KR20140034645A - Pharmaceutical composition comprising the extract of arctium lappa 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 supplement for preventing or treating thrombosis which include Arctium lappa extract as an active ingredient and, more specifically, to a pharmaceutical composition and a health supplement for preventing or treating thrombosis which include ethanol extract of the Arctium lappa extract as an active ingredient. The Arctium lappa extract as the active ingredient of the pharmaceutical composition and health supplement for preventing or treating thrombosis of the present invention is prepared by successively fractioning using hexane, ethyl acetate, and butanol after preparing an extract by extracting the Arcrium lappa root area using ethanol as being proved through an embodiment of the specification. The ethyl acetate fraction which has antithrombotic activities by suppressing thrombin direction inhibition, prothrombin inhibition and blood coagulation factor inhibition effects can efficiently suppress thrombosis and can be used for preventing and treating thrombosis such as ischemic stroke and cerebral hemorrhage through improvement of blood flow. Especially, the Arctium lappa extract of the present invention does not show oral acute toxic, has enhanced thermal stability, does not show loss of thrombosis under pH2 acid condition and inside plasma in order to be prepared in forms which can be taken at any time by being processed into various forms such as extract, powder, pill, tablet, and the likes so that the Arctium lappa extract is useful invention in food and drug industries.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF ARCTIUM LAPPA AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention is Burdock ( Arctium The present invention relates to a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing lappa ) extract as an active ingredient. More specifically, the ethyl acetate fraction of burdock ethanol extract, in particular, burdock ethanol extract is effective. It relates to a pharmaceutical composition for the prevention or treatment of thrombosis containing as a component and a health functional food comprising the extract or fraction.

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 a number of blood coagulation factors to finally produce a fibrin monomer from fibrinogen. The 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 thrombosis pathway, so that specific inhibition of blood coagulation factors is also important. . Until now, various anticoagulants such as heparin, coumarin, aspirin, and europine have been used for the prevention and treatment of thrombotic diseases. However, they are very expensive and have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.

Meanwhile, burdock ( Arctium lappa ) is a taxonomic two-year-old plant of the dicotyledonous plant Campanula Asteraceae. It is native to Europe and widely distributed in Siberia, Manchuria, and Korea. It is cultivated in Korea mainly in Gyeongnam, and it is a root vegetable that boils young sprouts or roots because of its unique aroma and chewing taste. It is usually eaten without salting, salted, boiled in vinegar or soy sauce. In oriental medicine, the roots are called right roots, the fruit is allies, and the stems are called the subventricular tube. Roric acid, lignin, myristic acid, propionic acid, cytosterol, stigmasterol and are known to contain a large amount of minerals such as copper, chromium, manganese, phosphorus, selenium, silicon, zinc.

To date, major researches on burdock have included burdock cultivation, peeling and cleaning of roots (Jin Jin-woong et al., 2003. Korean Society for Food and Distribution, Rice Society, International Symposium, P-23, p145: P-100, p202) It is concentrated on post browning control (Moon, Kwang-deok et al., 2005, Kor. J. Food Preserv. 12: 489-495), and burdock kimchi (Cho, Mi-Jung Choi, 1999, J. Kor. Soc. Food Sci. Nutr. 27: 618-). 624) and processed food research (eg, Mi-Kyung Kim, 2010, Kor. J. Food Cookery Sci. 26: 575-585), such as the preparation of muffins with burdock flour, have been reported.

