KR102120490B1 - Pharmaceutical composition for prevention or treatment of thrombosis comprising the ethylacetate fraction of shiitake mushroom extract as an effective component and health functional food comprising the same - Google Patents

Abstract

The present invention relates to an antithrombotic composition containing an ethyl acetate fraction of shiitake mushroom (Shiitake mushroom, Lentinus edodes ) extract as an active ingredient, and more specifically, containing an ethyl acetate fraction of shiitake mushroom hot water extract as an active ingredient. A pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood clotting and inhibition of platelet aggregation, and a health functional food. Ethyl acetate fraction of shiitake mushroom extract as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis of the present invention, as demonstrated through the examples herein, thrombus production-related enzyme inhibition and blood clotting factors It exhibits strong antithrombotic activity and platelet aggregation inhibitory activity by inhibition of, and at the same time, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, and has an enzyme and blood coagulation related to thrombus production even in acidic conditions and plasma of pH 2 Since there is no loss of factor inhibitory effect and platelet aggregation inhibitory effect, it is expected to be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredient is an extract, powder, pill, tablet It is a very useful invention in the pharmaceutical industry and the food industry because it has an excellent effect that can be prepared in a form that can be always taken by being processed in various forms such as.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF THROMBOSIS COMPRISING THE ETHYLACETATE FRACTION OF SHIITAKE MUSHROOM EXTRACT AS AN EFFECTIVE COMPONENT AND HEALTHTHAL FOOD COMPRISING THE SAME}

The present invention relates to an antithrombotic composition containing an ethyl acetate fraction of shiitake mushroom (Shiitake mushroom, Lentinus edodes ) extract as an active ingredient, and more specifically, containing an ethyl acetate fraction of shiitake mushroom hot water extract as an active ingredient. A pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood clotting and inhibition of platelet aggregation, and a health functional food.

Blood as a component of the human body has various important functions such as transport function and buffer 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 regulation of blood pressure, and biological defense. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolytic 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.

The production of fibrin thrombus is a thrombin that is involved in fibrin coagulation through several step reactions of a number of blood coagulation factors, finally producing fibrin monomer from fibrinogen, and the fibrin monomers are polymerized by calcium to platelet and endothelial cells. It binds and forms 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, an active inhibitor of thrombin can be used as a very useful preventive and therapeutic agent for various thrombotic diseases caused by excessive blood clotting abnormalities.

Meanwhile, in the endogenous thrombus production 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, and thus specific inhibition of the blood clotting factor is also important. It has become a target for the development of therapeutic agents for sexual diseases, and in the case of the exogenous thrombus production pathway, thrombus generation according to activation of factor II (prothrombin), factor V, factor VII, and factor X is known.

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 these are not only very expensive, but also have bleeding side effects and gastrointestinal disorders and irritability. The use of the reaction is limited.

On the other hand, mushrooms belong to the fungi taxonomy, but unlike the general fungi, they are characterized by making large fruiting bodies to form spores. Mushrooms are excellent in flavor and contain various nutrients such as carbohydrates, proteins, lipids, minerals, vitamins and minerals, and are foods that have been widely used for edible and medicinal purposes by producing bioactive substances. In addition, β-glucan of mushrooms has been reported to have an anti-tumor effect that recognizes and attacks mutant cells by stimulating the function of immune active agents, antioxidant capacity, regeneration and healing of biological tissues, antibiotics, antibacterial, antiviral and macrophages. have.

In particular, Shiitake Mushroom is an edible mushroom belonging to the genus Pleurotus japonica genus Pleurotus and Pine nut, which has the most vitamin C content and contains a large amount of various amino acids and ergosterol. In terms of health function, anti-cancer, lung and gastrointestinal diseases are prevented, immune enhancement against viruses, antibody production is promoted, blood pressure is lowered, arteriosclerosis is prevented, blood lipid concentration control function, diabetes inhibitory effect are known.

