KR102013288B1 - 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 is a rear light lamp (Tree of the tree stem: stem of Actinidia arguta ) relates to an antithrombotic composition containing an extract as an active ingredient, and more particularly, to prevent or treat / improve thrombosis through inhibiting blood coagulation, which contains an extract prepared by hydrothermally extracting flush water selected as an active ingredient. It relates to pharmaceutical compositions and nutraceuticals. The trailing light extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis of the present invention and the health functional food, as demonstrated through the examples of the present specification, is potent by inhibiting the blood clotting factor-related enzyme inhibition and blood coagulation factor. It exhibits antithrombotic activity, shows no hemolytic activity against human erythrocytes, has excellent thermal stability, and shows no loss of the effect of inhibiting blood clotting factor-related enzymes and coagulation factors even in acidic conditions at pH 2 and in plasma. The improvement is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke, and the active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc. Very effective in the pharmaceutical and food industries It is.

Description

Pharmaceutical composition and health functional food for prevention or treatment of thrombosis containing taillight extract as an active ingredient }

The present invention is a rear light lamp (Tree of the tree stem: stem of Actinidia arguta ) relates to an antithrombotic composition containing an extract as an active ingredient, and more particularly, to prevent or treat / improve thrombosis through inhibiting blood coagulation, which contains an extract prepared by hydrothermally extracting flush water selected as an active ingredient. It relates to pharmaceutical compositions and nutraceuticals.

Blood as a human component has various important functions such as transporting and buffering oxygen, nutrients, and wastes, maintaining body temperature, controlling osmotic pressure and ion balance, maintaining moisture, controlling fluid, maintaining and regulating blood pressure, and defending the body. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolytic reaction system in the body are complementarily regulated. Among them, the mechanism of the blood coagulation reaction system forms platelet thrombus due to the adhesion of platelets to the blood vessel walls and aggregation. In addition, it has been reported that the blood coagulation system is activated to form fibrin clots around platelet aggregates.

On the other hand, the generation of fibrin thrombus is a multi-step reaction of numerous blood coagulation factors that activates thrombin, which is involved in fibrin coagulation, and finally generates fibrin monomers from fibrinogen, and the fibrin monomers are polymerized by calcium, thereby forming platelets and endothelial cells. It binds to cells and creates permanent thrombi, forming fibrin polymers cross-linked by factor XIII. In addition, thrombin plays a pivotal role in thrombus formation by activating platelets, factor V and factor VII to promote blood coagulation. Therefore, thrombin activity inhibitors can be used as a prophylactic and therapeutic agent that is very useful for various thrombotic diseases that occur due to excessive blood clotting. On the other hand, in the endogenous thrombus generation pathway, sequential activation of factor XII, factor XI, factor XII, factor IX and factor X is followed by activation of prothrombin to finally activate thrombin. It has been a development target for the treatment of sexual diseases, and in the case of exogenous thrombus generation pathway, thrombus generation is known according to the activation of factor II (prothrombin), factor V, factor VII, and factor X. To date, various anticoagulants such as heparin, coumarin, aspirin, urokinase, antiplatelet agents, thrombolytic agents, etc. have been used for the prevention and treatment of thrombotic diseases. The use is limited by reaction etc.

Meanwhile, Actinidia arguta is a temperate deciduous fruit of the genus Actinidiaceae, which is a deciduous vine that grows in the mountains of Korea. The growing environment grows well in the shade of mountain forests and hiking trails. The height is about 2 ~ 5m, and the leaves are wide ovate and oval with thin and sharp teeth on the edge. There are five varieties known to tuna, and domestic tuna is a Hayward cultivar with a large fruit size, excellent shelf life, and excellent taste and aroma. In addition to the true larvae in Korea, the dogs, rodents and islands are growing wild.

The fruit of the tuna is attracting attention as a health food with a combination of sweet and sour taste, and Kiwifruit: Actinidia chinensis ), that is, its shape and taste are similar. In addition, the taraxacum sprouts are eaten as herbs or pickles, and are known as spring herbs along with the bamboo shoots. On the other hand, Sesame tree buds are called "mifudo" or "mifuri" in the herbal medicine, it is used to reduce heat and stop thirst, diuretic effect, vomiting prevention and indigestion treatment. In addition, the stem of the tree is called "backlight" in Chinese medicine, it is used for the prevention and treatment of insomnia, esophageal cancer, breast cancer, bronchitis, arthritis.

