KR101358203B1 - Composition comprising the extract of Cornus officinalis for prevention or control of thrombosis - Google Patents

Abstract

The invention Cornus (Cornus officinalis ) The present invention relates to a pharmaceutical composition for preventing and treating thrombin-inhibited thrombosis containing an extract as an active ingredient, and the extract of the present invention is applicable to water, alcohol, ethanol, methanol, etc. After extraction with a variety of solvents, the extract is obtained by sequential organic solvent fractionation does not show acute oral toxicity and chronic toxicity, excellent thermal stability, do not show a loss of thrombin inhibitory activity in acidic conditions and plasma of pH 2 The pharmaceutical composition containing this as an active ingredient exhibits thrombin inhibitory activity and platelet aggregation inhibitory activity, and thus can effectively suppress thrombus generation.It can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through blood circulation improvement. It is processed into various forms such as extract, powder, pill and tablet When there is an excellent effect that can be prepared by taking a dosage form as possible.

Description

Composition comprising the extract of Cornus officinalis for prevention or control of thrombosis}

The present invention relates to a pharmaceutical composition for preventing or treating thrombin-inhibited thrombosis containing a cornus extract as an active ingredient, and more specifically, a pharmaceutical for preventing or treating thrombin-inhibited thrombosis, characterized by containing a cornus extract as an active ingredient. A composition and functional health food composition for improving thrombin inhibitory thrombosis.

Human blood has a variety of important functions such as oxygen, nutrients, waste transport and buffering function, body temperature maintenance, osmotic pressure control and ion balance, hydration constant maintenance, fluid control action, blood pressure maintenance and control, biological defense. 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 thrombi undergoes a multi-step reaction of numerous factors, activating thrombin that is directly involved in fibrin coagulation, resulting in the formation of fibrin monomers from fibrinogen, which are polymerized by calcium, binding to platelets and endothelial cells. Persistent thrombus formation is achieved by forming fibrin polymers cross-linked by Factor XIII. In addition, thrombin plays a pivotal role in thrombus formation by activating platelets, factor V and factor VII to promote blood coagulation.

Therefore, thrombin activity inhibitors can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood coagulation, aiming to develop thrombin direct inhibitors worldwide. In particular, with the development of society and the aging of the population, thrombotic diseases, namely cerebrovascular diseases and heart diseases related to excessive thrombosis, have been rapidly increasing in recent years, and development of safe antithrombotic drugs is urgently needed. Various anticoagulants such as heparin, coumarin, aspirin, urokinase, antiplatelets, and thrombolytic agents 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 reactions. As a result, its use is limited.

Cornus ( Cornus) officinalis ) is a medicinal plant belonging to the genus Comaceae, whose fruit is in the shape of a central circular shape of about 1.5cm in length, and is known as Zhuchocho because of its sour taste, and it is widely used as a natural food with nourishing tonic effect. Currently, the products related to cornus oil in the market are mostly 1) dried product dried steamed cornus oil, 2) cornus milk extract liquid drink using dry cornus oil, and 3) cyclic product using cornus powder. Because of the sour taste, development of products that preserve the unique flavor and aroma of cornus oil is still insignificant.

Cornus oils include crystalline organic acids, molar acids, malic acid, and uric acid. Iridoid glycosides such as moronisid and loganan are known. Known to the public, there are diuretic effect, blood pressure strengthening effect, antibacterial effect, hepatoprotective effect. In oriental medicine, nourishment tonic, astringents to see the kidneys, frequent sweating, urine is used a little often.

As a patent document on cornus function, Korean Patent Publication No. 1020030073346 (published: 2003.09.19, patent application No. 10-2002-0012877, filing date: 2002.03.11) 'contains cornus extract with skin aging inhibitory effect Cosmetic composition and its manufacturing method 'and Korean Patent Publication No. 1020100110978 (published: October 14, 2010, patent application No. 10-2009-0029299, filing date: 2009.04.06)' functionality and lipid lowering by using Bacillus subtilis and cornus milk Methods for producing fermented soy milk with improved performance and fermented soy milk for preventing atherosclerosis obtained therefrom are known.

However, there has been no report on the antithrombotic activity of cornus extract and the antithrombotic effect through direct inhibition of human thrombin enzyme.

Therefore, the present inventors aimed at developing a functional food material exhibiting antithrombotic and blood flow improving activity, and after preparing cornus extract, evaluating thrombinogenesis inhibitory activity according to thrombin direct inhibition using human plasma and human thrombin, and human concentrated platelets. The platelet aggregation inhibitory activity of cornus milk was measured, and the potent antithrombotic activity was confirmed in the cornus extract and its sequential fractions.

