extract of adzuki beans as an active ingredient. The present invention further relates to a functional health food including the same. More specifically, the present invention relates to a pharmaceutical composition and a functional health food for preventing or treating thrombotic disease, containing, as an active ingredient, a hot water extract of adzuki beans (steamed liquid), a fraction derived from the steamed liquid sequentially treated with various polar organic solvents, and water-soluble water residues after the organic solvent fraction.

Description

[0001] PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF PHASEOLUS RADIATUS L. FOR PREVENTION AND CONTROL OF THROMBOSIS [0002] The present invention relates to a pharmaceutical composition for preventing or treating thrombotic diseases,

The present invention relates to a pharmaceutical composition for the prevention or treatment of a thrombotic disease containing hot water extract of red bean ( Phaseolus radiatus L.) as an active ingredient, and a health functional food containing the same, and more particularly, (Drip-proofing liquid) and a fraction prepared by using the various polar organic solvents as the preparation liquid and a pharmaceutical composition and health functional food for preventing or treating thrombotic diseases containing the water-soluble residue after the organic solvent fraction as an active ingredient .

As a constituent of human body, blood has various functions such as oxygen, nutrients, waste function and buffering action, maintenance of body temperature, control of osmotic pressure and ion balance, maintenance of moisture, regulation of fluidity, maintenance and regulation of blood pressure, . Therefore, blood and blood circulation are essential for the survival of human beings. In case of damage of blood vessels, blood clots should be rapidly generated to prevent blood loss. However, excessive blood clot formation in the blood vessels interferes with blood circulation, and in severe cases, blood vessels are blocked to cause serious damage to cardiovascular system and brain tissue, resulting in thrombosis. Such thrombosis is caused by the following: 1) abnormal vascular endothelial cells, 2) excessive coagulation activity of blood coagulation factors (XII factor, XI factor, IX factor, X factor etc.) and blood coagulation enzyme (thrombin, prothrombin enzyme) 4) The abnormal function of the thrombus dissolution system is known to be responsible for these causes. These causes are caused individually or in combination, resulting in excessive blood clot formation in the blood vessels. For the prevention and treatment of thrombotic diseases, currently 1) various anticoagulants such as heparin and coumarin which inhibit the activity of blood coagulation factors to inhibit thrombogenesis, 2) anti-platelet aggregation inhibitors such as aspirin Thrombocytopenia, and 3) a thrombolytic agent such as europaea, which inhibits thrombus formation by dissolving already generated thrombus. However, they are not only very expensive, but also have limited use due to hemorrhagic side effects, gastrointestinal disorders and hypersensitivity reactions.

On the other hand, red bean ( Phaseolus radiatus L.) is an annual plant of rosemary beans and is mainly cultivated in Northeast Asia such as Korea, China and Japan. The shape and structure of red beans is similar to that of beans, but it has a red luster and unlike beans, it has a white band at the center. It also contains 8 to 9% water, 68% carbohydrate and 20% protein and is nutritionally excellent. Especially, red bean contains various vitamins such as vitamin B1, minerals such as potassium, calcium and phosphorus, and lysine which is insufficient in rice, so it is effective for recovery of each disease and fatigue, And it has been used as a food material for nutritional supplementation because it exhibits a beneficial function of promoting carbohydrate metabolism. Red beans have a unique thermostable protein on the outside of the cell wall, and they are thermally denatured at the time of heating. Therefore, they have an unusual structure that can not be used as a paste but can form a sediment, and is mainly used for producing red beans, red beans, Recently, it has been developed as a variety of processed food such as red bean beverage and red bean fermented food.

