KR101404165B1 - Pharmaceutical composition comprising the extract of angelica acutiloba as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The invention ildanggwi (Angelica The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis containing an extract of acutiloba as an active ingredient and more particularly to a pharmaceutical composition for preventing or treating thrombosis, And to a health functional food comprising the extract. The pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the hyperproliferative extract as an active ingredient of the health functional food can be prepared by extracting the underside of the hypercholesterol with ethanol or the like to prepare an extract, After the fractionation, the fraction was sequentially fractionated using ethyl acetate. Among these, the ethyl acetate fraction showing the antithrombotic activity by the excellent anticoagulant effect has an effect of effectively inhibiting thrombogenesis, There is an excellent effect that can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke. In particular, the crude extract of the present invention does not exhibit acute oral toxicity, has excellent thermal stability, and does not show any loss of blood coagulation factor inhibition and activation effect even in an acidic condition of pH 2 and in plasma. Thus, the extract, powder, And it can be prepared in a form that can be taken at any time. Therefore, it is a very useful invention in the pharmaceutical industry and the food industry.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing an extract of Angelica keiskei koidz. As an active ingredient.

The invention ildanggwi (Angelica The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis containing an extract of acutiloba as an active ingredient and more specifically to a pharmaceutical composition and a health functional food containing an extract of Angelicae giganteus, A pharmaceutical composition for preventing or treating thrombosis, and a health functional food containing 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, activation of prothrombin after sequential activation of factor 12, factor 11, factor 9, and factor 10 in the endogenous thrombosis pathway ultimately activates thrombin, and 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.

In Korea, Angelica is a domestic native Angelica Angelica gigas Nakai), the origins of Japanese origin ( Angelica acutiloba ), Chinese Angelica ( Angelica sinensis ) are sold. Among them, Chrysanthemum angustifolia and Rhododendron are mainly distributed. It is known that Angelica gigas is mainly used for the prevention and treatment of anemia, and is effective for various gynecological diseases and cardiovascular diseases. In particular, in Dongbokgi, it is useful for blood-related diseases by showing both umbilical cord blood and hemostatic . However, there have been no studies on how to show the function of promoting thrombus formation (hemostatic function) and the function of inhibiting thrombogenesis (hematopoietic function) in Angelica gigas. In particular, there is no report on the inhibition of thrombogenesis.

Angelica acutiloba is a perennial herb that belongs to the dragonfly family. It is a dried root of Angelica gigas. It is also called Angelica gigas. Its taste is sweet and very sweet, and its quality is warm. Although it has been introduced and cultivated for the first time to export to Japan, it has recently been featured as a functional sweetener with a characteristic fragrance of leaves and rich minerals and vitamins (Yoo Hongseon et al., 2004, Herb Medicine, 12: 43-46).

It is known that it is more similar to Chinese oriental ginseng than Korean native oriental oriental oriental oriental oriental oriental angelica. In fact, the indicator material of oriental oriental oriental orientalis is coumarin type decursin and decursinol, but the indicator substance of the oriental oriental oriental phalanges is ligustilide and butylidine phthalide It is known that the main ingredient is different (Chung, Myeong-hee, et al., 2004, Korean Journal of Food Storage and Distribution, 11: 364-372).

However, in terms of efficacy, it was reported that the inhibitory effect of angiotensin converting enzyme was slightly weaker than that of Angelica gigas Nakai (Angelica sinensis, 1996. Korean Society for Applied Microbiology and Biotechnology, 24: 624-629) (2004), and the antioxidant effect of Angelica gigas Nakai (2011), Korean Journal of Applied Biology, 26: 67-74) (2004), Korean Journal of Food Preservation and Distribution, 11: 364-372), analgesic and anti-inflammatory effects (Kim, Min-Jung et al., 2004, Herbal Medicine Application 4: 1-13) have.

The other antioxidative effects of the extracts of Angelica gigas Nakai and the hexane fraction (Kimara et al., 2009, Korea Life Science Association 19: 117-122), fatty acids and amino acids were similar to those of Angelica gigas Nakai (Lee, JJ et al., 2009, Korean Journal of Food Preservation, 16: 94-100) and antioxidant and anticancer activities are similar to each other (Lee et al., 2008, Korean Society of Pathology, 22: 1158-1162) (Hwang, Jongok et al., The Korean Society of Horticulture, 21: 434-439).

Thus, there have been few studies on the regulation of blood circulation in the monkeys, and it has been reported that these compounds inhibit the platelet aggregation inhibition of the courmarin-based compounds of Angelica gigas Nakai (Lee et al., 2003, Arch Pharm. Res 26: 723-726 However, there is no report on the inhibition of thrombogenesis by inhibition of human thrombin, prothrombin, and blood coagulation factors in the extracts and fractions of A. persimilis.