1. Republic of Korea Patent Publication No. 1020030073346 (published: September 19, 2003) 2. Republic of Korea Patent Publication No. 1020100110978 (Published: 2010.10.14)

Accordingly, an object of the present invention is to provide a thrombin inhibitor by extracting and purifying the thrombin inhibitory active material with water or organic solvent from cornus oil, and using the same as an active ingredient.

The above object of the present invention is to determine the optimum extraction solvent and extraction conditions of thrombin inhibitory active material from cornus oil, purify the active ingredient of 30 kDa or less through the sequential organic solvent fraction and amicon molecular sieve of the extract, After investigating the chemical properties and stability of the material, the material was orally administered to rats to achieve acute and chronic toxicity tests.

Hereinafter, the configuration of the present invention will be described in detail.

As also proved through the examples herein, the cornus extract and the composition of the present invention is extracted with various solvents such as water, spirits, methanol, ethanol, and the like to obtain the extract using a sequential organic solvent fraction and amicon molecular sieve In addition, it exhibits an excellent thrombin inhibitory activity, has the effect of effectively inhibiting thrombus generation, and has an excellent effect that can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improved blood circulation. In addition, the cornus extract of the present invention does not show acute oral toxicity and chronic toxicity, excellent thermal stability, no loss of thrombin inhibitory activity in acidic conditions and plasma of pH 2, extracts, powders, pills, tablets It is a very useful invention in the food industry and pharmaceutical industry because it has an excellent effect that can be processed into various forms such as can be taken in a form that can be taken at all times.

1 is a graph showing the antithrombotic effect (thrombin time, prothrombin time, and apititime time) of the cornus fruit extract and its fractions, wherein-●-is an ethanol extract,-○-is a hexane fraction,- ▼-represents the ethyl acetate fraction,-▽-represents the butanol fraction, and-■-represents the water residue.

The present invention relates to a pharmaceutical composition for preventing and treating thrombin-inhibited thrombosis containing cornus extract. More specifically, after extraction with a variety of solvents such as water, alcohol, ethanol, methanol from cornus oil, sequential organic solvent fractions and water residues are prepared with hexane, ethyl acetate, butanol, and in particular, oral acute in the ethyl acetate fraction. Unlike conventional thrombogenic inhibitors without toxicity and chronic toxicity, and exhibits excellent antithrombotic activity, excellent thermal stability and acid stability, and relates to a substance having a molecular weight of 30 kDa or less that can be processed orally in various forms.

The present invention is to determine the optimum extraction solvent and extraction conditions of thrombin inhibitory active material from cornus milk; Purification of the active ingredient through the sequential organic solvent fraction of cornus extract and amicon molecular sieve; Examining the chemical properties and stability of the material; And testing the acute and chronic toxicity of the substance.

"Horse extract" included in the composition of the present invention means that obtained by extracting from various organs, such as fruit, leaves, stems, bark, throat of cornus, preferably means an extract obtained from the fruit of mature cornus.

Cornus fruit containing the thrombin inhibitory substance of the present invention is registered as a food raw material in the domestic food industry, low shelf life, boiled in boiling water immediately after harvesting and then dried and distributed. To date, the Korean Food and Drug Administration has not conducted food ingredient analysis, but as a result of general ingredient analysis on dry ingredients of commercial cornus oil (water content 50-55%), protein content 2.3%, crude fat 4.5%, carbohydrate 88.6%, ash 4.6 Known as% (404.9 kcal / 100g) (2006 Rural Food and Nutrition Analysis). The main production areas in Korea are Gurye, Jeonnam and Uiseong, Gyeongbuk. The annual output is about 600 tons. The wholesale price was 9,000-11,000 won / 600g in 2006-2008, but has soared to 30,000 won / 600g in 2010-2011 due to reduced production due to abnormal weather.

In order to edible cornus fruits, seeds that are known to be toxic should be removed. Generally, the cornus fruits, which have been boiled in boiling water immediately after harvesting, are removed using automated machinery. Damaged or poorly marketed cornus during the harvest and seed removal phases is being discarded. Therefore, the present invention can prepare a pharmaceutical composition for preventing and treating thrombin-inhibited thrombosis from cornus milk, including the old discarded or low-commercial cornus fruit.

