KR20200122778A - Pharmaceutical composition comprising the extract of heracleum moellendorffi 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 a pharmaceutical composition and health functional food for preventing or treating thrombosis containing an extract of Heracleum moellendorffii as an active component. More specifically, the present invention relates to a pharmaceutical composition and health functional food for preventing or treating/alleviating thrombosis by inhibiting blood coagulation and inhibiting platelet aggregation, which contain a leaf or root extract of Heracleum moellendorffii as an active component. The extract of Heracleum moellendorffii, as the active component of the pharmaceutical composition and health functional food for preventing or treating thrombosis of the present invention exhibits strong antithrombotic activity due to an anti-aggregation effect of platelets, which play role in the inhibition of thrombogenesis-related enzymes and blood clotting factors, and initiation of thrombogenesis, and at the same time, has excellent thermal stability, and does not exhibit the loss of a blood coagulation factor inhibitory effect and a thrombogenesis-related enzyme inhibitory effect even in acidic conditions of pH 2 and plasma, thereby being expected to be used for preventing and treating thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. In addition, the active component is a very useful invention in the pharmaceutical industry and the food industry since the active component has an excellent effect of being processed in various forms such as extracts, powders, pills, and tablets, which are prepared in a form that can be taken at any time.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing Eosuri extract as an active ingredient {PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF HERACLEUM MOELLENDORFFI AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing an extract of Eosuri ( Heracleum moellendorffi ) as an active ingredient, and more specifically, a leaf or root extract of Eosuri as an active ingredient. It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation, and a health functional food.

As a component of the human body, blood has a variety of important functions such as transporting and buffering oxygen, nutrients, and waste products, maintaining body temperature, controlling osmotic pressure and maintaining ionic balance, maintaining moisture schedule, controlling fluid, maintaining and controlling blood pressure, and defending the body. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombosis dissolution system are complementarily regulated in the body. Among them, the mechanism of the blood coagulation reaction system is after platelets adhere and aggregate on the vessel wall to form platelet thrombi. , It has been reported that the blood coagulation system is activated and fibrin thrombus is formed around the platelet aggregate.

On the other hand, the formation of fibrin thrombi causes thrombin, which is involved in fibrin coagulation, through several step reactions of numerous blood coagulation factors, and finally produces fibrin monomers from fibrinogen, and fibrin monomers are polymerized by calcium, platelets and endothelium. It binds to the cell and forms a fibrin polymer cross-linked by factor XIII, creating a permanent blood clot. In addition, thrombin plays a central role in the formation of blood clots by activating platelets, factors V, and VII to promote blood clotting reactions. Therefore, the thrombin activity inhibitory substance can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood coagulation. On the other hand, in the endogenous thrombogenic pathway, the sequential activation of factor XII, factor XI, factor IX, and factor X, followed by prothrombin activation, is known to ultimately activate thrombin, and specific inhibition of blood coagulation factors is also important. Being a target. To date, various anticoagulants such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases, but they are not only very expensive, but also hemorrhagic side effects, gastrointestinal disorders, and irritability. The situation is limited to such use.

Heracleum moellendorffi ( Heracleum moellendorffi ) is a perennial plant of the dicotyledonous umbel , Ranunculus , and is distributed in Korea, Japan, and China. In Korea, it is commonly found in mountains and fields nationwide, and young leaves are used for food. The stem of the stalk is 70~150cm in height, straight, has a hollow cylinder shape, has vertical lines, has coarse hairs, and thick branches are split. Leaves are alternate, pinnate compound leaves composed of 3~5 small leaves. They have hairs, and petioles become shorter as they rise above the stem, and the lower part is wide to wrap the stem. The small leaf attached to the end is heart-shaped and divided into three, and the small leaf attached to the side is wide egg-shaped or triangular, 7-20cm long, divided into 2-3, and has a serrated deeply recessed at the edge. Flowers bloom in white from July to August, and the fruit has a branch shape, and a flat egg of 7mm in length is upside down and has a unique pattern on the upper part. In oriental medicine, Eosuri is called Northeast wubangpung and dog poison bow, has analgesic and anticonvulsant effects, and has been used to treat chronic bronchitis and hypertension.

