KR20200015030A - Pharmaceutical composition comprising the extraction of spent mushroom substrate for lentinus edodes 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 prevention or treatment of thrombosis comprising, as an active ingredient, a spent mushroom substrate (SMS) extract which is generated as a by-product when cultivating Lentinus edodes. More specifically, the present invention relates to a pharmaceutical composition and health functional food for prevention or treatment/amelioration of thrombosis through potent blood coagulation inhibition comprising, as active ingredients, a hot water extract of the SMS obtained after inoculating and culturing a Lentinus edodes spawn in a medium for artificial cultivation of Lentinus edodes and harvesting Lentinus edodes, an ethyl acetate fraction and a butanol fraction of the hot water extract, and a water residue after sequential fractioning of an organic solvent. The SMS extract generated as the by-product during artificial cultivation of Lentinus edodes as an active ingredient of a pharmaceutical composition and health functional food for prevention or treatment of thrombosis according to the present invention exhibits potent antithrombotic activities due to inhibition of thrombus formation-related enzymes and inhibition of blood coagulation factors, and does not exhibit hemolytic activities to human red blood cells at all at the same time, has excellent thermal stability, and does not exhibit a decrease in inhibition activities on blood coagulation factors and thrombus formation-related enzymes even in an acid environment at pH 2 and in blood plasma, and thus may be expected to be used for preventing and treating thrombosis such as ischemic stroke and hemorrhagic stroke through improving blood circulation. Additionally, the active ingredient has an excellent effect of processing the active ingredient in various forms including extract, powder, pills, tablets and others such that the active ingredient is prepared in such a form that can be administered at all time. Therefore, the pharmaceutical composition and health functional food for prevention or treatment of thrombosis are inventions that are extremely useful in drug and food industries.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACTION OF SPENT MUSHROOM SUBSTRATE FOR LENTINUS EDODES AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention is a pharmaceutical composition and health functional food for preventing or treating thrombosis (thrombosis) containing as an active ingredient the extract of the mushroom (spent mushroom substrate, SMS) generated as a by-product when cultivated shiitake mushroom ( Lentinus edodes ) More specifically, after inoculating and cultivating shiitake mushroom seedlings in a culture medium for shiitake mushroom cultivation, the hydrothermal extract of the remaining waste medium and the ethyl acetate fraction, butanol fraction and sequential organic solvent fractions after harvesting the shiitake mushroom A pharmaceutical composition for the prevention or treatment / improvement of thrombosis through strong blood coagulation inhibition using water residue as an active ingredient, and a health functional food.

As a constituent of human body, blood has various important functions such as transporting and buffering oxygen, nutrients, and wastes, maintaining body temperature, controlling osmotic pressure and ion balance, keeping moisture constant, controlling liquid, maintaining and controlling blood pressure, and defending the body. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolytic reaction system in the body complement each other, and among them, the mechanism of the blood coagulation reaction system forms platelet thrombus by adhering and agglomerating platelets to blood vessel walls. In addition, it has been reported that the blood coagulation system is activated to form fibrin clots around platelet aggregates. Thrombotic disease occurs in abnormal cases due to various factors, such as the coagulation of platelets and plasma in the blood, fibrin breakdown, and the control and balance of coagulation inhibition processes. Excessive thrombus blocks blood vessels and impedes blood flow, leading to cerebral infarction and stroke. And myocardial infarction.

On the other hand, the generation of fibrin thrombi undergoes a multi-step reaction of numerous blood coagulation factors, which activates thrombin, which is involved in fibrin coagulation, and finally generates fibrin monomers from fibrinogen, which are polymerized by calcium, It binds to endothelial cells and creates permanent thrombus, forming fibrin polymers cross-linked by factor XIII. In addition, thrombin plays a pivotal role in thrombus generation by activating platelets, factor V and factor VII to promote blood coagulation. Therefore, thrombin's activity inhibitory substance can be used as a prophylactic and therapeutic agent which is very useful for various thrombotic diseases caused by excessive blood clotting. Meanwhile, in the endogenous thrombus generation pathway, sequential activation of XII, XI, IX and X factors is followed by the activation of prothrombin, which is known to activate thrombin. It is targeted. Until now, various anticoagulants, antiplatelets, thrombolytics such as heparin, coumarin, aspirin, urokinase, etc. have been used for the prevention and treatment of thrombotic diseases, but they are very expensive, and hemorrhagic side effects, gastrointestinal disorders and hypersensitivity reactions Its use is limited to such circumstances.

Shiitake mushroom is a edible mushroom belonging to the genus Pleurotus eryngii and pine nuts, which have the highest content of vitamin C and contain a large amount of various amino acids and ergosterol. In terms of health function, anti-cancer, lung disease and gastrointestinal disease prevention, virus immunity enhancement, antibody production promotion, blood pressure lowering, arteriosclerosis prevention, blood lipid concentration control function, diabetes suppression effect and the like are known.

