KR20240048690A - Pharmaceutical composition comprising the extract of ganoderma gibbosum fruiting body 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 the prevention or treatment/improvement of thrombosis containing the extract of the fruiting body of Ganoderma gibbosum as an active ingredient, and more specifically, to the fruiting body of Ganoderma gibbosum. It relates to a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis by inhibiting blood coagulation, containing ethanol extract as an active ingredient. The extract of the fruiting body of Chivalry mushroom as an active ingredient in the pharmaceutical composition and health functional food for preventing or treating thrombosis of the present invention exhibits strong antithrombotic activity by inhibiting enzymes and blood coagulation factors related to thrombosis, and has excellent thermal stability. Since there is no loss of the blood coagulation factor inhibitory effect and the enzyme inhibitory effect related to thrombus formation even under acidic conditions of pH 2 and in plasma, it is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke by improving blood circulation. . In addition, the fruiting body extract of the Chilhwang mushroom is used for food, is not toxic, and has excellent effects in the pharmaceutical and food industries as it can be processed into various forms such as extracts, powders, pills, tablets, etc. and prepared in a form that can be taken on a regular basis. It is a very useful invention.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing the extract of the fruiting body of Chillium mushroom as an active ingredient SAME}

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment/improvement of thrombosis containing the extract of the fruiting body of Ganoderma gibbosum as an active ingredient, and more specifically, to the fruiting body of Ganoderma gibbosum. It relates to a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis by inhibiting blood coagulation, containing ethanol extract as an active ingredient.

As a component of the human body, blood has a variety of important functions, including transport of oxygen, nutrients, and waste, buffering action, maintenance of body temperature, regulation of osmotic pressure and maintenance of ionic balance, maintenance of moisture content, fluid regulation, maintenance and control of blood pressure, and biological defense. there is. Normal blood circulation facilitates blood circulation through complementary regulation of the blood coagulation response system and the thrombolysis response system in the body. Among these, the mechanism of the blood coagulation response system is that platelets adhere and aggregate to the blood vessel wall to form a platelet thrombus. It has been reported that the blood coagulation system is activated and fibrin clots are formed centered on platelet aggregates.

The formation of a fibrin clot goes through a multi-step reaction of numerous blood coagulation factors, and thrombin, which is involved in fibrin coagulation, is activated, ultimately producing fibrin monomers from fibrinogen. The fibrin monomers are polymerized by calcium to form platelets and endothelial cells. They combine to form 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, factor V, and factor VII to promote blood coagulation. Therefore, substances that inhibit the activity of thrombin can be used as a very useful preventive and therapeutic agent for various thrombotic diseases caused by excessive blood coagulation abnormalities.

In the endogenous thrombus formation pathway, sequential activation of factor XII, factor It is becoming. To date, various anticoagulants, antiplatelet agents, and thrombolytics such as heparin, coumarin, aspirin, and urokinase have been used to prevent and treat thrombotic diseases, but these are not only very expensive, but also cause hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions. Due to this, its use is limited.

Meanwhile, Ganoderma gibbosum is a very unique type of reishi mushroom that grows only on poisonous lacquer trees. According to Korea Patent No. 10-0963826, the leaves and stems of lacquer and lacquer trees are boiled in boiling water and then dried. A method of cultivating the plant by undergoing a lacquer detoxification process, which is characterized in that it is performed, and then inoculating and cultivating the mycelium of Chilhwang mushrooms (International Accession for Microorganisms No. KCTC 11549BP) is disclosed. In order to cultivate chilhwang mushrooms, it is necessary to remove the toxicity by denaturing urushiol, a toxic component, using the lacquer tree, which is used to remove the toxicity of lacquer. In fact, in the private sector, when lacquer poisoning occurs, it is washed with water boiled in lacquer tree to remove the side effects of lacquer. has been treated.

