KR20140148141A - Pharmaceutical composition comprising the extract of hippophae rhamnoides l. leaf 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 containing an ethyl acetate fraction of an ethanol extract of Hippophae rhamnoids L. leaf or remaining water after fraction with an organic solvent as an active ingredient for prevention or treatment of thrombosis and a health food including the extract or fraction. The Hippophae rhamnoids L. leaf extract of the present invention is prepared by sequentially fractioning by using hexane, ethyl acetate, and butanol after preparing an extract by extracting Hippophae rhamnoids L. leaf with ethanol and the like. The ethyl acetate fraction and water residue having antithrombotic activity caused by an excellent effect of inhibiting blood coagulation factors have an effect of efficiently controlling thrombosis.

Description

[0001] PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF HIPPOPHAE RHAMNOIDES L. [0002] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, LEAF AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to the use of vitamin C ( Hippophae The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis which contain rhamnoides L. leaf extract as an active ingredient and more specifically to an ethanol extract of vitamin tree leaves, As an active ingredient, and a health functional food containing the extract or fraction. The present invention also relates to a pharmaceutical composition for preventing or treating thrombosis, 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 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.

Vitamin tree ( Hippophae rhamnoides ) is a deciduous shrub belonging to the Elaeagnaceae family originating in China and Mongolia. It grows widely in China, Europe, Russia, Mongolia and the Himalayas, . It is a very strong cold tolerant plant with small fruit similar to orange or yellow. It has been called by various names such as wild wood, Siberian pineapple, historical drama, sajee, sea buckthorn and sea berry. Vitamin Nut is rich in carbohydrate, protein, organic acid, and vitamin B group and vitamin C. Generally, it contains up to 2,500 mg per 100 g of fruit. It is a fruit and vegetable such as strawberry, kiwi, orange, (Bermath and Foldesi 1992). Vitamin tree fruits, leaves and roots also contain polyphenols, tocophenols, carotenoids, quercetin, gallic acid, tannins and other flavonoids. (Lee et al., 2010, Kor, J. Pharamcogn. 41: 308-312; Zu et al., 2006. Journal of Pharmaceutical and Biomedical Analysis 41: 714-19) (Kim et al., 2010). In addition, it has been reported that it has also been shown to be effective in treating wounds, burns and inflammation (Park et al., 2010. Journal of the Korean Society of Food Science and Nutrition, 39: 980-985; Park et al., 2011. J. Pharm. Sci. 24: 345-351). Vitamin tree oil is rich in omega-3 and omega-6 fatty acids and has been reported to alleviate the symptoms of dry eye syndrome. Other skin problems such as atopic eczema, deficiency regeneration, skin damaged by UV rays, dry mouth, , Ulcer disease, urinary tract infections, cervicitis, gonadal inflammation, and sinus inflammation (Javinen et al., 2011. Cornea, 30 (9): 1013-1019). Recently, it has been reported that the inhibition of platelet aggregation of oil extracted from vitamin tree branches (Johansson et al., 2000, J. Nutr, Biochem. 11: 491-495) -2271). However, the activity of inhibiting human thrombin, prothrombin and blood coagulation factors of vitamin A extract has not been known.

On the other hand, patent documents related to vitamin trees include Korean Patent No. 10-1223464, a method of cultivating mushroom using vitamin wood, a culture cultured by the method, Korean Patent No. 10-1121590, 7-rhamnoside as an active ingredient, Korean Patent No. 10-1224196, a method for producing omega-3 containing vitamin-tree extract and herbal medicine, Korean Patent No. 10- 1007001 Vitamin tree extract and its fractions (antioxidants, antibiotics and antidiabetic compositions containing vitamin tree extract and fractions thereof as an active ingredient), Korean Patent No. 10-1225623 Kimchi, A preparation method thereof, Korean Patent No. 10-1166852, a composition for improving lipid in the body including vitamin tree leaf powder or extract thereof , Korean Patent No. 10-1149711 A cosmetic composition for skin whitening containing a low molecular weight fucoidan, vitamin tree extract and niacinamide and a preparation method thereof, Korean Patent Registration No. 10-0995321 Vitamin tree fruit and leaf tea tea tea Korean Patent Registration No. 10-1045752 discloses a method for producing fermented beverage using vitamin nut fruit extract, and Korean Patent Laid-Open No. 10-2013-0049679 discloses a method of aging a batter dough mixed with vitamin tree leaf powder Korean Patent Laid-Open No. 10-2013-0031590 discloses a health functional food for preventing skin aging and improving wrinkles comprising vitamin nut extract, collagen, blueberry extract, hyaluronic acid and honey as active ingredients, Korean Patent Laid- -2012-0129168 Composition for the prevention and treatment of skin inflammation diseases containing hocassarinin, Patent No. 10-2012-0020858 A method for producing makgeolli using vitamin tree, calcium tree and / or blueberry fruit, Korean Patent Laid-Open No. 10-2012-0081880, Patent No. 10-2011-0108848 discloses methods of processing fish using vitamin tree extract. However, up to now, there has not been reported a direct inhibition of human thrombin enzyme, prothrombin enzyme, blood coagulation factors, and inhibition of platelet aggregation through inhibition of platelet aggregation in vitamin tree leaf extracts.

