Composition for preventing or treating amebiasis, comprising, as active ingredient, extract of xanthium orientale l., fraction thereof or compound isolated therefrom

Abstract

The present invention relates to a composition for preventing or treating amebiasis, comprising, as an active ingredient, an extract of Xanthium orientale L., a fraction thereof, or a compound isolated therefrom. The composition for preventing or treating amebiasis, comprising an extract of Xanthium orientale L., a fraction thereof, or a compound isolated therefrom, of the present invention, induces morphological changes in Naegleria fowleri, reduces the survival rate of Naegleria fowleri, and ultimately induces the death thereof. Therefore, the extract of Xanthium orientale L., the fraction thereof or the compound isolated therefrom, according to the present invention, can be effectively used in a pharmaceutical composition or a health functional food composition for preventing or treating amebiasis.

Description

Composition for preventing or treating amoebic infection comprising extract of large-headed snail, fraction thereof or compound isolated therefrom as an active ingredient

[0001] The present invention relates to a composition for preventing or treating amoebic infection, comprising a large-scale extract of the plant, a fraction thereof, or a compound isolated therefrom as an active ingredient.

[0002] Naegleria fowleri is a free-living amoeba that inhabits various terrestrial environments, including soil and freshwater. It is a highly pathogenic protozoan that causes primary amoebic meningoencephalitis (PAM) in humans as an opportunistic infection. The main site of infection of Naegleria fowleri is the central nervous system, and clinical symptoms after nasal infection include severe headache, loss of appetite, nausea, vomiting, high fever (38-40℃), dysfunction of limbs, signs of meningeal irritation, and encephalitis. Due to the specificity of the site of infection, early differential diagnosis is difficult, and the progression of the disease is very rapid, leading to death within 10 days after infection, with a mortality rate of over 97%.

[0003] Although the incidence of PAM is low worldwide, the number of deaths due to PAM is continuously increasing. Due to changes in the Earth's environment caused by global warming, the habitat of Naegleria fowleri is gradually expanding, and Naegleria fowleri has been confirmed to exist in swimming pools and wet soil in addition to reservoirs, lakes, rivers, and hot springs. Recently, a case of a Korean person infected abroad dying in Korea has been reported. Despite the high mortality rate of PAM, there is currently no early diagnosis method or effective treatment. Currently, amphotericin B and miltefosine are considered as possible agents, but their therapeutic efficacy is uncertain. Therefore, the development of an effective treatment for PAM is very urgent.

[0004] Accordingly, the present inventors conducted repeated research to develop a composition for preventing or treating Naegleria fowleri infection, and as a result, confirmed that a large-scale extract, a large-scale fraction, or a compound isolated therefrom has an effect of killing Naegleria fowleri, and confirmed that a large-scale extract (Xanthium orientale L.), a fraction thereof, or a compound isolated therefrom induces morphological changes in Naegleria fowleri, reduces the survival rate of Naegleria fowleri, and induces changes in the microscopic cell structure, thereby completing the present invention.

[0005] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating amoebic infection, which comprises as an active ingredient an extract of Xanthium orientale L., a fraction thereof, or a compound represented by the following chemical formula 1 or 2 separated therefrom.

[0006] [Chemical Formula 1]

[0007]

[0008] [Chemical Formula 2]

[0009]

[0010] In addition, another object of the present invention is to provide a health functional food composition for preventing or improving amoebic infection, which comprises as an active ingredient an extract of Xanthium orientale L., a fraction thereof, or a compound represented by the chemical formula 1 or 2 separated therefrom.

[0011] In addition, another object of the present invention is to provide a method for preventing or treating amoebic infection, comprising a step of administering the pharmaceutical composition to a subject.

[0012] In order to achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating amoebic infection, which comprises as an active ingredient an extract of Xanthium orientale L., a fraction thereof, or a compound represented by the following chemical formula 1 or 2 separated therefrom.

[0013] [Chemical Formula 1]

[0014]

[0015] [Chemical Formula 2]

[0016]

[0017] Next, the present invention provides a health functional food composition for preventing or improving amoebic infection, which comprises as an active ingredient an extract of Xanthium orientale L., a fraction thereof, or a compound represented by the chemical formula 1 or 2 separated therefrom.

[0018] Furthermore, the present invention provides a method for preventing or treating amoebic infection, comprising administering the pharmaceutical composition to a subject.

[0019] The composition for preventing or treating amoebic infection comprising the extract of the present invention, a fraction thereof, or a compound isolated therefrom induces morphological changes in Naegleria fowleri, reduces the survival rate of Naegleria fowleri, and ultimately induces death. Therefore, the extract of the present invention, a fraction thereof, or a compound isolated therefrom can be usefully used as a pharmaceutical composition or a health functional food composition for preventing or treating amoebic infection.

[0020] Figure 1 is a schematic diagram showing a process for producing a large-flowered dandelion extract and fraction according to one embodiment of the present invention.

