KR20240048685A - Pharmaceutical composition comprising the extract of songhwa mushroom 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 of thrombosis containing extract of the fruiting body of Songhwa mushroom as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition and health functional food containing extract of the fruiting body of Songhwa mushroom as an active ingredient. It relates to a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis by inhibiting blood coagulation, containing fruiting body extract of cultivated shiitake mushrooms (Songflower: Republic of Korea Patent Publication No. 10-2022-0073141) as an active ingredient. . Songhwa mushroom fruiting body extract 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 formation 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 pine flower mushroom fruiting body extract is used for edible purposes and is not toxic. It is processed into various forms such as extracts, powders, pills, tablets, etc. and has excellent effects in the pharmaceutical and food industries. It is a very useful invention.

Description

Pharmaceutical composition and health functional food for preventing or treating thrombosis containing extract of fruiting body of pine flower mushroom as an active ingredient SAME}

The present invention relates to a pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing extract of the fruiting body of Songhwa mushroom as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition and health functional food containing extract of the fruiting body of Songhwa mushroom as an active ingredient. It relates to a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis by inhibiting blood coagulation, containing fruiting body extract of cultivated shiitake mushrooms (Songflower: Republic of Korea Patent Publication No. 10-2022-0073141) 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, mushrooms taxonomically belong to the fungi, but unlike general fungi, they are characterized by producing large fruiting bodies to form spores, and most are higher fungi belonging to the Basidiomycota and Ascomycota. Mushrooms not only have excellent flavor and contain a variety of nutrients such as carbohydrates, proteins, lipids, minerals, vitamins and minerals, but they also produce physiologically active substances, making them a food that has been widely used for edible and medicinal purposes since ancient times. In addition, mushroom β-glucan is reported to have immune activator functions, antioxidant capacity, biological tissue regeneration and healing functions, antibiotic, antibacterial, antiviral, and antitumor effects that stimulate macrophages to recognize and attack mutant cells. there is.

In particular, shiitake mushrooms ( Lentinula edodes ) are edible mushrooms belonging to the Basidiomycetes class, Oysteraceae family, and Pine mushroom genus. They have the highest content of vitamin C among mushrooms and contain a large amount of various amino acids and ergosterol. In terms of health functionality, shiitake mushrooms are well known for their anti-cancer, prevention of lung and gastrointestinal diseases, enhancement of immunity against viruses, promotion of antibody production, lowering of blood pressure, prevention of arteriosclerosis, regulation of blood lipid concentration, and anti-diabetes effect. , 2010. Master's thesis, Sungkyunkwan University; Jeong-Im Lee et al., 2020. Journal of Life Sciences 30: 877-885).

Recently, shiitake mushrooms grown with fermented sulfur have been developed and are widely sold on the market under the name “pine mushrooms.” Pine mushrooms have a stronger flavor than existing shiitake mushrooms and have an excellent texture due to their hard mushroom structure. , it has good storage properties, and is known to have excellent antioxidant properties, resulting in excellent skin whitening, anti-aging, and wrinkle improvement effects (Korean Patent Publication No. 10-2022-0073141). As of August 2022, the consumer market for pine mushrooms is rapidly expanding, with more than 3,100 outlets in famous domestic online shopping malls.

Patents related to shiitake mushrooms include Republic of Korea Patent No. 10-1839507 [Method for manufacturing seasoned Changnan Jeotgal containing shiitake mushrooms and fermented shiitake mushrooms], No. 10-1830763 [Method for manufacturing rice soybean paste containing shiitake mushroom extract] and rice soybean paste manufactured therefrom], No. 10-1823460 [Method for manufacturing semi-dried rockfish using shiitake mushroom powder], No. 10-1759312 [Method for manufacturing shiitake mushroom powder, manufacturing nutritional bar using shiitake mushroom powder Method and nutritional bar manufactured thereby], No. 10-1673573 [Manufacturing method of shiitake mushroom tea and shiitake mushroom tea manufactured thereby] are disclosed, and Republic of Korea Patent Publication No. 10-2004-0038957 [Shiitake mushroom powder] [Food on the subject of manufacturing seaweed (laver) containing laver] has been disclosed.