Research related to chilhwang mushrooms includes G-DNA extraction and ITS nrDNA region analysis results for mushroom identification, Ganoderma gibbosum (Blume & T. Nees) Pat. It is believed to be, and it has been confirmed to be most similar to the mushroom collected under the name ‘balloon elixir of age’ in the area around Mt. Baekdu (Mushroom Encyclopedia of Mt. Baekdu 2, 2014, Jo Deok-hyeon) (National Institute of Horticultural and Herbal Science, 2016). In addition, research has been conducted on mass production cultivation technology, search for functional substances, and product commercialization of lacquer mushrooms, which are parasitic on lacquer trees (Wonchilhwang Agricultural Corporation, 2015), and the cancer cell growth inhibition effect of Lanostane-type Triterpenoid isolated from Chilhwang mushrooms. (Kim Dong-hwa et al., 2020, Journal of Herbal Medicine 51: 36-40), Immune-enhancing activity of lanostane-type triterpenoids isolated from Chilhwang mushroom fruiting bodies (Pu, D. B et al., 2017. Highly oxygenated lanostane-type triterpenoids and their bioactivity from the fruiting body of Ganoderma gibbosum 119: 1-7) and antifungal active substances (Pu, D. B et al., 2019. Triterpenoids from Ganoderma gibbosum: A Class of Sensitizers of FLC-Resistant Candida albicans to Fluconazole. J. Natural products 82 : 2067-2077) is known, and recently, artificial culture and nutritional evaluation of wild chili pepper mushrooms (Yu, Changxia et al., 2020, J. Science Society of Thailand 46: 548) have been reported.

Patents related to chilhwang mushrooms include Republic of Korea Patent No. 10-0963826 [New Chilhwang Mushroom and Method for Culturing the Same], No. 10-0931526 [Fermented Food Using Chilhwang Mushroom Mycelium and Manufacturing Method], No. 10 -No. 1002256 [Abalone drying method using chilhwang mushrooms and far-infrared rays], No. 10-1303544 [Natural dye for clothing and its manufacturing method], No. 10-1477078 [Natural deodorizing and disinfectant containing herbal ingredients and their Manufacturing method], No. 10-1541872 [Livestock feed using food waste leachate and manufacturing method thereof], No. 10-1625937 [Seed treatment method and seeds treated with the above method], and No. 10-1996384 [Method for manufacturing functional cheonggukjang block] using chilhwang mushrooms is disclosed.

However, to date, there are no known studies or patents related to the strong anticoagulant activity of Chilhwang mushroom fruiting body extract.

KR 10-1996384 B

Dong-Hwa Kim et al., 2020, Journal of Herbal Medicine 51: 36-40 Inhibitory Effect on Cancer Cell Growth of Lanostane-type Triterpenoid Isolated from Chilhwang Mushroom

The present invention was created to solve the problems of the prior art as described above, and the problem to be solved by the present invention is to prevent thrombosis through strong inhibition of blood coagulation using the fruiting body extract of Ganoderma gibbosum as an active ingredient. The aim is to provide pharmaceutical compositions and health functional foods for treatment/improvement.

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

It is preferable that the Chilhwang mushroom fruiting body extract is an ethanol extract of the Chilhwang mushroom fruiting body.

Additionally, the present invention provides a blood coagulation inhibitor containing Ganoderma gibbosum fruiting body extract as an active ingredient.

It is preferable that the Chilhwang mushroom fruiting body extract is an ethanol extract of the Chilhwang mushroom fruiting body.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing a fruiting body extract of Ganoderma gibbosum .

It is preferable that the Chilhwang mushroom fruiting body extract is an ethanol extract of the Chilhwang mushroom fruiting body.

The extract of the fruiting body of Chivalry mushroom as an active ingredient in the pharmaceutical composition and health functional food for preventing or treating thrombosis of the present invention exhibits strong antithrombotic activity by inhibiting enzymes and blood coagulation factors related to thrombosis, and has excellent thermal stability. Since there is no loss of the blood coagulation factor inhibitory effect and the enzyme inhibitory effect related to thrombus formation even under acidic conditions of pH 2 and in plasma, it is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke by improving blood circulation. . In addition, the fruiting body extract of the Chilhwang mushroom is used for food, is not toxic, and has excellent effects in the pharmaceutical and food industries as it can be processed into various forms such as extracts, powders, pills, tablets, etc. and prepared in a form that can be taken on a regular basis. It is a very useful invention.

Figure 1 is a photograph of the chilhwang mushroom fruiting body and its powder of the present invention.

Hereinafter, the present invention will be described in detail.

In order to test the anti-thrombotic efficacy of Chilhwang mushrooms, the inventors of the present invention prepared an ethanol extract of Chilhwang mushroom fruiting bodies by a certain method and then evaluated its anti-thrombotic activity, and found that the extract had excellent anti-thrombotic activity as well as heat stability and acidity. By confirming that it has excellent stability, the extract of the fruiting body of the Chillahwang mushroom was intended to be used as a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis.