Patents related to shiitake mushrooms include Korean Registered Patent No. 10-1839507 [Manufacturing method of seasoned raw salted shiitake containing shiitake mushrooms and fermented shiitake mushrooms], No. 10-1830763 [Method for manufacturing rice miso containing shiitake mushroom extract And rice miso prepared therefrom], No. 10-1823460 [Method for producing semi-dried rug using shiitake mushroom powder], No. 10-1759312 [Method for preparing shiitake mushroom powder, preparation of nutrition bar using shiitake mushroom powder] Method and nutrition bar produced thereby], No. 10-1673573 [Method of manufacturing shiitake mushroom tea and shiitake mushroom tea prepared therefrom] is disclosed, and Republic of Korea Patent Publication No. 10-2004-0038957 [Shiitake mushroom powder Foods with the theme of manufacturing laver) are published. Most of the patents known to date are related to shiitake mushroom cultivation methods and various food preparations using shiitake mushrooms, and patents on useful physiological activity of shiitake mushrooms are very limited.

In addition, as a patent related to useful physiological activity of shiitake mushrooms, Korean Patent No. 10-1487742 [Food composition having antioxidant efficacy including shiitake mushrooms and cheonma extract], No. 10-1806808 [fermentation using shiitake mushroom mycelium Composition for the prevention, improvement or treatment of menopausal symptoms of women, containing fermented red pepper sewage as an active ingredient, No. 10-1611947 [Health food composition for preventing and improving vascular disease and its manufacturing method], No. 10-1266528 [Moisturizing and/or whitening cosmetic composition containing polysaccharide extracted from shiitake mushroom mycelium culture as an active ingredient] is disclosed, and Patent Publication No. 10-2017-0036912 [Atopy containing shiitake mushroom extract as an active ingredient And composition for preventing and improving contact dermatitis], Patent Publication No. 10-2003-0056753 [Shiitake mushroom mycelium, Agaricus mycelium, health food composition for controlling obesity containing water-soluble chitosan], No. 10-2008-0110212 Compositions to help bone length growth using hydrothermal extracts are disclosed. In particular, the Republic of Korea Registered Patent No. 10-1611947 related to thrombosis is characterized by including Heeheung, Hasuo, Seokchangpo, Wonji, Shiitake mushrooms and Mushrooms as active ingredients [Health food composition for preventing and improving vascular disease and its Manufacturing method] is disclosed, but to date, a safe and practically available antithrombotic composition through strong blood clotting inhibition derived from shiitake mushrooms is known as a main component of medicinal disorders.

KR 10-1611947 B

The present invention has been devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to prepare a fat-soluble ethyl acetate fraction from shiitake mushroom extract and to contain an antithrombotic pharmaceutical composition containing it as an active ingredient. And to provide health functional food.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, containing the ethyl acetate fraction of shiitake mushroom extract as an active ingredient.

The shiitake mushroom extract is preferably a shiitake mushroom hot water extract.

In addition, the present invention provides a health functional food for preventing or improving thrombosis comprising the ethyl acetate fraction of the shiitake mushroom extract of the present invention.

Ethyl acetate fraction of shiitake mushroom extract as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis of the present invention, as demonstrated through the examples herein, thrombus production-related enzyme inhibition and blood clotting factors It exhibits strong antithrombotic activity and platelet aggregation inhibitory activity by inhibition of, and at the same time, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, and has an enzyme and blood coagulation related to thrombus production even in acidic conditions and plasma of pH 2 Since there is no loss of factor inhibitory effect and platelet aggregation inhibitory effect, it is expected to be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredient is an extract, powder, pill, tablet It is a very useful invention in the pharmaceutical industry and the food industry because it has an excellent effect that can be prepared in a form that can be always taken by being processed in various forms such as.