[0021] FIG. 2 is a diagram showing the chemical structures of three compounds derived from a large-flowered dandelion extract in one embodiment of the present invention.

[0022] Figure 3 is a diagram showing the effect of a large-scale extract of the present invention on killing free amoeba Fowleri in one embodiment of the present invention.

[0023] FIG. 4 is a diagram showing the results of measuring the killing effect of a large-scale extract of amoebae fowleri according to the induction of morphological changes in the amoeba.

[0024] FIG. 5 is a diagram showing the effect of a large-scale fraction of the present invention on killing free amoeba Fowleri in one embodiment of the present invention.

[0025] FIG. 6 is a diagram showing the results of confirming the anti-Fowler's amoeba efficacy and cytotoxicity of a compound derived from a large-scale extract of the present invention.

[0026] FIG. 7 is a diagram showing the results of confirming the effect of a compound derived from a large-scale extract of the present invention on inducing apoptosis in Naegleria fowleri through an apoptosis assay in one embodiment of the present invention.

[0027] FIG. 8 is a diagram showing the results of an apoptosis assay in which a compound derived from a large-scale extract of the present invention does not induce apoptosis of C6 cells, which are control cells, in one embodiment of the present invention.

[0028] FIG. 9 is a diagram showing the results of confirming the effect of a compound derived from a large-scale extract of the present invention on inducing apoptosis in Naegleria fowleri through a TUNEL assay in one embodiment of the present invention.

[0029] FIG. 10 is a diagram showing the results of a TUNEL assay confirming that a compound derived from a large-scale extract of the present invention does not induce apoptosis of C6 cells, which are control cells, in one embodiment of the present invention.

[0030] FIG. 11 is a diagram showing the results of confirming the effect of a compound derived from a large-scale extract of the present invention on increasing reactive oxygen species (ROS) in Naegleria fowleri, according to one embodiment of the present invention.

[0031] FIG. 12 is a diagram showing the results of analyzing the membrane electrochemical changes of the mitochondria of Naegleria fowleri treated with a compound derived from a large-scale extract of the present invention.

[0032] FIG. 13 is a diagram showing the results of analyzing changes in ATP production of mitochondria of Naegleria fowleri treated with a compound derived from a large-scale extract of the present invention.

[0033] Hereinafter, the present invention will be described in detail with reference to the attached drawings and exemplary embodiments thereof. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0034] In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0035] Throughout this specification, '%' used to indicate the concentration of a particular substance means solid / solid (w / w) %, solid / liquid (w / v) %, and liquid / liquid (v / v) %, unless otherwise stated.

[0036] In one aspect, the present invention provides a pharmaceutical composition for preventing or treating amoebic infection, comprising as an active ingredient an extract of Xanthium orientale L., a fraction thereof, or a compound represented by the following chemical formula 1 or 2 separated therefrom.

[0037] [Chemical Formula 1]

[0038]

[0039] [Chemical Formula 2]

[0040]

[0041] In one embodiment of the present invention, the molecular formula of the compound represented by the chemical formula 1 is C 15 H 20 Its chemical name is O2, its IUPAC name is (3aR,5S,6S,7aR)-6-Ethenylhexahydro-6-methyl-3-methylene-5-(1-methylethenyl)-2(3H)-benzofuranone, and its molecular weight is 232.1463 g / mol.

[0042] In one embodiment of the present invention, the molecular formula of the compound represented by the chemical formula 2 is C 15 H 18 Its IUPAC name is Naphtho[2,3-b]furan-2,7-dione,3,3a,4,4a,5,6,9,9a-octahydro-4a,5-dimethyl-3-methylene-,[3aR-(3aα,4aβ,5β,9aα)] and its molecular weight is 246.1256 g / mol.

[0043] The term “Xanthium orientaeL.,” used in the present invention, is a naturalized plant native to eastern North America, and is an annual herb growing 0.5 to 2 m tall on roadsides, abandoned vacant lots, and beaches. Xanthium orientaeL. is also called spear daisy.

[0044] The extract according to the present invention can be obtained by extracting and separating from nature using extraction and separation methods known in the art, and the "extract" defined in the present invention is extracted from a large-sized snail using an appropriate solvent, and includes, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. Any pharmaceutically acceptable organic solvent may be used as a suitable solvent for extracting the extract from the above-mentioned large-boiled eggplant, and water or an organic solvent may be used, and is not limited thereto, for example, purified water, alcohols having 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane, and various solvents may be used alone or in combination. As an extraction method, any one of hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and pressing may be selected and used. Additionally, the desired extract may be subjected to additional conventional fractionation processes and purified using conventional purification methods.

[0045] There is no limitation on the method for producing the extract of the present invention, and any known method can be used. For example, the extract included in the composition of the present invention can be produced in a powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying of the primary extract extracted by the above-mentioned hot water extraction or solvent extraction method. In addition, the primary extract can be obtained by further purifying fractions using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. Therefore, in the present invention, the extract is a concept that includes all extracts, fractions, and purified products obtained at each stage of extraction, fractionation, or purification, as well as their dilutions, concentrates, or dried products.