In one room, red beans are called red beetles and are used to treat diarrhea, dysentery, species, and obesity. In the private sector, red beans have been used as water for swelling and diet drinks by promoting diuretic and bowel movements. The shell of bean contains not only a large amount of cyanidin glycosides and saponins which give an arine flavor but also contains a large amount of growth inhibitory factors such as a trypsin inhibitor, , Boiled for 10 to 20 minutes at a temperature of 90 ° C or higher, and after removing the first mulled solution, the red beans are recovered and then boiled to a high temperature and high pressure to prepare boiled red beans, which are then dried in a suitable manner, At this time, boiled water is used as a beverage in a second high temperature.

Studies on domestic red beans have been gradually transformed into studies on useful physiological functions of red beans and studies on development of bean processed products in studies on seed improvement and cultivation of red beans. Studies on red bean skin pigment for using natural anthocyanin type pigments, , Inhibition of carcinogenesis of red bean ethanol extract, inhibition of arthritis, and antibacterial effect on oral bacteria of bean hot water extract and inhibition of DNA damage have been reported.

The activity related to thrombosis was found to be similar to that of plasmin in the case of acid treatment (pH 3.0, 1 min) and heat treatment (100 ℃, 10 min) after extracting red bean with distilled water at room temperature (Isoflavin content, antioxidant and thrombolytic activity of red bean and mung bean, Kor, J. Soc. Food Cookery Sci. 19 : 263-270), and evaluation of antithrombotic activity of red bean and boiled red bean , But the ethanol extract of boiled bean ethanol did not exhibit any thrombogenic effect at all, while the extract of infant ethanol extract showed strong inhibition of blood coagulation enzyme and blood coagulation factor at 2.5 ~ 5.0 mg / ml concentration (Lee, Kyung-Gil, 2014. Comparison of Composition and Physiological Activity of Red Bean and Red Bean, Korean J. Microbiol. Biotechnol. 42: 162-169). However, there has been no known anti-thrombotic activity due to inhibition of blood coagulation enzymes and blood coagulation factors and inhibition of platelet aggregation in hot water extract of red beans, that is, juice and its controlled preparations.

Korean Patent No. 10-0786213 discloses a patent for fermented soybean food having excellent fibrinolytic activity and antioxidative activity, fermented with various soybean and red bean, -0112747 A patent for manufacturing a well-being walnut confectionery including natto or chongkukjang is known, but there is no patent for strong antithrombotic activity of red bean juice.

KR 10-0786213 B

 Lee, WK, et al., 2014. Comparison of components and physiological activities of red beans and red beans. Korean J. Microbiol. Biotechnol. 42: 162-169.  Oh, Hee-Sook et al., 2003. Isoprolavin content, antioxidant and thrombolytic activity of red bean and mung bean. Kor. J. Soc. Food Cookery Sci. 19: 263-270

Accordingly, a problem to be solved by the present invention is to extract red bean, which has been freed from foreign matter by screening and rinsing, with hot water to prepare a cooking liquid, which is an extract of bean juice, and sequentially fractionating the lean solution with an organic solvent of hexane, ethyl acetate and butanol Ethyl acetate fractions, butanol fractions and water-soluble residue were recovered to confirm strong anti-blood coagulant activity and human platelet aggregation inhibitory activity of bean hydrothermal extract. These extracts and fractions were confirmed to have no hemolytic activity on human erythrocytes, And to provide a pharmaceutical composition exclusively for hemostasis and a health functional food containing the active ingredient.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating thrombotic diseases, which comprises hot-water extract of red bean ( Phaseolus radiatus L.) as an active ingredient.

The bean hydrothermal extract is preferably at least one selected from the group consisting of ethyl acetate fraction, butanol fraction and water residue obtained by successively fractionating the bean hydrothermal extract with hexene, ethyl acetate and butanol.

In addition, the present invention provides a pharmaceutical composition for inhibiting blood clotting, which comprises hot water extract of red bean ( Phaseolus radiatus L.) as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for inhibiting platelet aggregation, which comprises red hot water extract of red bean ( Phaseolus radiatus L.) as an active ingredient.

