KR101378527B1 - Composition comprising the extract of Cedrela sonensis A. Juss for prevention or control of thrombosis - Google Patents

Abstract

The present invention is the oak tree ( Cedrela sinensis A. The present invention relates to a pharmaceutical composition for preventing and treating thrombin-inhibited thrombosis containing Juss) as an active ingredient, and the extract of the oak tree of the present invention is directed 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 formation. Processed in various forms such as extracts, powders, pills, tablets, etc. There are excellent effects that can be prepared in the available formulations.

Description

Composition comprising the extract of Cedrela sonensis A. Juss for prevention or control of thrombosis}

The present invention relates to a composition for preventing or treating thrombin-inhibited thrombosis containing oak extract as an active ingredient, and more specifically, to a pharmaceutical composition and thrombin for preventing or treating thrombin-inhibited thrombosis by containing oak extract as an active ingredient. It relates to a functional health food composition for improving 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 clot involves multiple steps of a number of factors, activating thrombin directly involved in fibrin clotting, ultimately producing fibrin monomers from fibrinogen, fibrin monomers being polymerized by calcium to bind platelets and endothelial cells And forms a cross-linked fibrin polymer by Factor XIII, producing permanent thrombosis. 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.

Meanwhile the oak tree ( Cedrela sinensis A. Juss.) Is a taxonomy belonging to the genus Mulberry, and is used for food in Korea and China. Due to its unique fragrance, it has strong characteristics against pests and contains various nutrients, and has been recently studied as a health functional ingredient. In addition, the leaves and berries have been used as a herbal medicine with excellent pharmacological action, and recently, the activity of antioxidant, anti-inflammatory, immune enhancing, etc. has been studied. The main studies to date are cut or powdered oak leaves, extracted with hot water, ethanol, and methanol, and the extracts were examined for immune enhancing activity, antimicrobial activity, anti-inflammatory and analgesic activity. In addition, the flavonoid content of the oak leaves in summer and the flavonoid content of the quarcetin (quercitrin), Afzelin (afzelin) according to the season has been reported.

Patent documents related to oak wood include Korean Patent Publication No. 1020060028826 (published: 2006.04.04, patent application No. 10-2004-0077685, filed: 2004.09.30) 'Method of extract and powder of oak and leather trees And a food composition added thereto and Korean Patent Publication No. 1020080062794 (published: 2008.07.03, patent application No. 10-2006-0138911, filing date: December 29, 2006) 'Food and health function using the oak leaf and its extract Food 'is known, and in fact, various studies on the activity of oak tree have been made, but the antithrombotic activity of oak tree extract and the direct inhibition of human thrombin enzyme have not been reported.

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

1. Republic of Korea Patent Publication No. 1020060028826 (Published: 2006.04.04) 2. Republic of Korea Patent Publication No. 1020080062794 (Published: 2008.07.03)

Accordingly, an object of the present invention is to provide a thrombin inhibitor by separating and purifying a thrombin inhibitory active material from water from an oak tree with water or an organic solvent, and providing a thrombus generating inhibitor using the same as an active ingredient.

The object of the present invention is to determine the optimum extraction solvent and extraction conditions of the thrombin inhibitory active material from the oak tree, to purify the polymer active ingredient of 30 kDa or more through the sequential organic solvent fraction and amicon molecular sieve of the extract, After examining the properties and stability, 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 demonstrated in the present Example, the oak extract and composition of the present invention can be extracted with various solvents such as water, alcohol, methanol, ethanol, and the extract can be obtained 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 oak 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, Processed in a variety of forms, such as tablets, because it is an excellent effect that can be prepared in a form that can be taken at all times is a very useful invention in the food industry and pharmaceutical industry.

1 is a graph comparing the antithrombotic effects (thrombin time, prothrombin time, and apiity time) of the oak extract and its fraction, 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 the oak extract. More specifically, after extracting from edible oak leaves and stems with various solvents such as water, spirits, ethanol, methanol, etc., sequential organic solvent fractions and water residues are prepared with hexane, ethyl acetate, butanol, and among them. Unlike the conventional thrombogenic inhibitors, the ethyl acetate fraction does not show oral acute toxicity and chronic toxicity, and shows excellent antithrombotic activity, and has excellent thermal stability and acid stability, which can be processed into various forms and have a molecular weight of 30 kDa or more. It relates to a polymer material.