As for Eosuri-related studies, studies on Eosuri's potent antioxidant activity and whitening effect have been most commonly conducted. Recently, Melanin Synthesis Inhibition and Antioxidant Effects of Eosuri Ethanol Extract (Seol-A Park, 2015, Master's Thesis at Wonkwang University), Isolation and Content Analysis of Flavonoids Exhibiting Peroxynitrite Scavenging Activity from Eosuri Young Leaves (Park Hee-Jun et al., 210, Korean J. Plant Resources 23, 393-398), Analysis of antioxidant activity and components of Eosuri according to extraction conditions (Bang Eun, 2009, Journal of Food Storage and Distribution, 16, 765-771), Antioxidant activity and anti-melanin effect of Eosuri leaf extract (Seo Beom-ju, 2015, Kyungpook National University's master's thesis), whitening effect by regulating the expression of ERK1/2-mediated MITF in Eosuri leaves (Alam MB et al., 2016, Int J Mol Sci. 17, E1844). Research on Eosuri's use of cosmetics include functional exploration of Eosuri leaf extract for the development of health functional foods and cosmetic materials (Lee So-Hyun, 2018, Master's thesis from Andong University), Eosuri's functional natural product development study (Kim Hee-Kwang, 2016, Master of Hoseo University) Thesis), etc. are known.

In addition, the antibacterial and antifungal activity of Eosuri (Ha Eun-su, 2004, Master's thesis of Chungnam National University), herbicidal activity against Dolpi (Geon-woo Kim, 2007, Kor. J. Weed Sci. 27, 285-295), and cancer cell growth inhibitory activity were reported. (Nakano Y et al., 1998, Biol Pharm Bull. 21, 257-261), and a study on the quality characteristics and storage properties of noodles added with Eosuri powder (Nam Yuhwa, 2010, Korean Journal of Food Storage and Distribution, 17, 602-607) Although also progressed, studies on Eosuri are very insignificant to date, and there are no known studies on antithrombotic activity in Eosuri.

As a patent related to Eosuri, Republic of Korea Patent Registration No. 10-1901133 [a cosmetic composition for improving skin wrinkles containing Eosuri extract as an active ingredient] is known, and in No. 10-1857651 [Alcoholic brain containing Eosuri extract Pharmaceutical composition for the prevention or treatment of nervous system diseases], No. 10-1862836, [Pharmaceutical composition for preventing or treating stress-related diseases], No. 10-1708250 [Esuri extract is effective Pharmaceutical composition for relieving hangover and protecting liver containing as an ingredient], No. 10-1313255 shows [a fragrance composition containing natural essential oil extracted from the leaves of Eosuri], and No. And a baking and a method of manufacturing the same containing Angelicae extract] is disclosed, and Korean Patent Laid-Open Publication No. 10-2015-0106088 discloses a [natural disinfectant fragrance composition] using Eosuri extract as an active ingredient. However, until now, no patents related to antithrombotic activity against Eosuri have been known.

KR 10-1901133 B KR 10-1857651 B KR 10-1862836 B

The present invention was conceived to solve the problems of the prior art as described above, and the problem to be solved in the present invention is a pharmaceutical composition and health function for the prevention or treatment/improvement of thrombotic diseases containing Eosuri extract as an active ingredient They want to provide food.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing an extract of Heracleum moellendorffi as an active ingredient.

The eosuri ( Heracleum moellendorffi ) extract is preferably a leaf extract of eosuri or a root extract of eosuri .

In addition, the present invention provides a health functional food for preventing or improving thrombosis, containing an extract of Heracleum moellendorffi as an active ingredient.

The eosuri ( Heracleum moellendorffi ) extract is preferably a leaf extract of eosuri or a root extract of eosuri .

Eosuri extract as an active ingredient of a pharmaceutical composition for preventing or treating thrombosis and a health functional food of the present invention has an inhibitory effect on the aggregation of platelets, which plays a role in the initiation of thrombus formation along with inhibition of thrombus formation-related enzymes and blood coagulation factors. It exhibits strong antithrombotic activity, has excellent thermal stability, and does not lose blood coagulation factor inhibitory effects and blood clotting-related enzyme inhibitory effects even in acidic conditions of pH 2 and plasma.Thus, ischemic stroke and hemorrhagic stroke through improvement of blood circulation It is expected to be used for the prevention and treatment of thrombosis such as, and the active ingredient is processed into various forms such as extracts, powders, pills, and tablets, and has excellent effects that can be prepared in a form that can be taken at any time, so the pharmaceutical industry And it is a very useful invention in the food industry.

Figure 1 shows the human platelet aggregation inhibitory activity of ethanol extracts of leaves or roots of Eosulis: 1: solvent control (DMSO), 2: aspirin (0.25mg/ml), 3: aspirin (0.125mg/ml), 4 : Eosuri leaf ethanol extract (0.25mg/ml), 5: Eosuri root ethanol extract (0.25mg/ml).

