KR102172558B1 - Pharmaceutical composition comprising the extract of pueraria flos 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 health functional food for preventing or treating thrombosis containing an extract of Pueraria flos as an active component. More specifically, the present invention relates to a pharmaceutical composition and health functional food for preventing or treating/alleviating thrombosis by inhibiting blood coagulation and inhibiting platelet aggregation, which contains a water residue obtained after fractionation of various organic solvents from an ethanol extract of Pueraria flos, which is a flower bud of Pueraria montana as an active component. The extract of Pueraria flos, as the active component of the pharmaceutical composition and health functional food for preventing or treating thrombosis of the present invention exhibits strong antithrombotic activity by an effect of inhibiting the aggregation of platelets, which play a role in the initiation of thrombus formation along with inhibition of thrombus formation-related enzymes and blood coagulation factors, and at the same time, does not exhibit any hemolytic activity on human red blood cells, has excellent thermal stability, and does not exhibit loss of an inhibitory effect on blood coagulation factors and an inhibitory effect on thrombus formation-related enzymes, thereby being able to expect to be used for preventing and treating thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. In addition, the active component is processed into various forms such as extracts, powder, pills, tablets, etc., and has an excellent effect of being prepared in a form that can be taken at any time, thereby being a very useful invention in the pharmaceutical and food industries.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing brown flower extract as an active ingredient {PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF PUERARIA FLOS AS 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 health functional food for the prevention or treatment of thrombosis containing an extract of Pueraria flos as an active ingredient, and more specifically, various from the ethanol extract of Galhwa, which is a bud of arrowroot. It relates to a pharmaceutical composition for preventing or treating/improving thrombosis by inhibiting blood coagulation and inhibiting platelet aggregation, containing a water residue obtained after fractionation of an organic solvent as an active ingredient, and a health functional food.

Blood as a component of the human body has various important functions such as transporting and buffering oxygen, nutrients and waste products, maintaining body temperature, maintaining osmotic pressure and maintaining ionic equilibrium, maintaining moisture schedule, controlling liquid properties, maintaining and controlling blood pressure, and defending the body. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombosis dissolution system are complementarily regulated in the body. Among them, the mechanism of the blood coagulation reaction system is after platelets adhere and aggregate on the vessel wall to form platelet thrombi. , It has been reported that the blood coagulation system is activated and fibrin thrombus is formed around the platelet aggregate.

The formation of fibrin thrombi leads to activation of thrombin, which is involved in fibrin coagulation, through several step reactions of numerous blood coagulation factors. Finally, fibrin monomers are produced from fibrinogen, and fibrin monomers are polymerized by calcium, resulting in platelets and endothelial cells. It binds and forms a fibrin polymer that is cross-linked by factor XIII, creating a permanent blood clot. In addition, thrombin plays a central role in the formation of blood clots by activating platelets, factors V, and VII to promote blood clotting reactions. Therefore, the thrombin activity inhibitory substance can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood coagulation. On the other hand, in the endogenous thrombogenic pathway, the sequential activation of factor XII, factor XI, factor IX, and factor X, followed by prothrombin activation, is known to ultimately activate thrombin, and specific inhibition of blood coagulation factors is also important. Being a target. To date, various anticoagulants such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases, but they are not only very expensive, but also bleeding side effects, gastrointestinal disorders and irritability. The situation is limited to such use.

On the other hand, arrowroot ( Pueraria lobata or Pueraria montana var.lobata) is a perennial deciduous vine plant that grows naturally in the temperate and temperate regions of Korea, China, Japan, and Taiwan. It grows well in barren land and salty beaches. The root is enlarged as a tuber and grows more than 1m, the stem usually extends longer than 20m, and the flower blooms in August in a reddish purple color in a butterfly shape. The fruit is a narrow fruit, 4~9cm long, and ripens around September~October. Arrowroot roots contain 10-15% starch and various nutrients, and have been edible as oral crops for a long time. Recently, isoflavone active substances such as daidzin, genistin and puerarin, various saponins, vegetable sterols and phenol compounds It has been confirmed that physiologically active substances are included (Jung-suk Kim et al., 2002, Korean Journal of Food Science and Technology 164:710-718). Therefore, as various effects such as prevention of osteoporosis, improvement of insomnia, and prevention of hypertension by various physiologically active substances of arrowroot root are known, they are newly used as health foods in Guhwang foods. Various processed foods are being developed.

