WO2024063550A1 - Composition for prevention, amelioration or treatment of diseases caused by particulate matter comprising citri tangerinae semen extract and use thereof - Google Patents

Composition for prevention, amelioration or treatment of diseases caused by particulate matter comprising citri tangerinae semen extract and use thereof Download PDF

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WO2024063550A1
WO2024063550A1 PCT/KR2023/014337 KR2023014337W WO2024063550A1 WO 2024063550 A1 WO2024063550 A1 WO 2024063550A1 KR 2023014337 W KR2023014337 W KR 2023014337W WO 2024063550 A1 WO2024063550 A1 WO 2024063550A1
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composition
disease
fine dust
extract
tangerine
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PCT/KR2023/014337
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French (fr)
Korean (ko)
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박종길
조민지
조영래
이장욱
이남경
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한국생명공학연구원
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    • 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)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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

Definitions

  • the present invention relates to a composition for preventing, improving or treating diseases caused by fine dust containing Citri tangerinae Semen extract and its use.
  • Fine dust is defined as particulate matter (PM) with a diameter of 10 ⁇ m or less among the total floating dust in the air, and is divided into fine dust with a diameter of less than 10 ⁇ m (PM10) and ultrafine dust with a diameter of less than 2.5 ⁇ m (PM2.5). Divided. Fine dust particles are very small in size, so they penetrate deeper into the human body and cause various inflammatory reactions in the human body (Seagrave J., Toxicology and applied pharmacology 2008; 232(3): 469-77.), and there are various health hazards of fine dust. Evaluation studies have reported an association with increased mortality, effects on the cardiovascular system, effects on the respiratory system, and increased cancer incidence (Yu-Ran et al., Journal of Korean Oriental Medicine, Volume 40, No. 3, 2019).
  • fine dust is closely related to negative effects on human health, such as endotoxin.
  • fine dust may contain various biological elements such as endotoxins and mold.
  • fine dust is known to play a role in various respiratory problems such as asthma, fever, chills, joint pain, and cardiovascular disease by mediating inflammation through endotoxin, a major component of the outer cell membrane of Gram-negative bacteria (Jing Li et al. al., Atmospheric Environment 212 (2019) 305-315).
  • Citri tangerinae Semen refers to the ripe seeds of the tangerine tree Citrus unshiu Marcorvich or Citrus reticulata Blanco (Rutaceae). In Korea, it is registered as a herbal medicine in the Korean Pharmacopoeia Specification for Herbal Medicine (Herbal Medicine).
  • Citri tangerinae Semen extract suppresses the inflammatory response and increased expression of cell adhesion molecules caused by fine dust or endotoxin contained in fine dust in the body, and that the Citri tangerinae Semen extract is included in fine dust or fine dust.
  • the present invention was completed by confirming that it can be applied to the prevention, improvement, or treatment of diseases caused by endotoxin.
  • One object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by fine dust, containing Citri tangerinae Semen extract as an active ingredient.
  • Another object of the present invention is to provide a method for preventing or treating diseases caused by fine dust, comprising administering the pharmaceutical composition of the present invention to an individual.
  • Another object of the present invention is to provide a food composition for preventing or improving fine dust-induced diseases, including tangerine core extract.
  • Another object of the present invention is to provide a feed composition for preventing or improving fine dust-induced diseases, including tangerine core extract.
  • the Citri tangerinae Semen extract of the present invention suppresses the inflammatory response and increased expression of cell adhesion molecules caused by fine dust in the body, and can be applied to the prevention, improvement, or treatment of diseases caused by fine dust.
  • Figure 1 is a diagram showing the inhibitory effect of Citri tangerinae Semen extract on the production of nitric oxide (NO) generated in macrophages by fine dust.
  • FIG. 2 shows the fine dust generated in macrophages by the tangerine core extract. This diagram shows the effect of suppressing the expression of IL (interleukin)-1 ⁇ and TNF- ⁇ (tumor necrosis factor- ⁇ ).
  • IL interleukin
  • TNF- ⁇ tumor necrosis factor- ⁇
  • Figure 3 is a diagram showing the cytotoxicity of the tangerine core composition.
  • FIG. 4 shows the inhibitory effect on the increase in expression of cell adhesion molecules [ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1)] generated in vascular endothelial cells by fine dust of tangerine core extract.
  • IAM-1 internal adhesion molecule-1
  • VCAM-1 vascular cell adhesion molecule-1
  • Figure 5 is a diagram showing the inhibitory effect of tangerine core extract on the increase in the expression of cell adhesion molecules in vascular endothelial cells caused by harmful substances derived from lung tissue epithelial cells treated with fine dust.
  • Figure 6 is a diagram showing the inhibitory effect of tangerine core extract and tangerine peel extract on NO production generated in macrophages by fine dust.
  • Figure 7 is a diagram showing the inhibitory effect of tangerine core extract and tangerine peel extract on the increase in expression of cell adhesion molecules (ICAM-1 and VCAM-1) generated in vascular endothelial cells by fine dust.
  • IAM-1 and VCAM-1 cell adhesion molecules
  • Figure 8 is a diagram showing the inhibitory effect of endotoxin (LPS) of tangerine core extract on the increase in expression of cell adhesion molecules occurring in vascular endothelial cells.
  • LPS endotoxin
  • Figure 9 shows the anti-inflammatory effect of tangerine core extract in an endotoxin-injected mouse model.
  • One aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases caused by fine dust, comprising Citri tangerinae Semen extract as an active ingredient.
  • Citri tangerinae Semen refers to the ripe seeds of a tangerine tree. In Korea, it is registered as a herbal medicine in the Korean Pharmacopoeia Specification for Herbal Medicine (Herbal Medicine).
  • the tangerine tree is not particularly limited as long as it is a tangerine tree known in the art, but examples include Citrus unshiu Marcorvich and Citrus reticulata Blanco (Rutaceae).
  • extract refers to a liquid component obtained by immersing the desired material in various solvents and then extracting it at room temperature, low temperature, or heated state for a certain period of time, and a liquid component obtained by removing the solvent from the liquid component. It refers to the result of solid content, etc.
  • it can be comprehensively interpreted to include all dilutions of the results, concentrates thereof, crude preparations, purifications, etc. of the above results.
  • the extract may be an extract of tangerine tree seeds, that is, tangerine core.
  • the extract can be obtained by extraction with water or various organic solvents such as media and alcohol.
  • the organic solvent used is not particularly limited as long as the extract can be obtained, but may specifically be water, a polar solvent, or a non-polar solvent, and more specifically, water, a medium, or a low-grade solvent having 1 to 6 carbon atoms. It may be alcohol [C1 to 6, for example, methanol, ethanol (ethyl alcohol), propanol, or butanol, etc.], or a mixed solvent thereof.
  • the method for obtaining the extract is not particularly limited, as long as the extract of the tangerine core can be obtained, and extraction may be performed according to a method commonly used in the art.
  • the method includes hot water extraction, ultrasonic extraction, filtration, reflux extraction, supercritical extraction, etc., and may be performed alone or in combination of two or more methods, but is not limited thereto.
  • the tangerine core can be purchased and used commercially, or collected or cultivated from nature.
  • the tangerine core of the present invention may be included in the composition of the present invention in the form of not only its extract, but also the tangerine core itself, its dried product, fermented broth, or fraction, but is not limited thereto.
  • the composition of the present invention may not exhibit cytotoxicity.
  • the tangerine core extract did not show cytotoxicity even at high concentration (200 ⁇ g/mL) (FIG. 3).
  • the composition of the present invention has one or more effects selected from the group consisting of inhibiting nitric oxide production in immune cells, reducing IL (interleukin)-1 ⁇ expression, and TNF- ⁇ (tumor necrosis factor- ⁇ ) expression. It may be something to have.
  • the NO level of the macrophage culture medium treated with fine dust significantly increased compared to the NO level of the macrophage culture medium not treated with fine dust.
  • the NO level of macrophage culture medium was confirmed to significantly decrease in a tangerine core extract concentration-dependent manner ( Figure 1).
  • the expression levels of IL-1 ⁇ and TNF- ⁇ were significantly increased in macrophages treated with fine dust compared to IL-1 ⁇ and TNF- ⁇ in macrophages not treated with fine dust.
  • the expression levels of IL-1 ⁇ and TNF- ⁇ in macrophages were confirmed to significantly decrease in a tangerine core extract concentration-dependent manner ( Figure 2).
  • composition of the present invention may inhibit increased expression of cell adhesion molecules in vascular endothelial cells.
  • the cell adhesion molecule may be one or more selected from ICAM-1 (intercellular adhesion molecule-1) or VCAM-1 (vascular cell adhesion molecule-1), but is not limited thereto.
  • the expression levels of ICAM-1 and VCAM-1 were significantly increased in the vascular endothelial cell model treated with fine dust compared to the control model, but when treated with tangerine core extract, ICAM-1 and It was confirmed that the expression level of VCAM-1 was significantly decreased in a concentration-dependent manner of the tangerine core extract (Figure 4).
  • the culture medium containing harmful substances derived from lung tissue epithelial cells treated with fine dust increased the expression levels of ICAM-1 and VCAM-1 in vascular endothelial cells compared to the control culture medium.
  • the expression levels of ICAM-1 and VCAM-1 were significantly decreased in a concentration-dependent manner of tangerine core extract ( Figure 5).
  • composition of the present invention may exhibit the effect of preventing, improving, or treating diseases caused by fine dust due to the above-mentioned effects.
  • the tangerine core extract which is the active ingredient of the composition of the present invention, has an inhibitory effect on nitric oxide production in immune cells and/or cell adhesion molecules in vascular endothelial cells more than extracts from other parts of the tangerine other than the tangerine core, such as the peel part of the tangerine (tangerine peel). Since the effect of suppressing the increase in the expression of
  • both tangerine core extract and tangerine peel extract inhibited NO production in macrophage culture medium, but it was confirmed that the NO production inhibition ability of tangerine core extract was higher than that of tangerine peel extract (FIG. 6).
  • the tangerine peel extract did not inhibit the increase in ICAM-1 and VCAM-1 expression in the fine dust-treated vascular endothelial cell model, whereas the tangerine core extract increased the expression of ICAM-1 and VCAM-1. It was confirmed that it was inhibited (Figure 7).
  • the term "disease caused by fine dust” may include without limitation any disease caused by the inflow of fine dust into the body, for example, a disease caused by inflammation caused by the inflow of fine dust into the body. It may be, but is not limited to this.
  • diseases caused by fine dust may include inflammatory diseases, cardiovascular diseases, metabolic diseases, nervous system diseases, respiratory system diseases, and eye diseases.
  • inflammatory diseases include, but are not limited to, pneumonia, tracheitis, chronic respiratory diseases (COPD), and dermatitis.
  • COPD chronic respiratory diseases
  • cardiovascular diseases include, but are not limited to, high blood pressure, cardiac arrhythmia, ischemic heart disease, myocardial infarction, heart failure, and cardiac arrhythmia.
  • Examples of the metabolic disease include, but are not limited to, diabetes.
  • neurological diseases include, but are not limited to, stroke, neurodevelopmental disorders, Alzheimer's disease, and worsening mental disorders.
  • respiratory diseases include, but are not limited to, lung cancer, lung growth disorders, and upper respiratory tract infections.
  • eye disease examples include, but are not limited to, dry eye syndrome and retinal microvascular damage.
  • the disease caused by fine dust may be an inflammatory disease, but is not limited thereto, and diseases caused by inflammation caused by the inflow of fine dust into the body may also be included within the scope of the present invention. there is.
  • the 'disease caused by fine dust' of the present invention may be, but is not limited to, various inflammatory diseases caused by endotoxin contained in fine dust. In the present invention, it can be used interchangeably with ‘disease caused by endotoxin contained in fine dust’.
  • endotoxin may be lipopolysaccharide (LPS) derived from Gram-negative bacteria, a derivative thereof, or a compound containing lipid A.
  • LPS lipopolysaccharide
  • the endotoxin may be an inflammatory agent such as a cytokine. The argument can be activated.
  • the fine dust of the present invention may cause diseases caused by inflammation through endotoxin, a major component of the outer cell membrane of Gram-negative bacteria, mediating inflammation, but is not limited to this.
  • the disease caused by fine dust and/or the disease caused by endotoxin contained in fine dust may include inflammatory disease, cardiovascular disease, metabolic disease, nervous system disease, respiratory system disease, and eye disease.
  • prevention refers to all actions that suppress or delay the onset of diseases caused by fine dust by administration of the composition
  • treatment refers to diseases caused by fine dust by administration of the composition. It refers to any action that improves or changes symptoms to a benefit.
  • the pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions.
  • the content of tangerine core extract, which is an active ingredient contained in the pharmaceutical composition is not particularly limited, but may include 0.0001% by weight to 10% by weight, specifically 0.001% by weight to 1% by weight, based on the total weight of the composition. there is.
  • the concentration of the tangerine core extract, which is an active ingredient included in the pharmaceutical composition is not particularly limited, but is 50 ⁇ g/mL or more, 60 ⁇ g/mL or more, 70 ⁇ g/mL or more, 80 ⁇ g/mL or more, 90 ⁇ g/mL or more. It may be included at a concentration of mL or more or 100 ⁇ g/mL or more, and specifically, it may be included at a concentration of 100 to 200 ⁇ g/mL.