Specifically, the present inventors have developed a pharmaceutical composition for preventing or treating/improving thrombosis using chilhwang mushrooms, which are known to be effective in treating various diseases such as skin diseases, bacterial infectious diseases, vascular and circulatory systems, digestive systems, and metabolic systems, and In order to develop health functional foods, various solvent extracts were prepared from the fruiting bodies of Chilhwang mushrooms, and their antithrombotic activity was measured by direct thrombin inhibition of human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin Time), and active portion thrombin. As a result of evaluating the activated Partial Thromboplastin Time (aPTT), it was confirmed that the extract of the fruit body of the Chillia Mushroom had excellent thrombin inhibition, prothrombin inhibition, and thrombosis inhibition activity by inhibiting blood coagulation factors.

Therefore, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing a fruiting body extract of Ganoderma gibbosum as an active ingredient.

It is preferable that the Chilhwang mushroom fruiting body extract is an ethanol extract of the Chilhwang mushroom fruiting body.

Additionally, the present invention provides a blood coagulation inhibitor containing Ganoderma gibbosum fruiting body extract as an active ingredient.

It is preferable that the Chilhwang mushroom fruiting body extract is an ethanol extract of the Chilhwang mushroom fruiting body.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing a fruiting body extract of Ganoderma gibbosum .

It is preferable that the Chilhwang mushroom fruiting body extract is an ethanol extract of the Chilhwang mushroom fruiting body.

Hereinafter, the production method and efficacy test of the chilhwang mushroom fruiting body extract of the present invention will be described in more detail.

The present invention includes the steps of preparing an extract through solvent extraction from the fruiting body of Chilhwang mushroom; A step of evaluating the antithrombotic activity of the extract; It includes a step of investigating the stability of the ethanol extract of the fruiting body of Chilhwang mushroom.

The "chillwang mushroom fruiting body extract" included in the composition of the present invention is a process of harvesting the chilwang mushroom fruiting body, drying it, cutting it into 2-3 cm pieces, extracting it with an organic solvent, and using a filter net of 0.06 mm or less for the extract. It can be obtained by filtering and concentrating it under reduced pressure.

Organic solvents used in the present invention include water (cold water, hot water), hexene, methylene chloride, acetone, alcohol, anhydrous or hydrous lower alcohols with 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), and the lower alcohols above. A mixed solvent of ethanol and water can be used, and 95% ethanol extraction is preferred.

The ethanol extract can be sequentially or separately fractionated with organic solvents 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, the concentration of the fruiting body extract of Chilhwang mushroom was adjusted to 5 mg/ml and the thrombin time, prothrombin time, and AP time were measured. As a result, the ethanol extract showed very strong thrombin inhibition, prothrombin inhibition, and blood coagulation factor inhibition. Confirmed. The anti-thrombotic activity of this active extract was superior to that of aspirin (1.5 mg/ml), which is used clinically as an anti-thrombotic agent. In addition, the extract has no hemolytic activity against human red blood cells and has excellent heat and acid stability, so it can be eaten and drunk in various forms, so it was confirmed that it can be practically used as an agent for preventing or treating/improving thrombosis. .

The fruiting body extract of Chilhwang mushroom of the present invention can be manufactured into powder through a conventional powdering process such as reduced pressure drying, freeze drying, or spray drying. They are not decomposed by various decomposition enzymes in plasma, and remain active even when heat treated at 100°C and at pH 2 in the human stomach.

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

The pharmaceutical composition containing the active ingredient of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injectable solutions according to conventional methods to suit each purpose of use. It can be formulated and used in various forms, and can be administered through various routes, including oral administration, intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

These pharmaceutical compositions may additionally contain 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, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

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

As a preferred embodiment, oral liquid preparations may include suspensions, oral solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Fragrances, preservatives, etc. may be included.

As a preferred embodiment, preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injectables may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. 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 singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person 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, gender, and weight, and is generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg per day. Alternatively, it can be administered every other day or divided into 1 to 3 doses per day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

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

In the present invention, “administration” means providing a predetermined substance to a patient by any appropriate 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. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

In the present invention, “subject” includes, but is not particularly limited to, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits or guinea pigs. And, preferably, it means a mammal, and more preferably, a human.