In oriental medicine, the root of arrowroot is called "galgeun", and it is used as a precious medicine that protects health. Donguibogam said, "The property is flat and cool. The taste is sweet and there is no poison. The richness of the head heals the soreness of the head, and the sweat is released to release the mark, open the pores, and relieve alcohol poisoning. Stop alternating to improve the taste and digestion. It is explained, "It removes heat from the chest, makes the small intestine better, and heals the injuries from the iron. It also creates the essence and quenches the thirst."

Studies related to arrowroot include the antibacterial effect of root extract and the cell protective effect against oxidative stress (Shin Yusu et al., 2012. Korean Society of Medicinal Crop Science Spring Conference 307-308), immune cell activation effect (Kim Jongjin et al., 2012. Journal of Life Science 22: 1107-1113), Effect of Arrowroot on Lipid Metabolism and Bone Metabolism in Ovariectomized Mice (Heejun Cho, 2010, Master's Thesis, Korea University), Lipids in Diabetic Rats Induced by Streptozotocin Extract Effect on Metabolism (So-Jung Hwang, 2008. Master's Thesis at Kosin University), Antibacterial Effect of Arrowroot Extract on Oral Microorganisms (Hyunok Lee et al., 2004. Journal of Dental Hygiene Science 4: 45-48), On the noodle making properties of noodles added with arrowroot starch Research (Young-soon Lee et al., Korean Journal of Culinary Research 16: 681-688), Quality Characteristics and Isoflavone Content of Cheonggukjang Added with Arrowroot Isoflavone (Myung-Ye Lee et al., 2010. East Asian Society for Dietary Life, 20: 543-550), Arrowroot Extract Cheonggukjang The anti-obesity effect of capsules on ob/ob Mice (Kilsoo Kim et al., 2012. Journal of the Korean Society of Food Science and Nutrition 41: 782-789) is known, and changes in antioxidant activity and puerarin content of arrowroot according to roasting conditions (Koohee Choi et al. , 2017. Korean Journal of Food Storage and Distribution 24: 440-445) has been reported to appear.

As for the patents related to arrowroot, Korean Patent No. 10-1434163 [Composition with antioxidant, anti-inflammatory and whitening effect containing arrowroot extract as an active ingredient], No. 10-1177508 [Arrow for the prevention and treatment of menopausal women's health] Extract composition], No. 10-1064139 [Method for producing functional arrowroot lactobacillus fermented milk enriched with isoflavones], No. 10-0891165 [Isoflavone-enhanced arrowroot chunggukjang and health supplement composition for preventing osteoporosis containing the same as an active ingredient] , No. 10-0996952 [Isoflavone-fortified arrowroot Cheonggukjang sikhye and its manufacturing method], No. 10-0557006 [Arrow lactic acid bacteria fermented product enhanced with isoflavone, a vegetable female hormone, and its manufacturing method], No. 10-0345798 [ Hangover-relieving functional food and its manufacturing method using a mixture of earth japonica, alder, arrowroot], No. 10-1256352 [Vinegar manufacturing method using brown root], No. 10-0183436 [functional beverage composition containing arrowroot starch and The manufacturing method], No. 10-1105391 [Food composition containing the enzyme digestion liquid of arrowroot and its manufacturing method], No. 10-0771706 [Method for producing functional fermented wine using brown flour], No. 10-0557333 [Functionality A method of manufacturing arrowroot jelly], No. 10-1083507 [Method for producing a odor-removing extract using arrowroot and Sukjihwang] and No. 10-0953800 [A composition for preventing white hair and treating vitiligo containing brown root] are disclosed. In addition, Korean Patent Application Publication No. 10-2017-0068029 [Pharmaceutical composition for treating malignant melanoma containing arrowroot extract as an active ingredient], No. 10-2013-0112840 [Arrow extract or fractions thereof] Antimicrobial composition containing a light irradiation for Korean light], No. 10-2015-0104782 [Textile sheet for improving atopy including nanocapsules of arrowroot and arrowroot extract], No. 10-2018-0086028 [Fermented vinegar using arrowroot Method of manufacturing], No. 10-2018-0032800 [Arrow powder for hangover and a method of manufacturing gum using the same] is known.