  • the pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, freeze-dried preparations, and suppositories. It can be in various oral or parenteral dosage forms. When formulated, it is 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.
  • Solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc.
  • excipients such as starch, calcium carbonate, sucrose, or lactose
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate.
  • injectable esters such as ethyl oleate.
  • As a base for suppositories witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.
  • composition of the present invention can be administered in a pharmaceutically effective amount.
  • the term "pharmaceutically effective amount” refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, and severity of the disease. It can be determined based on factors including the type, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times.
  • the preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but for desirable effects, the composition of the present invention is administered in a specific dosage of 0.0001 to 500 mg/kg per day. It is recommended to administer from 0.001 to 200 mg/kg. Administration may be administered once a day, or may be administered several times.
  • composition can be administered to various mammals such as rats, livestock, and humans by various routes, and the method of administration includes without limitation any method conventional in the art, for example, orally, rectally, intravenously, intramuscularly, or intravenously. It may be administered by subcutaneous, intrauterine, intrathecal, or intracerebrovascular injection.
  • composition of the present invention can be used not only in the form of a medicine for humans, but also in the form of an animal medicine.
  • animals are a concept that includes livestock and companion animals.
  • Another aspect of the present invention provides a method for preventing or treating diseases caused by fine dust, comprising the step of administering the pharmaceutical composition of the present invention to an individual.
  • the disease caused by fine dust may be an inflammatory disease, but is not limited thereto. This is the same as described above in the previous aspect.
  • the “administration” means introducing the composition of the present invention into an individual by any appropriate method, and the composition may be administered through any general route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, locally, or intranasally, but is not limited thereto.
  • the “individual” refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs other than humans who have or may develop diseases caused by fine dust. This means all animals, including The type of subject is not limited as long as the above disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.
  • Another aspect of the present invention provides a food composition for preventing or improving fine dust-induced diseases, comprising a tangerine core extract.
  • tangerine core extract which is an active ingredient of the present invention, can be added to food compositions.
  • the active ingredient of the present invention can be added as is or used together with other foods or ingredients, and can be used appropriately according to conventional methods.
  • the mixing amount of the active ingredient can be appropriately determined depending on the purpose of use, and the content of the tangerine core extract, which is an active ingredient included in the food composition, is not particularly limited, but is preferably 0.0001% by weight to 10% by weight based on the total weight of the composition. Typically, it may include 0.001% by weight to 1% by weight.
  • the concentration of the tangerine core extract which is an active ingredient contained in the composition, is not particularly limited, but is 50 ⁇ g/mL or more, 60 ⁇ g/mL or more, 70 ⁇ g/mL or more, 80 ⁇ g/mL or more, and 90 ⁇ g/mL or more. Alternatively, it may be included at a concentration of 100 ⁇ g/mL or more, and specifically, it may be included at a concentration of 100 to 200 ⁇ g/mL.
  • Examples of foods to which the above active ingredients can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, and drink preparations. , alcoholic beverages and vitamin complexes, etc., and may include all foods in the conventional sense, and may include foods used as feed for animals.
  • the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, and alcohol.
  • it may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.
  • the food can be manufactured in dosage forms such as tablets, granules, powders, capsules, liquid solutions, and pills according to known manufacturing methods.
  • other ingredients there is no particular limitation on other ingredients, and various common flavoring agents or natural carbohydrates may be included as additional ingredients.
  • Another aspect of the present invention provides a feed composition for preventing or improving fine dust-induced diseases, comprising a tangerine kernel extract.
  • tangerine core extract which is an active ingredient of the present invention, can be added to feed compositions or feed additives.
  • the term “feed” refers to a substance that supplies organic or inorganic nutrients necessary for maintaining the life of an individual and raising the individual.
  • the feed may contain nutrients such as energy, protein, lipid, vitamins, and minerals required by the individual consuming the feed, but is not particularly limited thereto, and may include grains, roots and fruits, food processing by-products, algae, and fiber.
  • vegetable feed such as fats and oils, starches, crustaceans, and grain by-products
  • animal feed such as proteins, inorganic substances, oils, minerals, single-cell proteins, zooplankton, and fish meal.
  • the feed may contain additional feed additives such as amino acids, vitamins, enzymes, flavoring agents, non-protein nitrogen compounds, silicate agents, buffers, extractants, and oligosaccharides for promoting individual growth and disease prevention.
  • additional feed additives such as amino acids, vitamins, enzymes, flavoring agents, non-protein nitrogen compounds, silicate agents, buffers, extractants, and oligosaccharides for promoting individual growth and disease prevention.
  • feed additive refers to a general term for substances added in small amounts to feed for nutritional or specific purposes. In the present invention, it refers to substances added for the purpose of preventing or improving fine dust-induced diseases.
  • the feed additives may further include binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, and amino acids, vitamins, enzymes, probiotics, flavoring agents, non-protein nitrogen compounds, etc. added to increase utility. It may additionally include silicate agents, buffering agents, colorants, extractants, oligosaccharides, etc. In addition, it may additionally include feed mixtures, etc., but is not limited thereto.
  • the content of tangerine core extract, which is an active ingredient contained in the feed composition or feed additive is not particularly limited, but may include 0.0001% by weight to 10% by weight, specifically 0.001% by weight to 1% by weight, based on the total weight of the composition. there is.
  • the concentration of the tangerine core extract, which is an active ingredient contained in the composition is not particularly limited, but is 50 ⁇ g/mL or more, 60 ⁇ g/mL or more, 70 ⁇ g/mL or more, 80 ⁇ g/mL or more, and 90 ⁇ g/mL or more. Alternatively, it may be included at a concentration of 100 ⁇ g/mL or more, and specifically, it may be included at a concentration of 100 to 200 ⁇ g/mL.
  • the object refers to an object of breeding and includes without limitation any living organism that can consume the feed of this application, and includes, for example, livestock and companion animals.
  • Another aspect of the present invention provides a quasi-drug composition for preventing or improving fine dust-induced diseases, comprising a tangerine core extract.
  • tangerine core extract which is an active ingredient of the present invention, can be added to a quasi-drug composition.
  • the term "quasi-drug” refers to fibers, rubber products or similar products used for the purpose of treating, alleviating, treating or preventing diseases in humans or animals, which have a weak effect on the human body or do not act directly on the human body, and devices. Or, it is an article that is not a machine or similar, or an agent used for sterilization, insecticide, and similar purposes to prevent infection, and is used for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases in humans or animals. It refers to articles other than instruments, machines, or devices among articles used for the purpose of having a pharmacological effect on the structure and function of humans or animals, excluding articles for external use on the skin and personal hygiene. Also includes supplies.
  • the skin external preparation is not particularly limited thereto, but may be specifically prepared and used in the form of ointment, lotion, spray, patch, cream, powder, suspension, gel or gel.
  • the personal hygiene products include, but are not limited to, soap, cosmetics, wet tissues, toilet paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, makeup, foundation, blush, mascara, eye shadow, and sunscreen lotion. , it may be a hair care product, air freshener gel or cleaning gel.
  • other examples of the quasi-drug composition of the present invention include disinfectant cleaners, shower foams, garnishes, wet tissues, detergent soaps, hand washes, humidifier fillers, masks, ointments, or filter fillers.
  • the active ingredient of the present invention When adding the active ingredient of the present invention to a quasi-drug composition for the purpose of preventing or improving diseases caused by fine dust, the active ingredient can be added as is or used together with other quasi-drug ingredients, and can be used appropriately according to conventional methods. .
  • the mixing amount of the active ingredient can be appropriately determined depending on the purpose of use, and the content of the tangerine core extract, which is an active ingredient included in the quasi-drug composition, is not particularly limited, but is 0.0001% by weight to 10% by weight based on the total weight of the composition. It may include 0.001% by weight to 1% by weight.
  • the concentration of the tangerine core extract which is an active ingredient contained in the composition, is not particularly limited, but is 50 ⁇ g/mL or more, 60 ⁇ g/mL or more, 70 ⁇ g/mL or more, 80 ⁇ g/mL or more, and 90 ⁇ g/mL or more. Alternatively, it may be included at a concentration of 100 ⁇ g/mL or more, and specifically, it may be included at a concentration of 100 to 200 ⁇ g/mL.
  • the tangerine core extract was obtained by adding 95% ethanol (ethyl alcohol) (guaranteed reagent, GR grade) to the dried and powdered tangerine core in the shade and using an ultrasonic extractor (SDN-900H, Sd-ultrasonic co.). Extraction was performed at room temperature with 30 cycles (40 KHz, 1500 W, 15 minutes sonication - 120 minutes per cycle). The obtained product was filtered (Qualitative Filter No. 100, Hyundai micro co.), dried under reduced pressure, and then dissolved in DMSO (Dimethyl sulfoxide) before use. It can be purchased from the Korean Plant Extract Bank of the Korea Research Institute of Bioscience and Biotechnology under the sales number CA01-020.
  • DMSO Dimethyl sulfoxide
  • Example 2 Effect of suppressing the production of nitric oxide (NO), an inflammatory substance generated in macrophages
  • macrophages RAW264.7 were purchased from the Korea Cell Line Bank (Korea) and incubated with 10% (v/v) bovine calf serum (BCS, Gibco), 100 U/mL penicillin, and 100 mL of penicillin.
  • the cells were cultured at 37°C and 5% CO 2 (v/v) using DMEM (Dulbecco's modified Eagle's medium, HyClone) supplemented with ⁇ g/mL streptomycin (Gibco). Culture medium was changed every 2 to 3 days, and cells were cultured until 80 to 90% confluency in all experiments.
  • DMEM Dulbecco's modified Eagle's medium, HyClone
  • Macrophages RAW264.7 were seeded in a 12-well plate at a density of 5 200 ⁇ g/mL) and cultured for 24 hours. The group treated only with fine dust was used as a positive control group, and the untreated group was used as a negative control group.
  • the obtained culture supernatant was mixed with an equal amount of Griess reagent solution (Sigma-Aldrich Chemical Co.), and the absorbance was measured at 550 nm using a microplate reader.
  • the NO concentration of each experimental group was calculated by referring to a standard curve generated with known sodium nitrate (NaNO 2 ) concentration.
  • the NO level of the macrophage culture medium treated with fine dust significantly increased to 18.630 ⁇ M compared to the NO level of the macrophage culture medium (3.557 ⁇ M) that was not treated with fine dust.
  • the NO level of macrophage culture medium significantly decreased from 16.988 ⁇ M to 9.842 ⁇ M in a concentration-dependent manner of the tangerine core extract ( Figure 1).
  • Example 3 Effect of suppressing the expression of inflammatory cytokines occurring in macrophages
  • inflammatory cytokines IL interleukin-1 ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • macrophages were cultured by treating fine dust and tangerine core extracts at different concentrations in the same manner as in Example 1 above.
  • the group treated only with fine dust was used as a positive control group, and the untreated group was used as a negative control group.
  • the obtained cells were washed with cold 1 , 0.5% NP-40) was added and dissolved. After centrifuging the cell lysate at 14,000 rpm and 4°C for 20 minutes, only the supernatant was taken and the protein was quantified using a BCA (bicinchoninic acid) protein assay kit. The quantified protein was mixed in sample buffer (1M Tris-HCl (pH 6.8), 50% glycerol, 10% SDS, 1% bromophenol blue, and 5% mercaptoethanol) and boiled at 100°C for 5 minutes. Next, electrophoresis was performed on a 15% acrylamide gel.
  • sample buffer (1M Tris-HCl (pH 6.8), 50% glycerol, 10% SDS, 1% bromophenol blue, and 5% mercaptoethanol
  • the electrophoresed proteins were transferred to a methanol-activated polyvinylidene difluoride (PVDF) membrane at 30 V overnight.
  • PVDF polyvinylidene difluoride
  • the membrane was separated and blocked with 3% BSA (bovine serum albumin) for 2 hours, then added to primary antibody diluted in 3% BSA and incubated for 2 hours. Afterwards, the membrane was washed three times for 10 minutes each with 1 The membrane was again washed three times for 10 minutes each with 1 At this time, anti-goat-IL-1 ⁇ (AF401-NA, R&D systems), anti-rabbit-TNF- ⁇ (ab9739, Abcam), and anti-mouse- ⁇ -actin (ab6276, Abcam) were used as primary antibodies.
  • BSA bovine serum albumin
  • Donkey anti-Goat IgG, HRP (Invitrogen), Donkey anti-Rabbit IgG, HRP (Invitrogen), and Donkey anti-Mouse IgG, HRP (Invitrogen) were used.
  • the expression levels of IL-1 ⁇ and TNF- ⁇ were calculated as the fold of the expression levels of IL-1 ⁇ and TNF- ⁇ in each experimental group based on the untreated group.
  • a cell viability test was performed to determine whether the tangerine core extract exhibited cytotoxicity.
