KR102092960B1 - Pharmaceutical composition comprising ethylacetate fraction fractionated from ethanol extract of abeliophyllum distichum stem 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 containing the ethyl acetate fraction of the stem ethanol extract of Abeliophyllum distichum as an active ingredient, and more specifically, the ethyl acetate fraction of the ethanol extract prepared from the stem of the rice plant tree as the active ingredient. The present invention relates to a pharmaceutical composition for preventing or treating / improving thrombosis through inhibition of blood clotting and inhibition of platelet aggregation and health functional food. The ethylacetate fraction of the stalk of stalk ethanol extract as an active ingredient of the pharmaceutical composition and health functional food for preventing or treating thrombosis of the present invention, as evidenced through the examples herein, inhibits thrombus production-related enzymes and blood It exhibits strong antithrombotic activity by inhibiting coagulation factors and inhibiting platelet aggregation, and at the same time, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, is an enzyme in the blood, acidic conditions at pH 2, and plasma-related enzymes, blood Since there is no loss of the effect of inhibiting clotting factors and inhibiting platelet aggregation, it is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredients are extracts, powders, pills, Processed in various forms, such as tablets, which can be taken at all times Since the excellent results that can be prepared to be very useful inventions the pharmaceutical industry and the food industry.

Description

PHARMACEUTICAL COMPOSITION COMPRISING ETHYLACETATE FRACTION FRACTIONATED FROM ETHANOL EXTRACT OF ABELIOPHYLLUM DISTICHUM STEM AS AN EFFECTIVE COMPONENT PRE OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention is Abeliophyllum distichum ) relates to an antithrombotic composition containing the ethyl acetate fraction of the stem ethanol extract as an active ingredient, and more specifically, inhibiting blood clotting containing the ethyl acetate fraction of the ethanol extract prepared from the trunk of the stalk tree as an active ingredient, and The present invention relates to a pharmaceutical composition for preventing or treating / improving thrombosis through inhibition of platelet aggregation and a dietary supplement.

Blood as a component of the human body has various important functions such as transport function and buffering function of oxygen, nutrients, wastes, maintenance of body temperature, regulation of osmotic pressure and maintenance of ion balance, maintenance of moisture, fluid control, maintenance and control of blood pressure, and biological defense. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolysis reaction system in the body are complementarily regulated, and among these, the mechanism of the blood coagulation reaction system is that platelets adhere to and adhere to blood vessel walls to form platelet thrombus. , It has been reported that the blood coagulation system activates to form a fibrin thrombus around a platelet aggregate.

On the other hand, the production of fibrin thrombus is activated by thrombin, which is involved in fibrin coagulation through several stage reactions of numerous blood coagulation factors, to finally produce fibrin monomer from fibrinogen, and the fibrin monomers are polymerized by calcium, thereby causing platelets and endothelial It binds to cells and creates a permanent thrombus, forming a fibrin polymer cross-linked by factor XIII. In addition, thrombin plays a pivotal role in thrombus production, such as promoting blood clotting reactions by activating platelets, factor V and factor VII. Therefore, the activity inhibitor of thrombin can be used as a very useful preventive and therapeutic agent for various thrombotic diseases caused by excessive blood clotting abnormalities. On the other hand, in the endogenous thrombus generation pathway, it is known that activation of prothrombin following sequential activation of factor XII, factor XI, factor XII, factor IX, factor X finally activates thrombin. It has become a target for the development of therapeutic agents for sexual diseases, and in the case of exogenous thrombus production pathways, clot generation is known according to activation of factor II (prothrombin), factor V, factor VII, and factor X. To date, various anticoagulants such as heparin, coumarin, aspirin, and eurokinase, antiplatelet agents, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases, but they are not only very expensive, but also have bleeding side effects and gastrointestinal disorders and irritability. The use of the reaction is limited.

Meanwhile, Abeliophyllum distichum ) is a rare plant of the first genus in the world, and is a Korean plant native to Korea. The Korean Ministry of Environment has designated and protected the American native tree as a protected wild plant. It is native to Jincheon, Goesan, Yeongdong, and Buan in North Jeolla Province, and is rare enough to be designated as a natural monument. The stalk tree is a shrub tree belonging to the family of the ash family, about 1m high, the branches are spread out in all directions, drooping downwards, purplish, quadrangular, and the bones are stair-shaped. The leaves are opposite, ovate or elliptical, with narrow ends, flat edges, and short stems. The flowers bloom before the leaves and hang on the inflorescence, and the shape of the seed resembles a fan, so it is called a thorn tree.