Figure 1 shows the activity of human platelet aggregation inhibitory activity of shiitake mushroom hot water extract and its sequential organic solvent fraction. 1: solvent control (DMSO), 2: aspirin (0.25 mg/ml), 3: aspirin (0.125 mg/ml), 4: shiitake hot water extract (0.25 mg/ml), 5: ethyl acetate of shiitake hot water extract Fraction (0.25mg/ml), 6: Ethyl acetate fraction of shiitake mushroom hot water extract (0.125mg/ml), 7: Ethyl acetate fraction of shiitake mushroom hot water extract (0.063mg/ml) 8: Butanol fraction of shiitake mushroom hot water extract (0.25mg/ml), 9: Water residue (0.25mg/ml) after the organic solvent fraction of shiitake mushroom hot water extract.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepare hexene, ethyl acetate, butanol fractions and water residues from shiitake mushroom hot water extracts prepared by a certain method in order to test the antithrombogenic efficacy of shiitake mushroom extracts, and the fractions and water residues. By evaluating the antithrombotic activity, the ethyl acetate fraction of the shiitake mushroom hot water extract was recovered as an antithrombotic component, and the fraction showed excellent hemolytic activity against human red blood cells, and showed excellent thermal stability and acid stability. The fraction was intended to be used as a pharmaceutical composition and health functional food for prevention or treatment/improvement of thrombosis.

Specifically, the present inventors use shiitake mushroom extract 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, firstly, hot water extract from shiitake mushroom and ethanol Preparation of extracts, antithrombotic activity, thrombin time, prothrombin time, and blood coagulation factor inhibition (active partial thromboplastin time: aPTT) and platelets The aggregation inhibitory activity was evaluated. As a result, it was confirmed that these extracts had little antithrombotic activity. Next, the present inventors prepared a sequential organic solvent fraction for shiitake mushroom hot water extract to evaluate blood clotting inhibitory ability and platelet aggregation inhibitory ability, and unlike the extracts showed little antithrombotic activity, blood clotting inhibitory activity and platelet aggregation The present invention was completed by identifying the ethyl acetate fraction capable of effectively inhibiting all inhibitory activity.

The efficiency of the hot water extract of shiitake mushroom was 34.7%, the ethyl acetate fractionation efficiency of the extract was 0.5%, and the ethyl acetate fraction per 100 g of shiitake mushrooms was able to recover 0.17 g, thereby producing an antithrombotic agent in a very pure state. The fractions are 3.32 times and 3.12 times, respectively, at 5 mg/ml concentrations, compared to no additives. The thrombin time, prothrombin time, and apity time extended by 2.18 times or more were shown, and especially thrombin time extended by 15 times or more compared to the additive-free group at 6 mg/ml concentration was confirmed. In addition, the fraction showed a platelet aggregation inhibitory activity of 36.6% at a concentration of 0.25 mg/ml, which corresponds to a better platelet aggregation inhibitory activity than aspirin of the same concentration. It was also confirmed that the ethyl acetate fraction of shiitake mushroom hot water extract exhibiting such excellent antithrombotic activity did not cause hemolytic activity against human red blood cells and thus 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 shiitake mushroom extract as an active ingredient.

Shiitake mushroom extract, which is an active ingredient of the pharmaceutical composition, is preferably shiitake mushroom hot water extract.

In addition, the present invention provides a health functional food for preventing or improving thrombosis, including the ethyl acetate fraction of shiitake mushroom extract.

Shiitake mushroom extract, which is an active ingredient of the health functional food, is preferably shiitake mushroom hot water extract.

Hereinafter, a method for producing an ethyl acetate fraction of shiitake mushroom extract of the present invention and an efficacy test will be described in more detail.

In order to achieve the object of the present invention, the inventors of the present invention prepare a extract from pickled and washed shiitake mushrooms; Fractionating the organic solvent from the extract to prepare a hexene, ethyl acetate, butanol fraction and water residue; Experiments of the anti-thrombotic activity evaluation of the fraction and water residue and stability evaluation of the active material were performed.

As a preferred embodiment, the "ethyl acetate fraction of shiitake mushroom extract" contained in the composition of the present invention comprises the steps of extracting shiitake mushrooms with a solvent, fractionating the extract sequentially with an organic solvent, and filtering the fraction with a filter network of 0.06 mm or less. And filtered, and concentrated under reduced pressure.

The solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), mixed solvents of the lower alcohol and water, etc. It can be used and is preferably hot water.