In addition, as a patent relating to the indole grise, a whitening cosmetic composition containing an extract of Tianjin extract of Korean Patent No. 10-0345226 (registered on July 6, 2002), Korea Patent No. 10-0345225 (registered date: 2002 Jul. 6, 2006) A cosmetic composition for whitening containing a crude oil extract, Korean Registered Patent No. 10-0571851 (registered on Apr. 11, 2006) Why a lower alcohol solvent extract extracted from Angelica gigas or a nicotine Pharmaceutical compositions for the prevention and treatment of addiction and withdrawal symptoms are known. On the other hand, the patent for the preparation method of livestock feed (Korean Patent No. 10-0500396, date of registration June 30, 2005, 0533062, registered on November 25, 2005, a method for producing animal feeds and feeds).

However, there are no patents related to the antithrombotic and hemostatic promoting activities of the extracts and fractions of A. equisetum and the patent for the anticoagulant activity of the intrinsic pathway of the mutant strains associated with this study, It is not known.

Korean Patent No. 10-0784339

DISCLOSURE Technical Problem Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a method for inhibiting endogenous blood coagulation factors containing, as an active ingredient, A pharmaceutical composition for preventing or treating thrombotic diseases, and a health functional food comprising the extract.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, comprising an extract of Angelicae gigantis as an active ingredient.

Preferably, the crude extract is a crude extract.

Also, it is preferable that the crude extract is the ethylacetate fraction of the crude extract.

The ethyl acetate fraction is preferably obtained when the ethanol extract is fractionated successively with hexane fraction followed by ethyl acetate.

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

The pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the hyperproliferative extract as an active ingredient of the health functional food can be prepared by extracting the underside of the hypercholesterol with ethanol or the like to prepare an extract, After the fractionation, it is prepared as a sequential fraction of ethyl acetate. The ethyl acetate fraction showing antithrombotic activity by the excellent anticoagulant effect has an effect of effectively inhibiting the thrombogenesis, and ischemic stroke There is an excellent effect that can be used for prevention and treatment of thrombosis such as hemorrhagic stroke. In particular, the crude extract of the present invention does not exhibit acute oral toxicity, is excellent in thermal stability, and does not show a loss of the blood coagulation factor inhibitory effect even in an acidic condition of pH 2 and plasma, and thus can provide a variety of extracts, powders, It is an extremely useful invention in the pharmaceutical industry and the food industry because it has excellent effect that it can be prepared in a form that can be taken at any time.

The present invention relates to a pharmaceutical composition for preventing or treating thrombosis and a health functional food having a blood coagulation factor inhibitory activity containing a monosaccharide extract. More specifically, the inventors of the present invention have found that an antithrombotic active ingredient is recovered from a crude extract obtained from a certain method, and that these ingredients have characteristics of excellent thermal stability and acid stability without showing oral acute toxicity The extract was used as a pharmaceutical composition for preventing or treating thrombosis and as a health functional food.

Specifically, the present inventors prepared a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis using a crude extract, and prepared an ethanol extract of the extract of Angelicae monocytogenes and fractionated it with an organic solvent to obtain a hexane fraction, an ethyl acetate fraction, a butanol fraction, Water residues and the like were obtained and evaluated for thrombin time, prothrombin time and activated partial thromboplastin time (aPTT) for human plasma and human thrombin, respectively . As a result, the thrombin time prolonging activity (thrombin inhibitory activity) and the active partial thromboplastin time prolonging activity (specific inhibitory activity of blood coagulation factor) were confirmed in the crude extract of Angelicae monocytogenes. Among the organic solvent fractions, the ethyl acetate fraction , The present inventors have completed the present invention by confirming that the active site thromboplastin time is prolonged by at least 15 times as compared with the untreated site without changing the thrombin time.

In order to confirm such an effect, the inventors of the present invention prepared a crude extract and an organic solvent fraction, analyzed their useful components such as total polyphenol and total flavonoid, and found that the extract and fraction had a direct inhibitory effect on human thrombin, (8, 9, 11, and 12 factors) of the active thromboplastin time extension inhibitory effect of the present invention and the antithrombotic aspirin Thrombocytopenic activity of the present invention is confirmed, the human erythrocyte hemolytic activity does not appear, the heat and acid stability of the substance are investigated, and the substance is orally administered to rats to conduct an acute toxicity test, It was confirmed that there was almost no toxicity to human body.

