Pharmaceutical composition comprising the extract of ganoderma lucidum fruiting body as an effective component for prevention or treatment of thrombosis and health functional food comprising the same

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis containing an extract of the fruiting body of Ganoderma lucidum as an active ingredient. More specifically, the invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through inhibition of blood coagulation containing an ethanol extract of the fruiting body of Ganoderma lucidum as an active ingredient. The extract of the fruiting body of Ganoderma lucidum, which serves as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis according to the present invention, exhibits strong antithrombotic activity by inhibiting blood clot-forming related enzymes and blood coagulation factors. It also has excellent thermal stability and does not show loss of blood coagulation factor inhibitory effects and blood clot-forming related enzyme inhibitory effects even under acidic conditions of pH 2 and in plasma. Therefore, it is expected to be usable for the prevention and treatment of thrombosis, such as ischemic stroke and hemorrhagic stroke, through the improvement of blood circulation. Furthermore, the above-mentioned Reishi mushroom fruiting body extract is edible, does not exhibit toxicity, and has the excellent effect of being able to be processed into various forms such as extract, powder, pills, and tablets for regular consumption; therefore, it is a highly useful invention for the pharmaceutical and food industries.

Description

Technology Field

[0001] The present invention relates to the fruiting body of the Reishi mushroom. Ganoderma lucidum The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis containing an extract of a Reishi mushroom fruiting body as an active ingredient, and more specifically, to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through inhibition of blood coagulation containing an ethanol extract of a Reishi mushroom fruiting body as an active ingredient. The present invention is the result of the research service for the "evaluation of antithrombotic / antioxidant activity of Pine mushroom." Background Technology

[0002] As a component of the human body, blood possesses various important functions, including the transport of oxygen, nutrients, and waste products, buffering, maintaining body temperature, regulating osmotic pressure and ion balance, maintaining constant fluid levels, regulating humors, maintaining and regulating blood pressure, and providing biological defense. Normal blood circulation is facilitated by the complementary regulation of the blood coagulation and thrombolytic systems within the body. Among these, the mechanism of the blood coagulation system is reported to involve platelets adhering to and aggregating on the blood vessel walls to form a platelet thrombus, after which the blood coagulation system is activated to form a fibrin thrombus centered around the platelet aggregate.

[0003] The formation of fibrin clots involves a multi-step reaction of numerous blood coagulation factors, through which thrombin, which is involved in fibrin coagulation, is activated. This ultimately leads to the generation of fibrin monomers from fibrinogen. These fibrin monomers are polymerized by calcium and bind to platelets and endothelial cells, forming a permanent blood clot by creating a fibrin polymer cross-linked by Factor XIII. Furthermore, thrombin plays a pivotal role in clot formation by activating platelets, Factor V, and Factor VII to promote blood coagulation reactions. Therefore, substances that inhibit the activity of thrombin can be used as highly useful preventive and therapeutic agents for various thrombotic diseases resulting from excessive blood coagulation abnormalities.

[0004] It is known that the endogenous thrombosis pathway involves the sequential activation of Factor XII, Factor XI, Factor IX, and Factor X, followed by the activation of prothrombin, which ultimately leads to the activation of thrombin; therefore, the specific inhibition of blood coagulation factors is also an important target for the development of treatments for thrombotic diseases. To date, various anticoagulants, antiplatelet agents, and thrombolytics such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases; however, their use is currently limited due to their very high cost, as well as hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions.

[0005] Meanwhile, Reishi mushrooms ( Ganoderma lucidumIt is an annual mushroom of the family Ganodermataceae that grows on broad-leaved trees in the summer, and is also called Yeongjicho, Jicho, or Bullocho. The stem is about 10 cm tall, and the cap is heart-shaped or circular, with a luster on the surface of the cap and stem resembling lacquer. Initially, it is egg-yolk white, but it changes to yellowish-brown or reddish-brown; as it matures, the entire surface becomes hard and leathery corky, with a reddish-brown or purplish-brown sheen. It has two layers, upper and lower; the upper layer is almost white, while the lower layer containing the pores is light orange. The cap is semicircular, kidney-shaped, or fan-shaped, with a flat surface and concentric grooves.