[0046] The term "fraction" of the present invention means a result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture containing various components.

[0047] In the present invention, the fractionation method for obtaining the above fraction is not particularly limited and can be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing the fraction through an ultrafiltration membrane having a certain molecular weight cut-off value, a chromatographic fractionation method performed by performing various chromatographies (designed for separation according to size, charge, hydrophobicity, or affinity), and combinations thereof. Specifically, a method of obtaining a fraction from an extract obtained by extracting Polyscias fruticosa of the present invention by treating the extract with a predetermined solvent can be exemplified.

[0048] In the present invention, the type of fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohols having 1 to 4 carbon atoms; non-polar solvents such as hexane, ethyl acetate, chloroform, dichloromethane, benzene, toluene, acetone, carbon tetrachloride, ether, methylene, etc.; or mixed solvents thereof. These may be used alone or in combination of one or more thereof, but are not limited thereto. Preferably, n-hexane, chloroform, ethyl acetate, n-butanol, and more preferably, n-hexane can be used, but are not limited thereto.

[0049] In one embodiment of the present invention, the fraction may be obtained by fractionating a large-scale extract of the plant with a solvent selected from the group consisting of n-hexane, chloroform, ethyl acetate, n-butanol, and a mixed solvent thereof, more preferably n-hexane, but is not limited thereto.

[0050] In one embodiment of the present invention, the amoebic infection may be, but is not limited to, primary amoebic meningoencephalitis caused by Naegleria fowleri.

[0051] In one embodiment of the present invention, the composition may have an effect of killing Naegleria fowleri, but is not limited thereto.

[0052] In one embodiment of the present invention, the composition may induce morphological changes in Naegleria fowleri, but is not limited thereto.

[0053] In one embodiment of the present invention, the morphological change may be, but is not limited to, a reduction in the size and a reduction in the number of food vacuoles of Naegleria fowleri.

[0054] In one embodiment of the present invention, the composition may reduce the survival rate of Naegleria fowleri, but is not limited thereto.

[0055] In one embodiment of the present invention, the composition may induce changes in the microscopic cell structure of Naegleria fowleri, but is not limited thereto.

[0056] In one embodiment of the present invention, the microscopic cellular structural change may be, but is not limited to, deformation of cell organelles or functional impairment of mitochondria.

[0057] In one embodiment of the present invention, the composition may induce apoptosis of Naegleria fowleri, but is not limited thereto.

[0058] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete or incomplete adjuvant may be further included to increase immunogenicity.

[0059] The pharmaceutical composition according to the present invention can be prepared in a form in which a large-bodied extract, a fraction thereof, or a compound represented by the above chemical formula 1 or 2 isolated therefrom is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0060] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0061] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0062] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0063] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0064] The above pharmaceutical composition can be formulated into various oral or parenteral dosage forms.

[0065] Oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, and granules. These dosage forms may further contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). In addition, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidine, and, if desired, disintegrants or effervescent mixtures such as starch, agar, alginic acid or its sodium salt, and / or absorbents, coloring agents, flavoring agents, and sweetening agents. The above formulation can be prepared by conventional mixing, granulating or coating methods.

[0066] In addition, representative parenteral administration formulations include injectable preparations, and solvents for injectable preparations include water, Ringer's solution, isotonic saline solution, or suspensions. Sterile fixed oils for the injectable preparations can be used as solvents or suspension media, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.

[0067] Additionally, the above injectable formulation may use a fatty acid such as oleic acid.

[0068] In one aspect, the present invention provides a health functional food composition for preventing or improving amoebic infection, comprising as an active ingredient an extract of Xanthium orientale L., a fraction thereof, or a compound represented by the following chemical formula 1 or 2 separated therefrom.

[0069] [Chemical Formula 1]

[0070]

[0071] [Chemical Formula 2]

[0072]

[0073] The term "health functional food" used in the present invention is the same as food for special health use (FoSHU), and refers to a food with high medical and healthcare effects that has been processed to efficiently exhibit a bioregulatory function in addition to providing nutrition. Here, "function" means regulating nutrients for the structure and function of the human body or obtaining a useful effect for health purposes such as physiological action. The health functional food of the present invention can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a food. The health functional food of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material, and is highly portable, so the health functional food of the present invention can be taken as a supplement to enhance the effect of improving blood circulation.

[0074] The above health functional foods refer to foods that have a more active health maintenance or promotion effect than regular foods, while health supplement foods refer to foods intended for health supplementation. In some cases, the terms health functional foods, health foods, and health supplements are used interchangeably.

[0075] Specifically, the health functional food means a food product manufactured by adding the composition of the present invention to food materials such as beverages, teas, spices, gums, and confectionery, or by manufacturing it in the form of encapsulation, powder, suspension, etc., and which brings about a specific health effect when consumed, but unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs made from food as a raw material.

