A composition comprising an extract of fomitopsis pinicola as an active ingredient for treating or preventing ischemic brain disease and the use thereof.

Abstract

The present invention relates a composition comprising an extract of Fomitopsis pinicola as an active ingredients for the treatment or prevention of ischemic brain diseases and the use thereof. The inventive extract of Fomitopsis pinicola showed potent treating effect on ischemic brain disease through various experiments, for example, (1) ASIC1 ion channel inhibition assay using a patch clamp (Experimental Example 1) on the extract of Fomitopsis pinicola of the present invention; and neuroprotective effect analysis experiment using a neuroblastoma cell line (sh-sy5y) to inhibit ASIC1 and inhibit apoptosis, and the results of these experiments confirm that the inventive extract of the present invention can be used as a treatment or adjuvant for ischemic brain disease, and confirm that the composition is useful as a pharmaceutical composition, health functional food and dietary supplement for the prevention or treatment of ischemic brain disease.

Description

[Rectified under Rule 91, 01.02.2024]A COMPOSITION COMPRISING AN EXTRACT OF FOMITOPSIS PINICOLA AS AN ACTIVE INGREDIENT FOR TREATING OR PREVENTING ISCHEMIC BRAIN DISEASE AND THE USE THEREOF.

[0001] The present invention is related to a composition comprising an extract of Fomitopsis pinicola as an active ingredient for treating or preventing ischemic brain disease and the use thereof.

[0002]

[0003] The majority of strokes are ischemic strokes, which account for 105,000 patients every year in Korea, and are the fourth leading cause of death. There are two types of stroke, and about 85% of ischemic strokes are caused by blockage of blood vessels in the brain due to blood clots, and it is also called cerebral infarction. In other cases, hemorrhagic strokes are caused by rupture of blood vessels, also known as cerebral hemorrhage.[Tapuwa D. Musuka et. al. Diagnosis and management of acute ischemic stroke: speed is critical. CMAJ. 2015 Sep 8; 187(12): 887-893.]

[0004] When ischemia occurs due to an ischemic stroke, the oxygen supply to the surrounding tissues is cut off from the blood flow, causing damage to the nerve cells in the brain, resulting in permanent disability or death.[ Qiu Jing Wu et. al. Targeting NMDA receptors in stroke: new hope in neuroprotection. Molecular Brain (2018) 11:153]

[0005] Numerous studies have been conducted on the treatment of ischemic stroke, but to date, the only treatment used after cerebral infarction is tissue plasminogen activator (tPA), a thrombolytic drug.[ Qiu Jing Wu et. al. Targeting NMDA receptors in stroke: new hope in neuroprotection. Molecular Brain (2018) 11:153]

[0006] Recently, various studies have been conducted to prevent the death of brain nerve cells after ischemic stroke, and there has been a growing interest in traditional natural resources.

[0007]

[0008] When ischemic stroke occurs, various pathways and biochemical changes contribute to the damage of brain nerve cells, but apoptosis due to increased intracellular calcium is known to be the main cause. The increase in intracellular calcium can be introduced through a variety of pathways, and the most notable pathway in brain neurons is the NMDA receptor [ Qiu Jing Wu et. al. Targeting NMDA receptors in stroke: new hope in neuroprotection. Molecular Brain (2018) 11:15]. However, studies targeting NMDA receptors have shown little effect on ischemia-induced cranial neuroprotection.

[0009]

[0010] When ischemic stroke occurs, another pathway for intracellular calcium increase is acid-sensing ion channels; ASIC) is being discussed. ASICs are activated by a decrease in pH and are known to cause the influx of calcium and apoptosis. When ischemic stroke occurs, pathophysiological features include acidosis due to the accumulation of lactic acid and the production and release of hydrogen ions. At this time, ASIC1 activity occurs due to the lowered pH, and apoptosis occurs due to the increase in calcium concentration in cells, and apoptosis increases. Various previous studies have reported that the use of psalmotoxin, an inhibitor of ASIC1α, or the reduction of damage to brain neurons caused by ischemia in animal models with ASIC1 knocked out.[ Xin Qi et. al. Pharmacological Validation of ASIC1a as a Druggable Target for Neuroprotection in Cerebral Ischemia Using an Intravenously Available Small Molecule Inhibitor. Front. Pharmacol., 24 March 2022 Sec. Pharmacology of Ion Channels and Channelopathies Volume 13 - 2022; Zhi-Gang Xiong et. al. The role of ASICs in cerebral ischemia. Wiley Interdisciplinary Reviews: Membrane Transport and Signaling Volume 1, Issue 5 Sep 2012P ages533-683; Jing-Jing Wang et. al. Disruption of auto-inhibition underlies conformational signaling of ASIC1a to induce neuronal necroptosis. Nature Communications volume 11, Article number: 475 (2020) ]