Cheng J, Kondo K, Suzuki Y, Ikeda Y, Meng X, Umemura K. 2003. Inhibitory effects of total flavones of Hippophae rhamnoides L on thrombosis in mouse femoral artery and in vitro platelet aggregation. Life Sci. 72 (20): 2263-2271 Johansson AK, Korte H, Yang B, Stanley J C, Kallio HP. 2000. Sea buckthorn berry oil inhibits platelet aggregation. J Nutr Biochem. 11 (10): 491-495

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to provide a method for inhibiting direct thrombin inhibition, A pharmaceutical composition for preventing or treating thrombotic diseases caused by inhibition of endogenous blood coagulation factors and inhibition of platelet aggregation, 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, which comprises vitamin extract ( Hippophae rhamnoides L.) leaf extract as an active ingredient.

The vitamin tree leaf extract is preferably an ethanol extract of vitamin tree leaves.

The vitamin tree leaf extract is preferably an ethyl acetate fraction or a water residue obtained by successively fractionating an ethanol extract of a vitamin tree leaf with an organic solvent.

The ethyl acetate fraction or the water residue is preferably obtained by successively fractionating the ethanol extract of the vitamin tree leaf with hexene, ethyl acetate and butanol.

The present invention also provides a health functional food comprising the vitamin tree leaf extract of the present invention.

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the vitamin tree leaf extract as an active ingredient of the health functional food can be prepared by extracting vitamin tree leaves with ethanol or the like to prepare an extract Ethyl acetate fractions and water residues exhibiting antithrombotic activity due to the excellent anticoagulant effect are effective to inhibit thrombogenesis efficiently. The anticoagulant activity of the anticoagulant, 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 vitamin tree leaf extract of the present invention is free from acute oral toxicity, has no human erythropoietic activity, has excellent thermal stability, and is capable of inhibiting direct inhibition of human thrombin, prothrombin inhibition, blood coagulation factor inhibition It is an extremely useful invention in the pharmaceutical industry and the food industry because it can be prepared in various forms such as extract, powder, ring, and tablet, and can be prepared at any time.

The present invention relates to a pharmaceutical composition for preventing or treating thrombosis having a direct inhibition of human thrombin, a prothrombin inhibitor and an endogenous blood coagulation factor inhibitory activity, which comprises vitamin tree leaf extract, and a health functional food.

The inventors of the present invention recovered the antithrombotic active ingredient from the vitamin tree leaf extract obtained by a certain method, and confirmed that these components have excellent thermal stability and acid stability without showing oral acute toxicity, A pharmaceutical composition for preventing or treating thrombosis, and a health functional food.

Specifically, in order to develop a pharmaceutical composition and a health functional food for preventing or treating thrombosis using a vitamin tree leaf, the present inventors prepared an ethanol extract of a vitamin tree leaf and sequentially fractionated it with an organic solvent to obtain a hexane fraction , Ethyl acetate fraction, butanol fraction, and water residue, and analyzing useful components such as total polyphenol, total flavonoid, etc., and analyzing them for thrombin time and thrombin time for human plasma and human thrombin, respectively, Prothrombin time and activated partial thromboplastin time (aPTT), and platelet aggregation inhibition were evaluated.

As a result, thrombin time, prothrombin time, and apathy time were prolonged by 15 times or more as compared with the no-added group at a concentration of 5 mg / ml of ethanol extract of vitamin tree leaf. Thrombin time and apathy time were extended more than 15 times, and prothrombin time was extended by 1.37 times as compared with no-added period.