  • macrophages were seeded in a 12-well plate at a density of 5 . After incubation, 5 mg/ml of MTT (3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltrazolium Bromide) solution was added to each well and further cultured for 2 hours. Formazan crystals present in each well were dissolved in DMSO, and absorbance was measured at 540 nm using a microplate reader. Cell survival rate was calculated at each tangerine core extract concentration based on the cell survival rate of macrophages not treated with tangerine core extract.
  • MTT 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltrazolium Bromide
  • Example 5 Effect of suppressing expression of cell adhesion molecules occurring in vascular endothelial cells
  • vascular endothelial cells human umbilical artery endothelial cells, HUAECs
  • HUAECs human umbilical artery endothelial cells
  • the primary antibodies used were anti-rabbit-ICAM-1 (sc-7891, Santa Cruz), anti-rabbit-VCAM-1 (sc-8304, Santa Cruz), and anti-mouse- ⁇ -actin (ab6276, Abcam). ) was used, and as secondary antibodies, Donkey anti-Rabbit IgG, HRP (Invitrogen) and Donkey anti-Mouse IgG, HRP (Invitrogen) were used.
  • Example 6 Inhibitory effect on the expression of cell adhesion molecules generated in vascular endothelial cells by harmful substances derived from lung tissue epithelial cells
  • Western blot analysis was performed on the expression levels of cell adhesion molecules ICAM-1 and VCAM-1 according to tangerine core extract treatment in culture media containing harmful substances generated from lung tissue epithelial cells treated with fine dust.
  • lung tissue epithelial cells (Beas-2B) were grown at a density of 8X10 5 cells/ml (fine dust treatment group) or 3X10 5 cells/ml (control group) at 60 mm. After seeding the plate and pre-cultivating for 16 hours, fine dust (PM2.5, 200 ⁇ g/mL) or PBS (control) was added to DMEM/F12 medium (1:1 mixture of F-12 Nutrient Mixture and DMEM medium). were treated and cultured for 48 hours. 2 mL of culture medium upon completion of culture was obtained.
  • HUAECs Newly seeded HUAECs were seeded in 3ml on a 60 mm plate at a density of 4 was added to each well and cultured for 18 hours, and then the expression levels of ICAM-1 and VCAM-1 were analyzed by Western blot in the same manner as in Example 3. At this time, the same antibody as in Example 5 was used.
  • culture medium containing harmful substances derived from lung tissue epithelial cells treated with 200 ⁇ g/mL of fine dust increased the expression levels of ICAM-1 and VCAM-1 in HUAECs compared to control culture medium, but treatment with tangerine core extract
  • the expression levels of ICAM-1 and VCAM-1 were significantly decreased in a concentration-dependent manner of the tangerine core extract ( Figure 5).
  • Macrophages were treated with tangerine core extract (200 ⁇ g/mL) or tangerine peel extract (200 ⁇ g/mL) in the same manner as in Example 2, and the amount of NO in the culture medium was analyzed.
  • the levels of ICAM-1 and VCAM-1 expressed when fine dust (PM2.5, 200 ⁇ g/mL) was treated with fine dust-treated vascular endothelial cell model in the same manner as in Example 5 above were measured in tangerine core extract (0 , 100 or 200 ⁇ g/mL) or tangerine peel (0, 100 or 200 ⁇ g/mL) extract treatment.
  • the control model was treated with DMSO.
  • Example 8 Inhibitory effect of tangerine core extract on the increase in cell adhesion molecule expression occurring in vascular endothelial cells caused by endotoxin (LPS)
  • vascular endothelial cell model that simulates the direct effect of endotoxin (LPS, lipopolysaccharide) infiltrating the body on vascular endothelial cells
  • cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM
  • LPS lipopolysaccharide
  • human umbilical artery endothelial cells (HUAECs) were seeded on a 60 mm plate at a density of 4X10 5 cells/ml and pre-cultured for 16 hours. , treated with endotoxin (LPS, 50 ng/mL) or PBS (control), treated with tangerine core extract at different concentrations (50, 100, or 200 ⁇ g/mL) and cultured for 4 hours, then ICAM-1 and VCAM- The expression level of 1 was analyzed by Western blot in the same manner as in Example 3 above.
  • the primary antibodies used were anti-rabbit-ICAM-1 (sc-7891, Santa Cruz), anti-rabbit-VCAM-1 (sc-8304, Santa Cruz), and anti-mouse- ⁇ -actin (ab6276, Abcam). ) was used, and as secondary antibodies, Donkey anti-Rabbit IgG, HRP (Invitrogen) and Donkey anti-Mouse IgG, HRP (Invitrogen) were used.
  • the expression levels of ICAM-1 and VCAM-1 were significantly increased in the endotoxin-treated vascular endothelial cell model treated with 50 ng/mL of endotoxin compared to the control model, but when treated with tangerine core extract, ICAM-1 and VCAM It was confirmed that the expression level of -1 was significantly decreased in a concentration-dependent manner of the tangerine core extract (FIG. 8).
  • mice After orally administering 100 mg/kg of tangerine core extract to mice, the expression levels of inflammatory cytokines in the plasma of mice treated with endotoxin were analyzed.
  • mice of the C57BL6/J strain were used. Tangerine core extract was administered orally once at 100 mg/kg, and DMSO was used in the control group. 30 minutes after administration, 5 mg/kg of LPS was administered intraperitoneally, and phosphate buffer solution was administered intraperitoneally to the control group. 24 hours after administration, blood was collected from the orbital vein of the mouse using a capillary blood collection tube. After blood collection, it was stored on ice for 30 minutes and centrifuged at 3000 rpm for 15 minutes to separate plasma. A cytokine array was performed to simultaneously observe changes in 111 types of cytokines in separated plasma. The kit used at this time was Proteome Profiler Mouse XL Cytokine Array (R&D Systems, ARY028), and was performed according to the method suggested by the manufacturer.
  • the tangerine core extract suppresses the inflammatory response and the increased expression of cell adhesion molecules in the body, and this can be applied to the prevention, improvement or treatment of diseases caused by fine dust.

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Abstract

The present invention relates to: a composition for the prevention, amelioration or treatment of diseases caused by particulate matter or endotoxin contained in particulate matter, the composition comprising a Citri tangerinae Semen extract; and use thereof. The Citri tangerinae Semen extract of the present invention inhibits, in vivo, inflammatory responses caused by particulate matter or endotoxin contained in particulate matter, and increased expression of cell adhesion molecules, and thus can be applied to the prevention, amelioration or treatment of diseases caused by particulate matter or endotoxin contained in particulate matter.

Description

귤핵 추출물을 포함하는 미세먼지로 유발된 질환의 예방, 개선 또는 치료용 조성물 및 이의 용도Composition for preventing, improving or treating diseases caused by fine dust containing tangerine core extract and use thereof
본 발명은 귤핵(Citri tangerinae Semen) 추출물을 포함하는 미세먼지로 유발된 질환의 예방, 개선 또는 치료용 조성물 및 이의 용도에 관한 것이다.The present invention relates to a composition for preventing, improving or treating diseases caused by fine dust containing Citri tangerinae Semen extract and its use.
미세먼지에 대한 높은 노출로 인해 인간, 식물 및 동물의 건강에 해로운 영향을 미치는 것은 전세계적으로 잘 알려져 있다.It is well known worldwide that high exposure to fine dust has detrimental effects on the health of humans, plants and animals.
미세먼지는 공기 중의 총 부유분진 중 직경 10μm 이하의 먼지(PM, particulate matter)로 정의되며, 지름이 10μm 보다 작은 미세먼지(PM10)와 지름이 2.5μm보다 작은 초미세먼지(PM2.5)로 나뉜다. 미세먼지는 입자의 크기가 매우 작아 인체 내로 더 깊숙이 침투하여 다양한 인체 내 염증반응을 일으키며(Seagrave J., Toxicology and applied pharmacology 2008; 232(3): 469-77.), 여러 미세먼지의 건강유해평가 연구들에서 사망률 증가, 심혈관계에의 영향, 호흡기계에의 영향, 암 발병률 증가 등에 대한 관련성이 보고되었다(유이란 외, 대한한방내과학회지 제40권 3호, 2019.). Fine dust is defined as particulate matter (PM) with a diameter of 10 μm or less among the total floating dust in the air, and is divided into fine dust with a diameter of less than 10 μm (PM10) and ultrafine dust with a diameter of less than 2.5 μm (PM2.5). Divided. Fine dust particles are very small in size, so they penetrate deeper into the human body and cause various inflammatory reactions in the human body (Seagrave J., Toxicology and applied pharmacology 2008; 232(3): 469-77.), and there are various health hazards of fine dust. Evaluation studies have reported an association with increased mortality, effects on the cardiovascular system, effects on the respiratory system, and increased cancer incidence (Yu-Ran et al., Journal of Korean Oriental Medicine, Volume 40, No. 3, 2019).
특히, 미세먼지는 내독소와 같은 인간의 건강에 부정적인 영향을 미치는 것과 밀접한 관련이 있다. 즉, 미세먼지에는 내독소나 곰팡이와 같은 여러 생물학적 요소가 포함되기도 한다. 예를 들어, 미세먼지는 그람 음성균의 외부 세포막의 주요 구성 성분인 내독소가 염증을 매개하여 천식, 발열, 오한, 관절통, 심혈관 질환과 같은 다양한 호흡기 문제에 역할을 하는 것으로 알려져 있다(Jing Li et al., Atmospheric Environment 212 (2019) 305-315). In particular, fine dust is closely related to negative effects on human health, such as endotoxin. In other words, fine dust may contain various biological elements such as endotoxins and mold. For example, fine dust is known to play a role in various respiratory problems such as asthma, fever, chills, joint pain, and cardiovascular disease by mediating inflammation through endotoxin, a major component of the outer cell membrane of Gram-negative bacteria (Jing Li et al. al., Atmospheric Environment 212 (2019) 305-315).
또한, 2013년 세계보건기구(World Health Organization, WHO) 유럽지구 보고서에 따르면 PM2.5에 장기간(수개월 이상) 노출시, PM2.5 농도가 10 μg/m3 증가할 때마다 심장질환 및 폐질환으로 인한 사망률이 6~13% 증가한다고 보고되었다(WHO regional office for Europe. 2013. Health effects of particulate matter.).In addition, according to the 2013 World Health Organization (WHO) European Region report, when exposed to PM2.5 for a long period of time (several months or more), heart disease and lung disease occur every time the PM2.5 concentration increases by 10 μg/m 3 . It has been reported that the mortality rate due to this increases by 6-13% (WHO regional office for Europe. 2013. Health effects of particulate matter.).
한편, 귤핵(Citri tangerinae Semen)은 귤나무인 Citrus unshiu Marcorvich 또는 Citrus reticulata Blanco (운향과 Rutaceae)의 잘 익은 씨를 의미한다. 한국에서는 대한민국약전외한약(생약)규격집에 생약으로써 등재되어 있다.Meanwhile, Citri tangerinae Semen refers to the ripe seeds of the tangerine tree Citrus unshiu Marcorvich or Citrus reticulata Blanco (Rutaceae). In Korea, it is registered as a herbal medicine in the Korean Pharmacopoeia Specification for Herbal Medicine (Herbal Medicine).
본 발명자들은 체내에서 미세먼지 또는 미세먼지에 포함된 내독소에 의해 유발되는 염증 반응 및 세포 부착 분자의 발현 증가를 귤핵(Citri tangerinae Semen) 추출물이 억제시켜, 귤핵 추출물을 미세먼지 또는 미세먼지에 포함된 내독소로 유발된 질환의 예방, 개선 또는 치료에 적용할 수 있음을 확인함으로써 본 발명을 완성하였다.The present inventors have shown that Citri tangerinae Semen extract suppresses the inflammatory response and increased expression of cell adhesion molecules caused by fine dust or endotoxin contained in fine dust in the body, and that the Citri tangerinae Semen extract is included in fine dust or fine dust. The present invention was completed by confirming that it can be applied to the prevention, improvement, or treatment of diseases caused by endotoxin.
본 발명의 하나의 목적은 귤핵(Citri tangerinae Semen) 추출물을 유효성분으로 포함하는, 미세먼지로 유발된 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.One object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by fine dust, containing Citri tangerinae Semen extract as an active ingredient.
본 발명의 다른 하나의 목적은 본 발명의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 미세먼지로 유발된 질환의 예방 또는 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing or treating diseases caused by fine dust, comprising administering the pharmaceutical composition of the present invention to an individual.
본 발명의 또 다른 하나의 목적은 귤핵 추출물을 포함하는, 미세먼지 유발 질환의 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving fine dust-induced diseases, including tangerine core extract.
본 발명의 또 다른 하나의 목적은 귤핵 추출물을 포함하는, 미세먼지 유발 질환의 예방 또는 개선용 사료 조성물을 제공하는 것이다.Another object of the present invention is to provide a feed composition for preventing or improving fine dust-induced diseases, including tangerine core extract.
본 발명의 귤핵(Citri tangerinae Semen) 추출물은 체내에서 미세먼지에 의해 유발되는 염증 반응 및 세포 부착 분자의 발현 증가를 억제시켜, 미세먼지로 유발된 질환의 예방, 개선 또는 치료에 적용할 수 있다.The Citri tangerinae Semen extract of the present invention suppresses the inflammatory response and increased expression of cell adhesion molecules caused by fine dust in the body, and can be applied to the prevention, improvement, or treatment of diseases caused by fine dust.