[0011] "Fomitopsis pinicola", the scientific name of the pine butterfly mushroom, has been reported to show anti-cancer, anti-inflammatory, antibacterial, tonic, and glycemic control effects have been reported.[ KS Bishop et. al. Characterisation of Extracts and Anti-Cancer Activities of Fomitopsis pinicola Nutrients 2020, 12(3), 609; J.E. Haight et. al. Phylogeny of Fomitopsis pinicola: a species complex. Mycologia, 108 (2016), pp. 925-938; Robert Rogers, The Fungal Pharmacy: The Complete Guide to Medicinal Mushrooms and Lichens of North America, Berkeley, Calif.: North Atlantic Press, 2011, pp. 158-162; Christopher Hobbs, Medicinal Mushrooms, 1996(3rd ed.), pp. 92-93.]

[0012]

[0013] As prior arts, Korean Patent Publication No. 10-2004-0039195 (Immune Enhancer); Korea Patent Publication No. 10-2007-0078911 (Pine Butterfly Mushroom Extract and Its Uses with Anti-Inflammatory Effect); Korea Patent Publication No. 10-2007-0078404 (Extract of pine butterfly mushroom with high antioxidant capacity and its use); Korean Patent Publication No. 10-2007-0072433 (Pine Butterfly Mushroom Extract and Its Use, which has the effect of inhibiting the production of free radicals due to diabetes) etc have been disclosed.

[0014] However, there has been not reported or disclosed about the extract of Fomitopsis pinicola for treating or preventing ischemic brain disease in the above cited literatures, the disclosures of which are incorporated herein by reference.

[0015]

[0016] To investigate the preventing and treating activity of the extract of Fomitopsis pinicola for ischemic brain disease, the inventors of the present invention conducted (1) ASIC1 ion channel inhibition assay using a patch clamp (Experimental Example 1) on the extract of Fomitopsis pinicola of the present invention; and neuroprotective effect analysis experiment using a neuroblastoma cell line (sh-sy5y) to inhibit ASIC1 and inhibit apoptosis, and the results of these experiments confirm that the inventive extract of the present invention can be used as a treatment or adjuvant for ischemic brain disease, and confirm that the composition is useful as a pharmaceutical composition, health functional food and dietary supplement for the prevention or treatment of ischemic brain disease, and complete the present invention.

[0017]

[0018] The technical solution to solve the problem of the background art is for the development of effective treating agent of ischemic brain diseases from natural resource.

[0019] Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising an extract ofFomitopsis pinicola as an active ingredients for the treatment or prevention of ischemic brain diseases.

[0020] It is an object of the present invention to provide an ASIC (Acid-sensing Ion channel) 1 inhibitor comprising an extract ofFomitopsis pinicola as an active ingredient.

[0021] The term "Fomitopsis pinicola" disclosed herein comprises a fruiting body of the naturally grown distributed in Japan, Korea, China etc, cultivated or artificial　cultured Fomitopsis pinicola.

[0022] The term "extract" disclosed herein comprises crude extract, polar solvent soluble extract and non-polar solvent soluble extract of Fomitopsis pinicola.

[0023]

[0024] Specifically, the term "crude extract" disclosed herein comprises the extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, or the mixtures thereof, preferably spirit, ethanol or the mixture thereof, more preferably, 30 -100% (v / v) spirit or ethanol.

[0025]

[0026] The term "non-polar solvent soluble extract" disclosed herein can be prepared by extracting the above described crude extract with non-polar solvent, for example, hexane, methylene chloride, ethyl acetate or chloroform.