In addition, the hexane fraction, ethyl acetate fraction, butanol fraction, and water residue obtained from the vitamin tree leaf ethanol extract were all at least 15 times longer than the no-added group at the concentration of 5 mg / ml of thrombin time, prothrombin time and apathy time And the vitamin tree leaf extracts were found to contain various kinds of thrombogenic inhibitors.

In particular, the ethyl acetate fraction of the fractions showed the most potent thrombogenesis inhibitory activity at a concentration of 1.5 mg / ml, with the prolongation of thrombin time, prothrombin time, and apathy time by 15 times or more as compared with the no-added group. On the other hand, the water residue showed strong antithrombogenic activity after the fraction of ethyl acetate in the fraction. In this case, the thrombin time, prothrombin time and apathy time were extended more than 15 times as compared with the no-addition period even at the concentration of 2.5 mg / ml Respectively.

Furthermore, the toxicity and stability of the ethyl acetate fraction and the water residue of the vitamin-tree leaf extract of the present invention were evaluated. The substances showed no hemolytic activity on human erythrocytes and were excellent in heat and acid stability. And the extract was found to be almost free of human toxicity by conducting an acute toxicity test.

Accordingly, the invention is vitamin tree (Hippophae The present invention provides a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing the rhamnoides L. leaf extract as an active ingredient.

Hereinafter, the production method and efficacy test of the vitamin tree leaf extract of the present invention will be described in more detail.

The present invention relates to a method for preparing an active extract, comprising: preparing an active extract from commercially available dried vitamin tree leaves; Adjusting an organic solvent fraction such as hexane, ethyl acetate, butanol and the like to a water residue from a vitamin tree leaf extract; Examining the antithrombotic efficacy and stability of the extract and fraction; And an acute toxicity testing step of the fraction.

"Vitamin tree leaf extract" contained in the composition of the present invention is prepared by washing vitamin tree leaves, drying, extracting the dried leaves with an organic solvent, filtering the extract 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 And 70-95% ethanol is most preferred.

In the present invention, when the concentration of ethanol extract of vitamin A leaves was 5.0 mg / ml, thrombin time, prothrombin time, and apathy time were measured. As a result, ethanol extract of vitamin tree leaves 15 times or more stronger Thrombogenic activity. At the concentration of 2.5 mg / ml, thrombin time, prothrombin time, and apathy time were 15 times, 1.37 times, and 15 times longer than those of no-added, respectively. Aspirin, which is used as an antithrombotic agent, was prolonged by 8.6 times, 1.8 times, and 1.9 times at 2.5 mg / ml, respectively, and 2.5 times, 1.6 times, and 1.6 times longer at aspirin 1.5 mg / , And vitamin tree leaf extract showed better antithrombotic effect than aspirin.

In addition, the sequential organic solvent fractions of the ethanol extract of vitamin tree showed excellent antithrombotic effect. Particularly, in the ethyl acetate fraction at the concentration of 1.5 mg / ml, thrombin time, prothrombin time and apathy time were 15 times And the water residue at the concentration of 1.5 mg / ml was 4.9 times as much as that of the no-added solution at thrombin time, prothrombin time, and apathy time. 7.26 times and over 15 times, the ethyl acetate fraction and water residues showed more potent antithrombotic activity than ethanol tree extract of vitamin. These results indicate that the ethyl acetate fraction and water residues in the vitamin tree leaves are highly effective in inhibiting thrombogenesis by acting on human thrombin, prothrombin, and blood coagulation factors, and the adverse effects such as aspirin Suggesting that thrombogenic inhibitors may be substituted.

The vitamin tree leaf extract of the present invention, preferably the vitamin tree leaf ethanol extract, its ethylacetate fraction or the water residue, can be made into a 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 vitamin tree leaf 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 containing the vitamin tree leaf extract of the present invention may be formulated into oral compositions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, sterilized injection solutions And the like, and they can 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 dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of vitamin A leaf 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 May 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, 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 vitamin tree leaf extract of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they can be used in the form of powder, granule, tablet, capsule or beverage .

The vitamin tree leaf extract of the present invention may be added in an amount of 0.01 to 15% by weight of the whole food, and the health beverage composition may be added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, have.