However, research on the useful physiological activity of burdock has been very active in recent years, and the anti-inflammatory effect of burdock root extract (Ye-Jin Kim et al., 2012, Korean Journal of Plant Resources, 25: 1-6), arctiin was isolated from Diabetic activity (Lu et al., 2012. Planta Med. 2012.) and cancer cell killing activity (Susanti et al., 2012, J. Nat. Med. 66: 614-621), antioxidant effect of burdock caffeoylquinic acids (Liu et al., 2012) , J. Agric.Food Chem. 60: 4067-4075), Burdock arctiin alleviates the endoplasmic reticulum stress (Gu et al., 2012, Acta Pharmacol. Sin. 33: 941-952). Effect (Jian et al., 2012, BMC Complement Altern. Med. 12: 8), and gastric ulcer prevention effect (de Almeida et al., 2012. J. Med. Food. 15: 378-383) have been reported, the most medicinal It is emerging as a plant. In addition, anti-inflammatory activity of burdock seed in Korea (Park So-young et al., 2005, Nat. Product Sci. 11: 85-88), lard thermal oxidation and autooxidative effect of ethyl acetate and hexene extract of burdock (Kim, Moon et al., 2004, 13: 460-466), Antioxidative Effects of Burdock Sprout Vegetables (Lee Mu-Yeul et al., 2009, Korean Journal of Plant Resources, 22: 1304-311) and Antioxidative and Antimutagenic Effects of Burdock Ethanol Extracts (Lee Mi-sook, 2011, Korean Journal of Food and Nutrition, 24: 713-719.

On the other hand, patent documents related to burdock, natural products containing cosmetics effective in the treatment and prevention of acne (Korea Patent No. 10-0357824, registered date 2002. 10.9), cosmetic composition for pore reduction using burdock extract fermentation (Korea Registered Patent No. 10-1002796, date of registration December 14, 2010), astringent cosmetic composition using Burdock extract as an active ingredient (Korea Patent No. 10-0874113, registered date 2008. 12. 9), Burdock extract and cold persimmon Cosmetic composition for reducing pores containing ingredients (Korean Patent No. 10-0767974, registered date 2007. 10. 11), Cosmetic composition containing a mixed extract of natural plants having anti-inflammatory and skin irritation-releasing effect (Korea Patent Registration No. 10-1000695, registration date 2010. 12. 6), shampoo composition having a hair loss prevention function of the main ingredient of Angelica and Burdock extract (Korea Patent No. 10-0609210, registered date 2006. 7. 2 7), cosmetic composition for removing blackheads (Korean Patent No. 10-0962344, registered date June 1, 2010), herbal cosmetics composition for atopic dermatitis mainly comprising herbal extracts, especially burdock extract (Korea Patent No. 10- Patents related to anti-inflammatory and cosmetic compositions, such as 0905386, registration date June 23, 2009), and other nicotine removal compositions (Korea Patent No. 10-0960305, registration date 2010. 5. 20) And a hemostatic composition (registered patent No. 10-0628429) patent containing a burdock root as an active ingredient is known. However, to date, no antithrombotic activity of burdock extract, direct thrombin enzyme inhibition, and blood clotting factor inhibition have been reported.

Therefore, the present inventors aimed at developing a functional food material exhibiting anti-thrombotic and blood flow improving activity, and after preparing burdock extract, evaluating thrombinogenesis-inhibiting activity according to thrombin direct inhibition using human plasma and human thrombin, and human concentrated platelets. The platelet aggregation inhibitory activity of the burdock was measured using the burdock extract, and the potent antithrombotic activity was confirmed in the sequential fraction thereof, thereby completing the present invention.

Republic of Korea Patent No. 10-062842, Hemostatic composition containing burdock root as an active ingredient, the registration date September 19, 2006

The present invention has been made in order to solve the problems of the prior art as described above, the problem to be solved in the present invention is directly inhibiting human thrombin containing the extract of burdock root which is used for edible and medicinal, inhibits prothrombin And it is to provide a health functional food comprising the pharmaceutical composition and the extract for the prevention or treatment of thrombotic disease caused by the inhibition of endogenous blood coagulation factor.

In order to solve the above problems, the present invention burdock ( Arctium lappa ) provides a pharmaceutical composition for preventing or treating thrombosis containing an extract as an active ingredient.

The burdock extract is preferably a burdock ethanol extract.

The burdock extract is preferably an ethyl acetate fraction of burdock ethanol extract.

The ethyl acetate fraction is preferably obtained by fractionating the ethanol extract with hexane and then sequentially fractionating with ethyl acetate.

In addition, the present invention provides a health functional food comprising the burdock extract of the present invention.