[0027]

[0028] The term "polar solvent soluble extract" disclosed herein can be prepared by extracting the above described crude extract after removing the non-polar solvent soluble extract with polar solvent, for example, water, lower alcohol such as methanol, ethanol, preferably butanol and the like, or the mixtures thereof.

[0029]

[0030] The term "ischemic brain diseases" disclosed herein comprises stroke such as derebral　infarction or cerebral hemorrhage, specifically, stroke such as derebral　infarction or cerebral hemorrhage, which can be treated or inhibited through the inhibition of neuronal cell death by ASIC1 inhibition.

[0031] Hereinafter, the present invention is described in detail.

[0032] An inventive extract of the present invention can be prepared in detail by following procedures.

[0033]

[0034] For example, Fomitopsis pinicola is dried, cut, crushed, and added to 1 to 20-fold, preferably, approximately 1 to 7-fold volume of water, spirit, lower alcohols such as methanol, ethanol, butanol, or the mixtures thereof, preferably spirit, ethanol or the mixture thereof, more preferably, 30 -100% (v / v) spirit or ethanol; the solution is treated with hot water at the temperature ranging from 30℃∼150℃, preferably, 50℃∼100℃, for the period ranging from 30 mins to 48 hours, preferably, 1 to 36 hours with the extraction method by the extraction with hot water, cold water, reflux extraction, or ultra-sonication extraction, preferably, extraction with reflux extraction; the extraction step is repeated 1 to 20 times, preferably, 2 - 10 times; the extract is collected with filtration, concentrated under reduced pressure and dried to obtain an crude extract of the present invention.

[0035] At then, the crude extract prepared by above step, is suspended in 0.0005 to 500 fold, preferably, 0.05 to 5 fold volume of water based on the weight of the crude extract (v / w), and then is mixed with non-polar solvent such as hexane, chloroform, or ethyl to fractionate into non-polar solvent soluble extract and polar solvent soluble extract soluble in methanol, ethanol, butanol, or the mixtures thereof.

[0036]

[0037]

[0038] Also, the above-described procedures may be modified or subjected to further step to fractionate or isolate more potent fractions or compounds by conventional procedure well- known in the art, for example, the procedure disclosed in the literature (Harborne J. B. Phytochemical methods:A guide to modern techniques of plant analysis, 3rdEd. pp6-7, 1998).

[0039]

[0040] It is an object of the present invention to provide the above-described preparation method and the composition comprising the extract prepared by the above-described preparation method useful in the treatment or prevention of ischemic brain diseases.

[0041]

[0042] It is an object of the present invention to provide a use of extract ofFomitopsis pinicola for the manufacture of therapeutic agent for the treatment or prevention of ischemic brain diseases in human or mammal.

[0043]

[0044] It is an object of the present invention to provide a method of treating or preventing ischemic brain diseases in a mammal comprising administering to said mammal an effective amount of an extract ofFomitopsis pinicola together with a pharmaceutically acceptable carrier thereof.

[0045]

[0046] It is another object of the present invention to provide a health food or food additives comprising an extract ofFomitopsis pinicola together with a sitologically acceptable additive for the prevention or improvement of ischemic brain diseases.

[0047]

[0048] The pharmaceutical composition of the present invention can contain about 0.01 ~ 50 % by weight of the above extract based on the total weight of the composition.

[0049]

[0050] The health food of the present invention comprises above extracts as 0.01 to 80 %, preferably 1 to 50 % by weight based on the total weight of the composition.

[0051]

[0052] Above health food can be contained in health food, health beverage etc, and may be used as powder, granule, tablet, chewing tablet, capsule, beverage etc.

[0053]

[0054] The term "prevent" disclosed herein comprises any act to inhibit or postpone the occurrence of certain disease or disorder disclosed herein by way of administrating the inventive composition; and the term "treat" disclosed herein comprises any act to alleviate or favorably changing the symptom associated with certain disease or disorder disclosed herein by way of administrating the inventive composition.