Cornus milk that exhibits inhibitory activity against human thrombin in the present invention can be extracted using ethanol from various sites such as fruit, leaves or stems. Thrombin time, prothrombin time and aptity time (aPTT) were measured at 5 mg / mL cornus ethanol extract, resulting in more than 15 times longer thrombin time and prothrombin time. (prothrombin time) and affinity time (aPTT). This is a very potent antithrombotic effect, given that the thrombin time, prothrombin time and aPTT are extended by 2.49, 1.25 and 1.44 times, respectively, at a concentration of 1.5 mg / mL of purified aspirin. It was confirmed that there is. In addition, thrombin time, prothrombin time and apititime were extended at least 15 times, 1.1 times, and 1.0 times, respectively, at 2.5 mg / mL of cornus ethanol extract, indicating that thrombin-specific inhibition was observed at relatively low concentrations. . In the ethyl acetate fraction of Cornus ethanol extract, thrombin time, prothrombin time and apititime were extended by 15 times, 1.2 times and 1.6 times, respectively, at 1.25 mg / mL concentrations, resulting in better antithrombotic activity than aspirin. It can be seen that.

On the other hand, as a result of evaluating platelet aggregation inhibitory activity in human platelet concentrate, the cornus ethanol extract and its ethyl acetate fraction showed the same platelet aggregation inhibitory activity as that of aspirin 0.5 mg / mL at 0.25 mg / mL. The activity was followed by butanol fraction, hexene fraction and water residue. These results indicate that cornus extract can effectively replace anticoagulants such as aspirin, and it is expected that cornus extract will help blood circulation by preventing thrombus formation.

"Horse extract" in the present invention can be prepared using a variety of extraction solvents. For example, water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, and the like, "Horse extract" can be prepared. Preferably extracts using ethanol or alcohol, shows the maximum yield of active material. Cornus extract of the present invention may be prepared into a powder through a conventional powdering process such as distillation and freeze-drying, or spray drying.

Cornus extract prepared using the above extraction solvents such as alcohol, water, methanol, ethanol, and the like, after suspension in water, may be sequentially partitioned using hexene, ethyl acetate and butanol, and divided into remaining water fractions. In this case, thrombin Inhibitory actives are strongly identified in the hexene fraction, the ethyl acetate fraction, and the butanol fraction, and are relatively weakly detected in the water residue. These results indicate that there are thrombin inhibitors with various properties such as fat solubility and water solubility in the cornus extract. Ethyl acetate fraction showing the strongest activity can be prepared into powder through conventional powdering process such as distillation, lyophilization or spray drying, which is dissolved in ethanol and then microcon (microcon YM-30, Millipore) The molecular weight can be divided into less than 30,000 and more than 30,000 substances, anti-thrombotic activity is confirmed only in the molecular weight of less than 30,000. Therefore, the adjusted substance is negative for the ninhydrin reaction and positive for the anthrone reaction, and it can be seen that the polysaccharide having a molecular weight of 30 kDa or less containing a phenolic substance. They maintain activity at thermal stability and pH in the human stomach at pH 2, and do not show hemolytic activity against human erythrocytes up to 1 mg / mL.

In the present invention, the term "active ingredient" refers to a substance or a group of substances (a medicinal agent whose pharmacologically active ingredient is not known, etc.) which is expected to express the efficacy or effect of the drug directly or indirectly by inherent pharmacological action. It means containing a main component).

In the present invention, "functional health food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means a human body It means the ingestion for the purpose of obtaining a useful effect in health use such as nutrient control or physiological action on the structure and function of.

Carriers, excipients and diluents that may be included in the pharmaceutical composition comprising the cornus extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or lactose, gelatin, or the like is mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The preferred dosage of the pharmaceutical composition according to the present invention depends on the condition and body weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Cornus extract according to the present invention may be added to food or beverage for the purpose of improving thrombin inhibitory thrombosis. Food or beverages to which the cornus extract may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health functional foods, powders, granules, tablets, capsules or beverages. At this time, the amount of the cornus extract in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition is based on 100 ml in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g Can be added.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example  1: from cornus fruit, leaves, seeds and stems Thrombin  Extraction of Inhibitors