Patents related to shiitake mushrooms include Korean Registered Patent No. 10-1839507 [Method for preparing seasoned salted fish sauce containing shiitake mushroom and shiitake mushroom fermentation], No. 10-1830763 [Manufacturing method of rice miso including shiitake mushroom extract] And rice miso prepared therefrom, No. 10-1823460 [Method for preparing semi-dried urok using shiitake mushroom powder], No. 10-1759312 [Method for preparing shiitake mushroom powder, preparation of nutrition bar using shiitake mushroom powder] Method and nutrition bar prepared thereby, No. 10-1673573 [Preparation method of shiitake mushroom tea and shiitake mushroom tea prepared therefrom], Korean Patent Publication No. 10-2004-0038957 Foods based on the manufacture of laver containing. Most of the patents known to date are for cultivating shiitake mushrooms and preparing various foods using shiitake mushrooms, and patents on useful physiological activities of shiitake mushrooms are very limited.

In addition, patents related to useful physiological activity of shiitake mushrooms are registered in Korean Patent No. 10-1487742 [Food composition having antioxidant efficacy including shiitake mushroom and cheonma extract], No. 10-1806808 [Fermentation using shiitake mushroom mycelium] A composition for the prevention, improvement or treatment of women's menopausal symptoms containing the prepared dropwise fermentation product as an active ingredient], No. 10-1611947 [Health food composition for preventing and improving vascular disease and preparation method thereof], No. 10-1266528 [Moisturizing and / or whitening cosmetic composition containing a polysaccharide extracted from shiitake mushroom mycelium culture as an active ingredient] is disclosed, and atopy containing shiitake mushroom extract as an active ingredient. And composition for preventing and improving contact dermatitis], Patent Publication No. 10-2003-0056753 [Health for the control of obesity containing shiitake mycelium, Agaricus mycelium, water-soluble chitosan Product composition], the No. 10-2008-0110212 [composition to aid in bone growth using the mushroom hot-water extract; this is disclosed. In particular, the Republic of Korea Patent No. 10-1611947 associated with thrombosis [Health food composition for preventing and improving vascular disease and its characteristics, characterized in that it comprises Happiness, Sewao, Seokchangpo, Wonji, Shiitake, Blossom mushroom as an active ingredient [Method of manufacture] is disclosed, but the anti-thrombotic composition that is safe and practically available through the inhibition of potent blood coagulation derived from shiitake mushroom has been known as a main ingredient of medicinal herb.

Meanwhile, the annual total production of domestic edible mushrooms is reported from 173,354 tons in 2013 to 162,292 tons in 2017.In 2017, 58,784 tons of oyster mushrooms, 48,588 tons of mushrooms of mushrooms, 38,092 tons of mushrooms of mushrooms, and 10,173 of mushrooms of mushrooms Tons were produced, and shiitake mushroom production is estimated at 25,000 tons (Ministry for Food, Agriculture, Forestry and Livestock, 2016. Production of specialty crops). Such mushroom cultivation is gradually changing in a natural medium using a specific artificial medium. As the artificial cultivation and production of mushrooms increases, the emissions of SMS are also rapidly increasing. Given that 5 kg of mushroom medium is typically consumed to produce 1 kg of mushrooms, domestic SMS is estimated to emit more than 2 million tons per year. Especially for shiitake mushrooms, various wood sawdust badges are used.In 2015, sawdust badges imported from China were 36,285 tons, more than doubled in three years compared to 11,825 tons in 2012 (KCS import and export statistics; weekly Shipbuilding, April 11, 2016).

If artificial media are used, mushroom cultivation is known to require 20-25% of nutrients (Williams et al, 2001. Biores Technol. 79: 227-230 .; Bae et al, 2006. J Anim Sci & Technol Kor. 48: 237-246). Therefore, a considerable amount of nutrients remain in the SMS. In addition, since SMS is already cultured in mushroom mycelium, a variety of enzymes such as β-glucosidase and cellulase derived from mushroom fruiting bodies and mycelium, and various physiological activities such as plant growth promoting active ingredients, antibacterial substances, disease resistant derivatives, etc. Metabolites are known to be included (Hautzel and Anke., 1990, Z Naturforsch. 45: 1093-1098; Lim et al., 2012. Mycobiology. 41: 160-166; Parada et al., 2012 Crop Protection 30 : 443-450; Suess, 2006, Agriculture and Lands.pp. 1-101). Therefore, SMS can be used as a nutritionally and bioactively superior material. However, until now, research into the development of high value-added functional materials using SMS has been very limited, and attention has been focused on research on efficient reuse of SMS.

In Korea, some of the SMS has been used for feed (Jung Jong-cheon et al., 2012. J. Mushroom Sci. Prod. 10: 174-178), and it is reused as artificial medium by adding 10-30% to mushroom cultivation (Namgil et al., 2015. J. Mushrooms, 13: 310-313) However, if the artificial medium has a high sawdust content, it is not used for feed or mushroom cultivation and is almost used as compost. Recently, studies have been actively conducted to efficiently purify livestock waste using various microorganisms and enzymes of SMS (Luo X et al., 2018, Bioresour. Technol. 250: 611-620; Meng L et al., 2018. Bioresour.Technol. 253: 197-203; Meng X et al., 2018.Bioresour.Technol. 251: 22-30), future SMS is suggested as a new environmental purifier.