Most of the researches on soybean trees known to date have been reported on the native habitats and cultivation and growth characteristics of the soybean trees (Park Seong-hyuk, 2016. Korean Master's degree in Yeongpung University, "Characteristics of vegetation structure according to the management method of the native tree of the Korean native tree) "; Lee Na-shim et al., 2014, Journal of Plant Biotechnology 41: 94-99), recently analyzed the composition of leaves and stems and anti-cancer, anti-inflammatory, anti-oxidation and whitening of leaves and immature seeds. Nam-Young Kim et al., 2015. Korean journal of medicinal crop science 23: 155-160; Jang Tae-won et al., 2017.Journal of life science 27: 536-544; Park Jae-ho, 2011, The Korea journal of herbology 26: 95-99), anti-wrinkle Activity (Nam-Young Kim et al., 2015. Korean journal of medicinal crop science 23: 231-236) has been reported. However, until now, strong antithrombotic activity by inhibiting blood clotting and inhibiting platelet aggregation in the upper part of the myrtle tree is not known at all.

In addition, most of the patents related to the stalk tree known to date are patents on cosmetics using the scent of the stalk tree and its antioxidant and anti-inflammatory activity. In Korean Patent No. 10-1729210, [Atopy containing the extract of stalk tree is improved. Product Composition], Registered Patent No. 10-1560672, [Deodorant Composition Containing Wisteria Tree Extract], Registered Patent No. 10-1181998, [Cosmetic Composition for Eliminating Odorous Odors Containing Wisteria Tree Extract as an Active Ingredient], Registered Patent No. 10-1470887 [Fragrance composition reproducing the smell of a wisteria tree], Registered Patent No. 10-1773090 [Fragment composition reproducing the scent of a wisteria flower and a composition for external application for skin containing the same], Patent No. 10 [1191225] [Cosmetic composition], registered patent No. 10-0954695 [Cosmetic composition containing the extract of saffron as an active ingredient] is disclosed, and in 2007, registered patent No. 10-0656287 known as [Anti-inflammatory agent containing hedge tree extract], and Patent No. 10-0706131 [Anti-cancer agent containing hedge tree extract] is known. In addition, recently disclosed in the Republic of Korea Patent Publication No. 10-2017-0122317 [Shampoo composition for preventing hair loss using the extract from the hedgehog tree], Patent Publication No. 10-2017-0122317 [Shampoo composition for preventing hair loss using the extract from the hedgehog tree], published [Patent No. 10-2017-0122315 [Cosmetic composition for skin using extract of hedgenut tree], Patent Publication No. 10-2015-0068643 [Special smell removing agent and method of production of livestock products and seafood using hedgehog tree extract], published patent In 10-2013-0074172, [Composition for cosmetic products using fermented pear tree] is disclosed. However, no antithrombotic effect by strong blood clotting inhibition and platelet aggregation inhibition of the myrtle extract has been known until now in any academic research or patent literature.

KR 10-1729210 B KR 10-2017-0122317 A

The present invention was devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is an antithrombogenic composition containing an ethyl acetate fraction of an ethanol extract prepared from the trunk of a sapling tree as an active ingredient. Is to provide.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, which contains the ethyl acetate fraction of the ethanol extract of the trunk tree stalk as an active ingredient.

In addition, the present invention provides a blood clotting inhibitor containing the ethyl acetate fraction of the ethanol extract of the trunk tree trunk as an active ingredient.

In addition, the present invention provides a platelet aggregation inhibitor that contains the ethyl acetate fraction of the ethanol extract of the trunk tree stem as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving thrombosis, which contains the ethyl acetate fraction of the ethanol extract of the myrtle stem as an active ingredient.

