WO2023136405A1 - Composition comprising eucommia ulmoides leaf fraction for preventing or treating brain diseases caused by particulate matter - Google Patents
Composition comprising eucommia ulmoides leaf fraction for preventing or treating brain diseases caused by particulate matter Download PDFInfo
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- WO2023136405A1 WO2023136405A1 PCT/KR2022/008463 KR2022008463W WO2023136405A1 WO 2023136405 A1 WO2023136405 A1 WO 2023136405A1 KR 2022008463 W KR2022008463 W KR 2022008463W WO 2023136405 A1 WO2023136405 A1 WO 2023136405A1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/46—Eucommiaceae (Eucommia family), e.g. hardy rubber tree
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/302—Foods, ingredients or supplements having a functional effect on health having a modulating effect on age
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
Definitions
- the present invention relates to a composition for preventing, improving or treating brain diseases caused by fine dust, comprising a leaf fraction of Eucommia ulmoides as an active ingredient. More specifically, it relates to a composition for preventing, improving or treating brain neurological diseases among brain diseases caused by ultrafine dust containing Eucommia leaf fraction.
- the present invention relates to a composition for preventing, improving or treating brain diseases caused by fine dust containing Eucommia ulmoides leaf fraction as an active ingredient, and more particularly, ultrafine dust containing Eucommia ulmoides leaf fraction as an active ingredient It relates to a composition for preventing, improving or treating brain nerve diseases caused by.
- Types of the cranial nerve disease include stroke, Alzheimer's disease (AD), dementia and Parkinson's disease.
- Ultrafine dust refers to fine dust with an aerodynamic diameter of 2.5 ⁇ m or less
- secondary air pollutants NO 3 - , SO 4 - , NH4 - , polyacromatichydrocarbon (PAH), quinone, etc.
- PAH polyacromatichydrocarbon
- PM 2.5 is a stronger risk factor than this. It has been reported to work as
- Dust with a diameter of 5 to 10 ⁇ g/m 3 or less can be absorbed into the body through the nasal mucosa, 2 to 5 ⁇ g/m 3 or less passes through the respiratory tract, and 0.1 to 1 ⁇ g/m 3 to damage the alveoli. will cause When fine dust is inhaled into the human body, it can be deposited in tissues by various mechanisms such as collision, gravitational sedimentation, diffusion, and electrostatic adsorption, and some circulate throughout the body along the blood.
- Eucommia ulmoides is a deciduous tree belonging to the Eucommia family Eucommia, which has high medicinal value and is widely cultivated in Korea, China, and Japan. It is reported that the bark of Eucommia has an effect on blood pressure control, and the leaves of Eucommia have an effect on hyperlipidemia and fatty liver disease. There are phenolics, lignans, iridoids and triterpenes in the leaves and bark of Eucommia. In particular, iridoids derivatives and lignan glycosides are known to have excellent anti-obesity and anti-cardiovascular disease effects.
- Eucommia leaves and bark have been reported to have antioxidant, anti-inflammatory and neuroprotective effects. Therefore, the efficacy of the ethyl acetate fraction of Eucommia leaves in improving cognitive function in the oxidative stress and inflammatory response caused by PM 2.5 was evaluated.
- the cytoprotective effect against cytotoxicity generated in brain nerve cells was verified, and through animal experiments, the composition containing the extract of Eucommia leaves was used to prevent, improve, or treat brain diseases caused by ultrafine dust. It was confirmed that it can be used as, and the present invention has been completed.
- the present invention aims to provide a composition for preventing, improving or treating brain diseases caused by ultrafine dust containing a leaf fraction of Eucommia ulmoides as an active ingredient, More specifically, the cytoprotective effect of Eucommia leaf fraction against cytotoxicity generated in brain nerve cells was verified, and the present invention was completed by confirming the efficacy of preventing, improving or treating brain diseases caused by ultrafine dust directly through animal experiments did
- the present invention provides a health functional food composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia leaf fraction as an active ingredient.
- the present invention provides a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust containing a leaf fraction of Eucommia ulmoides as an active ingredient.
- the present invention provides a cosmetic composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia ulmoides leaf fraction as an active ingredient.
- the present invention provides a method for preventing or treating brain diseases caused by ultrafine dust comprising administering the pharmaceutical composition to a subject.
- the Eucommia ulmoides leaf fraction of the present invention has excellent preventive, ameliorative or therapeutic effects on brain diseases caused by ultrafine dust, so it is a health functional food composition for preventing or improving brain diseases caused by ultrafine dust, especially brain nerve diseases .
- a food composition for preventing or improving brain diseases caused by ultrafine dust, especially brain nerve diseases, a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust, especially brain nerve diseases, or brain diseases caused by ultrafine dust, especially brain can be usefully used as a cosmetic composition for preventing or improving neurological diseases.
- 1 is a diagram showing the results of measuring the total phenol and flavonoid content of the Eucommia leaf fraction of the present invention.
- Figure 2 is a diagram showing the antioxidant activity measurement results (ABTS radical scavenging activity and DPPH radical scavenging activity) of the Eucommia leaf fraction of the present invention.
- Figure 3 is a diagram showing the effect of the Eucommia leaf fraction of the present invention inhibiting the production of intracellular reactive oxygen species (ROS) in brain neurons.
- ROS reactive oxygen species
- Figure 4 is a diagram showing the effect of inhibiting apoptosis in brain neurons of the Eucommia leaf fraction of the present invention.
- 5 is a diagram showing the spatial learning and memory improvement effects of the Eucommia leaf fraction of the present invention using the Y-maze test.
- Figure 6 is a diagram showing the long-term learning and memory improvement effect of the Eucommia leaf fraction of the present invention using the Morris water maze experiment.
- FIG. 7 is a diagram showing the effect of improving SOD activity in brain tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
- FIG. 8 is a diagram showing the effect of improving GSH activity in brain tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
- FIG. 9 is a diagram showing the effect of improving MDA activity in brain tissue induced by ultrafine dust (PM 2.5 ) of the Eucommia leaf fraction of the present invention.
- FIG. 10 is a diagram showing the effect of improving cholinergic system damage in brain tissue induced by ultrafine dust (PM 2.5 ) of the Eucommia leaf fraction of the present invention.
- FIG. 11 is a diagram showing the effect of improving mitochondrial activity in brain tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
- FIG. 12 is a diagram showing the effect of improving inflammation in the olfactory bulb tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
- FIG. 13 is a diagram showing the effect of improving inflammation in hippocampus tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
- the present invention is Eucommia ulmoides
- An object of the present invention is to provide a health functional food composition for preventing or improving brain diseases caused by ultrafine dust containing a leaf fraction as an active ingredient. More preferably, it aims to provide a health functional food composition for preventing or improving brain nerve diseases caused by ultrafine dust.
- an object of the present invention is to provide a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust containing Eucommia leaf fraction as an active ingredient. More preferably, it aims to provide a pharmaceutical composition for preventing or treating brain nerve diseases caused by ultrafine dust.
- an object of the present invention is to provide a cosmetic composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia leaf fraction as an active ingredient. More preferably, it aims to provide a cosmetic composition for preventing or improving brain nerve diseases caused by ultrafine dust.
- an object of the present invention is to provide a method for preventing or treating brain diseases caused by ultrafine dust comprising administering the pharmaceutical composition to a subject. More preferably, furthermore, the present invention aims to provide a method for preventing or treating brain nerve disease caused by ultrafine dust comprising the step of administering the pharmaceutical composition to a subject.
- the present invention relates to a health functional food composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia ulmoides leaf fraction as an active ingredient.
- the fraction of the present invention may be a water fraction, an n-hexane fraction, a chloroform fraction, or an ethyl acetate fraction, preferably an ethyl acetate fraction, but is not limited thereto.
- the brain disease of the present invention is characterized in that it is a cranial nerve disease, and the cranial nerve disease is any one of stroke, Alzheimer's disease (AD), dementia, and Parkinson's disease. It may be characterized as being, but is not limited thereto.
- the composition of the present invention may be characterized in that ABTS radical scavenging activity or DPPH radical scavenging activity in brain tissue is increased, but is not limited thereto.
- the composition of the present invention may be characterized by an effect of inhibiting the production of malondialdehyde (MDA), a lipid peroxide in brain tissue induced by ultrafine dust, but is limited thereto it is not going to be
- MDA malondialdehyde
- the composition of the present invention may be characterized by an effect of inhibiting the generation of reactive oxygen species (ROS) in brain tissue induced by ultrafine dust, but is not limited thereto no.
- ROS reactive oxygen species
- the composition of the present invention may be characterized by an effect of inhibiting cell death in brain tissue induced by ultrafine dust, but is not limited thereto.
- the composition of the present invention may be characterized by a tissue protective effect on the inflammatory reaction in brain tissue induced by ultrafine dust, but is not limited thereto.
- the composition of the present invention may be characterized in that it improves spatial recognition deterioration due to ultrafine dust, but is not limited thereto.
- the composition of the present invention may be characterized by improving short-term learning and short-term memory deteriorated due to ultrafine dust, but is not limited thereto.
- the composition of the present invention may be characterized in that it improves long-term learning and long-term memory deteriorated due to ultrafine dust, but is not limited thereto.
- the composition of the present invention may be characterized in that it improves cognitive function deterioration in brain tissue due to ultrafine dust, but is not limited thereto.
- the composition of the present invention may be characterized in that it improves functional degradation of the cholinergic system in brain tissue due to ultrafine dust, but is not limited thereto.
- the extract according to the present invention may be obtained by extraction and separation from nature using an extraction and separation method known in the art, and the "extract" defined in the present invention is extracted from Eucommia leaves using an appropriate solvent.
- an appropriate solvent for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract are all included.
- any food- or pharmaceutically-acceptable organic solvent may be used, and water or an organic solvent may be used, but is not limited thereto, for example , purified water, alcohol having 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, etc., acetone, ether, benzene ( benzene), chloroform (chloroform), ethyl acetate (ethyl acetate), methylene chloride (methylene chloride), hexane (hexane) and cyclohexane (cyclohexane) can be used alone or in combination.
- purified water alcohol having 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, etc.
- acetone ether
- benzene benzene
- chloroform chloroform
- ethyl acetate ethyl acetate
- any one of methods such as hot water extraction, cold brew extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used.
- the desired extract may be additionally subjected to a conventional fractionation process or may be purified using a conventional purification method.
- the extract included in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying the primary extract extracted by the hot water extraction or solvent extraction method described above.
- a fraction further purified from the primary extract using various chromatography such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography you can also get Therefore, in the present invention, the extract is a concept that includes all extracts, fractions, and purified products obtained in each step of extraction, fractionation, or purification, and dilutions, concentrates, or dried products thereof.
- the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food compositions.
- natural carbohydrates examples include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol.
- natural flavoring agents thaumatin
- stevia extracts eg rebaudioside A, glycyrrhizin, etc.
- synthetic flavoring agents sacharin, aspartame, etc.
- the food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods.
- Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. there is
- the food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, in addition to extracts as active ingredients. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like.
- the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.
- the health functional food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like.
- 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionalities for the human body according to Health Functional Food Act No. 6727, and the structure and function of the human body It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.
- the health functional food of the present invention may contain ordinary food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards.
- Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
- chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid
- natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum
- mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
- a health functional food in the form of a tablet is obtained by granulating a mixture obtained by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives in a conventional manner, and then adding a lubricant or the like to compression molding, or as described above.
- the mixture can be directly compression molded.
- the health functional food in the form of a tablet may contain a flavoring agent and the like as needed.
- hard capsules can be prepared by filling a mixture in which the active ingredient of the present invention is mixed with additives such as excipients in a normal hard capsule. It can be prepared by filling the mixture mixed with gelatin in a capsule base.
- the soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.
- the health functional food in the form of a pill can be prepared by molding a mixture in which the active ingredient of the present invention, excipients, binders, disintegrants, etc. are mixed with a conventionally known method, and if necessary, it can be coated with white sugar or other coating agents, Alternatively, the surface may be coated with a material such as starch or talc.
- Health functional food in the form of granules can be prepared in granular form by a conventionally known method of mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention, and if necessary, flavoring agents, flavoring agents, etc. can
- the present invention relates to a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust comprising a leaf fraction of Eucommia ulmoides as an active ingredient.
- the pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient.
- an adjuvant in addition to the active ingredient.
- any one may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect of the present invention.
- the pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field.
- Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
- the pharmaceutical composition of the present invention may 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 according to conventional methods, respectively. .
- Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.
- Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc.
- compositions for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories.
- Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
- a base for suppositories witepsol, tween 61, cacao paper, laurin paper, glycerogelatin and the like may be used.
- composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
- the pharmaceutical composition may be formulated into various oral or parenteral dosage forms.
- Formulations for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. chlorose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol).
- lubricants eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol.
- the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid or a disintegrant or effervescent mixture, such as its sodium salt, and/or absorbents, colorants, flavors, and sweeteners.
- a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid or a disintegrant or effervescent mixture, such as its sodium salt, and/or absorbents, colorants, flavors, and sweeteners.
- the formulation may be prepared by conventional mixing, granulating or coating methods.
- a typical formulation for parenteral administration is an injection formulation, and water, Ringer's solution, isotonic physiological saline or suspension may be used as a solvent for the injection formulation.
- Sterile fixed oils of the above injectable preparations may be used as a solvent or suspension medium, and any bland fixed oil may be used for this purpose, including mono- and di-glycerides.
- the formulation for injection may use a fatty acid such as oleic acid.
- the present invention relates to a method for treating brain diseases caused by ultrafine dust, comprising administering the pharmaceutical composition to a subject.
- the term "subject” refers to a subject requiring a method for preventing, controlling, or treating a disease, and may be used without limitation, such as humans, dogs, monkeys, cats, rodents, such as mice and genetically engineered mice. . More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.
- the pharmaceutical composition of the present invention can be administered in a therapeutically effective amount or a pharmaceutically effective amount.
- the term "therapeutically effective amount” refers to an amount of a pharmaceutically acceptable salt of a composition effective for preventing or treating a target disease
- a therapeutically effective amount of the composition of the present invention may include several factors, such as For example, it may vary depending on the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in an appropriate amount considering both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are discussed, 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.
- the term "pharmaceutically effective amount” means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's health
- the condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to
- the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
- the present invention relates to a cosmetic composition for preventing or improving brain diseases caused by ultrafine dust comprising a leaf fraction of Eucommia ulmoides as an active ingredient.
- the "cosmetic composition" of the present invention may be prepared by including a cosmetically effective amount of the extract extracted from the leaves of Eucommia of the present invention and a cosmetically acceptable carrier.
- the term "cosmetically effective amount” means an amount sufficient to achieve the skin regeneration effect through the proliferation of epidermal keratinocytes of the composition of the present invention described above.
- the appearance of the cosmetic composition contains a cosmetic or dermatologically acceptable medium or base.
- a cosmetic or dermatologically acceptable medium or base are all formulations suitable for topical application, for example solutions, gels, solids, pasty anhydrous products, emulsions obtained by dispersing an oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and It may be provided in the form of a non-ionic follicular dispersant, or in the form of a cream, toner, lotion, powder, ointment, spray or conceal stick.
- These compositions can be prepared according to conventional methods in the art.
- the composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.
- the cosmetic composition according to an embodiment of the present invention is not particularly limited in its dosage form, for example, softening lotion, astringent lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, eye essence, cleansing It can be formulated into cosmetics such as cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil and body essence.
- the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fibers, vegetable fibers, wax, paraffin, starch, tracanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. this can be used
- lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluorohydro propellants such as carbon, propane/butane or dimethyl ether.
- a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.
- a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.
- the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate , fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives, or ethoxylated glycerol fatty acid esters.
- carrier components aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate , fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols,
- the cosmetic composition of the present invention can be applied to skin, lotion, cream, essence, pack, foundation, color cosmetics, sunscreen, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, lip gloss, lip fix, eyebrow pencil , It can be applied to cosmetics such as lotion and detergents such as shampoo and soap.
- the cosmetic composition according to an embodiment of the present invention may further include functional additives and components included in general cosmetic compositions in addition to the extract extracted from the leaves of Eucommia.
- the functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extracts.
- the cosmetic composition of the present invention may further contain components included in general cosmetic compositions as needed.
- Ingredients other than those included include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation accelerators, cooling agents, antiperspirants, purified water and the like.
- Eucommia leaves used in this experiment were purchased and used in April 2019 from a farmhouse located in Yeongcheon-si, Gyeongsangbuk-do and dried in hot air at 40 ° C. 20 g of powdered Eucommia leaves were mixed with 1 L of 40% ethanol solvent, which had the highest total phenol content through previous studies, and extracted by reflux cooling at 40 ° C. for 2 hours. Thereafter, the extract obtained after filtration using filter paper was fractionated using the same amount of n -hexane, chloroform, and ethyl acetate. The obtained ethyl acetate fraction was concentrated using a rotary vacuum concentrator (NN series, Eyela Co., Tokyo, Japan), and lyophilized to use in this experiment.
- a rotary vacuum concentrator N series, Eyela Co., Tokyo, Japan
- the brain neurons (MC-IXC) used in this experiment were purchased from ATCC and maintained in minimum essential medium (MEM) medium containing 10% FBS and 1% penicillin (50 units/mL)/streptomycin (100 ⁇ g/mL).
- MEM minimum essential medium
- BV-2 Microglial cells
- FBS fetal bovine serum
- penicillin 50 units/mL
- streptomycin 100 ⁇ g/mL
- Example 1 Determination of total phenolic compounds and total flavonoid content in Eucommia leaf fractions
- the sample was mixed with Folin &Ciocalteau's phenol reagent, left at room temperature for 5 minutes, and then mixed with 7% Na 2 CO 3 . Then, after constant use with tertiary distilled water, the mixture was reacted at room temperature for 2 hours and the absorbance at 760 nm (UV-1800, Shimadzu, Kyoto, Japan) was measured. The measured absorbance indicated the content of total phenolic compounds in a calibration curve prepared using gallic acid.