Arrowroot flower buds, "brown flower", have been used to treat hangovers, headache, fever, thirst, loss of appetite, abdominal distension, and vomiting, and research related to browning is induced by UVB of ethanol extract of brown flower in hairless mice. Anti-wrinkle effect on wrinkled wrinkle formation (Miyoung Yoon, 2018, Korean Journal of Aesthetics, 521-525), A study on the hepatoprotective effect of browning and puerarin in experimental non-alcoholic fatty liver model and its mechanism of action (Hyung-Chil Hwang, 2014, Wonkwang University Ph.D. dissertation), the regulation of browning when the function of macrophages activated by lipopolysaccharide derived from Gram-negative bacteria is performed (Senting et al., 2009, Korean journal of medicinal crop science 17: 8-14), Puerariae Flos extract induces hyperlipidemia in rats Effect on the antioxidant activity and lipid concentration of (Park, Kun-Jin et al., 2009, Journal of the Korean Society of Food Science and Nutrition, 38: 846-851), Analysis of Components and Antioxidant Activity of Brown Flower (Shin Eon-hwan, 2009, Journal of the Korean Society of Food Science and Nutrition 38: 1139-1144 ), antimutagenic activity of galwha (Jung Youngjae et al., 2004, Journal of Korean Oriental Internal Medicine 25: 106-118), effect of active ingredient of galwha on streptozotocin diabetic hyperlipidemia (Myunghee Shin, 2002, Master's thesis at Kyungsung University) Antioxidant and cytoprotective effects of one isoflavone (Kyung-Tae Lee et al., 1999, Journal of Pharmacy, 43: 736-742), reduction of macrophage-mediated inflammatory response (Ting Shen et al., 2009. Korean J Medicinal Crop Sci.17:8?14 ) And anti-inflammatory activity of lactic acid-fermented brown flower (Won, Kyung-Hwa, 2015, Master's thesis, Kyung Hee University) is known.

In addition, patents related to browning include Korean Patent Registration No. 10-1357119 [Pharmaceutical composition for the prevention and treatment of endometriosis containing brown flower extract as an active ingredient], No. 10-0545382 [Galhwa extract with anti-stress effect] ], No. 10-0552995 [Brain function improving effect of brown flower extract and its manufacturing method], No. 10-0733336 [Composition for preventing or treating obesity or hyperlipidemia containing fermented product of brown root or brown flower extract], Article No. 10-0394148 [Treatment for gastric ulcer and duodenal ulcer using irisoridone isolated from the flower of the brown flower] is disclosed. In the food field, No. 10-0540788 [Health food containing brown flower extract] and No. 10-1752571 [Cultured and aged cheonggukjang using brown flower extract and a method for producing brown red pepper paste using the same] is known.

In addition, Korean Patent Application Publication No. 10-2018-0078942 [Composition for preventing or treating oral diseases containing brown flower extract], No. 10-2015-0104916 [For skin regeneration containing essential oil extracted from arrowroot flowers] External preparation and cosmetic composition], No. 10-2011-0029317 [Pharmaceutical composition for preventing or treating obesity or hyperlipidemia], No. 10-2016-0118740 [Composition for innate immunity enhancement and antiviral containing Galhwa extract as an active ingredient ], No. 10-2015-0103847 [Composition for the prevention or treatment of non-alcoholic fatty liver disease containing brown flower extract as an active ingredient], No. 10-1996-0000088 [Beverage for removing hangovers using brown flower], No. 10 -2010-0075211 [Galhwaju and its manufacturing method] is known.

Regarding antithrombotic activity, it has been reported that puerarin contained in prickly pear and brown stalk exhibits platelet aggregation inhibitory activity to prevent and improve thrombosis (Liu R et al., Am J Chin Med 34: 1037-1045), Until now, antithrombotic activity by potent inhibition of platelet aggregation of water residues after organic solvent fractionation of brown flowers extract without puerarin as an active substance has not been known.

KR 10-0552995 B

Liu R, Xing D, Lu H, Wu H, Du L. 2006. Pharmacokinetics of puerarin and ginsenoside Rg1 of CBN injection and the relation with platelet aggregation in rats. Am J Chin Med 34: 1037-1045

The present invention was conceived to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to provide a pharmaceutical composition and health functional food for the prevention or treatment of thrombotic diseases containing brown flower extract as an active ingredient. I want to provide.

In order to solve the above problems, the present invention is a pharmaceutical composition for the prevention or treatment of thrombosis containing as an active ingredient a water residue obtained by sequentially fractionating the ethanol extract of Pueraria flos with hexene, ethyl acetate and butanol Provides.

