KR101440574B1 - Pharmaceutical composition comprising the extract of nardostachys chinenesis as an effective component for prevention or treatment of diseases related to thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to the use of Nardostachys chinensis The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating a thrombotic disease containing an extract of Batal as an active ingredient and more specifically to a pharmaceutical composition and a health functional food for preventing or treating thrombotic diseases A pharmaceutical composition for preventing or treating diseases, and a health functional food containing the extract. The pharmaceutical composition for preventing or treating the thrombotic disease of the present invention and the ginseng extract as an active ingredient of the health functional food can be prepared by extracting the ginseng root with hot water or the like to prepare an extract The hexane fraction exhibiting antithrombotic activity by the inhibition of excellent blood coagulation factor and inhibition of platelet aggregation has an effect of effectively inhibiting thrombogenesis and is effective in improving ischemic stroke and hemorrhage There is an excellent effect that can be used for prevention and treatment of thrombosis such as stroke. In particular, the ginseng extract of the present invention does not exhibit acute oral toxicity, is excellent in thermal stability, does not show loss of antithrombotic effect even in an acidic condition of pH 2 and plasma, and can be used in various forms such as 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 and a health functional food for preventing or treating thrombotic diseases containing an extract of Safflower oil as an effective ingredient and a health functional food. COMPRISING THE SAME}

The present invention relates to the use of Nardostachys chinensis The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating a thrombotic disease containing an extract of Batal as an active ingredient and more particularly to a pharmaceutical composition and a health functional food for preventing or treating thrombotic diseases, The present invention also relates to a pharmaceutical composition for preventing or treating thrombotic diseases 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. The production of fibrin clot involves multiple steps of multiple clotting factors, activating thrombin involved in fibrin clotting, resulting in fibrin monomers from fibrinogen, fibrin monomers polymerized by calcium, and bound to platelets and endothelial cells And forms a cross-linked fibrin polymer by factor XIII, producing a permanent thrombus. Therefore, the activity inhibitor of thrombin can be used as a prophylactic and therapeutic agent very useful for various thrombotic diseases caused by excessive blood coagulation.

On the other hand, prothrombin activation after sequential activation of factor XII, factor XI, factor IX and factor X is known to activate thrombin in the endogenous thrombogenesis pathway, so that specific inhibition of blood coagulation factors is also important. It is becoming a target. 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.

On the other hand, Nathostachys chinensis Batal refers to the roots and roots of the perennials of the matariaceae, or the plants of the same species, It is known to be effective in relieving pain and headache, neurogenic gastritis, eliminating eosinophils, promoting blood circulation, and having a great effect on sedation of the central nervous system. It is distributed in Nepal and China near the Himalayas. In China, it is distributed in Nardostachys chinensis Batal), Nardostachys jatamansi ) as a medicinal herb. Gamsang-hyang has been used as an analgesic from ancient times due to its peculiar direction and refreshing feelings, and is being used as an aroma for Jewish wedding. It is not cultivated or distributed in Korea. It is not registered as raw material of food in Korea Food Code and can be used for medicinal use (http://fse.foodnara.go.kr/).

The results of recent studies on ginseng extracts suggest that the antioxidant and anti-inflammatory effects of ginseng extract [Baek, S., J., et al., 2009, Korean J. Orient . Physiol . Pathol . 23: 853-859], acute chronic inflammatory diseases and dendritic cell maturation inhibitory activity [O, KW, et al., 2010. The J. Orient. Obstrics . Gynecol . 23: 14-25], neuronal protective and anti-inflammatory effects [Hwang, JS, et al., 2012, Bioorg . Med . Chem . Lett . 22: 706-708], memory and learning augmentation effects [Joshi, H., and M. Parle. 2006. J. Med . Food . 9: 113-118], anti-bacterial activity against Listeria monocytogenes [Han, JS, and DH Shin. 2001. Kor . J. Food Sci . Technol . 33: 774-783], mitigation effects of atopic dermatitis in mice [Min, DL, and EJ Park. 2010. J. Kor . Orient . Pediatr . 26: 13-24], the inhibitory effect of essential oils on central nervous system [Koo, BS, et al., 2006. J. Life Sci . 16: 156-161.], Promoting leukemic cell differentiation [Kim, JK, et al., 2011. Korean J. Orient . Physiol. Pathol . 25: 29-36] and the treatment effect of acute intestinal inflammation [Bae, GS, et al . , 2012. Exp . Ther . Med . 4: 533-537], endotoxin shock inhibition [Bae, GS, et al., 2011. J. Nat . Med . 65: 63-72] and inhibition of melanin synthesis [Jang JY, et al., 2011. J. Ethnopharmacol . 137: 1207-1214], but there is no known anti-thrombotic activity.