Burdock extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis of the present invention and health functional food, as shown in the examples of the present specification, after extracting the burdock root with ethanol or the like to prepare the extract, hexene , Ethyl acetate and butanol were sequentially prepared and fractionated. Among them, ethyl acetate fraction showing antithrombotic activity due to inhibitory effect of blood coagulation factor has the effect of effectively inhibiting blood clot production and improving blood circulation. There is an excellent effect that can be used for the prevention and treatment of thrombosis, such as ischemic stroke and hemorrhagic stroke. In particular, the burdock extract of the present invention does not show acute oral toxicity, excellent thermal stability, direct inhibition of human thrombin, prothrombin inhibition, blood coagulation factor inhibitory effect in acidic conditions and plasma of pH 2, extract, It is a very useful invention for the pharmaceutical industry and the food industry because it has an excellent effect that can be processed into various forms such as powder, pills, tablets, etc., so that it can be always taken.

1 is a graph showing the results of human platelet aggregation inhibitory activity of the burdock ethanol extract and fractions. From the left of Figure 1 1: solvent control (DMSO), 2: aspirin (0.50 mg / ml), burdock ethanol extract (0.25 mg / ml), 4: burdock hexene fraction (0.25 mg / ml), 5: burdock Ethyl acetate fractions (0.25 mg / ml), 6: burdock butanol fraction (0.25 mg / ml) and 7: burdock water residue (0.25 mg / ml) were shown sequentially, respectively.

Hereinafter, the present invention will be described in detail.

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

Specifically, the present inventors prepared a ethanol extract of burdock to develop a pharmaceutical composition for the prevention or treatment of thrombosis and health functional food using burdock, and then sequential organic solvent fractions hexene fraction, ethyl acetate fraction, butanol fraction And water residues, and the like, which are treated with human thrombin, thrombin time, prothrombin time and activated partial thromboplastin time (aPTT), and platelets, respectively. The aggregation inhibitory ability was evaluated. As a result, thrombin time, prothrombin time and active partial thromboplastin time prolonging activity were hardly recognized in the burdock ethanol extract, but thrombin time stronger than aspirin (commercial antithrombotic agent) in the organic solvent fraction, especially ethyl acetate fraction. The present invention was completed by confirming the prolonging effect and prothrombin time similar to aspirin, active partial thromboplastin time extending effect, and platelet aggregation inhibitory effect.

On the other hand, the water residue after the sequential fractionation of the organic solvent confirmed the effect of promoting thrombus formation, which is consistent with the previous Korean patent (Hemostatic composition containing burdock root as an active ingredient, Republic of Korea Patent No. 10-0628429) The thrombus promoting effect of burdock was confirmed. This is the hemostatic action of the water fraction that occupies most of the burdock extract, but ethyl acetate fraction occupies 0.4% in the burdock extract is a very unusual case showing a strong antithrombotic activity.

In order to confirm the effect of such burdock, the inventors of the present invention to prepare a burdock extract and organic solvent fractions, and analyze the useful components of its total polyphenols, total flavonoids, etc., the direct inhibitory effect of human thrombin of the extracts and fractions, To evaluate the effect of prothrombin inhibitory effect and the prolonged effect of thromboplastin time by the inhibition of blood coagulation factors (factors VIII, IX, XI and XII), the extract of the present invention and the like have been used commercially It was confirmed that it had better antithrombotic activity than aspirin (trade name: protector), did not show human erythrocyte hemolytic activity, and investigated the thermal and acid stability of the substance, followed by oral administration of the substance to rats, and then subjected to an acute toxicity test. As a result, it was confirmed that the extract or fraction has little human toxicity.

Therefore, the present invention is characterized by providing a pharmaceutical composition and health functional food for preventing or treating thrombosis containing burdock extract as an active ingredient.

In a preferred embodiment, the burdock extract is preferably a burdock ethanol extract.

As another preferred embodiment, the burdock extract is preferably an ethyl acetate fraction in an organic solvent fraction of burdock ethanol extract. The ethyl acetate fraction can be obtained by fractionating the ethanol extract with hexane and then sequentially fractionating with ethyl acetate.

Hereinafter, the preparation method and efficacy test of the burdock extract and the organic solvent fraction of the present invention will be described in more detail.