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

In a preferred embodiment, the crude lipase extract is preferably a crude lipase ethanol extract.

In another preferred embodiment, the crude extract is preferably the ethyl acetate fraction of the organic solvent fraction of the crude extract. The ethyl acetate fraction can be obtained by sequentially fractionating the ethanol extract after the hexane fraction with ethyl acetate.

Hereinafter, the method for producing the crude extract of the present invention and the efficacy experiments will be described in more detail.

The present invention relates to a method for preparing an active ingredient, Adjusting the organic solvent fraction such as hexene, ethyl acetate, and butanol from the crude extract; Checking the efficacy and stability of the extract and fraction; And an acute toxicity testing step of the fraction.

The "virgin extract" contained in the composition of the present invention is prepared by washing the roots of the roots, shrunk, extracting the roots with an organic solvent, filtering the roots using a filter net of 0.06 mm or less, Followed by 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, thrombin time, prothrombin time, and apitime time were measured at a concentration of 5 mg / ml of the crude extract of Angelicae giganteus. The results showed that the ethanol extract of Angelicae giganteus produced 1.7 times, 1.0 times, and 1.3 times Inhibitory activity. Thus, it was proved that the ethanol extract of Angelica gigas Nakai had an anti-thrombotic effect as a whole. Meanwhile, the hexane fraction of the fractions elongated the thrombin time 1.4 times at the concentration of 5 mg / ml, but did not affect the prothrombin time and the apathy time at 1.1 times and 0.9 times, respectively. On the other hand, in the case of the ethyl acetate fraction, thrombin time, prothrombin time, and apathy time were extended by 1.1, 1.5 and 15 times, respectively, at the concentration of 5 mg / ml. These results indicate that the ethyl acetate fraction of A. monocytogenes has the effect of inhibiting the blood clotting factor by inhibiting the prothrombin - From these results, it can be seen that the crude extract of Phellodendron griseus, which has been used for the removal of eosinophils from the private sector, contains a thrombogenic inhibitory substance. Especially, the ethyl acetate fraction of Pseudomonas aeruginosa can replace the anticoagulant such as conventional aspirin do.

The crude extract and its ethylacetate fraction of the present invention can be made into powders through conventional powdering processes such as vacuum distillation and freeze drying or spray drying. They maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The unreasonable 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 crude extract of the present invention may be formulated into tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, injections of sterilized injection solutions And may be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

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

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

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

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

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

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

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

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

In addition, the health functional food of the present invention can be variously used for foods and beverages effective for prevention or improvement of thrombosis. Examples of foods containing the crude extract of the present invention include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used as powder, granule, tablet, capsule or beverage .

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

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

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

Examples of the flavoring 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 gdmf per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the crude 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  One: Unreasonable  Extracts and organic solvents Fraction  pharmacy

In the year 2011, the undergrowth of the ginseng grown in Andong, Gyeongsang province was purchased, and 5 times of distilled water was added to the ginseng, and the mixture was heated at 100 ° C for 2 hours and then cooled. The mixture was filtered and concentrated under reduced pressure to prepare a hot water extract .

Ethanol extracts were extracted with ethanol (95%, Duksan, Korea) 10 times by volume for 2 hours at room temperature for 8 hours. The extract was concentrated under reduced pressure to give a powder, which was 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.

The yield of hot water extract was 28.56 ± 0.7%, which was similar to that of hot water extraction of 30.1 ± 0.6%. The yield of ethanol extract was 4.3 ± 0.3%, which was 10.2 ± 0.5% Of the total. This indicates that the ginseng contains less lipophilic components than Angelica gigas. The yields of the hexane fraction, ethylacetate fraction, butanol fraction and water residue of the ethanol extract were 22.7%, 3.4%, 10.1% and 62.1%, respectively. Ethyl acetate fraction showed 38% of total ethanol extract and showed no antithrombotic activity. Ethyl acetate fraction showed 3.4% of ethanol extract but showed strong antithrombotic activity. Therefore, it can be seen that the antithrombotically active substance is different from the antioxidant activity of Angelica gigas Nakai, and the composition of the lipid-soluble substance is different.