- the total flavonoid content was measured by mixing diethylene glycol and sodium hydroxide in 1 mL of Eucommia leaf extract, followed by reaction at 30 ° C. for 60 minutes.
- the absorbance of the final reactant was measured at 420 nm, and the total flavonoid content was calculated by substituting it into a calibration curve prepared using rutin, which was expressed as rutin equivalent (mg RE (rutin equivalent)/g of dried weight).
- ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity was tested by adding 2.45 mM potassium persulfate and 100 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride. After mixing 7 mM ABTS reagent and leaving it in a constant temperature water bath at 68° C. for 15 minutes, it was filtered using a water-soluble filter and stored in a refrigerator for 24 hours to generate ABTS radicals and used.
- the ABTS radical solution adjusted the absorbance value (0.70 ⁇ 0.02) at 734 nm, and the ABTS solution and the sample were mixed and reacted at 37° C. for 10 minutes. Radical scavenging activity was calculated by measuring absorbance at 734 nm using a spectrophotometer.
- DPPH radical scavenging activity was adjusted so that the absorbance value at 517 nm of 0.1 mM DPPH solution dissolved in 80% methanol was 1.00 ⁇ 0.02, and the DPPH solution and the sample were mixed and reacted for 30 minutes at room temperature and in the dark.
- the absorbance of the final reactant was measured using a spectrophotometer.
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice Male, 4 weeks
- mice an experimental animal supplier
- This animal experiment was conducted after the approval of the Animal Ethics Review Committee of Gyeongsang National University (Gyeongsang National University Animal Experiment License Number: GNU-120831-M0067).
- Eucommia leaf fraction at a concentration of 500 ⁇ g/mL showed radical scavenging activity similar to that of vitamin C at a concentration of 200 ⁇ g/mL.
- the IC 50 values of vitamin C were 111.75 ⁇ g/mL and 117.91 ⁇ g/mL, respectively, and the IC 50 values of the Eucommia leaf fraction were 212.80 ⁇ g/mL and 359.13 ⁇ g/mL, respectively.
- the lipid peroxide inhibitory activities of Eucommia leaf fraction at 100 ⁇ g/mL concentration and catechin were 87.40% and 85.07%, respectively, and the IC 50 values of catechin and Eucommia leaf fractions When compared, they were 25.29 ⁇ g/mL and 33.18 ⁇ g/mL, respectively.
- the excellent antioxidant activity in mouse brain tissue of Eucommia leaf fraction is due to the correlation with the content of total phenolic compounds in Example 1, and the functional damage of cells caused by oxidative stress It is believed to help reduce and protect
- Example 3 Inhibition of intracellular production of ultrafine dust-induced oxidative stress in brain nerve cells of Eucommia leaf fraction
- MC-IXC cells were regularly dispensed into a 96 well plate at a concentration of 1 ⁇ 10 4 cells/well, and BV-2 cells were dispensed at a concentration of 0.5 ⁇ 10 4 cells/well, and 24 Cells were attached by culturing in an incubator for a period of time.
- MC-IXC cells were reacted for 24 hours and BV-2 cells were reacted for 12 hours, and then treated with 50 ⁇ M DCF-DA and reacted for 40 minutes. Fluorescence intensity was measured at excitation wave 485 nm and emission wave 535 nm using a fluorescence photometer (Infinite F200, Tecan, Mannedorf, Swiss).
- ROS When MC-IXC and BV-2 cells were treated with PM 2.5 , ROS increased to 646.99% and 341.71%, respectively, but decreased to 75.27% and 93.03%, respectively, when treated with 10 ⁇ g/mL of Eucommia leaf fraction, respectively, in the normal control group. showed similar or lower ROS levels.
- Eucommia leaf fraction exhibits a cytoprotective effect by inhibiting the excessive production of intracellular ROS induced by ultrafine dust.
- Example 4 Effect of ultrafine dust-induced apoptosis inhibition in brain nerve cells of Eucommia leaf fraction
- cells were pretreated under the same conditions as those for measuring intracellular ROS content, and samples and fine dust were also treated under the same conditions.
- MC-IXC cells were reacted for 24 hours and BV-2 cells were reacted for 12 hours, and then all the culture medium treated with the medium was removed. Absorbance was measured at 570 nm (determination wave) and 690 nm (reference wave).
- Ultrafine dust is known to induce cell function deterioration and death by increasing oxidative stress within cells and promoting inflammatory responses (Guo et al., 2015), and ultrafine dust induction in brain nerve cells of Eucommia leaf fraction The results of measuring the apoptosis inhibitory effect are shown in FIG. 4 .
- the group treated with 10 ⁇ g/mL of Eucommia leaf fraction showed a cell survival rate similar to or higher than that of the positive control group. Therefore, it is confirmed that it exhibits a cytoprotective effect from oxidative stress induced by PM 2.5 .
- Eucommia leaf extract is a health functional food composition, food composition, pharmaceutical composition or cosmetic composition that can help improve function and treat brain nerve cells from cell damage that can be induced by ultrafine dust. Its potential for use is expected.
- a Y-maze was conducted to test the alternating behavioral ability, and a Y-maze made of black plastic made of Y-shape was used for the experiment. After designating each arm as A, B, and C, the path the mouse moved for 8 minutes was recorded with a smart 3.0 video tracking system (Panlab, Barcelona, Spain). The mouse moves freely in the Y-shaped maze for 8 minutes, and the correct behavior is evaluated when the mouse sequentially moves alternately on the three arms.
- FIG. 5 The results of evaluating the spontaneous alternating behavioral ability of mice due to PM 2.5 exposure are shown in FIG. 5 .
- Figure 5A shows the total distance that the mice moved the Y-maze for 8 minutes, and the behavioral abilities of the mice were the same in all groups, meaning that there was no behavioral disorder of the mice.
- Figure 5B shows the mouse alternation behavior, and as a result, it was confirmed that the PM 2.5 group showed a low alternation behavior level of 22.56%, but the ability of voluntary alternation behavior increased to 33.56% and 29.41% in the 20 and 40 groups of Eucommia leaf fractions. As a result, it was confirmed that the shift behavior ability was effectively improved.
- a Morris water maze experiment was conducted to evaluate long-term learning and memory abilities, and mice were allowed to swim freely for 1 minute in a water tank divided into four quadrants during the experiment.
- the water tank is divided into east, west, south, and north, and a platform is placed on one side. Therefore, the Morris water maze test consists of a visible test (Day 1) in which the platform is visible, a hidden test (Day 2-5) in which the platform is invisible, and a probe test (Day 6) in which the platform is completely removed. proceeded.
- Day 1 the visible test
- a hidden test Day 2-5) in which the platform is invisible
- a probe test Day 6 in which the platform is completely removed. proceeded.
- the hidden test the time for the mouse to find the hidden platform and escape was measured, and in the probe test, the time the mouse stayed at the place where the platform was located was measured.
- FIG. 6 The results of evaluating the long-term memory ability of mice due to PM 2.5 exposure are shown in FIG. 6 .
- Figure 6A is the result of measuring the time taken for the mouse to escape to the platform during the visible test and hidden test of the Morris water maze test. As a result, no change in escape time was observed in the PM 2.5 group, but the experiment was conducted in the 20 and 40 groups of the leaf fractions. It was confirmed that the escape time was shortened as much as possible.
- 6B shows the result of measuring the time for mice to stay in the W zone where the platform was present in the last probe test, and showed a low level of 29.11% in the PM 2.5 group, and 55.85% and 75.16% in the 20 and 40 groups of Eucommia leaf fractions, respectively. showed higher levels.
- Brain tissue excised from the mouse was homogenized using phosphate buffered saline (PBS), and the obtained homogenate was centrifuged at 400 ⁇ g for 5 minutes to obtain a pellet.
- the obtained pellet was extracted using 1 ⁇ cell extraction buffer (10 ⁇ SOD buffer 1.0 mL, 20% triton X-100 0.2 mL, distilled water 8.8 mL, phenylmethanesulfonyl fluoride in ethanol 10 ⁇ L).
- the results showing the SOD content in the brain tissue of the mouse model induced by PM 2.5 exposure are shown in FIG. 7 .
- the SOD content in brain tissue of mice exposed to PM 2.5 was 3.50 units/mg of protein, and 4.46 units/mg of protein in 40 groups of Eucommia leaf fractions, respectively. Therefore, it was confirmed that intake of Eucommia leaf fraction could improve the reduction of SOD level caused by exposure to PM 2.5 .
- Brain tissues extracted from mice were homogenized using 10 mM sodium phosphate buffer with 1 mM EDTA (pH 6.0), and the homogenate was centrifuged at 10,000 ⁇ g for 15 minutes. After protein quantification was performed with the obtained supernatant, the supernatant was mixed with 5% metaphosphoric acid at a ratio of 1:1 and centrifuged at 2,000 ⁇ g for 2 minutes, and the experiment was conducted with the obtained supernatant.
- the reduced GSH content in brain tissue of mice exposed to PM 2.5 is shown in FIG. 8 .
- Chronic exposure to PM 2.5 reduced GSH in brain tissue to 74.92% when the normal control group was 100%, and the content increased to 95.89% in the 40 groups of Eucommia leaf fractions. Therefore, it was confirmed that the degradation of the antioxidant system of mouse brain tissue induced by chronic exposure to PM 2.5 was improved by ingesting Eucommia leaf fraction.
- the brain tissue excised from the mouse was homogenized using PBS, and the obtained homogenate was centrifuged at 5,000 rpm for 10 minutes, and the supernatant obtained was used in the experiment.
- the supernatant was mixed with 1% phosphoric acid and 0.67% thiobarbituric acid (TBA) and reacted in a constant temperature water bath at 95°C for 1 hour.
- TSA thiobarbituric acid
- the absorbance of the reaction solution was measured at 532 nm using a microplate reader (Epoch2, BioTek).
- FIG. 9 The result of evaluating the content of lipid peroxide in brain tissue of mice chronically exposed to PM 2.5 is shown in FIG. 9 .
- chronic exposure to PM 2.5 in brain tissue increased the lipid peroxide content, which was 4.32 nmole/mg of protein in the PM 2.5 group and 3.11 nmole/mg of protein in the 40 group of Eucommia leaf fractions. appear. Therefore, it was confirmed that PM 2.5 induces lipid peroxidation in mouse brain tissue, and Eucommia leaf fraction inhibits lipid peroxidation from PM 2.5 .
- Brain tissue excised from the mouse was homogenized using PBS, which was centrifuged at 12,000 rpm for 30 minutes to obtain the supernatant.
- the obtained supernatant was used to measure acetylcholine (ACh) content and acetylcholinesterase (AChE) activity after protein quantification.
- ACh acetylcholine
- AChE acetylcholinesterase
- AChE activity the supernatant was mixed with 50 mM sodium phosphate buffer and reacted at 37°C for 15 minutes. Thereafter, an AChE solution in which acetylthiocholin and DTNB were combined was added and reacted at 37° C. for 5 minutes, and the absorbance was measured at 405 nm using a microplate reader (Epoch2, BioTek).
- FIG. 10 The result of evaluating the effect on the cholinergic system of mice by chronic exposure of PM 2.5 is shown in FIG. 10 .
- 10 (A) and (B) show the results of evaluating the content of ACh and the activity of AChE in mouse brain tissue. Exposure to PM 2.5 reduced the content of ACh, a neurotransmitter, to 1.67 mM/mg of protein, The activity of AChE to degrade ACh was increased to 108.52% when the normal control was 100%. Accordingly, in the 40 groups of Eucommia leaf fractions, the content of ACh was 1.95 mM/mg of protein, and the activity of AChE was confirmed to be reduced to 83.35%.
- the PM 2.5 group increased by 1.56 times compared to the normal control group, and the Eucommia leaf fraction 40 group decreased by 0.71 times compared to the PM 2.5 group, similar to the control group. was confirmed to decrease.
- mitochondria were extracted from brain tissue.
- Brain tissue was homogenized using an isolation buffer (pH 7.2) consisting of 215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin (BSA), 20 mM HEPES (Na + ) and a buffer mixed with 20 mM EGTA, and the homogenate was centrifuged at 1,300 ⁇ g for 5 minutes. The supernatant obtained through this was centrifuged at 13,000 ⁇ g for 10 minutes, and a pellet was obtained.
- BSA bovine serum albumin
- Isolation buffer containing 20 mM EGTA and 0.1% digitonin was added to the resulting pellet, left on ice for 5 minutes, and centrifuged at 13,000 ⁇ g for 15 minutes. The obtained pellet was mixed with isolation buffer and centrifuged at 10,000 ⁇ g for 10 minutes, and mitochondrial activity was evaluated using the finally obtained pellet.
- Mitochondrial extract extracted from brain tissue was used to measure mitochondrial membrane potential (MMP) activity. Mitochondrial extract was mixed with mitochondrial isolation buffer containing 5 mM pyruvate and 5 mM malate, and then 1 ⁇ M JC-1 was added. and reacted in the dark for 20 minutes. The reaction solution was measured at excitation wave 530 nm and emission wave 590 nm using a fluorescence photometer (Infinite F200, Tecan).
- the PM 2.5 group showed 73.60% loss of membrane potential in mouse brain tissue, when the normal control group was 100%, and the 40 group of Eucommia leaf fractions showed a loss of membrane potential by 152.17%. Protected against loss of potential.
- ATP levels in mitochondrial extracts extracted from brain tissue were centrifuged at 10,000 ⁇ g for 10 minutes, and the obtained pellets were mixed with 1% trichloroacetic acid and reacted on ice for 10 minutes. Then, it was mixed with 25 mM Tris-acetate buffer (pH 7.7), centrifuged at 10,000 ⁇ g for 15 minutes, and the obtained supernatant was used to measure the ATP level, which was obtained using an ATP assay kit (Promega, Madison, WI, USA). was measured using a luminometer (GloMax-Multi Detection System, Promega, Corp., Madison, WI, USA).
- amyloid beta pathway proteins and proteins involved in inflammatory responses was measured using hippocampus and olfactory bulb tissues isolated from brains excised from mice. Proteins were extracted from the excised tissues using cell/tissue lysis buffer with 1% protease inhibitor added. The protein extract was centrifuged at 13,000 rpm for 10 minutes to obtain a supernatant, which was quantified using the bradford method. Tissue proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a poly-vinylidene difluoride (PVDF) membrane. Thereafter, blocking was performed using 5% skim milk for 1 hour, followed by washing.
- SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
- PVDF poly-vinylidene difluoride
- the primary antibody diluted 1:1000 in TBST solution containing 0.5% BSA and 0.1% sodium azide was reacted overnight, and then washed and the secondary antibody was reacted at room temperature for 1 hour.
- the membrane after the secondary antibody reaction was reacted with ECL solution to develop color, and the luminescence density was detected using ChemiDoc (iBrightTM CL1000 instrument, Invitrogen, Carlsbad, CA, USA).
- the density of the band was quantified using ibright analysis software (Thermo Fisher, Waltham, Middlesex, MA, USA), and the density value of each factor was expressed by dividing it by the density value of ⁇ -actin.
- FIG. 12 The results of measuring the expression levels of proteins involved in the amyloid beta pathway and inflammatory factors in olfactory bulb tissues isolated from mouse brain tissues are shown in FIG. 12 . It has been reported that the olfactory bulb is one of the pathways for PM 2.5 to penetrate into brain tissue, and that when PM 2.5 accumulates in the olfactory bulb, the inflammatory response is accelerated. It has also been reported that the olfactory bulb exhibits amyloid beta accumulation and hyperphosphorylation of tau protein in the early stages of cognitive impairment. Therefore, in the results of FIG.
- FIG. 13 The expression levels of amyloid-beta and p -tau in the hippocampal tissue were increased 1.56-fold and 3.32-fold, respectively, compared to the normal control group in the PM 2.5 group, and 1.73-fold and 2.83-fold, respectively, compared to the PM 2.5 group in the 40 group of eutrophic leaf fractions, similar to the control. It was confirmed that it was improved (Fig. 13B-C).
- the expression levels of p -I ⁇ B ⁇ , caspase-1, IL-1 ⁇ , and TNF- ⁇ factors in the PM 2.5 group were increased by 1.84, 1.65, 1.50, and 1.93 times compared to the normal control group. Compared to the 2.5 group, the expression level was reduced by 0.65 times, 0.62 times, 0.67 times, and 0.61 times (FIG. 13D-G).
- UPLC Q-TOF/MS 2 analysis was performed to analyze the physiologically active substances contained in the leaf fractions of Eucommia.
- Eucommia leaf fraction was dissolved using 100% methanol, and filtered using a filter before use. This was analyzed in negative ion mode using an Acquity UPLC BEH C 18 column, and the transfer rate was 0.35 mL/min.
- Mobile phase solvents were 0.1% formic acid in distilled water (A) and 0.1% formic acid in acetonitrile (B), 1% B at 0-1 min, 1-100% B at 1-7min, 100% B at A solvent gradient was performed under conditions of 7-8 min, 100-1% B at 8-8.2 min, and 1% B at 8.2-10 min.
- UPLC Q-TOF/MS 2 analysis was performed to analyze the physiologically active substances contained in the leaf fraction of Eucommia used in the present invention (FIG. 14).
- the identified peaks are compound 1: 707 m/z (RT: 2.84 min), compound 2: 609 m/z (RT: 3.15 min), compound 3: 463 m/z (RT: 3.22 min), compound 4: 505 m/z (RT 3.28 min), compound 5: 489 m/z (RT: 3.44 min), compound 6: 301 m/z (RT 3.84 min), which are respectively 5-O-caffeoylquinic acid, They were identified as rutin, quercetin-O-hexoside, quercetin-O-acetylhexoside (isomer), luteolin-O-acetylhexoside, and quercetin (Table 1).