The patent literature related to the physiological activity of ginseng root to date includes Korean Patent No. 10-1023780 (registered on Mar. 23, 2011) "Pharmaceuticals for the Prevention and Treatment of Acute Pancreatitis Containing Gamma- Quot; composition, "Korean Registered Patent No. 10-0546735 (registered on January 19, 2006), skin whitening composition, Korean Registered Patent No. 10-1142541 (registered on Apr. 26, 2012) Patent No. 10-0581316 (Registration Date: May 05, 2006) "Composition for preventing hair loss and promoting hair growth" includes patent application pending composition and method for extracting the same, No. 10-2011-0029418 (filed on March 31, 2011) "Composition and cosmetic composition for the treatment of pancreatic cancer containing ginseng root extract", Application No. 10-2011-0029412 (filed on March 31, 2011) Kidney cancer, including extract (Application Date: 2011.03.31) "Composition and cosmetic composition for the treatment of lung cancer comprising extract of ginseng root," Application No. 10-2011-0012201 : 2011.02.11) "Composition and cosmetic composition for treatment of brain cancer containing ginseng root extracts" have been reported. These patents are patents based on suppression of growth of human cancer cells and antioxidant and whitening activity of ginseng root, There is no report on extracts and organic solvent fractions derived from ginseng root having activity.

KR 10-1023780 B

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the problems of the prior art as described above, and an object of the present invention is to provide a method for inhibiting endogenous blood coagulation factor and platelet aggregation inhibitor A pharmaceutical composition for preventing or treating thrombotic diseases according to activity, and a health functional food comprising the extract.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of a thrombotic disease comprising an extract of Ganoderma lucidum as an active ingredient.

It is preferable that the extract of the persimmon extract is a hot-water extract of persimmon extract.

Preferably, the extract of the ginseng flavor is a hexane fraction of a ginseng flavored hot-water extract.

The thrombotic disorder may be selected from the group consisting of acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dysesthesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, pulmonary thrombosis, deep vein thrombosis, , Pain, acute peripheral arterial atresia, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous thrombosis and central retinal vein occlusion.

The present invention also provides a health functional food comprising the ginseng extract of the present invention.

The pharmaceutical composition for preventing or treating the thrombotic disease of the present invention and the ginseng extract as an active ingredient of the health functional food can be prepared by extracting the ginseng root with hot water or the like to prepare an extract After that, hexane, ethyl acetate and butanol were sequentially used to prepare the fraction. The hexane fraction exhibiting a strong inhibitory effect on blood platelet aggregation and exhibiting inhibition of human platelet aggregation can effectively inhibit thrombus formation , There is an excellent effect that can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. In particular, the ginseng extract of the present invention does not exhibit acute oral toxicity, is excellent in thermal stability, and exhibits no loss of blood coagulation factor inhibitory effect even in an acidic condition of pH 2 and in plasma. Thus, the extract, powder, It is an extremely useful invention in the pharmaceutical industry and the food industry because it has an excellent effect that it can be prepared in various forms and can be prepared at any time.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating a thrombotic disease having an inhibitory effect on endogenous blood coagulation factor and platelet aggregation inhibitory activity containing ginseng root extract. More specifically, the inventors of the present invention have found that the antithrombotic active ingredient is recovered from the ginseng extract obtained by a certain method, and that these ingredients have excellent thermal stability and acid stability without showing oral acute toxicity Thereby making use of the extract as a pharmaceutical composition and health functional food for the prevention or treatment of thrombotic diseases.