In addition, the present invention provides a health functional food for preventing or ameliorating a thrombotic disease containing hot water extract of red bean ( Phaseolus radiatus L.) as an active ingredient.

The juice of the bean jugular hot water extract prepared by hot water extraction of the red bean paste of the present invention, the sequential organic solvent fraction thereof and the water residue obtained after the fractionation of the beverage of the present invention showed strong inhibition of blood coagulation enzyme and inhibition of blood coagulation factor and human platelet aggregation And exhibits antithrombotic activity by its inhibitory activity and has an effect of effectively inhibiting thrombogenesis and has an excellent effect that can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. In addition, the bean hydrothermal extract of the present invention and its active fractions can be safely used because of no hemolytic activity against human erythrocytes, excellent thermal stability, no acidic condition at pH 2, and no loss of activity even in human plasma, , Cream, powder, ring, tablet, etc., and can be very usefully used in the pharmaceutical industry and the food industry.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an absorbance analysis chart of the bean jugular water extract of the present invention at 200 to 700 nm. FIG. The maximum absorption wavelength is 210 to 215 nm, and the specific absorption wavelength is 275 to 278 nm.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for the prevention and treatment of thrombosis, which comprises a red bean juice extract of red bean, more specifically a fraction thereof or a water residue after an organic solvent fraction. The inventor of the present invention has found that red bean extract The extract was resuspended in sterilized water, and then fractionated with an organic solvent of hexane, ethyl acetate and butanol in order to prepare an organic solvent fraction and a water residue, and their blood coagulation enzyme inhibitory activity , Coagulation factor inhibitory activity and platelet aggregation inhibitory activity were confirmed. In addition, they were found to be free of human hemolytic hemolytic activity, so that they were found to be free of toxicity, and exhibited excellent thermal stability, acid resistance at pH 2, and plasma safety, thus confirming that conventional antithrombotic agents can be substituted.

Accordingly, the present invention provides a pharmaceutical composition and a health functional food for the prevention or treatment of a thrombotic disease which contains bean juice extract as an active ingredient.

The bean hydrothermal extract showing strong antithrombotic activity without the hemolytic activity of the present invention and the organic solvent fractions or water residues thereof can be used for the prevention or treatment of various diseases related to thrombosis. Such diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, , Deep vein thrombosis, lower limb edema, pain and acute peripheral artery occlusion. Vein thrombosis can include deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.

In addition, the present invention provides a pharmaceutical composition for inhibiting blood coagulation, which comprises bean hot-water extract as an active ingredient.

The pharmaceutical use of the blood coagulation inhibition can be used as an anticoagulant which acts as a mechanism for inhibiting the activities of blood coagulation enzymes and blood coagulation factors that are the causes of thrombosis.

In addition, the present invention provides a pharmaceutical composition for inhibiting platelet aggregation comprising an adzuki bean hydrothermal extract as an active ingredient.

The pharmaceutical use of the platelet aggregation inhibition can be used as an anti-platelet agent which acts as a mechanism for inhibiting aggregation of platelets which is a cause of thrombus formation.

In a preferred embodiment, the present invention relates to a method for preparing a bean jam hot water extract (extracting active material); Purifying the active ingredient through sequential organic solvent fractions; The stability of the active ingredient; And evaluating the human erythrocyte hemolytic activity.

The bean paste hydrothermal extract (edible juice) can be prepared by adding 1.1 to 1.15 times of water to the red bean which has been freed of foreign substances by screening, washing with water, boiling at 90 ° C or higher for 1 hour and cooling at room temperature for about 4 hours The recovery rate of the juice is preferably about 75 to 95% of the weight of the bean used.

* The prepared red bean juice liquid of the present invention can be pulverized and then further fractionated with various organic solvents. As a preferable example, sequential organic solvent fractions using hexene, ethyl acetate and butanol can be recovered, and the organic solvent fractions and water residues are recovered to evaluate the blood coagulation inhibiting activity and platelet aggregation inhibiting activity of the present invention .