도 1은 귤핵(Citri tangerinae Semen) 추출물의 미세먼지에 의해 대식세포에서 발생하는 일산화질소(nitric oxide, NO) 생성 억제 효과를 나타낸 도이다.Figure 1 is a diagram showing the inhibitory effect of Citri tangerinae Semen extract on the production of nitric oxide (NO) generated in macrophages by fine dust.
도 2는 귤핵 추출물의 미세먼지에 의해 대식세포에서 발생하는 IL(interleukin)-1β 및 TNF-α(tumor necrosis factor-α) 발현 억제 효과를 나타낸 도이다.Figure 2 shows the fine dust generated in macrophages by the tangerine core extract. This diagram shows the effect of suppressing the expression of IL (interleukin)-1β and TNF-α (tumor necrosis factor-α).
도 3은 귤핵 조성물의 세포 독성을 나타낸 도이다.Figure 3 is a diagram showing the cytotoxicity of the tangerine core composition.
도 4는 귤핵 추출물의 미세먼지에 의해 혈관 내피세포에서 발생하는 세포 부착 분자[ICAM-1(intercellular adhesion molecule-1) 및 VCAM-1(vascular cell adhesion molecule-1)]의 발현 증가에 대한 억제 효과를 나타낸 도이다.Figure 4 shows the inhibitory effect on the increase in expression of cell adhesion molecules [ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1)] generated in vascular endothelial cells by fine dust of tangerine core extract. This is a diagram showing .
도 5는 귤핵 추출물의 미세먼지 처리된 폐 조직 상피세포 유래 유해 물질에 의한 혈관 내피세포에서의 세포 부착 분자의 발현 증가에 대한 억제 효과를 나타낸 도이다.Figure 5 is a diagram showing the inhibitory effect of tangerine core extract on the increase in the expression of cell adhesion molecules in vascular endothelial cells caused by harmful substances derived from lung tissue epithelial cells treated with fine dust.
도 6은 귤핵 추출물 및 귤피 추출물의 미세먼지에 의해 대식세포에서 발생하는 NO 생성 억제 효과를 나타낸 도이다.Figure 6 is a diagram showing the inhibitory effect of tangerine core extract and tangerine peel extract on NO production generated in macrophages by fine dust.
도 7은 귤핵 추출물 및 귤피 추출물의 미세먼지에 의해 혈관 내피세포에서 발생하는 세포 부착 분자(ICAM-1 및 VCAM-1)의 발현 증가에 대한 억제 효과를 나타낸 도이다.Figure 7 is a diagram showing the inhibitory effect of tangerine core extract and tangerine peel extract on the increase in expression of cell adhesion molecules (ICAM-1 and VCAM-1) generated in vascular endothelial cells by fine dust.
도 8은 귤핵 추출물의 내독소(LPS)에 의해 혈관 내피세포에서 발생하는 세포 부착 분자의 발현 증가에 대한 억제효과를 나타낸 도이다.Figure 8 is a diagram showing the inhibitory effect of endotoxin (LPS) of tangerine core extract on the increase in expression of cell adhesion molecules occurring in vascular endothelial cells.
도 9는 귤핵 추출물의 내독소 주입 마우스모델에서 항염증 효과를 나타낸 것이다.Figure 9 shows the anti-inflammatory effect of tangerine core extract in an endotoxin-injected mouse model.
본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered to be limited by the specific description described below.
본 발명의 하나의 양태는 귤핵(Citri tangerinae Semen) 추출물을 유효성분으로 포함하는, 미세먼지로 유발된 질환의 예방 또는 치료용 약학적 조성물을 제공한다. One aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases caused by fine dust, comprising Citri tangerinae Semen extract as an active ingredient.
본 발명에서 용어, "귤핵(Citri tangerinae Semen)"은 귤나무의 잘 익은 씨를 의미한다. 한국에서는 대한민국약전외한약(생약)규격집에 생약으로써 등재되어 있다. 상기 귤나무는 당업계에 알려진 귤나무라면 특별히 제한되지 않으나, 일 예로 Citrus unshiu Marcorvich 및 Citrus reticulata Blanco (운향과 Rutaceae) 등일 수 있다.In the present invention, the term " Citri tangerinae Semen " refers to the ripe seeds of a tangerine tree. In Korea, it is registered as a herbal medicine in the Korean Pharmacopoeia Specification for Herbal Medicine (Herbal Medicine). The tangerine tree is not particularly limited as long as it is a tangerine tree known in the art, but examples include Citrus unshiu Marcorvich and Citrus reticulata Blanco (Rutaceae).
본 발명에서 용어, "추출물(extract)"은, 목적하는 물질을 다양한 용매에 침지한 다음, 상온, 저온 또는 가온 상태에서 일정시간 동안 추출하여 수득한 액상성분, 상기 액상성분으로부터 용매를 제거하여 수득한 고형분 등의 결과물을 의미한다. 뿐만 아니라, 상기 결과물에 더하여, 상기 결과물의 희석액, 이들의 농축액, 이들의 조정제물, 정제물 등을 모두 포함하는 것으로 포괄적으로 해석될 수 있다.In the present invention, the term "extract" refers to a liquid component obtained by immersing the desired material in various solvents and then extracting it at room temperature, low temperature, or heated state for a certain period of time, and a liquid component obtained by removing the solvent from the liquid component. It refers to the result of solid content, etc. In addition, in addition to the above results, it can be comprehensively interpreted to include all dilutions of the results, concentrates thereof, crude preparations, purifications, etc. of the above results.
본 발명에 있어서, 상기 추출물은 귤나무 씨앗, 즉 귤핵의 추출물일 수 있다. 상기 추출물은 물 또는 배지, 알코올 등의 다양한 유기용매 등으로 추출하여 수득할 수 있다. 이때, 사용되는 유기용매는 추출물을 수득할 수 있는 한, 특별히 이에 제한되지 않으나, 구체적으로는 물, 극성용매 또는 비극성용매가 될 수 있고, 보다 구체적으로는 물, 배지, 탄소수 1 내지 6의 저급 알코올[C1 내지 6, 예를 들어, 메탄올, 에탄올(에틸 알코올), 프로판올 또는 부탄올 등], 또는 이들의 혼합용매 일 수 있다.In the present invention, the extract may be an extract of tangerine tree seeds, that is, tangerine core. The extract can be obtained by extraction with water or various organic solvents such as media and alcohol. At this time, the organic solvent used is not particularly limited as long as the extract can be obtained, but may specifically be water, a polar solvent, or a non-polar solvent, and more specifically, water, a medium, or a low-grade solvent having 1 to 6 carbon atoms. It may be alcohol [C1 to 6, for example, methanol, ethanol (ethyl alcohol), propanol, or butanol, etc.], or a mixed solvent thereof.
또한, 상기 추출물을 수득하기 위한 방법은 상기 귤핵의 추출물을 수득할 수 있는 한, 특별히 이에 제한되지 않으며, 당해 기술분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 예를 들어, 상기 방법은 열수 추출법, 초음파 추출법, 여과법, 환류 추출법, 초임계 추출법 등을 포함하며 이를 단독으로 수행하거나 2종 이상의 방법을 병용하여 수행할 수 있으나, 이에 제한되지 않는다.Additionally, the method for obtaining the extract is not particularly limited, as long as the extract of the tangerine core can be obtained, and extraction may be performed according to a method commonly used in the art. For example, the method includes hot water extraction, ultrasonic extraction, filtration, reflux extraction, supercritical extraction, etc., and may be performed alone or in combination of two or more methods, but is not limited thereto.
상기 귤핵은 상업적으로 판매되는 것을 구입해서 사용하거나, 자연에서 채취 또는 재배된 것을 사용할 수 있다.The tangerine core can be purchased and used commercially, or collected or cultivated from nature.
본 발명의 귤핵은 이의 추출물뿐만 아니라, 귤핵 그 자체, 이의 건조물, 발효액 또는 분획물 등의 형태로 본 발명의 조성물에 포함될 수 있으나, 이에 제한되지 않는다.The tangerine core of the present invention may be included in the composition of the present invention in the form of not only its extract, but also the tangerine core itself, its dried product, fermented broth, or fraction, but is not limited thereto.
본 발명에 있어서, 본 발명의 조성물은 세포 독성을 나타내지 않는 것일 수 있다.In the present invention, the composition of the present invention may not exhibit cytotoxicity.
본 발명의 일 구현예에서, 귤핵 추출물은 고농도(200 μg/mL)에서도 세포 독성이 나타나지 않았다(도 3).In one embodiment of the present invention, the tangerine core extract did not show cytotoxicity even at high concentration (200 μg/mL) (FIG. 3).
본 발명에 있어서, 본 발명의 조성물은 면역세포에서 일산화질소 생성 억제, IL(interleukin)-1β 발현 감소 및 TNF-α(tumor necrosis factor-α) 발현 감소로 이루어지는 군으로부터 선택되는 어느 하나 이상의 효과를 갖는 것일 수 있다.In the present invention, the composition of the present invention has one or more effects selected from the group consisting of inhibiting nitric oxide production in immune cells, reducing IL (interleukin)-1β expression, and TNF-α (tumor necrosis factor-α) expression. It may be something to have.
본 발명의 일 구현예에서, 미세먼지를 처리하지 않은 대식세포 배양액의 NO 수준 대비 미세먼지를 처리한 대식세포 배양액의 NO 수준이 크게 증가하였다. 반면, 귤핵 추출물과 미세먼지를 동시 처리한 경우 대식세포 배양액의 NO 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소함을 확인하였다(도 1).In one embodiment of the present invention, the NO level of the macrophage culture medium treated with fine dust significantly increased compared to the NO level of the macrophage culture medium not treated with fine dust. On the other hand, when tangerine core extract and fine dust were simultaneously treated, the NO level of macrophage culture medium was confirmed to significantly decrease in a tangerine core extract concentration-dependent manner (Figure 1).
본 발명의 다른 일 구현예에서, 미세먼지를 처리하지 않은 대식세포에서의 IL-1β 및 TNF-α 대비 미세먼지를 처리한 대식세포에서 IL-1β 및 TNF-α의 발현 수준이 크게 증가하였다. 반면, 귤핵 추출물과 미세먼지를 동시 처리한 경우 대식세포에서의 IL-1β 및 TNF-α의 발현 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소함을 확인하였다(도 2).In another embodiment of the present invention, the expression levels of IL-1β and TNF-α were significantly increased in macrophages treated with fine dust compared to IL-1β and TNF-α in macrophages not treated with fine dust. On the other hand, when tangerine core extract and fine dust were simultaneously treated, the expression levels of IL-1β and TNF-α in macrophages were confirmed to significantly decrease in a tangerine core extract concentration-dependent manner (Figure 2).
본 발명에 있어서, 본 발명의 조성물은 혈관 내피세포에서 세포 부착 분자의 발현 증가를 억제하는 것일 수 있다.In the present invention, the composition of the present invention may inhibit increased expression of cell adhesion molecules in vascular endothelial cells.
상기 세포 부착 분자는 ICAM-1(intercellular adhesion molecule-1) 또는 VCAM-1(vascular cell adhesion molecule-1) 중 선택되는 어느 하나 이상일 수 있으나, 이에 제한되지 않는다.The cell adhesion molecule may be one or more selected from ICAM-1 (intercellular adhesion molecule-1) or VCAM-1 (vascular cell adhesion molecule-1), but is not limited thereto.
본 발명의 일 구현예에서, 미세먼지를 처리한 미세먼지 처리 혈관 내피세포 모델에서 대조군 모델에 비해 ICAM-1 및 VCAM-1의 발현 수준이 현저히 증가하였으나, 귤핵 추출물을 처리한 경우 ICAM-1 및 VCAM-1의 발현 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소함을 확인하였다(도 4).In one embodiment of the present invention, the expression levels of ICAM-1 and VCAM-1 were significantly increased in the vascular endothelial cell model treated with fine dust compared to the control model, but when treated with tangerine core extract, ICAM-1 and It was confirmed that the expression level of VCAM-1 was significantly decreased in a concentration-dependent manner of the tangerine core extract (Figure 4).
본 발명의 다른 일 구현예에서, 미세먼지를 처리한 폐 조직 상피세포에서 유래한 유해 물질을 포함하는 배양액은 대조군 배양액에 비해 혈관 내피세포에서 ICAM-1 및 VCAM-1의 발현 수준을 증가시켰으나, 귤핵 추출물을 처리한 경우 ICAM-1 및 VCAM-1의 발현 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소함을 확인하였다(도 5).In another embodiment of the present invention, the culture medium containing harmful substances derived from lung tissue epithelial cells treated with fine dust increased the expression levels of ICAM-1 and VCAM-1 in vascular endothelial cells compared to the control culture medium. When treated with tangerine core extract, it was confirmed that the expression levels of ICAM-1 and VCAM-1 were significantly decreased in a concentration-dependent manner of tangerine core extract (Figure 5).