[0055] The present inventors conducted (1) ASIC1 ion channel inhibition assay using a patch clamp (Experimental Example 1) on the extract of Fomitopsis pinicola of the present invention; and neuroprotective effect analysis experiment using a neuroblastoma cell line (sh-sy5y) to inhibit ASIC1 and inhibit apoptosis, and the results of these experiments confirm that the inventive extract of the present invention can be used as a treatment or adjuvant for ischemic brain disease, and confirm that the composition is useful as a pharmaceutical composition, health functional food and dietary supplement for the prevention or treatment of ischemic brain disease.

[0056]

[0057] Accordingly, in accordance with the other aspect of the present invention, present invention provide a pharmaceutical composition comprising an extract ofFomitopsis pinicola as an active ingredient for the treatment or prevention of ischemic brain diseases.

[0058] Present invention provide a pharmaceutical composition comprising an extract ofFomitopsis pinicolaas an active ingredient and the pharmaceutically acceptable carriers or excipients, for the treatment or prevention of ischemic brain diseases.

[0059] In accordance with another aspect of the present invention, there is also provided a use of an extract ofFomitopsis pinicolafor manufacture of medicines employed for treating or preventing ischemic brain diseases.

[0060] The term "pharmaceutically acceptable carriers or excipients" defined herein comprises "pharmaceutical additives, the inactive ingredients used to make up a medication. They include dyes, flavors, binders, emollients, fillers, lubricants, preservatives, and many more classifications. Common excipients include cornstarch, lactose, talc, magnesium stearate, sucrose, gelatin, calcium stearate, silicon dioxide, shellac and glaze, which has been well-known in the art (See, Home-page of Food and Drug Administration:www.fda.govor drug information online:www.drugs.com) or previous literature (for example, Rowe, Raymond C et al., Handbook of Pharmaceutical Excipients, Pharmaceutical Press, 7th Edition, 2012)

[0061] In accordance with another aspect of the present invention, there is also provided a method of treating or preventing ischemic brain diseases in mammals, wherein the method comprises administering a therapeutically effective amount of an extract ofFomitopsis pinicola, into the mammal suffering from ischemic brain diseases.

[0062] The inventive composition for treating and preventing ischemic brain diseases may comprises above extracts as 0.1 ~ 99%, preferably, 0.1 ~ 50% by weight based on the total weight of the composition.

[0063] The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

[0064] For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

[0065] Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).

[0066] The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[0067] The desirable dose of the inventive extract varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.0001 to 1000mg / kg, preferably, 0.001 to 100mg / kg by weight / day of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day.

[0068] The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human)viavarious routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection.

[0069] The inventive extract of the present invention also can be used as a main component or additive and aiding agent in the preparation of various functional health food and health care food.

[0070] Accordingly, it is the other object of the present invention to provide a health functional food comprising an extract ofFomitopsis pinicola for the prevention or alleviation of ischemic brain diseases.

[0071] The term "a functional health food" defined herein" the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the extract of the present invention to conventional food to prevent or improve the purposed diseases in human or mammal.

[0072] It is the other object of the present invention to provide a health care food comprising an extract ofFomitopsis pinicola together with a sitologically acceptable additive for the prevention or alleviation of ischemic brain diseases.

[0073] The term "a health care food" defined herein "the food containing the extract of the present invention showing no specific intended effect but general intended effect in a small amount of quantity as a form of additive or in a whole amount of quantity as a form of powder, granule, capsule, pill, tablet etc.

[0074] The term "a sitologically acceptable additive" defined herein comprises "any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food", and can be classified into three groups according to its origin, i.e., (1) chemically synthetic additive such as ketones, glycine, potassium citrate, nicotinic acid, etc; (2) natural additive such as persimmon dye, licorice extract, crystalline cellulose, gua dum etc; (3) the mixed additive therewith such as sodium L-glutamate, preservatives, tar dye etc, or various categories according to its function in the food, for example, thickening agent, maturing agent, bleaching agent, sequestrant, humectant, anti-caking agent, clarifying agents, curing agent, emulsifier, stabilizer, thickener, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetener, preservative agent, anti-oxidant, etc, which has been well-known in the art or previous literature (See, "Codex General Standard for Food Additives" (GSFA, Codex STAN 192-1995) in Home-page of GSFA Online: www.codexalimentarius.net / gsfaonline / index.html).