The ethylacetate fraction of the stalk of stalk ethanol extract as an active ingredient of the pharmaceutical composition and health functional food for preventing or treating thrombosis of the present invention, as evidenced through the examples herein, inhibits thrombus production-related enzymes and blood It exhibits strong antithrombotic activity by inhibiting coagulation factors and inhibiting platelet aggregation, and at the same time, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, is an enzyme in the blood, acidic conditions at pH 2, and plasma-related enzymes, blood Since there is no loss of the effect of inhibiting clotting factors and inhibiting platelet aggregation, it is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredients are extracts, powders, pills, Processed in various forms, such as tablets, which can be taken at all times Since the excellent results that can be prepared to be very useful inventions the pharmaceutical industry and the food industry.

1 is a photograph of the ground portion of the hedge tree used in the embodiment of the present invention. Here, 1: terrestrial part, 2: leaf, and 3: stem are shown, respectively.
Figure 2 shows the activity of human platelet aggregation inhibitory activity of the stalk leaf and stem extract and its sequential organic solvent fraction. Here, 1: Solvent control (DMSO), 2: Aspirin (0.25mg / ml), 3: Aspirin (0.125mg / ml), 4: Wisteria leaf ethanol extract (0.25mg / ml), 5: Wisteria leaf Hexene fraction of ethanol extract (0.25 mg / ml), 6: ethylacetate leaf ethylacetate fraction of ethanol extract (0.25 mg / ml) 7: butane leaf fraction of ethanol extract (0.25 mg / ml) 8: pear leaf Water residue (0.25 mg / ml) after fractionation of organic solvent of ethanol extract, 9: hedgehog stem ethanol extract (0.25 mg / ml), 10: hexene fraction of hedgehog stem ethanol extract (0.25 mg / ml), 11: Ethyl acetate fraction of coleopteran stem ethanol extract (0.25mg / ml), 12: Butanol fraction of coleopteran stem ethanol extract (0.25mg / ml), 13: Water residue (0.25) after the organic solvent fraction of coleopteran ethanol extract mg / ml).

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared an ethanol extract of the myrtle leaf and stem prepared by a certain method in order to test the anti-thrombotic efficacy against the myrtle extract, which were sequentially fractionated with organic solvent by hexene, ethyl acetate, butanol, Finally, a water residue after fractionation was prepared, and the anti-thrombotic activity of the extract and the fraction was evaluated to recover the ethyl acetate fraction of the ethanol extract of the American barberry stem as an antithrombotic component, and the fraction had no hemolytic activity against human red blood cells. Without showing, it was intended to utilize the fraction as a pharmaceutical composition of antithrombotic activity and health functional food by confirming that it has excellent thermal stability and acid stability.

Specifically, the present inventors use stalk tree, which is known to have various physiological activities in the private sector, to develop a pharmaceutical composition and health functional food for prevention or treatment / improvement of thrombosis, using ethanol extract as a target for stalk leaf and stem Each was prepared, and sequential organic solvent fractions and finally water residues after fractionation were prepared with hexene, ethyl acetate, and butanol for these extracts. Thrombin time and prothrombin inhibition of antithrombotic activity were targeted for the samples. (Prothrombin Time), blood coagulation factor inhibition (activated Partial Thromboplastin Time: aPTT) and platelet aggregation inhibition were evaluated, and the ethyl acetate fraction of the stalk ethanol extract was used for anticoagulation and platelet aggregation inhibition. It was confirmed that the anti-thrombotic activity was excellent.

It is expected that the ethanol extract of the myrtle stem can recover 5.8 g per 100 g of the stem, and the ethyl acetate fraction of the stem ethanol extract can recover 0.7 g, making it possible to manufacture an antithrombotic agent very economically. The ethanol extract of S. aureus showed weak blood coagulation enzymes (thrombin and prothrombin) and blood coagulation factor inhibitory activity, but its ethyl acetate fraction increased 1.24 times, 1.14 times, and 1.35 times compared to no additives at a concentration of 5 mg / ml. It showed thrombin time, prothrombin time, and apity time, and showed concentration-dependent inhibitory activity, showing increased thrombin time, prothrombin time, and apity time by 1.61, 1.55, and 1.77 times higher than the non-additive at 7 mg / ml concentration. Showed strong anticoagulant activity. In addition, it was confirmed that the ethyl acetate fraction of the ethanol extract of the myrtle tree trunk inhibited human platelet aggregation in a concentration-dependent manner, and did not exhibit hemolytic activity against human red blood cells and did not cause acute toxicity.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, which contains the ethyl acetate fraction of the ethanol extract of the myrtle stem as an active ingredient.