In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis containing as an active ingredient a water residue obtained by sequentially fractionating the ethanol extract of Pueraria flos with hexene, ethyl acetate and butanol.

In addition, the present invention provides a blood coagulation inhibitor containing as an active ingredient a water residue obtained by sequentially fractionating the ethanol extract of Pueraria flos with hexene, ethyl acetate, and butanol.

In addition, the present invention provides a platelet aggregation inhibitor containing as an active ingredient a water residue obtained by sequentially fractionating the ethanol extract of Pueraria flos with hexene, ethyl acetate and butanol.

The pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the extract as an active ingredient in health functional foods are inhibited by the inhibitory effect of platelet aggregation, which plays a role in the initiation of thrombus formation, along with inhibition of thrombus formation-related enzymes and blood clotting factors. At the same time, it exhibits strong antithrombotic activity, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, and shows a loss of blood coagulation factor inhibitory effect and blood clot formation-related enzyme inhibitory effect even in acidic conditions of pH 2 and plasma. Therefore, it is expected that it can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredient is processed into various forms such as extract, powder, pills, tablets, etc. It is a very useful invention in the pharmaceutical industry and food industry because it has an excellent effect that can be prepared with.

Figure 1 shows the human platelet aggregation inhibitory activity of the ethanol extract and the organic solvent fraction thereof. 1: solvent control (DMSO), 2: aspirin (0.25mg/ml), 3: aspirin (0.125mg/ml), 4: browned ethanol extract (0.25mg/ml), 5: hexene fraction of browned ethanol extract (0.25 mg/ml), 6: ethyl acetate fraction of browned ethanol extract (0.25mg/ml), 7: butanol fraction of browned ethanol extract (0.25mg/ml), 8: water residue after organic solvent fraction of browned ethanol extract ( 0.25 mg/ml).
Figure 2 shows the chemical structural formula of puerarin.
Figure 3 shows the TLC results of brown flower extract and puerarin. 1: puerarin (2 μg/spot), 2: ethyl acetate fraction of browned ethanol extract (10 μg/spot), 3: butanol fraction of browned ethanol extract (10 μg/spot), 4: residual water after organic solvent fractionation of browned ethanol extract Water (10 μg/spot).

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy against browning, the inventors of the present invention prepared a browning extract and a sequential organic solvent fraction thereof by a certain method, and evaluated their antithrombotic activity, and the residual water after the organic solvent fraction of browning ethanol extract Water was recovered as an active ingredient, and the water residue did not show any hemolytic activity against human red blood cells, but it was confirmed that it had excellent thermal stability and acid stability, thereby preventing or treating/improving thrombosis. It was intended to be used as a pharmaceutical composition and health functional food.

Specifically, the inventors of the present invention use the known effective in treating hangover removal, headache, fever, thirst, anorexia, abdominal distension, vomiting, etc. in the private sector, using a pharmaceutical composition for preventing or treating/improving thrombosis and health functions In order to develop a food product, an ethanol extract of browned ethanol and a sequential organic solvent fraction thereof and a water residue after the organic solvent fraction were prepared, and their antithrombotic activity was directly inhibited by human plasma and human thrombin (Thrombin Time: TT), Prothrombin inhibition (Prothrombin Time: PT) and active partial thromboplastin time (activated Partial Thromboplastin Time: aPTT) were evaluated to show strong anticoagulant activity in the hexene and ethyl acetate fractions of the browned ethanol extract, and the butanol fraction of the browned ethanol extract. And excellent anticoagulant activity and strong platelet aggregation inhibitory activity in water residues. In addition, as a result of thin layer chromatography (TLC) analysis of the water residue, it was confirmed that the antithrombotic active substance is different from the previously known puerarin. In addition, it was confirmed that, unlike the hexene fraction and ethyl acetate fraction, the water residue of the browned ethanol extract did not show hemolytic activity against human red blood cells.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing as an active ingredient a water residue obtained by sequentially fractionating the ethanol extract of Pueraria flos with hexene, ethyl acetate and butanol.

In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis containing as an active ingredient a water residue obtained by sequentially fractionating the ethanol extract of Pueraria flos with hexene, ethyl acetate and butanol.

In addition, the present invention provides a blood coagulation inhibitor containing as an active ingredient a water residue obtained by sequentially fractionating the ethanol extract of Pueraria flos with hexene, ethyl acetate, and butanol.

