KR20190085730A - Pharmaceutical composition comprising the extracts from stem of actinidia arguta as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to an antithrombotic composition comprising an extract from the stem of Actinidia arguta as an active ingredient, and more specifically, to a pharmaceutical composition and a functional health food composition, comprising an extract as an active ingredient, the extract produced by extracting washed and sorted stems of Actinidia arguta with hot water, for preventing or treating/alleviating thrombosis through inhibiting blood coagulation. As the active ingredient of the pharmaceutical composition and the functional health food composition for preventing or treating thrombosis of the present invention, the extract from the stem of Actinidia arguta, as proven through the embodiments of the present specification, exhibits potent antithrombotic activity due to inhibition on thrombus formation-associated enzymes and inhibition on blood coagulation factors, without showing no hemolytic activity to human red blood cells; shows excellent thermal stability; and even in acidic condition at pH 2 and serum, does not show a decrease in the inhibitory effect on thrombus formation-associated enzymes and blood coagulation factors, and thus is promising to be used for preventing and treating thrombosis, such as ischemic stroke and hemorrhagic stroke, through promoting blood circulation. In addition, the active ingredient is advantageous in that it can be processed in various forms including a liquid extract, powder, a pill, a tablet, etc., so as to be formulated in a form that can be conveniently taken anytime, and thus is an extremely useful invention in the drug and food industry.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the prevention or treatment of thrombosis, which contains, as an active ingredient, }

The present invention is based on the finding that the stem of Actinidia The present invention relates to an anti-thrombogenic composition containing an extract of arguta as an active ingredient. More particularly, the present invention relates to an anti-thrombotic composition comprising an extract obtained by hot-water extraction of a water- A pharmaceutical composition and a health functional food.

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

On the other hand, the production of fibrin thrombus is activated by thrombin involved in fibrin clotting through several steps of many blood coagulation factors to finally produce fibrin monomers from fibrinogen. Fibrin monomers are polymerized by calcium, Cells to form a cross-linked fibrin polymer by factor XIII and produce a permanent thrombus. In addition, thrombin plays a pivotal role in thrombus formation by activating platelet, V factor, and Factor VII to promote blood coagulation. Therefore, the activity inhibitor of thrombin can be used as a prophylactic and therapeutic agent very useful for various thrombotic diseases caused by excessive blood coagulation. On the other hand, activation of prothrombin after sequential activation of factor XII, factor XI, factor XII, factor IX and factor X is finally known to activate thrombin in the endogenous thrombosis pathway, and the specific inhibition of blood clotting factor In the extrinsic thrombosis pathway, thrombogenesis is known to be caused by the activation of factor II (prothrombin), factor V, factor VII, and factor X. Up to now, various anticoagulants such as heparin, coumarin, aspirin, and europine have been used for the prevention and treatment of thrombotic diseases. However, these anticoagulants and thrombolytic agents are not only very expensive but also have hemorrhagic side effects, The use thereof is limited due to the reaction and the like.

On the other hand, Actinidia arguta is a deciduous tree which grows in the mountain of each place of our country, as the warm autumn leaves and water of Actinaidiaceae. The growth environment grows well in mountain forests and half-shaded areas of hiking trails. Its height is about 2 ~ 5m. Leaves are wide ovate and oval, with sharp and sharp sawtooth on edge. There are five varieties known to be the quail, and the domestic variety is Hayward, which is large in fruit size, excellent in storage, and superior in flavor and aroma than other varieties. In addition to the stamina, there are native species such as mackerel, ruddrae, and island dwarf.

The fruit of kiwifruit is attracted attention as health food by the harmony of sweetness and sour taste, and kiwifruit ( Actinidia chinensis ), which is similar in both morphology and taste to both. It is also known as a herb that represents spring together with a foxtail tree. On the other hand, it is called "Mifudo" or "Mifuri" in the oriental medicine, and it is used for treatment of diuretic action, vomiting prevention and indigestion which stops fever and stops thirst. In addition, the stamina tree is called "Mifu" in the oriental medicine and is used for the prevention and treatment of insomnia, esophageal cancer, breast cancer, bronchitis and arthritis.