따라서, 본 발명의 조성물은 전술한 효과에 의해 미세먼지로 유발된 질환의 예방, 개선 또는 치료 효과를 나타내는 것일 수 있다.Therefore, the composition of the present invention may exhibit the effect of preventing, improving, or treating diseases caused by fine dust due to the above-mentioned effects.
특히, 본 발명 조성물의 유효성분인 귤핵 추출물은 귤핵이 아닌 귤의 다른 부위, 예컨대 귤의 껍질 부위(귤피)의 추출물보다 면역세포에서의 일산화질소 생성 억제 효과 및/또는 혈관 내피세포에서 세포 부착 분자의 발현 증가 억제 효과가 현저한바, 동일한 귤이라도 부위에 따라 미세먼지로 유발된 질환의 예방, 개선 또는 치료 효과가 상이함을 확인한 것에 의의가 있다.In particular, the tangerine core extract, which is the active ingredient of the composition of the present invention, has an inhibitory effect on nitric oxide production in immune cells and/or cell adhesion molecules in vascular endothelial cells more than extracts from other parts of the tangerine other than the tangerine core, such as the peel part of the tangerine (tangerine peel). Since the effect of suppressing the increase in the expression of
본 발명의 일 구현예에서, 귤핵 추출물 및 귤피 추출물 모두 대식세포 배양액에서 NO 생산을 억제하였으나, 귤핵 추출물의 NO 생산 억제능이 귤피 추출물에 비하여 높음을 확인하였다(도 6).In one embodiment of the present invention, both tangerine core extract and tangerine peel extract inhibited NO production in macrophage culture medium, but it was confirmed that the NO production inhibition ability of tangerine core extract was higher than that of tangerine peel extract (FIG. 6).
본 발명의 다른 일 구현예에서, 귤피 추출물에서는 미세먼지 처리 혈관 내피세포 모델에서 ICAM-1 및 VCAM-1 발현 증가 억제가 확인되지 않은 반면, 귤핵 추출물에서는 ICAM-1 및 VCAM-1의 발현 증가가 억제됨을 확인하였다(도 7).In another embodiment of the present invention, the tangerine peel extract did not inhibit the increase in ICAM-1 and VCAM-1 expression in the fine dust-treated vascular endothelial cell model, whereas the tangerine core extract increased the expression of ICAM-1 and VCAM-1. It was confirmed that it was inhibited (Figure 7).
본 발명에서 용어, "미세먼지로 유발된 질환"은 미세먼지의 체내 유입에 의해 발생하는 질환이라면 제한 없이 포함할 수 있으며, 일 예로, 미세먼지의 체내 유입에 의해 발생하는 염증이 원인이 되는 질환일 수 있으나, 이에 제한되지 않는다. 그 예로, 상기 미세먼지로 유발된 질환은 염증성 질환, 심혈관계 질환, 대사성 질환, 신경계 질환, 호흡기계 질환 및 안질환 등일 수 있다.In the present invention, the term "disease caused by fine dust" may include without limitation any disease caused by the inflow of fine dust into the body, for example, a disease caused by inflammation caused by the inflow of fine dust into the body. It may be, but is not limited to this. For example, diseases caused by fine dust may include inflammatory diseases, cardiovascular diseases, metabolic diseases, nervous system diseases, respiratory system diseases, and eye diseases.
상기 염증성 질환의 예로, 폐렴, 기도염, 만성호흡기질환(Chronic Respiratory Diseases, COPD) 및 피부염 등이 있으나, 이에 제한되지 않는다. Examples of the inflammatory diseases include, but are not limited to, pneumonia, tracheitis, chronic respiratory diseases (COPD), and dermatitis.
상기 심혈관계 질환의 예로, 고혈압, 심부정맥, 허혈성 심질환, 심근경색, 심부전 및 심부정맥 등이 있으나, 이에 제한되지 않는다.Examples of the cardiovascular diseases include, but are not limited to, high blood pressure, cardiac arrhythmia, ischemic heart disease, myocardial infarction, heart failure, and cardiac arrhythmia.
상기 대사성 질환의 예로, 당뇨병 등이 있으나, 이에 제한되지 않는다.Examples of the metabolic disease include, but are not limited to, diabetes.
상기 신경계 질환의 예로, 뇌졸중, 신경발달장애, 알츠하이머병 및 정신질환 악화 등이 있으나, 이에 제한되지 않는다.Examples of the neurological diseases include, but are not limited to, stroke, neurodevelopmental disorders, Alzheimer's disease, and worsening mental disorders.
상기 호흡기계 질환의 예로, 폐암, 폐성장 장애 및 상기도 감염 등이 있으나, 이에 제한되지 않는다.Examples of the respiratory diseases include, but are not limited to, lung cancer, lung growth disorders, and upper respiratory tract infections.
상기 안질환의 예로, 안구건조증 및 망막미세혈관손상 등이 있으나, 이에 제한되지 않는다.Examples of the eye disease include, but are not limited to, dry eye syndrome and retinal microvascular damage.
본 발명에 있어서, 상기 미세먼지로 유발된 질환은 염증성 질환일 수 있으나, 이에 제한되는 것은 아니며, 상기 미세먼지의 체내 유입에 의해 발생하는 염증이 원인이 되는 질환도 본 발명의 범위에 모두 포함될 수 있다.In the present invention, the disease caused by fine dust may be an inflammatory disease, but is not limited thereto, and diseases caused by inflammation caused by the inflow of fine dust into the body may also be included within the scope of the present invention. there is.
또한, 본 발명의 '미세먼지로 유발된 질환'은 미세먼지에 포함된 내독소에 의한 다양한 염증성 질환일 수 있으나, 이에 제한되지 않는다. 본 발명에서는 '미세먼지에 포함된 내독소에 의해 유발된 질환'과 혼용되어 사용될 수 있다. In addition, the 'disease caused by fine dust' of the present invention may be, but is not limited to, various inflammatory diseases caused by endotoxin contained in fine dust. In the present invention, it can be used interchangeably with ‘disease caused by endotoxin contained in fine dust’.
본 발명에서 용어, "내독소(endotoxin)”는 그람음성 세균 유래 지질다당류(Lipopolysaccharide, LPS), 이의 유도체, 또는 지질 A(lipid A) 함유 화합물일 수 있다. 상기 내독소는 사이토카인과 같은 염증성 인자를 활성화할 수 있다. In the present invention, the term "endotoxin" may be lipopolysaccharide (LPS) derived from Gram-negative bacteria, a derivative thereof, or a compound containing lipid A. The endotoxin may be an inflammatory agent such as a cytokine. The argument can be activated.
본 발명의 미세먼지는 그람 음성균의 외부 세포막의 주요 구성 성분인 내독소가 염증을 매개하여 염증이 원인이 되는 질환을 유발할 수 있으나, 이에 제한되지 않는다. 그 예로, 상기 미세먼지로 유발된 질환 및/또는 미세먼지에 포함된 내독소에 의해 유발된 질환은 염증성 질환, 심혈관계 질환, 대사성 질환, 신경계 질환, 호흡기계 질환 및 안질환 등일 수 있다.The fine dust of the present invention may cause diseases caused by inflammation through endotoxin, a major component of the outer cell membrane of Gram-negative bacteria, mediating inflammation, but is not limited to this. For example, the disease caused by fine dust and/or the disease caused by endotoxin contained in fine dust may include inflammatory disease, cardiovascular disease, metabolic disease, nervous system disease, respiratory system disease, and eye disease.
본 발명의 다른 일 구현예에서, 내독소 처리 혈관 내피세포 모델에서 처리한 경우 ICAM-1 및 VCAM-1의 발현 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소함을 확인하였다(도 8).In another embodiment of the present invention, it was confirmed that when treated in an endotoxin-treated vascular endothelial cell model, the expression levels of ICAM-1 and VCAM-1 were significantly reduced in a concentration-dependent manner of the tangerine core extract (FIG. 8).
본 발명의 다른 일 구현예에서, 내독소가 처리된 마우스의 혈장 내 염증성 사이토카인의 발현량을 분석한 결과, 통계적으로 유의미하게 발현된 상위 6개의 혈장 단백질을 동정하였다. In another embodiment of the present invention, as a result of analyzing the expression level of inflammatory cytokines in the plasma of mice treated with endotoxin, the top six statistically significantly expressed plasma proteins were identified.
본 발명에서 용어, "예방"은 상기 조성물의 투여에 의해 미세먼지로 유발된 질환을 억제하거나 발병을 지연시키는 모든 행위를 의미하며, "치료"는 상기 조성물의 투여에 의해 미세먼지로 유발된 질환에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. In the present invention, the term "prevention" refers to all actions that suppress or delay the onset of diseases caused by fine dust by administration of the composition, and "treatment" refers to diseases caused by fine dust by administration of the composition. It refers to any action that improves or changes symptoms to a benefit.
본 발명의 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형체 또는 희석제를 추가로 포함할 수 있다. 이때, 상기 약학적 조성물에 포함되는 유효성분인 귤핵 추출물의 함량은 특별히 이에 제한되지 않으나, 조성물 총 중량에 대하여 0.0001 중량% 내지 10 중량%로, 구체적으로 0.001 중량% 내지 1 중량%를 포함할 수 있다. 또한, 상기 약학적 조성물에 포함되는 유효성분인 귤핵 추출물의 농도는 특별히 이에 제한되지 않으나, 50 μg/mL 이상, 60 μg/mL 이상, 70 μg/mL 이상, 80 μg/mL 이상, 90 μg/mL 이상 또는 100 μg/mL 이상의 농도로 포함할 수 있으며, 구체적으로 100 내지 200 μg/mL의 농도로 포함할 수 있다.The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions. At this time, the content of tangerine core extract, which is an active ingredient contained in the pharmaceutical composition, is not particularly limited, but may include 0.0001% by weight to 10% by weight, specifically 0.001% by weight to 1% by weight, based on the total weight of the composition. there is. In addition, the concentration of the tangerine core extract, which is an active ingredient included in the pharmaceutical composition, is not particularly limited, but is 50 μg/mL or more, 60 μg/mL or more, 70 μg/mL or more, 80 μg/mL or more, 90 μg/mL or more. It may be included at a concentration of mL or more or 100 μg/mL or more, and specifically, it may be included at a concentration of 100 to 200 μg/mL.
상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 동결건조제제 및 좌제으로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있으며, 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구 투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, freeze-dried preparations, and suppositories. It can be in various oral or parenteral dosage forms. When formulated, it is 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. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.
본 발명의 조성물은 약학적으로 유효한 양으로 투여할 수 있다.The composition of the present invention can be administered in a pharmaceutically effective amount.
본 발명에서 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 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 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다. 본 발명의 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르지만, 바람직한 효과를 위해서, 본 발명의 조성물은 1일 0.0001 내지 500mg/kg으로, 구체적으로 0.001 내지 200mg/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 조성물은 쥐, 가축, 인간 등의 다양한 포유동물에 다양한 경로로 투여할 수 있으며, 투여의 방식은 당업계의 통상적인 방법이라면 제한없이 포함하며, 예를 들어, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다. In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, and severity of the disease. It can be determined based on factors including the type, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art. The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but for desirable effects, the composition of the present invention is administered in a specific dosage of 0.0001 to 500 mg/kg per day. It is recommended to administer from 0.001 to 200 mg/kg. Administration may be administered once a day, or may be administered several times. The composition can be administered to various mammals such as rats, livestock, and humans by various routes, and the method of administration includes without limitation any method conventional in the art, for example, orally, rectally, intravenously, intramuscularly, or intravenously. It may be administered by subcutaneous, intrauterine, intrathecal, or intracerebrovascular injection.
또한, 본 발명의 약학적 조성물은 인간에 적용되는 의약품뿐만 아니라, 동물 의약품의 형태로도 사용될 수 있다. 여기에서, 동물이란 가축 및 반려동물을 포함하는 개념이다.In addition, the pharmaceutical composition of the present invention can be used not only in the form of a medicine for humans, but also in the form of an animal medicine. Here, animals are a concept that includes livestock and companion animals.
본 발명의 다른 하나의 양태는, 본 발명의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 미세먼지로 유발된 질환의 예방 또는 치료 방법을 제공한다. Another aspect of the present invention provides a method for preventing or treating diseases caused by fine dust, comprising the step of administering the pharmaceutical composition of the present invention to an individual.
여기에서 사용되는 용어는 전술한 바와 같다.The terms used here are the same as described above.
상기 미세먼지로 유발된 질환은 일 예로, 염증성 질환일 수 있으나, 이에 제한되지 않는다. 이에 대해서는 이전 양태에서 전술한 바와 같다.For example, the disease caused by fine dust may be an inflammatory disease, but is not limited thereto. This is the same as described above in the previous aspect.
상기 "투여"는 어떠한 적절한 방법으로 개체에게 본 발명의 조성물을 도입하는 것을 의미하며, 상기 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 복강 내 투여, 정맥 내 투여, 근육 내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비 내 투여될 수 있으나, 이에 제한되지 않는다.The “administration” means introducing the composition of the present invention into an individual by any appropriate method, and the composition may be administered through any general route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, locally, or intranasally, but is not limited thereto.