In addition, the present invention provides a blood clotting inhibitor containing the ethyl acetate fraction of the ethanol extract of the trunk tree trunk as an active ingredient. The pharmaceutical use is based on the inhibitory activity of blood coagulation enzyme and blood coagulation factor of the ethyl acetate fraction of the ethanol extract of the myrtle stem of the present invention.

In addition, the present invention provides a platelet aggregation inhibitor that contains the ethyl acetate fraction of the ethanol extract of the trunk tree stem as an active ingredient. The pharmaceutical use is based on the inhibitory activity of platelet aggregation of the ethyl acetate fraction of the ethanol extract of the myrtle stem of the present invention.

In addition, the present invention provides a health functional food for preventing or improving thrombosis, which contains the ethyl acetate fraction of the ethanol extract of the myrtle stem as an active ingredient.

Hereinafter, a method and an efficacy test of the ethyl acetate fraction of the stalk of stalk tree ethanol extract of the present invention will be described in more detail.

In order to achieve the object of the present invention, the inventors of the present invention are prepared by separating the leaves and stems from the ground parts of the selected and washed stalks; Preparing an ethanol extract of the myrtle leaf and stem; Preparing a sequential organic solvent fraction and finally a water residue after fractionation with hexene, ethyl acetate, and butanol for the extract; The extracts and fractions were tested for antithrombotic activity evaluation and stability evaluation of the active substance.

The "plum tree stem ethanol extract" contained in the composition of the present invention is a step of preparing and extracting the stem tree trunk, extracting the stem with ethanol, and filtering the extract using a filter network of 0.06 mm or less, which is concentrated under reduced pressure. Ethyl acetate fraction can be obtained as an active substance by sequential organic solvent fractionation of the stalk of stalk tree ethanol extract followed by recovery of ethyl acetate fraction and concentration under reduced pressure.

In the present invention, the activity of promoting platelet aggregation was confirmed in all samples except the hexene fraction and the ethyl acetate fraction of the stem, among the ethanol extract of the pine tree leaf and the stem and the sequential organic solvent fraction thereof, in particular, the ethyl acetate fraction of the stem was 0.25 At a concentration of mg / ml, it showed that the aggregation degree of 71.5% was higher than that of the additive-free group, indicating that it exhibited excellent platelet aggregation inhibitory activity. In addition, the ethyl acetate fraction of the ethanol extract of the trunk tree stalk showed prolonged thrombin time, prothrombin time and apity time of 1.24 times, 1.14 times, and 1.35 times longer, respectively, compared to the additive-free concentration at a concentration of 5 mg / ml, confirming good anticoagulant activity. Did. This is excellent anticoagulant activity considering that aspirin (trade name: PROTECT), which is used clinically as an antithrombotic agent, extends thrombin time, prothrombin time, and apity time by 1.95, 1.40, and 1.63 times, respectively, at a concentration of 1.5 mg / ml. You can see that

The ethyl acetate fraction of the ethanol extract of the trunk tree stalk of the present invention may be prepared as a powder through a conventional powdering process such as drying under reduced pressure and freeze drying, or spray drying. They are not degraded by various degrading enzymes in plasma, and they maintain activity even at a heat treatment of 100 ° C and a pH of 2 in the human stomach.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases related to thrombosis. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, motor abnormalities, sensory abnormalities, personality changes, decreased vision, epilepsy seizures, pulmonary thrombosis , Deep vein thrombosis, swelling of the lower extremities, pain and acute peripheral artery occlusion, and intravenous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, brain venous sinus thrombosis and central retinal vein occlusion.

The pharmaceutical composition containing the active ingredient of the present invention is an oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, injection of sterile injectable solution according to a conventional method according to each purpose of use. It can be formulated and used in various forms, such as oral administration or intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

The pharmaceutical composition may further include a carrier, excipient or diluent, 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 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, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., such solid preparations comprising at least one excipient in the pharmaceutical composition, for example starch, calcium carbonate, It is formulated by mixing sucrose, lactose, and gelatin. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

As a preferred embodiment, a liquid preparation for oral use may be exemplified by suspensions, solvents, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, Fragrances, preservatives, and the like may be included.