Studies have shown that the study of kiwifruit has been focused on cultivation methods and prevention of pest and disease (Lee, 2002, Research in Plant Disease, 22: 168-177) (Korean Journal of Food Science and Technology 14: 100-104), and fermentation of wine (Korean Journal of Food Science and Technology, 14: 100-104) (Gang, Sang-Dong et al., 2011. Journal of Life Science 21: 509-514). A study on the useful physiological activity of kiwifruit showed that the antioxidant activity of fruit (Kim, Na et al., Korea Food Research Journal, No. 22: 408-420), the neurotoxic protective effect (Kim JH, 171), antimicrobial and lung cancer cell proliferation inhibitory effects (YongSeo et al., 2009. Journal of the East Asian Society of Dietary Life 19: 202-209), nitrite scavenging activity (Park, Yongseo et al., 2008 Korean Journal of Horticultural Science & Technology 26: 495 , And PC-12 neuronal cell protection effects (Lee et al., 2015, Korean journal of horticultural science & technology 33: 259-267). Mihu et al. 7 cells on the inflammatory response "(Kim, Young-joon, 2015. Master's thesis, Dong-A University). Therefore, the strong antithrombotic activity due to the inhibition of thrombogenesis-related enzymes and blood coagulation factors such as Mifu has not been known to date.

On the other hand, patents related to kiwifruit fruit and Mifu which have been known so far include Korea Patent No. 10-0536495 [food additive, animal feed additive or cosmetic composition containing extracts having anti-allergic and anti-inflammatory activity] Patent No. 10-1368293 discloses an anti-inflammatory agent composition and a skin lightening agent composition using a fermented fruit of kiwifruit, and a skin lightening agent composition using a leaf extract of a zinnia tree in Patent No. 10-1181998, [Patent No. 10-1246093 discloses a tannin-enriched high-quality quince wine and its preparation method], and in particular, in relation to Mifu and the like, Japanese Patent Registration No. 10-1342497 discloses an extract of Bombyx mori L. having alpha-glucosidase inhibitory activity and a composition for lowering blood glucose containing the same, which is disclosed in Japanese Patent No. 10-1382412 10-1196486 discloses a herbal composition for treating allergic diseases and Patent No. 10-1663971 discloses a composition for promoting the differentiation of human mesenchymal stem cells Composition]. Therefore, up to now, no scientific research or patent literature has been known about the potent thrombin generation-related enzymes such as Mifu and the antithrombotic effect by the inhibition of blood coagulation factors.

KR 10-1342497 B KR 10-1382412 B

 Effect of Mifu extract on the inflammatory response of LPS-induced RAW264.7 cells, Young-Joon Kim, 2015, Master's thesis

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the problems of the prior art as described above, and an object of the present invention is to provide an antithrombotic composition containing a hot- .

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which comprises a stem of Actinidia arguta hot-water extract as an active ingredient.

The present invention also relates to the use of the stem of Actinidia arguta ) hot-water extract as an effective ingredient.

The present invention also relates to the use of the stem of Actinidia The present invention provides a health functional food for preventing or ameliorating thrombosis which contains hot water extract as an active ingredient.

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the extract of Mifu etc. as an active ingredient of the health functional food can be used for the prevention and treatment of thrombosis such as thrombogenesis-related enzyme inhibition and strong inhibition of blood coagulation factors Exhibits antithrombotic activity, exhibits no hemolytic activity against human erythrocytes, exhibits excellent thermal stability, and does not exhibit the effect of inhibiting thrombogenesis-related enzymes and blood coagulation factors in acidic conditions of pH 2 and plasma, And is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through the improvement of blood pressure and blood pressure. The active ingredient can be processed into various forms such as extract, powder, Can be very useful in the pharmaceutical and food industries It is.

