KR101432220B1 - Pharmaceutical composition comprising the extract of betel nut of Areca catechu L. for prevention and control of thrombosis - Google Patents

Abstract

The present invention relates to an Acer The present invention relates to a pharmaceutical composition for preventing and treating thrombin-inhibiting thrombosis, which comprises an extract of Okamotoanum as an active ingredient. The extract of the present invention is extracted with various solvents such as water, alcohol, ethanol and methanol, Which is obtained by fractionation, does not show acute oral toxicity and chronic toxicity, is excellent in thermal stability, does not show loss of thrombin inhibitory activity even in an acidic condition of pH 2 and plasma, and the pharmaceutical composition containing it as an active ingredient has a thrombin inhibitory activity And can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. Therefore, it can be processed into various forms such as extract, powder, ring and tablet, Can be prepared.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for preventing and treating thrombin-inhibiting thrombosis,

The present invention relates to a pharmaceutical composition for preventing and treating thrombin-inhibiting thrombosis, which comprises an umbrella tree extract.

As a constituent of human body, blood has various important functions such as oxygen, nutrients, waste function and buffering function, maintaining body temperature, controlling osmotic pressure and ion balance, maintaining water constant, controlling liquid, maintaining and controlling blood pressure, have. Normal blood circulation facilitates blood circulation by complementary regulation of the blood coagulation system and thrombolysis system in the body. Among them, the mechanism of the blood coagulation system is that the platelets adhere to the blood vessel walls and coagulate to form platelet thrombus , It is reported that the blood coagulation system is activated and fibrin thrombus is formed centering on the platelet aggregation mass.

The production of fibrin clot involves multiple steps of 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 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, and thus aims to develop a thrombin direct inhibitor worldwide. In particular, as the development of society and the aging of the population, the development of thrombotic diseases, that is, cerebrovascular diseases or heart diseases associated with excessive thrombosis, is rapidly increasing, and thus it is in desperate condition to develop a safe antithrombotic agent. Until now, various anticoagulants such as heparin, coumarin, aspirin, and europine have been used for the prevention and treatment of thrombotic diseases. However, they are very expensive and have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.

On the other hand, in Korea, there is a tradition of drinking tree sap such as god roots, birch and bamboo sap as health drinks. Actually, these tree saps are effective for diuretic, constipation, gastrointestinal diseases, gout, It has been known. In particular, in the case of ganoderma sap, domestic production is the largest, and the ganoderma sap is used not only directly as a beverage but also in a variety of products for use as a soy sauce manufacturing water to be fermented using its useful minerals and various components. There are about 10 types of noodle trees in Korea, and there are known noodle trees, red noodle trees, umbrella noodle trees, Manchurian noodle trees, long noodle trees, king noodle trees, mountain noodles, tongs noodles and noodles.

It is known that umbrella gonorrhoea, which is known to have the highest efficacy among the gonorrhea trees, is a native species only in Ulleungdo, Korea, and its activity to inhibit growth of cancer cells (Qadir et al., 2007, Kor, J. Medicnal Crop Sci. 15, 405-410) , Immunological enhancement and antioxidant action (Jin et al., 2008, Kor. J. Medicnal Crop Sci., 16, 427-433), skin whitening action (Jeong et al., J. Kor. Recently, Cleomiscosin A and Cleomiscosin C have been reported which have antioxidative activity from leaves and stems (Jin et al., 2007. Arch Pharm. Res. 30, 275 -281). However, the antithrombotic activity and the inhibitory effect of the human thrombin enzyme on the thrombogenesis inhibition of the umbrella nodule extract have not been reported.

Therefore, the present inventors evaluated the activity of inhibiting thrombus formation following direct inhibition of thrombin by using human plasma and human thrombin after preparing umbrella nodosaccharide extract for the purpose of developing a functional food material exhibiting antithrombotic and blood flow improving activity. As a result, Extremely potent antithrombotic activity was observed in the umbellate larvae extract and sequential fractions.

Accordingly, an object of the present invention is to provide a thrombogenic inhibitor comprising a thrombin inhibitory active substance extracted and purified from water, an organic solvent, and the like, as an effective ingredient.

The above object of the present invention can be achieved by a method for determining the optimal extraction solvent and extraction conditions of a thrombin inhibitory substance from an umbrella nodule and purifying the active ingredient through a sequential organic solvent fraction of the extract and investigating the chemical properties and stability of the substance, Materials were orally administered to rats to achieve acute and chronic toxicity tests.