In addition, the present invention provides a platelet aggregation inhibitor containing as an active ingredient a water residue obtained by sequentially fractionating the ethanol extract of Pueraria flos with hexene, ethyl acetate and butanol.

Hereinafter, the manufacturing method and efficacy experiment of the brown flower extract of the present invention will be described in more detail.

The present invention comprises the steps of obtaining an ethanol extract and a sequential organic solvent fraction thereof and a water residue from browning; Evaluating the antithrombotic activity of the extract and fraction or water residue; And investigating the stability of the active substance. TLC analysis step.

The "brown flower extract" included in the composition of the present invention is obtained by extracting the buds of arrowroot that have just started to bloom with an organic solvent and filtering the extract using a filter net of 0.06 mm or less, and concentrating it under reduced pressure. I can.

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

As a preferred embodiment, the ethanol extract of the present invention may be used by extracting the brown flower with ethanol. Here, the ethanol extract may be sequentially fractionated with an organic solvent of hexene, ethyl acetate and butanol to obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

In the present invention, as a result of measuring thrombin time, prothrombin time, and AP time at a concentration of 5 mg/ml of the brown ethanol extract and fraction, the hexene fraction and ethyl acetate fraction exhibited excellent anticoagulant activity, and concentration-dependent activity As a result, the hexene fraction at 7mg/ml concentration extended the thrombin time, prothrombin time, and APT time by 1.63 times, 1.90 times and 2.17 times, respectively, compared to the additive-free group.At 7mg/ml concentration, the ethylacetate fraction was thrombin compared to the non-added group. Time, prothrombin time, and AP time were extended by 2.25 times, 3.67 times and 2.76 times, respectively. At this time, the antithrombotic aspirin (1.5mg/ml) used as a control was extended by 1.68 times, 1.41 times, and 1.47 times, respectively, confirming strong anticoagulant activity. Meanwhile, at the concentration of 7mg/ml, the butanol fraction prolonged thrombin time, prothrombin time, and AP time by 1.48 times, 1.25 times and 1.35 times, respectively, compared to the non-added group, and the water residue was thrombin time, prothrombin time, and The AP time was extended by 1.25 times, 1.22 times and 1.44 times, respectively, confirming excellent anticoagulant activity.

In addition, as a result of confirming the platelet aggregation inhibitory activity by adjusting the browned ethanol extract and the fraction to a concentration of 0.25 mg/ml, the butanol fraction and the water residue of the browned ethanol extract showed platelet aggregation capacity of 51.4% and 49.4%, respectively. It was confirmed that there is a strong platelet aggregation inhibitory activity. On the other hand, the ethanol extract, the hexene fraction and the ethyl acetate fraction showed rather platelet aggregation promoting activity. Aspirin used as a control showed 32.5% platelet aggregation at a concentration of 0.25mg/ml. These results imply that the butanol fraction and water residues of the ethanol extract of brown halides exhibit very strong antithrombotic activity, suggesting that anticoagulants and antiplatelet agents such as aspirin, which have a high risk of side effects, can be replaced.

The browned ethanol extract and fractions of the present invention may be prepared into a powder through a conventional powdering process such as vacuum drying, freeze drying, or spray drying. They are not decomposed by various degrading enzymes in plasma, and maintain their activity even at a heat treatment of 100°C and a pH of 2 in the stomach.

The active ingredient of the present invention can be used for prevention or treatment of various diseases related to thrombosis. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, motor abnormalities, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis. , Deep vein thrombosis, lower extremity swelling, pain, and acute peripheral arterial atresia. As venous thrombosis, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral sinus thrombosis, and central retinal vein occlusion.

In addition, the active ingredient of the present invention can also be used in pharmaceutical applications for blood coagulation inhibitors (anticoagulants) and platelet aggregation inhibitors (antiplatelet agents).

Pharmaceutical compositions containing the active ingredients of the present invention are oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to a conventional method for each purpose of use, and injections of sterile injectable solutions It may be formulated and used in various forms such as, and may be administered orally or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further include a carrier, excipient, or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil And the like. In addition, the pharmaceutical composition of the present invention may further contain a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

In a preferred embodiment, solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid formulations include at least one excipient, such as starch, calcium carbonate, and the like, in the pharmaceutical composition. It is formulated by mixing sucrose, lactose, gelatin, and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used.