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 these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the vitamin tree leaf 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: Vitamin tree  Leaf extract and organic solvent Fraction  pharmacy

In 2011, cultivated and dried vitamin tree leaves were cultivated in Andong, Gyeongsang Province, Korea. Vitamin tree leaves were extracted with ethanol (80%, Duksan, Korea) 10 times at room temperature for 8 hours. The extract was recovered after removing impurities by using a filter machine, and concentrated under reduced pressure to prepare powder, which was kept at low temperature until use. In the case of the organic solvent sequential fraction, the ethanol extract was suspended in distilled water, and then fractionated with hexane, ethyl acetate and butanol sequentially to obtain a final water residue.

The yields of the ethanol extracts were 17.2 ± 1.5%, and the yields of the hexane fraction, ethyl acetate fraction, butanol fraction and water residue of the ethanol extracts were 2.89%, 22.46%, 38.52% and 31.37%, respectively. In particular, the amount of ethyl acetate fraction was much higher than that of leaf extracts of other plants, and the hexene fraction was relatively small.

Example  2: Vitamin tree  Component analysis of leaf extract and Anti-thrombosis  Activity evaluation

The total flavonoid, total polyphenol, total sugar, and reducing sugar content of the prepared vitamin tree leaf 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 Analysis of Ethanol Extracts from Vitamin Tree Leaves  extract Extraction efficiency (%) Content (mg / g) Total polyphenol Total flavonoid Total Reducing sugar 80% ethanol extract 17.2 ± 1.5 135.85 + - 0.46 71.14 + - 2.81 228.71 ± 17.10 97.48 ± 2.77

Vitamin tree leaf ethanol extracts showed significant amounts of total polyphenols and total flavonoids, which were found to be associated with various antioxidant, anti-inflammatory, antibacterial and cancer cell growth inhibitory activities.

Next, the antithrombotic activity of vitamin A leaf extracts 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, the prothrombin time, and the apathy time were measured by the following procedure.

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.

Apathy  time ( 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 value of three repeated experiments.

The results of the thrombin time, the prothrombin time and the apitime measurement according to the above-described measurement method of antithrombotic activity are shown in the following [Table 2].

Vitamin tree leaf ethanol extract antithrombotic activity Final concentration
(mg / mL) Thrombin Time (sec) Prothrombin time (seconds) Apathy Time (seconds) ethanol
extract aspirin ethanol
extract aspirin ethanol
extract aspirin 0 32.1 ± 0.5 32.1 ± 0.5 18.1 ± 0.3 18.1 ± 0.3 55.1 ± 1.4 55.1 ± 1.4 0.62 32.1 ± 0.8 - 18.2 ± 0.3 - 67.2 ± 2.1 - 1.25 31.5 ± 0.6 - 17.8 ± 0.5 - > 800 - 1.5 51.4 ± 6.1 80.1 ± 1.6 18.1 ± 0.5 30.0 ± 3.2 > 800 87.6 ± 4.5 2.5 > 480 277.1 ± 11.9 24.7 ± 6.1 32.8 ± 3.8 > 800 104.3 ± 7.2 5.0 > 480 > 480 > 270 > 270 > 800 > 800

Aspirin used as a control showed a good antithrombotic effect at a concentration of 5 mg / ml. The ethanol extract of vitamin-tree leaves prolonged thrombin time by more than 15 times at a concentration of 2.5 mg / ml, Was elongated more than 15 times at the concentration of 1.25 mg / ml compared to the no-added solution. However, prothrombin time showed little extension at 2.5 mg / ml concentration. Therefore, it was confirmed that the vitamin tree leaf extract showed strong antitumor activity due to strong inhibition of human thrombin and blood coagulation factors.

Example  3: Vitamin tree  Sequential organic solvent of leaf extract Fraction  Component analysis and evaluation of antithrombotic activity

The components and antithrombotic activities of each fraction and water residues of ethanol tree extract of vitamin tree with excellent antithrombotic activity were examined. The total flavonoid, total polyphenol, total sugar, and reducing sugar content of the fractions and residues were measured, and the results are shown in Table 3 below and the antithrombotic activity is shown in [Table 4].