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Abstract
The purpose of the present invention is to provide a composition comprising a (Eucommia ulmoides leaf fraction as an active ingredient for preventing, palliating, or treating brain diseases caused by ultrafine particulate matter. More specifically, a Eucommia ulmoides leaf fraction was verified to have an excellent cytoprotective effect against the cytotoxicity generated in brain nerve cells and identified to be directly preventive, palliative, or therapeutic for brain nerve diseases caused by ultrafine particulate matter through animal experiments, which has led to the present invention. The present invention can be usefully utilized as a health functional food composition for preventing or palliating brain diseases caused by ultrafine particulate matter, as a food composition for preventing or palliating brain diseases caused by ultrafine particulate matter, as a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine particulate matter, or as a cosmetic composition for preventing or palliating brain diseases caused by ultrafine particulate matter.
Description
본 발명은 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 미세먼지로 인한 뇌질환의 예방, 개선 또는 치료용 조성물에 관한 것이다. 더욱 구체적으로는 두충 잎 분획물을 포함하는 초미세먼지로 인한 뇌질환 중 뇌 신경 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, improving or treating brain diseases caused by fine dust, comprising a leaf fraction of Eucommia ulmoides as an active ingredient. More specifically, it relates to a composition for preventing, improving or treating brain neurological diseases among brain diseases caused by ultrafine dust containing Eucommia leaf fraction.
본 발명은 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 미세먼지로 인한 뇌질환의 예방, 개선 또는 치료용 조성물에 관한 것으로서, 더욱 상세하게는 두충 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌 신경 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다. 상기 뇌 신경 질환의 종류로는 뇌졸중, 알츠하이머 병 (Alzheimer’s disease; AD), 치매 (dementia) 및 파킨슨병 등이 있다.The present invention relates to a composition for preventing, improving or treating brain diseases caused by fine dust containing Eucommia ulmoides leaf fraction as an active ingredient, and more particularly, ultrafine dust containing Eucommia ulmoides leaf fraction as an active ingredient It relates to a composition for preventing, improving or treating brain nerve diseases caused by. Types of the cranial nerve disease include stroke, Alzheimer's disease (AD), dementia and Parkinson's disease.
한편, 최근 미세먼지 (particulate matter, PM)의 농도가 증가하고 지속기간이 증가됨에 따라 미세먼지에 의한 인체 영향성에 대한 관심이 증가되고 있는 추세이다(Kim 등, 2017). 미세먼지는 우리나라의 오랜 역사와 함께 해왔으며, 예로부터 몽고와 중국 사막지역 및 황화강 유역에서 발원된 황사의 영향을 받았고, 최근에는 우리나라 및 동북아시아 국가들의 급격한 산업화가 미세먼지의 주요 발생 원인으로 보고되고 있다(Myung, 2016).Meanwhile, as the concentration of particulate matter (PM) increases and the duration increases, interest in the effects of fine dust on the human body is increasing (Kim et al., 2017). Fine dust has been with Korea for a long history, and has been affected by yellow dust originating from the desert regions of Mongolia and China and the Yellow River basin since ancient times. Recently, rapid industrialization in Korea and Northeast Asian countries has become a major cause of fine dust. It has been reported (Myung, 2016).
초미세먼지(PM2.5)는 공기역학적 지름이 2.5 μm 이하인 미세먼지를 의미하며, 대기 오염 발생원에서 직접 배출되어지는 1차 오염 물질과 대기에서 반응하여 생성되는 2차 대기오염 물질 (NO3
-, SO4
-, NH4-, polyacromatichydrocarbon (PAH), quinone등)이 주를 이룬다(Kim 등, 2017). 세계보건기구(World Health Organization, 2013)에 의하면, PM10 (공기역학적 지름이 10 μm 이하인 미세먼지)에 장기 노출될 경우 호흡기관 관련 질환과 사망률이 증가하게 되는데, PM2.5는 이보다 더 강한 위험 인자로 작용한다고 보고하였다.Ultrafine dust (PM 2.5 ) refers to fine dust with an aerodynamic diameter of 2.5 μm or less, and secondary air pollutants (NO 3 - , SO 4 - , NH4 - , polyacromatichydrocarbon (PAH), quinone, etc.) are the main ones (Kim et al., 2017). According to the World Health Organization (2013), long-term exposure to PM 10 (fine dust with an aerodynamic diameter of 10 μm or less) increases respiratory tract-related diseases and mortality, but PM 2.5 is a stronger risk factor than this. It has been reported to work as
지름 5~10 μg/m3 이하의 먼지는 코 점막을 통해 체내에 흡수가 가능하며 2~5 μg/m3 이하는 기도 (호흡기)를 통과하고, 0.1~1 μg/m3는 폐포 손상까지 유발하게 된다. 미세먼지가 인체에 흡입되었을 경우, 충돌·중력침강·확산·정전기적 흡착 등과 같은 다양한 기전에 의해서 조직에 침착될 수 있으며, 일부는 혈액을 따라서 전신을 순환하기도 한다. Dust with a diameter of 5 to 10 μg/m 3 or less can be absorbed into the body through the nasal mucosa, 2 to 5 μg/m 3 or less passes through the respiratory tract, and 0.1 to 1 μg/m 3 to damage the alveoli. will cause When fine dust is inhaled into the human body, it can be deposited in tissues by various mechanisms such as collision, gravitational sedimentation, diffusion, and electrostatic adsorption, and some circulate throughout the body along the blood.
특히, 체내에 침착된 미세먼지는 산화적 스트레스와 염증 반응을 유도하고 호흡계 및 순환계 질환의 급성 악화 등을 유발하는 것으로 보고되고 있다(Myung, 2016). 또한, 후각 상피세포를 거치면 혈관 뇌 장벽(Blood-Brain Barrier; BBB)을 거치지 않고도 뇌로 직접 도달할 수 있으며, 자성을 띄는 입자의 경우 뇌 조직에 직접 침착됨으로써 미세아교세포(Microglia)를 활성화하여 pro-inflammatory cytokines (IL-1β, TNF-α 및 IFN-γ) 등을 생산함으로써 뇌신경 염증을 유도하고 더 나아가 인지장애를 유도하는 것으로 보고되었다(Block, 2004). 따라서, 이러한 미세먼지로 유도될 수 있는 인체 내 산화적 스트레스 억제 및 염증 반응을 억제시켜 줄 수 있는 천연 식품 소재에 대한 연구가 필요하다고 사료된다.In particular, it has been reported that fine dust deposited in the body induces oxidative stress and inflammatory reactions and causes acute exacerbation of respiratory and circulatory diseases (Myung, 2016). In addition, when passing through olfactory epithelial cells, it can reach the brain directly without passing through the Blood-Brain Barrier (BBB), and in the case of magnetic particles, they are directly deposited in brain tissue, activating microglia and pro- It has been reported that by producing -inflammatory cytokines (IL-1β, TNF-α and IFN-γ), etc., inflammation of the cranial nerve is induced and further cognitive impairment is induced (Block, 2004). Therefore, it is thought that there is a need for research on natural food materials that can suppress oxidative stress and inflammatory reactions in the human body that can be induced by such fine dust.
한편 두충(Eucommia ulmoides)은 두충나무과 두충속의 낙엽교목으로써 높은 약용가치를 나타내고 있어 한국, 중국, 일본 등에서 널리 재배되고 있다. 두충의 수피는 혈압 조절을 효능을 지니고 있으며, 두충 잎은 고지혈증과 지방간 질환에 대한 효능을 지닌다고 보고되어 진다. 두충의 잎과 수피에는 phenolics, lignan, iridoids 및 triterpene 등이 존재하고, 특히 iridoids 유도체와 lignan 배당체는 항비만, 항심혈관질환에 우수한 효능을 나타내는 것으로 알려져 있다. 나아가 두충 잎과 수피는 항산화, 항염증 및 신경 보호 효과에 대해 보고되어 있다. 따라서 두충 잎 아세트산 에틸 분획물이 PM2.5로 인한 산화적 스트레스 및 염증 반응에서 인지기능 개선 효능을 평가하고자 하였다.On the other hand, Eucommia ulmoides is a deciduous tree belonging to the Eucommia family Eucommia, which has high medicinal value and is widely cultivated in Korea, China, and Japan. It is reported that the bark of Eucommia has an effect on blood pressure control, and the leaves of Eucommia have an effect on hyperlipidemia and fatty liver disease. There are phenolics, lignans, iridoids and triterpenes in the leaves and bark of Eucommia. In particular, iridoids derivatives and lignan glycosides are known to have excellent anti-obesity and anti-cardiovascular disease effects. Furthermore, Eucommia leaves and bark have been reported to have antioxidant, anti-inflammatory and neuroprotective effects. Therefore, the efficacy of the ethyl acetate fraction of Eucommia leaves in improving cognitive function in the oxidative stress and inflammatory response caused by PM 2.5 was evaluated.
따라서, 본 연구에서는 뇌 신경세포에서 발생 되는 세포 독성에 대한 세포보호 효과를 검증하였고, 동물실험을 통해 두충 잎 분획물을 포함하는 조성물이 초미세먼지로 인한 뇌질환에 대해 예방, 개선 또는 치료용 조성물로 사용될 수 있음을 확인하여 본 발명을 완성하기에 이르렀다.Therefore, in this study, the cytoprotective effect against cytotoxicity generated in brain nerve cells was verified, and through animal experiments, the composition containing the extract of Eucommia leaves was used to prevent, improve, or treat brain diseases caused by ultrafine dust. It was confirmed that it can be used as, and the present invention has been completed.
본 발명은 상기와 같은 문제를 해결하기 위해, 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환의 예방, 개선 또는 치료용 조성물을 제공하는 것을 목적으로 하며, 더욱 상세하게는 두충 잎 분획물이 뇌 신경세포에서 발생되는 세포 독성에 대한 세포보호 효과를 검증하였으며, 동물실험을 통해 직접적으로 초미세먼지로 인한 뇌질환의 예방, 개선 또는 치료 효능을 확인하여 본 발명을 완성하였다.In order to solve the above problems, the present invention aims to provide a composition for preventing, improving or treating brain diseases caused by ultrafine dust containing a leaf fraction of Eucommia ulmoides as an active ingredient, More specifically, the cytoprotective effect of Eucommia leaf fraction against cytotoxicity generated in brain nerve cells was verified, and the present invention was completed by confirming the efficacy of preventing, improving or treating brain diseases caused by ultrafine dust directly through animal experiments did
상기한 과제를 해결하기 위하여, 본 발명은 두충 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물을 제공한다.In order to solve the above problems, the present invention provides a health functional food composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia leaf fraction as an active ingredient.
또한, 본 발명은 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust containing a leaf fraction of Eucommia ulmoides as an active ingredient.
아울러, 본 발명은 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 개선용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia ulmoides leaf fraction as an active ingredient.
나아가, 본 발명은 상기 약학 조성물을 개체에 투여하는 단계를 포함하는 초미세먼지로 인한 뇌질환 예방 또는 치료방법을 제공한다.Furthermore, the present invention provides a method for preventing or treating brain diseases caused by ultrafine dust comprising administering the pharmaceutical composition to a subject.
본 발명의 두충(Eucommia ulmoides) 잎 분획물은 초미세먼지로 인한 뇌질환에 대해 예방, 개선 또는 치료 효과가 뛰어나므로 초미세먼지로 인한 뇌질환 특히 뇌 신경 질환에 대한 예방 또는 개선용 건강기능성식품 조성물, 초미세먼지로 인한 뇌질환 특히 뇌 신경 질환에 대한 예방 또는 개선용 식품 조성물, 초미세먼지로 인한 뇌질환 특히 뇌 신경 질환에 대한 예방 또는 치료용 약학 조성물 또는 초미세먼지로 인한 뇌질환 특히 뇌 신경 질환에 대한 예방 또는 개선용 화장료 조성물로 유용하게 이용할 수 있다. The Eucommia ulmoides leaf fraction of the present invention has excellent preventive, ameliorative or therapeutic effects on brain diseases caused by ultrafine dust, so it is a health functional food composition for preventing or improving brain diseases caused by ultrafine dust, especially brain nerve diseases , A food composition for preventing or improving brain diseases caused by ultrafine dust, especially brain nerve diseases, a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust, especially brain nerve diseases, or brain diseases caused by ultrafine dust, especially brain It can be usefully used as a cosmetic composition for preventing or improving neurological diseases.
도 1은 본 발명의 두충 잎 분획물의 총 페놀 및 플라보노이드 함량 측정 결과를 나타낸 도이다.1 is a diagram showing the results of measuring the total phenol and flavonoid content of the Eucommia leaf fraction of the present invention.
도 2는 본 발명의 두충 잎 분획물의 항산화 활성 측정 결과(ABTS 라디칼 소거 활성 및 DPPH 라디칼 소거 활성)를 나타낸 도이다.Figure 2 is a diagram showing the antioxidant activity measurement results (ABTS radical scavenging activity and DPPH radical scavenging activity) of the Eucommia leaf fraction of the present invention.
도 3은 본 발명의 두충 잎 분획물이 뇌 신경세포에서의 세포 내 활성산소 (reactive oxygen species, ROS)의 생성 억제 효과를 나타낸 도이다.Figure 3 is a diagram showing the effect of the Eucommia leaf fraction of the present invention inhibiting the production of intracellular reactive oxygen species (ROS) in brain neurons.
도 4는 본 발명의 두충 잎 분획물의 뇌 신경세포에서의 세포사멸을 억제시키는 효과를 나타낸 도이다.Figure 4 is a diagram showing the effect of inhibiting apoptosis in brain neurons of the Eucommia leaf fraction of the present invention.
도 5는 본 발명의 두충 잎 분획물의 공간 학습 및 기억능력 개선 효과를 Y-maze test를 이용해 나타낸 도이다.5 is a diagram showing the spatial learning and memory improvement effects of the Eucommia leaf fraction of the present invention using the Y-maze test.
도 6은 본 발명의 두충 잎 분획물의 장기 학습 및 기억능력 개선 효과를 Morris water maze 실험을 이용해 나타낸 도이다.Figure 6 is a diagram showing the long-term learning and memory improvement effect of the Eucommia leaf fraction of the present invention using the Morris water maze experiment.
도 7은 본 발명의 두충 잎 분획물의 초미세먼지(PM2.5)로 유도된 뇌 조직에서 SOD 활성 개선 효과를 나타낸 도이다.7 is a diagram showing the effect of improving SOD activity in brain tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
도 8은 본 발명의 두충 잎 분획물의 초미세먼지(PM2.5)로 유도된 뇌 조직에서 GSH 활성 개선 효과를 나타낸 도이다.8 is a diagram showing the effect of improving GSH activity in brain tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
도 9는 본 발명의 두충 잎 분획물의 초미세먼지(PM2.5)로 유도된 뇌 조직에서 MDA 활성 개선 효과를 나타낸 도이다.9 is a diagram showing the effect of improving MDA activity in brain tissue induced by ultrafine dust (PM 2.5 ) of the Eucommia leaf fraction of the present invention.
도 10은 본 발명의 두충 잎 분획물의 초미세먼지(PM2.5)로 유도된 뇌 조직에서의 콜린성 시스템 손상 개선 효과를 나타낸 도이다.10 is a diagram showing the effect of improving cholinergic system damage in brain tissue induced by ultrafine dust (PM 2.5 ) of the Eucommia leaf fraction of the present invention.
도 11은 본 발명의 두충 잎 분획물의 초미세먼지(PM2.5)로 유도된 뇌 조직에서 미토콘드리아 활성 개선 효과를 나타낸 도이다.11 is a diagram showing the effect of improving mitochondrial activity in brain tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
도 12는 본 발명의 두충 잎 분획물의 초미세먼지(PM2.5)로 유도된 olfactory bulb 조직에서의 염증 개선 효과를 나타낸 도이다.12 is a diagram showing the effect of improving inflammation in the olfactory bulb tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
도 13은 본 발명의 두충 잎 분획물의 초미세먼지(PM2.5)로 유도된 hippocampus 조직에서의 염증 개선 효과를 나타낸 도이다.13 is a diagram showing the effect of improving inflammation in hippocampus tissue induced by ultrafine dust (PM 2.5 ) of the leaf fraction of Eucommia of the present invention.
도 14는 본 발명의 두충 잎 분획에 포함된 생리 활성 물질 분석 결과를 나타낸 도이다.14 is a diagram showing the analysis results of physiologically active substances contained in Eucommia leaf fractions of the present invention.
본 발명은 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물 제공을 목적으로 한다. 보다 바람직하게는, 초미세먼지로 인한 뇌 신경 질환 예방 또는 개선용 건강기능성식품 조성물 제공을 목적으로 한다.The present invention is Eucommia ulmoides An object of the present invention is to provide a health functional food composition for preventing or improving brain diseases caused by ultrafine dust containing a leaf fraction as an active ingredient. More preferably, it aims to provide a health functional food composition for preventing or improving brain nerve diseases caused by ultrafine dust.
또한, 본 발명은 두충 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 치료용 약학 조성물 제공을 목적으로 한다. 보다 바람직하게는, 초미세먼지로 인한 뇌 신경 질환 예방 또는 치료용 약학 조성물 제공을 목적으로 한다.In addition, an object of the present invention is to provide a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust containing Eucommia leaf fraction as an active ingredient. More preferably, it aims to provide a pharmaceutical composition for preventing or treating brain nerve diseases caused by ultrafine dust.
아울러, 본 발명은 두충 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 개선용 화장료 조성물 제공을 목적으로 한다. 보다 바람직하게는, 초미세먼지로 인한 뇌 신경 질환 예방 또는 개선용 화장료 조성물 제공을 목적으로 한다.In addition, an object of the present invention is to provide a cosmetic composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia leaf fraction as an active ingredient. More preferably, it aims to provide a cosmetic composition for preventing or improving brain nerve diseases caused by ultrafine dust.