The present invention is the step of determining the optimum extraction solvent and extraction conditions of thrombin inhibitory active material from the oak wood; Purification of the active ingredient through the sequential organic solvent fraction of the oak extract and Amicon molecular sieve; Examining the chemical properties and stability of the material; And testing the acute and chronic toxicity of the substance.

"Oak tree extract" included in the composition of the present invention means that it is obtained by extracting from various organs, such as leaves, stems, bark, neck of the oak tree, preferably edible rather than edible leaves, stems, rough to edible Means extracts from mature oak leaves and stems showing texture.

The tender shoots of the oleander tree containing the thrombin active inhibitor of the present invention are registered as a food raw material in the domestic food industry, as well as harvesting sprouts in the new spring in the spring, lightly boiled in boiling water, or processed and pickled and distributed do. However, in July-August-September, where the antithrombotic activity becomes stronger, mature leaves and stems are poorly eaten and are almost discarded. To date, the Korean Food and Drug Administration has not conducted food ingredient analysis, but as a result of analyzing the general ingredients of young bamboo shoots, moisture content 72.1%, protein content 9.4%, crude fat 1.95%, carbohydrate 12.17%, dietary fiber 2.39%, ash 4.6 Contains 83% kcal / 100g and is known as a low calorie health food.

Although it is designated as a target of idle land reforestation support designated by the Korea Forest Service, it is currently planted nationwide, but it is a problem because it is known as a host plant of Chinese cicada that causes great damage to fruit farming. Meanwhile, young shoots are sold as expensive health foods, but they can be harvested for a limited time, and most mature leaves and stems are almost discarded. Therefore, the present invention can manufacture a pharmaceutical composition for preventing and treating thrombin-inhibited thrombosis from oak leaves including oak leaves and stems and sprouts of low commercial value due to the existing discarded or rough texture.

In the present invention, the oak tree exhibiting inhibitory activity against human thrombin can be extracted using ethanol from various sites such as leaves, stems, necks, and bark. The thrombin time, prothrombin time and apti-time were measured at 5 mg / mL of ethanol extract of oak tree, and they were 2.21 times, 1.44 times and 1,96 times longer than the non-added households, respectively. The thrombin time, the prothrombin time and the activity time. This resulted in an excellent antithrombotic effect comparable to that of aspirin, considering that the thrombin time, prothrombin time, and apititime were extended 2.49, 1.25, and 1.44 times, respectively, at a concentration of 1.5 mg / ml of purified aspirin. In particular, when ethanol extract of oak tree was prepared with sequential fractions of hexene, ethyl acetate, butanol, and water, hexene and ethyl acetate fractions showed stronger thrombin inhibitory activity than extracts, especially ethyl acetate fractions at 5 mg / ml. Thrombin time, prothrombin time and activity time, respectively, were extended 15 times or more than the spheres. Given the continued use of aspirin for gastrointestinal upset and limited user use, these results suggest the possibility of oak extract and its ethylacetate fraction to replace antithrombotic agents such as aspirin.

On the other hand, as a result of evaluating platelet aggregation inhibitory activity in human platelet concentrates, strong platelet aggregation inhibitory activity was obtained comparable to that of aspirin 0.5 mg / mL at 0.25 mg / mL of oak butanol fraction, and hexene and ethyl acetate fractions. Good platelet aggregation inhibitory activity was also confirmed in. Therefore, the oak extract is expected to be able to efficiently replace anticoagulants such as aspirin, and the oak extract is expected to help blood circulation by inhibiting blood clot production.

In the present invention, "oak-tree extract" can be prepared using various extraction solvents. For example, using 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, "Oak Tree Extract" can be prepared. Preferably extracts using ethanol or alcohol, shows the maximum yield of active material. The oak extract of the present invention may be prepared as a powder through a conventional powdering process such as distillation under reduced pressure and lyophilization or spray drying.