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy in Eosuri, the inventors of the present invention prepared the leaf extract and the root extract of Eosuri in a certain way and recovered them as antithrombotic active ingredients, and these extracts have excellent properties of heat stability and acid stability. By confirming that it has, the extract was intended to be used as a pharmaceutical composition and health functional food for the prevention or treatment/improvement of thrombosis.

Specifically, the inventors of the present invention to develop a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis using eosuri, which is known to be effective in various diseases such as vascular and circulatory system, digestive system, metabolic system, aging, etc. , Ethanol extracts were prepared from leaves and roots of Esuridae, and their antithrombotic activity was directly inhibited in human plasma and human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin Time), and active part thromboplastin time. (activated Partial Thromboplastin Time: aPTT) was evaluated to confirm that the leaf and root extracts had very strong antithrombotic activity by inhibiting blood coagulation and inhibiting platelet aggregation.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing an extract of Heracleum moellendorffi as an active ingredient.

The eosuri extract is preferably a leaf extract of eosuri or a root extract of eosuri.

In addition, the present invention provides a health functional food for preventing or improving thrombotic diseases containing Eosuri extract as an active ingredient.

The eosuri extract is preferably a leaf extract of eosuri or a root extract of eosuri.

Hereinafter, a method for preparing the Eosuri extract of the present invention and an efficacy experiment will be described in more detail.

The present invention comprises the steps of preparing an extract from leaves or roots of Eosulis; Evaluating the antithrombotic activity of the extract; And it includes the step of investigating the stability of the active substance of the leaf and root extract.

The "Eosuri extract" included in the composition of the present invention may be obtained by extracting the leaves or roots of Eosuri with an organic solvent and filtering the extract using a filter net of 0.06 mm or less, and concentrating it under reduced pressure. .

The organic solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc. Can be used, hot water, or 70% ethanol extraction is most preferred.

As a preferred embodiment, the eosuri extract of the present invention may be used by extracting eosuri with hot water or ethanol. Here, the hot water or ethanol extract may be sequentially or fractionated with an organic solvent of hexene, ethyl acetate, and butanol to additionally obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

In the present invention, as a result of measuring thrombin time, prothrombin time, and AP time by using a leaf extract and a root extract of Eosuri at a concentration of 5 mg/ml, a blood coagulation factor similar to aspirin (1.5 mg/ml) known as an antithrombotic agent It showed inhibition and showed significant thrombin and prothrombin inhibition. The thrombin time, prothrombin time, and AP time are all concentration-dependently prolonged, so the thrombin time, prothrombin time, and AP time were 1.60 times, 1.32 times and 1.81 times, respectively, at the concentration of 7mg/ml in the case of Esuri leaf extract. At 7mg/ml concentration of Eosuri root extract, thrombin time, prothrombin time, and APT time were 1.48 times, 1.21 times and 1.93 times, respectively, compared to the non-added group.

In addition, as a result of evaluating the inhibition of platelet aggregation using Eosuri extract at a concentration of 0.25mg/ml, the leaves and root extracts showed an aggregation rate of 71.36% and 75.5%, respectively, similar to 0.125mg/ml of aspirin used as an antithrombotic agent. Platelet aggregation inhibitory activity was shown. These results show excellent antithrombotic activity even though the leaves and root extracts of Eosuri are unrefined.In particular, considering that Eosuri leaves are edible as herbs in large quantities, anticoagulants such as aspirin, which have high concerns about side effects. , Suggests that antiplatelet drugs can be substituted.

The Eosuri extract of the present invention may be prepared as a powder through a conventional powdering process such as vacuum drying, freeze drying, or spray drying. They are not decomposed by various degrading enzymes in plasma, and maintain their activity even at a heat treatment of 100°C and a pH of 2 in the stomach.

The active ingredient of the present invention can be used for 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, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis. , Deep vein thrombosis, lower extremity swelling, pain, and acute peripheral arterial atresia. As venous thrombosis, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral sinus thrombosis, and central retinal vein occlusion.

Pharmaceutical compositions containing the active ingredients of the present invention are oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to a conventional method for each purpose of use, and injections of sterile injectable solutions It may be formulated and used in various forms such as, and may be administered orally or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions 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, gum acacia, 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 contain a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

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

As a preferred embodiment, as a liquid formulation for oral use, a suspension, a liquid formulation, an emulsion, a syrup, and the like may be exemplified, and various excipients other than water and liquid paraffin, which are commonly used simple diluents, such as wetting agents, sweetening agents, Fragrances, preservatives, etc. may be included.