Specifically, the present inventors prepared a hot-water extract of Gamso-hyang to prepare a pharmaceutical composition and a health functional food for preventing or treating thrombotic diseases using a ginseng flavor, sequenced organic solvent fraction thereof, and fractionated with hexane fraction, ethyl acetate Fractions, butanol fractions, water residues and the like, and analyzing their useful components such as total polyphenols and total flavonoids, respectively, and comparing them to thrombin time and prothrombin time for human plasma and human thrombin, respectively ), Activated Partial Thromboplastin Time (aPTT) and platelet aggregation inhibition were evaluated. As a result, it was confirmed that the active part of thromboplastin time (aPTT) prolongation activity superior to aspirin (commercial antithrombotic agent), which is currently used in the ginseng hydrothermal extract, and the inhibitory effect of platelet aggregation more effective than aspirin used as antiplatelet agent Respectively.

On the other hand, the hot-water extract and hexene fractions of Gamsoo-hyang did not show hemolytic activity on human erythrocytes and showed excellent heat and acid stability, and did not show acute toxicity when administered orally to rats. Thereby completing the present invention.

Accordingly, the present invention provides a pharmaceutical composition and a health functional food for the prevention or treatment of a thrombotic disease containing an extract of Safflower oil as an active ingredient.

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

The present invention relates to a method for preparing an active extract, An organic solvent fraction such as hexane, ethyl acetate, butanol, etc., and a water residue from the ginseng root extract; Checking the efficacy and stability of the extract and fraction; And an acute toxicity testing step of the fraction.

The "safflower extract" contained in the composition of the present invention is obtained by extracting commercial dried perspiration with hot water and organic solvent, filtering the extract using a filter net of 0.06 mm or less and concentrating it under reduced pressure . The solvent to be used in the present invention may be selected from the group consisting of water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol), a mixed solvent of the lower alcohol and water And hot water at 100 ° C is most preferable.

In the present invention, thrombin time, prothrombin time and apathy time were measured by preparing a ginseng hydrothermal extract at a concentration of 5 mg / ml. As a result, the ginseng hydrothermal extract showed 1.9 times longer thrombin time and 1.2 times longer prothrombin time However, apathy time, which is involved in endogenous thrombogenesis, showed a very strong inhibitory activity of blood coagulation factor, which was 15 times longer than that of non - additive. This strong coagulation factor inhibitory activity was confirmed to be stronger than that of aspirin (1.5 mg / ml). On the other hand, the sequential organic solvent fractions of the hydrothermal extracts showed slightly lower thrombin time and prothrombin time than aspirin at the concentration of 5 mg / ml, but the apathy times involved in endogenous thrombus formation were all excellent. In particular, the hexane fraction exhibited almost the same antithrombotic activity as that of the hot - water extract, confirming that the active ingredient of the hot - water extract was almost transferred to the hexene fraction, and that the major antithrombotic active ingredient of Gam - sunhyang was a liposoluble substance extracted in hexene.

In the evaluation of platelet aggregation activity, the hexane fraction showed the most potent activity and the weak activity was confirmed in the ethyl acetate fraction among the ginseng hydrothermal extract and its fractions. Especially, the hexene fraction showed about twice as much as the aspirin inhibitory activity. These results indicate that the hydrothermal extract of Gamso-hyang and its hexane fraction act on blood clotting factors and human platelets to inhibit thrombogenesis and replace the antithrombotic agents such as aspirin, It can be done.

The ginseng hydrothermal extract of the present invention and its hexane fraction can be prepared into powder by a conventional powdering process 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 ginseng root extract of the present invention can be used for the prevention or treatment of various diseases related to thrombotic diseases. 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 containing the ginseng extract of the present invention may be formulated into tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, sterile injectable solutions Injections, and the like, and they can be administered through various routes including oral administration, intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

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

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

In a preferred embodiment of the present invention, the effective amount of the ginseng root 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 It can be administered daily 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 or intracerebroventricular injection.

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 preventing or improving thrombotic diseases. Examples of foods containing the ginseng extract of the present invention include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used in the form of powders, granules, tablets, capsules or beverages have.