The useful substance content of the SMS will depend on the medium composition before the mushroom inoculation and the type and condition of the cultured mushrooms. To date, most of the research has been focused on top mushroom SMS (Jung Mushroom, et al., 2012. J. Mushroom Sci. Prod. 10: 174-178), but recently, SMS of snail mushroom (Lee Sang-yeop, 2015, The Korean Journal of Mycology 43: 185-190) and beta-glucan content of shiitake sawdust medium (Young-Ae Park, 2016. Korean Journal of Mycology, 44: 296-299). To date, the known useful physiological activities of SMS include the growth of pepper extract, shiitake mushroom, and locust mushroom in water, at room temperature, to promote the growth of red pepper and inhibit the plague (Kwak Amin et al., 2015. J. Mushrooms, 13: 16 -20; Kang DS et al., 2017. Plant Pathology J. 33: 264-275) and anti-inflammatory effects of water extracts of Oyster mushroom SMS are known (Rivero-Perez N. et al., 2016. Indian J Pharmacol. 48: 141 -144). In addition, water soluble polymer polysaccharide (polysaccharide) has been mainly reported as the main active ingredient of SMS, and recently, an antioxidant active polymer polysaccharide has been reported in shiitake mushroom SMS (Zhu H et al., 2018, Int. J. Biol. 112: 976-978), acidic polysaccharides that inhibit cancer cell growth (Zhang Y et al., 2017. Int. J. Med. Mushrooms. 19: 395-403) and antimicrobial polysaccharides (Zhu H, 2012). , Int. J. Biol.Macromol. 50: 840-843). However, no strong antithrombotic activity has been reported in shiitake SMS.

In addition, until recently, all of the SMS-related patents were manufactured for fuel pellets using SMS (Korean Patent No. 10-1728233, a method for producing pellets for fuel using waste medium or waste wood after mushroom cultivation), and feed production (Korea Patent Publication) No. 10-2014-0066036, feed using a mushroom waste medium and its manufacturing method), and the preparation of glycosylation enzyme (Korean Patent No. 10-1458628, a method for producing a glycosylase from mushroom waste medium) The potent antithrombotic activity of mushroom SMS is unknown.

KR 10-1611947 B

The present invention has been made to solve the problems of the prior art as described above, the problem to be solved in the present invention is for the prevention or treatment of thrombosis containing the extract of SMS generated as a by-product during shiitake artificial cultivation as an active ingredient It is to provide a pharmaceutical composition and dietary supplement.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing Lentinus edodes (Spent Mushroom Substrate, SMS) after harvest as an active ingredient.

The extract is preferably a hot water extract.

The extract is preferably selected from the group consisting of ethyl acetate fraction, butanol fraction and water residue obtained by sequentially fractionating hot water extract with hexene, ethyl acetate and butanol.

The present invention also provides a dietary supplement for the prevention or improvement of thrombosis containing Lentinus edodes (Spent Mushroom Substrate, SMS) after harvest as an active ingredient.

The extract is preferably a hot water extract.

The extract is preferably selected from the group consisting of ethyl acetate fraction, butanol fraction and water residue obtained by sequentially fractionating hot water extract with hexene, ethyl acetate and butanol.

Extract of SMS generated as a by-product during the artificial cultivation of shiitake mushroom as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis of the present invention and a health functional food is a potent antithrombosis caused by inhibition of enzymes related to blood clotting and inhibition of blood coagulation factors At the same time, it exhibits no hemolytic activity against human erythrocytes, excellent thermal stability, and no loss of blood coagulation factor inhibition and thrombus formation related enzyme inhibitory effects even in acidic conditions and plasma at pH 2, thereby improving blood circulation. Through it is expected to be used for the prevention and treatment of thrombosis, such as ischemic stroke and hemorrhagic stroke, the active ingredient is processed into various forms such as extract, powder, pills, tablets can be prepared in a form that can be taken at all times Its outstanding effect makes it very useful for the pharmaceutical and food industries It is a person.

1 is a sterilization medium for shiitake mushroom cultivation used in the experiment from left to right, inoculation and incubation of shiitake spawn seedlings in sterilization medium, SMS after harvesting shiitake mushrooms once, SMS after harvesting 3 times, and harvesting A photograph of shiitake mushrooms.
Figure 2 is a sterilization medium for shiitake mushroom cultivation used in the experiment from left to right, inoculation and incubation of shiitake spawn seedlings in the sterilization medium SMS after harvesting shiitake mushrooms once, SMS after three harvests, and harvested Photograph showing the hydrothermal extract of each shiitake mushroom.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

In order to verify the antithrombotic efficacy of shiitake mushroom SMS, the inventors of the present invention inoculated and cultured with shiitake mushroom seedlings in sterilization medium (new medium before inoculation) and sterilization medium for cultivation of shiitake mushrooms, and harvested shiitake mushrooms once. Thereafter, SMS, three crops after harvesting, and dried powder of each of the harvested shiitake mushrooms were prepared. Subsequently, the hydrothermal extracts were prepared by a constant method, and their antithrombotic activity was evaluated. As a result, the anti-coagulation inhibitory activity, which is not found in shiitake mushroom powder and sterilization medium, was found in SMS after one harvest and SMS after three harvests. After that, ethyl acetate fraction, butanol fraction and water residue after organic solvent fraction were recovered from each extract as an antithrombotic active ingredient, and the extract and fraction were thermally stable without showing any hemolytic activity against human erythrocytes. By confirming that the peracid stability has excellent characteristics, the extracts and fractions were intended to be utilized as pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of thrombosis.