The research on solanum known to date is focused on cultivation methods and pest prevention (Cho, Ki-Suk et al., 2016. Research in Plant Disease. 22: 168-177), and some studies have been conducted on processed juices using the fruit of the sesame (Lee Jin-won et al., 2003, Korea). Korean Journal of Food Science and Technology 35: 628-634), Jelly (Oh, Hyun-Jung et al., 2013. Korean Journal of Culinary Research, 19: 110-120), Vinegar (Woo, Seung-Mi et al., 2007. Journal of Korean Society for Food Preservation 14: 100-104), and Wine Fermentation (Kang Sang-dong et al., 2011. Journal of Life Science 21: 509-514). Studies on the useful physiological activity of the sea stalks have shown that the antioxidant activity of fruits (Kim, A Na et al., 2015. Korean Journal of Food Preservation and Distribution 22: 408-420), the neurotoxic protective effect (Kim, Jung-Hee et al. 2010, Korean Journal of plant resources 23: 165- 171), antimicrobial and lung cancer cell proliferation inhibitory effect (Park Yong-seo et al., 2009. Journal of the East Asian Society of Dietary Life 19: 202-209), nitrite scavenging activity (Park Yong-seo et al., 2008 Korean journal of horticultural science & technology 26: 495 -500), and PC-12 neuronal cell protective effects (Lee et al., 2015, Korean journal of horticultural science & technology 33: 259-267), and studies on trailing-light have been described as "LPS-induced LPS-induced RAW264. 7 "Influence on the inflammatory response of cells" is the only situation (Kim Young-jun, 2015. Master's thesis, Dong-Eui University). Therefore, until now, no strong antithrombotic activity by the inhibition of thrombus production-related enzymes and coagulation factors has been known.

On the other hand, patents related to the fruit of the sesame and sour light known to date, such as [food additives, animal feed additives or cosmetic composition containing a worm extract having anti-allergic and anti-inflammatory activity] in Korea Patent No. 10-0536495, In Patent No. 10-1368293 [Anti-inflammatory composition and skin lightening composition using a sesame fruit fermentation], Patent No. 10-1181998 [Skin whitening composition using a sesame leaf extract], Patent No. 10-1315975 No. [An exfoliating effect composition containing a sesame seed extract] is disclosed in [Tanin-reinforced high-quality sesame wine and its manufacturing method] in Korean Patent No. 10-1246093, in particular, Patent No. 10-1342497 [Trapeum stem extract having alpha-glucosidase inhibitory activity and a composition for lowering blood sugar containing the same] is disclosed in [Patent No. 10-1382412]. [Composition containing the extract of the taraxacum having activating activity], [A Chinese herbal composition for the treatment of allergic diseases] is registered in Patent No. 10-1196486, [Registration No. 10-1663971] for promoting the differentiation of human mesenchymal stem cells Composition] is disclosed. Therefore, there is no known antithrombotic effect by inhibition of the enzyme and blood coagulation factor related to the strong thrombus generation in any academic research or patent literature.

KR 10-1342497 B KR 10-1382412 B

 Effect of Mihu Lung Extract on Inflammatory Response of LPS-Induced RAW264.7 Cells, Young-Jun Kim, 2015, Master's Thesis, Dong-Eui University

The present invention has been made in order to solve the problems of the prior art as described above, the problem to be solved in the present invention is to prepare an anti-thrombotic composition containing a hydrothermal extract, such as a tail, as an active ingredient .

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing a stem of Actinidia arguta hydrothermal extract as an active ingredient.

In addition, the present invention is the stem of Actinidia arguta ) provides a blood coagulation inhibitor containing hot water extract as an active ingredient.

In addition, the present invention is the stem of Actinidia arguta ) Provides a dietary supplement for the prevention or improvement of thrombosis containing hot water extract as an active ingredient.