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

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

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

As the flavor, natural flavoring agents such as tautatin and stevia such as rebaudioside A or glycyrrhizin, and synthetic flavorings 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 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 ginseng root 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  One: Gam-cho  Extracts and organic solvents Fraction  pharmacy

The dried ginseng imported from China was pulverized and pulverized, and 20 times volume of water was added to the ginseng powder, and the mixture was heated to 100 ° C and extracted three times for 30 minutes. The hot - water extract was concentrated into a powder by vacuum concentration and stored at low temperature until use. For the preparation of the fractions, the extract was suspended in distilled water and then sequentially fractionated with hexene, ethyl acetate and butanol to finally obtain a water residue.

The yield of hydrothermal extract was 11.2 ± 0.2%. The yields of hexane fraction, ethylacetate fraction, butanol fraction and water residue of ginseng hydrothermal extract were 4.13%, 13.30%, 5.05% and 76.4%, respectively. Unlike conventional hot water extracts of herb medicines, the amount of ethyl acetate fraction was very high, and a large amount of hexene fraction was obtained. Particularly, in the case of the hexene fraction, it means that about 0.46 g is obtained for 100 g of sorghum, which is judged to be a highly purified fraction of the oil state.

The total flavonoid, total polyphenol, total sugar and reducing sugar content of the prepared ginseng extract and fractions were measured. To determine total flavonoid content, each sample was stirred for 18 hours in methanol, 400 ml of 90% diethylene glycol was added to 400 μl of the filtered extract, 40 μl of 1 N NaOH was added, and the absorbance at 420 nm was measured at 37 ° C for 1 hour. As a standard reagent, rutin was used. Total polyphenol content was determined 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 fraction analysis of hot water extracts and fractions of ginseng broth (unit: mg / g) Extract / fraction Extraction and Fraction Efficiency (%) Content (mg / g) Total polyphenol Total flavonoid Total Reducing sugar Hot water extract 11.2 ± 0.2 82.53 + - 1.16 69.42 ± 3.13 394.77 ± 12.84 137.20 ± 11.14 Hexene fraction 4.13 ± 0.1 62.89 + - 0.55 78.93 + - 3.92 72.07 + - 5.24 56.46 ± 3.93 Ethyl acetate fraction 13.3 ± 0.3 174.37 + - 7.21 112.35 + - 5.33 96.96 + - 4.45 70.76 ± 3.97 Butanol fraction 5.1 ± 0.3 169.13 + - 6.75 114.27 + - 4.27 576.33 + - 13.5 207.42 + - 11.65 Water residue 76.4 ± 3.5 74.91 + - 2.35 51.69 + - 3.51 442.58 ± 9.71 157.32 + - 6.26

As a result, the yield of organic solvent fraction of the hot water extract was in the order of water residues> ethyl acetate fraction> butanol fraction> hexene fraction, total polyphenol contents were in the order of ethyl acetate = butanol> water> hexene fraction, total flavonoid content Ethyl acetate = butanol> hexene> order of water residue, total sugar and reducing sugar content were in the order of butanol> water> ethyl acetate> hexane fraction. Thus, the ginseng extracts contained very high amounts of polyphenols and flavonoids, and ethyl acetate and butanol fractions contain significant amounts of polyphenols and flavonoids. It is more than 10 ~ 100 times higher than that of ordinary edible fruits and is associated with strong antioxidant activity of ginseng (no result).

Example  2: Gam-cho  The extract and Fraction Anti-thrombosis  Activity evaluation

The antithrombotic activity of the ginseng root extract and the organic solvent fraction of Example 1 was examined. Thrombin time, prothrombin time, and apathy time were measured.

Antithrombotic activity 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 apathy time. Plasma was prepared from the whole blood of the applicant, and immediately after collection, the plasma was separated by centrifugation at 5,000g 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 measurements were performed as follows.

Thrombin  time( Thrombin Time )

50 [mu] l 0.5 U thrombin (Sigma Co., USA) and 20 mM CaCl ₂ 50 μl, and 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 time measurement was expressed as the average value of the experiments repeated three or more times.

Prothrombin  time( prothrombin time )

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample solution to tubes of Amelung coagulometer KC-1A (Japan), incubate at 37 ° C for 3 minutes, add 130 μl of PT reagent, The time from the start of the experiment to the start of the experiment was expressed as the average value of the experiments repeated three times. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 18.1 sec. The prothrombin time measurement was expressed as the mean value of the experiments repeated three or more times.

aPTT  ( activated Partial Thromboplastin Time )

After adding 100 μl of plasma and 10 μl of various concentrations of the sample to a tube of Amelung coagulometer KC-1A (Japan), the mixture was heated at 37 ° C for 3 minutes, then 50 μl of aPTT reagent (Sigma, ALEXIN ^™ ) Min. After the addition of 50 μl CaCl ₂ (35 mM), the time until the plasma coagulated was measured. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 55.1 seconds. The results of aPTT were expressed as the average value of three repeated experiments.