상기 "개체"는 미세먼지로 유발된 질환이 발병하였거나 발병할 수 있는 인간을 제외한 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함한 모든 동물을 의미한다. 본 발명의 약학적 조성물을 개체에게 투여함으로써 상기 질환을 효과적으로 예방 또는 치료할 수 있다면 개체의 종류는 제한되지 않는다.The “individual” refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs other than humans who have or may develop diseases caused by fine dust. This means all animals, including The type of subject is not limited as long as the above disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.
본 발명의 또 다른 하나의 양태는 귤핵 추출물을 포함하는, 미세먼지 유발 질환의 예방 또는 개선용 식품 조성물을 제공한다.Another aspect of the present invention provides a food composition for preventing or improving fine dust-induced diseases, comprising a tangerine core extract.
여기에서 사용되는 용어는 전술한 바와 같다.The terms used here are the same as described above.
구체적으로, 미세먼지 유발 질환의 예방 또는 개선을 목적으로 본 발명의 유효성분인 귤핵 추출물을 식품 조성물에 첨가할 수 있다. Specifically, for the purpose of preventing or improving fine dust-induced diseases, tangerine core extract, which is an active ingredient of the present invention, can be added to food compositions.
본 발명의 유효성분을 식품 첨가물로 사용할 경우, 상기 유효성분을 그대로 첨가하거나 다른 식품 또는 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합량은 사용 목적에 따라 적합하게 결정할 수 있으며, 상기 식품 조성물에 포함되는 유효성분인 귤핵 추출물의 함량은 특별히 이에 제한되지 않으나, 조성물 총 중량에 대하여 0.0001 중량% 내지 10 중량%로, 바람직하게는 0.001 중량% 내지 1 중량%를 포함할 수 있다. 또한, 상기 조성물에 포함되는 유효성분인 귤핵 추출물의 농도는 특별히 이에 제한되지 않으나, 50 μg/mL 이상, 60 μg/mL 이상, 70 μg/mL 이상, 80 μg/mL 이상, 90 μg/mL 이상 또는 100 μg/mL 이상의 농도로 포함할 수 있으며, 구체적으로 100 내지 200 μg/mL의 농도로 포함할 수 있다.When using the active ingredient of the present invention as a food additive, the active ingredient can be added as is or used together with other foods or ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use, and the content of the tangerine core extract, which is an active ingredient included in the food composition, is not particularly limited, but is preferably 0.0001% by weight to 10% by weight based on the total weight of the composition. Typically, it may include 0.001% by weight to 1% by weight. In addition, the concentration of the tangerine core extract, which is an active ingredient contained in the composition, is not particularly limited, but is 50 μg/mL or more, 60 μg/mL or more, 70 μg/mL or more, 80 μg/mL or more, and 90 μg/mL or more. Alternatively, it may be included at a concentration of 100 μg/mL or more, and specifically, it may be included at a concentration of 100 to 200 μg/mL.
본 발명의 식품의 종류에는 특별한 제한은 없다. 상기 유효성분을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있고, 통상적인 의미에서의 식품을 모두 포함할 수 있으며, 동물을 위한 사료로 이용되는 식품을 포함할 수 있다.There is no particular limitation on the type of food of the present invention. Examples of foods to which the above active ingredients can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, and drink preparations. , alcoholic beverages and vitamin complexes, etc., and may include all foods in the conventional sense, and may include foods used as feed for animals.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄 산화제 등을 함유할 수 있다. 그밖에 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 또한, 상기 식품은 공지의 제조방법에 따라 정제, 과립, 분말, 캅셀, 액상의 용액 및 환 등의 제형으로도 제조될 수 있다. 본 발명에 따른 유효성분을 포함하는 것 이외에는 다른 성분에는 특별한 제한이 없으며, 통상의 여러 가지 향미제 또는 천연 탄수화물 등을 추가성분으로 포함할 수 있다.In addition to the above, the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, and alcohol. , may contain carbonation agents used in carbonated beverages, etc. In addition, it may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. In addition, the food can be manufactured in dosage forms such as tablets, granules, powders, capsules, liquid solutions, and pills according to known manufacturing methods. Other than containing the active ingredient according to the present invention, there is no particular limitation on other ingredients, and various common flavoring agents or natural carbohydrates may be included as additional ingredients.
본 발명의 또 다른 하나의 양태는 귤핵 추출물을 포함하는, 미세먼지 유발 질환의 예방 또는 개선용 사료 조성물을 제공한다.Another aspect of the present invention provides a feed composition for preventing or improving fine dust-induced diseases, comprising a tangerine kernel extract.
여기에서 사용되는 용어는 전술한 바와 같다.The terms used here are the same as described above.
구체적으로, 미세먼지 유발 질환의 예방 또는 개선을 목적으로 본 발명의 유효성분인 귤핵 추출물을 사료 조성물 또는 사료 첨가제에 첨가할 수 있다.Specifically, for the purpose of preventing or improving fine dust-induced diseases, tangerine core extract, which is an active ingredient of the present invention, can be added to feed compositions or feed additives.
본 발명에서 용어, "사료"는 개체의 생명을 유지하고 상기 개체를 사육하는데 필요한 유기 또는 무기 영양소를 공급하는 물질을 의미한다. 상기 사료는 사료를 섭취하는 개체가 필요로 하는 에너지, 단백질, 지질, 비타민, 광물질 등 영양소를 포함할 수 있으며, 특별히 이에 제한되지 않으나, 곡물류, 근과류, 식품가공부산물류, 조류, 섬유질류, 유지류, 전분류, 박류, 곡물부산물류 등의 식물성 사료 또는 단백질류, 무기물류, 유지류, 광물성류, 단세포 단백질, 동물성 플랑크톤류, 어분 등의 동물성 사료가 될 수 있다.In the present invention, the term “feed” refers to a substance that supplies organic or inorganic nutrients necessary for maintaining the life of an individual and raising the individual. The feed may contain nutrients such as energy, protein, lipid, vitamins, and minerals required by the individual consuming the feed, but is not particularly limited thereto, and may include grains, roots and fruits, food processing by-products, algae, and fiber. , vegetable feed such as fats and oils, starches, crustaceans, and grain by-products, or animal feed such as proteins, inorganic substances, oils, minerals, single-cell proteins, zooplankton, and fish meal.
상기 사료에는 개체의 성장촉진, 질병 예방 등을 위한 아미노산제, 비타민제, 효소제, 향미제, 비단백질태질소화합물, 규산염제, 완충제, 추출제, 올리고당 등의 추가적인 사료 첨가제가 포함될 수 있다.The feed may contain additional feed additives such as amino acids, vitamins, enzymes, flavoring agents, non-protein nitrogen compounds, silicate agents, buffers, extractants, and oligosaccharides for promoting individual growth and disease prevention.
본 발명에서 용어, "사료 첨가제"는 영양적 또는 특정 목적을 위하여 사료에 미량으로 첨가되는 물질을 총칭하는 것으로, 본 발명에서는 미세먼지 유발 질환의 예방 또는 개선을 목적으로 첨가되는 물질을 의미한다. In the present invention, the term “feed additive” refers to a general term for substances added in small amounts to feed for nutritional or specific purposes. In the present invention, it refers to substances added for the purpose of preventing or improving fine dust-induced diseases.
상기 사료 첨가제에는 품질 저하를 방지하기 위해 첨가되는 결착제, 유화제, 보존제 등을 추가로 포함할 수 있고, 효용 증대를 위하여 첨가되는 아미노산제, 비타민제, 효소제, 생균제, 향미제, 비단백태 질소화합물, 규산염제, 완충제, 착색제, 추출제, 올리고당 등을 추가로 포함할 수 있으며, 그 외에도 사료 혼합제 등을 추가로 포함할 수 있으며, 이에 한정된 것은 아니다.The feed additives may further include binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, and amino acids, vitamins, enzymes, probiotics, flavoring agents, non-protein nitrogen compounds, etc. added to increase utility. It may additionally include silicate agents, buffering agents, colorants, extractants, oligosaccharides, etc. In addition, it may additionally include feed mixtures, etc., but is not limited thereto.
상기 사료 조성물 또는 사료 첨가제에 포함되는 유효성분인 귤핵 추출물의 함량은 특별히 이에 제한되지 않으나, 조성물 총 중량에 대하여 0.0001 중량% 내지 10 중량%로, 구체적으로 0.001 중량% 내지 1 중량%를 포함할 수 있다. 또한, 상기 조성물에 포함되는 유효성분인 귤핵 추출물의 농도는 특별히 이에 제한되지 않으나, 50 μg/mL 이상, 60 μg/mL 이상, 70 μg/mL 이상, 80 μg/mL 이상, 90 μg/mL 이상 또는 100 μg/mL 이상의 농도로 포함할 수 있으며, 구체적으로 100 내지 200 μg/mL의 농도로 포함할 수 있다.The content of tangerine core extract, which is an active ingredient contained in the feed composition or feed additive, is not particularly limited, but may include 0.0001% by weight to 10% by weight, specifically 0.001% by weight to 1% by weight, based on the total weight of the composition. there is. In addition, the concentration of the tangerine core extract, which is an active ingredient contained in the composition, is not particularly limited, but is 50 μg/mL or more, 60 μg/mL or more, 70 μg/mL or more, 80 μg/mL or more, and 90 μg/mL or more. Alternatively, it may be included at a concentration of 100 μg/mL or more, and specifically, it may be included at a concentration of 100 to 200 μg/mL.
상기 개체는 사육 대상을 의미하는 것으로 본 출원의 사료를 섭취할 수 있는 생명체라면 제한 없이 포함되며, 예를 들면, 가축 및 반려동물을 포함하는 개념이다.The object refers to an object of breeding and includes without limitation any living organism that can consume the feed of this application, and includes, for example, livestock and companion animals.
본 발명의 또 다른 하나의 양태는 귤핵 추출물을 포함하는, 미세먼지 유발 질환의 예방 또는 개선용 의약외품 조성물을 제공한다.Another aspect of the present invention provides a quasi-drug composition for preventing or improving fine dust-induced diseases, comprising a tangerine core extract.
여기에서 사용되는 용어는 전술한 바와 같다.The terms used here are the same as described above.
구체적으로, 미세먼지 유발 질환의 예방 또는 개선을 목적으로 본 발명의 유효성분인 귤핵 추출물을 의약외품 조성물에 첨가할 수 있다.Specifically, for the purpose of preventing or improving fine dust-induced diseases, tangerine core extract, which is an active ingredient of the present invention, can be added to a quasi-drug composition.
본 발명에서 용어, "의약외품"은 사람이나 동물의 질병을 치료, 경감, 처치 또는 예방할 목적으로 사용되는 섬유, 고무제품 또는 이와 유사한 것, 인체에 대한 작용이 약하거나 인체에 직접 작용하지 아니하며, 기구 또는 기계가 아닌 것과 이와 유사한 것, 감염 예방을 위하여 살균, 살충 및 이와 유사한 용도로 사용되는 제제 중 하나에 해당하는 물품으로서, 사람이나 동물의 질병을 진단, 치료, 경감, 처치 또는 예방할 목적으로 사용하는 물품 중 기구, 기계 또는 장치가 아닌 것 및 사람이나 동물의 구조와 기능에 약리학적 영향을 줄 목적으로 사용하는 물품 중 기구, 기계 또는 장치가 아닌 것을 제외한 물품을 의미하며, 피부 외용제 및 개인위생용품도 포함한다.In the present invention, the term "quasi-drug" refers to fibers, rubber products or similar products used for the purpose of treating, alleviating, treating or preventing diseases in humans or animals, which have a weak effect on the human body or do not act directly on the human body, and devices. Or, it is an article that is not a machine or similar, or an agent used for sterilization, insecticide, and similar purposes to prevent infection, and is used for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases in humans or animals. It refers to articles other than instruments, machines, or devices among articles used for the purpose of having a pharmacological effect on the structure and function of humans or animals, excluding articles for external use on the skin and personal hygiene. Also includes supplies.
상기 피부 외용제는 특별히 이에 제한되지 않으나, 구체적으로 연고제, 로션제, 스프레이제, 패취제, 크림제, 산제, 현탁제, 겔제 또는 젤의 형태로 제조되어 사용될 수 있다. 상기 개인위생용품에는 특별히 이에 제한되지 않으나, 구체적으로 비누, 화장품, 물티슈, 휴지, 샴푸, 피부 크림, 얼굴 크림, 치약, 립스틱, 향수, 메이크업, 파운데이션, 볼터치, 마스카라, 아이섀도우, 선스크린 로션, 모발 손질 제품, 에어프레쉬너 겔 또는 세정 겔일 수 있다. 또한, 본 발명의 의약외품 조성물의 또 다른 예로 소독청결제, 샤워폼, 가그린, 물티슈, 세제비누, 핸드워시, 가습기 충진제, 마스크, 연고제 또는 필터충진제가 있다.The skin external preparation is not particularly limited thereto, but may be specifically prepared and used in the form of ointment, lotion, spray, patch, cream, powder, suspension, gel or gel. The personal hygiene products include, but are not limited to, soap, cosmetics, wet tissues, toilet paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, makeup, foundation, blush, mascara, eye shadow, and sunscreen lotion. , it may be a hair care product, air freshener gel or cleaning gel. Additionally, other examples of the quasi-drug composition of the present invention include disinfectant cleaners, shower foams, garnishes, wet tissues, detergent soaps, hand washes, humidifier fillers, masks, ointments, or filter fillers.