The present invention comprises the steps of preparing an active extract from the burdock underground; A step of adjusting each fraction and water residue from a burdock extract using an organic solvent such as hexene, ethyl acetate, butanol, and the like; Checking the efficacy and stability of the extract and fraction; And an acute toxicity testing step of the fraction.

"Burdock extract" included in the composition of the present invention, after washing the root portion of burdock, drying, extracting the dried root with an organic solvent and filtering the extract using a filter network of 0.06 mm or less, and It can be obtained by the step of concentration under reduced pressure. The organic solvent used in the present invention may be any organic solvent such as water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol (methanol, ethanol, alcohol, propanol, butanol etc.) having 1 to 4 carbon atoms, a mixed solvent of the lower alcohol and water , With 80-95% ethanol being the most preferred.

In the present invention, the results of the measurement of thrombin time, prothrombin time and epitaxial time with the concentration of burdock ethanol extract 5 mg / ml, the burdock ethanol extract is very weakly extended by 1.09 times, 1.14 times and 1.01 times more than the non-added, respectively It showed one antithrombotic inhibitory activity. However, the hexene fraction in the sequential organic solvent fractions extended thrombin time by 15-fold at 5 mg / ml concentration and prothrombin time and epitaxial time by 2.52- and 1.1-fold, respectively. Therefore, the strong thrombin inhibitory activity of the hexene fraction was confirmed. On the other hand, ethyl acetate fraction at 5 mg / ml concentration of thrombin time, prothrombin time, apitime all extended more than 15 times compared to the non-added group, it was confirmed that inhibiting the thrombus generation at all stages. In particular, the ethyl acetate fraction extended thrombin time by 15 times or more at 1.25 mg / ml concentration, showing superior antithrombotic activity than the antithrombotic aspirin used in the clinic.

In the case of butanol fraction, thrombin time, prothrombin time, and apitime were extended by 1.68 times, 1.23 times, and 1.68 times, respectively, at 5 mg / ml. The tee time was shortened by 0.74 times compared to no additions to promote thrombus generation. These results suggest that the ethyl acetate fraction of burdock has the effect of inhibiting thrombogenesis by acting on thrombin, prothrombin, and blood coagulation factors, and it can replace anticoagulants such as aspirin, which have reported side effects such as gastrointestinal disorders. do.

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

Burdock extract 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 can include deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition comprising the burdock extract of the present invention is powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, sterile injectable solutions, etc. It may be formulated and used in various forms, and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

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

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

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

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

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage 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 burdock extract in the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg body weight daily. Or every other day or divided into 1 to 3 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 the food containing the burdock extract of the present invention include 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. .

Burdock extract of the present invention may generally be added at 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 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 agent include tau martin; Natural flavoring agents such as stevia such as rebaudioside A or 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 proportion of such additives is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of burdock extract of the present invention.

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

[ Example ]

Example  1: Burdock Extract and Organic Solvent Fraction  pharmacy

In 2011, 10 times of ethanol (95%, Deoksan, Korea) was added to burdock, which was grown in Uiseong, Gyeongbuk, Korea, and extracted twice a day at room temperature. In addition, only the thin shell and the peeled edible portion removed when used for food were collected separately in the same manner as described above, each extract was concentrated under reduced pressure to prepare a powder and stored at low temperature until use. Thereafter, the ethanol extract was suspended in distilled water, and then fractionated with hexane, ethyl acetate and butanol in order to finally obtain a water residue.

At this time, the skin portion and the peeled edible portion of the whole root showed 87.5% and 12.5% weight ratio, respectively, and the yields of ethanol extracts of the whole root, shelled and peeled edible portion were 10.25 ± 0.47% and 4.99 ± 0.32, respectively. %, 10.94 ± 0.55%. Therefore, in the case of burdock, it is generalized that it is commonly sold after harvesting by mechanical grinding after harvesting farmhouses, and that the edible part after peeling is effective in view of the yield efficiency.

The yields of hexene fraction, ethyl acetate fraction, butanol fraction and water residue of ethanol extract of burdock edible fraction were 1.62%, 0.42%, 8.98% and 85.38%, respectively. In particular, the amount of ethyl acetate fraction was very low, which means that the ethyl acetate fraction was obtained about 46 mg for 100 g of burdock, and the ethyl acetate fraction was considered to be considerably purified.