Specifically, the present inventors, in order to develop a pharmaceutical composition for the prevention or treatment / improvement of thrombosis and health functional food in SMS after mass culture of mycelia and immature fruiting bodies of shiitake mushrooms known to have excellent health functional effects, After the harvest of shiitake mushrooms, SMS, and the nutritional analysis and physicochemical characterization of SMS after harvesting shiitake mushrooms 3 times, hydrothermal extracts were prepared for the samples, and antithrombotic activity was evaluated. After the 1st and 3rd harvests, the SMS hot water extracts were subjected to sequential organic solvent fractionation with hexene, ethyl acetate and butanol, respectively, and finally water residues were prepared after the fractionation. The antithrombotic activity of each extract and fractions was evaluated for Thrombin Time, Prothrombin Time and Activated Partial Thromboplastin Time (aPTT) against human plasma and human thrombin. As a result, strong anticoagulant activity was confirmed in SMS hydrothermal extract and ethyl acetate fraction, butanol fraction and water residue after 1 and 3 times of shiitake mushroom harvesting. This antithrombotic activity was a potent antithrombotic activity that surpassed aspirin, which is used in the clinic as an antithrombotic agent. In addition, the SMS hydrothermal extract and active fractions after harvesting during shiitake mushroom cultivation did not show hemolytic activity against human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing shiitake mushroom harvest as an active ingredient.

The extract is preferably a hot water extract.

The extract is preferably selected from the group consisting of ethyl acetate fraction, butanol fraction and water residue obtained by sequentially fractionating hot water extract with hexene, ethyl acetate and butanol.

The present invention also provides a health functional food for preventing or improving thrombosis containing shiitake mushroom harvest as an active ingredient.

The extract is preferably a hot water extract.

The extract is preferably selected from the group consisting of ethyl acetate fraction, butanol fraction and water residue obtained by sequentially fractionating hot water extract with hexene, ethyl acetate and butanol.

Hereinafter, the method of producing and extracting the SMS extract and each active fraction material during the shiitake mushroom artificial cultivation of the present invention will be described in more detail.

The present invention is a sterilization medium for artificial cultivation of shiitake mushrooms, inoculation and culture of shiitake mushroom seedlings in sterilization medium, SMS after harvesting shiitake mushrooms once, SMS after harvesting three times, and each of the harvested shiitake mushrooms Preparing a powder; Preparing an extract from the four samples; Evaluating the antithrombotic activity of the extract; Preparation of sequential organic solvent fractions of hexene, ethyl acetate and butanol from post-harvest SMS extracts during shiitake artificial cultivation exhibiting strong antithrombotic activity and subsequent water residues; And evaluating the antithrombotic activity of the fractions and investigating the stability of the active substances of the extracts and active fractions.

"SMS extract after harvesting shiitake mushroom cultivation" included in the composition of the present invention is a step of inoculating and cultivating shiitake mushrooms, and recovering and discarding the SMS that are discarded after harvesting shiitake mushrooms 1 to 3 times; Extracting the ground SMS with a solvent and filtering the extract using a filtering network of 0.06 mm or less, and concentrating it under reduced pressure.

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

The extract, preferably hot water extract, may be sequentially or separately fractionated with an organic solvent of hexene, ethyl acetate and butanol to additionally obtain a water residue after hexene fraction, ethyl acetate fraction, butanol fraction and organic solvent fraction.

In the present invention, the sterilized medium extract for shiitake mushroom cultivation, the SMS extract after one harvest, the SMS extract after three harvests, and the extract of the shiitake mushroom dry powder at the concentration of 5 mg / ml, respectively, As a result, the bactericidal medium extract and shiitake powder extract showed weak antithrombotic activity, whereas after one and three harvests, the SMS extract showed very strong thrombin inhibition and coagulation factor inhibition. After the harvest, the SMS extract showed a very strong blood coagulation inhibitory effect by prolonging both thrombin time and epitaxial time by more than 15 times compared to the non-added group at 6 mg / ml concentration, even at 2.5 mg / ml concentration. The thrombin time was 2.35 ~ 3.84 times longer and the 2.95 ~ 3.46 times longer time.