Analysis of Fractional Efficiency and Components of Organic Solvent Fractions of Vitamin tree Leaf Extracts extract Fraction efficiency
(%) Content (mg / g) Total polyphenol Total flavonoid Total Reducing sugar Hexene fraction 2.89 70.27 1.52 45.72 + 2.81 244.96 +/- 14.55 59.54 + - 2.95 Ethyl acetate fraction 22.46 246.15 + 16.39 139.02 + - 4.78 159.58 ± 5.62 130.55 + - 0.74 Butanol fraction 38.52 132.82 + - 8.50 71.54 + - 2.25 224.74 ± 5.87 96.96 ± 1.29 Water residue 31.37 77.23 + - 0.91 41.70 ± 2.81 251.62 ± 9.19 92.66 +/- 1.48

The organic solvent fraction of ethanol extract of vitamin tree leaves was fractionated in the order of butanol fraction, water residue and ethyl acetate fraction. The fraction of hexane fraction was only 2.9%. Unusually, all the fractions contained very large amounts of polyphenols and flavonoids, which were 10 times higher than those of Omija and Cherry, which are known to contain large amounts of useful physiologically active ingredients. In addition, each fraction contained 159 to 251 mg / g of sugar, and it was confirmed that the components of the fractions contained hexane-soluble substance, ethyl acetate-soluble substance, butanol-soluble substance and water-soluble substance, .

As shown in Table 4, the hexane fraction and the butanol fraction were weaker than the antithrombogenic effect of aspirin 1.5 mg / ml in the case of thrombin and prothrombin inhibition, but the blood coagulation The apathy time extension effect was shown by the factor inhibition. On the other hand, the ethyl acetate fraction and the water residue showed a stronger thrombogenic effect than the aspirin-identical concentration, and the ethyl acetate fraction showed an antithrombotic effect equivalent to aspirin 5 mg / ml at the concentration of 1.5 mg / ml ([ Table 2] and [Table 4]). Considering that the ethyl acetate fraction and the water residue are not purified, this result indicates that the purified active material exhibits more potent antithrombotic activity.

Antithrombotic activity of organic solvent fractions and water residues of vitamin A leaf extract 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 1.5 80.1 ± 1.6 30.0 ± 3.2 87.6 ± 4.5 Hexene fraction 5.0 > 480 > 280 > 800 2.5 55.8 ± 4.7 26.3 ± 5.4 > 800 1.25 35.5 ± 3.5 18.4 ± 1.5 99.1 ± 8.9 0.5 32.9 ± 1.1 17.8 ± 2.4 71.6 ± 3.4 Ethyl acetate
Fraction 5.0 > 480 > 280 > 800 2.5 > 480 > 280 > 800 1.5 > 480 > 280 > 800 1.25 28.6 ± 3.2 19.7 ± 0.3 > 800 0.5 28.2 ± 2.5 18.2 ± 1.5 66.1 ± 6.7 Butanol fraction 5.0 > 480 > 280 > 800 2.5 163.1 + - 10.8 > 280 > 800 1.5 32.1 ± 0.5 22.3 ± 0.2 > 800 0.6 29.3 ± 2.5 18.5 ± 1.9 71.6 ± 11.2 Water residue 5.0 > 480 > 280 > 800 2.5 > 480 > 280 > 800 1.5 157.3 ± 12.8 131.4 ± 24.9 > 800 1.25 62.9 ± 3.3 24.1 ± 0.7 > 800 0.5 32.3 ± 3.2 18.9 ± 0.5 50.7 ± 3.5

Example  4: Vitamin tree  Ethanol extract of leaves, sequential organic solvent Fraction  Human platelet aggregation inhibitory activity

The anti-aggregation inhibitory activity against human platelets was evaluated as an evaluation of antithrombotic activity of the vitamin-tree leaf extract and each fraction, and the results are shown in Table 5. First, aspirin showed excellent human platelet aggregation inhibitory activity and ascertained concentration dependent coagulation inhibitory activity. The ethanol extract of vitamin A leaves exhibited a potency of platelet agglutination inhibition twice as much as that of aspirin at the concentration of 0.25 mg / ml, and its hexane fraction and ethyl acetate fraction showed more potent activity than aspirin. In addition, aspirin - like platelet aggregation inhibitory activity was also observed in water residues. From these results, the ethyl acetate fraction and the water residue sequentially fractionated from the ethanol extract of the vitamin tree leaf of the present invention have a strong thrombin inhibition and platelet aggregation inhibitory activity more than the aspirin, so that the aspirin is reduced to 1/2 to 1/4 Concentration in the culture medium. 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, centrifuged at 1,100 rpm for 10 minutes, and platelet rich plasma (PRP) to _{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) and washed once. After resuspending in 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 several minutes, 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 agarose (Chrono-log, USA).