Total flavonoid, total polyphenol, total sugar and reducing sugar contents of the prepared burdock extracts were measured. To determine the total flavonoid content, each sample was stirred for 18 hours in methanol, and 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extract. 40 μl of 1 N NaOH was added thereto, followed by reaction at 37 ° C for 1 hour, Absorbance was measured. As a standard reagent, rutin was used. The total polyphenol content is 400 μl of the extraction sample, 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ Saturated solution was added and left at room temperature for 1 hour, and then absorbance was measured at 725 nm. Tannic acid was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was quantified by phenol-sulfuric acid method. The results are shown in Table 1 below.

Yield and Analysis of Ethanol Extracts of Whole Burdock Root, Skin and Peeled Edible Part (Unit: mg / g)  extract Weight ratio (%) Extraction efficiency (%) Total polyphenol Total flavonoid Total Reducing sugar Root full 100 10.25 ± 0.47 5.15 ± 0.14 0.99 ± 0.16 660.02 ± 15.11 249.94 ± 4.23 Shell 12.5 4.99 ± 0.32 6.17 ± 0.05 1.00 ± 0.06 418.46 ± 14.42 100.87 ± 7.34 After peeling
Edible part 87.5 10.94 ± 0.55 5.01 ± 0.05 0.96 ± 0.04 694.53 ± 18.58 271.24 ± 1.38

The skin part of burdock was about 20% higher in total polyphenols and the total flavonoid content was similar to that of the edible parts. However, total sugars and reducing sugars were 60% and 37% lower than those of the edible portion, respectively.

Next, the antithrombotic activity of the burdock extract was evaluated. Antithrombotic activity was assessed in accordance with previously reported methods (Sohn et < RTI ID = 0.0 > al ., 2004. Kor . J. Pharmacogn 35, 52-61; Kwon et al ., 2004. J. Life Science , 14: 509-513; Ryu et al. 2010. J. Life Science , 20. 922-928), thrombin time, prothrombin time and apathy time were measured. Plasma was prepared from the whole blood of the volunteer, and immediately after the blood collection, the plasma was separated by centrifugation at 5,000 g at 4 ° C. for 5 minutes, frozen and stored (fresh frozen plasma), and thawed at room temperature if necessary. The thrombin time, prothrombin time and apathy measurement were performed as follows.

Thrombin  time( Thrombin Time )

50 μl of 0.5 U thrombin (Sigma Co., USA) and 20 mM CaCl ₂ 50 μl, 10 μl of various concentrations of sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) and allowed to react for 2 minutes. Then, 100 μl of plasma was added and the time until the plasma coagulated was measured. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO had a solidification time of 32.1 seconds. In the case of thermal stability measurement, the sample solution of various concentrations was heat-treated at 100 ° C. for 30 minutes, allowed to cool at room temperature for 1 hour, and then the remaining activity was measured. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the coagulation time at the time of sample addition divided by the coagulation time of the solvent control was expressed as% by multiplying by 100.

Prothrombin  time( prothrombin time )

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of the sample to tubes of Amelung coagulometer KC-1A (Japan), heat at 37 ° C for 3 minutes, add 130 μl of PT reagent The time until the plasma coagulated was expressed as the average value of the experiment which was 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.

aPTT  ( activated Partial Thromboplastin Time )

100 μl of plasma and 10 μl of various concentrations of the sample extract were added to a tube of Amelung coagulometer KC-1A (Japan), warmed at 37 ° C. for 3 minutes, and then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added again. Incubate for 3 minutes at ℃. Then 50 μl CaCl ₂ (35 mM) was added to measure the time until the plasma coagulated. DMSO was used as a solvent control instead of the sample, in which case it showed a solidification time of 55.1 seconds. The results of aPTT were expressed as the average of three replicate experiments. The value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control was multiplied by 100 and expressed in%.