In addition, patents related to the beneficial physiological activities of shiitake mushrooms include Republic of Korea Patent No. 10-1487742 [Food composition with antioxidant efficacy containing shiitake mushrooms and apricot root extract], No. 10-1806808 [Fermentation using shiitake mushroom mycelium] Composition for preventing, improving or treating female menopausal symptoms containing fermented fermented sewage as an active ingredient], No. 10-1611947 [Health food composition for preventing and improving vascular diseases and method for manufacturing the same], No. 10-1266528 [Cosmetic composition for moisturizing and/or whitening containing polysaccharides extracted from shiitake mushroom mycelium culture as an active ingredient] is disclosed, and Patent Publication No. 10-2017-0036912 [Atopic dermatitis containing shiitake mushroom extract as an active ingredient] is disclosed. and Composition for preventing and improving contact dermatitis], Publication Patent No. 10-2003-0056753 [Health food composition for controlling obesity containing shiitake mycelium, Agaricus mycelium, and water-soluble chitosan], No. 10-2008-0110212 [Shiitake mushroom A composition that helps bone length growth using hot water extract] has been disclosed. In particular, with regard to thrombosis, Patent No. 10-2120490 [Pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing the ethyl acetate fraction of shiitake mushroom extract as an active ingredient] and No. 10-2128625 [ Pharmaceutical compositions and health functional foods for the prevention or treatment of thrombosis containing as an active ingredient the extract of the culture medium after harvesting shiitake mushrooms are known.

Among studies related to pine flower mushrooms, there is one report on dermatitis that occurred after consuming pine flower mushrooms (Myeong-jin Park et al., 2019. Journal of the Korean Society of Dermatology 57: 342-343), but no specific research has been conducted.

Patents related to pine mushrooms include Republic of Korea Patent No. 10-1944397 [Gochujang containing sulfur shiitake mushrooms and manufacturing method thereof], and No. 10-2060312 [Manufacture of pine flower mushroom spawn using liquid medium containing turmeric extract] Method] and No. 10-2322070 [Kimchi using cutlassfish and pine flower mushrooms and its manufacturing method] are disclosed, and Patent Publication No. 10-2022-0073141 [Shiitake mushrooms with added fermented sulfur and their cultivation method] are disclosed. and [Method for cultivating sulfur-containing shiitake mushrooms and oyster mushrooms] is disclosed in No. 10-2020-0142655. Additionally, No. 10-2022-0073141 [Shiitake mushrooms with added fermented sulfur and their cultivation method] reports that pine mushrooms have excellent antioxidant activity, skin whitening, anti-aging, and wrinkle improvement effects.

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

KR 10-2022-0073141 A

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 or prevent thrombosis through strong inhibition of blood coagulation using Songhwa mushroom fruiting body extract 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 Songhwa mushroom fruiting body extract as an active ingredient.

The extract is preferably an ethanol extract of pine flower mushroom fruiting body.

Additionally, the present invention provides an anticoagulant containing Songhwa mushroom fruiting body extract as an active ingredient.

The extract is preferably an ethanol extract of pine flower mushroom fruiting body.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing Songhwa mushroom fruiting body extract as an active ingredient.

The extract is preferably an ethanol extract of pine flower mushroom fruiting body.

Songhwa mushroom fruiting body extract 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 formation 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 pine flower mushroom fruiting body extract is used for edible purposes and is not toxic. It is processed into various forms such as extracts, powders, pills, tablets, etc. and has excellent effects in the pharmaceutical and food industries. It is a very useful invention.

Figure 1 shows shiitake mushroom and pine flower mushroom fruiting bodies.

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic effect of pineflower mushrooms, the inventors of the present invention prepared an ethanol extract of pineflower mushroom fruiting body by a certain method and then evaluated its antithrombotic activity by comparing it with that of shiitake mushrooms, and found that the pineflower mushroom fruiting body extract had excellent antithrombotic activity. By confirming that it has excellent heat stability and acid stability, the extract 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 and In order to develop health functional foods, various solvent extracts were prepared from the fruiting bodies of pine flower 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 pine flower mushroom fruiting body extract had excellent thrombin inhibition, prothrombin inhibition, and blood clotting factor inhibition activities.

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

The extract is preferably an ethanol extract of pine flower mushroom fruiting body.

Additionally, the present invention provides an anticoagulant containing Songhwa mushroom fruiting body extract as an active ingredient.

The extract is preferably an ethanol extract of pine flower mushroom fruiting body.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing Songhwa mushroom fruiting body extract as an active ingredient.