[0075] If a substance is added to a food for a specific purpose in that food, it is referred to as a direct additive and indirect food additives are those that become part of the food in trace amounts due to its packaging, storage or other handling.

[0076] The term "health care foods or health functional foods" disclosed herein can be contained in food, health beverage, dietary supplement etc, and may be formulated into a form of pharmaceutically dosing form such as a powder, granule, tablet, suspension, emulsion, syrup, chewing tablet, capsule, beverage etc; or the food form, for example, bread, rice cake, dry fruit, candy, chocolate, chewing gum, ice cream, milk such as low-fat milk, lactose-hydrolyzed milk, goat-milk, processed milk, milk product such as fermented milk, butter, concentrated milk, milk cream, butter oil, natural cheese, processed cheese, dry milk, milk serum etc, processed meat product such as hamburger, ham, sausage, bacon etc, processed egg product, fish meat product such as fish cake etc, noodle products such as instant noodles, dried noodles, wet noodles, fried noodles, non-fried noodles, gelatinized dry noodles, cooked noodles, frozen noodles, Pasta etc, tea product such as tea bag, leached tea etc, health drinks such as fruit drinks, vegetable drinks, carbonated soft drinks, soymilk drinks, lactic beverage mixed beverage, etc, seasoning food such as soy sauce, soybean paste, red pepper paste, chunjang (a kind of fermented soybean product colored by caramel), cheonggukjang (natural fermented soybean by B. subtillis), mixed paste, vinegar, sauce, ketchup, curry, dressing etc, margarine, shortening, pizza etc, but not intended herein to limit thereto, for preventing or improving of purposed disease.

[0077] Also, above described extract can be added to food or beverage for prevention and improvement of purposed disorder. The amount of above described extract in food or beverage as a functional health food or health care food may generally range from about 0.01 to 100 w / w % of total weight of food for functional health food composition. In particular, although the preferable amount of the extract of the present invention in the functional health food, health care food or special nutrient food may be varied in accordance to the intended purpose of each food, it is preferably used in general to use as an additive in the amount of the extract of the present invention ranging from about 0.01 to 5% in food such as noodles and the like, from 40 to 100% in health care food on the ratio of 100% of the food composition.

[0078] Providing that the health beverage composition of present invention contains above described extract or compound as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartame et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 ㎖ of present beverage composition.

[0079] The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese, chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w / w % per 100 w / w % present composition. Examples of addable food comprising aforementioned extract or compound therein are various food, beverage, gum, vitamin complex, health improving food and the like.

[0080] Inventive extract of the present invention has no toxicity and adverse effect therefore; they can be used with safe.

[0081] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[0082] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

[0083]

[0084] As described in the present invention, the present inventors demonstrated that an extract ofFomitopsis pinicola showed potent treating effect on ischemic brain disease through various experiments, for example, (1) ASIC1 ion channel inhibition assay using a patch clamp (Experimental Example 1) on the extract of Fomitopsis pinicola of the present invention; and neuroprotective effect analysis experiment using a neuroblastoma cell line (sh-sy5y) to inhibit ASIC1 and inhibit apoptosis, and the results of these experiments confirm that the inventive extract of the present invention can be used as a treatment or adjuvant for ischemic brain disease, and confirm that the composition is useful as a pharmaceutical composition, health functional food and dietary supplement for the prevention or treatment of ischemic brain disease.

[0085]

[0086] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[0087] Fig. 1 shows the scheme of preparation of inventive extract;

[0088] Fig. 2 represents the inhibitory effect of the inventive extract (EtOH) on ASIC1 ion channel;

[0089] Fig. 3 represents the inhibitory effect of the inventive extract (EA) on ASIC1 ion channel;

[0090] Fig. 4 depicts the inhibitory effect of the inventive extract (MC) on ASIC1 ion channel;

[0091] Fig. 5 depicts the summary of inhibitory effect of various inventive extract on ASIC1 ion channel;

[0092] Fig. 6 presents the inhibitory effect of various inventive extract on apoptosis in neuroblastoma cell line (H-SY5Y cell).