The oak extract prepared using the above extraction solvents such as alcohol, water, methanol, ethanol, and the like can be divided into the remaining water fractions after suspending them in water, sequentially using hexene, ethyl acetate and butanol, and in this case, Thrombin inhibitors are strongly identified in ethyl acetate fractions and relatively weakly detected in hexene, butanol fractions and water residues. Ethyl acetate fraction showing strong activity can be prepared as a powder through a conventional powdering process such as distillation under reduced pressure, lyophilization, or spray drying, which is dissolved in ethanol and then used as a microcon (microcon YM-30, Millipore). Molecular weight 30,000 or less and can be divided into more than 30,000 substances, anti-thrombotic activity is confirmed only in the molecular weight 30,000 or more layer. Therefore, the crude (crude) purified material is negative for the ninhydrin reaction, positive for the antron reaction, it can be seen that the polymer polysaccharide having a molecular weight of 30 kDa or more containing a phenolic material. They maintain activity at thermal stability and pH within 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 extract of the oak tree 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.

The oak 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 oak extract can 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 oak extract in the food or beverage may be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is based on 100 ml 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 oak leaves and stems Thrombin  Extraction of Inhibitors

The shoots, middle leaves, mature leaves and stems of various dates from April to September 2010 in Uiseong, Gyeongbuk were extracted with ethanol and distilled water. First, in the case of solvent extraction using spring leaves and stems appearing in April, 5 times ethanol or distilled water was added to each sample, and then extracted twice at room temperature for 8 hours. The extract was concentrated under reduced pressure to give a powder, which was stored at low temperature until use. On the other hand, the total flavonoid and total polyphenol content, total sugar and reducing sugar content of the prepared oak 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 again, and reacting at 37 ° C for 1 hour and absorbance at 420 nm. Was 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 and 100 μl of saturated Na ₂ CO ₃ solution were left at room temperature for 1 hour, and then absorbance was measured at 725 nm. Tannic acid was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was quantified by phenol-sulfuric acid method.

Table 1 and Table 2 show the extraction efficiency and useful component analysis results of ethanol extraction and water extraction of leaves, stems, and leaves and stems (in the case of edible shoots). The highest yield was 4.46-9.23% in the leaves, and the lowest yield was 2.49-2.92% in the stems, and the leaf and stem mixtures, which were edible in new shoots, showed the highest yields in the water extraction. 1.5 times higher. Total flavonoid and total polyphenol contents were 1.3-2.5 times higher in ethanol extract than water extract.

Analysis of Ethanol Extraction Efficiency and Extract Components in Various Parts of Oak Tree sample Extraction rate (%) Content (mg / g) Total polyphenol Total flavonoid  Total Reducing sugar leaf 4.46 72.19 ± 5.93 120.01 ± 1.04 54.99 ± 0.66 29.13 ± 0.87 stem 2.49 1.84 ± 0.52 47.68 ± 0.24 151.63 ± 1.73 134.34 ± 2.85 Leaf, stem mixture 2.89 31.46 ± 2.27 87.36 ± 0.4 93.41 ± 2.32 67.26 ± 5.39

Analysis of Water Extraction Efficiency and Extract Components in Various Parts of Oak Tree sample Extraction rate (%) Content (mg / g) Total polyphenol Total flavonoid  Total Reducing sugar leaf 9.23 8.33 ± 0.09 48.78 ± 2.28
29.34 ± 0.93 5.71 ± 0.12 stem 2.92 1.90 ± 0.66 36.14 ± 0.44 145.9 ± 1.63 108.48 ± 9.05 Leaf, stem mixture 4.42 7.65 ± 0.84 37.76 ± 0.64 97.26 ± 2.99 91.73 ± 5.95

Example  2: of oak leaf and stem extract Anti-thrombosis  Activity evaluation

The extracts of oak leaves, stems, and mixed portions of edible leaves (edible) were dissolved in DMSO (dimethylsulfoxide) and diluted to appropriate concentrations to measure 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%.