 FIG. 1 shows human platelet aggregation inhibitory activity of hot-water extracts and ethanol extracts of the fruit of kiwifruit and Mifu used in the examples of the present invention. Herein, 1: solvent control (DMSO), 2: aspirin (0.25 mg / ml), 3: aspirin (0.125 mg / ml), 4: hot water extract (0.25 mg / ml), 6: hot water extract (0.25 mg / ml) such as Mifu, and 7: Mifu ethanol extract (0.25 mg / ml).

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic effect of the extract of the present invention, the inventors of the present invention prepared an extract of the fruit of Q. pertussis and Mifu etc. prepared by a certain method and evaluated the antithrombotic activity of the extract, The extracts of the present invention were found to have excellent thermal stability and acid stability without showing any hemolytic activity against human erythrocytes. Thus, the above extracts can be used as pharmaceutical compositions for preventing or treating / improving thrombosis, Health functional foods.

Specifically, the inventors of the present invention conducted a study to develop a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis using azalea known to have various physiological activities in the private sector. (Thrombin time), prothrombin time (prothrombin time) and inhibition of blood coagulation factor (activated partial thromboplastin time: aPTT) were measured by using the extracts , And it was confirmed that the hydrothermal extract of Mifu and the like had excellent antithrombotic activity due to strong anticoagulant inhibition.

Mihu and other hot water extracts can recover 1.08 g per 100 g of Mifu and can be produced as an antithrombotic agent very economically. At 5 mg / ml concentration of the extract, the thrombin time is extended 15 times or more, Prothrombin time and 1.6-fold prolonged apathy time, indicating strong antithrombotic activity. In addition, it has been confirmed that the Mihu isothermal water extract exhibiting antithrombotic activity does not show hemolytic activity on human erythrocytes and does not cause acute toxicity to human body. No other antithrombotic effect was observed in the extracts of hot water, ethanol, and mihu of the other fruit of kiwifruit. In addition, in the evaluation of platelet aggregation inhibition activity using human platelets, other extracts except for the hot water extracts of Mifu and the like showed promoting effects on human platelet aggregation. Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, which comprises, as an active ingredient, a Mihu-like hot-water extract.

The Mihu iso-hydrothermal extract showed a strong prolongation effect on thrombin time, prothrombin time and apitime time, and thus showed strong blood coagulation activity. Therefore, it can be applied as an anticoagulant, i.e., a blood coagulation inhibitor. As an effective ingredient, an anti-coagulant agent for blood clotting.

The antithrombotic activity of such Mihuo hot-water extract can be used as a material for functional foods. Accordingly, the present invention provides a health functional food for preventing or improving thrombosis, which contains Mihuo's hot-water extract as an active ingredient.

Hereinafter, the method for producing the Mihu-like hot-water extract of the present invention and the efficacy experiments will be described in more detail.

In order to achieve the object of the present invention, Mifu and the like; Experiments of the antithrombotic activity evaluation of the extract and the evaluation of the stability of the active substance were carried out.

The "Mifu etc. extract" contained in the composition of the present invention is prepared by preparing Mifu and the like, extracting Mifu and the like with a solvent, filtering the extract using a filter net of 0.06 mm or less and concentrating it under reduced pressure Can be obtained.

The solvent to be used in the present invention may be selected from the group consisting of water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol), a mixed solvent of the lower alcohol and water And hot water extraction is most preferred.

In the present invention, thrombin time, prothrombin time, and apitime time were measured at a concentration of 5 mg / ml of Mifu's hot-water extract. As a result, thrombin time was extended 15 times or more, prothrombin time was extended 1.3 times, 1.6 times longer apitate time, indicating strong antithrombotic activity. Considering that thrombin time, prothrombin time, and apathy time are extended by 1.95 times, 1.40 times, and 1.61 times, respectively, at 1.5 mg / ml concentration of aspirin (product name: Protect) Activity.