From the above results, the organic solvent fractions and water residues of SMS extract after 1 and 3 harvests, which were by-products after 1 and 3 harvests of shiitake mushrooms, were prepared, and their blood coagulation inhibitory activity was evaluated. The strong thrombin inhibition and coagulation factor inhibition were confirmed in the ethyl acetate fraction and water residue of the SMS extract after the harvest, and the very strong thrombin inhibition in both the ethyl acetate fraction, butanol fraction and the water residue of the SMS extract after the third harvest. By confirming the inhibition of coagulation factor, it was confirmed that SMS after shiitake mushroom harvest contained various antithrombotic active substances with various solubilities. In addition, the anti-thrombotic activity is weak in the early sterilization medium, but as the lung medium after the first harvest after three harvests shows a strong antithrombotic activity, it is a complex such as biological conversion of shiitake mycelium, fruiting body and medium components It is understood as the enhancement of activity exhibited by the change.

In particular, the ethyl acetate fraction of the SMS extract after the first harvest showed very strong thrombin inhibition, prothrombin inhibition and coagulation factor inhibition, which was confirmed to be superior to aspirin in antithrombotic activity. Therefore, it was confirmed that the post-harvest SMS extract and its active fractions, which are generated as a by-product of shiitake mushroom cultivation, could be developed as anticoagulants (blood coagulation inhibitors) that can replace aspirin, which has high side effects such as gastrointestinal disorders.

On the other hand, the antimicrobial inhibitory effect on human platelets was confirmed by using a concentration of 0.25 mg / ml prepared sterilized culture medium for shiitake mushroom cultivation, SMS extract after one harvest, SMS extract after three harvests and shiitake mushroom dry powder. As a result, cultivated sterilized medium extract showed 72.6% platelet aggregation compared to no addition group, and the other extract samples showed 95.9 ~ 118.6% aggregation. Since aspirin used as a control showed a cohesiveness of 24.9% at a concentration of 0.25 mg / ml and 85.9% at a concentration of 0.125 mg / ml, the extract samples other than the culture sterilization medium did not recognize platelet aggregation inhibitory activity.

In addition, as a result of confirming inhibition of human platelet aggregation of organic solvent fraction and water residues of the extract of SMS after one and three harvests of prepared shiitake mushrooms, ethyl acetate fraction (0.25 mg / ml) and butanol of SMS extract after one harvest The platelet aggregation ability of 55.1% and 76.5% was confirmed in the fraction (0.25 mg / ml), and the platelet aggregation ability of 95.0% was confirmed in the ethyl acetate fraction (0.25 mg / ml) of SMS extract after three harvests. Although this activity has a lower aggregation inhibitory effect than the same concentration of aspirin, it was confirmed that side effects such as aspirin can be used as a platelet aggregation inhibitor as a useful fermentation material to replace the known anti-thrombotic agents.

After the mushroom harvest during shiitake mushroom artificial cultivation of the present invention, the SMS extract and its active fractions may be prepared into powder through a conventional powdering process such as vacuum drying and freeze drying, or spray drying. They are not degraded by various degrading enzymes in plasma and remain active even at a heat treatment of 100 ° C. and pH in the human stomach at pH 2.

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

The pharmaceutical composition comprising the active ingredient of the present invention may be prepared in accordance with conventional methods for the purpose of use, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and injectables of sterile injectable solutions. It may be formulated and used in various forms, and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further 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 rubber, 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 a filler, an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like may also be used.

As a preferred embodiment, oral liquid preparations may be exemplified by suspending agents, solvents, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, Fragrances, preservatives and the like.

As a preferred embodiment, the preparation for parenteral administration may be sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized, suppository and the like. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, activity of the drug, Sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one 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 age, sex, and weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg body weight daily. Or every other day or divided into 1 to 3 times daily. However, since the dose may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

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

As used herein, "administration" means providing a patient with any substance by any suitable method, wherein the route of administration of the pharmaceutical composition of the present invention is oral or parenteral via all common routes as long as the target tissue can be reached. Oral administration. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

"Subject" in the present invention is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And preferably mammals, and more preferably humans.

In addition, the health functional food of the present invention can be used in a variety of foods and beverages and the like effective in preventing or improving thrombosis. Examples of the food containing the active ingredient of the present invention include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, 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 in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 10 g, preferably 0.3 to 1 g based on 100 ml.

In addition to containing the compound as an essential ingredient in the indicated proportions, the health functional food of the present invention may contain as food additives food additives such as natural carbohydrates and various flavoring agents.

Examples of the natural carbohydrate include conventional 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, taumartin; Natural flavors such as stevia such as rebaudioside A or glycyrzin and synthetic flavors such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally used from about 1 to 20 g, preferably from about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals, synthetic flavors and natural flavoring agents, colorants and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids Thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for preparing natural fruit juice, fruit juice beverage, vegetable beverage and the like. These components can be used independently or in combination. The proportion of such additives is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

EXAMPLE

Example 1: Physicochemical Characterization and Color Analysis of Shiitake Mushroom Sterilization Medium, SMS after One Harvest, SMS and Shiitake Mushroom After Three Harvests

Shiitake mushroom cultivation medium (composition: oak 80%, bran 19%, edible gypsum 1%) prepared in 2017 at the Chinese cash register was press-sealed and sterilized at 100 ℃ for 8 hours to prepare a sterile medium, 808 was inoculated and incubated in the culture room for about 120 days, and then incubated in a grower for 40 days at 23 ° C. and 70% humidity conditions, and then generated. Thereafter, the resulting shiitake mushrooms were harvested once (1 cycle harvest) and then SMS was prepared after 1 harvest. Then, the shiitake mushrooms were re-generated 2 times (2 cycles harvesting) and 3 times (3 cycles harvesting). SMS was recovered after the remaining three harvests (FIG. 1).