As a preferred embodiment, the formulation for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injections can 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, "a 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 is the type of patient's disease, severity, activity of the drug, Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-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, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal 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 patient's age, sex, and weight, and generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg daily Or it can be administered every other day or divided into 1 to 3 times a day. However, since the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., 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 dura mater or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient in any suitable way, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to target cells.

“Subject” in the present invention includes, but is not particularly limited to, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, mice, rabbits, or guinea pigs, for example. And, preferably, a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in various ways, such as food and beverages effective in preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, and dietary supplements, 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 at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

The dietary supplement of the present invention may contain, as an essential ingredient in the indicated proportions, the above compound as an essential ingredient, and may additionally contain, as an additional ingredient, food additives, such as natural carbohydrates and various flavoring additives.

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

As the flavoring agent, natural flavoring agents such as stevia such as taumatin, rebaudioside A or glycyrrhizine, and synthetic flavoring agents such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally used in about 1 to 20 g, preferably about 5 to 12 g per 100 ml of 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 flavoring agents, flavoring agents such as natural flavoring agents, coloring agents and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective colloids It may contain a thickening agent, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like.

In addition, the health functional food of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The ratio of these 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 through 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 following examples.

[Example]

Example  1: Ethanol extract and its object from the leaves and stems of the myrtle tree Fraction  Preparation and component analysis of each sample

After purchasing the above-ground parts of Misun trees grown in Goesan, Chungcheongbuk-do, Korea (Uncheon Farm: Endangered wild animals and plants storage notification: No. 2012-2), after removing foreign substances, they were divided into leaves and stems and used to prepare ethanol extracts. When preparing the ethanol extract, 10 times ethanol (95%, Deoksan, Korea) was added to each sample, extracted three times at room temperature, and the extract was collected and filtered, and then concentrated under reduced pressure to prepare a powder. Subsequently, the ethanol extract was subjected to sequential organic solvent fractionation with hexene, ethyl acetate, and butanol, and finally the water residue was recovered. The extraction efficiency of ethanol extraction, the efficiency of fractionation of organic solvents, and the results of component analysis of extracts / fractions are shown in Table 1. As a component analysis of the prepared ethanol extract and fractions, total polyphenols, total flavonoids, total sugars, and reducing sugars were measured. The total polyphenol content was 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ saturated solution in 400 μl of the extracted sample solution, allowed to stand at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol stirring for 18 hours, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample, 40 μl of 1N NaOH was added again, and the absorbance at 420 nm was measured after reacting at 37 ° C. for 1 hour. Rutin was used as a standard reagent. The reducing sugar was quantified using the DNS method and the total sugar was phenol-sulfuric acid method.

As a result, as shown in Table 1, the ethanol extract of the myrtle leaf showed 25.9%, and the ethanol extract of the stem showed an extraction efficiency of 5.8%, showing that the leaves were 4.5 times higher than the stem. Among the fractions of the leaf extract, the highest amount was butanol fraction (57.9% of extract), followed by ethyl acetate fraction (33.7%), hexene fraction (9.9%) and water residue (3.5%). On the other hand, in the case of the stem extract, butanol fraction (50.5% of the extract), water residue (26.6%), hexene fraction (20.6%) and ethyl acetate fraction (12.2%) was fractionated in this order. As a result of the component analysis, the total flavonoid and total flavonoid contents were higher in the leaf extract than in the stem extract, and in the case of the fraction, the leaves showed a high total flavonoid and total flavonoid content in the ethyl acetate fraction and the stem in the butanol fraction. On the other hand, in the case of total sugar and reducing sugar content, the leaf showed the highest content in the butanol fraction and the stem the water residue. These results indicate that the leaves and stems of the stalk tree are composed of distinctly different components.