[0076] The above health functional food may additionally include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the relevant technical field may be used.

[0077] In addition, the health functional food may include additional ingredients commonly used in foods to improve odor, taste, appearance, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and amino acids such as lysine, tryptophan, cysteine, and valine.

[0078] In addition, the above health functional food may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), sterilizers (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glucose), film agents, gum bases, foam suppressants, solvents, and improvers. The above additives can be selected according to the type of food and used in an appropriate amount.

[0079] As an example of the health functional food of the present invention, it can be used as a health beverage composition, and in this case, it can contain various flavoring agents or natural carbohydrates as additional ingredients like a regular beverage. The above-mentioned natural carbohydrates can be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol and erythritol. The sweetener can be a natural sweetener such as thaumatin and stevia extract; a synthetic sweetener such as saccharin and aspartame, etc. The proportion of the natural carbohydrate can be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

[0080] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, it may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly important, but is typically selected within the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

[0081] In one aspect, the present invention provides a method for preventing or treating an amoebic infection comprising administering the pharmaceutical composition to a subject.

[0082] The term "subject" as used in the present invention refers to a subject requiring a method for preventing, controlling, or treating a disease, and may be used without limitation as a human, dog, monkey, cat, rodent, such as a mouse, genetically modified mouse, etc. More specifically, it refers to a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc.

[0083] The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount or a pharmaceutically effective amount.

[0084] The term "therapeutically effective amount" used in the present invention means the amount of a pharmaceutically acceptable salt of a composition effective in preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, the condition of the patient, etc. Therefore, the dosage for use in humans should be determined as an appropriate amount by taking both safety and efficacy into consideration. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal testing. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0085] The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be determined according to factors including the patient's health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent use of drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0086] As described above, specific embodiments of the present invention have been described in detail. However, those skilled in the art who understand the spirit of the present invention will be able to easily suggest other inventions that are backwards or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the scope of the same spirit. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description described above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0087] <Preparation Example 1> Cell culture

[0088] 1-1. Cultivation of free amoeba Fowleri

[0089] Naegleria fowleri used in the present invention was obtained from ATCC, USA, as Carter NF69 strain (ATCC No. 30215). Naegleria fowleri was cultured in Nelson medium (Nelson and Jones, 1970) containing 5% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin / streptomycin (Sigma, USA).

[0090] 1-2. Control cell culture

[0091] C6 rat glial cells (C6, ATCC CCL-107) were used as a control group in the present invention. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin / streptomycin (Gibco).

[0092] <Example 1> Preparation of large-leaved cod extract

[0093] 10 L of 70% ethanol was added to 350 g of dried powder of Xanthium orientae L., extracted at room temperature for 24 hours, and then ultrasonicated for 1 hour. The extract was filtered through filter paper (Whatman No. 2), and the remaining residue was re-extracted and filtered. The filtrate was concentrated under reduced pressure and freeze-dried to obtain a solid ethanol extract.

[0094] <Example 2> Preparation of large-scale fraction of scutellaria baicalensis

[0095] Referring to FIG. 1, the ethanol extract prepared in Example 1 was suspended in water, and then sequentially fractionated into equal amounts of n-hexane, chloroform, ethyl acetate, and n-butanol to obtain a hexane fraction (Hx), a chloroform fraction (CHCl3), an ethyl acetate fraction (EA), a butanol fraction (BuOH), and a residual water layer fraction (water).

[0096] <Example 3> Isolation of compounds derived from large-leaved clam extract

[0097] The effective substances were purified using high-performance liquid chromatography (HPLC) from the hexane (n-hexan) fraction of Example 2, and the chemical structures of the three purified effective substances (rp2-rp3, rp2-rp5, rp3-rp2) were identified by nuclear magnetic resonance analysis (NMR) (see Fig. 2).

[0098] <Experimental Example 1> Confirmation of the efficacy of large-scale extracts of the amoeba fowleri in killing the amoeba.

[0099] 1-1. Morphological changes and survival rate analysis

[0100] In order to confirm the killing efficacy of the large-scale extract of the above-mentioned codonopsis lanceolata prepared in Example 1 against Naegleria fowleri, the large-scale extract of the codonopsis lanceolata (25, 50, 100 ㎍ / mL) was treated to Naegleria fowleri and the morphological changes and survival rate of Naegleria fowleri were measured.

[0101] Inoculate the trophotype of free-living amoeba Fowleri cultured in Preparation Example 1-1 into a 96-well microplate (10 5 After 100 μg / mL of the extract of Codonopsis lanceolata (cells / well), the cells were cultured in a 37°C incubator. The cultured Naeba fowleri was treated with various concentrations (25, 50, 100 μg / mL, 0.1% DMSO) of the extract of Codonopsis lanceolata and cultured in a 37°C incubator for 48 hours. The group without the Codonopsis lanceolata extract used Naeba fowleri cultured with only 0.1% DMSO. As a control group, C6 rat glial cells cultured in Preparation Example 1-2 were inoculated (2 × 10) into a 96-well microplate. 4 After culturing (100 cells / well), the cells were cultured in a 37°C, 5% CO2 incubator and used. After culturing, the morphological changes of the Naegleria fowleri were observed under a light microscope, and the survival rate of the Naegleria fowleri was measured using CellTiter-Blue ® Cell viability assay (Promega, USA) was used for analysis according to the manufacturer's method. The analyzed morphological changes and viability are shown in Figure 3.