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

As can be seen from the above examples, the umbrella larva extract and composition of the present invention are extracted with various solvents such as water, alcohol, methanol, and ethanol, and the extract is obtained using sequential organic solvent fractions and amiconic molecular sieves And exhibits excellent thrombin inhibitory activity and has an effect of effectively inhibiting thrombogenesis and has 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 addition, the umbrella nodule extract of the present invention does not exhibit acute oral toxicity and chronic toxicity, is excellent in thermal stability, does not show loss of thrombin inhibitory activity even in an acidic condition of pH 2 and plasma, It can be prepared in various forms such as tablets, capsules, and tablets, and can be prepared at any time.

1 is a comparative diagram of the antithrombotic effect (thrombin time, prothrombin time, and apathy time) of the umbellate alopecia leaves leaf extract and the fractions thereof (in this case, the? - ethanol extract,? -Hexane fraction,? -Ethyl acetate fraction , -? - butanol fraction, -? - water residue).

The present invention relates to a pharmaceutical composition for preventing and treating thrombin-inhibiting thrombosis, which comprises an umbrella tree extract. More specifically, the extract is extracted from various leaves such as water, alcohol, ethanol and methanol from the leaves of Alaska pollack, followed by preparing sequential organic solvent fractions and water residues with hexane, ethyl acetate and butanol, , Which shows excellent antithrombotic activity unlike the conventional thrombus formation inhibitor without showing oral acute toxicity and chronic toxicity, and is excellent in thermal stability and acid stability, and can be processed into various forms and can be orally administered to a polymer substance having a molecular weight of 30 kDa or more .

The present invention relates to an optimal extraction solvent and an extraction condition determining step of a thrombin inhibitory substance from an umbrella nodule; A step of purifying the active ingredient through sequential organic solvent fraction of amylose hydrochloride extract and amiconic molecular sieve; Examining the chemical properties and stability of the material; And testing the acute and chronic toxicity of the substance.

The "umbrella nodogoshi extract" contained in the composition of the present invention refers to an extract obtained from various organs such as stem, bark, neck, and leaf of an umbrella noodle, and preferably refers to an extract obtained from the leaves of a mature umbrella noodle.

The umbrella gonorrhoeae containing the thrombin inhibitory substance of the present invention is a kind of gonorrhoi that is native to Ulleungdo in Korea and is a Korean native species designated as a subject to be approved for export. In Korea, the harvesting of umbrella gonorrhea has been approved and commercialized in 2002, and in 2009 about 116 tons were collected. Currently, in order to protect the limited umbrella goggles, [the umbrella gogolabusu gauging management guideline] has been collected by people who have been educated by the people who have taken the sap to protect the umbrella acorns. However, It causes problems such as microbial contamination of collected juice, limit of collection of hygiene of sap, and seasonal limit of harvesting and difficulty of mass production. However, leaves and stems of umbrella gonorrhoe can grow and grow very quickly after cutting, thus producing and recovering leaves and stalks continuously. The method of preparing leaves and stems, not wood perforations, can solve various problems mentioned above The physiologically active substance of the umbrella can be recovered.

In the present invention, the umbrella gonads that exhibit inhibitory activity against human thrombin can be extracted from various parts such as leaves or stems using ethanol. Thrombin time, prothrombin time, and aPTT were measured at 5 mg / mL of umbrella ethanol extract. The results were as follows: Thrombin time was prolonged more than 15 times, Prothrombin time and aptitude time (aPTT). Considering that thrombin time, prothrombin time and aPTT were extended by 2.49, 1.25 and 1.44 times, respectively, at the concentration of purified aspirin 1.5 mg / ml, a very strong antithrombotic effect . These results indicate that umbrella nodule extract can effectively replace anticoagulants such as conventional aspirin.

In the present invention, "umbrella hornet extract" can be produced using various extraction solvents. Examples of the solvent include water (cold water, hot water), alcohol, water, or a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, "Umbrella noodle extract" can be produced. Preferably extracts using ethanol or alcohol, shows the maximum yield of active material. The umbrella noodle extract of the present invention can be made into a powder by a conventional powdering process such as gamma-distillation and freeze-drying, or spray-drying.