The trailing light extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis of the present invention and the health functional food, as demonstrated through the examples of the present specification, is potent by inhibiting the blood clotting factor-related enzyme inhibition and blood coagulation factor. It exhibits antithrombotic activity, shows no hemolytic activity against human erythrocytes, has excellent thermal stability, and shows no loss of the effects of clotting factor-related enzymes and coagulation factor in acidic conditions and plasma of pH 2. The improvement is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke, and the active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc. Very effective in the pharmaceutical and food industries It is.

 Figure 1 shows the human platelet aggregation inhibitory activity of the water and ethanol extract of sesame fruit and humyeo used in the embodiment of the present invention. Wherein: 1: solvent control (DMSO), 2: aspirin (0.25 mg / ml), 3: aspirin (0.125 mg / ml), 4: hydrothermal extract of Cauliflower fruit (0.25 mg / ml), 5: The ethanol extract (0.25 mg / ml), 6: hydrothermal extract (0.25 mg / ml), etc., and the tail | light lamp | ramp, 7: ethanol extract (0.25 mg / ml) of the tail, etc. are shown, respectively.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The inventors of the present invention to prepare anti-thrombotic efficacy in the sesame seed extract, to prepare the sesame fruit extract and taillight extract prepared by a certain method, to evaluate the antithrombotic activity of the extract to anti-thrombotic hydrothermal extract The extract was recovered as a component, and the hydrothermal extract was found to have excellent heat stability and acid stability without showing any hemolytic activity against human red blood cells, thereby preventing the extract from being prevented or treated / improved by the pharmaceutical composition and It was intended to be used as a dietary supplement.

Specifically, the present inventors, in order to develop a pharmaceutical composition and health functional food for the prevention or treatment / improvement of thrombosis using sesame seeds known to have various physiological activities in the folklore, hot water extract and ethanol for the sesame fruit and after Extracts were prepared separately, and antithrombotic activity was determined for thrombin time, prothrombin time, and blood coagulation factor inhibition (activated partial thromplastin time, aPTT). In evaluation, it was confirmed that the taillight hot water extract has excellent antithrombotic activity due to strong anticoagulant inhibition.

The taillight hot water extract can recover 1.08g per 100g of taillight and can be manufactured very economically as an antithrombotic agent.The extract has a thrombin time that is 15 times longer than that of no addition at 5mg / ml and 1.3 times longer. Prothrombin time and 1.6-fold extended epitaxial time, indicating strong antithrombotic activity. In addition, it was confirmed that the taillight hydrothermal extract showing antithrombotic activity did not exhibit hemolytic activity against human erythrocytes and thus did not cause acute toxicity in humans. In addition, anti-thrombotic effects were not observed in ethanol extracts such as hydrothermal extract, ethanol extract, and hind cultivation of sesame fruit. In addition, in the evaluation of platelet aggregation inhibitory activity using human platelets, all the extracts except the late hydrothermal extract showed the effect of promoting human platelet aggregation. Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, which contains a taillight hot water extract as an active ingredient.

Since the rear light extract has a strong blood coagulation activity by showing an excellent prolonging effect in thrombin time, prothrombin time and apitime, it can be applied as an anticoagulant, that is, a blood coagulation inhibitor, therefore, the present invention Provided is a blood coagulation inhibitor containing an isothermal water extract as an active ingredient.

The antithrombotic activity of such taillight hot water extract can be used as a material for functional foods. Therefore, the present invention provides a health functional food for preventing or improving thrombosis containing a taillight hot water extract as an active ingredient.

Hereinafter, the production method and efficacy test of the taillight hot water extract of the present invention will be described in more detail.

The inventors of the present invention, to achieve the object of the present invention, preparing a selected, washed tail, etc .; Preparing a back light extract; Experiments were performed to evaluate the antithrombotic activity of the extract and to evaluate the stability of the active substance.

"Two taillight extract" included in the composition of the present invention is prepared by the step of preparing a taillight, extracting the taillights with a solvent and the extract is filtered using a filter net of 0.06mm or less, and concentrated under reduced pressure Can be obtained.

The solvent used in the present invention includes water (cold water, hot water), spirits, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), mixed solvents of the lower alcohols with water, and the like. Hydrothermal extraction is most preferred.