As a preferred embodiment, as a liquid formulation for oral use, a suspension, a liquid formulation, an emulsion, a syrup, and the like may be exemplified, and various excipients other than water and liquid paraffin, which are commonly used simple diluents, such as wetting agents, sweetening agents, Fragrances, preservatives, etc. may be included.

As a preferred embodiment, the preparation for parenteral administration may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized agent, a suppository, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyloleate. Injectables may contain conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

As a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kilogram of body weight daily Alternatively, it may be administered every other day or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount is not limited by any method.

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

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to target cells.

In the present invention, the "object" is not particularly limited, but includes, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, mouse, rabbit, or guinea pig And, 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 in preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules or beverages. .

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

The health functional food of the present invention may contain the compound as an essential component in the indicated ratio as an additional component, as well as food supplementary additives acceptable for food, such as natural carbohydrates and various flavoring agents.

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

Taumatin as the flavoring agent; Natural flavoring agents such as stevia, such as rebaudioside A or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame, may be used. 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 includes a variety of nutrients, vitamins, minerals, synthetic flavors, and flavoring agents such as natural flavors, colorants and thickeners, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juices, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

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

[Example]

Example 1: Preparation of brown flower extract and fractions thereof and analysis of useful components thereof

Brown flowers harvested in Andong in the late summer of 2018 were purchased and used to prepare ethanol extracts. Specifically, after removing foreign substances after harvesting the freshly bloomed brown flower, 10 times ethanol was added to the weight of the brown flower, extracted three times at room temperature, collected and filtered, concentrated under reduced pressure to prepare a powder, and an ethanol extract was prepared. Was prepared.

The content of total polyphenols, total flavonoids, total sugars and reducing sugars was measured by component analysis of the brown flower extract. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample, and allowed to stand at room temperature for 1 hour, and the absorbance was measured at 725 nm. Tannic acid was used as a standard reagent. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and then 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample solution, 40 μl of 1 N NaOH was added, and the absorbance was measured at 420 nm after reaction at 37° C. for 1 hour. Rutin was used as a standard reagent. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Analysis of extraction efficiency and useful components of brown flower extract and fractions

As shown in Table 1, the extraction efficiency of browned ethanol was 4.7%, and most of the fraction was transferred to the butanol fraction, followed by ethyl acetate fraction> hexene fraction> water residue. With respect to 100 g of browning used, 3.21 g of butanol fraction and 0.193 g of water residue were recovered.

As a result of the analysis of useful components of the extracts and fractions, the total polyphenol content was 48.1mg/g, the highest in the ethylacetate fraction, followed by water residue> hexene fraction> butanol fraction. As a result of measuring the total flavonoid content, the ethyl acetate fraction showed the highest 24.7mg/g, in the order of hexene fraction> butanol fraction> water residue. Therefore, it is considered that the ethyl acetate fraction contains puerarin, which has been intensively studied, and the fraction is expected to exhibit various useful activities such as antioxidant and antibacterial activity.

Example 2: Evaluation of blood coagulation inhibitory activity of brown flower extract and fractions

The blood coagulation inhibitory activity of the browned ethanol extract and its fractions of Example 1 was evaluated, and the results are shown in Table 2. At this time, the method for evaluating the blood coagulation inhibitory activity of Galhwa extract was evaluated according to the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time and AP time were measured. Plasma was used as a commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China). Thrombin time, prothrombin time, and AP time were measured by the following procedure.

Thrombin Time

50 μl of 0.5U thrombin (Sigma Co., USA) at 37°C, 50 μl of 20 mM CaCl ₂ , and 10 μl of sample extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes, and then 100 μl of plasma was mixed. After addition, the time until plasma coagulation was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 32.1 seconds. The thrombin inhibitory effect was expressed as the average value of experiments repeated three or more times, and the thrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

Prothrombin time

70μl of standard plasma (MD Pacific Co., China) and 10μl of sample solution of various concentrations were added to the tube of Amelung coagulometer KC-1A(Japan), heated at 37℃ for 3 minutes, and then 130μl of PT reagent was added and plasma coagulated. The time until the result was expressed as the average value of the experiment repeated three times. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 18.1 seconds. The prothrombin inhibitory activity was expressed by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

aPTT(activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of sample extracts of various concentrations were added to a tube of Amelung coagulometer KC-1A (Japan), heated at 37° C. for 3 minutes, 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added, and again at 37° C. Incubated for 3 minutes. Thereafter, after 50 μl CaCl ₂ (35 mM) was added, the time until plasma coagulation was measured. DMSO was used instead of the sample as a solvent control, and in this case, a coagulation time of 55.1 seconds was shown. The result of aPTT was expressed as the average value of the experiment repeated three times, and the blood coagulation factor inhibitory activity was expressed by dividing the aPTT at the time of sample addition by the aPTT of the solvent control.