The results of the thrombin time, the prothrombin time, and the apitime measurement are shown in Table 2 below. Aspirin used as a control showed excellent antithrombotic effect at 5 mg / ml concentration, and ethanol extracts of whole root, skin and edible parts of burdock were found in thrombin time, prothrombin time and api up to 5 mg / ml concentration. It did not change the time significantly. Therefore, it would be difficult to detect antithrombotic activity when preparing anti-thrombotic activity by preparing burdock extract.

Antithrombotic Activity of Ethanol Extracts from Whole Burdock Root, Peel and Peeling Final concentration
(mg / mL) 0 1.5 2.5 5.0 Thrombin
time
(second) Whole root 32.1 ± 0.5 30.9 ± 1.9 35.1 ± 2.1 35.6 ± 2.4 Shell 32.1 ± 0.5 31.2 ± 0.6 35.2 ± 1.1 37.3 ± 1.4 After peeling
Edible part 32.1 ± 0.5 35.6 ± 2.4 35.6 ± 2.4 34.5 ± 1.7 aspirin 32.1 ± 0.5 86.3 ± 6.6 277.1 ± 11.9 > 480 Prothrombin Time
(second) Whole root 18.1 ± 0.3 17.1 ± 0.5 18.5 ± 1.1 20.1 ± 0.4 Shell 18.1 ± 0.3 20.1 ± 0.6 20.0 ± 0.4 19.7 ± 1.1 After peeling
Edible part 18.1 ± 0.3 17.9 ± 0.7 19.5 ± 0.8 20.6 ± 1.8 aspirin 18.1 ± 0.3 28.1 ± 2.4 32.8 ± 3.8 > 270 APT
time
(second) Whole root 55.1 ± 1.4 55.6 ± 2.4 57.5 ± 2.9 57.1 ± 2.3 Shell 55.1 ± 1.4 53.9 ± 1.5 57.3 ± 3.8 58.4 ± 1.9 After peeling
Edible part 55.1 ± 1.4 55.1 ± 2.0 53.1 ± 3.3 55.6 ± 0.8 aspirin 55.1 ± 1.4 75.5 ± 5.1 104.3 ± 7.2 > 800

Example  2: Sequential Organic Solvents of Burdock Extract Fraction  Component analysis and Anti-thrombosis  Activity evaluation

After the solvent fractionation of Burdock ethanol extract of Example 1, the components and antithrombotic activity of the fractions were investigated. The total flavonoid, total polyphenol, total sugar and reducing sugar content of the fractions were measured and the results are shown in Table 3.

Fractionation Efficiency of Organic Solvent Fraction from Burdock Extract and Its Component Analysis extract Fractionation efficiency (%) Content (mg / g) Total polyphenol Total flavonoid Total Reducing sugar Hexene fraction 1.62 2.48 ± 0.01 3.25 ± 0.42 161.05 ± 5.86 41.81 ± 0.92 Ethyl acetate fraction 0.42 4.59 ± 0.01 147.36 ± 2.89 85.23 ± 0.45 68.74 ± 1.38 Butanol fraction 8.98 3.51 ± 0.00 8.16 ± 0.44 675.02 ± 5.63 494.51 ± 2.29 Water residue 85.38 2.27 ± 0.04 0.57 ± 0.03 532.85 ± 25.34 229.05 ± 4.13

The organic solvent fraction of burdock ethanol extract was mostly water residue, and part of it was converted to butanol fraction. The hexene fraction and ethyl acetate fraction were very low at 1.62% and 0.42%, respectively. The water and butanol fractions were mostly composed of sugars, with low polyphenol content of 2.27-3.51 mg / g and flavonoid content of 0.57-8.16 mg / g. The hexene fraction also contained about 16% sugar, showing a polyphenol content of 2.48 mg / g and a relatively low flavonoid content of 3.25 mg / g. In the ethyl acetate fraction, however, the sugar content was 8.5%, but the polyphenol and 4.147 mg / g of flavonoid content were found to be very high at 4.59 mg / g. These results suggest that the ethyl acetate fraction of burdock is considered to have the most flavonoids with burdock, which is expected to show strong antioxidant and useful physiological activities, and actually shows strong antioxidant activity (not shown).