The organic solvent used in the present invention may be hexene, ethyl acetate and butanol, and each organic solvent fraction may be prepared through sequential or individual fractions.

The ethyl acetate fraction of the shiitake mushroom extract 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 in the human stomach of pH 2.

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, sensory abnormalities, personality changes, decreased vision, epilepsy attacks, pulmonary thrombosis , Deep vein thrombosis, swelling of the lower extremities, pain and acute peripheral artery occlusion, etc. Examples of venous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, brain venous sinus thrombosis and central retinal vein occlusion.

Since the active ingredient of the present invention has excellent blood coagulation inhibitory activity and platelet aggregation inhibitory activity as described above, it may be specified and used for pharmaceutical use as an anticoagulant that is a blood coagulation inhibitor or an antiplatelet agent that is a platelet aggregation inhibitor.

The pharmaceutical composition comprising 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 carrier, excipient, or diluent 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 suspending agents, solvent solutions, 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 preparation for parenteral administration may include sterilized 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 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, "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 drugs, 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, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., so that the 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 the active ingredient to target cells.

“Subject” in the present invention is not particularly limited, and 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 used in a variety of 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, 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 contain, as an additional ingredient, food additives, such as natural carbohydrates and various flavoring additives.

Examples of the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and sugars such as xylitol, sorbitol, and erythritol, and common sugars 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 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 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 beverage and vegetable beverage. 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, a 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: Preparation and component analysis of shiitake mushroom extract

A powder product of domestic shiitake mushrooms cultivated in Mungyeong, Gyeongbuk, was purchased and used to prepare hot water extract and ethanol extract. When preparing the hot water extract, 20 times of distilled water was added to the shiitake mushroom powder sample, and extracted at 100°C for 1 hour and 3 times, the extract was collected and filtered, and then concentrated under reduced pressure to prepare a powder. When preparing the ethanol extract, 10 times ethanol (95%, Deoksan, Korea) was added to the shiitake mushroom powder sample, extracted three times at room temperature, the extract was collected, filtered, and concentrated under reduced pressure to prepare a powder. Table 1 shows the results of each extraction efficiency and component analysis.

Total polyphenols, total flavonoids, total sugars, and reducing sugar content were measured by analyzing the components of the prepared extract. 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, and allowed to stand at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. The total flavonoid content was extracted by stirring each sample for 18 hours with methanol, 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 was measured at 420 nm 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.

[Table 1] Extraction efficiency and component analysis of shiitake mushroom extract

Example 2: Evaluation of blood coagulation inhibitory activity of shiitake mushroom extract

The coagulation inhibitory activity of shiitake mushroom extracts 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; Ryu et al. 2010. J. Life Science, 20. 922-928), were evaluated by measuring thrombin time, prothrombin time, and affinity time, but with shiitake mushroom hot water extract and ethanol The extract was found to have little activity at thrombin time, prothrombin time and apity time at a concentration of 5 mg/ml (Table 2).

Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China). Thrombin time, prothrombin time and aptitude measurement were performed as follows.

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

After adding 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations to the tube of Amelung coagulometer KC-1A (Japan), heating at 37° C. for 3 minutes, and then adding 130 μl of PT reagent and coagulating plasma 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. 3 Incubated for a minute. Then, after adding 50 μl CaCl ₂ (35 mM), the time until the plasma was clot was measured. DMSO was used as a solvent control instead of a sample, 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.

[Table 2] Evaluation of blood coagulation inhibitory activity of shiitake mushroom extract

Example 3: Evaluation of shiitake mushroom extract activity against human platelet aggregation

Platelets are discoid small cells that circulate through blood vessels with various blood cells, but instead of having nuclei, they have cytoplasmic granules containing high concentrations of various substances related to blood vessel damage protection and platelet aggregation, and when damage to the inner walls of blood vessels appears, It is an important cell that secretes agglutination factors and forms primary hemostatic plugs by combining with collagen exposed due to damage to endothelial cells to initiate thrombosis. Therefore, since platelet aggregation inhibition is a very important activity for preventing thrombus generation, human platelet aggregation inhibitory activity was evaluated for shiitake mushroom extracts of Example 1, and the results are shown in Table 3 and part of FIG. In the case, it was found that there was almost no inhibitory activity on platelet aggregation, and in the case of ethanol extract, it was confirmed that platelet aggregation promoting activity was observed.