The mihuacid hot-water extract of the present invention can be made into powders through a conventional powdering process such as vacuum drying and freeze drying, spray drying and the like. They are not degraded by various degradation enzymes in the plasma, and maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases associated with thrombosis. Such diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, , Deep vein thrombosis, lower limb edema, pain, and acute peripheral artery occlusion. Vein thrombosis includes deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition containing the active ingredient of the present invention may be formulated into tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, injections of sterilized injection solutions And may be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

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

In a preferred embodiment, the solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, for example starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used.

Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Perfumes, preservatives, and the like.

As a preferable specific example, the preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

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

As a preferable example, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight per day Or every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell.

In the present invention, the term "object" includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , Preferably a mammal, more preferably a human.

In addition, the health functional food of the present invention can be variously used for foods and beverages effective for prevention or improvement of thrombosis. Examples of foods containing the active ingredient of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they can be used in the form of powder, granule, tablet, capsule or beverage .

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

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

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

Examples of the flavoring agent include tau martin; Natural flavoring agents such as stevia such as rebaudioside A or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the specific method of the present invention will be described in more detail by way of examples. The following examples are only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example  One: Cherry  Fruit and Mifu, etc.  Preparation of extract

The fruits of kiwifruit cultivated in Cheongsong, Gyeongbuk, Korea were removed and used for the production of hot water and ethanol extracts. First, at the preparation of the hot water extract, 20 times of purified water was added to each sample, and the mixture was repeatedly extracted three times at 100 ° C for 1 hour. The extracts were collected, filtered and concentrated under reduced pressure. For preparing the ethanol extract, 10 times of ethanol (95%, Duksan, Korea) was added to each sample, and the extracts were collected three times at room temperature. The extracts were collected by filtration and concentrated under reduced pressure to give powder. Total polyphenols, total flavonoids, total sugars and reducing sugar content were determined by analysis of the constituents of the prepared extracts. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau and 100 μl of saturated Na ₂ CO ₃ solution to 400 μl of the extract solution, leaving it at room temperature for 1 hour and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. The total flavonoid content of each sample was measured by stirring for 18 hours in methanol. To the 400 μl of the filtered extract, 4 ml of 90% diethylene glycol was added and 40 μl of 1N NaOH was added. The reaction was carried out at 37 ° C. for 1 hour and then absorbance was measured at 420 nm. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

As a result, as shown in Table 1, the extraction efficiency of hot water extract was 9.95% and that of ethanol extract was 8.22%, and that of hot water extract was 1.08% and that of ethanol extract was 0.67% The efficiency of stem extract was lower than that of fruit extract regardless of extraction solvent. In addition, in the case of Mifu, the hot water extract was 1.6 times more than the ethanol extract. The total polyphenol contents of the extracts showed a very high total polyphenol content (155.8 mg / g) and a relatively low total flavonoid content (12.9 mg / g) in the Mihu hydrothermal extract. Mihu and other ethanol extracts showed a similar pattern and showed high total polyphenol content (125.1 mg / g) and low total flavonoid content (12.4 mg / g). On the other hand, in the case of the fruit extract of kiwifruit, the content of total polyphenols of 9.2 to 13.9 mg / g and the total flavonoid of 6.0 to 6.1 mg / g in the hydrothermal and ethanol extracts were estimated to show lower physiological activity than the stem of Mifu. Actual Mifu extracts showed strong antioxidant activity compared to the fruit extracts (no result). On the other hand, the contents of total sugar and reducing sugar were 3 ~ 4 times higher than those of Mifu extract.