나아가, 본 발명은 상기 약학 조성물을 개체에 투여하는 단계를 포함하는 초미세먼지로 인한 뇌질환 예방 또는 치료방법을 제공하는 것을 목적으로 한다. 보다 바람직하게는, 나아가, 본 발명은 상기 약학 조성물을 개체에 투여하는 단계를 포함하는 초미세먼지로 인한 뇌 신경 질환 예방 또는 치료방법을 제공하는 것을 목적으로 한다.Furthermore, an object of the present invention is to provide a method for preventing or treating brain diseases caused by ultrafine dust comprising administering the pharmaceutical composition to a subject. More preferably, furthermore, the present invention aims to provide a method for preventing or treating brain nerve disease caused by ultrafine dust comprising the step of administering the pharmaceutical composition to a subject.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현 예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허 청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다.Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following implementation examples are presented as examples of the present invention, and if it is determined that a detailed description of a well-known technology or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the description of the claims described later and equivalent scopes interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(Terminology)들은 본 발명의 바람직한 실시 예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 “포함”한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the contents throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 '%'는 별도의 언급이 없는 경우, 고체/고체는(w/w) %, 고체/액체는(w/v) %, 그리고 액체/액체는(v/v) %이다.Throughout this specification, '%' used to indicate the concentration of a particular substance is solid/solid (w/w) %, solid/liquid (w/v) %, and Liquid/liquid is (v/v) %.
일 측면에서, 본 발명은 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물에 관한 것이다.In one aspect, the present invention relates to a health functional food composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia ulmoides leaf fraction as an active ingredient.
본 발명의 일 실시예에 있어서, 본 발명의 분획물은 물 분획물, n-헥산 분획물, 클로로포름 분획물 또는 에틸아세테이트 분획물인 것일 수 있으며, 바람직하게는 에틸아세테이트 분획물인 것일 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the fraction of the present invention may be a water fraction, an n-hexane fraction, a chloroform fraction, or an ethyl acetate fraction, preferably an ethyl acetate fraction, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 뇌질환은 뇌 신경 질환인 것을 특징으로 하며, 상기 뇌 신경 질환은 뇌졸중, 알츠하이머병 (Alzheimer’s disease; AD), 치매 (dementia) 및 파킨슨병 중 어느 하나인 것으로 하는 것을 특징으로 할 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the brain disease of the present invention is characterized in that it is a cranial nerve disease, and the cranial nerve disease is any one of stroke, Alzheimer's disease (AD), dementia, and Parkinson's disease. It may be characterized as being, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 뇌 조직 내 ABTS 라디칼 소거 활성 또는 DPPH 라디칼 소거 활성이 증가된 것을 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized in that ABTS radical scavenging activity or DPPH radical scavenging activity in brain tissue is increased, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 유도된 뇌 조직 내에서의 지방질과산화물인 말론디알데히드(Malondialdehyde, MDA)의 생성을 억제하는 효과를 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized by an effect of inhibiting the production of malondialdehyde (MDA), a lipid peroxide in brain tissue induced by ultrafine dust, but is limited thereto it is not going to be
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 유도된 뇌 조직 내에서의 활성산소(reactive oxygen species, ROS)의 생성을 억제시키는 효과를 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized by an effect of inhibiting the generation of reactive oxygen species (ROS) in brain tissue induced by ultrafine dust, but is not limited thereto no.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 유도된 뇌 조직 내에서의 세포사멸을 억제시키는 효과를 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized by an effect of inhibiting cell death in brain tissue induced by ultrafine dust, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 유도된 뇌 조직 내에서의 염증 반응에 대한 조직 보호 효과를 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized by a tissue protective effect on the inflammatory reaction in brain tissue induced by ultrafine dust, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 인한 공간인지력 저하를 개선시키는 것을 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized in that it improves spatial recognition deterioration due to ultrafine dust, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 인해 저하된 단기 학습 및 단기 기억력을 개선시키는 것을 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized by improving short-term learning and short-term memory deteriorated due to ultrafine dust, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 인해 저하된 장기 학습 및 장기 기억력을 개선시키는 것을 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized in that it improves long-term learning and long-term memory deteriorated due to ultrafine dust, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 인해 뇌 조직에서의 인지기능 저하를 개선시키는 것을 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized in that it improves cognitive function deterioration in brain tissue due to ultrafine dust, but is not limited thereto.
본 발명의 일 실시예에 있어서, 본 발명의 조성물은 초미세먼지로 인한 뇌 조직에서 콜린성 시스템의 기능 저하를 개선시키는 것을 특징으로 할 수 있으나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the composition of the present invention may be characterized in that it improves functional degradation of the cholinergic system in brain tissue due to ultrafine dust, but is not limited thereto.
본 발명에 따른 추출물은 당업계에 공지된 추출 및 분리방법을 사용하여 천연으로부터 추출 및 분리하여 수득한 것을 사용할 수 있으며, 본 발명에서 정의된 "추출물"은 적절한 용매를 이용하여 두충 잎으로부터 추출한 것이며, 예를 들어, 조추출물, 극성용매 가용 추출물 또는 비극성용매 가용 추출물을 모두 포함한다. 상기 두충 잎으로부터 추출물을 추출하기 위한 적절한 용매로는 식품학적으로 또는 약학적으로 허용되는 유기용매라면 어느 것을 사용해도 무방하며, 물 또는 유기용매를 사용할 수 있으며, 이에 제한되지는 않으나, 예를 들어, 정제수, 메탄올 (methanol), 에탄올 (ethanol), 프로판올 (propanol), 이소프로판올 (isopropanol), 부탄올 (butanol) 등을 포함하는 탄소수 1 내지 4의 알코올, 아세톤 (acetone), 에테르 (ether), 벤젠 (benzene), 클로로포름 (chloroform), 에틸아세테이트 (ethyl acetate), 메틸렌클로라이드 (methylene chloride), 헥산 (hexane) 및 시클로헥산 (cyclohexane) 등의 각종 용매를 단독으로 혹은 혼합하여 사용할 수 있다. 추출 방법으로는 열수추출법, 냉침추출법, 환류냉각추출법, 용매추출법, 수증기증류법, 초음파추출법, 용출법, 압착법 등의 방법 중 어느 하나를 선택하여 사용할 수 있다. 또한, 목적하는 추출물은 추가로 통상의 분획 공정을 수행할 수도 있으며, 통상의 정제 방법을 이용하여 정제될 수도 있다.The extract according to the present invention may be obtained by extraction and separation from nature using an extraction and separation method known in the art, and the "extract" defined in the present invention is extracted from Eucommia leaves using an appropriate solvent. , For example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract are all included. As a suitable solvent for extracting the extract from the leaves of Eucommia, any food- or pharmaceutically-acceptable organic solvent may be used, and water or an organic solvent may be used, but is not limited thereto, for example , purified water, alcohol having 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, etc., acetone, ether, benzene ( benzene), chloroform (chloroform), ethyl acetate (ethyl acetate), methylene chloride (methylene chloride), hexane (hexane) and cyclohexane (cyclohexane) can be used alone or in combination. As an extraction method, any one of methods such as hot water extraction, cold brew extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used. In addition, the desired extract may be additionally subjected to a conventional fractionation process or may be purified using a conventional purification method.
본 발명의 추출물의 제조방법에는 제한이 없으며, 공지되어 있는 어떠한 방법도 이용될 수 있다. 예를 들면, 본 발명의 조성물에 포함되는 추출물은 상기한 열수 추출 또는 용매추출법으로 추출된 1차 추출물을, 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조할 수 있다. 또한 상기 1차 추출물을 실리카겔 컬럼 크로마토그래피 (silica gel column chromatography), 박층 크로마토그래피 (thin layer chromatography), 고성능 액체 크로마토그래피 (high performance liquid chromatography) 등과 같은 다양한 크로마토그래피를 이용하여 추가로 정제된 분획을 얻을 수도 있다. 따라서 본 발명에 있어서 추출물은 추출, 분획 또는 정제의 각 단계에서 얻어지는 모든 추출액, 분획 및 정제물, 그들의 희석액, 농축액 또는 건조물을 모두 포함하는 개념이다.There is no limitation on the preparation method of the extract of the present invention, and any known method may be used. For example, the extract included in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying the primary extract extracted by the hot water extraction or solvent extraction method described above. In addition, a fraction further purified from the primary extract using various chromatography such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography you can also get Therefore, in the present invention, the extract is a concept that includes all extracts, fractions, and purified products obtained in each step of extraction, fractionation, or purification, and dilutions, concentrates, or dried products thereof.
본 발명의 식품 조성물은 유효성분인 분획물을 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In addition to containing fractions as active ingredients, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food compositions.
상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 향미제는 천연 향미제 (타우마틴), 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 본 발명의 식품 조성물은 상기 약학 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등이 있다.Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agents described above, natural flavoring agents (thaumatin), stevia extracts (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. there is
또한, 상기 식품 조성물은 유효성분인 추출물 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, in addition to extracts as active ingredients. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.
본 발명의 건강기능성식품 조성물은, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공될 수 있다. 본 발명에서 '건강기능성식품 조성물'이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다. 본 발명의 건강기능성식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. 상기 '식품 첨가물 공전'에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료 제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다. 예를 들어, 정제 형태의 건강기능성식품은 본 발명의 유효성분을 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능성식품은 필요에 따라 교미제 등을 함유할 수도 있다. 캅셀 형태의 건강기능성식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분을 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 본 발명의 유효성분을 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다. 환 형태의 건강기능성식품은 본 발명의 유효성분과 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다. 과립 형태의 건강기능성식품은 본 발명의 유효성분의 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다.The health functional food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like. In the present invention, 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionalities for the human body according to Health Functional Food Act No. 6727, and the structure and function of the human body It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions. The health functional food of the present invention may contain ordinary food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations. For example, a health functional food in the form of a tablet is obtained by granulating a mixture obtained by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives in a conventional manner, and then adding a lubricant or the like to compression molding, or as described above. The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a flavoring agent and the like as needed. Among health functional foods in the form of capsules, hard capsules can be prepared by filling a mixture in which the active ingredient of the present invention is mixed with additives such as excipients in a normal hard capsule. It can be prepared by filling the mixture mixed with gelatin in a capsule base. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. The health functional food in the form of a pill can be prepared by molding a mixture in which the active ingredient of the present invention, excipients, binders, disintegrants, etc. are mixed with a conventionally known method, and if necessary, it can be coated with white sugar or other coating agents, Alternatively, the surface may be coated with a material such as starch or talc. Health functional food in the form of granules can be prepared in granular form by a conventionally known method of mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention, and if necessary, flavoring agents, flavoring agents, etc. can
일 측면에서, 본 발명은 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 치료용 약학 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust comprising a leaf fraction of Eucommia ulmoides as an active ingredient.
본 발명의 약학 조성물에는 유효성분 이외에 보조제 (adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있으나, 예를 들어 프로인트(Freund)의 완전 보조제 또는 불완전 보조제를 더 포함하여 본 발명의 효과를 증가시킬 수 있다.The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. As long as the adjuvant is known in the art, any one may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect of the present invention.
본 발명에 따른 약학 조성물은 유효성분을 약학적으로 허용된 담체에 혼입시킨 형태로 제조될 수 있다. 여기서, 약학적으로 허용된 담체는 제약 분야에서 통상 사용되는 담체, 부형제 및 희석제를 포함한다. 본 발명의 약학 조성물에 이용할 수 있는 약학적으로 허용된 담체는 이들로 제한되는 것은 아니지만, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition of the present invention may 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 according to conventional methods, respectively. .
제제화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 그러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘 카르보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 일반적으로 사용되는 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수용성용제, 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 트윈 (tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin and the like may be used.
본 발명에 따른 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식이 예상될 수 있는데, 예를 들면 경구, 정맥, 근육, 피하, 복강내 주사에 의해 투여될 수 있다.The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
상기 약학 조성물은 다양한 경구 또는 비경구 투여 형태로 제형화될 수 있다. The pharmaceutical composition may be formulated into various oral or parenteral dosage forms.
경구 투여용 제형으로는 예를 들면 정제, 환제, 경질, 연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제 등이 있는데, 이들 제형은 유효성분 이외에 희석제 (예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제 (예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/ 또는 폴리에틸렌 글리콜)를 추가로 포함할 수 있다. 또한, 상기 정제는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 트라가칸스, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제 및 감미제를 함유할 수 있다. 상기 제형은 통상적인 혼합, 과립화 또는 코팅 방법에 의해 제조될 수 있다.Formulations for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. chlorose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid or a disintegrant or effervescent mixture, such as its sodium salt, and/or absorbents, colorants, flavors, and sweeteners. The formulation may be prepared by conventional mixing, granulating or coating methods.
또한, 비경구 투여용 제형의 대표적인 것은 주사용 제제이며, 주사용 제제의 용매로서 물, 링거액, 등장성 생리식염수 또는 현탁액을 들 수 있다. 상기 주사용 제제의 멸균 고정 오일은 용매 또는 현탁 매질로서 사용할 수 있으며 모노-, 디-글리세라이드를 포함하여 어떠한 무자극성 고정오일도 이러한 목적으로 사용될 수 있다.In addition, a typical formulation for parenteral administration is an injection formulation, and water, Ringer's solution, isotonic physiological saline or suspension may be used as a solvent for the injection formulation. Sterile fixed oils of the above injectable preparations may be used as a solvent or suspension medium, and any bland fixed oil may be used for this purpose, including mono- and di-glycerides.
또한, 상기 주사용 제제는 올레산과 같은 지방산을 사용할 수 있다.In addition, the formulation for injection may use a fatty acid such as oleic acid.
일 측면에서, 본 발명은 상기 약학 조성물을 개체에 투여하는 단계를 포함하는, 초미세먼지로 인한 뇌질환 치료방법에 관한 것이다.In one aspect, the present invention relates to a method for treating brain diseases caused by ultrafine dust, comprising administering the pharmaceutical composition to a subject.
본 발명에서 사용되는 용어 "개체"란 질병의 예방, 조절 또는 치료방법을 필요로 하는 대상을 의미하고, 사람, 개, 원숭이, 고양이, 설치류, 예컨대 마우스, 유전자 조작된 마우스 등 제한 없이 사용될 수 있다. 보다 구체적으로는, 인간 또는 비-인간인 영장류, 생쥐 (mouse), 쥐 (rat), 개, 고양이, 말, 소 등의 포유류를 의미한다.As used herein, the term "subject" refers to a subject requiring a method for preventing, controlling, or treating a disease, and may be used without limitation, such as humans, dogs, monkeys, cats, rodents, such as mice and genetically engineered mice. . More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.
본 발명의 약학 조성물은 치료학적으로 유효한 양 또는 약학적으로 유효한 양으로 투여될 수 있다.The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount or a pharmaceutically effective amount.
본 발명에서 사용되는 용어 "치료학적으로 유효한 양"은 대상 질환을 예방 또는 치료하는데 유효한 조성물의 약학적으로 허용가능한 염의 양을 의미하며, 본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.에 기술되어있다.As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutically acceptable salt of a composition effective for preventing or treating a target disease, and a therapeutically effective amount of the composition of the present invention may include several factors, such as For example, it may vary depending on the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in an appropriate amount considering both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are discussed, 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.
본 발명에서 사용되는 용어 “약학적으로 유효한 양”은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.As used herein, the term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's health The condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
일 측면에서, 본 발명은 두충(Eucommia ulmoides) 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 개선용 화장료 조성물에 관한 것이다.In one aspect, the present invention relates to a cosmetic composition for preventing or improving brain diseases caused by ultrafine dust comprising a leaf fraction of Eucommia ulmoides as an active ingredient.
본 발명의 “화장료 조성물”은 상술한 본 발명의 두충 잎에서 추출한 추출물의 화장품학적 유효량 (cosmetically effective amount) 및 화장품학적으로 허용되는 담체를 포함하여 제조할 수 있다.The "cosmetic composition" of the present invention may be prepared by including a cosmetically effective amount of the extract extracted from the leaves of Eucommia of the present invention and a cosmetically acceptable carrier.
본 명세서에서 용어 “화장품학적 유효량”은 상술한 본 발명의 조성물의 표피 각질형성세포의 증식을 통한 피부 재생 효능을 달성하는 데 충분한 양을 의미한다.As used herein, the term "cosmetically effective amount" means an amount sufficient to achieve the skin regeneration effect through the proliferation of epidermal keratinocytes of the composition of the present invention described above.
화장료 조성물의 외형은 화장품학 또는 피부과학적으로 허용 가능한 매질 또는 기제를 함유한다. 이는 국소적용에 적합한 모든 제형으로, 예를 들면, 용액, 겔, 고체, 반죽 무수 생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는, 이온형 (리포좀) 및 비이온형의 소낭 분산제의 형태로, 또는 크림, 스킨, 로션, 파우더, 연고, 스프레이 또는 콘실 스틱의 형태로 제공될 수 있다. 이들 조성물은 당해 분야의 통상적인 방법에 따라 제조될 수 있다. 본 발명에 따른 조성물은 또한 포말 (foam)의 형태로 또는 압축된 추진제를 더 함유한 에어로졸 조성물의 형태로도 사용될 수 있다.The appearance of the cosmetic composition contains a cosmetic or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example solutions, gels, solids, pasty anhydrous products, emulsions obtained by dispersing an oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and It may be provided in the form of a non-ionic follicular dispersant, or in the form of a cream, toner, lotion, powder, ointment, spray or conceal stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.
본 발명의 일 실시예에 따른 상기 화장료 조성물은 그 제형에 있어서 특별히 한정되는 바가 없으며, 예를 들면, 유연화장수, 수렴화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 아이크림, 아이에센스, 클렌징크림, 클렌징폼, 클렌징워터, 팩, 파우더, 바디로션, 바디크림, 바디오일 및 바디에센스 등의 화장품으로 제형화될 수 있다.The cosmetic composition according to an embodiment of the present invention is not particularly limited in its dosage form, for example, softening lotion, astringent lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, eye essence, cleansing It can be formulated into cosmetics such as cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil and body essence.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fibers, vegetable fibers, wax, paraffin, starch, tracanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. this can be used
본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluorohydro propellants such as carbon, propane/butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액의 경우에는 담체 성분으로서 용매, 용매화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.
본 발명의 화장료 조성물의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 리놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate , fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives, or ethoxylated glycerol fatty acid esters.