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, it was suspended again in the 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 and reacted for 5 minutes, and platelet aggregation was measured at 37° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 3] Evaluation of platelet aggregation inhibitory activity of shiitake mushroom extract

Example 4: Evaluation of human erythrocyte hemolysis activity of shiitake mushroom extract

Shiitake mushrooms are widely used as foods by themselves, so it is judged that shiitake extracts do not show any specific toxicity. To evaluate the potential acute toxicity of shiitake mushroom extract, human red blood cell hemolytic activity was evaluated, and the results are shown in Table 4.

At this time, the hemolytic activity was evaluated according to the previous report (Hohn Son, 2014, Korean J. Microbiol. Biotechnol. 42: 285~292), and simply 100 μl of human red blood cells washed three times with PBS in a 96-well microplate. After adding 100 μl of sample solution of various concentrations and reacting for 30 minutes at 37° C., the reaction solution was centrifuged for 10 minutes (1,500 rpm) to transfer 100 μl of the supernatant to a new microtiter plate, and the degree of hemoglobin outflow due to hemolysis was 414 nm. It was 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 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 DMSO house furniture,

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

[Table 4] Human red blood cell hemolytic activity of shiitake mushroom extract

First, it was confirmed that DMSO and distilled water used as a control 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. The anticancer agent empoteracin B, which has been reported to have acute toxicity, showed hemolytic activity of more than 97% at a concentration of 0.025 mg/ml. On the other hand, shiitake mushroom hot water extract and ethanol extract did not show any hemolytic activity even at the concentration of 1mg/ml as expected.

Example 5: Preparation and component analysis of the organic solvent fraction of shiitake mushroom hot water extract

Despite the results of Example 2 and Example 3, the inventors of the present invention sequentially fractionated organic solvents with hexene, ethyl acetate, butanol as a target for hot water extract among shiitake mushroom extracts obtained from Example 1, and finally retained water Water was recovered. Each organic solvent fraction efficiency and fractional component analysis were performed in the same manner as in Example 1, and the results are shown in Table 5.

[Table 5] Fractional efficiency and component analysis of organic solvent fraction of shiitake mushroom hot water extract

As shown in Table 5, 81.3% of the hot water extract was transferred to the water residue, followed by butanol fraction (21.9% of extract), ethyl acetate fraction (0.5%) and hexene fraction (0.1%). The content of total polyphenols and total flavonoids among the components was highest in the ethyl acetate fraction, and it was found that even in the case of total sugar, it was present in a fairly high level in the ethyl acetate fraction. Accordingly, it is understood that the ethyl acetate fraction contains a very high content of polyphenols, flovonoids and glycoside-linked glycoside compounds.

Example 6: Organic solvent of shiitake mushroom hot water extract Fraction Evaluation of blood clotting inhibitory activity

The thrombin time, prothrombin time, and affinity time of the fractions of shiitake mushroom hot water extracts obtained from Examples 1 and 5 were measured for thrombin time, prothrombin time, and affinity time in the same procedure as in Example 2. Was evaluated.

[Table 6] Evaluation of blood coagulation inhibitory activity of organic solvent fraction of shiitake mushroom hot water extract

As a result, as shown in Table 6, among the organic solvent fractions sequentially fractionated from shiitake mushroom hot water extract, the ethyl acetate fraction showed strong anticoagulant inhibitory activity at both thrombin time, prothrombin time and apity time. In particular, the ethyl acetate fraction exhibited a thrombin time that was extended by 15 times or more at a concentration of 6 mg/ml, thus exhibiting a completely different thrombus production inhibition pattern than the hot water extract. These results indicate that the ethyl acetate fraction of shiitake mushroom hot water extract of the present invention contains a low molecular weight fat-soluble substance as an anticoagulant factor. In addition, this result suggests that the ethyl acetate fraction of shiitake mushroom hot water extract can be developed as a powerful antithrombotic agent, especially an anticoagulant, capable of replacing aspirin, which indicates gastrointestinal disorders.