[0006] Because Reishi mushrooms are as hard as wood, they cannot be consumed directly; instead, they are ground into a powder or dried for medicinal use. The general method of consumption involves boiling them for a long time and drinking the resulting liquid continuously. In traditional Korean medicine, they are used for nervous exhaustion, heart disease, hypertension, and various types of cancer due to their tonic, antitussive, and anti-inflammatory properties.

[0007] Studies related to Reishi mushrooms include the inhibitory effect of lanostane-type triterpenoids isolated from Reishi mushrooms on cancer cell growth (Kim Dong-hwa et al., 2020, Journal of the Korean Society of Pharmacognosy 51: 36-40) and the immunomodulatory activity of lanostane-type triterpenoids isolated from Reishi mushroom fruiting bodies (Pu, D. B et al., 2017. Highly oxygenated lanostane-type triterpenoids and their bioactivity from the fruiting body of Ganoderma Ganoderma lucidum. Fitoterapia 119: 1-7) and antifungal active substances (Pu, D. B et al., 2019. Triterpenoids from Ganoderma lucidum: A Class of Sensitizers of FLC-Resistant Candida albicansFluconazole. J. Natural products 82: 2067-2077) is known, and recently, artificial cultivation and nutritional evaluation of wild Reishi mushrooms (Yu, Changxia et al., 2020, J. Science Society of Thailand 46: 548) have been reported.

[0008] Patents related to Reishi mushrooms include Korean Registered Patent No. 10-0963826 [Novel Reishi Mushroom and Method for Cultivating the Same], No. 10-0931526 [Fermented Food Using Reishi Mushroom Mycelium and Method for Manufacturing the Same], No. 10-1002256 [Abalone Drying Method Using Reishi Mushrooms and Far-Infrared Rays], No. 10-1303544 [Natural Dye for Clothing and Method for Manufacturing the Same], No. 10-1477078 [Natural Deodorizing and Sterilizing Agent Containing Herbal Ingredients and Method for Manufacturing the Same], No. 10-1541872 [Livestock Feed Using Food Waste Leachate and Method for Manufacturing the Same], No. 10-1625937 [Seed Treatment Method and Seeds Treated by the Above Method], and No. 10-1996384 [Of a Functional Cheonggukjang Block Using Reishi Mushroom A [manufacturing method] is disclosed.

[0009] However, to date, no studies or patents related to the potent anticoagulant activity of Reishi mushroom fruiting body extracts are known. Prior art literature

[0010] (Patent Document 0001) KR 10-1996384 B

[0011] Kim, Dong-Hwa et al., 2020, Journal of the Korean Society of Pharmacognosy 51: 36-40 Inhibitory effect of lanostane-type triterpenoid isolated from Reishi mushrooms on cancer cell growth The problem to be solved

[0012] The present invention has been devised to solve the problems of the prior art as described above, and the problem to be solved by the present invention is Reishi mushroom ( Ganoderma lucidumThe aim is to provide a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis through strong inhibition of blood coagulation containing a fruiting body extract as an active ingredient. means of solving the problem

[0013] In order to solve the above problems, the present invention relates to a Reishi mushroom ( Ganoderma The present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing an extract of the fruiting body of *Lucidum lucidum* as an active ingredient.

[0014] It is preferable that the above-mentioned Reishi mushroom fruiting body extract is an ethanol extract of the Reishi mushroom fruiting body.

[0015] In addition, the present invention relates to Reishi mushrooms ( Ganoderma lucidum ) Provides a blood coagulation inhibitor containing a fruiting body extract as an active ingredient.

[0016] It is preferable that the above-mentioned Reishi mushroom fruiting body extract is an ethanol extract of the Reishi mushroom fruiting body.

[0017] In addition, the present invention relates to Reishi mushrooms ( Ganoderma lucidum Provides a health functional food for the prevention or improvement of thrombosis containing a fruiting body extract.