Human Platelet Aggregation Inhibitory Activity of Ethanol Extracts and Fractions of Vitamin Tree Leaves Sample / Control burglar
(Amplitude: ohm) inclination
(Slope) Extension time
(Lag time: seconds) Fall area
(Area under) DMSO (solvent: DMSO) 14 4 78 148.5 Aspirin (0.50 mg / mL) 5 One 184 21.7 Aspirin (0.25 mg / mL) 10 2 96 67.2 Ethanol extract (0.25 mg / mL) 4 One 207 22.3 The hexane fraction (0.25 mg / mL) 5 One 96 33.1 Ethyl acetate fractions (0.25 mg / mL) 2 0 215 13.0 Butanol fraction (0.25 mg / mL) 22 2 109 156.6 The water residue (0.25 mg / mL) 14 2 91 89.1

* The smaller the falling area, the stronger the cohesion inhibition.

Example  5: Vitamin tree  Leaf ethyl acetate Fraction  And the chemical properties and stability of the active substance of the water residue

Ethyl acetate fractions and water residues of the vitamin tree leaves obtained in Example 1 were subjected to pressure drying to prepare powders, and the recovered active substances were tested for hemolytic activity against human erythrocytes, plasma stability, thermal stability and acid stability. The regulated active substance was treated at 100 ° C for 2 hours, treated at pH 2 (0.01 M HCl) for 2 hours, treated for 2 hours in plasma, and showed no inhibition of antithrombotic activity and decreased activity of platelet aggregation inhibition, Respectively. In addition, hemolytic activity against human erythrocytes was not observed up to a concentration of 5 mg / ml (Table 6).

Human Erythrocyte Hemolytic Activity of Vitamin Tree Leaf Extracts and Fractions Samples (5 mg / ml) Human hemolytic hemolytic activity (%) Ethanol extract 0.10 ± 0.05 Hexene fraction 0.10 0.55 Ethyl acetate fraction 0.21 ± 0.82 Butanol fraction 1.57 ± 0.50 Water residue 2.83 ± 0.16 Triton X-100 (0.1% 100.00 ± 0.00 Amphotericin B 125 μg / ml 75.30 +/- 8.50

Example  6: Vitamin tree  Leaf ethyl acetate Fraction  And water residues Single oral toxicity  exam

Four-week-old rats (Slc, ICR Mouse) were supplied from the Central laboratory animals and maintained at a temperature of 23 ± 3 ° C, 50 ± 10% relative humidity, and 150 ~ 300 Lux at 12- Hour), the single oral toxicity of the ethyl acetate fraction and the water residue obtained from Example 1 was evaluated. Animal experiments were carried out with the prior approval of the Animal Experimentation Ethics Committee of Andong University (Industry Research Support Division -2132, May 18, 2012). After samples were dissolved in DMSO and water, 400, 800, 1500, 2000 mg / kg And the survival and pathological abnormalities were observed for one week after oral administration. 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 and gum residues of vitamin tree leaves that have been used for edible and medicinal purposes are classified as low toxic or non - toxic, and the use of low concentrations thereof is not likely to cause any additional problems.

Claims (5)

Vitamin tree ( Hippophae A pharmaceutical composition for prevention or treatment of thrombosis, comprising a leaf extract of rhamnoides L. as an active ingredient. The pharmaceutical composition for preventing or treating thrombosis according to claim 1, wherein the vitamin tree leaf extract is an ethanol extract of a vitamin tree leaf. The pharmaceutical composition for preventing or treating thrombosis according to claim 1, wherein the vitamin tree leaf extract is an ethyl acetate fraction or a water residue obtained by successively fractionating an ethanol extract of a vitamin tree leaf with an organic solvent. 4. The pharmaceutical composition according to claim 3, wherein the ethyl acetate fraction or the water residue is obtained by sequentially fractionating the ethanol extract of the vitamin tree leaf with hexene, ethyl acetate and butanol. A health functional food comprising the vitamin tree leaf extract of any one of claims 1 to 4.