The total flavonoid, total polyphenol, total sugar, and reducing sugar content of the prepared crude 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. Total polyphenol content is obtained by adding 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ The saturated solution was added 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. 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 yield analysis of the extracts of hot water and ethanol extracts (mg / g) extract Extraction efficiency (%) Total polyphenol Total flavonoid Total Reducing sugar Hot water extraction 28.56 ± 0.7 3.39 ± 0.13 4.74 ± 0.11 648.60 ± 2.43 112.05 ± 2.8 Ethanol extraction 4.3 ± 0.3 39.59 + 1.41 17.17 + - 0.46 362.00 ± 40.77 70.38 + - 0.52

The total extracts of polyphenols and total flavonoids were 8.6% and 27.6% higher than those of ethanol extracts, respectively. On the other hand, the ethanol extract contains a very high polyphenol component, which is considered to exhibit excellent antioxidative activity and useful physiological activity, which is a part of the antidiabetic, antioxidant and anticancer effects of the previously reported dietary supplements.

Next, the antithrombotic activity of the crude 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 applicant, and immediately after collection, the plasma was separated by centrifugation at 5,000 g for 5 minutes at 4 ° C and stored frozen (fresh frozen plasma) and thawed at room temperature if necessary. The thrombin time, prothrombin time and apathy measurement were performed as follows.

Thrombin  time( Thrombin Time )

50 μl of 0.5 U thrombin (Sigma Co., USA) 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 showed a clotting time of 32.1 sec. In the case of thermal stability measurement, sample solutions at various concentrations were heat-treated at 100 ° C for 30 minutes, allowed to stand at room temperature for 1 hour, and residual activity was measured. The thrombin inhibitory effect was expressed as the average value of the experiments repeated three or more times. The value obtained by dividing the solidification time at the time of adding the sample by the solidification time of the solvent control was multiplied by 100 and expressed as%.

Prothrombin  time( prothrombin time )

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of 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 )

After adding 100 μl of plasma and 10 μl of various concentrations of the sample extract to the tube of Amelung coagulometer KC-1A (Japan) and heating at 37 ° C for 3 minutes, add 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) Lt; 0 > C for 3 minutes. After the addition of 50 μl of CaCl ₂ (35 mM), the time until plasma coagulation was measured. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 55.1 seconds. The results of aPTT were expressed as the average of three replicate experiments. The value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control was multiplied by 100 and expressed in%.

The 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 the concentration of 5 mg / ml, and the hot water extract of Angelica gigas Nakai did not change the thrombin time, prothrombin time, or apathy until the concentration of 5 mg / ml. On the other hand, the ethanol extracts increased the thrombin time and apathy by about 1.7 times and 1.3 times, respectively. This suggests that the anti-thrombotic effect equivalent to 1.5 to 2.0 mg / ml of aspirin can be used as an antithrombotic agent. Considering that the gastrointestinal disorder represented by aspirin and the ethanol extract of crude extract are not purified, It may be commercially useful in securing the thrombogenic material.

Anti-thrombotic activity of extracts of hot water and extracts final
density
(mg / mL) Thrombin Time (sec) Prothrombin time (seconds) Apathy Time (seconds) Heat number ethanol aspirin Heat number ethanol aspirin Heat number ethanol aspirin 0 32.1 32.1 32.1 18.1 18.1 18.1 55.1 55.1 55.1 1.5 ND ND 79.9 ND ND 22.6 ND ND 65.0 2.5 34.1 ± 1.7 31.1 ± 1.3 282.4 17.9 ± 2.4 18.1 ± 1.0 28.3 56.8 ± 2.7 63.2 ± 3.5 85.4 5.0 33.2 ± 3.3 54.6 ± 3.2 > 480 18.3 ± 0.5 18.3 ± 1.3 > 270 55.1 ± 10.5 71.6 ± 6.6 > 800

ND: Not Determined

Example  2: Unreasonable  The sequential organic solvent of the extract Fraction  Component analysis and Anti-thrombosis  Activity evaluation

The ethanol extract of Angelicae giganteus (Example 1), in which antithrombotic activity was confirmed, was subjected to solvent fractionation, and the components and antithrombotic activity of the fractions were examined. The total flavonoid, total polyphenol, total sugar and reducing sugar content of the fractions were measured and the results are shown in Table 3.

Analysis of Fractional Efficiency of Organic Solvent Fractions of Angelica gigas Nakai Extract and Their Components extract Fraction efficiency
(%) Content (mg / g) Total polyphenol Total flavonoid Total Reducing sugar Hexene fraction 22.72 15.65 + - 0.43 4.14 + - 0.10 76.29 ± 1.22 18.89 ± 0.26 Ethyl acetate fraction 3.33 112.74 + - 0.98 47.69 +/- 1.45 194.72 + - 11.44 73.47 ± 1.40 Butanol fraction 10.10 108.14 + - 11.16 11.02 + - 0.82 464.42 + 52.95 127.80 ± 6.29 Water residue 62.08 25.71 + - 2.76 2.65 ± 0.22 666.68 + - 31.04 139.40 ± 5.24

Ethanol content of ethyl acetate>butanol>water> hexane fraction, total flavonoid contents were in the order of ethyl acetate>butanol> ethyl acetate>hexane>butanol> ethyl acetate fraction. >Hexene> The order, total sugar, and sugar content of the water fractions were in the order of water>butanol> ethyl acetate> hexene fraction. These results suggest that the butanol and ethyl acetate fractions are expected to exhibit strong antioxidant activity and useful physiological activity.