[0102] As shown in Fig. 3a, when the extract of the great aster was applied to Naegleria fowleri, morphological changes were measured. As a result, it was confirmed that the Naegleria fowleri transformed into an abnormal shape, including a decrease in size and a decrease in food vacuoles, and that this induced morphological changes and death. In addition, it was confirmed that morphological changes occurred in a concentration-dependent manner with the extract of the great aster. In contrast, the control cells showed no morphological changes.

[0103] As shown in Figure 3b, when the survival rate of Naegleria fowleri was measured by treating Naegleria fowleri with the extract of large-scale spores, it was confirmed that the survival rate of Naegleria fowleri decreased in a concentration-dependent manner. In contrast, the survival rate of the control cells did not change.

[0104] Through this, it was confirmed that the extract of large-scale amoebae has an excellent effect in killing Naegleria fowleri by inducing morphological changes in Naegleria fowleri and reducing the survival rate.

[0105] 1-2. Analysis of changes in microscopic cell structure

[0106] In order to confirm the killing efficacy of the large-scale extract of the above-mentioned Example 1 against Naegleria fowleri, 50 ㎍ / mL of the large-scale extract of the above-mentioned Naegleria fowleri was treated to Naegleria fowleri and changes in the microscopic cell structure of Naegleria fowleri were measured.

[0107] After treating the Naegleria fowleri with 50 μg / mL of the extract of greater scutellariae, the cells were cultured at room temperature for 12 and 24 hours. The Naegleria fowleri were collected, fixed with a 2% glutaraldehyde solution, and further fixed with a postfixative (osmium tetroxide, OsO4). The Naegleria fowleri were dehydrated with a mixture of propylene and epon, embedded with epoxy resin, and blocks were made. Sections were prepared at a thickness of 1 μm using an ultramicrotome, applied to copper grids, and observed using a Talos L120C Cryo Bio 120kV Transmission Electron Microscope (Thermo). This is shown in Figure 4. In Figure 4, NC means negative control.

[0108] As shown in Figure 4, when the extract of the large-scale snail was applied to the amoeba fowleri and the changes in the microscopic cell structure were measured, it was found that deformation of the cell organelles and an increase in abnormal mitochondria were induced in the amoeba fowleri.

[0109] Through this, it was confirmed that the extract of large-scale amoebae interferes with the normal physiological mechanism of Naegleria fowleri and induces death.

[0110] <Experimental Example 2> Confirmation of the efficacy of the large-scale fraction of the snail to kill Naegleria fowleri.

[0111] In order to confirm the killing efficacy of the large-scale fraction of the snail prepared in Example 2 on Naegleria fowleri, the large-scale fraction was treated at various concentrations to Naegleria fowleri and the morphological changes and survival rate of Naegleria fowleri were measured.

[0112] The experimental method was the same as in Experimental Example 1-1 above, and the hexane fraction (Hx), ethyl acetate fraction (EA), butanol fraction (BuOH), and residual water layer fraction (water) of the great amoebae were treated at concentrations of 10, 20, 40, 50, and 80 μg / mL, and the chloroform fraction (CHCl3) of the great amoebae was treated at concentrations of 0.1, 0.5, 1, 5, and 1 μg / mL. The results of measuring the morphological changes and survival rates after treating the great amoebae fractions to Naegleria fowleri are shown in Figure 5. In Figure 5, NC means negative control.

[0113] As shown in Fig. 5, the extract of the large-scaled spore-forming plant was re-fractionated into a hexane fraction (Hx) (Fig. 5a), a chloroform fraction (CHCl3) (Fig. 5e), an ethyl acetate fraction (EA) (Fig. 5b), a butanol fraction (BuOH) (Fig. 5c), and a residual water layer fraction (water) (Fig. 5d), and the anti-N. ameba efficacy of each fraction was analyzed. As a result, excellent anti-N. ameba efficacy was observed in the hexane fraction (Hx). The ethyl acetate fraction (EA), butanol fraction (BuOH), and residual water layer fraction (water) did not exhibit anti-N. ameba efficacy, and the chloroform fraction (CHCl3) exhibited anti-N. ameba efficacy even at low concentrations, but exhibited strong cytotoxicity against the control cells.

[0114] Through this, it was confirmed that the hexane fraction (Hx) of the large-scale extract of the amoeba fowleri has an excellent killing effect on the amoeba fowleri by inducing morphological changes in the amoeba and reducing its survival rate.