In the present invention, after measuring the thrombin time, prothrombin time, apititime with a concentration of 5 mg / ml of the taillight hot water extract, the thrombin time, prolonged 1.3 times prolonged prolonged, 1.6-fold extended epitaxial time was shown, indicating strong antithrombotic activity. This is a very potent anticoagulant, considering that aspirin (trade name), which is used clinically as an antithrombotic agent, extends thrombin time, prothrombin time, and epitaxial time by 1.95, 1.40, and 1.61 times, respectively, at a concentration of 1.5 mg / ml. It can be seen that it is active.

The taillight hot water extract of the present invention may be prepared into a powder through a conventional powdering process such as vacuum drying and freeze drying, or spray drying. They are not degraded to various degrading enzymes in plasma and remain active at 100 ° C. heat treatment and pH 2 in the human stomach.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases associated with thrombosis. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis , Deep vein thrombosis, lower extremity edema, pain, and acute peripheral arterial obstruction. Examples of venous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous thrombosis, and central retinal vein occlusion.

The pharmaceutical composition comprising the active ingredient of the present invention may be orally formulated as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It may be formulated and used in various forms, and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further include carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include a filler, an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

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

As a preferred embodiment, oral liquid preparations may be exemplified by suspensions, solvents, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, Fragrances, preservatives and the like.

As a preferred embodiment, the preparation for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories and the like. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters 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 an effective dose level means the type, severity, activity of the drug, Sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, and generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg daily. Or every other day or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

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

As used herein, "administration" means providing a patient with any substance by any suitable method, wherein the route of administration of the pharmaceutical composition of the present invention is oral or parenteral via all common routes as long as the target tissue can be reached. Oral administration. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

"Subject" in the present invention is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And preferably mammals, and more preferably humans.

In addition, the health functional food of the present invention can be used in a variety of foods and drinks effective for preventing or improving thrombosis. Examples of the food containing the active ingredient of the present invention include various foods, beverages, gums, teas, vitamin complexes, dietary supplements, and the like, and can be used in the form of powders, granules, tablets, capsules, or beverages. .

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

In addition to containing the compound as an essential ingredient in the indicated ratios, the health functional food of the present invention may contain food-acceptable food supplement additives such as natural carbohydrates and various flavoring agents as additional ingredients.

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

As the flavoring agent, taumartin; Natural flavors such as stevia such as rebaudioside A or glycyrzin and synthetic flavors such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally used in the range of 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 includes various nutrients, vitamins, minerals, synthetic flavors and natural flavoring agents, colorants and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids Thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the health functional food of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage, vegetable beverage and the like. These components can be used independently or in combination. The proportion of such additives is generally selected from 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 with reference to Examples. The following examples are only one 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: Blue sky  Berries and Taillight  Preparation of Extract

The foreign substances were removed from the fruit and tail of cultivated in Cheongsong, Gyeongbuk, Korea, and used to prepare hot water and ethanol extract. First, when preparing hot water extract, 20 times of purified water was added to each sample, and extracted three times at 100 ° C. for 1 hour. The extracts were collected and filtered, and concentrated under reduced pressure to prepare a powder. Meanwhile, when preparing the ethanol extract, 10 times ethanol (95%, Deoksan, Korea) was added to each sample, extracted three times at room temperature, the extracts were collected and filtered, and concentrated under reduced pressure to prepare a powder. Component analysis of the prepared extracts determined the total polyphenols, total flavonoids, total sugars and reducing sugars. The total polyphenol content was added to 400μl of the extracted sample solution, 50μl of Folin-ciocalteau, 100μl of Na ₂ CO ₃ saturated solution, and left at room temperature for 1 hour and absorbance at 725nm. Tannic acid was used as the standard reagent. For the total flavonoid content, each sample was extracted by stirring for 18 hours with methanol, 4 ml of 90% diethylene glycol was added to 400 µl of the filtered extract sample, 40 µl of 1N NaOH was added thereto, and the absorbance was measured at 420 nm after 1 hour of reaction at 37 ° C. Rutin was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was determined by phenol-sulfuric acid method.