Example  2: Cherry  Fruit and Mifu, etc.  Evaluation of blood coagulation inhibitory activity of extract

The blood coagulation inhibitory activity (thrombogenesis inhibitory activity) of the extracts of the fruit of kiwifruit and Mifu prepared in Example 1 was evaluated and compared with the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35, 52-61 As in Kwon et al., 2004. J. Life Science, 14. 509-513; and Li et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time, Respectively. Plasma was measured using a commercially available control plasma (MD Pacific Technology Co., Ltd., Huayuan Industrial Area, China), and thrombin time, prothrombin time and apitime time were measured in the following manner.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA) and 50 μl of 20 mM CaCl ₂ and 10 μl of various concentrations of the sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes at 37 ° C., After the addition, the time until the plasma coagulated was measured. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a solidification time of 30.5 sec. The thrombin inhibitory effect was expressed as the average value of the experiments repeated three or more times. The thrombin inhibitory activity was expressed by the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

Prothrombin time ( prothrombin  time)

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample solution to tubes of Amelung coagulometer KC-1A (Japan), heat at 37 ° C for 3 minutes, add 130 μl of PT reagent, The time from the start of the experiment to the start of the experiment was expressed as the average value of the experiments repeated three times. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 16.7 seconds. The prothrombin inhibitory activity was expressed as the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

aPTT  (activated Partial Thromboplastin  Time)

Add 50 μl of aPTT reagent (Sigma, ALEXIN ^™ ) to the tubes of Amelung coagulometer KC-1A (Japan) and incubate for 3 minutes at 37 ° C. Add 100 μl of plasma and 10 μl of various concentrations of sample extract Min. After the addition of 50 μl CaCl ₂ (35 mM), the time until the plasma coagulated was measured. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 58.1 seconds. The results of aPTT were expressed as the mean value of three repeated experiments. The activity of inhibiting blood coagulation factor was represented by aPTT divided by the aPTT of the solvent control when the sample was added.

As a result, as shown in Table 2, the hot water and ethanol extracts of the fruit of kiwifruit showed weak thrombin inhibition and blood coagulation factor inhibition at the concentration of 5 mg / ml, and the thrombin time was 1.15 to 1.23 times and the apathy time was 1.21 to 1.27 times Extended. However, the hot-water extracts of Mifu and the like exhibited potent antithrombotic activity and a concentration-dependent activity by prolonging thrombin time by 15 times, prothrombin time by 1.3 times, and apathy time by 1.6 times, , And Mifu showed 1.16 times, 1.13 times, and 1.28 times longer thrombin time, prothrombin time, and apathy time at the concentration of 5 mg / ml, respectively. At this time, aspirin used as a control was lengthened by 1.95 times, 1.40 times, and 1.61 times of thrombin time, prothrombin time, and apathy time at a concentration of 1.5 mg / ml. These results suggest that only hydrothermal extracts such as Mifu can be developed as a potent antithrombotic agent that can replace gastrointestinal aspirin.

Example  3: Cherry  Fruit and Mifu, etc.  The activity of human platelet aggregation inhibitory activity of the extract

The activity of inhibiting human platelet aggregation inhibition by the extract of each region and solvent of the kiwifruit of Example 1 was evaluated and the results are shown in Table 3 and FIG. Platelets are cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentration instead of nucleus, and circulating blood vessels together with various blood cells. It is an important cell that secretes factors and binds to collagen exposed by damage of endothelial cells to form a primary hemostatic plug to initiate thrombogenesis. Therefore, inhibition of platelet aggregation is a very important activity to prevent thrombogenesis. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity (Platelet aggregation inhibition activity)

Human platelets were used as platelets and washed once with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, the cells were resuspended in a suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 0.49 mM MgCl ₂ , 0.25% gelatin, pH 7.4) and incubated at 3,000 rpm for 10 minutes After centrifugation, the cells were resuspended in a suspending buffer and the platelet count was adjusted to 4 × ^{10 9} / ml. Then, 2.5 μl of collagen was added to 1 ml of the suspension, and the mixture was reacted for 5 minutes. Platelet aggregation was measured at 37 ° C using a whole-blood aggregator (Chrono-log, USA).