[0115] <Experimental Example 3> Confirmation of the anti-N. fowleriformis efficacy of compounds derived from the extract of large-leaved clams

[0116] In order to confirm the anti-amoeba Fowleri efficacy (amoeba killing efficacy) of the three compounds isolated in Example 3 above, the morphological changes and survival rate of amoeba Fowleri treated with the compounds were measured.

[0117] Specifically, the trophotype of free-living amoeba Fowler cultured in the above Preparation Example 1-1 was inoculated (10) into a 96-well microplate. 5 After culturing, the cultured N. fowleri was treated with compounds (rp2-rp3, rp2-rp5, rp3-rp2) at various concentrations (1–100 μg / mL, 0.1% DMSO) and cultured in a 37°C incubator for 48 hours. The N. fowleri cultured with only 0.1% DMSO without adding compounds derived from the extract of the great aster was used as a control group (survival rate 100%). The morphological changes of the N. fowleri after culture were observed under a light microscope, and the survival rate of the N. fowleri was determined by CellTiter-Blue ® Cell viability assay (Promega, USA) was used for analysis according to the manufacturer's method.

[0118] As a result, as shown in Fig. 6a, when various concentrations of compounds derived from the extract of Codonopsis japonica were treated to Naegleria fowleri, morphological changes and death of Naegleria fowleri were induced. In addition, as shown in Fig. 6b, the survival rate of Naegleria fowleri decreased as the concentration of the compound derived from the extract of Codonopsis japonica treated increased. In particular, rp2-rp3 showed remarkably excellent anti-Negleria fowleri efficacy, and rp2-rp5 and rp3-rp2 showed similar levels of anti-Negleria fowleri efficacy.

[0119] Through this, it was confirmed that three compounds derived from the extract of large-scale amoebae had excellent anti-fowlerian amoeba efficacy by inducing morphological changes in amoeba fowleri and reducing the survival rate.

[0120] <Experimental Example 4> Confirmation of cytotoxicity of compounds derived from large-scale extracts of the Korean dandelion

[0121] In order to confirm the cytotoxicity of the three compounds isolated in Example 3 above, the survival rate of control cells (C6 rat glial cells) treated with the compounds was measured.

[0122] Specifically, the control cells cultured in the above preparation example 1-2 were inoculated into a 96-well microplate (2 × 10 4 After 100 cells / well, the cells were cultured in a 37°C, 5% CO2 incubator. The cultured cells were treated with compounds (rp2-rp3, rp2-rp5, rp3-rp2) at various concentrations (1–100 μg / mL, 0.1% DMSO) and cultured in a 37°C incubator for 48 hours. Cells cultured without adding compounds derived from the extract of the great aster were treated with only 0.1% DMSO and used as a control group (survival rate 100%). The survival rate of the control and experimental group cells was measured using CellTiter-Blue ® Cell viability assay (Promega, USA) was used for analysis according to the manufacturer's method.

[0123] As a result, as shown in Fig. 6c, when C6 cells, which are control cells, were treated with various concentrations of compounds derived from the extract of large-scale snails, rp2-rp3 showed low CC concentrations. 50 The SI value was 4.36, which was good. rp3-rp2 did not show significant cytotoxicity against C6 cells, and the SI value was 4.89 or higher. On the other hand, rp2-rp5 showed partial cytotoxicity against C6 cells, and the SI value was low at 2.17.

[0124] Therefore, two compounds (rp2-rp3, rp3-rp2) that showed significant anti-amoeba activity against Naegleria fowleri and had an SI value of 4 or higher were selected and further analyzed for their anti-amoeba activity.

[0125] <Experimental Example 5> Confirmation of the apoptosis-inducing effect of compounds derived from the extract of large-leaved clams

[0126] 5-1. Confirmation of cell apoptosis induction effect through apoptosis assay

[0127] In order to confirm the cell death induction effect of two compounds (rp2-rp3, rp3-rp2) isolated from the large-scale extract of the selected plant in Experimental Example 4 above, an apoptosis assay was performed.

[0128] Specifically, the free amoeba Fowler cultured in the above Preparation Example 1-1 was inoculated (5 × 10) into a 96-well microplate (black / clear bottom). 4 cells / well) and IC 50After treatment with compounds derived from the extract of the large-sized dandelion, the cells were cultured at 37℃ for 24 or 48 hours. As a control, Naegleria fowleri treated with only 0.1% DMSO and cultured under the same conditions were used. After culture, Naegleria fowleri was treated with apopxin green, cytocalcein, and 7-aminoactinomycin D (7-AAD) and further cultured at room temperature for 2 hours. After culture, Naegleria fowleri was washed three times with phosphate buffered saline (PBS, pH 7.4) and observed under a fluorescence microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific, USA) (Apoxin: Ex / Em=490 / 525 nm, 7-ADD: Ex / Em=550 / 650 nm: CytoCalcein Violet 450: Ex / Em=405 / 450 nm).