As a result, as shown in Table 1, the extract of T. alba fruit extract showed 9.95% of hot water extract, 8.22% of ethanol extract, and the taillights showed 1.08% of hot water extract and 0.67% of ethanol extract. Regardless of the extraction solvent, the stem extract efficiency was lower than the fruit extraction rate. In addition, the hydrothermal extract showed more than 1.6 times the amount of ethanol extract in the case of Mihu. The total polyphenol content of the extract showed very high total polyphenol content (155.8 mg / g) and relatively low total flavonoid content (12.9 mg / g). The ethanol extracts of the posterior light showed similar patterns, showing high total polyphenol content (125.1 mg / g) and low total flavonoid content (12.4 mg / g). On the other hand, the extract of T. oleracea showed the total polyphenols of 9.2 to 13.9 mg / g and the total flavonoid content of 6.0 to 6.1 mg / g in hot water and ethanol extract, which was supposed to have lower physiological activity than dorsum. Indeed, the hindlight extracts showed stronger antioxidant activity than the fruit extracts (not shown). On the other hand, the total sugar and reducing sugar content of the fruit extract was 3-4 times higher than the taillight extract.

Example  2: Blue sky  Berries and Taillight  Evaluation of Blood Coagulation Inhibitory Activity of Extracts

The anticoagulant inhibitory activity (thrombogenicity inhibitory activity) of the extracts of Cauliflower and oleander extract prepared in Example 1 was evaluated, and the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61 Kwon et al., 2004. J. Life Science, 14. 509-513; and R. et al. 2010. J. Life Science, 20. 922-928), measuring thrombin time, prothrombin time, and aptitime Evaluated. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and thrombin time, prothrombin time and apiity time measurements were performed as follows.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA), 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, followed by 100 μl of plasma. After addition, the time until plasma coagulates was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. DMSO showed a solidification time of 30.5 seconds. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the thrombin inhibitory activity was expressed as the coagulation time when the sample was added divided by the coagulation time of the solvent control.

Prothrombin Time prothrombin  time)

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample solution were added to a tube of Amelung coagulometer KC-1A (Japan), warmed at 37 ° C. for 3 minutes, and then 130 μl of PT reagent was added and the plasma coagulated. The time until was expressed as the average value of the experiment repeated three times. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. DMSO showed a solidification time of 16.7 seconds. The prothrombin inhibitory activity was expressed as the coagulation time at the time of sample addition divided by the coagulation time at the solvent control.

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), warmed at 37 ° C. for 3 minutes, and then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added again to 3 ° C. at 37 ° C. Incubate for minutes. Since 50 μl CaCl ₂ (35mM) was added to measure the time until the plasma coagulate. DMSO was used as a solvent control instead of the sample, in which case it showed a solidification time of 58.1 seconds. The results of aPTT were shown as the average value of the experiment repeated three times, and the blood coagulation factor inhibitory activity was expressed as aPTT at the time of sample addition divided by aPTT of the solvent control.

As a result, as shown in Table 2, the hot water and ethanol extract of the lychee fruit showed weak thrombin inhibition and coagulation factor inhibition at the concentration of 5 mg / ml, the thrombin time is 1.15 ~ 1.23 times, the epitaxial time is 1.21 ~ 1.27 times Extended. However, Mihu et al hydrothermal extracts showed potent antithrombotic activity, showing more than 15-fold extended thrombin time, 1.3-fold extended prothrombin time, and 1.6-fold extended epitaxial time compared to no additives, showing concentration-dependent activity. The ethanol extracts of, and after showed 1.16 fold, 1.13 fold and 1,28 fold extended thrombin time, prothrombin time, and epitaxial time at 5 mg / ml concentrations, respectively. At this time, the aspirin used as a control was extended by 1.95 times, 1.40 times and 1.61 times the thrombin time, prothrombin time, apitime at 1.5 mg / ml concentration, respectively. These results suggest that only hydroponic extracts in the back of the tuna can be developed as a powerful antithrombotic agent that can replace aspirin, which indicates gastrointestinal disorders.

Example  3: Blue sky  Berries and Taillight  Human Platelet Aggregation Inhibitory Activity of Extracts

Human platelet aggregation inhibitory activity was evaluated in extracts of different parts and solvents of the sesame seeds of Example 1, and the results are shown in Table 3 and FIG. 1. Platelets are discoid small cells that circulate blood vessels along with various blood cells. They have a cytoplasmic granule that contains high concentrations of various substances related to vascular damage protection and platelet aggregation instead of the nucleus. It is an important cell that secretes factors and binds collagen and the like exposed to damage of endothelial cells to form a primary hemostatic plug to initiate thrombus formation. Thus, platelet aggregation inhibition is a very important activity for preventing thrombus formation. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Platelets were human concentrated platelets, which were 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). After resuspending in suspending buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 0.49mM MgCl ₂ , 0.25% gelatin, pH 7.4), and then resuspended at 3,000 rpm for 10 minutes. After centrifugation and resuspended in the suspending buffer, the platelet count was adjusted to 4x10 ⁹ / ml. Thereafter, 2.5 μl 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).