The extract is preferably an ethanol extract of pine flower mushroom fruiting body.

Below, the manufacturing method and efficacy test of the pine flower mushroom fruiting body extract of the present invention will be described in more detail.

The present invention includes the steps of preparing an extract from pine mushroom fruiting bodies through solvent extraction; A step of evaluating the antithrombotic activity of the extract; It includes steps to investigate the stability and safety of the ethanol extract of pine flower mushroom fruiting bodies.

The "pineflower mushroom fruiting body extract" included in the composition of the present invention is a process of harvesting the pineflower mushroom fruiting body, drying it, cutting it into 2-3cm pieces, extracting it with an organic solvent, and using a filter net of 0.06mm 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 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 pine mushroom fruiting body extract 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 (coagulation), unlike shiitake mushrooms. factor) inhibition was confirmed. The thrombus formation inhibitory activity of this active extract was an excellent anticoagulant activity comparable to that of aspirin (1.5 mg/ml), which is used clinically as an antithrombotic 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 pine flower mushroom fruiting body extract 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.

In addition to preventing or treating the above-mentioned thrombosis-related diseases, the active ingredient of the present invention can also be used as an anticoagulant (also called an anticoagulant, anticoagulant, blood coagulation inhibitor, or blood coagulation inhibitor, etc.).

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: Obtaining Songhwa and Shiitake Mushroom Fruiting Bodies

Fruiting body of Songhwa mushroom and fruiting body of Ganoderma lucidum were provided by Gold Farm Bio Co., Ltd., Mungyeong, Gyeongbuk, and each fruiting body is shown in Figure 1. Songhwa mushrooms refer to shiitake mushrooms cultivated by adding fermented sulfur, and the cultivation method is described in detail in Republic of Korea Patent Publication No. 10-2022-0073141 [Shiitake mushrooms with added fermented sulfur and their cultivation method]. To briefly explain the pine mushroom cultivation method, the medium sterilization step is to make and sterilize the shiitake mushroom sawdust medium; An inoculation step of inoculating shiitake mushroom spawn into the medium sterilized in the medium sterilization step; A culture step of cultivating mushrooms by supplying a fermented sulfur solution to the medium inoculated with shiitake mushroom spawn in the inoculation step; and a browning step of browning the shiitake mushrooms cultured in the culturing step so that they appear colored, wherein the fermented sulfur solution contains 10 parts by weight of sulfur per 100 parts by weight of water. After mixing 30 parts by weight, 10 to 30 parts by weight of sodium hydroxide, 1 to 2 parts by weight of phyllite, 1 to 2 parts by weight of red clay, 1 to 2 parts by weight of solar salt, and 1 to 2 parts by weight of zeolite, the mixture was stirred for 1 to 3 hours to dissolve sulfur. A mixing step of preparing a mixture; A filtration step of preparing a sulfur solution by allowing the sulfur mixture prepared in the mixing step to stand for 1 to 3 days and then filtering the precipitate; And manufacturing a fermented sulfur solution in which the sulfur solution prepared in the filtration step is passed through a sieve once or twice, mushroom mycelia are added to the sieve sulfur solution, and fermented at 20 to 25° C. for 5 to 10 days to produce a fermented sulfur solution. It is a method of cultivating shiitake mushrooms with added fermented sulfur, characterized in that it consists of steps. Songhwa mushrooms have a stronger flavor than shiitake mushrooms, have a harder texture, have an excellent texture, and have good storage properties. The actual moisture content of pine flower mushrooms and shiitake mushrooms was 70.5±1.5% and 80.3±2.4%, respectively, showing a significant difference.

Example 2: Preparation of ethanol extract of pine flower and shiitake mushroom fruiting bodies and analysis of components of the extract

The pine flower and shiitake mushroom fruiting bodies obtained in Example 1 were shade-dried, cut into pieces, and ethanol extracts were prepared from them. 10 times the amount of ethanol (95%, Deoksan, Korea) was added to each sample, and incubated at room temperature. After repeated extraction twice, the extracts were collected, filtered, and concentrated under reduced pressure to prepare a powder. The extraction yields of pine flower and shiitake mushroom fruiting bodies and their useful content analysis results are shown in Table 1.