Cornus fruits, leaves, seeds, and stems harvested in December 2010 in Uiseong, Gyeongbuk, Korea, were extracted using ethanol and hot water. In case of ethanol extraction, 5 times of ethanol was added to each sample, and extracted three times at room temperature for 24 hours. In case of hot water extraction, 5 times of distilled water was added to each sample and extracted at 100 ° C. for 30 minutes. The extract was concentrated under reduced pressure to give a powder, which was stored at low temperature until use. Meanwhile, the total flavonoid and total polyphenol content, total sugar and reducing sugar content of the prepared Cornus extract were measured. The total flavonoid content was measured by stirring and extracting each sample for 18 hours with methanol stirring, adding 4 mL of 90% diethylene glycol to 400 μl of the filtered extraction sample, adding 40 μl of 1 N NaOH, and reacting at 37 for 1 hour. Measured. As a standard reagent, rutin was used. The total polyphenol content was added to 400 μl of the extracted sample solution, 50 μl of Folin-ciocalteau, 100 Na ₂ CO ₃ saturated solution, and left at room temperature for 1 hour, followed by measurement of absorbance 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.

In the case of ethanol extraction, the extraction efficiencies of the fruits, leaves, seeds and stems were 31.49%, 13.73%, 18.33% and 1.18%, respectively, showing the highest extraction rate in the fruit and very little in the stem. Extraction efficiency and component analysis results of fruits, leaves and seeds using ethanol and hot water are shown in [Table 1] and [Table 2].

Ethanol Extraction Efficiency and Component Analysis from Cornus Fruits, Leaves and Seeds sample Extraction rate (%) Content (mg / g) Total polyphenol Total flavonoids  Total Reducing sugar Fruit 31.49 1.36 ± 0.59 15.70 ± 0.32 276.76 ± 10.53 242.00 ± 4.81 leaf 17.43 33.54 ± 1.51 98.22 ± 2.55 317.32 ± 5.31 275.93 ± 1.49 Seed 18.33 4.92 ± 0.01 560.59 ± 12.75 133.05 ± 9.14 95.96 ± 0.30

Efficiency and Component Analysis of Hot Water Extract from Cornus Fruits, Leaves and Seeds sample Extraction rate (%) Content (mg / g) Total polyphenol Total flavonoids  Total Reducing sugar Fruit 30.61 3.44 ± 0.41 58.7 ± 0.00 680.90 ± 7.80 644.78 ± 5.77 leaf 26.83 32.87 ± 1.19 215.63 ± 10.98 218.34 ± 12.02 208.40 ± 0.55 Seed 16.53 4.68 ± 0.38 591.15 ± 10.63 163.50 ± 3.02 163.25 ± 3.35

For ethanol extraction, the total flavonoid content was in order of seed>leaf> fruit and the total polyphenol content was in order of leaf>seed> fruit. The pattern was the same in the case of hot water extraction, and the ethanol and hot water extraction efficiencies were similar in the fruit and the seed, but in the case of the leaves, the hot water extract showed a high extraction rate of 9.4%. Therefore, the cornus fruit used as a food raw material may be extracted with water or ethanol, but the extract of the cornus oil is considered to be more efficient.

Example  2: of cornus extract Anti-thrombosis  Activity evaluation

Cornus extracts were dissolved in DMSO (dimethylsulfoxide), diluted to appropriate concentrations, and measured thrombin time, prothrombin time and activity time using human plasma. 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 aPTT. 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 method were as follows.

Thrombin  time( Thrombin Time )

At 37 ℃ 0.5 U thrombin (Sigma Co., USA) 50 ㎕ and 20 mM CaCl ₂ 50 μl and 10 μl of sample extracts at various concentrations 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. Aspirin (Sigma Co., USA) and heparin (heparin; Sigma Co., USA) were used as controls, and DMSO was used instead of the sample as a solvent control. DMSO had a solidification time of 32.1 seconds. 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 )

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample were added to a tube of Amelung coagulometer KC-1A (Japan), and the mixture was heated at 37 ° C. for 3 minutes. Then, 130 μl of PT reagent was added 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 solidification time of 18.1 seconds.

aPTT  ( activated Partial Thromboplastin Time )

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

Cornus fruit, leaf, and seed ethanol extracts showed thrombin times more than 15 times, 1.9 times, and 1.1 times higher than the non-addition at 5 mg / mL, respectively. The concentrations showed more than 5.5-fold, 15-fold and 1.1-fold increase in thrombin time, respectively, compared to the no addition group. On the other hand, aspirin used as an antithrombotic agent exhibited an increased thrombin time of 2.49 times at a concentration of 1.5 mg / mL, confirming high antithrombotic activity in the ethanol extract of the cornus fruit and the hydrothermal extract of the leaf [Table 3].