As a preferred embodiment, the preparation for parenteral administration may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized agent, a suppository, 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 ethyloleate. Injectables may contain conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to 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 disease, severity, drug activity, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

As 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 body weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kilogram of body weight daily Alternatively, it may be administered every other day or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount is not limited by any method.

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

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, 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.

In the present invention, the "object" is not particularly limited, but includes, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, mouse, rabbit, or guinea pig And, 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 in preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules or beverages. .

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

The health functional food of the present invention may contain the compound as an essential component in the indicated ratio as an additional component, as well as food supplementary additives acceptable for food, such as natural carbohydrates and various flavoring agents.

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

Taumatin as the flavoring agent; Natural flavoring agents such as stevia, such as rebaudioside A or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame, may be used. 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 includes a variety of nutrients, vitamins, minerals, synthetic flavors, and flavoring agents such as natural flavors, colorants and thickeners, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juices, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion 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 present invention will be described in more detail through examples. The following examples are only one preferred specific example of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example 1: Preparation of Eosuri extract and analysis of useful components thereof

Eosuri harvested in Andong, Gyeongbuk in 2018 was purchased and used to prepare ethanol extract. Specifically, leaves and roots of Eosuridae were separated, 10 times ethanol (70% ethanol) was added to each sample, and extraction was repeated twice at room temperature. Next, the extract was collected, filtered, and concentrated under reduced pressure to prepare a powder to prepare an ethanol extract.

The content of total polyphenols, total flavonoids, total sugars and reducing sugars was measured by component analysis of the Eosuri extract. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample, and allowed to stand at room temperature for 1 hour, and the absorbance was measured at 725 nm. Tannic acid was used as a standard reagent. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample solution, 40 μl of 1 N NaOH was added, and the absorbance was measured at 420 nm after 1 hour reaction at 37°C. Rutin was used as a standard reagent. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Analysis of extraction efficiency and useful components of extracts by part of Eosuri

As shown in Table 1, Esuri ethanol extraction efficiency was found to be about 4% higher in leaves than roots, and as a result of analysis of useful components of extracts by site, the total polyphenol content of leaf extracts was 5.4 times higher than that of root extracts at 85.7mg/ g. The total flavonoid content was also 113.1mg/g, which was about 24 times higher than that of the root extract in the leaf extract. These results are similar to the previous report on the strong antioxidant activity of leaf extracts, and were understood as the basis for the excellent physiological activity of Essuri leaves. On the other hand, as a result of the total sugar analysis, 856.1mg/ml in the root extract was 1.3 times higher than that of the leaf extract, but in the case of reducing sugar, the leaf extract was 222.4mg/ml, 1.27 times higher than the root extract. Therefore, it was found that the root extract contained a large amount of non-reducing sugar, while the leaf extract contained a significant amount of reducing sugar.

Example 2: Evaluation of blood coagulation inhibitory activity of Eosuri extracts

The blood coagulation inhibitory activity of Eosuri ethanol extract of Example 1 was evaluated, and the results are shown in Table 2. At this time, the method of evaluating the blood coagulation inhibitory activity of Eosuri extract was evaluated according to 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 and AP time were measured. Plasma was used as a commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and the thrombin time, prothrombin time, and AP measurement methods were performed as follows.

Thrombin Time

50 μl of 0.5U thrombin (Sigma Co., USA) at 37°C, 50 μl of 20 mM CaCl ₂ , and 10 μl of sample extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes, and then 100 μl of plasma was mixed. After addition, the time until plasma coagulation was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. In the case of DMSO, the coagulation time was 32.1 seconds. The thrombin inhibitory effect was expressed as the average value of experiments repeated three or more times, and the thrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

Prothrombin time

70μl of standard plasma (MD Pacific Co., China) and 10μl of sample solution of various concentrations were added to the tube of Amelung coagulometer KC-1A(Japan), heated at 37℃ for 3 minutes, and then 130μl of PT reagent was added and plasma coagulated. The time until the result 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. In the case of DMSO, the coagulation time was 18.1 seconds. The prothrombin inhibitory activity was expressed by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

aPTT(activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of sample extracts of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37°C for 3 minutes, 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added, and then 3 at 37°C. Incubated for minutes. Thereafter, after 50 μl CaCl ₂ (35 mM) was added, the time until plasma coagulation was measured. DMSO was used instead of the sample as a solvent control, and in this case, the coagulation time was 55.1 seconds. The result of aPTT was expressed as the average value of the experiment repeated three times, and the blood coagulation factor inhibitory activity was expressed by dividing the aPTT at the time of sample addition by the aPTT of the solvent control.