[Table 1] Yield and useful content analysis of pine flower and shiitake mushroom fruiting body extracts

As shown in Table 1, the extraction efficiency of pine flower mushroom fruiting bodies was 9.1%, which is 3.5 times higher than that of shiitake mushrooms, showing that pine flower mushrooms contain more diverse bioactive components than shiitake mushrooms. To analyze useful components of pine flower mushroom fruiting body extract, total polyphenol, total flavonoid, and total sugar contents 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 analyzing the total polyphenol content, the pine flower mushroom extract showed a very high polyphenol content of 62.7 mg/g, which was about 14.3 times higher than the shiitake mushroom extract (4.4 mg/g). In the total flavonoid content analysis, the pine flower mushroom extract showed a very high content of 3.6 mg/g, which was about 12.0 times higher than the shiitake mushroom extract (0.3 mg/g). As a result of analyzing the total sugar content, the pine flower mushroom extract showed 21.3 mg/g, which was about 3.3 times higher than the shiitake mushroom extract (6.4 mg/g). Therefore, it is expected that Songhwa mushroom fruiting body extract will exhibit various useful physiological activities compared to shiitake mushrooms.

Example 3: Evaluation of blood coagulation inhibitory activity of pine flower and shiitake mushroom fruiting body extracts

The blood coagulation inhibitory activity of the pine flower and shiitake mushroom fruiting body extracts of Example 2 was evaluated, and the results are shown in Table 2. At this time, the blood coagulation inhibitory activity evaluation method of the 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 2] Blood coagulation inhibitory activity of pine mushroom and shiitake mushroom fruiting body extracts

As shown in Table 2, the thrombin time, prothrombin time, and APTI time were measured using the ethanol extract of the prepared pine flower mushroom fruiting body at a concentration of 5 mg/ml, and the results were 1.36 times, 1.45 times, and 3.03 times, respectively, compared to the solvent control. It showed extended thrombin time, prothrombin time, and AP time, showing very excellent antithrombotic activity compared to shiitake mushroom extract. Shiitake mushroom fruiting body extract was not found to have antithrombotic activity when compared to the solvent control. These results show that it has excellent antithrombotic activity compared to aspirin (1.5mg/ml), which is used clinically as an antithrombotic agent, and it was judged that it could replace aspirin, which has severe side effects such as gastrointestinal disorders, in the future. Considering that pineflower mushroom fruiting bodies are currently consumed as tea and beverages in addition to being eaten in cooking, pineflower mushroom fruiting body extracts are believed to be able to effectively inhibit the formation of blood clots through inhibition of various blood coagulation factors, thrombin and prothrombin.

Example 4: Evaluation of antioxidant activity and nitrite scavenging ability of pine flower and shiitake mushroom fruiting body extracts

The pine flower and shiitake mushroom fruiting body extracts prepared in Example 2 were evaluated for their antioxidant and nitrite scavenging abilities related to blood clot formation, and the results are shown in Table 3. At this time, the extract was dissolved in DMSO (dimethylsulfoxide) and then diluted to an appropriate concentration to obtain DPPH (1,1-diphenyl-2-picryl hydrazyl) anion radical scavenging ability, ABTS [2,2-azobis (3-ethylbenzo thiazoline-6-sulfonate)] It was used to measure cation radical scavenging ability, nitrite scavenging ability, and reducing power.

First, in the case of DPPH scavenging ability, ³⁸⁰ μl of 2 Absorbance was measured using Hitech, Expert96, Asys Co., Austria). DPPH radical scavenging ability was expressed as a percentage of the sample added and the sample not added. In the case of ABTS scavenging ability, 5ml of 7mM ABTS (Sigma Co., USA) and 88ml of 140mM potassium persulfate were mixed and light was blocked for 16 hours at room temperature to form ABTS cations. This solution was then diluted with ethanol to obtain an absorbance value of 1.5 at 414nm. It was diluted with . After mixing 190 ml of the prepared diluted solution and 10 ml of samples prepared at various concentrations, reacted at room temperature for 6 minutes, absorbance was measured at 734 nm, and ABTS radical scavenging ability was calculated using the following equation.

ABTS radical scavenging ability (%) = [(C-S)/C] x 100,

C: Absorbance upon addition of solvent control DMSO

S: Absorbance upon sample addition.