Thrombin Inhibitory Activity of Cornus Fructus, Leaf and Seed Extracts Ethanol extract
(mg / mL) Thrombin time
(x control) Hot water extract
(mg / mL) Thrombin time
(x control) Fruit 5.0 > 15 Fruit 5.0 5.5 ± 0.42 2.5 > 15 2.5 4.5 ± 0.24 1.25 1.8 ± 0.19 1.25 1.1 ± 0.08 leaf 5.0 1.9 ± 0.25 leaf 5.0 > 15 2.5 1.3 ± 0.01 2.5 > 15 1.25 1.1 ± 0.01 1.25 1.61 ± 0.06 Seed 5.0 1.1 ± 0.15 Seed 5.0 1.1 ± 0.15 2.5 1.1 ± 0.11 2.5 1.1 ± 0.11 1.25 1.1 ± 0.01 1.25 1.1 ± 0.01 aspirin 1.5 2.49

Example  3: cornus ethanol extract and Fraction  Ingredient survey

Cornus ethanol extract of Example 1 having the best yield and antithrombotic activity was partitioned into hexene, ethyl acetate and butanol, and finally water residue was obtained. The yield and component analysis of each fraction are shown in [Table 4], and the total polyphenol content was very high in all cornus extracts and fractions except water residues, and in the hexene fraction and ethyl acetate fraction, High total flavonoid content. Total and reducing sugars showed high content in butanol fraction and water residue.

Fraction Efficiency and Component Analysis of Cornus Ethanol Extracts Fraction Fraction rate (%) Total polyphenol Total flavonoids  Total Reducing sugar Hexene fraction 1.88 11.02 ± 1.17 30.78 ± 0.32 60.76 ± 0.78 39.94 ± 5.79 Ethyl acetate fraction 4.85 10.64 ± 0.18 50.49 ± 0.74 50.83 ± 0.78 32.97 ± 1.68 Butanol fraction 53.70 1.92 + - 0.44 18.32 ± 0.00 208.07 ± 20.29 126.00 ± 8.66 Water residue 37.14 0.54 ± 0.18 3.46 ± 0.32 283.66 ± 10.14 232.92 ± 9.62

Example  4: Cornus ethanol extract and Fraction Anti-thrombosis  activation

The antithrombotic activity of the Cornus ethanol extracts and the organic solvent fractions of Examples 1 and 3 was investigated. The results of measuring thrombin time, prothrombin time, and epitaxial time are shown in FIG. 1, and the results of evaluating platelet aggregation inhibitory activity are shown in Table 5.

As shown in FIG. 1, the ethyl acetate fraction extended thrombin time, prothrombin time, and epitaxial time more than 15 times at 2.5 mg / ml concentration, and thrombin time, prothrombin time, and epitaxial time at 1.25 mg / ml concentration. Were extended by at least 15, 1,2 and 1.6 times, respectively. This effect is more than twice as strong anti-thrombotic effect than the original ethanol extract, it was confirmed that the potency compared to aspirin. The hexene fraction, on the other hand, selectively extended only thrombin time and the water residue showed no activity.

As shown in Table 5, the cornus ethanol extract and the organic solvent fractions were also excellent in human platelet aggregation inhibitory activity. In particular, the ethanol extract and ethyl acetate fraction showed a platelet aggregation inhibitory effect corresponding to aspirin of 0.5 mg / mL at 0.25 mg / mL concentration. The aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibitory activity ( Platelet aggregation inhibition activity )

Platelets were mixed with a 3.2% sodium citrate solution in a ratio of 1: 9 from whole human blood, and then centrifuged at 1,100 rpm for 10 minutes to obtain PRP (platelet rich plasma) in the upper layer, which was washed with buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Then, resuspend 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 , 0.25% gelatin, pH 7.4) and then at 3,000 rpm. After centrifugation for 10 minutes and resuspended in suspending buffer, the platelet count was adjusted to 4x10 ⁹ / ml. Then, 2.5 ul collagen was added to the 1 ml suspension for 5 minutes, and platelet aggregation was measured at 37 ° C. using a whole-blood aggregometer (Chrono-log, USA).