[0018] It is preferable that the above-mentioned Reishi mushroom fruiting body extract is an ethanol extract of the Reishi mushroom fruiting body. Effects of the invention

[0019] The Reishi mushroom fruiting body extract, which serves as an active ingredient in the pharmaceutical composition and health functional food for the prevention or treatment of thrombosis according to the present invention, exhibits strong antithrombotic activity by inhibiting blood clot-forming enzymes and blood coagulation factors. It also has excellent thermal stability and does not lose its inhibitory effects on blood coagulation factors and blood clot-forming enzymes even under acidic conditions of pH 2 and in plasma. Therefore, it is expected to be usable for the prevention and treatment of thrombosis, such as ischemic stroke and hemorrhagic stroke, through the improvement of blood circulation. Furthermore, the Reishi mushroom fruiting body extract is edible, does not exhibit toxicity, and has excellent effects in that it can be processed into various forms such as extracts, powders, pills, and tablets, allowing for regular consumption. Thus, it is a highly useful invention for the pharmaceutical and food industries. Brief explanation of the drawing

[0020] Figure 1 is a photograph of the fruiting body of the Reishi mushroom and the powder thereof according to the present invention. Specific details for implementing the invention

[0021] The present invention will be described in detail below.

[0022] The inventors of the present invention intended to utilize the Reishi mushroom fruiting body extract as a pharmaceutical composition and health functional food for the prevention or treatment / improvement of thrombosis by preparing an ethanol extract of the Reishi mushroom fruiting body by a certain method to test the antithrombotic efficacy of the Reishi mushroom, and then evaluating its antithrombotic activity and confirming that the extract has excellent antithrombotic activity along with excellent thermal stability and acid stability.

[0023] Specifically, in order to develop pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of thrombosis using Reishi mushrooms, which are known in folk medicine to be effective against various diseases such as skin diseases, bacterial infections, vascular and circulatory systems, digestive systems, and metabolic systems, the inventors prepared various solvent extracts from the fruiting bodies of Reishi mushrooms and evaluated their antithrombotic activity by measuring direct thrombin inhibition (Thrombin Time), prothrombin inhibition (Prothrombin Time), and activated partial thromboplastin time (aPTT) against human thrombin. As a result, it was confirmed that the Reishi mushroom fruiting body extracts exhibited excellent thrombin inhibition, prothrombin inhibition, and thrombus formation inhibition activity through inhibition of blood coagulation factors.

[0024] Therefore, the present invention relates to the Reishi mushroom ( Ganoderma lucidum The present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing a fruiting body extract as an active ingredient.

[0025] It is preferable that the above-mentioned Reishi mushroom fruiting body extract is an ethanol extract of the Reishi mushroom fruiting body.

[0026] In addition, the present invention relates to Reishi mushrooms ( Ganoderma lucidum ) Provides a blood coagulation inhibitor containing a fruiting body extract as an active ingredient.

[0027] It is preferable that the above-mentioned Reishi mushroom fruiting body extract is an ethanol extract of the Reishi mushroom fruiting body.

[0028] In addition, the present invention relates to Reishi mushrooms ( Ganoderma lucidum Provides a health functional food for the prevention or improvement of thrombosis containing a fruiting body extract.

[0029] It is preferable that the above-mentioned Reishi mushroom fruiting body extract is an ethanol extract of the Reishi mushroom fruiting body.

[0030] Below, the method for preparing the Reishi mushroom fruiting body extract and efficacy experiments of the present invention will be explained in more detail.

[0031] The present invention comprises the steps of: preparing an extract from a Reishi mushroom fruiting body through solvent extraction; evaluating the antithrombotic activity of the extract; and investigating the stability of the Reishi mushroom fruiting body ethanol extract.

[0032] The "Reishi mushroom fruiting body extract" included in the composition of the present invention can be obtained by the steps of harvesting the Reishi mushroom fruiting body, drying it, cutting it into pieces 2 to 3 cm in size, and extracting it with an organic solvent, and filtering the extract using a filter mesh of 0.06 mm or less and concentrating it under reduced pressure.

[0033] The organic solvent used in the present invention may be water (cold water, hot water), hexene, methylene chloride, acetone, ethanol, anhydrous or aqueous lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, ethanol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc., and 95% ethanol extraction is preferred.

[0034] The above ethanol extract can be further obtained by sequentially or separately fractionating it with organic solvents of hexene, ethyl acetate, and butanol to obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

[0035] In the present invention, when the thrombin time, prothrombin time, and APT time were measured by adjusting the Reishi mushroom fruiting body extract to a concentration of 5 mg / ml, it was confirmed that the ethanol extract exhibited very strong thrombin inhibition, prothrombin inhibition, and coagulation factor inhibition. The antithrombotic activity of this active extract was superior to that of aspirin (1.5 mg / ml), which is used as an antithrombotic agent in clinical practice. Furthermore, it was confirmed that the extract has no hemolytic activity against human red blood cells and possesses excellent thermal and acid stability, allowing it to be consumed or drunk in various forms, thus enabling practical use as a preventive or therapeutic / improving agent for thrombosis.