The umbrella noodle extract prepared using the above-mentioned extraction solvent such as alcohol, alcohol, water, methanol, ethanol and the like can be successively fractionated by using hexane, ethyl acetate and butanol, and then separated into water fractions after suspension in water. In this case The active substance is strongly identified in the hexane fraction and the ethyl acetate fraction and relatively weakly in the butanol fraction and water residue. The ethyl acetate fraction exhibiting the most potent activity can be prepared as a powder through a conventional powdering process such as carcass distillation, lyophilization, or spray drying. The fraction is dissolved in ethanol, and then microcon YM-30 (Millipore) And can be classified into substances having a molecular weight of 30,000 or less and 30,000 or more. Antithrombotic activity is confirmed only in a substance layer having a molecular weight of 30,000 or more. Therefore, the modulated substance is negative for the ninhydrin reaction and positive for the anthrone reaction, and is a polymer polysaccharide having a molecular weight of 30 kDa or more including a phenolic substance. They maintain their thermal stability and activity even at pH in the human body at pH 2.

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

Example  One: Umbrella bulletin board  Human using ethanol from leaves and stems Thrombin  Extraction of inhibitory substances

The leaves and stems of umbrella gonorrhea collected from Ulleungdo in July 2010 and July 2011 were extracted with ethanol and then fractionated with solvents such as hexane, ethyl acetate, butanol and water. The extracts and fractions were concentrated under reduced pressure to prepare powders, kept at low temperature until use, dissolved in dimethylsulfoxide (DMSO), diluted to an appropriate concentration, and human thrombin inhibitory activity was measured using human plasma.

The inhibitory activity of thrombin was evaluated according to the previously reported method (Sohn et < RTI ID = 0.0 > al ., 2004. Kor . J. Pharmacogn 35, 52-61; Kwon et al ., 2004. J. Life Science , 14: 509-513; (2010. J. Life Science , 20. 922-928), thrombin time, prothrombin time and aPTT were measured. Plasma was prepared from the whole blood of the applicant, and immediately after collection, the plasma was separated by centrifugation at 5,000 g for 5 minutes at 4 ° C and stored frozen (fresh frozen plasma) and thawed at room temperature if necessary. The thrombin time, prothrombin time and apathy measurement 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. As a control, aspirin (Sigma Co., USA) and heparin (Sigma Co., USA) were 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 )

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 clotting time of 18.1 sec.

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. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 55.1 seconds. The results of aPTT were expressed as the average of three replicate experiments. The value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control was multiplied by 100 and expressed in%.

The ethanol extracts of umbrella stalks and leaves showed 1.33 times and 15 times more thrombin time at the concentration of 5 mg / mL, respectively. Aspirin showed an increased thrombin time of 2.49 times at the concentration of 1.5 mg / mL, Of the leaves showed particularly high antithrombotic activity.

Example  2: Umbrella bulletin board  Leaf ethanol extract preparation and extract concentration of thrombin time, Prothrombin  Time and Aptity  Measure

The umbrella leaf leaf extract of the present invention was prepared as follows.

First, the leaves of mature umbrella gonorrhea were washed with purified water and dried. Then, 1 kg of umbrella leaf nodule leaf was added to 3 L of ethanol and extracted for 15 hours at room temperature. Then, 3 L of ethanol was added again and extracted secondly under the same conditions. The extract was filtered with a filter paper (Adventec.No.2), and the filtrate was concentrated by 3 L / hour with a rotary evaporator (Rotavapor R-220, BUCHI) and vacuum dried at 50 ° C. under a reduced pressure of 650 mmHg for 3 days To obtain 118.6 g of an umbrella nodule extract. The extracts were dissolved in DMSO at a suitable concentration, and the measured thrombin time, prothrombin time, and apitity results are shown in Table 1. As an anticoagulant, aspirin was used as a control.

As shown in Table 1, the ethanol extract of umbrella noodle leaf showed a thrombin time of 39.3 seconds at the concentration of 1500 ㎍ / mL and a thrombin time of 99.5 seconds at the concentration of 1,500 ㎍ / mL of the aspirin, Ethanol extracts showed stronger thrombin inhibitory activity. However, at the concentration of 5,000 mg / mL, thrombin time was prolonged more than 15 times. On the other hand, in the case of prothrombin time and apathy time related to exogenous and endogenous thrombosis, the ethanol extract of umbrella gonorrhoeae showed a stronger antithrombotic effect than the purified aspirin.