The results of anti-thrombotic measurements of Gamso-Hwang are shown in Table 2 below. At 5 mg / ml concentration, aspirin (Product name: Protect) used as a control increased to more than 280 seconds in both thrombin time and prothrombin time and apathy time, At the concentration of 5 mg / ml, the anti - thrombotic activity was selectively extended by 15 times or more only on the apical time, and it was confirmed that the suppressive fragrance selectively activated the blood coagulation factors.

The antithrombotic activity of the fraction was confirmed by the hexane fraction, the ethyl acetate fraction, the butanol fraction and the water residue, respectively. Especially, the hexane fraction showed 15 times or more the APT And it was found that the main antithrombotic active ingredient of the ginseng root was a fat-soluble oil component. These results suggest that the curative used in medicinal herbs in Korea can be developed as a safe antithrombotic agent without side effects because of its antithrombotic activity through inhibition of blood coagulation factors.

Anti-thrombotic activity of hot-water extract and its fractions sample Concentration (mg / ml) Thrombogenic time (seconds) Thrombin time Prothrombin time Apathy time DMSO (solvent) - 32.1 ± 0.5 18.1 ± 0.3 55.1 ± 1.4 aspirin 5.0 > 480 > 280 > 800 1.5 83.4 ± 3.6 30.7 ± 1.8 77.1 ± 5.1 Hot water extract 5.0 60.9 ± 2.3 21.7 ± 1.3 > 800 2.5 51.4 ± 2.1 18.0 ± 0.6 82.6 ± 4.1 Hexene fraction 5.0 38.5 ± 0.7 21.7 ± 1.3 > 800 2.5 32.1 ± 1.2 18.7 ± 0.5 82.6 ± 4.9 Ethyl acetate fraction 5.0 41.7 ± 1.6 23.5 ± 3.6 148.7 ± 11.3 2.5 32.0 ± 1.9 18.4 ± 1.0 77.1 ± 5.2 Butanol fraction 5.0 51.4 ± 2.2 19.9 ± 3.6 121.2 ± 9.6 2.5 32.3 ± 0.5 18.2 ± 0.4 55.1 ± 2.8 Water residue 5.0 64.2 ± 2.9 23.5 ± 1.8 93.6 ± 8.1 2.5 54.5 ± 2.7 18.5 ± 0.2 53.7 ± 4.4

Example  3: Gam-cho  Extract, sequential organic solvent Fraction  Human platelet aggregation inhibitory activity

The anti-aggregation inhibitory activity against human platelets was evaluated as an antithrombotic activity evaluation of the extracts and fractions of ganoderma lucidum. The results are shown in Table 3. First, aspirin showed excellent human platelet aggregation inhibitory activity and ascertained concentration dependent coagulation inhibitory activity. In the case of hot water extracts, there was little effect on platelet aggregation, but each fraction had a large effect on platelet aggregation at a concentration of 0.25 mg / ml. First, the hexane fraction exhibited a better inhibitory activity than the aspirin at the same concentration, and the ethyl acetate fraction showed weak activity. On the other hand, the butanol fraction and the water residue were found to exhibit platelet aggregation promoting activity. Therefore, it was confirmed that hot - water extracts and hexane fractions of ginseng hydrolyzate can replace conventional aspirin as a platelet aggregation inhibitor. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet Aggregation Inhibitory Activity Platelet aggregation inhibition activity )

Platelets were mixed with 3.2% sodium citrate solution in a ratio of 1: 9 from healthy human whole blood, and centrifuged at 1,100 rpm for 10 minutes to obtain upper layer platelet rich plasma (PRP). The platelets were washed with a washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, the cells were resuspended in a suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 0.49 mM MgCl ₂ , 025% gelatin, pH 7.4) and incubated at 3,000 rpm for 10 minutes After centrifugation, the cells were resuspended in a suspending buffer and the platelet count was adjusted to ⁴ × ^{10 9} / ml. Then platelet aggregation was measured at 37 ° C using a whole-blood agarometer (Chrono-log, USA) after 2.5 μl of collagen was added to 1 ml of suspension and reacted for 5 minutes.