Example  2: Ethanol leaf and stem ethanol extract and its sequential organic solvent minute Evaluation of human platelet aggregation inhibitory activity

The activity of inhibiting the activity of human platelet aggregation was evaluated for the ethanol extract of S. japonica and stem of Example 1 and fractions thereof, and the results are shown in Table 2 and FIG. 2. Platelets are disk-shaped small cells that circulate blood vessels with various blood cells, but instead of nucleus, they have cytoplasmic granules containing high concentrations of various substances related to vascular damage protection and platelet aggregation, and aggregation when damage to the vascular lining appears It is an important cell that secretes factors and forms a primary hemostatic plug by combining with collagen and the like exposed by damage of endothelial cells to initiate thrombosis, so inhibition of platelet aggregation is very important to prevent thrombus production Active. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Human platelets were used as 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). Thereafter, 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), 10 min at 3,000rpm After centrifugation, the suspension was suspended again in a suspending buffer, and the platelet number was adjusted to be 4x10 ⁹ / ml. Thereafter, 2.5 μl collagen was added to the 1 ml suspension for 5 minutes to react, and platelet aggregation was measured at 37 ° C. using a whole-blood aggregometer (Chrono-log, USA).

As a result, as shown in Table 2, in the case of DMSO, a solvent control, human platelets were rapidly and strongly aggregated by collagen addition, and aspirin, a platelet aggregation inhibitor, strongly inhibited platelet aggregation in a concentration-dependent manner. At this time, aspirin showed a degree of aggregation of 27.5% at a concentration of 0.25 mg / ml and a degree of aggregation of 58.3% at a concentration of 0.125 mg / ml, confirming the basis for clinical use as an antithrombotic agent. On the other hand, the stalk leaf and stem extract exhibited platelet aggregation of 155.8% and 179.3% at 0.25 mg / ml concentration, respectively, thereby facilitating thrombus generation, and the sequential organic solvent fraction of the leaf extract also showed stronger platelet aggregation activity. In the stem extract, butanol fraction and water residue showed platelet aggregation promoting activity, and the hexene fraction did not affect platelet aggregation. However, the ethyl acetate fraction of the stem extract showed a platelet aggregation degree of 71.5% at a concentration of 0.25 mg / ml, confirming that it strongly inhibits platelet aggregation.

Example  3: Ethanol leaf and stem ethanol extract and its object Fraction  Blood coagulation inhibitory activity evaluation

The blood coagulation inhibitory activity (thrombus production inhibitory activity) of the ethanol extract of the cactus leaf and stem prepared in Example 1 and their fractions 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; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time, The AP time was measured and evaluated. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and thrombin time, prothrombin time and apity time measurement were performed in the following process.

Thrombin Time

50 μl of 0.5U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ and 10 μl of sample extract of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) at 37 ° C. for 2 minutes, and then 100 μl of plasma was added. The time from addition to plasma clotting was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of a sample as a solvent control. In the case of DMSO, a coagulation time of 30.5 seconds was shown. 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 value of the solidification time when the sample was added divided by the solidification time of the solvent control.

Prothrombin time ( prothrombin  time)

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations are added to a tube of Amelung coagulometer KC-1A (Japan), heated at 37 ° C. for 3 minutes, then 130 μl of PT reagent is added and plasma is coagulated. Time until it 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 a sample as a solvent control. In the case of DMSO, a coagulation time of 16.7 seconds was shown. The prothrombin inhibitory activity was expressed as a value obtained by dividing the coagulation time when the sample was added by the coagulation time of the solvent control.

aPTT (activated Partial Thromboplastin  Time)

100 μl of plasma and 10 μl of sample extract of various concentrations are added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37 ° C. for 3 minutes, then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) is added, and again at 37 ° C. Incubated for 3 minutes. Thereafter, 50 μl CaCl ₂ (35 mM) was added, and then the time until plasma clot was measured. DMSO was used instead of the sample as a solvent control, and in this case, a coagulation time of 58.1 seconds was exhibited. The result of aPTT was expressed as the average value of the experiment repeated 3 times, and the blood coagulation factor inhibitory activity was expressed by dividing the aPTT at the time of adding the sample by the aPTT of the solvent control.