Example  3: Umbrella bulletin board  leaf Antithrombotic  Selection of extraction solvent and examination of extraction efficiency

For the efficient extraction of the antithrombotic active ingredient of umbrella leaf, 100 g of leaf was extracted with various solvents such as water, alcohol, ethanol and methanol, and water was extracted at 60 ° C. The extraction time was 20 hours for organic solvent and 2 hours for water extraction at 60 ℃. [Table 2] shows the extraction efficiency of each solvent and the antithrombotic activity of the extract by measuring thrombin time. As a result, it was confirmed that extraction using ethanol or alcohol was efficient, and ethanol extraction was most effective in water percentage.

Example  4: Umbrella bulletin board   Sequential Organic Solvent Active Fraction of Leaf Ethanol Extract and Fraction  Active measurement

100 g of the ethanol extract obtained in Example 2 was suspended in distilled water, and sequential organic solvent fractions were carried out in the order of hexane, ethyl acetate and butanol. As a result, 26.4 g of the hexane fraction, 45.8 g of the ethyl acetate fraction and 15.2 g of the butanol fraction were recovered, and 16.8 g of the water residue was recovered. The antithrombotic activity of each of the fractions was shown in the ethyl acetate fraction, which occupies the most amount of the most activity, as shown in the following [Table 3], [Table 4] and [Table 5]

Example  5: Umbrella bulletin board   Chemical properties and stability of active materials

The ethyl acetate fraction obtained in Example 4 was subjected to high pressure drying to prepare a powder. 10 mg of the powder was dissolved in 1 ml of ethanol, and the mixture was subjected to centrifugation using Amicon 30 (Millipore co.) And Amicon The material layer was separated by 30 kDa or less. As a result of measuring the activity of each material layer, it was confirmed that the antithrombotic active substance was a substance of 30 kDa or more (Table 6).
Thrombin Inhibitory Effect of Molecular Size-Adjusted Compounds on the Ethyl Acetate Fraction of Ethanol Extract of Umbrella Leaf Leaf division Umbrella almond tree 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) > 480 38.4 ± 3.2 79.9 ± 5.7 32.1 ± 1.5 Prothrombin
Time (seconds) > 270 22.8 ± 3.2 22.6 ± 1.4 18.1 ± 1.1 APT (sec) > 800 65.1 ± 10.7 55.0 ± 8.1 55.1 ± 5.5
Various coloring reactions, thermal stability and acid stability of the recovered active materials were confirmed. The chromogenic reactions were anthrone reaction for qualitative detection of carbohydrate, ninhydrin reaction for amino acid colorimetric determination, diethylene glycol reaction for detection of polyphenol, and flavonoid detection reaction. As a result, the active substance was identified as a polymer containing polyphenol and flavonoid components, partially in the form of a carbohydrate complex free of amino acids (Table 7). In addition, the regulated active substance showed high stability because it was treated at 100 ° C for 2 hours, treated for 2 hours at pH 2 (0.01 M HCl), and decreased for 2 hours at plasma.
analysis Anthron reaction Ninhydrin
reaction Diethylene glycol
reaction Flavonoid
Detection reaction Ethyl acetate
Fraction
30KDa or more
Active substance + - + + glucose + - - - Glutamic
Acid - + - - Routine - - + +

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

Four-week-old rats were fed with Sprague-Dawley rats in an experimental animal breeding room of the Safety Research Center of the Korea Research Institute of Chemical Technology and were given 12-hour intervals (8 am to 8 pm) with a temperature of 23 ± 3 ℃, a relative humidity of 50 ± 10% Day to 8:00 pm) for 14 days and then divided into 10 dogs to evaluate single oral toxicity of the ethyl acetate fraction of Example 4, 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, the survival rate was 100%. No sign of pathologic abnormality or long - term bleeding was observed. Serum biochemical tests of GPT and GOT were not found to be significant compared to the control. Therefore, it was concluded that the use of low concentration of umbrella nodule extract did not cause additional problems.

INDUSTRIAL APPLICABILITY As described above, the present invention is an extremely useful invention in the biopharmaceutical industry because it has an excellent effect of obtaining a pharmaceutical composition for prevention and treatment of thrombosis from the leaves of A. umbellata.

Claims (5)

A pharmaceutical composition for preventing or treating thrombosis comprising an ethyl acetate fraction obtained from an ethanol extract of an umbrella tree leaf as an active ingredient. 2. The pharmaceutical composition according to claim 1, wherein the ethyl acetate fraction is a substance layer having a molecular weight of 30 kDa or more. delete delete delete

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