The photographs of the sterilization medium, SMS after one harvest, SMS after three harvests, and harvested shiitake mushrooms are shown in FIG. 1, and the crude protein, carbohydrate, crude lipid, ash and moisture content thereof were measured according to food regulations. The calories are shown in Table 1.

[Table 1] Nutritional analysis of sterilization medium, SMS after 1 harvest, SMS and shiitake mushroom after 3 harvests

Moisture content was changed from 46.8% at the beginning to 52.6% after the first harvest and 54.0% after the third harvest. The crude protein was 3.1% in the sterile medium, but increased more than twice from 6.75% to 7.5% in the SMS after harvesting. Ash content was also 6.55% in sterilization medium, but after harvest, it increased rapidly from 14.77 to 14.93% in SMS. Ash content of shiitake mushroom was 0.8%. On the other hand, crude lipid content was minimal and crude carbohydrates decreased rapidly from 43.3% to 25.7% after the first harvest and 23.2% after the third harvest. This is judged to be the result of the medium after primary and tertiary harvest containing a large amount of shiitake mycelium and fruiting body.

On the other hand, as a result of the color analysis of each sample after grinding the shiitake mushroom medium and shiitake mushroom, the sterilization medium showed relatively low brightness, redness, yellowness, and the color difference was 73.3. Post SMS showed 46.3 and 44.75, respectively, showing similar brightness, redness and yellowness (Table 2). At this time, the color difference analysis was measured using a Hunter Color Difference meter (Super color SP-80 Colormeter, Tokyo Denshoku Co., Japan), brightness (white 100 ~ 0 black), redness (red 100 ~ -80 green), Yellowness (yellow 70-80 black) was measured. In this case, the chromaticity of the standard whiteboard was determined by L. value of 92.44, a value of -0.06, and b value of 1.35. The average value was obtained by measuring three times per sample, and the color difference (△ E) was expressed by the following equation. Calculated using.

[Table 2] Color difference analysis of sterilization medium, SMS after one harvest, SMS and shiitake powder after three harvests

Example 2: Preparation of sterile medium, SMS after one harvest, SMS and shiitake hot water extract after three harvests, and physicochemical characterization thereof

A hydrothermal extract was prepared for the sterilization medium prepared in Example 1, SMS after one harvest, SMS after three harvests, and shiitake mushroom dry powder. 10 times of distilled water was added to each sample, and extracted at 100 ° C. for 1 hour, and then the pH, brix, and color difference of the obtained extract were measured, and the results are shown in Table 3.

[Table 3] Physicochemical Properties of Sterilized Medium, SMS after 1st Harvest, SMS after 3rd Harvest, Shiitake Powder Hydrothermal Extract

The initial pH of the sterile medium was 5.6, but decreased to 4.6 for SMS after 1 and 3 harvests. In addition, the initial brix of the sterilization medium increased from 6.0 to 16.0 and 20.0 after 1 and 3 harvests, and the acidity also increased to 0.83 and 1.40 after 1 and 3 harvests. On the other hand, the color analysis of the hydrothermal extract showed that the lightness, redness and yellowness increased with increasing number of harvests, and the color difference increased from 76.8 in early sterilization medium to 78.2 in SMS after the third harvest. In other words, the longer the culture of shiitake mushrooms, the more noticeable the increase in acidity, brix, and yellowness of SMS were (Figure 2).

Example 3: Component analysis of sterilization medium, SMS after one harvest, SMS after three harvests, and shiitake hot water extract

After filtering the hot water extract prepared in Example 2, and concentrated under reduced pressure to prepare a powder, the results of each extraction efficiency and component analysis of the extract is shown in Table 4. Component analysis determined total polyphenols, total flavonoids, total sugars and reducing sugars content. In the total polyphenol content, 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ saturated solution was added to 400 μl of the extraction sample solution, and the absorbance was measured at 725 nm after standing at room temperature for 1 hour. Tannic acid was used as the standard reagent. For the total flavonoid content, each sample was extracted by stirring for 18 hours with methanol, 4 ml of 90% diethylene glycol was added to 400 µl of the filtered extract sample, and 40 µl of 1 N NaOH was added again. Absorbance was measured at. Rutin was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was determined by phenol-sulfuric acid method.

[Table 4] Component analysis of sterilized medium, SMS after one harvest, SMS and shiitake hot water extract after three harvests

As shown in Table 4, the extraction efficiency was very low (1%) in the sterilization medium, but SMS showed a high yield of 5.1-9.2%, and SMS after the third harvest (9.2) was higher than SMS (5.1%) after the first harvest. %) Showed about 1.8 times higher extraction yield. The extraction efficiency of the hydrothermal extract of shiitake dry powder was very high as 34.7%. This is believed to be due to the increase in soluble components due to decomposition of the sterilization medium as the shiitake cultivation proceeds.