[0093]

[0094]

[0095] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[0096] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

[0097]

[0098] EXAMPLES

[0099] The following Comparative Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

[0100]

[0101] Example 1. The preparation of inventive extract (Fig.1)

[0102] 1-1. Preparation of crude extract (EtOH)

[0103] 480g of dried fruiting body of Fomitopsis pinicola (purchased from CHUNCHEOWANGCHO Co. Ltd., Rm 1410, 606dong, 161, 14, Sesil-ro, Chuncheon-si, Gangwon-do, Republic of Korea) was cut into small pieces and mixed with 4.8L of 70% ethanol (v / w). The mixture was extracted with reflux extraction at 65℃ for 2 hours to collect the filtrate, three times. The extract was filtered with filtration paper (5㎛, TY2-090, ADVANTEC) to remove the debris, five times. The collected filtrate was concentrated by rotary vacuum evaporator (Buchi, Ratavapor, R-300) at 40 to 50℃ under reduced pressure (-700 to - 760mmHg) and dried with freezing dryer to obtain 7.584g of dried crude extract (designated as 'EtOH extract" hereinafter).

[0104]

[0105] 1-2. Preparation of respective fractions

[0106] 400mL of concentrated crude extract was suspended in 1000mL of distilled water and performed to conventional fractionation process with n-hexane, dichloromethane (CH2Cl2), ethylacetate (EtOAc), and n-butanol, successively, to obtain respective dried n-hexane fraction (11.30mg, designated as "hexane extract" hereinafter., dichloromethane fraction (10.83mg, designated as " MC extract" hereinafter), ethylacetate fraction (11.25mg, designated as "EA extract" hereinafter) and n-butanol fraction (10.97mg, designated as 'BuOH extract" hereinafter).

[0107] Experimental Example 1. inhibition effect on ASICI ion channel using by patch clamp

[0108] In order to determine the inhibitory activity of inventive extract from ischemic stokes, following inhibition test on ASICI ion channel using by patch clamp was performed according to the procedure disclosed in the literature (Biology 2022; 11(2), 167, Cell 2004, 118, 687-698).

[0109]

[0110] When an ischemic stroke occurs, acidosis occurs in the lesions, which can lead to activation of ASIC1 (Acid-sensing Ion Channels; ASIC) and the activation of ASIC1 increases intracellular calcium concentration, resulting in the induction of apoptosis (Biology 2020, 9(12), 460).

[0111] The inhibition effect on ASIC ion channel which can increase intracellular calcium concentration, resulting in the induction of apoptosis, has been performed as follows.

[0112] The extracellular fluid for patch clamp analysis was prepared to contain 149mM NaCl, 4mM KCl, 1mM MgCl2, 2mM CaCl2, 5mM D-glucose, 10mM HEPES(H3375, Sigma) and adjusted to pH 7.4 with NaOH.

[0113] In accordance with the characteristics of ASIC1 that is activated at low pH, the composition of the extracellular fluid for activation is the same as above, and it was prepared with a solution with a pH of 6.0.

[0114] The intracellular fluid for patch clamp analysis was prepared to contain 135mM KCl, 10mM EGTA, 4mM MgATP, 0.3mM Na2GTP, 20mM HEPES (H3375, Sigma) and adjusted to pH 7.4 with KOH.

[0115] The ASIC1 electric current was determined by using Axopatch 200B amplifier (Axon Axopatch 200B Microelectrode Amplifier, Molecular Devices) and Digidata 1440A(Axon digidata 1440A, Molecular Devices) and the data was analyzed by pClamp10.4 (pClamp10.4, Molecular Devices).

[0116] The voltage of cellular membrane was fixed to -70mM to record, repeatedly.

[0117] The analysis of the inhibition rate of ASIC1 was performed by comparing the ASIC1 current increased by pH 6.0 with the current increased by pH 6.0 after the treatment of the sample.

[0118] As can be seen in Fig. 2, it has been confirmed that the activated ASIC1 electric current in the group treated with the inventive extract (EtOH) has been unexpectedly inhibited (42.62±8.147 %) and that of respective other test samples, i.e., MCfraction (64.23±10.96 %) and EA fraction (47.9±8.639 %) was also unexpectedly inhibited (SeeFigs. 3 and 4). The inhibition ratio of each test sample was shown in Fig. 5.