본 발명의 화장료 조성물은 스킨, 로션, 크림, 에센스, 팩, 파운데이션, 색조화장품, 선크림, 투웨이케이크, 페이스파우더, 콤팩트, 메이크업베이스, 스킨커버, 아이쉐도우, 립스틱, 립글로스, 립픽스, 아이브로우 펜슬, 화장수 등의 화장품 및 샴푸, 비누 등의 세정제에 적용될 수 있다.The cosmetic composition of the present invention can be applied to skin, lotion, cream, essence, pack, foundation, color cosmetics, sunscreen, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, lip gloss, lip fix, eyebrow pencil , It can be applied to cosmetics such as lotion and detergents such as shampoo and soap.
본 발명의 일 실시예에 따른 화장료 조성물에는 상기 두충 잎에서 추출한 추출물 이외에 기능성 첨가물 및 일반적인 화장료 조성물에 포함되는 성분이 추가로 포함될 수 있다. 상기 기능성 첨가물로는 수용성 비타민, 유용성 비타민, 고분자 펩티드, 고분자 다당, 스핑고 지질 및 해초 엑기스로 이루어진 군에서 선택된 성분을 포함할 수 있다.The cosmetic composition according to an embodiment of the present invention may further include functional additives and components included in general cosmetic compositions in addition to the extract extracted from the leaves of Eucommia. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extracts.
본 발명의 화장료 조성물에는 또한, 상기 기능성 첨가물과 더불어 필요에 따라 일반적인 화장료 조성물에 포함되는 성분을 배합해도 된다. 이외에 포함되는 배합 성분으로서는 유지 성분, 보습제, 에몰리엔트제, 계면 활성제, 유기 및 무기 안료, 유기 분체, 자외선 흡수제, 방부제, 살균제, 산화 방지제, 식물 추출물, pH 조정제, 알콜, 색소, 향료, 혈행 촉진제, 냉감제, 제한 (制汗)제, 정제수 등을 들 수 있다.In addition to the above functional additives, the cosmetic composition of the present invention may further contain components included in general cosmetic compositions as needed. Ingredients other than those included include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation accelerators, cooling agents, antiperspirants, purified water and the like.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.
[준비예][Preparation example]
준비예 1. 두충 잎 분획물의 제조Preparation Example 1. Preparation of Eucommia leaf fraction
본 실험에 사용된 두충 잎은 경상북도 영천시에 위치한 농가로부터 2019년 4월에 40℃에서 열풍 건조된 두충 잎을 구입하여 사용하였다. 파우더 형태로 마쇄된 두충 잎 20 g은 사전 연구를 통해 총 페놀의 함량이 가장 우수하였던 40% 에탄올 용매 1 L에 혼합되어 40℃에서 2시간 동안 환류 냉각 추출하였다. 이 후, 여과지를 이용해 여과한 후 얻어진 추출물은 동일한 양의 n-헥세인, 클로로폼, 에틸아세테이트를 사용하여 분획을 실시하였다. 얻어진 에틸아세테이트 분획물은 회전진공농축기(N-N series, Eyela Co., Tokyo, Japan)를 이용하여 농축하고 동결건조하여 본 실험에 사용하였다.Eucommia leaves used in this experiment were purchased and used in April 2019 from a farmhouse located in Yeongcheon-si, Gyeongsangbuk-do and dried in hot air at 40 ° C. 20 g of powdered Eucommia leaves were mixed with 1 L of 40% ethanol solvent, which had the highest total phenol content through previous studies, and extracted by reflux cooling at 40 ° C. for 2 hours. Thereafter, the extract obtained after filtration using filter paper was fractionated using the same amount of n -hexane, chloroform, and ethyl acetate. The obtained ethyl acetate fraction was concentrated using a rotary vacuum concentrator (NN series, Eyela Co., Tokyo, Japan), and lyophilized to use in this experiment.
준비예 2. 실험에 사용된 세포 배양Preparation Example 2. Cell culture used in experiments
본 실험에 사용된 뇌 신경세포(MC-IXC)는 ATCC로부터 구입하여 10% FBS 및 1% penicillin (50 units/mL)/streptomycin (100 μg/mL)이 포함된 minimum essential medium (MEM) 배지를 사용하였다. 미세아교세포(BV-2)는 한국식품연구원으로부터 분양받아 10% fetal bovine serum (FBS) 및 1% penicillin (50 units/mL)/streptomycin (100 μg/mL)이 포함된 DMEM 배지를 배양액으로 사용하였고, 모든 세포는 37℃, 5% CO2 조건의 incubator에서 배양하였다.The brain neurons (MC-IXC) used in this experiment were purchased from ATCC and maintained in minimum essential medium (MEM) medium containing 10% FBS and 1% penicillin (50 units/mL)/streptomycin (100 μg/mL). used Microglial cells (BV-2) were purchased from the Korea Food Research Institute and used DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin (50 units/mL)/streptomycin (100 μg/mL) as a culture medium. All cells were cultured in an incubator at 37°C and 5% CO 2 conditions.
[실시예][Example]
실시예 1. 두충 잎 분획물의 총 페놀성 화합물 및 총 플라보노이드 함량 측정Example 1. Determination of total phenolic compounds and total flavonoid content in Eucommia leaf fractions
1-1. 실험 방법1-1. Experiment method
총 페놀성 화합물 함량을 측정하기 위하여 시료에 Folin & Ciocalteau’s phenol reagent를 혼합하여 실온에서 5분간 방치한 후 7% Na2CO3를 혼합하였다. 이후 3차 증류수를 이용해 정용한 후, 상온에서 2시간 동안 반응시켜 760 nm에서 흡광도(UV-1800, Shimadzu, Kyoto, Japan)를 측정하였다. 측정된 흡광도는 갈릭산을 이용해 작성된 검량 곡선으로 총 페놀성 화합물의 함량을 나타냈다.In order to measure the total phenolic compound content, the sample was mixed with Folin &Ciocalteau's phenol reagent, left at room temperature for 5 minutes, and then mixed with 7% Na 2 CO 3 . Then, after constant use with tertiary distilled water, the mixture was reacted at room temperature for 2 hours and the absorbance at 760 nm (UV-1800, Shimadzu, Kyoto, Japan) was measured. The measured absorbance indicated the content of total phenolic compounds in a calibration curve prepared using gallic acid.
또한, 총 플라보노이드 함량 측정은 두충 잎 추출물 1 mL에 다이에틸렌글리콜과 수산화나트륨을 혼합한 후, 30℃에서 60분 동안 반응시켰다. 최종반응물은 420 nm에서 흡광도를 측정하였고, 루틴을 사용하여 작성된 보정선에 대입하여 총 플라보노이드 함량을 계산하였고, 이를 루틴 당량(mg RE (rutin equivalent)/g of dried weight)으로 나타냈다.In addition, the total flavonoid content was measured by mixing diethylene glycol and sodium hydroxide in 1 mL of Eucommia leaf extract, followed by reaction at 30 ° C. for 60 minutes. The absorbance of the final reactant was measured at 420 nm, and the total flavonoid content was calculated by substituting it into a calibration curve prepared using rutin, which was expressed as rutin equivalent (mg RE (rutin equivalent)/g of dried weight).
1-2. 실험 결과1-2. Experiment result
두충 잎 40% 에탄올 추출물을 이용해 극성에 따라 n-헥세인, 클로로폼, 에틸아세테이트 용매로 순차적으로 분획을 실시하였으며, 얻어진 4개의 분획물 층을 이용해 총 페놀성 화합물 및 총 플라보노이드 함량을 측정하였다(도 1). n-헥세인, 클로로폼, 에틸아세테이트 및 증류수 층에서 총 페놀의 함량은 각각 18.25, 148.08, 353.75, 55.58 mg of GAE/g으로 나타났고, 총 플라보노이드 함량은 각각 9.74, 37.44, 463.46, 31.67 mg of RE/g으로 나타냈다.Using a 40% ethanol extract from Eucommia leaves, fractionation was performed sequentially with n-hexane, chloroform, and ethyl acetate solvents according to polarity, and the total phenolic compound and total flavonoid contents were measured using the obtained four fraction layers (Fig. One). In the n-hexane, chloroform, ethyl acetate and distilled water layers, the total phenol content was 18.25, 148.08, 353.75, and 55.58 mg of GAE/g, respectively, and the total flavonoid content was 9.74, 37.44, 463.46, and 31.67 mg of GAE/g, respectively. expressed as RE/g.
실시예 2. 뇌 조직에서의 라디칼 소거 활성 및 지방질과산화물 생성 억제 효과Example 2. Radical scavenging activity and inhibitory effect on lipid peroxide production in brain tissue
2-1. 실험 방법2-1. Experiment method
ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 라디칼 소거 활성은 150 mM 염화소듐을 포함한 100 mM 인산 완충용액 (pH 7.4)에 2.45 mM 과황산칼륨(potassium persulfate) 및 7 mM ABTS 시약을 혼합하여 68℃의 항온 수조에서 15분간 방치한 후, 수용성 필터를 이용해 여과하여 24시간 동안 냉장 보관하여, ABTS 라디칼을 생성시켜 사용하였다.ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity was tested by adding 2.45 mM potassium persulfate and 100 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride. After mixing 7 mM ABTS reagent and leaving it in a constant temperature water bath at 68° C. for 15 minutes, it was filtered using a water-soluble filter and stored in a refrigerator for 24 hours to generate ABTS radicals and used.
ABTS 라디칼 용액은 734 nm에서 흡광도 값 (0.70±0.02)을 조정하였고, ABTS 용액과 시료를 혼합하여 37℃에서 10분 동안 반응시켰다. 반응물은 분광광도계를 이용하여 734 nm에서 흡광도를 측정하여 라디칼 소거 활성을 계산하였다.The ABTS radical solution adjusted the absorbance value (0.70±0.02) at 734 nm, and the ABTS solution and the sample were mixed and reacted at 37° C. for 10 minutes. Radical scavenging activity was calculated by measuring absorbance at 734 nm using a spectrophotometer.
DPPH 라디칼 소거 활성은 80% 메탄올에 녹인 0.1 mM DPPH 용액을 517 nm에서 흡광도 값이 1.00±0.02가 되도록 조정하였고, DPPH 용액과 시료를 혼합하여 실온과 암실 조건에서 30분간 반응시켰다. 최종반응물은 분광광도계를 이용하여 흡광도를 측정하였다.DPPH radical scavenging activity was adjusted so that the absorbance value at 517 nm of 0.1 mM DPPH solution dissolved in 80% methanol was 1.00 ± 0.02, and the DPPH solution and the sample were mixed and reacted for 30 minutes at room temperature and in the dark. The absorbance of the final reactant was measured using a spectrophotometer.
마우스 뇌 조직을 이용한 지방질과산화물 (Malondialdehyde, MDA) 생성 억제 효과는 ICR (institute of cancer research) 마우스(male, 4 weeks)를 실험동물 공급업체 (Samtako, Osan, Korea)로부터 구입 후, 뇌를 적출하여 실험에 사용하였다. 본 동물실험은 경상대학교 동물윤리심의위원회 승인 후 진행하였다 (경상대학교 동물실험 인가번호: GNU-120831-M0067).The inhibitory effect of lipid peroxide (Malondialdehyde, MDA) production using mouse brain tissue was investigated by purchasing ICR (institute of cancer research) mice (male, 4 weeks) from an experimental animal supplier (Samtako, Osan, Korea), and extracting the brains. used in the experiment. This animal experiment was conducted after the approval of the Animal Ethics Review Committee of Gyeongsang National University (Gyeongsang National University Animal Experiment License Number: GNU-120831-M0067).
뇌 조직 무게 10배의 20 mM 트리스 염산 완충용액 (pH 7.4)를 첨가하여 조직파쇄기 (Next Advance Inc., NY, USA)를 사용하여 균질화하였다. 균질화 된 뇌 조직은 원심분리(5,000 x g, 20분, 4°C)하였고, 그 상층액을 실험에 사용하였다. 추출물, 상층액, 10 μM 황산철(Ⅱ), 0.1 mM 아스코브산을 혼합하여 37°C에서 1시간 동안 배양하였다. 배양 후, 30% 트라이클로로아세트산과 1% 싸이오바비투르산을 첨가하여 80℃의 항온 수조에서 20분 동안 가열하여 상층액을 532 nm에서 흡광도를 측정하였다.20 mM Tris HCl buffer solution (pH 7.4) 10 times the weight of the brain tissue was added and homogenized using a tissue disruptor (Next Advance Inc., NY, USA). The homogenized brain tissue was centrifuged (5,000 x g , 20 min, 4 °C), and the supernatant was used in the experiment. The extract, supernatant, 10 µM iron (II) sulfate, and 0.1 mM ascorbic acid were mixed and incubated at 37 °C for 1 hour. After incubation, 30% trichloroacetic acid and 1% thiobarbituric acid were added and heated in a constant temperature water bath at 80° C. for 20 minutes, and the supernatant was measured for absorbance at 532 nm.
2-2. 실험 결과2-2. Experiment result
두충 잎 분획물의 ABTS 및 DPPH 라디칼 소거 활성을 측정한 결과는 도 2와 같다. The results of measuring the ABTS and DPPH radical scavenging activities of Eucommia leaf fractions are shown in FIG. 2 .
양성대조군으로 사용된 비타민 C와 비교할 때 500 μg/mL 농도의 두충 잎 분획물은 200 μg/mL 농도의 vitamin C와 유사한 라디칼 소거능을 나타냈다. ABTS와 DPPH 라디칼 소거능에서 vitamic C의 IC50 값은 각각 111.75 μg/mL와 117.91 μg/mL으로 나타났고, 두충 잎 분획물의 IC50 값은 각각 212.80 μg/mL와 359.13 μg/mL으로 각각 나타났다. 또한 두충 잎 분획물의 지질과산화물 억제 활성을 측정한 결과는 100 μg/mL 농도의 두충 잎 분획물과 catechin의 지질과산화물 저해 활성은 각각 87.40%와 85.07%로 나타났고, catechin과 두충 잎 분획물의 IC50값을 비교하였을 때, 각각 25.29 μg/mL, 33.18 μg/mL로 나타났다. Compared to vitamin C used as a positive control, Eucommia leaf fraction at a concentration of 500 μg/mL showed radical scavenging activity similar to that of vitamin C at a concentration of 200 μg/mL. In terms of ABTS and DPPH radical scavenging activity, the IC 50 values of vitamin C were 111.75 μg/mL and 117.91 μg/mL, respectively, and the IC 50 values of the Eucommia leaf fraction were 212.80 μg/mL and 359.13 μg/mL, respectively. In addition, as a result of measuring the lipid peroxide inhibitory activity of Eucommia leaf fractions, the lipid peroxide inhibitory activities of Eucommia leaf fraction at 100 μg/mL concentration and catechin were 87.40% and 85.07%, respectively, and the IC 50 values of catechin and Eucommia leaf fractions When compared, they were 25.29 μg/mL and 33.18 μg/mL, respectively.
이러한 결과들을 고려할 때, 두충 잎 분획물의 마우스 뇌 조직에서 나타나는 우수한 항산화 활성은 실시예 1의 총 페놀성 화합물의 함량과의 상관관계에서 기인된 것이며, 산화적 스트레스로 인하여 발생되는 세포의 기능적 손상을 감소시켜 보호하는데 도움이 될 것으로 판단된다.Considering these results, the excellent antioxidant activity in mouse brain tissue of Eucommia leaf fraction is due to the correlation with the content of total phenolic compounds in Example 1, and the functional damage of cells caused by oxidative stress It is believed to help reduce and protect
실시예 3. 두충 잎 분획물의 뇌 신경세포에서의 초미세먼지 유도성 산화적 스트레스의 세포 내 생성 억제 효과Example 3. Inhibition of intracellular production of ultrafine dust-induced oxidative stress in brain nerve cells of Eucommia leaf fraction
3-1. 세포 내 산화적 스트레스 측정 방법3-1. Methods for measuring oxidative stress in cells
세포 내 산화적 스트레스 측정을 위해 MC-IXC 세포는 1×104 cells/well의 농도로 96 well plate에 일정하게 분주하였으며, BV-2 세포는 0.5×104 cells/well 농도로 분주하여, 24시간 동안 incubator에서 배양하여 세포를 부착시켰다. To measure intracellular oxidative stress, MC-IXC cells were regularly dispensed into a 96 well plate at a concentration of 1×10 4 cells/well, and BV-2 cells were dispensed at a concentration of 0.5×10 4 cells/well, and 24 Cells were attached by culturing in an incubator for a period of time.
이후 농도별 시료를 각 well에 처리하고 30분 뒤 MC-IXC 세포는 100 μg/mL 농도의 PM2.5를 처리하였고 BV-2 세포는 50 μg/mL 농도의 PM2.5를 처리하였다.Afterwards, samples for each concentration were treated in each well, and after 30 minutes, MC-IXC cells were treated with 100 μg/mL PM 2.5 , and BV-2 cells were treated with 50 μg/mL PM 2.5 .
이후 MC-IXC 세포는 24시간, BV-2 세포는 12시간 반응시킨 후 50 μM DCF-DA를 처리하여 40분간 반응시켰다. 이는 형광 광도계(Infinite F200, Tecan, Mannedorf, Swiss)를 사용하여 excitation wave 485 nm 및 emission wave 535 nm에서 형광강도를 측정하였다.Thereafter, MC-IXC cells were reacted for 24 hours and BV-2 cells were reacted for 12 hours, and then treated with 50 μM DCF-DA and reacted for 40 minutes. Fluorescence intensity was measured at excitation wave 485 nm and emission wave 535 nm using a fluorescence photometer (Infinite F200, Tecan, Mannedorf, Swiss).
3-2. 실험 결과3-2. Experiment result
PM2.5로 인한 MC-IXC, BV-2 세포에 대한 ROS 생성과 이에 대한 두충 잎 분획물의 세포 보호 효과를 확인한 결과는 도 3과 같다. The results of confirming the generation of ROS on MC-IXC and BV-2 cells caused by PM 2.5 and the cytoprotective effect of the leaf fractions on this are shown in FIG. 3 .