The results of antithrombotic activity at concentrations of 5 mg / mL of oak leaves, stems, and leaf and stem mixture (edible) extracts are shown in Tables 3 and 4. For edible shoots (leaf and stem mixture), ethanol extract (5 mg / mL) showed almost the same effect as aspirin (1.5 mg / mL). However, the water extract was less effective than aspirin. On the other hand, anti-thrombotic activity was highest in the leaves of ethanol extract, and the highest in the stem of water extract.

Comparison of Antithrombotic Activity of Oak Ethanol Extract (5 mg / mL Leaf, Stem and Leaf and Stem Mixture) and Aspirin (1.5 mg / mL) part Thrombin time (seconds) Prothrombin Time (sec) Affinity Time (sec) Sesame extract aspirin Sesame extract aspirin Sesame extract aspirin leaf 111.4
79.9 21.3
22.8 68.5
107.9 stem 55.5 28.2 120.9 Leaf, stem mixture 70.9 26.7 119.0

Comparison of Antithrombotic Activity of Water Extracts from Oak Tree (leaves, Stem and Leaf and Stem Mixtures Concentrated at 5 mg / mL) and Aspirin (1.5 mg / mL) part Thrombin time (seconds) Prothrombin Time (sec) Affinity Time (sec) Sesame extract aspirin Sesame extract aspirin Sesame extract aspirin leaf 44.9
79.9 20.9
22.8 65.1
107.9 stem 47.5 28.2 101.5 Leaf, stem mixture 31.3 23.5 84.3

Example  3: Organic Solvent of Oak Ethanol Extract Fraction  Ingredient survey

After extracting the edible part of the oak tree (leaf and stem mixture) with ethanol, the extract was partitioned into hexene, ethyl acetate, butanol, and finally water residue was obtained. The yield and component analysis of each fraction are shown in Table 5. 58.71% of the ethanol extracts were water residues and 3.17% of ethyl acetate fractions. Ethyl acetate fractions, although lower in yield, showed higher total polyphenol and flavonoid contents than other fractions.

Fractional Efficiency and Component Analysis of Oak Ethanol Extracts Fraction Fraction rate (%) Total polyphenol Total flavonoid  Total Reducing sugar Hexene fraction 18.01 44.49 ± 0.07 88.23 ± 2.62 60.76 ± 0.78 39.94 ± 5.79 Ethyl acetate fraction 3.17 478.33 ± 1.11 118.96 ± 3.14 53.80 ± 1.13 21.84 ± 0.68 Butanol fraction 13.60 77.58 ± 2.51 43.80 ± 1.57 102.56 ± 1.46 35.96 ± 3.35 Water residue 58.71 27.76 ± 1.77 0.48 ± 0.52 203.74 ± 4.31 129.34 ± 8.93

Example  4: oak leaf and stem ethanol extract and Fraction Anti-thrombosis  activation

The antithrombotic activity of the oak extract and organic solvent fractions of Example 1 and Example 3 was investigated. The results of measuring thrombin time, prothrombin time, and affinity time are shown in FIG. 1, and the results of evaluating platelet aggregation inhibitory activity are shown in Table 6 below.

As shown in FIG. 1, the ethyl acetate fraction extended thrombin time, prothrombin time, and epitaxial time by more than 15 times at 5 mg / mL or higher concentration, and thrombin time, prothrombin time, and epitaxial time at 2.5 mg / mL concentration. Were extended 1.99 times, 1.51 times and 1.88 times, respectively. This effect is a much stronger antithrombotic effect than the original ethanol extract and is similar to aspirin activity at a concentration of 1.5 mg / mL. The hexene fraction showed 1.5- and 1.3-fold increase in thrombin time and prothrombin time than ethanol extract, but decreased in apity time. Butanol fractions and water residues were less active than ethanol extracts (see Figure 1).