As a result of evaluating the antithrombotic activity of the fractions, the hexene fraction showed a 15-fold increased thrombin time and 2.5-fold increased prothrombin time at 5 mg / ml concentration, showing excellent thrombin inhibitory activity. However, when compared with the aspirin used in the same concentration at the clinical level showed a relatively weak inhibitory activity. This was similar in butanol fraction and water residues, and confirmed weak antithrombosis compared to aspirin. However, in the case of ethyl acetate fraction, the thrombin time, prothrombin time, and apitime were all increased by 15 times at 5 mg / ml concentration, confirming the very strong antithrombotic activity. In particular, even at a concentration of 1.25 mg / ml lower than 1.5 mg / ml of aspirin used in the experiment, it showed a 15-fold increase in thrombin time prolongation effect and 1.47-fold, 1.5-fold extended prothrombin time and apitime. It has been confirmed that it can be developed as an antithrombogenic composition better than aspirin. In addition, the present results suggest that the ethyl acetate sequential solvent fraction shows a very low fraction yield but strong activity, which is very efficient as a method for purifying the antithrombotic active material of burdock.

Antithrombotic Activity of Burdock Organic Solvent Fraction and Water Residue sample Concentration (mg / ml) Thrombus generation time (seconds) Thrombin time Prothrombin time Apathy time DMOS (Solvent) - 32.1 ± 0.5 18.1 ± 0.3 55.1 ± 1.4 aspirin 1.5 86.3 ± 6.6 28.1 ± 2.4 75.5 ± 5.1 Hexene fraction 5.0 > 480 46.5 ± 2.1 60.6 ± 2.8 2.5 52.9 ± 1.2 27.5 ± 1.8 58.1 ± 3.6 1.25 52.3 ± 1.5 20.7 ± 0.9 54.2 ± 3.3 0.5 50.4 ± 1.4 18.2 ± 0.8 54.7 ± 2.2 Ethyl acetate
Fraction 5.0 > 480 > 280 > 800 2.5 > 480 38.4 ± 1.7 148.7 ± 3.5 1.25 > 480 26.7 ± 1.5 82.7 ± 1.6 0.5 128.4 ± 3.7 26.9 ± 2.8 70.0 ± 2.7 0.25 76.7 ± 3.7 23.1 ± 1.1 67.8 ± 2.6 0.125 48.5 ± 2.4 20.2 ± 0.6 59.5 ± 1.3 Butanol fraction 5.0 53.9 ± 4.3 22.3 ± 2.0 92.6 ± 7.3 2.5 36.9 ± 2.6 20.1 ± 1.2 66.1 ± 4.1 1.25 36.0 ± 1.9 22.2 ± 0.9 65.4 ± 5.7 0.5 32.9 ± 0.6 20.5 ± 2.5 55.5 ± 3.7 Water residue 5.0 39.1 ± 3.2 19.5 ± 1.3 40.7 ± 2.4 2.5 35.7 ± 2.4 18.2 + 1.7 45.5 ± 1.9 1.25 36.1 ± 3.3 18.5 ± 2.1 52.4 ± 3.3

Example  3: Burdock extract, sequential organic solvent Fraction  Human platelet aggregation inhibitory activity

As part of the antithrombotic activity evaluation of the burdock extract and fractions, the aggregation inhibitory activity on human platelets was evaluated, and the results are shown in FIGS. 1 and 5. First, aspirin showed excellent human platelet aggregation inhibitory activity and ascertained concentration dependent coagulation inhibitory activity. Burdock ethanol extract and organic solvent fractions showed excellent platelet aggregation inhibitory activity at 0.25 mg / ml concentration, which was comparable to the aggregation inhibitory activity indicated by 0.25 mg / ml aspirin concentration, especially hexene fraction and ethyl acetate fraction It showed more potent platelet aggregation inhibitory activity than the effect of 0.5 mg / ml concentration of aspirin at prolonged time and lowered area. Therefore, it was confirmed that burdock extract and fractions can replace aspirin with antithrombotic agents. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibitory activity ( Platelet aggregation inhibition activity )

Platelets were mixed with a 3.2% sodium citrate solution in a ratio of 1: 9 from whole human blood, and then centrifuged at 1,100 rpm for 10 minutes to obtain PRP (platelet rich plasma) in the upper layer, which was washed with buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, it was 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 , 025% gelatin, pH 7.4) After centrifugation for 10 minutes, the cells were resuspended in suspending buffer, and the platelet count was adjusted to ⁴ × ^{10 9} / ml. Then, 2.5 ul collagen was added to the 1 ml suspension for 5 minutes, and platelet aggregation was measured at 37 ° C. using a whole-blood aggregometer (Chrono-log, USA).