Platelet Aggregation Inhibitory Activity of Cornus Ethanol Extract and Fractions of Cornus Fruit Sample / Control burglar
(Amplitude: ohm) inclination
(Slope) Extension time
(Lag time: seconds) Descending area
(Area under) DMSO-1 16 4 78 148.5 DMSO-2 14 2 84 164.8 Aspirin 0.5 mg / mL 5 One 184 21.7 Cornus Extract 0.25 mg / mL 4 0 84 25.5 Hexene fraction 0.25 mg / mL 11 2 63 67.3 Ethyl acetate fraction 0.25 mg / mL 6 One 105 29.7 Butanol fraction 0.25 mg / mL 10 One 62 47.4 Water residue 0.25 mg / mL 11 One 106 67.7

Example  5: Chemical Properties and Stability of Cornus Active Material of the Present Invention

The ethyl acetate fractions obtained in Example 3 were dried under reduced pressure to prepare a powder, and then dissolved in 1 ml of ethanol in 10 mg of powder, and then used for the amicon molecular sieve (Amicon 30, Millipore co.) And a centrifuge. To separate material layers of 30 kDa and above. After measuring the activity of each material layer, it was confirmed that the antithrombotic active material is less than 30 kDa. Various color reactions, thermal stability, and acid stability were confirmed for the recovered active materials. Color reaction was carried out anthrone reaction for carbohydrate qualitative detection, ninhydrin reaction for amino acid colorimetric determination, diethylene glycol reaction for polyphenol detection, flavonoid detection reaction. As a result, the active substance was identified as a polymer containing polyphenol and flavonoid components, in part in the form of carbohydrate complexes not containing amino acids. In addition, the crude purified active material did not show a decrease in activity even after 2 hours treatment at 100 ℃, 2 hours treatment at pH 2 (0.01 M HCl), 2 hours treatment in plasma showed a high stability. Considering the antithrombotic activity of the fractions of Examples 3 and 4 and the component composition of the fractions, the antithrombotic active substance of cornus milk is presumed to be a polyphenolic compound.

On the other hand, both cornus extract and its fractions were expected to show no acute toxicity because there is no hemolytic activity against human erythrocytes at all to 1.0 mg / ml [Table 6].

Hemolytic Activity of Cornus Ethanol Extracts and Fractions of Cornus Fruits sample Human erythrocyte hemolytic activity Ethanol extract 0.64 ± 0.85 Hexene fraction 0.55 ± 0.46 Ethyl acetate fraction 0.00 ± 0.69 Butanol fraction 1.20 ± 3.70 Water residue 0.71 ± 0.87

Example  6: Example  4 ethyl acetate Fraction Single oral toxicity  exam

Four-week-old rats were supplied with Sprague-Dawley rats from the Laboratory of Animal Breeding in the Safety Research Center of the Korea Research Institute of Chemical Technology at 12 ± 3 ℃ temperature, 50 ± 10% relative humidity, and 150 ~ 300 Lux. After circulating for 14 days in an animal laboratory controlled at 8 pm), 10 animals were divided and evaluated for single oral toxicity of the ethyl acetate fraction of Example 3, respectively. First, each sample was dissolved in DMSO, 10 mice were orally administered at a concentration of 1,000 mg / kg, and survival and pathological abnormality were observed for one week. After that, autopsy was performed for bleeding and serum biochemical tests of various organs. Was carried out. Only DMSO was orally administered as a control. As a result, survival rate was 100%, no pathological abnormality or long-term bleeding was observed, and serum biochemical tests of GPT and GOT were not significant. Therefore, it was judged that the use of low concentration of cornus extract would not cause additional problems.

Claims (5)

Extracting selected from fruits, seeds or leaves from which cornus seeds are removed using hot water or ethanol; Sequentially fractionating the extract obtained above with hexene and ethyl acetate; Purifying the ethyl acetate fraction obtained by using an amicon 30 molecular sieve (molecular sieve) to recover only the polysaccharides of less than 30 KD, the purified water of the extract from the cornus extract fraction.
The cornus oil extract according to claim 1, wherein the organic solvent used as the extraction solvent in the extract of the cornus oil is selected from alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, and a mixed solvent of lower alcohol and water. Method for preparing a purified product of the fractions.
A purified product of an extract from the cornus extract, which is prepared according to the method of claim 1 or 2, which has acid stability and contains a polymer polysaccharide having a molecular weight of 30 kDa or less as an active ingredient.
A pharmaceutical composition for preventing or treating thrombosis-free thrombosis, characterized in that it contains a purified product of the extract of the cornus oil extract of claim 3 as an active ingredient.
Hemolytic phenomenon-free thrombosis-improving functional health food composition, characterized in that it contains a purified product of the cornus extract fraction of claim 3.

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