[0036] The Reishi mushroom fruiting body extract of the present invention can be prepared into a powder through conventional pulverization processes such as vacuum drying, freeze-drying, or spray drying. These are not degraded by various degrading enzymes in plasma and maintain their activity even under heat treatment at 100°C and at a pH of 2 in the human stomach.

[0037] The active ingredient of the present invention can be used for the prevention or treatment of various diseases related to thrombosis. The diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, motor abnormalities, sensory abnormalities, personality changes, visual impairment, epileptic seizures, pulmonary thrombosis, deep vein thrombosis, lower extremity edema, pain, and acute peripheral arterial occlusion, and venous thrombosis such as deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral sinus thrombosis, and central retinal vein occlusion.

[0038] A pharmaceutical composition containing the active ingredient of the present invention can be formulated into various forms according to conventional methods to suit each intended use, such as oral formulations like powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injectable formulations like sterile injectable solutions, and can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

[0039] These pharmaceutical compositions may additionally include carriers, excipients, or diluents, and examples of suitable carriers, excipients, or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical compositions of the present invention may additionally include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0040] As a preferred embodiment, solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid formulations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above pharmaceutical composition. In addition, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

[0041] As a preferred embodiment, oral liquid formulations may be exemplified as suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients in addition to commonly used simple diluents such as water and liquid paraffin, such as humectants, sweeteners, flavorings, preservatives, etc.

[0042] As a preferred embodiment, formulations for parenteral administration may be exemplified as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

[0043] 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 with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, 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 in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above-mentioned factors into consideration, and this can be easily determined by a person skilled in the art.

[0044] As 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, gender, and body weight, and generally, 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0045] The pharmaceutical composition of the present invention may be administered to a subject via various routes. Any mode of administration may be anticipated, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intradural, or intracerebroventricular injection.

[0046] In the present invention, "administration" means providing a specific substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention may be oral or parenteral through any general route capable of reaching the target tissue. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

[0047] In the present invention, "object" includes, but is not specifically limited to, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, and preferably means mammals, more preferably humans.

[0048] In addition, the health functional food of the present invention can be used in various ways in foods and beverages, etc., that are effective in preventing or improving thrombosis. Foods containing the active ingredients of the present invention include, for example, various types of food, beverages, chewing gum, tea, vitamin complexes, health supplements, etc., and can be used in the form of powder, granules, tablets, capsules, or beverages.

[0049] The active ingredient of the present invention can generally be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition can be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

[0050] In addition to containing the above compound as an essential component in the indicated proportions, the health functional food of the present invention may contain food-grade acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional components.

[0051] Examples of the above natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, as well as common sugars and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0052] As the above flavoring agents, natural flavoring agents such as stevia, thaumatin, rebaudioside A, or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of the above natural carbohydrates is generally about 1 to 20g, preferably about 5 to 12g, per 100ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various nutritional supplements, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the health functional food of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, vegetable beverages, etc. These ingredients may be used independently or in combination. The proportion of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

[0053] The present invention will be explained in more detail below through examples. The following examples are merely preferred embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[0054] [Example]

[0055] Example 1: Obtaining Reishi mushroom fruiting bodies and color difference analysis

[0056] Fruiting body of Reishi mushroom Ganoderma lucidum The samples were provided by Gold Pharm Bio Co., Ltd. in Mungyeong, Gyeongbuk, and the shape of the Reishi mushroom fruiting bodies and the color difference analysis after grinding are shown in Figure 1 and Table 1.

[0057] [Table 1] Color analysis of Reishi mushroom fruiting bodies

[0058]

[0059]

[0060] As a result, the pH of the Reishi mushroom was 4.8, and the brix, which indicates the concentration of water-soluble substances, was 3.6. In addition, the lightness (L) was 33.66, the redness (a) was 6.72, the yellowness (b) was 13.28, and the overall color difference was 60.19.