As a result of evaluating human platelet aggregation inhibitory activity of the oak extract and the organic solvent fraction, it was confirmed that the butanol fraction (0.25 mg / ml) very good activity. The ethyl acetate fraction, which had the strongest thrombin inhibitory activity, also showed platelet aggregation inhibitory activity following the butanol fraction, and the hexene fraction also showed good platelet aggregation inhibitory effect. 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 Extracts and Fractions from Oak Tree Sample / Control burglar
(Amplitude: ohm) inclination
(Slope) Extension time
(Lag time: seconds) Descending area
(Area under) DMSO 16 2 72 118.2 Aspirin 0.5 mg / ml 7 One 81 46.3 Aspirin 0.25 mg / ml 8 One 98 53.1 Oak Extract 0.25 mg / ml 13 One 64 25.5 Hexene fraction 0.25 mg / ml 10 One 75 67.3 Ethyl acetate fraction 0.25 mg / ml 10 One 79 29.7 Butanol fraction 0.25 mg / ml 8 One 85 47.4 Water residue 0.25 mg / ml 17 2 58 67.7

Example  5: Chemical properties and stability of the oak tree active material of the present invention

After drying the ethyl acetate fraction obtained in Example 3 under reduced pressure, 1 mL of ethanol was added to 10 mg of the powder to dissolve it, and then using 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 more than 30 kDa (Table 7).
Thrombin Inhibitory Effect of Modified Compounds of Ethyl Acetate Fractions from Oak Trees by Molecular Size division Oak Ethyl Acetate Fraction aspirin
(1.5 mg / ml) menstruum
Control
(DMSO) 30 KDa or more
(1.5 mg / ml) 30 KDa or less
(1.5 mg / ml) Thrombin Time (sec) 73.8 ± 6.4 34.5 ± 2.6 80.5 ± 7.2 32.1 ± 1.5
Meanwhile, various color reactions, thermal stability, and acid stability of the recovered active materials were confirmed. The chromogenic reactions were anthrone reaction for carbohydrate qualitative detection, ninhydrin reaction for amino acid colorimetric determination, diethylene glycol reaction for polyphenol detection, and 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 (Table 8).
analysis Anthron reaction Ninhydrin
reaction Diethylene glycol
reaction Flavonoids
Detection reaction Ethyl acetate
Fraction
30KDa or more
Active substance + - + + glucose + - - - Glutamic
Acid - + - - Routine - - + +
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 composition of the fractions, the active substance of 30 KDa or more of the ethyl acetate fraction of jujube is assumed to be a polyphenolic compound.

Meanwhile, both the oak extract and its fractions were expected to show no acute toxicity due to no hemolytic activity against human erythrocytes up to 1.0 mg / mL (see Table 9).

Human Erythrocyte Hemolytic Activity of Ethanol Extracts and Fractions of Sesame 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, and 10 mice were orally administered at a concentration of 1,000 mg / kg. After 1 week of survival and pathological abnormalities were observed, and then autopsied and bleeding and serum biochemical tests Respectively. 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 oak extract would not cause additional problems.

Claims (5)

Extracting oak leaves and stems using an organic solvent; Sequential organic solvent fractionation of hexene and ethyl acetate; The ethyl acetate fraction is further characterized in that the purification step by centrifugation with Amicon molecular sieve (Amicon 30)
A method for preparing an ethyl acetate fractional tablet having no hemolytic activity and thrombin inhibitory activity. The method of claim 1,
Characterized in that the organic solvent used as the extraction solvent in the oak extract step is selected from spirits, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms, and a mixed solvent of lower alcohols and water.
A method for preparing an ethyl acetate fractional tablet having no hemolytic activity and thrombin inhibitory activity. A polymerized polysaccharide fractional tablet having a molecular weight of 30 kDa or more prepared according to the method of claim 1 or 2, having thermal stability and acid stability, no hemolytic activity, and thrombin inhibitory activity. A pharmaceutical composition for preventing or treating thrombosis, comprising an ethyl acetate fractional tablet having thrombin inhibitory activity of the oak tree according to claim 3 as an active ingredient. A thrombin improving functional health food composition comprising an ethyl acetate fractional tablet having thrombin inhibitory activity of claim 3 as an active ingredient.

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