The pharmaceutical composition containing the bean jug hot water 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, Injections, and the like, and they can be administered through various routes including oral administration, 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.

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, the effective amount of each of the organic solvent fractions or water residues after the organic solvent fraction of the bean hydrothermal extract having excellent antithrombotic activity in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, May be administered daily or every other day, or one to three times per day, in an amount of 1 to 5,000 mg, preferably 100 to 3,000 mg per body weight. 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. In addition, the pharmaceutical composition of the present invention may 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 bean jam hot water extract of the present invention, its fractions or water residues may generally be added in an amount of 0.01 to 15% by 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 . 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 flavor agent include tau martin, rebaudioside A, glycyrrhizin, saccharin, and aspartame. The proportion of the above-mentioned flavoring agent 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 fraction of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples.

<Examples>

Example 1: Preparation and characterization of red bean juice

The domestic red bean (Chungju red bean) grown in Andong, Gyeongbuk Province in 2012 was selected, washed and washed to remove foreign matter. Afterwards, 4kg was added to the washing machine and washed 5 times. After that, it was put on a normal sieve for 30 minutes and dehydrated naturally. Then, 4.5 L of water was added and boiled at 90 ° C or more for 1 hour. Thereafter, the mixture was cooled at room temperature for about 4 hours, and 3.4 L of the juice was collected. At this time, the recovery rate of the cooked liquid was 85% of the weight of the used bean. The process of boiling ordinary red beans, that is, adding red bean juice 2.5 to 3 times the weight of water and boiling for several hours, causes an additional problem of concentrating the fermented juice, There is a problem that the sensibility is bad and it is not suitable.

The prepared juice was a pale yellowish clear liquid with a pH of 6.5, a solids content of 1.26% (water solids content of 1.2%), and a distinctive aroma taste remained (Table 1). The color difference measurement result showed a brightness of 12.6, a redness of 2.8 and a yellowness of 5.4, and a color difference of 80.0. The color difference measurement was performed by repeating the L value, lightness, a value, and redness values and the b value and yellowness values three times using a colorimeter (Chromatometer CR-300, Minota, Japan) And the color difference (E) was calculated. For the standard white plate, the L value was 92.39, the a value was -0.08, and the b value was 1.39, and the color difference ( E ) was calculated using the following equation.

[Table 1] Physicochemical properties and color difference of red bean juice

In addition, the juice contains 15 mg of crude protein per 100 g, 40 mg of lipid, 411 mg of carbohydrate, and 74 mg of aspirate, resulting in a very low calorie of 2.06 Kcal / 100 g, And it can be used as a swelling-removing food used in plastic surgery and the like.

On the other hand, as a result of measuring the absorbance at various wavelengths, the maximum absorbance of the dipping liquid was found at 210 to 215 nm, and the specific absorbance was 275 to 278 nm, indicating that the dipping liquid contained a large amount of aromatic compounds (for example, polyphenol component) (Fig. 1).

Example 2: Preparation of organic solvent fractions and water residues of red bean juice

The organic solvent fraction of the red bean juice prepared in Example 1 was sequenced using hexane, ethyl acetate and butanol, and finally the water residue was recovered. Table 2 shows the yields and the results of component analysis of each fraction relative to the weight of red bean juice. For the total flavonoid content, each sample was stirred for 18 hours in methanol, 400 μl of 90% diethylene glycol was added to 400 μl of the filtered extract, and 40 μl of 1 N NaOH was added. After reacting at 37 ° C for 1 hour, absorbance was measured at 420 nm Respectively. As a standard reagent, rutin was used. 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. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 2] Analysis of yield and composition of each fraction relative to the weight of red bean juice

As shown in Table 2, 38.3% of the juice was transferred to the butanol fraction, about 51.7% was transferred to the water residue, and the hexane and ethyl acetate fractions were less than 0.1% and 8.9%, respectively. Water soluble component. Total flavonoid contents were in the order of ethyl acetate> butanol> hexene fraction> water residue. Total polyphenol contents were in the order of butanol> ethyl acetate> water residue> hexene fraction. On the other hand, total sugar and reducing sugar contents were in the order of water residues> butanol> ethyl acetate> hexene fraction. Total polyphenol content, total polyphenol content, total sugar content and reducing sugar content were 75.5%, 407.4%, 59.2% and 67.4%, respectively, in the water residues.