[0061] Example 2: Preparation of ethanol extract of Reishi mushroom fruiting bodies and analysis of extract components

[0062] An ethanol extract was prepared from the Reishi mushroom fruiting body powder prepared in Example 1. Ten times the amount of ethanol (95%, Deoksan, Korea) was added to the sample, and the extract was extracted twice at room temperature. The extract was collected, filtered, and then concentrated under reduced pressure to produce a powder. The extraction yield and analysis results of the useful components of the Reishi mushroom fruiting body are shown in Table 2.

[0063] [Table 2] Yield and Analysis of Useful Components of Reishi Mushroom Fruiting Body Extract

[0064]

[0065] As shown in Table 2, the extraction efficiency of the Reishi mushroom fruiting bodies was 1.2%, suggesting a high content of wood. To analyze the beneficial components of the Reishi mushroom fruiting body extracts, the contents of total polyphenols, total flavonoids, total sugars, and reducing sugars were measured. For total polyphenol content, 50 μl of Folin-Ciocalteau and 100 μl of saturated Na2CO3 solution were added to 400 μl of the extract, left at room temperature for 1 hour, and the absorbance was measured at 725 nm. Tannic acid was used as the standard reagent. For total flavonoid content, each sample was subjected to ethanol-stirred extraction for 18 hours. To 400 μl of the filtered extract, 4 ml of 90% diethylene glycol was added, followed by 40 μl of 1 N NaOH. After reacting at 37°C for 1 hour, the absorbance was measured at 420 nm. Rutin was used as the standard reagent. Total sugars were quantified using the phenol-sulfuric acid method, and sucrose was used as the standard reagent. Reducing sugars were quantified using the DNS method, and glucose was used as the standard reagent.

[0066] Analysis of total polyphenol and total flavonoid content revealed a high polyphenol content of 59.1 mg / g and a flavonoid content of 6.1 mg / g. However, analysis of total sugar and reducing sugar content revealed a total sugar content of 49.7 mg / g and a reducing sugar content of 22.0 mg / g, suggesting that the Reishi mushroom fruiting body extract would exhibit various beneficial physiological activities.

[0067] Considering the extraction efficiency, it was expected that the fruiting body of the Reishi mushroom would contain 71 mg of polyphenols per 100 g.

[0068] Example 3: Evaluation of the blood coagulation inhibitory activity of Reishi mushroom fruiting body extract

[0069] The blood coagulation inhibitory activity of the Reishi mushroom fruiting body extract of Example 2 was evaluated, and the results are shown in Table 3. The blood coagulation inhibitory activity of the Reishi mushroom fruiting body samples was evaluated according to previously reported methods (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), and thrombin time, prothrombin time, and APT time were measured. Commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and the thrombin time, prothrombin time, and APT time measurements were performed as follows.

[0070] Thrombin Time

[0071] At 37°C, 50 μl of 0.5 U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl2, and 10 μl of sample extracts of various concentrations were mixed in the tube of an Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes. Afterward, 100 μl of plasma was added, and the time until the plasma coagulated was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used as the solvent control instead of the sample. In the case of DMSO, a coagulation time of 32.1 seconds was observed. The thrombin inhibitory effect was expressed as the average value of experiments repeated at least three times, and thrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time upon sample addition by the coagulation time of the solvent control.

[0072] Prothrombin time

[0073] 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solutions of various concentrations were added to the tubes of an Amelung coagulometer KC-1A (Japan). After heating at 37°C for 3 minutes, 130 μl of PT reagent was added, and the time until the plasma coagulated was recorded as the average of three repeated experiments. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used as the solvent control instead of the sample. In the case of DMSO, a coagulation time of 18.1 seconds was observed. Prothrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time upon sample addition by the coagulation time of the solvent control.

[0074] activated Partial Thromboplastin Time (aPTT)

[0075] 100 μl of plasma and 10 μl of sample extracts of various concentrations were added to the tube of an Amelung coagulometer KC-1A (Japan) and heated at 37°C for 3 minutes, after which 50 μl of aPTT reagent (Sigma, ALEXIN TM ) was added and incubated again at 37°C for 3 minutes. Afterwards, 50 μl of CaCl2 (35 mM) was added, and the time until plasma coagulation was measured. DMSO was used as the solvent control instead of the sample, and in this case, a coagulation time of 55.1 seconds was observed. The aPTT result was expressed as the average of three repeated experiments, and blood coagulation factor inhibitory activity was expressed as the value obtained by dividing the aPTT at the time of sample addition by the aPTT of the solvent control.