As shown in Table 4, the hexane fraction showed a 1.4-fold increased thrombin time at the concentration of 5 mg / ml, while the thrombin time and the apathy time were 1.1-fold and 0.9-fold, respectively, There was a change. The water residue selectively reduced the aprotic time by 0.7 times. On the other hand, the ethyl acetate fraction showed a strong antithrombotic activity by increasing the apathy time by 15 times and by increasing the prothrombin time by about 1.5 times, with little change of thrombin time at the concentration of 5 mg / ml. On the other hand, the butanol fraction showed a slight antithrombotic activity by increasing the thrombin time 1.5 times at the concentration of 5 mg / ml. Thus, the ethyl acetate fraction shows that it can be developed as a thrombogenic inhibitory composition due to its strong anticoagulant activity.

Antithrombotic activity of the organic solvent fraction and water residue sample density
(mg / ml) Thrombogenic time (seconds) Thrombin time Prothrombin time Apathy time water - 32.1 18.1 55.1 aspirin 1.5 79.9 22.6 65.0 Hexen
Fraction 5.0 45.0 ± 2.4 19.9 ± 1.3 49.6 ± 0.8 2.5 38.5 ± 1.6 19.9 ± 1.8 55.1 ± 1.6 1.0 31.1 ± 2.2 18.6 ± 1.4 55.4 ± 2.2 Ethyl acetate
Fraction 5.0 35.3 ± 2.1 27.1 ± 1.0 > 820 2.5 32.1 ± 1.8 19.9 ± 1.2 104.6 ± 5.1 1.0 32.3 ± 1.1 18.1 ± 0.8 82.6 ± 6.7 Butanol
Fraction 5.0 48.2 ± 4.7 18.1 ± 0.7 60.6 ± 5.3 2.5 35.3 ± 3.1 18.7 ± 1.1 55.1 ± 5.5 1.0 31.9 ± 0.7 18.3 ± 0.9 55.5 ± 4.2 water
Residue 5.0 32.1 ± 0.7 18.1 ± 0.4 38.6 ± 3.3 2.5 32.2 ± 1.5 18.1 ± 0.5 44.2 ± 3.1 1.0 31.8 ± 1.1 18.2 ± 0.8 54.7 ± 1.5

Example  3: Unreasonable  Ethyl acetate Fraction  Chemical properties and stability of active materials

The ethyl acetate fraction obtained in Example 1 was subjected to pressure-drying to prepare a powder, and the recovered active substance was tested for hemolytic activity, plasma stability, thermal stability and acid stability. The regulated active substances showed high stability because they did not show a decrease in the activity of inhibiting thrombogenicity of the ethyl acetate fraction even at 2 hours treatment at 100 ° C, 2 hours treatment at pH 2 (0.01 M HCl) or 2 hours treatment in plasma . In addition, hemolytic activity against human erythrocytes was not observed up to a concentration of 5 mg / ml (Table 5).

Hemolytic activity of human erythrocyte extracts and fractions Samples (5 mg / ml) Human hemolytic hemolytic activity (%) Ethanol extract 1.1 ± 2.7 Hexene fraction 3.8 ± 0.9 Ethyl acetate fraction 1.4 ± 1.5 Butanol fraction 2.1 ± 3.4 Water residue 3.9 ± 1.6 Triton X-100 (0.1% 100 ± 0.0 Amphotericin B 125 ug / ml 75.3 ± 8.5

Example  4: Unreasonable  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, it is considered that the ethyl acetate fraction of A. gingivalis which has been used for medicinal purposes is classified as low toxicity or non - toxic, and the use of its low concentration will not cause any additional problems.

Claims (5)

A pharmaceutical composition for preventing or treating thrombosis comprising an ethyl acetate fraction of Angelica acutiloba ethanol extract as an active ingredient.
delete delete The method according to claim 1,
Wherein the ethyl acetate fraction is obtained by fractionating the ethanol extract with hexane and then sequentially fractionating with ethyl acetate.
A health functional food for improving thrombosis comprising the ethyl acetate fraction of the Angelica acutiloba ethanol extract according to claim 1 or 4.