As a result of the total polyphenol analysis, the sterilization medium showed 20.5 mg / g, which was 2.5 times higher than shiitake mushroom, and after the first and the third harvest, SMS contained 34.9 ~ 37.5 mg / g. It was much higher than shiitake mushrooms. This pattern is the same in the analysis of total flavonoid content (Table 4). Therefore, after the first and third harvest, SMS was expected to show better physiological activity than mushroom sterilization medium and harvested shiitake mushrooms.

Meanwhile, total sugar showed the highest content of 144.9 mg / g in the sterilization medium, and reducing sugar showed the highest content of 53.4-54.0 mg / g in the SMS after the first and third harvests. On the other hand, shiitake extract showed very low total sugar (21.6 mg / g) and reducing sugar (19.7 mg / g) content, unlike other samples.

Example 4: Blood coagulation inhibitory activity of sterilization medium, SMS after one harvest, SMS and shiitake mushroom hot water extract after three harvests

Blood coagulation inhibitory activity was evaluated for various extracts prepared in Example 2, and the results are shown in Table 5. At this time, the evaluation method of blood coagulation inhibitory activity 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 episode time. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and thrombin time, prothrombin time and episode time measurements were performed as follows.

Thrombin Time (TT)

50 μl of 0.5 U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ , and 10 μl of various sample extracts were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes. After the addition of 100 μl of plasma, 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. DMSO showed a solidification time of 24.2 seconds. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the thrombin inhibitory activity was expressed as the coagulation time when the sample was added divided by the coagulation time of the solvent control.

Prothrombin Ttime (PT)

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations were added to a tube of Amelung coagulometer KC-1A (Japan), warmed at 37 ° C. for 3 minutes, and then 130 μl of PT reagent was added. The time to plasma coagulation is expressed as the average of three repeated experiments. Aspirin (Sigma Co., USA) was used as a control and DMSO was used instead of the sample as a solvent control. DMSO had a solidification time of 16.8 seconds. The prothrombin inhibitory activity was expressed by the solidification time of sample addition divided by the solidification time of the solvent control.

aPTT (activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of various concentrations of the sample extract were added to a tube of Amelung coagulometer KC-1A (Japan), warmed at 37 ° C. for 3 minutes, and then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added again. Incubated at 37 ° C. for 3 minutes. Thereafter, 50 μl CaCl ₂ (35 mM) was added and the time until the plasma coagulated was measured. DMSO was used as a solvent control instead of the sample, in which case it showed a solidification time of 42.2 seconds. The results of aPTT were shown as the average value of three repeated experiments, and the coagulation factor inhibitory activity was expressed as aPTT at the time of sample addition divided by aPTT of the solvent control.

[Table 5] Blood coagulation inhibitory activity of sterilized medium, SMS after 1 time harvest, SMS after 3 times harvest, hydrothermal extract of shiitake mushroom powder

As shown in Table 5, in the case of aspirin used as a control, TT, PT and aPTT were all extended by 15 times or more at 5 mg / ml compared with no addition, and TT, PT and aPTT even at 1.5 mg / ml. Was increased by 1.55 times, 1.39 times, and 1.46 times, respectively, to effectively inhibit blood coagulation. On the other hand, in the case of sterilized medium and shiitake extract, TT, PT and aPTT were increased by 1.05 times, 1.51 times, 1.25 times and 1.07 times, 0.93 times and 1.10 times, respectively, at 5 mg / ml. And blood coagulation factor inhibition. However, for SMS extracts after primary and tertiary harvest, TT, PT and aPTT were at least 15 times, 1.76 times, 4.97 times and 15 times higher, 1.05 times, and 13 times higher than the non-added at 5 mg / ml concentration, respectively. Increased to show good anticoagulant activity. In particular, post-harvest SMS extracts showed potent thrombin and coagulation factor inhibition at concentrations above 6 mg / ml, extending both TT and aPTT by more than 15-fold compared to no additions (Table 5). Although antithrombotic activity has been reported due to the inhibition of endogenous coagulation factor (aPTT) of shiitake mushrooms (Kang DS et al., 2017. Plant Pathol. J. 33, 264-275) Antithrombotic activity has not been known to date, and this activity is believed to be due to a substance newly produced by shiitake mycelium growth in sterile medium. The results suggest that shiitake SMS can be developed as a novel antithrombogenic material.

Example 5 Preparation and Component Analysis of Sequential Organic Solvent Fractions of SMS After One Harvest and SMS Hydrothermal Extract After Three Harvests

In order to confirm the active ingredients of the SMS after the first harvest and the SMS hot water extract after the third harvest prepared in Example 2, each extract was subjected to sequential organic solvent fractions using hexene, ethyl acetate, butanol, Finally water residue was recovered. Component analysis of each recovered fraction was analyzed in the same manner as in Example 3, the results are shown in Table 6.