[0119]

[0120] Experimental Example 2.neuro-protective effect using by human neuroblastoma cell (SH-SY5Y cell line)

[0121] In order to determine the neuro-protective activity of inventive extract, following test using of human neuroblastoma cell (SH-SY5Y cell line) was performed according to the procedure disclosed in the literature (Biology 2022; 11(2), 167, Cell 2004, 118, 687-698).

[0122]

[0123] Specifically, sh-sy5y cells (American Type Culture Collection; ATCC) appropriately inoculated into a 96-well plate, was incubated for 1 hr in a culture medium containing the extract of Example 1 of low pH 6.0, and then incubated for 1 hr. The culture was washed twice with DPBS (Dulbecco's Phosphate Buffered Saline. LB001-01, Welgene).

[0124] The culture medium was replaced with new culture medium (DMEM: Dulbecco's Modified Eagle's Medium. LM005-01, Welgene) to culture for 24 hrs. 24 hrs after the culture, 10 microliter of CCK-8 (Cell counting Kit 8. CK04, Dojindo) was treated thereto to react with together for 2 hours at 37 ℃ and final absorbance of the culture was determined at 450nm using microplate reader (Biotek).

[0125] The negative control group was incubated with pH6.0, and the test groups treated with the extract prepared in Examples was incubated in the culture medium of pH 6.0.

[0126] The extract prepared in Examples incubated in the culture medium (pH 7.4) has been still detected during the incubation for 24 hours after washing.

[0127] As can be seen in Fig. 6, it has been confirmed that the culture medium in the group treated with the inventive extract (100 μg / ml EtOH, 9.5±1.55 %), MCfraction (100 μg / ml 7.8±10.42 %) as well as EA fraction (100 μg / ml, 11.4±1.26 %) showed sharply reduced apoptosis whereas the negative control group (pH 6.0) showed the increased apoptosis (43.9±2.96 %).

[0128] Finally, it has been confirmed that all of the test groups treated with the inventive extract showed potent inhibitory activity from the increased apoptosis induced by acidic pH (pH 6.0) environment.

[0129]

[0130] Statistics analysis

[0131] All data were expressed in mean and standard deviation (mean±SD), and statistical significance verification was determined to be significant (P < 0.05) using one-way ANOVA and Dunnett's multiple comparison test as a post hoc test.

[0132]

[0133]

[0134] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

[0135]

[0136] Preparation of injection

[0137] EtOH extract: 100mg

[0138] Sodium metabisulfite : 3.0mg

[0139] Methyl paraben : 0.8mg

[0140] Propyl paraben : 0.1mg

[0141] Distilled water for injection : optimum amount

[0142] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2㎖ ample and sterilizing by conventional injection preparation method.

[0143]

[0144] Preparation of powder

[0145] Hexane extract: 500mg

[0146] Corn Starch : 100mg

[0147] Lactose : 100mg

[0148] Talc : 10mg

[0149] Powder preparation was prepared by mixing above components and filling sealed package.

[0150] Preparation of tablet

[0151] MCextract 200mg

[0152] Corn Starch 100mg

[0153] Lactose 100mg

[0154] Magnesium stearate optimum amount

[0155] Tablet preparation was prepared by mixing above components and entabletting.

[0156]

[0157] Preparation of capsule

[0158] EA extract : 100mg

[0159] Lactose : 50mg

[0160] Corn starch : 50mg

[0161] Talc : 2mg

[0162] Magnesium stearate optimum amount

[0163] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

[0164]

[0165] Preparation of liquid

[0166] BuOH extract : 1000mg

[0167] Sugar : 20g

[0168] Polysaccharide : 20g

[0169] Lemon flavor : 20g

[0170] Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000㎖ ample and sterilizing by conventional liquid preparation method.