Example 3: Evaluation of antithrombotic activity of red bean juice and fractions

The red ginseng juice and fractions were dissolved in dimethylsulfoxide (DMSO), diluted to an appropriate concentration, and thrombin time, prothrombin time and aPTT were measured using human plasma. At this time, the hexene fraction was almost not prepared and was excluded from the evaluation. Antithrombotic activity was evaluated according to the previously reported method (Ryu et al. 2010. J. Life Science , 20, 922-928). Plasma was purchased from commercial control plasma (MD Pacific Hemostasis, MD Pacific, China). Each blood coagulation inhibitory activity assay is as follows.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA) and 50 μl of 20 mM CaCl ₂ and 10 μl of various concentrations of sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes at 37 ° C., and 100 μl of plasma was added And the time until the plasma coagulated was measured. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 32.1 sec. In the case of the thermal stability measurement, sample solutions of various concentrations were heat-treated at 100 占 폚 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 times or more, and the coagulation time at the time of adding the sample was divided by the coagulation time of the solvent control.

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. 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 18.1 sec. The prothrombin inhibitory effect was expressed as the average value of the experiment repeated three times or more, and the solidification time at the time of adding the sample was divided 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. After the addition of 50 μl CaCl ₂ (35 mM), 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 value of three replicate experiments, and the solidification time at the time of sample addition was expressed as the value divided by the solidification time of the solvent control.

[Table 3] Evaluation of antithrombotic activity of red bean juice and fractions

As shown in Table 3, aspirin (1.5 mg / ml), which is used as an antithrombotic agent in clinical practice, showed good thrombin, prothrombin and blood coagulation factor inhibition, 1.7 times and 1.8 times Fold and 1.8 times longer. The thrombin time, prothrombin time and aTPT of the red ginseng juice and fractions (5 mg / ml) were measured to confirm a strong antithrombotic activity comparable to aspirin. Specifically, in the case of red bean juice, TT, PT and aPTT, which were extended more than 15 times at 2.5 mg / ml concentration, showed 15 times or more prolonged effect in PT and aPTT at the same concentration. Thrombin enzyme inhibition was the best in red bean juice, and the inhibition of blood clotting factor inhibition (aPTT) was the strongest in water residues. These results suggest that red bean juice and its fractions can efficiently inhibit thrombus formation in various pathways and replace conventional antithrombotic agents such as aspirin reported in gastrointestinal disorders.

Example 4: Inhibition activity of human platelet aggregation of red bean juice extract and fractions

The human platelet aggregation inhibitory activity was evaluated using the prepared red bean juice and fractions. The intravascular thrombus formation is caused by abnormalities of vascular endothelial cells, blood coagulation factors (XII factor, XI factor, IX factor, X factor, etc.) and excessive coagulant activity of blood coagulation enzyme (thrombin, prothrombin enzyme) It was evaluated not only for abnormal function but also for excessive platelet aggregation. 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 endothelial cell damage to form a primary hemostatic plug and initiates thrombus formation. Therefore, platelet aggregation inhibition is very important to prevent thrombogenesis It is active. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity (Platelet aggregation inhibition activity)

Platelets were mixed with 3.2% sodium citrate solution in a ratio of 1: 9 from healthy human whole blood, and centrifuged at 1.100 rpm for 10 minutes to obtain upper layer platelet rich plasma (PRP). The platelets were washed 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. Platelet aggregation was then measured at 37 ° C using a whole-blood agarose (Chrono-log, USA). The results of the platelet aggregation inhibition assay are shown in Table 4.