[0129] As a result, as shown in Figure 7, the free amoeba Fowleri that was not treated with the compound derived from the extract of the large-headed hawk moth was stained by cytocalcein, but IC 50 Naegleria fowleri treated with the same concentration of compounds derived from the extract of the great snail (rp2-rp3, rp3-rp2) were not stained by cytocalcein, but specifically stained by apopxin, which marks apoptosis. In contrast, 7-ADD staining, which indicates necrosis, was not observed. However, as shown in Figure 8, no apoptosis induction was observed in C6 cells treated with the same concentration of compounds derived from the extract of the great snail.

[0130] Through this, it was confirmed that rp2-rp3 and rp3-rp2, which are compounds derived from the extract of large-scale amoebae, induce selective apoptosis in Naegleria fowleri and induce cell death.

[0131] 5-2. Confirmation of apoptosis induction effect through TUNEL assay

[0132] In order to confirm the apoptosis-inducing effect of two compounds (rp2-rp3, rp3-rp2) isolated from the large-scale extract of the selected plant in Experimental Example 4 above, a TUNEL assay was performed.

[0133] Specifically, the free amoeba Fowleri (10) cultured in the above preparation example 1-1 6 After seeding 100 cells / well in a 6-well plate (Thermo Fisher Scientific, USA), IC 50 After treatment with compounds derived from the extract of the large-sized dandelion, the cells were cultured at 37°C for 12 and 24 hours. As a control, Naegleria fowleri, which was treated with only 0.1% DMSO and cultured under the same conditions, was used. After harvesting the cells, they were fixed with 3.7% formaldehyde for 20 minutes at room temperature. After fixation, Naegleria fowleri were washed twice with PBS and treated with 70% ethanol for 30 minutes. Naegleria fowleri were analyzed using the TUNEL fluorescein isothiocyanate (FITC) assay kit (Abcam, UK). That is, it was stained with terminal deoxynucleotidyl transferase dUTP end-labeling (TUNEL) and propidium iodide (PI) according to the manufacturer's method and observed under a fluorescence microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific, USA) (TUNEL: Ex / Em= 490 / 525 nm, PI: Ex / Em=550 / 650 nm).

[0134] As a result, as shown in Fig. 9, Naegleria fowleri treated with the compound derived from the extract of the great dandelion was stained by TUNEL and PI. On the other hand, as shown in Fig. 10, C6 cells treated with the same concentration of the compound derived from the extract of the great dandelion were not stained by TUNEL and PI.

[0135] Through this, it was confirmed that rp2-rp3 and rp3-rp2, compounds derived from the extract of large-scale amoebae, induce DNA fragmentation associated with selective apoptosis in Naegleria fowleri.

[0136] <Experimental Example 6> Confirmation of the effect of compounds derived from the extract of the large-leaved clam on increasing reactive oxygen species (ROS).

[0137] In order to analyze the level of production of reactive oxygen species (ROS) that induce cell death and mitochondrial dysfunction in two compounds (rp2-rp3, rp3-rp2) isolated from the large-scale extract of the selected plant in Experimental Example 4 above, a DCFDA / H2DCFDA-Cellular ROS assay kit (Abcam, UK) was used.

[0138] Specifically, the free amoeba Fowler cultured in the above preparation example 1-1 was inoculated (5 × 10) into a 96-well microplate. 4 cells / well) and IC 50 After treatment with compounds derived from the extract of the large-sized snail, the cells were cultured at 37℃ for 12 or 24 hours. As a control, Naegleria fowleri, which was treated with only 0.1% DMSO and cultured under the same conditions, was used. After culture, Naegleria fowleri was washed several times with PBS, treated with 2′,7′-dichlorofluorescein diacetate (DCFDA) solution (20 μM), and then cultured in the dark for 45 minutes. Naegleria fowleri was washed three times with PBS and observed under a fluorescence microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific, USA) (Ex / Em = 490 / 525 nm).

[0139] As a result, as shown in Fig. 11, ROS production in the free-living amoeba Fowler treated with the compound derived from the extract of the large-leaved clam was significantly increased compared to the control free-living amoeba Fowler.

[0140] Through this, it was confirmed that rp2-rp3 and rp3-rp2, which are compounds derived from the extract of large-scale amoebae, have the effect of increasing reactive oxygen species (ROS) in Naegleria fowleri, thereby inducing cell death and mitochondrial dysfunction.

[0141] <Experimental Example 7> Confirmation of the mitochondrial function-impairing effect of compounds derived from the extract of the great aster

[0142] In order to confirm the mitochondrial function-decreasing effect of two compounds (rp2-rp3, rp3-rp2) isolated from the large-scale extract of the selected plant in Experimental Example 4 above, the electrochemical changes in the mitochondrial membrane were measured.