In addition, the health functional food of the present invention can be used in a variety of foods and beverages that are 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, etc., 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.

In addition to containing the above compounds as essential ingredients in the indicated ratio, the health functional food of the present invention may contain foodologically acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional ingredients.

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

As the flavoring agent, natural flavoring agents such as thaumatin, rebaudioside A, or stevia such as 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 contains various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavors, colorants and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, etc. These ingredients can be used independently or in combination. The ratio of these 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.

Hereinafter, the present invention will be described in more detail through examples. The following example is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following example.

[Example]

Example 1: Securing Chilhwang Mushroom fruiting bodies and color difference analysis

The fruiting body of Ganoderma gibbosum was provided by Gold Farm Bio Co., Ltd. in Mungyeong, Gyeongsangbuk-do, and the shape of the fruiting body of Ganoderma gibbosum and color difference analysis after grinding are shown in Figure 1 and Table 1.

[Table 1] Color analysis of chilhwang mushroom fruiting bodies

As a result, the pH of chilhwang mushrooms was 6.8, and brix, which indicates the concentration of water-soluble substances, was 7.2. Additionally, the brightness (L) was 21.96, redness (a) was 6.22, and yellowness (b) was 8.27, and the overall color difference was 70.92, indicating a dark brown color.

Example 2: Preparation of ethanol extract of Chilhwang mushroom fruiting body and analysis of components of the extract

An ethanol extract was prepared from the fruiting body powder of Chilhwang mushroom prepared in Example 1. 10 times more ethanol (95%, Deoksan, Korea) was added to the sample, extraction was repeated twice at room temperature, and the extract was collected and filtered. , it was concentrated under reduced pressure and made into powder. The extraction yield and useful content analysis results of the Chilhwang mushroom fruiting body are shown in Table 2.

[Table 2] Yield and useful content analysis of Chilhwang mushroom fruiting body extract

As shown in Table 2, the extraction efficiency of the Chilhwang mushroom fruiting body was 9.1%, which could be expected to have a low xylem content. To analyze the useful components of the extract of the fruiting body of the Chilihwang mushroom, the contents of total polyphenols, total flavonoids, total sugars, and reducing sugars were measured. At this time, the total polyphenol content was measured by adding 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution to 400 μl of the extraction sample solution, leaving it at room temperature for 1 hour, and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. The total flavonoid content was determined by extracting each sample with ethanol for 18 hours, adding 4 ml of 90% diethylene glycol to 400 μl of the filtered extraction solution, adding 40 μl of 1 N NaOH, reacting at 37°C for 1 hour, and measuring the absorbance at 420 nm. Rutin was used as a standard reagent. Total sugar was quantified using the phenol-sulfuric acid method, and sucrose was used as the standard reagent. The DNS method was used to quantify reducing sugars, and glucose was used as the standard reagent.

As a result of analysis of total polyphenol and total flavonoid content, it showed a high polyphenol content of 58.7 mg/g and a flavonoid content of 6.9 mg/g. However, as a result of analyzing the total sugar and reducing sugar content, the total sugar content was 41.7 mg/g and the reducing sugar content was 2.3 mg/g.

Taking the extraction efficiency into account, the fruiting body of Chilhwang mushroom was expected to contain 534 mg of polyphenol components per 100 g and to exhibit various physiological activities.

Example 3: Evaluation of blood coagulation inhibitory activity of Chilhwang mushroom fruiting body extract

The blood coagulation inhibitory activity of the fruiting body extract of Chilhwang mushroom of Example 2 was evaluated, and the results are shown in Table 3. At this time, the blood coagulation inhibitory activity evaluation method of the Chilhwang mushroom fruiting body sample 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. As plasma, commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and thrombin time, prothrombin time, and AP time measurements were performed as follows.