Human Platelet Aggregation Inhibitory Activity of Burdock Ethanol Extract and Fraction Sample / Control burglar
(Amplitude: ohm) inclination
(Slope) Extension time
(Lag time: seconds) Descending area
(Area under) DMSO (Solvent: DMSO) 14 2 84 148.5 Aspirin (0.50 mg / mL) 5 One 184 21.7 Aspirin (0.25 mg / mL) 8 2 94 56.4 Ethanol Extract (0.25 mg / mL) 6 One 136 47.6 Hexene fraction (0.25 mg / mL) 4 0 160 21.0 Ethyl Acetate Fraction (0.25 mg / mL) 2 0 246 11.4 Butanol Fraction (0.25 mg / mL) 8 One 101 37.5 Water residue (0.25 mg / mL) 8 One 96 43.2

Example  4: Burdock ethyl acetate Fraction  Chemical Properties and Stability of Active Substances

The ethyl acetate fraction of burdock obtained in Example 1 was dried under pressure to prepare a powder, and then human erythrocyte hemolytic activity, plasma stability, thermal stability, and acid stability of the recovered active material were confirmed. The adjusted active substance was treated for 2 hours at 100 ° C, treated for 2 hours at pH 2 (0.01 M HCl), treated for 2 hours in plasma, and the ethyl acetate fraction showed no thrombin inhibition and reduced platelet aggregation inhibitory activity, Respectively. In addition, hemolytic activity against human erythrocytes was not seen up to 5 mg / ml concentration (Table 6).

Human Erythrocyte Hemolytic Activity of Burdock Extract and Fraction Sample (5 mg / ml) Human hemolytic hemolytic activity (%) Ethanol extract 2.70 ± 0.09 Hexene fraction 9.10 ± 0.75 Ethyl acetate fraction 5.63 ± 0.11 Butanol fraction 0.70 ± 0.28 Water residue 3.33 ± 1.18 Triton X-100 (0.1% 100.00 ± 0.00 Amphotericin B 125 ug / ml 75.30 ± 8.50

Example  5: Burdock ethyl acetate Fraction Single oral toxicity  exam

Four-week-old rats (Slc, ICR Mouse) were supplied from Central laboratory animals and were maintained at a temperature of 23 ± 3 ℃, relative humidity of 50 ± 10%, and illumination of 150 ~ Hour) for 14 days in an animal laboratory controlled by the method described in Example 1. The single oral toxicity of the ethyl acetate fraction obtained from Example 1 was then evaluated. First, the samples were dissolved in DMSO and water, and after oral administration at a dose of 400, 800, 1500 and 2000 mg / kg, respectively, the survival and pathological abnormalities were observed for one week. Only DMSO was orally administered as a control. As a result, the survival rate was 100% and there were no sign of pathological abnormality. Therefore, the ethyl acetate fraction of burdock, which has been used for medicinal purposes, is classified as low toxicity or non-toxic, and it was judged that the use of its low concentration would not cause additional problems.

Claims (5)

Burdock ( Arcium) lappa ) pharmaceutical composition for preventing or treating thrombosis containing an extract as an active ingredient. The method of claim 1,
The burdock extract is a pharmaceutical composition for preventing or treating thrombosis, characterized in that the burdock ethanol extract. The method of claim 1,
The burdock extract is a pharmaceutical composition for preventing or treating thrombosis, characterized in that the ethyl acetate fraction of burdock ethanol extract. The method of claim 3, wherein
The ethyl acetate fraction is a pharmaceutical composition for preventing or treating thrombosis, characterized in that the ethanol extract is obtained by hexene fractionation, and then sequentially fractionated with ethyl acetate. The health functional food containing the burdock extract of any one of Claims 1-4.

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