Human Platelet Aggregation Inhibitory Activity of Hot Water Extract and Its Fraction Sample / Control burglar
(Amplitude: ohm) inclination
(Slope) Extension time
(Lag time: seconds) Fall area
(Area under) DMSO (solvent: DMSO) 19 3 42 133.2 Aspirin (0.50 mg / mL) 4 One 67 25.3 Aspirin (0.25 mg / mL) 13 2 62 70.0 The hot-water extract (0.25 mg / mL) 20 3 34 134.2 The hexane fraction (0.25 mg / mL) 4 One 49 34.4 Ethyl acetate fractions (0.25 mg / mL) 14 2 30 105.3 Butanol fraction (0.25 mg / mL) 21 4 28 149.8 The water residue (0.25 mg / mL) 26 6 36 187.2

* Smaller fall area means less platelet aggregation

Example  4: Gam-cho Heat number  The extract and Hexen Fraction  stability

The thermal stability, acid stability and plasma stability of the hot-water extract and fractions obtained in Example 1 were confirmed. Each sample showed high stability because it did not show inhibition of blood clotting factor inhibition and platelet aggregation inhibition activity even at 2 hours treatment at 100 ° C, 2 hours treatment with pH 2 (0.01M HCl) or 2 hours treatment with plasma .

Example  5: Gam-cho Heat number  The extract and Fraction  Human red blood cells Hemolytic activity  And mouse single oral toxicity test

Table 4 shows the results of evaluating the hemolytic activity of human erythrocytes in the hot-water extracts and fractions obtained from Example 1 above. The hexane fraction showed about 17% hemolytic activity at a high concentration of 5 mg / ml, but the hemolytic activity was 3.1 ± 0.88% at the concentration of 0.25 mg / ml of the platelet aggregation inhibition concentration.

Hemolytic activity of human erythrocyte in hot water extracts and fractions Samples (5 mg / ml) Human hemolytic hemolytic activity (%) Hot water extract 0.6 ± 0.12 Hexene fraction 17.1 + - 2.54 Ethyl acetate fraction 0.3 ± 0.77 Butanol fraction 6.8 ± 1.08 Water residue 1.0 ± 0.34 Triton X-100 (0.1% 100.00 ± 0.00 Amphotericin B 125 ug / ml 75.30 +/- 8.50

In addition, a 4-week-old rat (Slc, ICR Mouse) was supplied from a Central laboratory animal and was kept at a temperature of 23 ± 3 ° C, a relative humidity of 50 ± 10% 8 hours) for 14 days, and the single oral toxicity of the hot-water extract and each fraction obtained from Example 1 was evaluated. First, the samples were dissolved in cone oil and water, and then administered at a dose of 400, 800, 1500, and 2000 mg / kg, respectively. The survival and pathological abnormalities were observed for one week. As a control, only cone oil was orally administered. As a result, the survival rate was 100% and there were no sign of pathological abnormality. Therefore, the hot - water extract and its hexane fraction of ginseng extract, which have been used for medicinal purposes, are classified as low - toxicity or non - toxic, and the use of low concentrations thereof does not cause additional problems.

Claims (5)

A pharmaceutical composition for the prevention or treatment of a thrombotic disease comprising an extract of Nardostachys chinensis as an active ingredient. The method according to claim 1,
The pharmaceutical composition for preventing or treating thrombotic diseases, wherein the ginseng extract is a ginseng hydrothermal extract. The method according to claim 1,
Wherein the ginseng root extract is a hexane fraction of a ginseng root hot-water extract. The method of claim 3,
The thrombotic disorder may be selected from the group consisting of acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dysesthesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, pulmonary thrombosis, deep vein thrombosis, , Pain, acute peripheral arterial atresia, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral sinus thrombosis and central retinal vein occlusion. A health functional food for preventing or ameliorating a thrombotic disease, comprising the ginseng extract of any one of claims 1 to 3.