[Table 2] Blood coagulation inhibitory activity of extracts and fractions

As a result, it was confirmed that the browning ethanol extract exhibited excellent anticoagulant activity by extending thrombin time, prothrombin time and AP time by 1.47 times, 1.29 times and 1.20 times compared to the non-added group at a concentration of 5 mg/ml, which showed a concentration-dependent activity. Indicated. Aspirin, which is currently used as an antithrombotic agent in clinical practice, extends thrombin time, prothrombin time, and APT time by 1.68 times, 1.41 times and 1.47 times, respectively, compared to the non-additives at 1.5mg/ml concentration. It was found that it exhibits anticoagulant activity.

On the other hand, the hexene fraction and ethyl acetate fraction of the browned ethanol extract showed stronger anticoagulant activity, and showed a concentration-dependent activity.At 7mg/ml concentration, the hexene fraction had thrombin time, prothrombin time, and APT compared to no additives. Time was extended 1.63 times, 1.90 times and 2.17 times, respectively, and the ethyl acetate fraction at a concentration of 7 mg/ml extended thrombin time, prothrombin time, and AP time by 2.25 times, 3.67 times and 2.76 times, respectively, compared to the additive-free group. Meanwhile, at the concentration of 7mg/ml, the butanol fraction prolonged thrombin time, prothrombin time, and AP time by 1.48 times, 1.25 times and 1.35 times, respectively, compared to the non-added group, and the water residue was thrombin time, prothrombin time, and The AP time was extended by 1.25 times, 1.22 times and 1.44 times, respectively, confirming excellent anticoagulant activity.

On the other hand, as a result of evaluating the anticoagulant activity of puerain, the main active substance of browning and browning, at a concentration of 0.5 mg/ml, the activity was not recognized.

Example 3: Platelet Aggregation Inhibitory Activity of Brown Flower Extracts and Fractions

The human platelet aggregation inhibitory activity of the browned ethanol extract and fraction of Example 1 was evaluated, and the results are shown in Table 3 and FIG. 1. Platelets are disk-shaped small cells that circulate in blood vessels with various blood cells. Instead of having a nucleus, platelets have cytoplasmic granules containing various substances related to blood vessel damage protection and platelet aggregation at high concentrations, and if damage to the inner wall of blood vessels appears, It is an important cell that secretes aggregation factors and initiates thrombus formation by binding to collagen exposed by damage to endothelial cells to form a primary hemostatic plug. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombus formation. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

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

[Table 3] Platelet Aggregation Inhibitory Activity of Galhwa Extract and Fractions

As shown in Table 3 and FIG. 1, first, aspirin exhibited 32.5% aggregation at 0.25 mg/ml concentration and 68.6% aggregation at 0.125 mg/ml concentration, showing a strong platelet aggregation inhibitory activity in a concentration-dependent manner. By confirming it, the evidence for use as an antithrombotic agent in clinical practice could be known.

On the other hand, as a result of confirming the platelet aggregation inhibitory activity by adjusting the brown flower extract and fractions to a concentration of 0.25mg/ml, it was confirmed that the above fractions exhibited a potent platelet aggregation inhibitory activity comparable to that of aspirin, showing platelet aggregation capacity of 51.4% and 49.4%, respectively. I did. On the other hand, the ethanol extract, the hexene fraction and the ethyl acetate fraction showed rather platelet aggregation promoting activity. These results imply that the butanol fraction and water residues of the ethanol extract of brown halides exhibit very strong antithrombotic activity, suggesting that anticoagulants and antiplatelet agents such as aspirin, which have a high risk of side effects, can be replaced.

Example 4: Human Erythrocyte Hemolytic Activity of Brown Flower Extracts and Fractions

Since brown flower has been drinking for a long time as a tea, it is a food ingredient that is secured for safety. Therefore, it is judged that the ethanol extract of browning will not cause a separate acute toxicity. In order to evaluate the possibility of acute toxicity of the ethanol extracts and fractions of brown halides, the hemolytic activity of human red blood cells was evaluated, and the results are shown in Table 4.