As a result, as shown in Table 3 and FIG. 1, in the case of DMSO as a solvent control, platelets rapidly and strongly aggregated by the addition of collagen, and aspirin, which is a platelet aggregation inhibitor, strongly inhibited platelet aggregation in a concentration-dependent manner. At this time, aspirin showed a coagulation degree of 27.5% at a concentration of 0.25 mg / ml and a coagulation degree of 58.3% at a concentration of 0.125 mg / ml, thus confirming the basis for use as an antithrombotic agent in clinical practice. On the other hand, hot - water extract and ethanol extract of kiwi fruit and ethanol extract of Mifu showed strong platelet aggregation promoting activity. The extracts exhibited 146%, 157% and 178% aggregation at 0.25 mg / ml concentration, respectively. On the other hand, hydrothermal extracts from Mifu had little effect on platelet aggregation. This is due to the fact that existing kiwi extracts inhibit platelet aggregation (Dizdarevic LL et al., Platelets. 2014, 25: 567-575; Karlsen A et al., J Hum Hypertens. , Platelets. 2004: 15: 287-292), the fruit extract of kiwifruit means promoting platelet aggregation, and even in the case of Mifu, only hydrothermal extract can exhibit antithrombotic activity without promoting platelet aggregation It means.

Example  4: Cherry  Fruit and Mifu, etc.  Evaluation of human erythrocyte hemolytic activity of extract

The fruit of kiwifruit and mihu has been used for edible and medicinal purposes for a long time, and the extracts such as fruit and mihu are not considered to have any specific toxicity. To evaluate the potential acute toxicity of hot-water extracts and ethanol extracts such as kiwifruit and mihu, human hemolytic hemolytic activity was evaluated and the results are shown in Table 4. At this time, hemolytic activity was evaluated in accordance with the existing report (Jongho Lee, 2014, Korean J. Microbiol. Biotechnol. 42: 285-292). In brief, 100 μl of human erythrocytes washed three times with PBS were dispensed into a 96-well microplate 100 μl of the supernatant was transferred to a new microtiter plate and centrifuged for 10 minutes at 1,500 rpm. Then, the hemoglobin efflux according to hemolysis was measured at 414 nm Respectively. Triton X-100 (1 mg / ml) was used as an experimental control for erythrocyte hemolysis. DMSO (2%) was used as a solvent control of the sample. Hemolytic activity was calculated using the following formula.

(%) Hemolysis = [(Abs.S-Abs.C) / (Abs.T-Abs.C)] 100

Abs. S: absorbance of sample added sphere,

Abs. C: Absorbance of DMSO furniture,

Abs. T: Absorbance of Triton X-100 added sphere.

First, DMSO and distilled water used as control did not have erythrocyte hemolytic activity, and Triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. On the other hand, the extracts of the fruits such as kiwifruit and mihu showed no hemolytic activity.

Example  5: Mifu, etc. Heat number  Evaluation of Plasma, Acid and Thermal Stability of Extracts

The plasma stability, thermal stability and acid stability of the anticoagulant activity of the Mihuo isotherm extracts obtained in Example 1 were confirmed. The extract retained its excellent activity without heat treatment at 100 ° C for 1 hour, 1 hour treatment with pH 2 (0.01M HCl), and 1 hour treatment with plasma without loss of anticoagulant activity. Therefore, in view of the component analysis results of Example 1 and the above stability results, the active substance of Mifu isotherm extract is expected to be a phenolic compound or a glycoside thereof. These results suggest that Mifu extract can be used as an antithrombotic agent and it may be possible to supplement and replace aspirin with adverse effects such as gastrointestinal complaints.

Claims (3)

Stem of Actinidia arguta) a pharmaceutical for the prevention and treatment of thrombosis comprising the hot-water extract as an active ingredient compositions. Stem of Actinidia arguta ) A blood coagulation inhibitor containing hot - water extract as an active ingredient. Stem of Actinidia arguta) dietary supplement for the prevention or improvement of thrombosis containing hot water extract as an active ingredient.

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