As a result, as shown in Table 3 and Figure 1, in the case of DMSO as a solvent control platelet was rapidly and strongly aggregated by the addition of collagen, and aspirin, a platelet aggregation inhibitor, strongly inhibited platelet aggregation in a concentration-dependent manner. At this time, aspirin showed a cohesiveness of 27.5% at a concentration of 0.25mg / ml and a concentration of 58.3% at a concentration of 0.125mg / ml, confirming the basis for use as an antithrombotic agent in the clinic. On the other hand, hot water extracts, ethanol extracts, and hind ethanol extracts of the lychee fruit showed potent platelet aggregation promoting activity. The extracts exhibited aggregation levels of 146%, 157% and 178% at 0.25 mg / ml concentrations, respectively. On the other hand, the late back hot water extract had little effect on platelet aggregation. This suggests that existing kiwi extracts inhibit platelet aggregation (Dizdarevic LL et al., Platelets. 2014, 25: 567-575; Karlsen A et al., J Hum Hypertens. 2013. 27: 126-130; Duttaroy AK et al. , Platelets. 2004. 15: 287-292), the extract of T. alba means that it promotes platelet aggregation, and even in the latter, only hydrothermal extract can show antithrombotic activity without promoting platelet aggregation. It means.

Example  4: Blue sky  Berries and Taillight  Evaluation of Human Erythrocyte Hemolytic Activity of Extracts

The fruit and the taillight of the tuna are not used as edible and medicinal for a long time. Human erythrocyte hemolysis activity was evaluated to evaluate the potential acute toxicity of the hot water extracts and the chylopods of the water and ethanol extracts, and the results are shown in Table 4. At this time, hemolytic activity was evaluated according to the existing report (Son Ho Yong, 2014, Korean J. Microbiol. Biotechnol. 42: 285 ~ 292), and 100 μl of human erythrocytes washed three times with PBS in a 96-well microplate. 100 μl of various sample solutions were added and reacted at 37 ° C. for 30 minutes. After that, the reaction solution was centrifuged for 10 minutes (1,500 rpm) to transfer 100 μl of the supernatant to a new microtiter plate. Measured at DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1 mg / ml) was used as an experimental control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

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

Abs. S: absorbance of the sample addition port,

Abs. C: absorbance of DMSO house furniture,

Abs. T: Absorbance of the Triton X-100 addition port.

First, DMSO and distilled water used as a control did not have erythrocyte hemolytic activity, Triton X-100 was confirmed that 100% hemolysis of red blood cells at a concentration of 1mg / ml. On the other hand, in the case of the extract of the fruit of the sesame seeds and the trailing tail did not show hemolytic activity.

Example  5: Taillight Hydrothermal  Evaluation of Plasma, Acid and Thermal Stability of Extracts

Plasma stability, thermal stability, and acid stability of the anticoagulant activity of the chylolight hydrothermal extract obtained in Example 1 were confirmed. The extract was maintained for 1 hour heat treatment at 100 ℃, 1 hour treatment at pH 2 (0.01M HCl), 1 hour treatment in plasma without loss of anticoagulant activity. Therefore, considering the component analysis results of Example 1 and the above stability results, the active substance of the taillight hydrothermal extract is expected to be a phenolic compound or glycoside thereof. The above results suggest that the taillight extract can be practically used as an antithrombotic agent, and it may be possible to supplement and replace aspirin reported side effects such as gastrointestinal disorders.

Claims (3)

Stem of Actinidia arguta ) A pharmaceutical composition for the prevention or treatment of thrombosis containing a hydrothermal extract as an active ingredient. delete Stem of Actinidia arguta ) Health functional food for the prevention or improvement of thrombosis containing hot water extract as an active ingredient.

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