[0143] Specifically, the analysis of electrochemical changes in the mitochondrial membrane of Naegleria fowleri treated with compounds derived from the extract of the great aster was performed using the JC-1 mitochondrial membrane potential assay kit (Abcam, UK) according to the manufacturer's method. Naegleria fowleri were inoculated (5 × 10) into a 96-well microplate (black / clear bottom). 4 cells / well) and IC 50 After treatment with compounds derived from the extract of the large-sized snail, the cells were cultured at 37°C for 24 or 48 hours. As a control, Naegleria fowleri, which was treated with only 0.1% DMSO and cultured under the same conditions, was used. Naegleria fowleri was washed three times with PBS, treated with JC-1 (1 μM), reacted for 30 minutes at room temperature, and then observed under a fluorescence microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific, USA) (JC-1 aggregate: Ex / Em=490 / 525 nm, JC-1 monomer: Ex / Em=550 / 650 nm).

[0144] Changes in ATP production were analyzed using an ATP assay kit (Abcam, UK) according to the manufacturer's method. Naegleria fowleri was IC 50 After treatment with compounds derived from the extract of the large-sized dandelion, the cells were cultured at 37°C for 24 or 48 hours. As a control, Naegleria fowleri, which was treated with only 0.1% DMSO and cultured under the same conditions, was used. Naegleria fowleri was harvested, suspended in ATP assay buffer (100 μl), and disrupted. The supernatant was mixed with the ATP reaction mixture, reacted at room temperature for 30 minutes, and then analyzed for absorbance at 570 nm using a Multiskan FC spectrophotometer (Thermo Fisher Scientific, USA).

[0145] As shown in Fig. 12, the analysis of membrane electrochemical changes in the mitochondria of Naegleria fowleri treated with compounds derived from the extract of Codonopsis lanceolata showed that in the control group Naegleria fowleri, JC-1 aggregates were formed normally as the normal mitochondrial membrane transition was maintained. On the other hand, in the mitochondria of Naegleria fowleri treated with compounds derived from the extract of Codonopsis lanceolata, monomeric JC-1 was observed due to damage to the membrane transition. In addition, as shown in Fig. 13, the analysis of changes in ATP production showed that ATP production was significantly reduced in Naegleria fowleri treated with compounds derived from the extract of Codonopsis lanceolata.

[0146] Through this, it was confirmed that rp2-rp3 and rp3-rp2, which are compounds derived from the extract of large-scale amoebae, have the effect of lowering mitochondrial function in Naegleria fowleri.

[0147] The present invention has been described above, focusing on preferred embodiments and experimental examples. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. A pharmaceutical composition for preventing or treating amoebic infection, comprising as an active ingredient an extract of Xanthium orientale L., a fraction thereof, or a compound represented by the following chemical formula 1 or 2 separated therefrom. [Chemical Formula 1] [Chemical Formula 2] 2. In paragraph 1, A pharmaceutical composition for preventing or treating amoebic infection, characterized in that the above-mentioned large-scale extract is extracted with a solvent selected from the group consisting of water, methanol, ethanol, hexane, butanol, ethyl acetate, chloroform and a mixed solvent thereof.

3. In paragraph 1, A pharmaceutical composition for preventing or treating amoebic infection, characterized in that the above fraction is obtained by fractionating a large-scale extract of the plant with a solvent selected from the group consisting of n-hexane, chloroform, ethyl acetate, n-butanol and a mixed solvent thereof.

4. In paragraph 1, A pharmaceutical composition for preventing or treating an amoebic infection, characterized in that the amoebic infection is primary amoebic meningoencephalitis caused by Naegleria fowleri.

5. In paragraph 1, A pharmaceutical composition for preventing or treating amoebic infection, characterized in that the composition has an effect of killing Naegleria fowleri.

6. In paragraph 1, A pharmaceutical composition for preventing or treating amoebic infection, characterized in that the composition induces morphological changes in Naegleria fowleri.

7. In paragraph 6, A pharmaceutical composition for preventing or treating amoebic infection, wherein the above morphological changes are a decrease in the size and decrease in the number of food vacuoles of Naegleria fowleri.

8. In paragraph 1, A pharmaceutical composition for preventing or treating amoebic infection, characterized in that the composition reduces the survival rate of Naegleria fowleri.

9. In paragraph 1, A pharmaceutical composition for preventing or treating amoebic infection, characterized in that the composition induces changes in the microscopic cell structure of Naegleria fowleri.

10. In paragraph 9, A pharmaceutical composition for preventing or treating amoebic infection, wherein the above microscopic cellular structural changes are deformation of cell organelles or damage to mitochondrial function.

11. In paragraph 1, A pharmaceutical composition for preventing or treating amoebic infection, characterized in that the composition induces apoptosis of Naegleria fowleri.

12. A health functional food composition for preventing or improving amoebic infection, comprising as an active ingredient an extract of Xanthium orientale L., a fraction thereof, or a compound represented by the following chemical formula 1 or 2 separated therefrom. [Chemical Formula 1] [Chemical Formula 2] 13. A method for preventing or treating amoebic infection, comprising administering to a subject the pharmaceutical composition of paragraph 1.

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