At this time, the hemolytic activity was evaluated according to the previous report (Ho-Yong Son, 2014 ㆍKorean J. Microbiol. Biotechnol. 42: 285-292), and simply 100 μl of human red blood cells washed three times with PBS was placed in a 96-well microplate. Then, 100 μl of sample solution of various concentrations was added and reacted for 30 minutes at 37°C. After that, the reaction solution was centrifuged for 10 minutes (1,500 rpm) to transfer 100 μl of the supernatant to a new microtiter plate, and the degree of hemoglobin outflow due to hemolysis was 414 nm. It was measured at. DMSO (2%) was used as a solvent control for the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 4] Hemolytic activity of human red blood cells of Galhwa extract and fractions

First, it was confirmed that DMSO and water used as control cells had no hemolytic activity, and triton X-100 hemolytic 100% red blood cells at a concentration of 1 mg/ml. In addition, it was confirmed that amphotericin B, which is used as an anticancer agent and antifungal agent, hemolyzes more than 50% of red blood cells at a concentration of 0.025mg/ml. It was confirmed that the ethanol extract of the present invention, the butanol fraction thereof, and the water residue did not show any red blood cell hemolysis up to a concentration of 1 mg/ml, and thus had no acute toxicity and red blood cell hemolytic activity. On the other hand, the hexene fraction and the ethyl acetate fraction exhibited hemolytic activities of 83.8% and 69.9% at a concentration of 1 mg/ml.

Example 5: Evaluation of whether the fractions of brown flower extract contain puerarin

The ethyl acetate fraction, butanol fraction, and water residue of the browned ethanol extract obtained in Example 1 were tested to determine whether they contained puerarin, which has known platelet aggregation inhibitory activity, through TLC. The chemical structural formula of puerarin for which platelet aggregation inhibitory activity is reported is shown in FIG. 2, and according to the dielectric constant according to the structure, the hexene fraction was excluded from the TLC analysis. At this time, Kieselgel 60 F254 (Merck CO., USA) was used as the TLC plate, and the lower layer of Chloroform: Methanol: Water = 52: 28: 8 (v/v/v) was used as the developing solvent. As a standard, 2 μg/spot of puerarin and 10 μg/spot of other fractions were spotted and developed, and separated spots were identified under UV of 254 nm (FIG. 3).

As a result, puerarin had an Rf value of 0.58, and 4 to 5 μg of puerarin was identified in the ethyl acetate fraction. This is consistent with the previous report that the ethyl acetate fraction of brown root and brown flower extracts contained a high concentration of puerarin. On the other hand, in the case of water residues, no spots corresponding to puerarin were found at all, and it was confirmed that the butanol fraction contained 1 μg or less of puerarin (less than 10% of the fraction). Therefore, it was found that the antithrombotic activity of the water residue was exhibited by substances other than puerarin.

Example 6: Plasma, acid, and thermal stability evaluation of water residue after organic solvent fractionation of brown flower extract

Plasma stability, thermal stability, and acid stability against antithrombotic activity were confirmed for the water residue after the organic solvent fractionation of the ethanol extract obtained in Example 1 above. As a result, the active ingredient has high stability because there is no significant loss of anticoagulation and platelet aggregation inhibitory activity even when heat treatment at 100°C for 1 hour, 1 hour treatment at pH 2 (0.01M HCl), and 1 hour treatment in plasma. Shown. Therefore, the water residue after the organic solvent fractionation of the browned ethanol extract is expected to maintain excellent antithrombotic activity even under various food processing processes.

Claims (4)

Gallhwa ( Pueraria flos ) A pharmaceutical composition for the prevention or treatment of thrombosis containing as an active ingredient a water residue obtained by sequentially fractionating an ethanol extract with hexene, ethyl acetate and butanol. Gallhwa ( Pueraria flos ) health functional food for the prevention or improvement of thrombosis containing as an active ingredient a water residue obtained by sequentially fractionating ethanol extract with hexene, ethyl acetate and butanol. Gallhwa ( Pueraria flos ) a blood coagulation inhibitor containing as an active ingredient a water residue obtained by sequentially fractionating an ethanol extract with hexene, ethyl acetate and butanol. A platelet aggregation inhibitor containing as an active ingredient a water residue obtained by sequentially fractionating an ethanol extract of Pueraria flos with hexene, ethyl acetate and butanol.

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