Example 7: Organic solvent of shiitake mushroom hot water extract Fraction Evaluation of human platelet aggregation inhibitory activity

The sequential organic solvent fractions of shiitake mushroom hot water extracts obtained from Examples 1 and 5 were evaluated for human platelet aggregation inhibitory activity in the same manner as in Example 3, and the results are shown in Table 7 and FIG. 1.

[Table 7] Evaluation of platelet aggregation inhibitory ability of organic solvent fraction of shiitake mushroom hot water extract

As shown in Table 7 and FIG. 1, 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 exhibited a cohesiveness of 39.3% at a concentration of 0.25 mg/ml and a cohesiveness of 44.7% at a concentration of 0.125 mg/ml, confirming the basis for clinical use as an antithrombotic agent. On the other hand, among the sequential organic solvent fractions of shiitake mushroom hot water extract, ethyl acetate fraction showed a cohesiveness of 36.6% superior to that of aspirin at a concentration of 0.25 mg/ml, confirming strong platelet aggregation inhibitory activity, and this hot water extract itself inhibited platelet aggregation. This is a very unusual result in light of showing little activity. Therefore, it is judged that the ethyl acetate fraction of shiitake mushroom hot water extract exhibits strong platelet aggregation inhibitory activity, so that it can be practically used as an antithrombotic agent, especially as an antiplatelet agent.

Example 8: Evaluation of human erythrocyte hemolysis activity of ethyl acetate fraction of shiitake mushroom hot water extract

As shown in Example 4, as a result of evaluating the potential acute toxicity of shiitake mushroom hot water extract, it is expected that there is no acute toxicity even in its ethyl acetate fraction by confirming that there is no human erythrocyte hemolytic activity. Human erythrocyte hemolysis activity of the ethyl acetate fraction of shiitake mushroom hot water extract prepared through Examples 1 and 5 was evaluated in the same manner as in Example 4, and the results are shown in Table 8.

[Table 8] Human erythrocyte hemolysis activity of ethyl acetate fraction of shiitake mushroom hot water extract

First, it was confirmed that DMSO and distilled water used as a control 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. The anticancer agent empoteracin B, which has been reported to have acute toxicity, showed hemolytic activity of more than 97% at a concentration of 0.025 mg/ml. On the other hand, the ethyl acetate fraction of shiitake mushroom hot water extract showed a weak hemolytic activity of about 34% at a concentration of 1 mg/ml, but it was a problem of practical use due to weak hemolytic activity of 1/160 times that of the control empoteracin B. It was judged not to be.

Example 9: Ethyl acetate of shiitake mushroom hot water extract Fraction Plasma, acid and thermal stability assessment

Plasma stability, thermal stability and acid stability of the ethyl acetate fraction of shiitake mushroom hot water extracts obtained in Examples 1 and 5 for anticoagulant activity and antiplatelet activity were confirmed. The fraction maintained excellent activity without loss of anticoagulant activity and platelet aggregation inhibitory activity even when heat treated at 100°C for 1 hour, treated for 1 hour at pH 2 (0.01M HCl), and treated for 1 hour in plasma. Therefore, considering the component analysis results of Example 5, the characteristics of the organic solvent fraction, and the stability results, the ethyl acetate fraction of shiitake mushroom hot water extract is expected to be a glycoside of a phenolic compound. The above results suggest that the ethyl acetate fraction of shiitake mushroom hot water extract can be practically used as an antithrombotic agent, more specifically, an anticoagulant or an antiplatelet agent, and can supplement and replace aspirin in which side effects such as gastrointestinal disorders have been reported. Is judged.

Claims (3)

A blood clotting inhibitor containing the ethyl acetate fraction of shiitake mushroom hot water extract as an active ingredient. delete delete

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