[Table 2] Anticoagulant Activity of Extracts by Eosuri Region

As shown in Table 2, as a result of measuring thrombin time, prothrombin time, and AP time using Eosuri leaf and root extracts at a concentration of 5 mg/ml, respectively, blood similar to aspirin (1.5 mg/ml) known as an antithrombotic agent It showed coagulation factor inhibition (prolonged AP time), and showed significant thrombin and prothrombin inhibition. The above thrombin time, prothrombin time, and AP time were all concentration-dependently prolonged, so the thrombin time, prothrombin time, and AP time were 1.60 times, 1.32 times and 1.81 times, respectively, at a concentration of 7 mg/ml in the case of Esuri leaf extract. At the concentration of 7mg/ml, Eosuri root extract exhibited thrombin time, prothrombin time, and APT time 1.48 times, 1.21 times and 1.93 times, respectively, compared to the non-added group. These results show excellent anticoagulant activity even though the leaves and root extracts of Eosuri are unrefined. Especially, considering that Eosuri leaves are edible as herbs in large quantities, anticoagulants such as aspirin, which have high concerns about side effects. Suggests that it can be replaced.

Example 3: Platelet Aggregation Inhibitory Activity of Eosuri Extract

The ethanol extract of Example 1 was evaluated for the activity of inhibiting human platelet aggregation, and the results are shown in Table 3 and FIG. 1. Platelets are disk-shaped small cells that circulate in blood vessels with various blood cells. Instead of having a nucleus, platelets have cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentrations, and aggregation when damage to the inner wall of blood vessels occurs. It is an important cell that secretes factors and forms a primary hemostatic plug by binding to collagen exposed due to damage to endothelial cells to initiate thrombus generation. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombus formation. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Platelets were concentrated human platelets, which were washed once with a washing buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 1mM EDTA, pH 6.5). Thereafter, re-suspended 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 at 3,000 rpm for 10 minutes After centrifugation, it was resuspended in the suspending buffer again, and at this time, the number of platelets was adjusted to be 4x10 ⁹ /ml. Thereafter, 2.5 μl collagen was added to the 1 ml suspension to react for 5 minutes, and platelet aggregation was measured at 37° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 3] Platelet Aggregation Inhibitory Activity of Extracts by Eosuri Region

As shown in Table 3 and FIG. 1, aspirin has a concentration of 32.5% at a concentration of 0.25 mg/ml and an aggregation rate of 68.6% at a concentration of 0.125 mg/ml, showing strong platelet aggregation inhibition in a concentration-dependent manner. The grounds used were known. On the other hand, as a result of evaluating the inhibition of platelet aggregation using Eosuri extract at a concentration of 0.25 mg/ml, platelets similar to 0.125 mg/ml of aspirin used as an antithrombotic agent showed an aggregation rate of 71.36% and 75.5%, respectively, in leaf and root extracts. It showed an anti-aggregation activity. These results can be understood as exhibiting excellent platelet aggregation inhibitory activity even though the leaves and root extracts of Eosuri are unrefined.In particular, considering that Esuri leaves are edible as herbs in large quantities, there are concerns about existing side effects. It has been suggested that antiplatelet drugs such as high aspirin can be replaced.

Example 4: Evaluation of Plasma, Acid and Thermal Stability of Essuri Leaf and Root Extract

Plasma stability, thermal stability, and acid stability for antithrombotic activity were confirmed for the extracts of Eosuri leaves and roots obtained in Example 1 above. Eosuri leaf and root extracts did not show a decrease in blood coagulation inhibition and platelet aggregation inhibitory activity even after 1 hour heat treatment at 100°C, 1 hour treatment at pH 2 (0.01M HCl), and 1 hour treatment in plasma. Therefore, it was confirmed that the extract of Eosuri contains antithrombotic active substances of various components having acid resistance and heat resistance.

Claims (4)

Eosuri ( Heracleum moellendorffi ) A pharmaceutical composition for preventing or treating thrombosis containing an extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the extract of Heracleum moellendorffi is a leaf extract of Eosuri or a root extract of Eosuri . Eosuri ( Heracleum moellendorffi ) A health functional food for preventing or improving thrombosis containing extract as an active ingredient. [4] The health functional food according to claim 3, wherein the Eosuri ( Heracleum moellendorffi ) extract is Eosuri leaf extract or Eosuri root extract.

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