MC-IXC, BV-2세포에 PM2.5를 처리하였을 때, ROS는 각각 646.99%, 341.71%로 증가하였으나, 두충 잎 분획물 10 μg/mL 처리하였을 때, 각각 75.27%, 93.03%로 감소하여 정상 대조군과 유사하거나 더 낮은 ROS의 수준을 나타내었다.When MC-IXC and BV-2 cells were treated with PM 2.5 , ROS increased to 646.99% and 341.71%, respectively, but decreased to 75.27% and 93.03%, respectively, when treated with 10 μg/mL of Eucommia leaf fraction, respectively, in the normal control group. showed similar or lower ROS levels.
이러한 결과를 바탕으로 보았을 때, 두충 잎 분획물은 초미세먼지로 유도된 세포 내 ROS의 과도한 생성을 억제하여 세포보호 효과를 나타내는 것으로 판단된다.Based on these results, it is judged that Eucommia leaf fraction exhibits a cytoprotective effect by inhibiting the excessive production of intracellular ROS induced by ultrafine dust.
실시예 4. 두충 잎 분획물의 뇌 신경세포에서의 초미세먼지 유도성 세포사멸 억제 효과Example 4. Effect of ultrafine dust-induced apoptosis inhibition in brain nerve cells of Eucommia leaf fraction
4-1. 세포 생존율 측정 방법4-1. How to measure cell viability
세포 생존율 측정을 위해 전처리는 세포 내 ROS 함량 측정과 동일한 조건으로 세포를 분주하였고, 시료와 미세먼지도 동일한 조건으로 처리하였다.To measure cell viability, cells were pretreated under the same conditions as those for measuring intracellular ROS content, and samples and fine dust were also treated under the same conditions.
이후 MC-IXC 세포는 24시간, BV-2 세포는 12시간 반응시킨 후 배지에 처리된 배양액을 모두 제거하였으며, 이후 다이메틸 일산화항 (dimethyl sulfoxide; DMSO)를 100 μL 처리하여 상온에서 반응시킨 뒤 570 nm (determination wave)와 690 nm (reference wave)에서 흡광도를 측정하였다.Then, MC-IXC cells were reacted for 24 hours and BV-2 cells were reacted for 12 hours, and then all the culture medium treated with the medium was removed. Absorbance was measured at 570 nm (determination wave) and 690 nm (reference wave).
4-2. 실험 결과4-2. Experiment result
초미세먼지는 세포 내에 산화적 스트레스를 증가시키고 염증 반응을 촉진 시킴으로써 세포의 기능 저하 및 사멸을 유도하는 것으로 알려져 있으며 (Guo 등, 2015), 두충 잎 분획물의 뇌 신경세포에서의 초미세먼지 유도성 세포사멸 억제 효과를 측정한 결과는 도 4와 같다.Ultrafine dust is known to induce cell function deterioration and death by increasing oxidative stress within cells and promoting inflammatory responses (Guo et al., 2015), and ultrafine dust induction in brain nerve cells of Eucommia leaf fraction The results of measuring the apoptosis inhibitory effect are shown in FIG. 4 .
도 4를 살펴보면 모든 세포에서 PM2.5의 처리는 세포의 사멸을 유도하는 것으로 확인되었고, MC-IXC, BV-2세포에 PM2.5를 처리하였을 때 세포 생존율은 각각 63.73%, 38.21%이며, 두충 잎 분획물 50 μg/mL 처리하였을 때, 각각 97.80%, 59.46%로 PM2.5로 인한 세포사멸로부터 세포를 보호하는 것으로 나타났다. Looking at Figure 4, it was confirmed that the treatment of PM 2.5 in all cells induces cell death, and when MC-IXC and BV-2 cells were treated with PM 2.5 , the cell viability was 63.73% and 38.21%, respectively, and the leaves of Eurymia When the fraction was treated with 50 μg/mL, 97.80% and 59.46%, respectively, were shown to protect cells from apoptosis caused by PM 2.5 .
또한 두충 잎 분획물 10 μg/mL 처리군에서는 양성 대조군와 유사하거나 더 높은 세포 생존률을 나타내었다. 따라서 PM2.5로 유도되는 산화적 스트레스로부터 세포보호 효과를 나타내는 것으로 확인된다.In addition, the group treated with 10 μg/mL of Eucommia leaf fraction showed a cell survival rate similar to or higher than that of the positive control group. Therefore, it is confirmed that it exhibits a cytoprotective effect from oxidative stress induced by PM 2.5 .
초미세먼지의 노출에 의한 세포 기능 저하는 다양한 메커니즘에 의한 것으로 그 중심에는 염증 반응의 활성화와 관련된 것으로 보고되고 있다. 초미세먼지는 세포막에서 직접적으로 반응하는데, lysosomal leakage 및 세포 표면 수용체인 toll-like 수용체와의 결합은 NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome의 형성을 유도한다. 이는 다시 IL-1β의 방출을 유도하게 되고 nuclear factor-κB (NF-κB) 및 mitogen-activated protein kinase (MAPK) 경로를 활성화시킴으로써 세포 내 ROS의 생성 유도 및 pro-inflammatory cytokine (IL-1 family, TNF-α 및 IL-18) 분비를 촉진하게 된다. 뿐만 아니라, 초미세먼지는 세포 표면에서 직접적으로 반응할 수 있으며, PAH 및 quinone과 같은 물질들은 산화-환원 관련 대사의 활성화를 통하여 ROS 및 반응성이 큰 친전자성 대사산물(O2
·-, H2O2, O2
·-및 ·OH)들을 생성함으로써 미토콘드리아의 손상을 유발하고, 세포의 기능 저하를 초래하게 된다(Guo 등, 2015). It has been reported that cell function deterioration due to exposure to ultrafine dust is caused by various mechanisms, at the center of which is related to the activation of an inflammatory response. Ultrafine dust reacts directly on the cell membrane, and lysosomal leakage and binding to toll-like receptors, which are cell surface receptors, induce the formation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasomes. This in turn induces the release of IL-1β and activates the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, thereby inducing the production of intracellular ROS and pro-inflammatory cytokines (IL-1 family, TNF-α and IL-18) secretion is promoted. In addition, ultrafine dust can directly react on the cell surface, and substances such as PAH and quinone can activate ROS and highly reactive electrophilic metabolites (O 2 - , H through oxidation-reduction related metabolism activation). 2 O 2 , O 2 ·- and · OH) causes damage to mitochondria and leads to cell function deterioration (Guo et al., 2015).
본 실험의 결과에서 나타난 두충 잎 추출물의 뇌 세포 내 ROS 생성 억제 효과 및 세포사멸 억제 효과는 두충에 함유되어 있는 다양한 생리 활성 물질에 의한 것으로 사료된다. 이러한 결과들을 종합해 볼 때, 두충 잎 추출물은 뇌 신경세포에서 초미세먼지로 유도될 수 있는 세포 손상으로부터 기능 개선 및 치료에 도움을 줄 수 있는 건강기능성식품 조성물, 식품 조성물, 약학 조성물 또는 화장료 조성물로서의 활용 가능성이 기대된다.It is believed that the inhibitory effect on ROS production and apoptosis in brain cells of Eucommia leaf extract shown in the results of this experiment is due to various physiologically active substances contained in Eucommia. Taken together these results, Eucommia leaf extract is a health functional food composition, food composition, pharmaceutical composition or cosmetic composition that can help improve function and treat brain nerve cells from cell damage that can be induced by ultrafine dust. Its potential for use is expected.
실시예 5. 뇌질환 개선효과를 보기위한 동물행동분석Example 5. Animal behavior analysis to see the effect of improving brain disease
5-1. 교대 행동 능력 평가 실험 (Y-maze)5-1. Alternate Behavioral Ability Evaluation Experiment (Y-maze)
교대 행동 능력을 실험하기 위하여 Y-maze를 실시하였고, Y모양으로 이루어진 검은색 플라스틱으로 제작한 Y-maze를 실험에 사용하였다. 각 arm을 각각 A와 B, C로 정한 후, 8분 동안 마우스가 움직인 경로를 smart 3.0 video tracking system (Panlab, Barcelona, Spain)으로 기록하였다. 마우스는 Y 모양으로 만들어진 미로에서 8분 동안 자유롭게 움직이며, 마우스가 3개의 팔에 순차적으로 교대하여 움직이는 것을 옳은 행동으로 평가한다.A Y-maze was conducted to test the alternating behavioral ability, and a Y-maze made of black plastic made of Y-shape was used for the experiment. After designating each arm as A, B, and C, the path the mouse moved for 8 minutes was recorded with a smart 3.0 video tracking system (Panlab, Barcelona, Spain). The mouse moves freely in the Y-shaped maze for 8 minutes, and the correct behavior is evaluated when the mouse sequentially moves alternately on the three arms.
PM2.5 노출로 인한 마우스의 자발적인 교대 행동 능력을 평가한 결과는 도 5에 나타내었다. 도 5A는 마우스가 8분 동안 Y-maze를 움직인 총 거리를 나타내었으며 모든 그룹에서 마우스의 행동 능력이 동일함에 따라 마우스의 행동 장애가 없음을 의미한다. 도 5B는 마우스 교대 행동을 나타내었으며, 그 결과 PM2.5그룹에서 22.56%로 낮은 교대 행동 수준을 나타내었으나 두충 잎 분획물 20과 40그룹에서 자발적 교대 행동의 능력이 33.56%와 29.41%로 증가하는 것을 확인하였는바 효과적으로 교대 행동 능력을 개선시키는 것을 확인하였다.The results of evaluating the spontaneous alternating behavioral ability of mice due to PM 2.5 exposure are shown in FIG. 5 . Figure 5A shows the total distance that the mice moved the Y-maze for 8 minutes, and the behavioral abilities of the mice were the same in all groups, meaning that there was no behavioral disorder of the mice. Figure 5B shows the mouse alternation behavior, and as a result, it was confirmed that the PM 2.5 group showed a low alternation behavior level of 22.56%, but the ability of voluntary alternation behavior increased to 33.56% and 29.41% in the 20 and 40 groups of Eucommia leaf fractions. As a result, it was confirmed that the shift behavior ability was effectively improved.
5-2. 장기 학습 및 기억능력 평가 실험 (Morris water maze)5-2. Long-term learning and memory test (Morris water maze)
장기 학습 및 기억능력을 평가하기 위하여 Morris water maze 실험을 실시하였고, 실험을 진행하는 동안 마우스는 4분면으로 나누어진 수조에서 1분 동안 자유롭게 수영하도록 하였다. 수조는 동, 서, 남, 북으로 나누어져 있고 한 쪽 분면에는 플랫폼을 위치시켰다. 따라서 Morris water maze test는 플랫폼을 보이게 하여 진행하는 visible test (Day 1)와 플랫폼을 보이지 않게 진행하는 hidden test (Day 2~5), 마지막으로 플랫폼을 완전히 제거하여 진행하는 probe test (Day 6)으로 진행하였다. Hidden test에는 마우스가 숨겨진 플랫폼을 찾아 탈출하기까지의 시간을 측정하였고, probe test에서는 마우스가 플랫폼이 위치 해있던 자리에 머무는 시간을 측정하였다. A Morris water maze experiment was conducted to evaluate long-term learning and memory abilities, and mice were allowed to swim freely for 1 minute in a water tank divided into four quadrants during the experiment. The water tank is divided into east, west, south, and north, and a platform is placed on one side. Therefore, the Morris water maze test consists of a visible test (Day 1) in which the platform is visible, a hidden test (Day 2-5) in which the platform is invisible, and a probe test (Day 6) in which the platform is completely removed. proceeded. In the hidden test, the time for the mouse to find the hidden platform and escape was measured, and in the probe test, the time the mouse stayed at the place where the platform was located was measured.
PM2.5 노출로 인한 마우스의 장기 기억 능력을 평가한 결과는 도 6에 나타내었다. 도 6A는 Morris water maze test의 visible test와 hidden test 동안 마우스가 플랫폼으로 탈출하기까지 걸리는 시간을 측정한 결과로 PM2.5그룹에서는 탈출 시간의 변화가 나타나지 않았으나 두충 잎 분획물 20과 40그룹에서는 실험이 진행될수록 탈출 시간이 짧아지는 것을 확인하였다. 도 6B는 마지막 probe test에서 플랫폼이 존재하던 W zone에 마우스가 머무르는 시간을 측정한 결과 PM2.5그룹에서 29.11%로 낮은 수준을 나타내었고, 두충 잎 분획물 20과 40그룹에서 각각 55.85%, 75.16%로 더욱 증가된 수준을 나타내었다.The results of evaluating the long-term memory ability of mice due to PM 2.5 exposure are shown in FIG. 6 . Figure 6A is the result of measuring the time taken for the mouse to escape to the platform during the visible test and hidden test of the Morris water maze test. As a result, no change in escape time was observed in the PM 2.5 group, but the experiment was conducted in the 20 and 40 groups of the leaf fractions. It was confirmed that the escape time was shortened as much as possible. 6B shows the result of measuring the time for mice to stay in the W zone where the platform was present in the last probe test, and showed a low level of 29.11% in the PM 2.5 group, and 55.85% and 75.16% in the 20 and 40 groups of Eucommia leaf fractions, respectively. showed higher levels.
실시예 6. 뇌 조직 내 항산화 시스템 개선 효과Example 6. Antioxidant system improvement effect in brain tissue
6-1. Superoxide dismutase (SOD) 활성 측정6-1. Superoxide dismutase (SOD) activity measurement
마우스로부터 적출된 뇌 조직은 phosphate buffer saline (PBS)를 이용하여 균질화하였고, 얻어진 균질액은 400 × g에서 5분 동안 원심분리하여 펠렛을 얻었다. 얻어진 펠렛은 1×cell extraction buffer (10×SOD buffer 1.0 mL, 20% triton X-100 0.2 mL, distilled water 8.8 mL, phenylmethanesulfonyl fluoride in ethanol 10 μL)를 이용하여 추출되었다. 이는 14,000 × g에서 5분 동안 원심분리되어 상등액을 얻었으며, 얻어진 상등액의 SOD 함량은 SOD assay kit (Sigma-Aldrich Chemical Co.)를 사용하여 측정하였으며 microplate reader (Epoch2, BioTek)를 통해 450 nm에서 흡광도를 측정하였다.Brain tissue excised from the mouse was homogenized using phosphate buffered saline (PBS), and the obtained homogenate was centrifuged at 400 × g for 5 minutes to obtain a pellet. The obtained pellet was extracted using 1×cell extraction buffer (10×SOD buffer 1.0 mL, 20% triton X-100 0.2 mL, distilled water 8.8 mL, phenylmethanesulfonyl fluoride in ethanol 10 μL). It was centrifuged at 14,000 × g for 5 minutes to obtain a supernatant, and the SOD content of the obtained supernatant was measured using a SOD assay kit (Sigma-Aldrich Chemical Co.) and was measured at 450 nm through a microplate reader (Epoch2, BioTek). Absorbance was measured.
PM2.5 노출로 유도된 마우스 모델의 뇌 조직에서 SOD 함량을 나타낸 결과는 도 7과 같다. PM2.5가 노출된 마우스의 뇌 조직에서 SOD 함량은 3.50 unit/mg of protein으로 나타났고, 두충 잎 분획물 40그룹에서는 각각 4.46 unit/mg of protein으로 나타났다. 따라서 두충 잎 분획물의 섭취는 PM2.5의 노출로 인한 SOD의 수준 감소를 개선 시킬 수 있음을 확인하였다.The results showing the SOD content in the brain tissue of the mouse model induced by PM 2.5 exposure are shown in FIG. 7 . The SOD content in brain tissue of mice exposed to PM 2.5 was 3.50 units/mg of protein, and 4.46 units/mg of protein in 40 groups of Eucommia leaf fractions, respectively. Therefore, it was confirmed that intake of Eucommia leaf fraction could improve the reduction of SOD level caused by exposure to PM 2.5 .
6-2. 환원형 글루타치온(reduced glutathione, reduced GSH) 활성 측정6-2. Measurement of reduced glutathione (reduced GSH) activity
마우스로부터 적출된 뇌 조직은 10 mM sodium phosphate buffer with 1 mM EDTA (pH 6.0)을 이용하여 균질화하였고, 균질액은 10,000 × g에서 15분 동안 원심분리하였다. 얻어진 상등액으로 단백질 정량을 진행한 후, 상등액과 5% metaphosphoric acid를 1:1로 혼합하여 2,000 × g에서 2분 동안 원심분리하여 얻어진 상등액으로 실험을 진행하였다. 상등액과 0.26 M Tris-HCl (pH 7.5), 0.65 N NaOH 및 OPT in methanol을 넣고 암실에서 15분 반응시킨 뒤, 형광 광도계 (Infinite F200, Tecan)를 사용하여 excitation wave 320 nm 및 emission wave 420 nm에서 측정하였다.Brain tissues extracted from mice were homogenized using 10 mM sodium phosphate buffer with 1 mM EDTA (pH 6.0), and the homogenate was centrifuged at 10,000 × g for 15 minutes. After protein quantification was performed with the obtained supernatant, the supernatant was mixed with 5% metaphosphoric acid at a ratio of 1:1 and centrifuged at 2,000 × g for 2 minutes, and the experiment was conducted with the obtained supernatant. After adding the supernatant, 0.26 M Tris-HCl (pH 7.5), 0.65 N NaOH, and OPT in methanol, and reacting in the dark for 15 minutes, using a fluorescence photometer (Infinite F200, Tecan), excitation wave 320 nm and emission wave 420 nm measured.
PM2.5에 노출된 마우스의 뇌 조직에서 reduced GSH 함량은 도 8과 같다. PM2.5의 만성적인 노출은 뇌 조직에서 reduced GSH는 정상 대조군을 100%로 하였을 때 74.92%로 감소 되었고, 두충 잎 분획물 40그룹에서는 95.89%로 그 함량이 증가 되었다. 따라서 PM2.5의 만성적인 노출로 유도된 마우스의 뇌 조직의 항산화 체계의 붕괴는 두충 잎 분획물을 섭취함으로써 개선되는 것을 확인하였다.The reduced GSH content in brain tissue of mice exposed to PM 2.5 is shown in FIG. 8 . Chronic exposure to PM 2.5 reduced GSH in brain tissue to 74.92% when the normal control group was 100%, and the content increased to 95.89% in the 40 groups of Eucommia leaf fractions. Therefore, it was confirmed that the degradation of the antioxidant system of mouse brain tissue induced by chronic exposure to PM 2.5 was improved by ingesting Eucommia leaf fraction.