As a result, as shown in Table 3, the ethanol extract of the myrtle leaf and stem showed weak thrombin inhibition and blood coagulation factor inhibition at a concentration of 5 mg / ml, so that the thrombin time was 1.17 to 1.22 times, and the AP time was 1.19 to 1.38. Times extended. However, of the various fractions, the hexene fraction and ethyl acetate fraction showed superior anticoagulant activity compared to the ethanol extract, and the butanol fraction and water residue showed weaker activity than the ethanol extract. Therefore, the concentration-dependent anticoagulant activity of the hexene fraction and ethyl acetate fraction of the hedgehog stem ethanol extract showing excellent platelet aggregation inhibitory activity in Example 2 among the hexene fractions and ethyl acetate fractions having excellent anticoagulant activity was measured. Table 4 shows the results.

 As a result, the ethyl acetate fraction of the stalk extract of S. japonica showed excellent activity as the concentration increased, and the thrombin time, prothrombin time, and apity time were increased by 1.61, 1.55, and 1.77 times compared to the additive-free group at a concentration of 7 mg / ml. , Showed excellent anticoagulant activity. At this time, the aspirin used as a control was extended to 1.95 times, 1.40 times, and 1.63 times the thrombin time, prothrombin time, and apity time, respectively, at a concentration of 1.5 mg / ml. These results suggest that the ethyl acetate fraction of the myrtle stalk extract can be developed as a powerful antithrombotic agent that can replace aspirin, which is a gastrointestinal disorder.

Example  4: Ethanol leaf and stem ethanol extract and its object Fraction  Evaluation of human red blood cell hemolysis activity

The leaves and stems of the stalk tree have been used for medicinal purposes for a long time, and they are also considered to have no specific toxicity since they have been used for drinking (Dong-A Ilbo, March 30, 2011). Human red blood cell hemolytic activity was evaluated to evaluate the potential acute toxicity of stalk leaf, stem extract, and their fractions, and the results are shown in Table 5. At this time, hemolytic activity was evaluated according to the previous report (Hohn Son, 2014, Korean J. Microbiol. Biotechnol. 42: 285 ~ 292), and simply added 100 μl of human red blood cells washed three times with PBS to a 96-well microplate. After adding 100 μl of the sample solution of various concentrations, the mixture was reacted at 37 ° C. for 30 minutes, and then the reaction solution was centrifuged (1,500 rpm) for 10 minutes to transfer 100 μl of the supernatant to a new microtiter plate, and the degree of hemoglobin outflow due to hemolysis at 414 nm. It was measured. 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 hemolysis of red blood cells. 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 the DMSO addition port,

Abs . T: Triton X-100 Additive  Absorbance.

First, it was confirmed that DMSO and distilled water used as controls did not have hemolytic activity of red blood cells, and Triton X-100 was hemolytically lysed red blood cells at a concentration of 1 mg / ml. In addition, in the case of empoteracin B used as an anticancer agent, hemolytic activity of 68% or more was observed even at a low concentration of 0.006 mg / ml. On the other hand, the ethanol extracts from the leaves and stems of the aster tree did not show any hemolytic activity at all, and hemolytic activity was weak only in the hexene fraction of the extract among the fractions. However, hemolytic activity was negligible at concentrations below 0.5 mg / ml. In particular, the ethyl acetate fraction of the trunk of the hedgehog tree does not show any hemolytic activity at all, so it is judged that it will not exhibit a separate problem even when used as an actual antithrombotic agent.

Example  5: Ethyl acetate of ethanol extract of S. japonica Fraction  Plasma, acid and thermal stability assessment

Plasma stability, thermal stability, and acid stability of the ethyl acetate fraction of the ethanol extract of the coleopteran stem obtained in Example 1 for anticoagulant activity and platelet aggregation inhibitory activity were confirmed. The extract maintained excellent activity without loss of anticoagulant activity and platelet aggregation inhibitory activity even when heat treated at 100 ° C for 1 hour, treated at pH 2 (0.01M HCl) for 1 hour, and treated for 1 hour in plasma. The above results suggest that the ethyl acetate fraction of the ethanol extract of the trunk tree stalk is practically available as an antithrombotic agent.

Claims (4)

A pharmaceutical composition for the prevention or treatment of thrombosis, which contains the ethyl acetate fraction of the ethanol extract of the myrtle stem as an active ingredient. delete A platelet aggregation inhibitor containing ethyl acetate fraction of the stalk stem ethanol extract as an active ingredient. A health functional food for preventing or improving thrombosis, which contains ethyl acetate fraction of ethanol extract from the myrtle stem as an active ingredient.

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