[0171]

[0172] Preparation of health food

[0173] EtOH extract: 1000mg

[0174] Vitamin mixture : optimum amount

[0175] Vitamin A acetate : 70g

[0176] Vitamin E : 1.0mg

[0177] Vitamin B10: 13mg

[0178] Vitamin B2:0.15mg

[0179] Vitamin B6 : 0.5mg

[0180] Vitamin B1 : 20.2g

[0181] Vitamin C : 10mg

[0182] Biotin : 10g

[0183] Amide nicotinic acid : 1.7mg

[0184] Folic acid : 50g

[0185] Calcium pantothenic acid : 0.5mg

[0186] Mineral mixture : optimum amount

[0187] Ferrous sulfate : 1.75mg

[0188] Zinc oxide : 0.82mg

[0189] Magnesium carbonate : 25.3mg

[0190] Monopotassium phosphate : 15mg

[0191] Dicalcium phosphate : 55mg

[0192] Potassium citrate : 90mg

[0193] Calcium carbonate : 100mg

[0194] Magnesium chloride : 24.8mg

[0195] The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

[0196]

[0197] Preparation of health beverage

[0198] BuOH extract: 1000mg

[0199] Citric acid : 1000mg

[0200] Oligosaccharide : 100g

[0201] Apricot concentration : 2g

[0202] Taurine : 1g

[0203] Distilled water : 900㎖

[0204] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85oC for 1 hour, filtered and then filling all the components in 1000mL ample and sterilizing by conventional health beverage preparation method.

[0205] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[0206]

[0207] As described in the present invention, the inventive extract ofFomitopsis pinicola showed potent treating effect on ischemic brain disease through various experiments, for example, (1) ASIC1 ion channel inhibition assay using a patch clamp (Experimental Example 1) on the extract of Fomitopsis pinicola of the present invention; and neuroprotective effect analysis experiment using a neuroblastoma cell line (sh-sy5y) to inhibit ASIC1 and inhibit apoptosis, and the results of these experiments confirm that the inventive extract of the present invention can be used as a treatment or adjuvant for ischemic brain disease, and confirm that the composition is useful as a pharmaceutical composition, health functional food and dietary supplement for the prevention or treatment of ischemic brain disease

Claims

1.A pharmaceutical composition comprising an extract ofFomitopsis pinicola as an active ingredients for the treatment or prevention of ischemic brain diseases.2.The pharmaceutical composition according to claim 1,wherein said Fomitopsis pinicola is a fruiting body of the naturally grown distributed in Japan, Korea, China etc, cultivated or artificial　cultured Fomitopsis pinicola.3.The pharmaceutical composition according to claim 1,wherein said extract is a crude extract, polar solvent soluble extract or a non-polar solvent soluble extract of Fomitopsis pinicola.4.The pharmaceutical composition according to claim 3,wherein said crude extract is the extract prepared by extracting plant material with water, lower alcohols or the mixtures thereof.5.The pharmaceutical composition according to claim 3,wherein said non-polar solvent soluble extract is the extract prepared by extracting said crude extract of claim 4 with non-polar solvent selected from hexane, methylene chloride, ethyl acetate or chloroform.6.The pharmaceutical composition according to claim 3,wherein said polar solvent soluble extract is the extract prepared by extracting said crude extract of claim 4 after removing the non-polar solvent soluble extract with polar solvent selected from water, lower alcohol or the mixtures thereof.7.The pharmaceutical composition according to claim 1,wherein said ischemic brain disease is a stroke.8.A health functional food comprising an extract ofFomitopsis pinicola as an active ingredients for the prevention or alleviation of ischemic brain diseases.9.The health functional food according to claim 8,wherein said health functional food is provided as powder, granule, tablet, capsule, pill, suspension, emulsion, syrup, tea bag, leached tea, or beverage type.10.A health care food comprising an extract ofFomitopsis pinicola as an active ingredients for the prevention or alleviation of ischemic brain diseases.11.An ASIC (Acid-sensing Ion channel) 1 inhibitor comprising an extract ofFomitopsis pinicola as an active ingredient.12.A use of an extract ofFomitopsis pinicola for manufacture of medicines employed for treating or preventing ischemic brain diseases.13.A method of treating or preventing ischemic brain diseases in mammals, wherein the method comprises administering a therapeutically effective amount of an extract ofFomitopsis pinicolainto the mammal suffering from ischemic brain diseases.

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