6-3. 지질과산화물(malondialdehyde, MDA) 함량 측정6-3. Lipid peroxide (malondialdehyde, MDA) content measurement
마우스로부터 적출된 뇌 조직은 PBS를 이용하여 균질화하였고, 얻어진 균질액을 5,000 rpm에서 10분 동안 원심분리하여 얻어진 상등액을 실험에 사용하였다. 상등액과 1% phosphoric acid 및 0.67% thiobarbituric acid (TBA)를 혼합하여 95℃ 항온 수조에서 1시간 동안 반응시켰다. 반응액은 microplate reader (Epoch2, BioTek)를 통해 532 nm에서 흡광도를 측정하였다.The brain tissue excised from the mouse was homogenized using PBS, and the obtained homogenate was centrifuged at 5,000 rpm for 10 minutes, and the supernatant obtained was used in the experiment. The supernatant was mixed with 1% phosphoric acid and 0.67% thiobarbituric acid (TBA) and reacted in a constant temperature water bath at 95℃ for 1 hour. The absorbance of the reaction solution was measured at 532 nm using a microplate reader (Epoch2, BioTek).
PM2.5를 만성적으로 노출 시킨 마우스의 뇌 조직에서 지질과산화물의 함량을 평가한 결과는 도 9와 같다. 결과를 보았을 때, 뇌 조직에서 PM2.5의 만성적인 노출은 지질과산화물의 함량을 증가시켰고, PM2.5그룹에서 4.32 nmole/mg of protein로 나타났고, 두충 잎 분획물 40그룹에서 3.11 nmole/mg of protein으로 나타났다. 따라서 PM2.5는 마우스의 뇌 조직에서 지질 과산화를 유도하며, 두충 잎 분획물은 PM2.5로부터 지질의 과산화를 억제하는 것을 확인하였다.The result of evaluating the content of lipid peroxide in brain tissue of mice chronically exposed to PM 2.5 is shown in FIG. 9 . Looking at the results, chronic exposure to PM 2.5 in brain tissue increased the lipid peroxide content, which was 4.32 nmole/mg of protein in the PM 2.5 group and 3.11 nmole/mg of protein in the 40 group of Eucommia leaf fractions. appear. Therefore, it was confirmed that PM 2.5 induces lipid peroxidation in mouse brain tissue, and Eucommia leaf fraction inhibits lipid peroxidation from PM 2.5 .
실시예 7. 콜린성 시스템 개선 효과Example 7. Cholinergic system improvement effect
마우스로부터 적출된 뇌 조직은 PBS를 이용하여 균질화하였고, 이는 12,000 rpm에서 30분 동안 원심분리하여 상등액을 얻었다. 얻어진 상등액은 단백질 정량 후 아세틸콜린(acetylcholine : ACh) 함량과 아세틸콜린에스터라제(acetylcholinesterase : AChE) 활성을 측정하는데 사용되었다. ACh의 함량 측정을 위해 상등액은 2 M hydroxylamin·HCl과 3.5 N NaOH를 1:1 비율로 혼합한 혼합액과 2분 동안 반응시켰다. 이후 0.5 N HCl와 0.37 M FeCl2·6H2O in 0.1 N HCl을 첨가하였고 이는 microplate reader (Epoch2, BioTek)를 이용하여 540 nm에서 흡광도 측정하였다. Brain tissue excised from the mouse was homogenized using PBS, which was centrifuged at 12,000 rpm for 30 minutes to obtain the supernatant. The obtained supernatant was used to measure acetylcholine (ACh) content and acetylcholinesterase (AChE) activity after protein quantification. To measure the content of ACh, the supernatant was reacted with a mixture of 2 M hydroxylamin HCl and 3.5 N NaOH in a 1:1 ratio for 2 minutes. Then, 0.5 N HCl and 0.37 M FeCl 2 ·6H 2 O in 0.1 N HCl were added, and the absorbance was measured at 540 nm using a microplate reader (Epoch2, BioTek).
AChE 활성을 측정을 위해 상등액은 50 mM sodium phosphate buffer와 혼합하여 37℃에서 15분 동안 반응시켰다. 이후 acetylthiocholin과 DTNB가 결합된 AChE solution을 첨가하고 37℃에서 5분간 반응시켰으며, 이는 microplate reader (Epoch2, BioTek)를 이용하여 405 nm에서 흡광도 측정하였다.To measure AChE activity, the supernatant was mixed with 50 mM sodium phosphate buffer and reacted at 37°C for 15 minutes. Thereafter, an AChE solution in which acetylthiocholin and DTNB were combined was added and reacted at 37° C. for 5 minutes, and the absorbance was measured at 405 nm using a microplate reader (Epoch2, BioTek).
PM2.5의 만성적인 노출로 마우스의 cholinergic system에 미치는 영향을 평가한 결과는 도 10과 같다. 도 10 (A)와 (B)는 마우스의 뇌 조직에서 ACh의 함량과 AChE의 활성을 평가한 결과로 PM2.5의 노출은 신경전달물질인 ACh의 함량을 1.67 mM/mg of protein으로 감소시켰고, ACh를 분해시키는 AChE의 활성은 정상 대조군을 100%으로 하였을 때 108.52%로 증가시켰다. 이에 두충 잎 분획물 40그룹에서 ACh의 함량은 1.95 mM/mg of protein으로 나타났고, AChE의 활성은 83.35%로 감소 되는 것으로 확인하였다. 또한 western blot assay를 통하여 마우스의 뇌 조직에서 AChE의 발현량을 측정한 결과 역시, PM2.5그룹에서 정상 대조군 대비 1.56배 증가되었고 두충 잎 분획물 40그룹에서는 PM2.5그룹 대비 0.71배 감소함으로써 대조군과 유사한 수준으로 감소되는 것을 확인하였다.The result of evaluating the effect on the cholinergic system of mice by chronic exposure of PM 2.5 is shown in FIG. 10 . 10 (A) and (B) show the results of evaluating the content of ACh and the activity of AChE in mouse brain tissue. Exposure to PM 2.5 reduced the content of ACh, a neurotransmitter, to 1.67 mM/mg of protein, The activity of AChE to degrade ACh was increased to 108.52% when the normal control was 100%. Accordingly, in the 40 groups of Eucommia leaf fractions, the content of ACh was 1.95 mM/mg of protein, and the activity of AChE was confirmed to be reduced to 83.35%. In addition, as a result of measuring the expression level of AChE in the mouse brain tissue through western blot assay, the PM 2.5 group increased by 1.56 times compared to the normal control group, and the Eucommia leaf fraction 40 group decreased by 0.71 times compared to the PM 2.5 group, similar to the control group. was confirmed to decrease.
실시예 8. 미토콘드리아 기능 개선 효과Example 8. Mitochondrial function improvement effect
8-1. 뇌 조직 내 미토콘드리아 분리8-1. Isolation of mitochondria in brain tissue
마우스 뇌 조직의 미토콘드리아 활성을 평가하기 위해 뇌 조직에서 미토콘드리아를 추출하였다. 215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin (BSA), 20 mM HEPES (Na+)로 이루어진 isolation buffer (pH 7.2)와 20 mM EGTA를 혼합된 버퍼를 이용해 뇌 조직은 균질화하였고, 균질액은 1,300 × g에서 5분 동안 원심분리하였다. 이를 통해 얻어진 상등액은 13,000 × g에서 10분 동안 원심분리하였고, 펠렛을 얻었다. 얻어진 펠렛에 20 mM EGTA와 0.1% digitonin이 함유된 isolation buffer를 첨가하여 ice에서 5분 동안 방치시킨 후, 이는 13,000 × g에서 15분 동안 원심분리하였다. 이를 통해 얻어진 펠렛은 isolation buffer와 혼합하여 10,000 × g에서 10분 동안 원심분리하였고, 최종적으로 얻어진 펠렛을 이용하여 미토콘드리아 활성을 평가하였다. To evaluate mitochondrial activity in mouse brain tissue, mitochondria were extracted from brain tissue. Brain tissue was homogenized using an isolation buffer (pH 7.2) consisting of 215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin (BSA), 20 mM HEPES (Na + ) and a buffer mixed with 20 mM EGTA, and the homogenate was centrifuged at 1,300 × g for 5 minutes. The supernatant obtained through this was centrifuged at 13,000 × g for 10 minutes, and a pellet was obtained. Isolation buffer containing 20 mM EGTA and 0.1% digitonin was added to the resulting pellet, left on ice for 5 minutes, and centrifuged at 13,000 × g for 15 minutes. The obtained pellet was mixed with isolation buffer and centrifuged at 10,000 × g for 10 minutes, and mitochondrial activity was evaluated using the finally obtained pellet.
8-2. 초미세먼지에 노출된 뇌 조직에서의 미토콘드리아 기능평가 (미토콘드리아 내 Reactive oxygen species; ROS 함량 측정)8-2. Mitochondrial function evaluation in brain tissue exposed to ultrafine dust (Reactive oxygen species in mitochondria; ROS content measurement)
뇌 조직으로부터 추출된 미토콘드리아 추출물에서 미토콘드리아 내 ROS 함량을 측정하기 위해, 25 μM DCF-DA 시약과 혼합하여 암흑에서 20분 동안 반응시켰다. 이 반응액은 형광 광도계 (Infinite F200, Tecan)를 사용하여 excitation wave 485 nm 및 emission wave 535 nm에서 측정하였다.In order to measure the ROS content in mitochondria in mitochondrial extracts extracted from brain tissue, they were mixed with 25 μM DCF-DA reagent and reacted in the dark for 20 minutes. This reaction solution was measured at excitation wave 485 nm and emission wave 535 nm using a fluorescence photometer (Infinite F200, Tecan).
PM2.5의 만성적인 노출로 유도된 마우스 뇌 조직의 미토콘드리아 내에서 ROS의 함량은 133.68%로 증가시켰으며, 두충 잎 분획물 40그룹에서는 73.12%로 감소시켰다(도 11A).The content of ROS in the mitochondria of mouse brain tissue induced by chronic exposure to PM 2.5 was increased to 133.68% and decreased to 73.12% in the 40 groups of Eucommia leaf fractions (FIG. 11A).
8-3. 초미세먼지에 노출된 뇌 조직에서의 미토콘드리아 기능평가 (미토콘드리아 막 전위 측정 Mitochondria membrane potential; MMP)8-3. Evaluation of mitochondrial function in brain tissue exposed to ultrafine dust (Mitochondria membrane potential; MMP)
뇌 조직으로부터 추출된 미토콘드리아 추출물은 mitochondrial membrane potential (MMP) 활성을 측정하기 위해 사용하였으며, 미토콘드리아 추출물은 5 mM pyruvate와 5 mM malate가 함유된 미토콘드리아 isolation buffer와 혼합하였고, 이후 1 μM JC-1을 첨가하여 20분 동안 암흑에서 반응시켰다. 반응액은 형광광도계(Infinite F200, Tecan)를 이용하여 excitation wave 530 nm 및 emission wave 590 nm에서 측정하였다.Mitochondrial extract extracted from brain tissue was used to measure mitochondrial membrane potential (MMP) activity. Mitochondrial extract was mixed with mitochondrial isolation buffer containing 5 mM pyruvate and 5 mM malate, and then 1 μM JC-1 was added. and reacted in the dark for 20 minutes. The reaction solution was measured at excitation wave 530 nm and emission wave 590 nm using a fluorescence photometer (Infinite F200, Tecan).
미토콘드리아의 막 전위를 평가한 결과에서 (도 11B), PM2.5그룹은 마우스의 뇌 조직에서 정상 대조군을 100%로 하였을 때, 73.60% 막 전위 손실을 나타내었고 두충 잎 분획물 40그룹에서 152.17%로 막 전위의 손실을 보호하였다.As a result of evaluating the mitochondrial membrane potential (FIG. 11B), the PM 2.5 group showed 73.60% loss of membrane potential in mouse brain tissue, when the normal control group was 100%, and the 40 group of Eucommia leaf fractions showed a loss of membrane potential by 152.17%. Protected against loss of potential.
8-4. 초미세먼지에 노출된 뇌 조직에서의 미토콘드리아 기능평가 (미토콘드리아 내 ATP 함량 측정)8-4. Evaluation of mitochondrial function in brain tissue exposed to ultrafine dust (measurement of ATP content in mitochondria)
뇌 조직으로부터 추출된 미토콘드리아 추출물에서 ATP level을 측정하기 위해, 추출물은 10,000 × g에서 10분 동안 원심분리하였고, 얻어진 펠렛은 1% trichloroacetic acid와 혼합하여 ice에서 10분 동안 반응시켰다. 이후 25 mM Tris-acetate buffer (pH 7.7)과 혼합한 뒤, 10,000 × g에서 15분 원심분리하였고 얻어진 상등액은 ATP level을 측정하는데 사용되었고, 이는 ATP assay kit (Promega, Madison, WI, USA)를 사용하여 luminometer (GloMax-Multi Detection System, Promega, Corp., Madison, WI, USA)를 이용하여 측정하였다.To measure ATP levels in mitochondrial extracts extracted from brain tissue, the extracts were centrifuged at 10,000 × g for 10 minutes, and the obtained pellets were mixed with 1% trichloroacetic acid and reacted on ice for 10 minutes. Then, it was mixed with 25 mM Tris-acetate buffer (pH 7.7), centrifuged at 10,000 × g for 15 minutes, and the obtained supernatant was used to measure the ATP level, which was obtained using an ATP assay kit (Promega, Madison, WI, USA). was measured using a luminometer (GloMax-Multi Detection System, Promega, Corp., Madison, WI, USA).
도 11C에서 미토콘드리아 내에 ATP level을 측정한 결과로 PM2.5의 만성적인 ATP 수준의 감소를 나타내었고, 두충 잎 분획물의 개선 효과는 미비하였다.As a result of measuring the ATP level in the mitochondria in FIG. 11C, the chronic decrease in the ATP level of PM 2.5 was shown, and the improvement effect of the Eucommia leaf fraction was insignificant.
8-5. 초미세먼지에 노출된 뇌 조직에서의 미토콘드리아 기능평가 (웨스턴블랏법을 활용한 단백질 발현측정)8-5. Mitochondrial function evaluation in brain tissue exposed to ultrafine dust (protein expression measurement using western blotting method)
마우스로부터 적출된 뇌에서 분리한 해마와 후각 망울 조직을 이용하여 아밀로이드 베타 경로 단백질과 염증 반응에 관여하는 단백질의 발현을 측정하였다. 적출된 조직들은 1% protease inhibitor가 첨가된 cell/tissue lysis buffer를 이용해 단백질을 추출하였다. 단백질 추출액은 13,000 rpm에서 10분 동안 원심분리하여 상등액을 얻었으며, 이는 bradford법을 이용하여 단백질 상대 정량하였다. 조직의 단백질은 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)로 분리하였고 이는 poly-vinylidene difluoride (PVDF) membrane으로 transfer하였다. 이후 5% skim milk를 이용하여 1시간 동안 blocking하고 세척하였다. 이후 0.5% BSA와 0.1% sodium azide를 포함하는 TBST solution에 1:1000으로 희석된 1차 항체를 overnight하여 반응하였으며, 이후 세척 하여 2차 항체를 실온에서 1시간 동안 반응시켰다. 2차 항체 반응이 종료된 membrane은 ECL solution에 반응시켜 발색시켰으며, 이는 ChemiDoc (iBrightTM CL1000 instrument, Invitrogen, Carlsbad, CA, USA)을 사용하여 발광 밀도를 검출하였다. 밴드의 밀도는 ibright analysis software (Thermo Fisher, Waltham, Middlesex, MA, USA)를 이용하여 수치화하였고, 각 인자의 밀도 값은 β-actin의 밀도 값으로 나누어서 나타냈다.The expression of amyloid beta pathway proteins and proteins involved in inflammatory responses was measured using hippocampus and olfactory bulb tissues isolated from brains excised from mice. Proteins were extracted from the excised tissues using cell/tissue lysis buffer with 1% protease inhibitor added. The protein extract was centrifuged at 13,000 rpm for 10 minutes to obtain a supernatant, which was quantified using the bradford method. Tissue proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a poly-vinylidene difluoride (PVDF) membrane. Thereafter, blocking was performed using 5% skim milk for 1 hour, followed by washing. Thereafter, the primary antibody diluted 1:1000 in TBST solution containing 0.5% BSA and 0.1% sodium azide was reacted overnight, and then washed and the secondary antibody was reacted at room temperature for 1 hour. The membrane after the secondary antibody reaction was reacted with ECL solution to develop color, and the luminescence density was detected using ChemiDoc (iBrightTM CL1000 instrument, Invitrogen, Carlsbad, CA, USA). The density of the band was quantified using ibright analysis software (Thermo Fisher, Waltham, Middlesex, MA, USA), and the density value of each factor was expressed by dividing it by the density value of β-actin.