Thrombin Time

At 37°C, 50μl of 0.5U thrombin (Sigma Co., USA), 50μl of 20mM CaCl ₂ , and 10μl of sample extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes, then 100μl of plasma was added. After that, the time until the plasma coagulated 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 coagulation time when adding the sample divided by the coagulation time of the solvent control.

prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan) and heated at 37°C for 3 minutes, then 130 μl of PT reagent was added and the plasma was coagulated. The time until this was achieved 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. Prothrombin inhibitory activity was expressed as the coagulation time upon sample addition divided by the coagulation time of the solvent control.

activated Partial Thromboplastin Time (aPTT)

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

[Table 3] Blood coagulation inhibitory activity of Chilhwang mushroom fruiting body extract

As shown in Table 3, as a result of measuring thrombin time, prothrombin time, and APTI time using the ethanol extract of the prepared fruiting body of Chilhwang mushroom at a concentration of 5 mg/ml, thrombin was extended by 1.80 times, 2.26 times, and >15 times, respectively. It showed very strong antithrombotic activity by showing thyme, prothrombin time and aptitude time. Even when the ethanol extract of the fruiting body of the Chilihwang mushroom was evaluated at a concentration of 2.5 mg/ml, the thrombin time, prothrombin time, and APTI time were extended by 1.56 times, 1.49 times, and 3.57 times, respectively, which is used clinically as an antithrombotic agent. It showed stronger antithrombotic activity than aspirin (1.5mg/ml).

Considering that Chilhwang mushroom fruiting bodies are currently mainly consumed as tea and beverages, Chilhwang mushroom fruiting body extracts can effectively inhibit the formation of blood clots by inhibiting various blood coagulation factors, thrombin and prothrombin, and can cause severe side effects such as gastrointestinal disorders. It was judged that it could replace aspirin.

Example 4: Human red blood cell hemolytic activity of Chilhwang mushroom fruiting body extract

In order to evaluate the acute toxicity potential of the fruiting body extract of Chilhwang mushroom prepared in Example 2, the hemolytic activity of human red blood cells was evaluated. At this time, hemolytic activity was evaluated according to an existing report (Ho-yong Son, 2014. Korean J. Microbiol. Biotechnol. 42: 285-292), and simply, 100 μl of human red blood cells washed three times with PBS were placed in a 96-well microplate. 100 μl of sample solutions of various concentrations were added and reacted at 37°C for 30 minutes. Afterwards, the reaction solution 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. It was measured in . DMSO (2%) was used as a solvent control for the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

[Table 4] Human red blood cell hemolytic activity of Chilhwang mushroom fruiting body extract

As shown in Table 4, DMSO and water used as controls had no hemolytic activity, and triton In addition, in the case of amphotericin B, which is used as an anticancer and antifungal agent, it was confirmed that more than 50% of red blood cells were hemolyzed at a concentration of 0.0032 mg/ml. Meanwhile, the Chilhwang mushroom fruiting body extract showed 55.0% red blood cell hemolysis at a concentration of 1 mg/ml and 33.7% hemolysis at a concentration of 0.5 mg/ml. It is understood that the hemolytic activity of Chilwang mushroom extract is caused by saponin, etc., and considering that it has been used for edible purposes (extracted tea, complex extract beverage) to date, it is expected that there will be no serious acute toxicity. In particular, compared to amphotericin B, which is known to have side effects, it was judged that acute toxicity would not occur because the extract of the fruiting body of Chillium elegans requires approximately 300 times higher concentration to show the same activity of red blood cell hemolytic activity.

Example 5: Evaluation of plasma, acid and heat stability of Chilhwang mushroom fruiting body extract

The extract of the fruiting body of the Chilhwang mushroom prepared in Example 2 was manufactured and consumed as ordinary teas, complex beverages, and alcoholic beverages, so it was judged that there would be no separate safety problems. Therefore, the plasma stability, heat stability, and acid stability of the Chilhwang mushroom fruiting body extract for inhibition of blood coagulation 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, and treated in plasma for 1 hour. Therefore, it was confirmed that the fruiting body extract of Chilhwang mushroom contains an anti-thrombotic active substance with acid resistance and heat resistance, confirming that it has high practical usability.

[R&D tasks]

- Research management organization: Andong University Industry-Academic Cooperation Foundation

- Contribution rate: 100%

- Research period: 2022. 03. 01 ~ 2022. 11. 30

- Research institution: Department of Food and Nutrition, Andong University

Claims (4)

A pharmaceutical composition for preventing or treating thrombosis containing a fruiting body extract of Ganoderma gibbosum as an active ingredient. The pharmaceutical composition according to claim 1, wherein the extract of the fruiting body of Chilhwang mushroom is an ethanol extract of the fruiting body of Chilhwang mushroom. A blood coagulation inhibitor comprising the active ingredient according to claim 1 or 2. A health functional food for preventing or improving thrombosis containing the active ingredient according to claim 1 or 2.