[0076] [Table 3] Inhibitory activity of Reishi mushroom fruiting body extract on blood coagulation

[0077]

[0078] As shown in Table 3, when the ethanol extract of the prepared Reishi mushroom fruiting body was prepared at a concentration of 5 mg / ml and the thrombin time, prothrombin time, and APT time were measured, the thrombin time, prothrombin time, and APT time were extended by 1.71 times, 1.74 times, and 3.05 times, respectively, demonstrating very potent antithrombotic activity. When the ethanol extract of the Reishi mushroom fruiting body was evaluated at a concentration of 2.5 mg / ml, the thrombin time, prothrombin time, and APT time were extended by 1.48 times, 1.45 times, and 1.91 times, respectively, showing stronger antithrombotic activity than aspirin (1.5 mg / ml), which is used as an antithrombotic agent in clinical practice.

[0079] Considering that Reishi mushroom fruiting bodies are currently mainly consumed as tea and beverages, it was determined that Reishi mushroom fruiting body extracts can effectively inhibit blood clot formation by inhibiting various blood coagulation factors, thrombin, and prothrombin, and can replace aspirin, which has severe side effects such as gastrointestinal disorders.

[0080] Example 4: Human erythrocyte hemolytic activity of Reishi mushroom fruiting body extract

[0081] To evaluate the potential for acute toxicity of the Reishi mushroom fruiting body extract prepared in Example 2, human erythrocyte hemolytic activity was evaluated. At this time, hemolytic activity was evaluated according to a previous report (Son Ho-yong, 2014. Korean J. Microbiol. Biotechnol. 42: 285-292). Simply put, 100 μl of human erythrocytes washed three times with PBS were placed in a 96-well microplate, 100 μl of sample solutions of various concentrations were added, and the mixture was reacted at 37°C for 30 minutes. Afterward, the reaction mixture was centrifuged (1,500 rpm) for 10 minutes, 100 μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. DMSO (2%) was used as the solvent control for the samples, and Triton X-100 (1 mg / ml) was used as the experimental control for erythrocyte hemolysis. Hemolytic activity was calculated using the following formula.

[0082]

[0083] [Table 4] Human erythrocyte hemolytic activity of Reishi mushroom fruiting body extract

[0084]

[0085] As shown in Table 4, DMSO and water, used as controls, showed no hemolytic activity, while Triton X-100 was confirmed to cause 100% hemolysis of red blood cells at a concentration of 1 mg / ml. Additionally, amphotericin B, which is used as an anticancer and antifungal agent, was confirmed to cause more than 50% hemolysis of red blood cells at a concentration of 0.0032 mg / ml. Meanwhile, the Reishi mushroom fruiting body extract showed 51.6% hemolysis of red blood cells at a concentration of 1 mg / ml. This hemolytic activity of the Reishi mushroom fruiting body extract is understood to be caused by saponins, etc., and considering that it has been used for food (extract tea, complex extract beverage) to date, it was expected that there would be no serious acute toxicity. In particular, compared to amphotericin B, which is known to have side effects, the Reishi mushroom fruiting body extract requires a concentration more than 300 times higher to exhibit the same erythrocyte hemolytic activity, so it was determined that acute toxicity in humans would not occur.

[0086] Example 5: Evaluation of Plasma, Acid, and Thermal Stability of Reishi Mushroom Fruiting Body Extract

[0087] The Reishi mushroom fruiting body extract prepared in Example 2 is consumed as a conventional tea, complex beverage, or alcoholic drink, so it was determined that there would be no separate safety issues. Accordingly, the plasma stability, thermal stability, and acid stability of the Reishi mushroom fruiting body extract regarding blood coagulation inhibition were confirmed. The extract did not show a decrease in blood coagulation inhibitory activity even when heat-treated at 100°C for 1 hour, treated at pH 2 (0.01M HCl) for 1 hour, or treated in plasma for 1 hour. Therefore, it was confirmed that the Reishi mushroom fruiting body extract contains antithrombotic active substances with acid resistance and heat resistance, confirming its high potential for practical use.

Claims

Claim 1 delete Claim 2 delete Claim 3 Reishi mushroom Ganoderma lucidum A blood coagulation inhibitor containing a fruiting body ethanol extract as an active ingredient. Claim 4 delete

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