마우스의 뇌 조직으로 분리한 olfactory bulb 조직에서 아밀로이드 베타 경로에 관여하는 단백질과 염증성 인자의 발현량을 측정한 결과는 도 12와 같다. Olfactory bulb는 PM2.5가 뇌 조직으로 침투하는 경로 중 하나로, PM2.5가 olfactory bulb에 축적되었을 때, 염증 반응의 가속화가 이루어진다고 보고되었다. 또한 olfactory bulb는 인지 손상의 초기 단계에서 아밀로이드 베타 축적과 타우 단백질의 과인산화를 나타낸다고 보고되었다. 따라서 도 12의 결과에서 PM2.5의 만성적인 노출은 마우스의 olfacotory bulb에서 amyloid-beta의 발현량을 정상 대조군 대비 1.70배 증가시켰고, 두충 잎 분획물 40그룹에서는 0.54배 감소하였으며, p-tau의 발현량을 통계적인 차이를 확인할 수 없었다(도 12B-C). 또한 p-IκBα, caspase-1, IL-1β, TNF-α의 발현량은 PM2.5그룹에서 정상 대조군 대비 각각 1.16배, 2.14배, 1.82배, 2.05배 증가시켰고, 두충 잎 분획물 40그룹에서는 PM2.5 그룹 대비 각각 0.55배, 0.51배, 0.53배, 0.60배 감소된 발현량을 나타내었다(도 12D-G).The results of measuring the expression levels of proteins involved in the amyloid beta pathway and inflammatory factors in olfactory bulb tissues isolated from mouse brain tissues are shown in FIG. 12 . It has been reported that the olfactory bulb is one of the pathways for PM 2.5 to penetrate into brain tissue, and that when PM 2.5 accumulates in the olfactory bulb, the inflammatory response is accelerated. It has also been reported that the olfactory bulb exhibits amyloid beta accumulation and hyperphosphorylation of tau protein in the early stages of cognitive impairment. Therefore, in the results of FIG. 12, chronic exposure to PM 2.5 increased the expression level of amyloid-beta in the olfacotory bulb of the mouse by 1.70 times compared to the normal control group, and decreased by 0.54 times in the 40 groups of Eucommia leaf fractions, and the expression level of p -tau No statistical difference could be confirmed (Fig. 12B-C). In addition, the expression levels of p -IκBα, caspase-1, IL-1β, and TNF-α increased 1.16-, 2.14-, 1.82-, and 2.05-fold, respectively, in the PM 2.5 group compared to the normal control group. The expression levels were reduced by 0.55-fold, 0.51-fold, 0.53-fold, and 0.60-fold, respectively, compared to the group (FIG. 12D-G).
마우스의 해마 조직은 기억과 학습에 중요한 역할을 하며, 따라서 마우스 해마 조직을 분리하여 아밀로이드 베타 경로 단백질과, 염증 경로에서 나타나는 단백질의 발현량을 측정한 결과는 도 13과 같다. 해마 조직 내에서 amyloid-beta와 p-tau의 발현량은 PM2.5그룹에서 정상 대조군 대비 각각 1.56배, 3.32배 증가되었고, 두충 잎 분획물 40그룹에서 PM2.5 그룹 대비 각각 1.73배, 2.83배로 control과 유사하게 개선되는 것을 확인하였다(도 13B-C). 또한 p-IκBα, caspase-1, IL-1β, TNF-α 인자의 PM2.5그룹에서의 발현량은 정상 대조군 대비 1.84배, 1.65배, 1.50배, 1.93배 증가되었으며, 두충 잎 분획물 40 그룹에서는 PM2.5 그룹 대비 0.65배, 0.62배, 0.67배, 0.61배 감소된 발현량을 나타내었다(도 13D-G).Mouse hippocampal tissue plays an important role in memory and learning. Therefore, the results of measuring the expression levels of amyloid beta pathway proteins and inflammatory pathway proteins by isolating mouse hippocampal tissues are shown in FIG. 13 . The expression levels of amyloid-beta and p -tau in the hippocampal tissue were increased 1.56-fold and 3.32-fold, respectively, compared to the normal control group in the PM 2.5 group, and 1.73-fold and 2.83-fold, respectively, compared to the PM 2.5 group in the 40 group of eutrophic leaf fractions, similar to the control. It was confirmed that it was improved (Fig. 13B-C). In addition, the expression levels of p -IκBα, caspase-1, IL-1β, and TNF-α factors in the PM 2.5 group were increased by 1.84, 1.65, 1.50, and 1.93 times compared to the normal control group. Compared to the 2.5 group, the expression level was reduced by 0.65 times, 0.62 times, 0.67 times, and 0.61 times (FIG. 13D-G).
실시예 9. 두충 잎 추출물의 주요 생리 활성 물질 분석Example 9. Analysis of main physiologically active substances in Eucommia leaf extract
두충 잎 분획물에 함유되어 있는 생리 활성 물질 분석을 위하여 UPLC Q-TOF/MS2 분석을 실시하였다. 두충 잎 분획물은 100% methanol을 이용하여 용해시킨 뒤, 필터를 이용하여 여과하여 사용하였다. 이는 Acquity UPLC BEH C18 컬럼을 이용하여 음이온 모드에서 분석을 실시하였고, 이동 속도는 0.35 mL/min으로 진행하였다. 이동상 용매는 0.1% formic acid in distilled water (A)와 0.1% formic acid in acetonitrile (B)를 사용하였으며, 1% B at 0-1 min, 1-100% B at 1-7min, 100% B at 7-8 min, 100-1% B at 8-8.2 min, and 1% B at 8.2-10 min 조건으로 용매 구배를 실시하였다.UPLC Q-TOF/MS 2 analysis was performed to analyze the physiologically active substances contained in the leaf fractions of Eucommia. Eucommia leaf fraction was dissolved using 100% methanol, and filtered using a filter before use. This was analyzed in negative ion mode using an Acquity UPLC BEH C 18 column, and the transfer rate was 0.35 mL/min. Mobile phase solvents were 0.1% formic acid in distilled water (A) and 0.1% formic acid in acetonitrile (B), 1% B at 0-1 min, 1-100% B at 1-7min, 100% B at A solvent gradient was performed under conditions of 7-8 min, 100-1% B at 8-8.2 min, and 1% B at 8.2-10 min.
본 발명에 사용된 두충 잎 분획물에 포함된 생리 활성 물질을 분석하기 위해 UPLC Q-TOF/MS2 분석을 실시하였다(도 14). 확인된 peak은 compound 1: 707 m/z (RT: 2.84 min), compound 2: 609 m/z (RT: 3.15 min), compound 3: 463 m/z (RT: 3.22 min), compound 4: 505 m/z (RT 3.28 min), compound 5: 489 m/z (RT: 3.44 min), compound 6: 301 m/z (RT 3.84 min)으로 나타났으며, 이들은 각각, 5-O-caffeoylquinic acid, rutin, quercetin-O-hexoside, quercetin-O-acetylhexoside (isomer), luteolin-O-acetylhexoside, quercetin으로 확인되었다(표 1).UPLC Q-TOF/MS 2 analysis was performed to analyze the physiologically active substances contained in the leaf fraction of Eucommia used in the present invention (FIG. 14). The identified peaks are compound 1: 707 m/z (RT: 2.84 min), compound 2: 609 m/z (RT: 3.15 min), compound 3: 463 m/z (RT: 3.22 min), compound 4: 505 m/z (RT 3.28 min), compound 5: 489 m/z (RT: 3.44 min), compound 6: 301 m/z (RT 3.84 min), which are respectively 5-O-caffeoylquinic acid, They were identified as rutin, quercetin-O-hexoside, quercetin-O-acetylhexoside (isomer), luteolin-O-acetylhexoside, and quercetin (Table 1).
[표 1][Table 1]
실시예 10. 통계처리Example 10. Statistical processing
모든 실험은 반복 실행 후, mean±SD로 나타냈다. 실험 결과의 통계적 유의성은 SAS software (version 9.1, SAS Institute Inc., Cary, NC, USA)를 이용하여 분산분석을 실시하였으며, 각 그룹 간의 유의성은 Duncan의 다중범위검정법 (Duncan’s new multiple-range test)으로 유의차를 5% 수준 (p<0.05)에서 검증하였다.All experiments were expressed as mean±SD after repeated runs. The statistical significance of the experimental results was analyzed using SAS software (version 9.1, SAS Institute Inc., Cary, NC, USA), and the significance between each group was determined using Duncan's new multiple-range test. A significant difference was verified at the 5% level ( p <0.05).
Claims (16)
- 두충 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.A health functional food composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia leaf fraction as an active ingredient.
- 제1항에 있어서,According to claim 1,상기 분획물은 물 분획물, n-헥산 분획물, 클로로포름 분획물 또는 에틸아세테이트 분획물인 것을 특징으로 하는, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The fraction is a water fraction, n-hexane fraction, chloroform fraction or ethyl acetate fraction, characterized in that, a health functional food composition for preventing or improving brain diseases caused by ultrafine dust.
- 제1항에 있어서,According to claim 1,상기 뇌질환은 뇌 신경 질환인 것을 특징으로 하며, 상기 뇌 신경 질환은 뇌졸중, 알츠하이머 병, 치매 및 파킨슨병 중 어느 하나인 것인, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The brain disease is characterized in that the brain nerve disease, the brain nerve disease is any one of stroke, Alzheimer's disease, dementia and Parkinson's disease, a health functional food composition for preventing or improving brain diseases caused by ultrafine dust.
- 제1항에 있어서,According to claim 1,상기 조성물은 뇌 조직 내 ABTS 라디칼 소거 활성 또는 DPPH 라디칼 소거 활성이 증가된 것을 특징으로 하는, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a health functional food composition for preventing or improving brain diseases caused by ultrafine dust, characterized in that the ABTS radical scavenging activity or DPPH radical scavenging activity in brain tissue is increased.
- 제1항에 있어서,According to claim 1,상기 조성물은 뇌 조직 내 지방질과산화물인 말론디알데히드 (Malondialdehyde, MDA)의 생성을 억제하는 효과를 가지는 것인, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a functional health food composition for preventing or improving brain diseases caused by ultrafine dust, which has an effect of inhibiting the production of malondialdehyde (MDA), a lipid peroxide in brain tissue.
- 제1항에 있어서, According to claim 1,상기 조성물은 뇌 조직 내 활성산소(reactive oxygen species, ROS)의 생성을 억제시키는 효과를 가지는 것인, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a health functional food composition for preventing or improving brain diseases caused by ultrafine dust, which has an effect of inhibiting the production of reactive oxygen species (ROS) in brain tissue.
- 제1항에 있어서,According to claim 1,상기 조성물은 뇌 조직 내 세포사멸을 억제시키는 효과를 가지는 것인, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a health functional food composition for preventing or improving brain diseases caused by ultrafine dust, which has an effect of inhibiting cell death in brain tissue.
- 제1항에 있어서,According to claim 1,상기 조성물은 뇌 조직 내 염증 반응에 대한 조직 보호 효과를 가지는 것인, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a functional health food composition for preventing or improving brain diseases caused by ultrafine dust, which has a tissue protective effect on the inflammatory response in brain tissue.
- 제1항에 있어서,According to claim 1,상기 조성물은 초미세먼지로 인한 공간인지력 저하를 개선시키는 것을 특징으로 하는, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a health functional food composition for preventing or improving brain disease caused by ultrafine dust, characterized in that it improves spatial recognition deterioration due to ultrafine dust.
- 제1항에 있어서,According to claim 1,상기 조성물은 초미세먼지로 인해 저하된 단기 학습 및 단기 기억력을 개선시키는 것을 특징으로 하는, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a health functional food composition for preventing or improving brain diseases caused by ultrafine dust, characterized in that for improving short-term learning and short-term memory deteriorated due to ultrafine dust.
- 제1항에 있어서,According to claim 1,상기 조성물은 초미세먼지로 인해 저하된 장기 학습 및 장기 기억력을 개선시키는 것을 특징으로 하는, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a health functional food composition for preventing or improving brain diseases caused by ultrafine dust, characterized in that it improves long-term learning and long-term memory deteriorated due to ultrafine dust.
- 제1항에 있어서,According to claim 1,상기 조성물은 초미세먼지로 인해 뇌 조직에서의 인지기능 저하를 개선시키는 것을 특징으로 하는, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a health functional food composition for preventing or improving brain disease caused by ultrafine dust, characterized in that it improves cognitive function deterioration in brain tissue due to ultrafine dust.
- 제1항에 있어서,According to claim 1,상기 조성물은 초미세먼지로 인한 뇌 조직에서 콜린성 시스템의 기능저하를 개선시키는 것을 특징으로 하는, 초미세먼지로 인한 뇌질환 예방 또는 개선용 건강기능성식품 조성물.The composition is a health functional food composition for preventing or improving brain diseases caused by ultrafine dust, characterized in that to improve the functional decline of the cholinergic system in brain tissue due to ultrafine dust.
- 두충 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 치료용 약학 조성물에서, 상기 뇌질환은 뇌 신경 질환인 것을 특징으로 하며, 상기 뇌 신경 질환은 뇌졸중, 알츠하이머 병, 치매 및 파킨슨병 중 어느 하나인 것인, 약학 조성물.In a pharmaceutical composition for preventing or treating brain diseases caused by ultrafine dust comprising Eucommia leaf fraction as an active ingredient, the brain disease is characterized in that the brain disease is a brain nerve disease, and the brain nerve disease is a stroke, Alzheimer's disease, dementia and Parkinson's disease Any one of the bottles, the pharmaceutical composition.
- 두충 잎 분획물을 유효성분으로 포함하는 초미세먼지로 인한 뇌질환 예방 또는 개선용 화장료 조성물에서, 상기 뇌질환은 뇌 신경 질환인 것을 특징으로 하며, 상기 뇌 신경 질환은 뇌졸중, 알츠하이머 병, 치매 및 파킨슨병 중 어느 하나인 것인, 화장료 조성물.In the cosmetic composition for preventing or improving brain diseases caused by ultrafine dust containing Eucommia leaf fraction as an active ingredient, the brain disease is characterized in that the brain disease is a brain nerve disease, and the brain nerve disease is a stroke, Alzheimer's disease, dementia and Parkinson's disease Any one of the bottles, the cosmetic composition.
- 제14항의 약학 조성물을 개체에 투여하는 단계를 포함하는, 초미세먼지로 인한 뇌질환 예방 또는 치료방법.A method for preventing or treating brain diseases caused by ultrafine dust, comprising administering the pharmaceutical composition of claim 14 to a subject.
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| KR20110002237A (en) * | 2009-07-01 | 2011-01-07 | 박현미 | A composition for preventing or treating neurological disorder comprising an extract of eucommia ulmoides |
| KR20140107992A (en) * | 2013-02-28 | 2014-09-05 | 성균관대학교산학협력단 | Pharmaceutical composition for prevention or treatment of Parkinson's disease comprising Eucommiae ulmoides extract or fraction thereof |
| KR20140137867A (en) * | 2013-05-24 | 2014-12-03 | 재단법인 산청한방약초연구소 | Composition comprising herbal mixture extract for treating or preventing inflammatory disease |
| CN106138168A (en) * | 2015-03-26 | 2016-11-23 | 南京海恒医药科技有限公司 | Cortex Eucommiae extract application in preparation prevention and treatment senile dementia |
-
2022
- 2022-01-17 KR KR1020220006389A patent/KR20230110891A/en unknown
- 2022-06-15 WO PCT/KR2022/008463 patent/WO2023136405A1/en unknown
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| KR20100043669A (en) * | 2008-10-20 | 2010-04-29 | 주식회사 유니베라 | Extract of eucommiae ulmoides for preventing or treating of learning or memory disorder, or dementia |
| KR20110002237A (en) * | 2009-07-01 | 2011-01-07 | 박현미 | A composition for preventing or treating neurological disorder comprising an extract of eucommia ulmoides |
| KR20140107992A (en) * | 2013-02-28 | 2014-09-05 | 성균관대학교산학협력단 | Pharmaceutical composition for prevention or treatment of Parkinson's disease comprising Eucommiae ulmoides extract or fraction thereof |
| KR20140137867A (en) * | 2013-05-24 | 2014-12-03 | 재단법인 산청한방약초연구소 | Composition comprising herbal mixture extract for treating or preventing inflammatory disease |
| CN106138168A (en) * | 2015-03-26 | 2016-11-23 | 南京海恒医药科技有限公司 | Cortex Eucommiae extract application in preparation prevention and treatment senile dementia |
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| KIM MIN JI, KANG JIN YONG, KIM JONG MIN, MOON JONG HYUN, LEE HYO LIM, JEONG HYE RIN, GO MIN JI, LEE UK, HEO HO JIN: "Effect of Ethyl Acetate Fraction from Eucommia ulmoides Leaves on PM2.5-Induced Inflammation and Cognitive Dysfunction", OXIDATIVE MEDICINE AND CELLULAR LONGEVITY, HINDAWI PUBLISHING CORPORATION, US, vol. 2022, 14 May 2022 (2022-05-14), US , pages 1 - 26, XP093078340, ISSN: 1942-0900, DOI: 10.1155/2022/7157444 * |
| KIM MIN JI, KANG JIN YONG, PARK SEON KYEONG, KIM JONG MIN, MOON JONG HYUN, KIM GIL HAN, LEE HYO LIM, JEONG HYE RIN, HEO HO JIN: "Protective effect of Eucommia ulmoides oliver leaves against PM2.5-induced oxidative stress in neuronal cells in vitro", KOREAN JOURNAL OF FOOD SCIENCE AND TECHNOLOGY, SEOUL, KR, vol. 53, no. 4, 1 June 2021 (2021-06-01), KR , pages 423 - 433, XP093078372, ISSN: 0367-6293, DOI: 10.9721/KJFST.2021.53.4.423 * |
| KIM, M. J. ET AL.: "Anti-inflammatory effect of ethyl acetate fraction from Eucommia ulmoides leaf on PM2.5-induced cognitive impairment", ABSTRACTS OF 2021 KOSFOST INTERNATIONAL SYMPOSIUM AND ANNUAL MEETING, P11-032, July 2021 (2021-07-01), pages 485 * |
| KIM, M. J. ET AL.: "Protective Effect of Ethyl Acetate Fraction of Eucommia ulmoides Leaves (EFEL) against PM2.5-induced Intestine Damage", PROCEEDINGS OF THE KOREAN SOCIETY OF FOOD SCIENCE AND NUTRITION CONFERENCE; THE KFN 50TH ANNIVERSARY 2021 KFN INTERNATIONAL SYMPOSIUM AND ANNUAL MEETING, October 2021 (2021-10-01), pages 531, XP009547708 * |
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|---|---|
| KR20230110891A (en) | 2023-07-25 |
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