본 발명의 유효성분을 미세먼지 유발 질환의 예방 또는 개선을 목적으로 의약외품 조성물에 첨가할 경우, 상기 유효성분을 그대로 첨가하거나 다른 의약외품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효 성분의 혼합량은 사용 목적에 따라 적합하게 결정할 수 있으며, 상기 의약외품 조성물에 포함되는 유효성분인 귤핵 추출물의 함량은 특별히 이에 제한되지 않으나, 조성물 총 중량에 대하여 0.0001 중량% 내지 10 중량%로, 구체적으로 0.001 중량% 내지 1 중량%를 포함할 수 있다. 또한, 상기 조성물에 포함되는 유효성분인 귤핵 추출물의 농도는 특별히 이에 제한되지 않으나, 50 μg/mL 이상, 60 μg/mL 이상, 70 μg/mL 이상, 80 μg/mL 이상, 90 μg/mL 이상 또는 100 μg/mL 이상의 농도로 포함할 수 있으며, 구체적으로 100 내지 200 μg/mL의 농도로 포함할 수 있다.When adding the active ingredient of the present invention to a quasi-drug composition for the purpose of preventing or improving diseases caused by fine dust, the active ingredient can be added as is or used together with other quasi-drug ingredients, and can be used appropriately according to conventional methods. . The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use, and the content of the tangerine core extract, which is an active ingredient included in the quasi-drug composition, is not particularly limited, but is 0.0001% by weight to 10% by weight based on the total weight of the composition. It may include 0.001% by weight to 1% by weight. In addition, the concentration of the tangerine core extract, which is an active ingredient contained in the composition, is not particularly limited, but is 50 μg/mL or more, 60 μg/mL or more, 70 μg/mL or more, 80 μg/mL or more, and 90 μg/mL or more. Alternatively, it may be included at a concentration of 100 μg/mL or more, and specifically, it may be included at a concentration of 100 to 200 μg/mL.
이하, 본 발명을 하기 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1. 귤핵 추출물 제조Example 1. Preparation of tangerine core extract
귤핵 추출물은 그늘에서 건조되고 분말화된 귤핵에 에탄올(에틸 알코올) 95%[특급 시약(guaranteed reagent), GR급]을 첨가하고, 초음파 추출기(SDN-900H, Sd-ultrasonic co.)를 사용하여 실온에서 30주기(40KHz, 1500W, 15분 초음파 처리-120분 주기당 방치)로 추출하였다. 상기 수득물은 여과(Qualitative Filter No.100, Hyundai micro co.) 및 감압 건조 후 DMSO(Dimethyl sulfoxide)에 용해시켜 사용하였다. 한국생명공학연구원의 한국식물추출물은행에서 분양번호 CA01-020로 분양받을 수 있다.The tangerine core extract was obtained by adding 95% ethanol (ethyl alcohol) (guaranteed reagent, GR grade) to the dried and powdered tangerine core in the shade and using an ultrasonic extractor (SDN-900H, Sd-ultrasonic co.). Extraction was performed at room temperature with 30 cycles (40 KHz, 1500 W, 15 minutes sonication - 120 minutes per cycle). The obtained product was filtered (Qualitative Filter No. 100, Hyundai micro co.), dried under reduced pressure, and then dissolved in DMSO (Dimethyl sulfoxide) before use. It can be purchased from the Korean Plant Extract Bank of the Korea Research Institute of Bioscience and Biotechnology under the sales number CA01-020.
실시예 2. 대식세포에서 발생하는 염증 유발 물질인 일산화질소(nitric oxide, NO)의 생성 억제 효과Example 2. Effect of suppressing the production of nitric oxide (NO), an inflammatory substance generated in macrophages
대식세포에 귤핵 추출물과 미세먼지를 동시 처리한 후 배양액 내 NO 양을 분석하였다.After simultaneously treating macrophages with tangerine core extract and fine dust, the amount of NO in the culture medium was analyzed.
구체적으로, 대식세포 RAW264.7는 한국 세포 은행(Korea Cell Line Bank, 한국)에서 구입하여, 10% (v/v) 우아 혈청(Bovine calf serum; BCS, Gibco), 100 U/mL 페니실린, 100 μg/mL 스트렙토마이신(Gibco)이 보충된 DMEM(Dulbecco's modified Eagle's medium, HyClone)을 이용하여 37℃ 및 5% CO2 (v/v)의 조건에서 배양하였다. 배양 배지를 2 ~ 3 일마다 교체하고, 모든 실험에서 세포를 80 ~ 90 % confluency가 될 때까지 배양하였다. Specifically, macrophages RAW264.7 were purchased from the Korea Cell Line Bank (Korea) and incubated with 10% (v/v) bovine calf serum (BCS, Gibco), 100 U/mL penicillin, and 100 mL of penicillin. The cells were cultured at 37°C and 5% CO 2 (v/v) using DMEM (Dulbecco's modified Eagle's medium, HyClone) supplemented with μg/mL streptomycin (Gibco). Culture medium was changed every 2 to 3 days, and cells were cultured until 80 to 90% confluency in all experiments.
대식세포 RAW264.7를 5X105 cells/ml의 밀도로 12 웰 플레이트에 시딩하여 16시간 동안 예비 배양한 후, 미세먼지(PM2.5, 200 μg/mL) 및 농도별 귤핵 추출물(50, 100 또는 200 μg/mL)를 처리하고 24시간 동안 배양하였다. 미세먼지만을 단독 처리한 군을 양성 대조군, 무처리군을 음성 대조군으로 하였다. Macrophages RAW264.7 were seeded in a 12-well plate at a density of 5 200 μg/mL) and cultured for 24 hours. The group treated only with fine dust was used as a positive control group, and the untreated group was used as a negative control group.
배양 완료후 수득한 배양 상등액을 동량의 Griess 시약용액(Sigma-Aldrich Chemical Co.)과 혼합하고, 마이크로 플레이트 판독기를 사용하여 550 nm에서 흡광도를 측정하였다. NO 농도는 알려진 질산나트륨(NaNO2) 농도로 생성된 표준 곡선을 참조하여 각 실험군의 NO 농도를 산출하였다.After completion of the culture, the obtained culture supernatant was mixed with an equal amount of Griess reagent solution (Sigma-Aldrich Chemical Co.), and the absorbance was measured at 550 nm using a microplate reader. The NO concentration of each experimental group was calculated by referring to a standard curve generated with known sodium nitrate (NaNO 2 ) concentration.
그 결과, 미세먼지를 처리하지 않은 대식세포 배양액의 NO 수준(3.557 μM) 대비 미세먼지를 처리한 대식세포 배양액의 NO 수준이 18.630 μM으로 크게 증가하였다. 반면, 귤핵 추출물과 미세먼지를 동시 처리한 경우 대식세포 배양액의 NO 수준이 귤핵 추출물 농도 의존적으로 16.988 μM부터 9.842 μM까지 유의하게 감소하였다(도 1).As a result, the NO level of the macrophage culture medium treated with fine dust significantly increased to 18.630 μM compared to the NO level of the macrophage culture medium (3.557 μM) that was not treated with fine dust. On the other hand, when tangerine core extract and fine dust were simultaneously treated, the NO level of macrophage culture medium significantly decreased from 16.988 μM to 9.842 μM in a concentration-dependent manner of the tangerine core extract (Figure 1).
실시예 3. 대식세포에서 발생하는 염증성 사이토카인의 발현 억제 효과Example 3. Effect of suppressing the expression of inflammatory cytokines occurring in macrophages
대식세포에 귤핵 추출물과 미세먼지를 동시 처리한 후 세포에서 염증성 사이토카인인 IL(interleukin)-1β 및 TNF-α(tumor necrosis factor-α)의 발현량을 웨스턴 블롯 분석하였다.After simultaneously treating macrophages with tangerine core extract and fine dust, the expression levels of inflammatory cytokines IL (interleukin)-1β and TNF-α (tumor necrosis factor-α) in the cells were analyzed by Western blot.
구체적으로, 상기 실시예 1과 동일한 방법으로 대식세포에 미세먼지 및 농도별 귤핵 추출물을 처리하여 배양하였다. 미세먼지만을 단독 처리한 군을 양성 대조군, 무처리군을 음성 대조군으로 하였다. Specifically, macrophages were cultured by treating fine dust and tangerine core extracts at different concentrations in the same manner as in Example 1 above. The group treated only with fine dust was used as a positive control group, and the untreated group was used as a negative control group.
배양 완료후 수득한 세포를 차가운 1 X PBS(phosphate-buffered saline)로 세척하고 분해 완충용액(50mM Tris-HCl(pH 8.0) 1mM EDTA, 150mM NaCl, 1%Sodium deoxy cholate, 1%Triton X-100, 0.5% NP-40)를 첨가하여 용해시켰다. 세포 용해물을 14,000rpm, 4℃에서 20분 동안 원심분리 후, 상층액만 취하여 BCA(bicinchoninic acid) 단백질 어세이 키트를 사용하여 단백질을 정량하였다. 정량한 단백질을 시료 완충용액(1M Tris-HCl (pH 6.8), 50% 글리세롤, 10% SDS, 1% 브로모페놀 블루 및 5%, 머캡토에탄올)에 넣어 혼합하고 100℃에서 5분 동안 끓인 다음, 15% 아크릴아미드 젤에서 전기영동하였다. 전기영동된 단백질을 메탄올로 활성화된 폴리비닐리덴 플루오라이드(polyvinylidene difluoride; PVDF) 멤브레인으로 30V에서 밤샘 트랜스퍼하였다. 멤브레인을 분리하여 3% BSA(bovine serum albumin)로 2시간 동안 블로킹한 후, 3% BSA에 희석한 1차 항체에 첨가하여 2시간 동안 배양하였다. 이후 멤브레인을 1 X TBS-T(0.1% Tween® 20 Detergent를 포함하는 Tris-buffered saline)로 10분씩 3회 세척하고, 3% BSA에 희석한 2차 항체를 첨가하여 1시간 동안 반응시켰다. 멤브레인을 다시 1 X TBS-T로 10분씩 3회 세척하고 ECL(enhanced chemiluminescence) 용액을 이용하여 단백질 발현량을 확인하였다. 이때, 1차 항체로는 anti-goat-IL-1β(AF401-NA, R&D systems), anti-rabbit-TNF-α(ab9739, Abcam) 및 anti-mouse-β-actin(ab6276, Abcam)을 사용하였고, 2차 항체로는 Donkey anti-Goat IgG, HRP(Invitrogen), Donkey anti-Rabbit IgG, HRP(Invitrogen) 및 Donkey anti-Mouse IgG, HRP(Invitrogen)를 사용하였다. IL-1β 및 TNF-α의 발현 수준은 무처리군을 기준으로 각 실험군의 IL-1β 및 TNF-α의 발현 수준을 배수(fold)로 산출하였다.After completion of incubation, the obtained cells were washed with cold 1 , 0.5% NP-40) was added and dissolved. After centrifuging the cell lysate at 14,000 rpm and 4°C for 20 minutes, only the supernatant was taken and the protein was quantified using a BCA (bicinchoninic acid) protein assay kit. The quantified protein was mixed in sample buffer (1M Tris-HCl (pH 6.8), 50% glycerol, 10% SDS, 1% bromophenol blue, and 5% mercaptoethanol) and boiled at 100°C for 5 minutes. Next, electrophoresis was performed on a 15% acrylamide gel. The electrophoresed proteins were transferred to a methanol-activated polyvinylidene difluoride (PVDF) membrane at 30 V overnight. The membrane was separated and blocked with 3% BSA (bovine serum albumin) for 2 hours, then added to primary antibody diluted in 3% BSA and incubated for 2 hours. Afterwards, the membrane was washed three times for 10 minutes each with 1 The membrane was again washed three times for 10 minutes each with 1 At this time, anti-goat-IL-1β (AF401-NA, R&D systems), anti-rabbit-TNF-α (ab9739, Abcam), and anti-mouse-β-actin (ab6276, Abcam) were used as primary antibodies. As secondary antibodies, Donkey anti-Goat IgG, HRP (Invitrogen), Donkey anti-Rabbit IgG, HRP (Invitrogen), and Donkey anti-Mouse IgG, HRP (Invitrogen) were used. The expression levels of IL-1β and TNF-α were calculated as the fold of the expression levels of IL-1β and TNF-α in each experimental group based on the untreated group.
그 결과, 미세먼지를 처리하지 않은 대식세포에서의 IL-1β 및 TNF-α 대비 미세먼지를 처리한 대식세포에서 IL-1β 및 TNF-α의 발현 수준이 크게 증가하였다. 반면, 귤핵 추출물과 미세먼지를 동시 처리한 경우 대식세포에서의 IL-1β 및 TNF-α의 발현 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소하였다(도 2).As a result, the expression levels of IL-1β and TNF-α were significantly increased in macrophages treated with fine dust compared to IL-1β and TNF-α in macrophages not treated with fine dust. On the other hand, when tangerine core extract and fine dust were simultaneously treated, the expression levels of IL-1β and TNF-α in macrophages were significantly decreased in a tangerine core extract concentration-dependent manner (Figure 2).