[Table 6] Preparation and Component Analysis of Sequential Organic Solvent Fractions of SMS After One Harvest and SMS Hot Water Extract After Three Harvests

As shown in Table 6, the SMS extract after one and three times the harvest was mostly transferred to the water residue after the organic solvent fraction, it can be seen that the hot water extract contains a significant amount of water-soluble components. After one harvest, the ethyl acetate fraction of SMS extract accounted for 5.7%, butanol fraction of 24.3%. After three harvests, the ethyl acetate fraction of SMS extract accounted for 8.1% and butanol fraction of 17.3%. That is, the ethyl acetate fractions were 0.29 g and 0.75 g, and the butanol fractions 1.24 g and 1.58 g, respectively. On the other hand, the hexene fraction of SMS after 1 and 3 harvests was very low, 0.2% and 0.1%, respectively, and thus was excluded from future antithrombotic activity evaluation.

As a result of analysis of the total polyphenol content of each fraction, the hexene fraction of SMS showed the highest 54.8 mg / g after one harvest, followed by ethyl acetate fraction> butanol fraction> water residue. However, after 3 harvests, the ethyl acetate fraction showed the highest 95.4 mg / g, followed by water residue> butanol fraction> hexene fraction. These results indicate that the SMS extract after one harvest and the SMS extract after three harvests are different.

Example 6 Blood Coagulation Inhibitory Activity of Sequential Organic Solvent Fractions of SMS After One Harvest and SMS Hot Water Extract After Three Harvests

The anticoagulant activity of the various fractions obtained in Example 5 was evaluated in the same manner as in Example 4. As shown in Table 7, the ethyl acetate fraction and water residue of SMS extract after one harvest showed strong anticoagulant activity, and the ethyl acetate fraction, butanol fraction and water residue of SMS extract after three harvests It exhibited strong anticoagulant activity.

[Table 7] Blood clotting inhibitory activity of sequential organic solvent fractions of SMS after one harvest and SMS hot water extract after three harvests

Example 7: Human red blood cell hemolysis activity of SMS after one harvest and SMS hydrothermal extract and three times after harvest

Shiitake mushrooms have been used for edible and medicinal purposes for a long time, and SMS has been used as feed after harvesting shiitake mushrooms. In the present invention, the human erythrocyte hemolytic activity test was performed to evaluate the acute toxicity of the SMS extract and their active fractions, and the results are shown in Table 8. At this time, hemolytic activity was evaluated according to the existing report (Jeong In Chang, Son Ho Yong, 2014, Korean J. Microbiol. Biotechnol. 42: 285-292), and simply, 100 μl of human red blood cells washed three times with PBS was 96- 100 μl of various sample solutions were added to the well microplate, and then reacted at 37 ° C. for 30 minutes. Then, the reaction solution was centrifuged for 10 minutes (1,500 rpm) to transfer 100 μl of the supernatant to a new microtiter plate. The degree of hemoglobin outflow was measured at 414 nm. DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1 mg / ml) was used as an experimental control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

[Table 8] Human red blood cell hemolysis activity of SMS hydrothermal extract and its active fractions after SMS and 3 times after harvest

First, DMSO and water used as a control did not have erythrocyte hemolytic activity, triton X-100 was confirmed that 100% hemolysis of red blood cells at a concentration of 1 mg / ml. On the other hand, SMS extract and its active fractions after 1 and 3 times harvesting did not show any hemolytic activity even at 1 mg / ml. Considering that the anticancer drug known as hemolytic activity, empoterasin B, exhibited 55.5% hemolytic activity at 0.00625 mg / ml and 93.7% at 0.05 mg / ml, the SMS extract and its active fractions after harvesting shiitake mushrooms It is judged that there is no activity.

Example 8 Evaluation of Plasma, Acid and Thermal Stability of SMS After One Harvest and SMS Hydrothermal Extract and Three After Harvest

Plasma stability, thermal stability and acid stability against antithrombotic activity of the shiitake mushrooms obtained in Examples 2 and 5 after harvesting SMS, SMS extracts after harvesting three times, and active fractions thereof were confirmed. The samples did not show a significant decrease in blood coagulation inhibitory activity even after 1 hour heat treatment at 100 ° C., 1 hour treatment at pH 2 (0.01 M HCl), and 1 hour treatment in plasma, indicating high stability. Therefore, the extracts and active fractions above are expected to maintain excellent antithrombotic activity during digestive absorption and food preparation.

Claims (4)

A pharmaceutical composition for the prevention or treatment of thrombosis containing Lentinus edodes (Spent Mushroom Substrate, SMS) after harvest as an active ingredient. The pharmaceutical composition of claim 1, wherein the extract is a hydrothermal extract. The method of claim 1, wherein the extract is at least one selected from the group consisting of ethyl acetate fraction, butanol fraction and water residue obtained by sequentially fractionating the hydrothermal extract into hexene, ethyl acetate and butanol Composition. A health functional food for preventing or improving thrombosis, comprising the active ingredient according to any one of claims 1 to 3.

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