[Table 4] Human Platelet Aggregation Inhibitory Activity of Red Bean Extracts and Fractions

As shown in Table 4, aspirin showed an inhibitory effect on platelet aggregation in a concentration dependent manner, and showed a coagulation degree of 59.2% (coagulation inhibition: 40.8%) at the concentration of 0.25 mg / ml compared to the non-additive. As a result of evaluating the degree of coagulation at the concentration of 0.25 mg / ml of the red bean juice and fractions, it was confirmed that the coagulation inhibition was stronger than that of aspirin in all samples except the hexene fraction. Especially, red ginseng juice and water residues inhibited 87 ~ 93% of platelet aggregation even at 0.125 mg / ml concentration and showed strong antithrombotic activity corresponding to aspirin several times. Platelet aggregation inhibition was in the order of water residues> red bean juice> ethyl acetate fraction> butanol fraction> hexene fraction. These results suggest that the red ginseng juice and its fractions can be used as anticoagulant food materials and functional foods in consideration with the thrombin generation enzyme (thrombin, prothrombin) and blood coagulation factor inhibitory activity of Example 3 have.

Example 5: Hemolytic activity of human erythrocyte of red bean juice and fraction

Table 5 shows the hemolytic activity of erythrocyte of red bean juice and fractions.

[Table 5] Red blood cell hemolytic activity of red bean juice and fraction

First, DMSO and water used as controls did not have hemolytic activity, and triton X-100 showed 100% hemolysis of red blood cells at a concentration of 0.1%. On the other hand, it was confirmed that there was no red blood cell hemolysis phenomenon at concentrations of 0.5 mg / ml in red bean juice, ethyl acetate fraction, butanol fraction, and water residues, so that acute toxicity and erythrocyte hemolytic activity were not observed. When the above results are summarized, the red ginseng juice and its ethyl acetate fraction, butanol fraction and water residue are excellent in antithrombotic effect by inhibition of human thrombin, prothrombin and blood coagulation factors, , Which is superior to the aspirin used in the present invention, and is expected to be developed as a natural antithrombotic agent having no side effects. Particularly, red kidney juice which shows inhibition of blood coagulation factor and inhibition of platelet aggregation and water residue after sequential organic solvent fraction, , Cosmetics, and pharmaceuticals industries.

Example 6: Stability of water residue after infusion of red bean juice and organic solvent

The red kidney juice obtained from the above Example 1 and Example 2 and the water residue after the sequential organic solvent fraction were treated at 100 ° C for 1 hour, treated at pH 2 (0.01M HCl) for 2 hours, treated with plasma for 2 hours As a result of evaluating the residual thrombin inhibitory activity, it was confirmed that all of the above treatments retained the initial activity. Thus, it was confirmed that the red stain juice and the water residue after the sequential organic solvent fraction showed high stability in acid, heat and plasma treatment Respectively.

Claims (5)

A pharmaceutical composition for the prevention or treatment of a thrombotic disease containing red ginseng liquid ( Phaseolus radiatus L.) as an active ingredient. The pharmaceutical composition according to claim 1, wherein the red bean juice is selected from the group consisting of ethyl acetate fraction, butanol fraction or water residue obtained by successively fractionating red bean juice with hexene, ethyl acetate and butanol. A pharmaceutical composition for inhibiting blood coagulation comprising red ginseng juice ( Phaseolus radiatus L.) as an active ingredient. A pharmaceutical composition for inhibiting platelet aggregation comprising red ginseng ( Phaseolus radiatus L.) as an active ingredient. Red bean ( Phaseolus radiatus L.) A health functional food for preventing or ameliorating a thrombotic disease containing an immersion liquid as an active ingredient.

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