Meanwhile, in the case of measuring nitrite scavenging ability, the sample solution was added to the nitrite solution (1mM), adjusted to pH 1.2 by adding 0.1N HCl, and reacted at 37°C for 1 hour using Griess reagent (Sigma Co., USA). was added and mixed. After leaving it at room temperature for 15 minutes, the absorbance was measured at 520 nm to measure the amount of remaining nitrite. The nitrite scavenging ability (%) was calculated using the following equation.

NSA (%) = [1-(A-C)/B] x100,

A: Absorbance after adding sample to 1mM nitrite solution and reacting for 1 hour

B: Absorbance of 1mM nitrite solution

C: Absorbance of mushroom extract sample

In the above antioxidant experiment, vitamin C (Sigma Co., USA) was used as a control, and DMSO was used as a solvent control. Each activity evaluation was expressed as the average and deviation of experiments repeated three times.

Meanwhile, in the case of reducing power evaluation, the method of Oyaizu et al. was modified (Ahn Seon-mi et al., 2011. J. Life Sci. 21: 576-583). To 2.5ml of sample dissolved in ethanol, 2.5ml of 0.2M sodium phosphate buffer (pH 6.6) and 2.5ml of 10% potassium ferricyanide were added, reacted at 50°C for 20 minutes, and then 2.5ml of 10% trichloroacetic acid was added to terminate the reaction. and centrifuged at 4000 rpm for 10 minutes to recover the supernatant. The recovered supernatant was diluted two-fold with distilled water, mixed with freshly prepared 0.1% ferric chloride solution in a 5:1 (v/v) ratio, and evaluated by measuring absorbance at 700 nm.

[Table 3] Antioxidant activity of pine mushroom and shiitake mushroom extracts

As shown in Table 3, the antioxidant power was evaluated by adjusting the concentration of the mushroom extract to 0.5 mg/ml. As a result, the DPPH anion scavenging ability of the pine flower mushroom fruiting body extract was almost similar to that of the shiitake mushroom extract, and the ABTS cation scavenging power and reducing power were 1.2 times higher. and showed a 1.17-fold increased activity. The biggest difference was in the nitrite scavenging ability, and when the antioxidant power was evaluated by adjusting the concentration of the mushroom extract to 0.5mg/ml, the pine flower mushroom fruiting body extract showed a scavenging ability of 41.4%, which was 4.4 times more powerful than the shiitake mushroom extract. It seemed. As a result of calculating the concentration (IC ₅₀ ) that can actually scavenge 50% of active radicals, the IC ₅₀ of pine flower and shiitake mushroom fruiting body extracts for nitrite were 365 and 1,851 mg/ml, respectively, which was 5.07 times lower for pine flower mushrooms. showed value. Oxidative stress, nitrite, peroxynitrite, NO, etc. are closely related to thrombus formation (Wang, L. 2017. Sci. Rep. 7: 12429; Martinez, M. 2013. Free Radic. Biol. Med. 65: 411- 418; Bijak, M. 2012. Thromb. Res. 130: e123-e128), it is believed that the excellent antioxidant and nitrite scavenging properties of pine mushroom extract contribute to antithrombotic activity.

Example 5: Human red blood cell hemolytic activity of pine flower and shiitake mushroom fruiting body extracts

To evaluate the acute toxicity potential of the pine flower and shiitake mushroom fruiting body extracts prepared in Example 2, human red blood cell hemolytic activity 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 pine flower and shiitake mushroom fruiting body extracts

As shown in Table 3, 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, pine flower mushroom fruiting body extract showed 68.6% red blood cell hemolysis at a concentration of 1 mg/ml and 49.9% hemolysis at a concentration of 0.5 mg/ml. On the other hand, in the case of shiitake mushrooms, red blood cell hemolysis was not observed even at a concentration of 1 mg/ml. It is understood that the hemolytic activity of pine flower 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 judged that there is no serious acute toxicity. In particular, compared to amphotericin B, which has recognized side effects, pine flower mushroom fruiting body extract requires a concentration of about 300 times higher to exhibit the same activity of red blood cell hemolysis, so it was judged that acute toxicity would not occur.

Example 6: Evaluation of plasma, acid and heat stability of Songhwa mushroom fruiting body extract

The pine flower mushroom fruiting body extract prepared in Example 2 is 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 pine flower mushroom fruiting body extract for inhibiting 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 pine flower mushroom fruiting body extract 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 Songhwa mushroom fruiting body extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the extract is an ethanol extract of pine flower mushroom fruiting body. An anticoagulant 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.