실시예 4. 귤핵 추출물에 의한 세포 독성 확인Example 4. Confirmation of cytotoxicity by tangerine core extract
귤핵 추출물이 세포 독성을 나타내는지 확인하기 위하여 세포 생존성 테스트를 수행하였다.A cell viability test was performed to determine whether the tangerine core extract exhibited cytotoxicity.
구체적으로, 대식세포를 5X105 cells/ml의 밀도로 12 웰 플레이트에 시딩하여 16시간 동안 예비 배양한 후, 농도별 귤핵 추출물(50, 100 또는 200 μg/mL)을 처리하고 24시간 동안 배양하였다. 배양 후 MTT(3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltrazolium Bromide) 용액 5 mg/ml를 각 웰에 첨가하여 2시간 동안 추가 배양하였다. 각 웰에 존재하는 포르마잔 결정(formazan crystal)을 DMSO에 용해시키고, 마이크로 플레이트 판독기를 사용하여 540 nm에서 흡광도를 측정하였다. 세포 생존율은 귤핵 추출물을 처리하지 않은 대식세포의 세포 생존율을 기준으로 각 귤핵 추출물 농도에서의 세포 생존율을 산출하였다.Specifically, macrophages were seeded in a 12-well plate at a density of 5 . After incubation, 5 mg/ml of MTT (3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltrazolium Bromide) solution was added to each well and further cultured for 2 hours. Formazan crystals present in each well were dissolved in DMSO, and absorbance was measured at 540 nm using a microplate reader. Cell survival rate was calculated at each tangerine core extract concentration based on the cell survival rate of macrophages not treated with tangerine core extract.
그 결과, 귤핵 추출물은 고농도인 200 μg/mL에서도 세포 독성이 나타나지 않았다(도 3).As a result, the tangerine core extract did not show cytotoxicity even at a high concentration of 200 μg/mL (Figure 3).
실시예 5. 혈관 내피세포에서 발생하는 세포 부착 분자의 발현 억제 효과Example 5. Effect of suppressing expression of cell adhesion molecules occurring in vascular endothelial cells
체내 침투한 미세먼지가 혈관 내피세포에 미치는 직접적인 영향을 모사한 미세먼지 처리 혈관 내피세포 모델에서 귤핵 추출물 처리에 따른 세포 부착 분자인 ICAM-1(intercellular adhesion molecule-1) 및 VCAM-1(vascular cell adhesion molecule-1)의 발현량을 웨스턴 블롯 분석하였다.In a fine dust-treated vascular endothelial cell model that simulates the direct effect of fine dust infiltrating the body on vascular endothelial cells, the cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell) resulting from tangerine core extract treatment were obtained. The expression level of adhesion molecule-1) was analyzed by Western blot.
구체적으로, 상기 미세먼지 처리 혈관 내피세포 모델을 제작하기 위해, 혈관 내피세포(human umbilical artery endothelial cells, HUAECs)를 4X105 cells/ml의 밀도로 60 mm 플레이트에 시딩하여 16시간 동안 예비 배양한 후, 미세먼지(PM2.5, 200 μg/mL) 또는 PBS (대조군)를 처리하고, 농도별 귤핵 추출물(100 또는 200 μg/mL)을 처리하여 4시간 동안 배양한 후, ICAM-1 및 VCAM-1의 발현량을 상기 실시예 3과 동일한 방법으로 웨스턴 블롯 분석하였다. 이때, 1차 항체로는 anti-rabbit-ICAM-1(sc-7891, Santa Cruz), anti-rabbit-VCAM-1(sc-8304, Santa Cruz) 및 anti-mouse-β-actin(ab6276, Abcam)을 사용하였으며, 2차 항체로는 Donkey anti-Rabbit IgG, HRP(Invitrogen) 및 Donkey anti-Mouse IgG, HRP (Invitrogen)를 사용하였다.Specifically, to produce the fine dust-treated vascular endothelial cell model, vascular endothelial cells (human umbilical artery endothelial cells, HUAECs) were seeded at a density of 4X105 cells/ml on a 60 mm plate and pre-cultured for 16 hours. , treated with fine dust (PM2.5, 200 μg/mL) or PBS (control group), treated with tangerine core extract at different concentrations (100 or 200 μg/mL) and cultured for 4 hours, then ICAM-1 and VCAM- The expression level of 1 was analyzed by Western blot in the same manner as in Example 3 above. At this time, the primary antibodies used were anti-rabbit-ICAM-1 (sc-7891, Santa Cruz), anti-rabbit-VCAM-1 (sc-8304, Santa Cruz), and anti-mouse-β-actin (ab6276, Abcam). ) was used, and as secondary antibodies, Donkey anti-Rabbit IgG, HRP (Invitrogen) and Donkey anti-Mouse IgG, HRP (Invitrogen) were used.
그 결과, 미세먼지 200 μg/mL를 처리한 미세먼지 처리 혈관 내피세포 모델에서 대조군 모델에 비해 ICAM-1 및 VCAM-1의 발현 수준이 현저히 증가하였으나, 귤핵 추출물을 처리한 경우 ICAM-1 및 VCAM-1의 발현 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소하였다(도 4).As a result, the expression levels of ICAM-1 and VCAM-1 were significantly increased in the fine dust-treated vascular endothelial cell model treated with 200 μg/mL of fine dust compared to the control model, but when treated with tangerine core extract, ICAM-1 and VCAM The expression level of -1 was significantly decreased in a concentration-dependent manner of the tangerine core extract (Figure 4).
실시예 6. 폐 조직 상피세포 유래 유해 물질에 의해 혈관 내피세포에서 발생하는 세포 부착 분자의 발현 억제 효과Example 6. Inhibitory effect on the expression of cell adhesion molecules generated in vascular endothelial cells by harmful substances derived from lung tissue epithelial cells
미세먼지가 처리된 폐 조직 상피세포에서 발생하는 유해 물질을 포함하는 배양액에서 귤핵 추출물 처리에 따른 세포 부착 분자인 ICAM-1 및 VCAM-1의 발현량을 웨스턴 블롯 분석하였다.Western blot analysis was performed on the expression levels of cell adhesion molecules ICAM-1 and VCAM-1 according to tangerine core extract treatment in culture media containing harmful substances generated from lung tissue epithelial cells treated with fine dust.
구체적으로, 상기 유해 물질을 포함하는 배양액을 제조하기 위해, 폐 조직 상피세포(Beas-2B)를 8X105 cells/ml(미세먼지 처리군) 또는 3X105 cells/ml(대조군)의 밀도로 60 mm 플레이트에 시딩하여 16시간 동안 예비 배양한 후, DMEM/F12 배지(F-12 Nutrient Mixture와 DMEM 배지를 1:1로 혼합)에서 미세먼지(PM2.5, 200 μg/mL) 또는 PBS(대조군)를 처리하고 48시간 동안 배양하였다. 배양이 완료된 배양액을 2mL 수득하였다.Specifically, in order to prepare a culture medium containing the above harmful substances, lung tissue epithelial cells (Beas-2B) were grown at a density of 8X10 5 cells/ml (fine dust treatment group) or 3X10 5 cells/ml (control group) at 60 mm. After seeding the plate and pre-cultivating for 16 hours, fine dust (PM2.5, 200 μg/mL) or PBS (control) was added to DMEM/F12 medium (1:1 mixture of F-12 Nutrient Mixture and DMEM medium). were treated and cultured for 48 hours. 2 mL of culture medium upon completion of culture was obtained.
새롭게 HUAECs을 4X105 cells/ml의 밀도로 60 mm 플레이트에 3ml로 시딩하여 16시간 동안 예비 배양한 후, 상기 수득한 배양액 2mL 및 농도별 귤핵 추출물(100 또는 200 μg/mL) 또는 DMSO(대조군)을 각 웰에 첨가하여 18시간 동안 배양한 후, ICAM-1 및 VCAM-1의 발현량을 상기 실시예 3과 동일한 방법으로 웨스턴 블롯 분석하였다. 이때, 항체는 상기 실시예 5와 동일한 항체를 사용하였다.Newly seeded HUAECs were seeded in 3ml on a 60 mm plate at a density of 4 was added to each well and cultured for 18 hours, and then the expression levels of ICAM-1 and VCAM-1 were analyzed by Western blot in the same manner as in Example 3. At this time, the same antibody as in Example 5 was used.
그 결과, 미세먼지 200 μg/mL를 처리한 폐 조직 상피세포에서 유래한 유해 물질을 포함하는 배양액은 대조군 배양액에 비해 HUAECs에서 ICAM-1 및 VCAM-1의 발현 수준을 증가시켰으나, 귤핵 추출물을 처리한 경우 ICAM-1 및 VCAM-1의 발현 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소하였다(도 5).As a result, culture medium containing harmful substances derived from lung tissue epithelial cells treated with 200 μg/mL of fine dust increased the expression levels of ICAM-1 and VCAM-1 in HUAECs compared to control culture medium, but treatment with tangerine core extract In one case, the expression levels of ICAM-1 and VCAM-1 were significantly decreased in a concentration-dependent manner of the tangerine core extract (Figure 5).
실시예 7. 귤 부위별 추출물의 미세먼지에 의한 항염증 효과 비교Example 7. Comparison of anti-inflammatory effects of extracts from different tangerine parts due to fine dust
7-1. NO 발현 억제 효과7-1. NO expression inhibition effect
상기 실시예 2와 동일한 방법으로 대식세포에 귤핵 추출물(200 μg/mL) 또는 귤피 추출물(200 μg/mL) 처리 후 배양액 내 NO 양을 분석하였다.Macrophages were treated with tangerine core extract (200 μg/mL) or tangerine peel extract (200 μg/mL) in the same manner as in Example 2, and the amount of NO in the culture medium was analyzed.
그 결과, 귤핵 추출물 및 귤피 추출물 모두 NO 생산을 억제하였으나 귤핵 추출물의 NO 생산 억제능이 귤피 추출물에 비하여 높았다(도 6).As a result, both the tangerine core extract and the tangerine peel extract inhibited NO production, but the NO production inhibition ability of the tangerine core extract was higher than that of the tangerine peel extract (FIG. 6).
7-2. 세포 부착 분자의 발현 억제 효과7-2. Effect of suppressing expression of cell adhesion molecules
상기 실시예 5와 동일한 방법으로 미세먼지 처리 혈관 내피세포 모델에 농도별 미세먼지(PM2.5, 200 μg/mL)를 처리하였을 때 발현되는 ICAM-1 및 VCAM-1의 수준이 귤핵 추출물(0, 100 또는 200 μg/mL) 또는 귤피(0, 100 또는 200 μg/mL) 추출물 처리에 의해 억제되는지를 확인하였다. 대조군 모델은 DMSO를 처리하였다.The levels of ICAM-1 and VCAM-1 expressed when fine dust (PM2.5, 200 μg/mL) was treated with fine dust-treated vascular endothelial cell model in the same manner as in Example 5 above were measured in tangerine core extract (0 , 100 or 200 μg/mL) or tangerine peel (0, 100 or 200 μg/mL) extract treatment. The control model was treated with DMSO.
그 결과, 귤피 추출물에서는 ICAM-1 및 VCAM-1 발현 증가 억제가 확인되지 않은 반면, 귤핵 추출물에서는 ICAM-1 및 VCAM-1의 발현 증가가 억제되었다(도 7).As a result, the inhibition of the increase in ICAM-1 and VCAM-1 expression was not confirmed in the tangerine peel extract, while the increase in the expression of ICAM-1 and VCAM-1 was inhibited in the tangerine core extract (Figure 7).
실시예 8. 귤핵 추출물의 내독소(LPS)에 의해 혈관 내피세포에서 발생하는 세포 부착 분자 발현 증가에 대한 억제 효과 Example 8. Inhibitory effect of tangerine core extract on the increase in cell adhesion molecule expression occurring in vascular endothelial cells caused by endotoxin (LPS)
체내 침투한 내독소(LPS, lipopolysaccharide)가 혈관 내피세포에 미치는 직접적인 영향을 모사한 내독소 처리 혈관 내피세포 모델에서 귤핵 추출물 처리에 따른 세포 부착 분자인 ICAM-1(intercellular adhesion molecule-1) 및 VCAM-1(vascular cell adhesion molecule-1)의 발현량을 웨스턴 블롯 분석하였다.In an endotoxin-treated vascular endothelial cell model that simulates the direct effect of endotoxin (LPS, lipopolysaccharide) infiltrating the body on vascular endothelial cells, cell adhesion molecules, ICAM-1 (intercellular adhesion molecule-1) and VCAM, are obtained by treatment with tangerine core extract. The expression level of -1 (vascular cell adhesion molecule-1) was analyzed by Western blot.
구체적으로, 상기 내독소 처리 혈관 내피세포 모델을 제작하기 위해, 혈관 내피세포(human umbilical artery endothelial cells, HUAECs)를 4X105 cells/ml의 밀도로 60 mm 플레이트에 시딩하여 16시간 동안 예비 배양한 후, 내독소(LPS, 50 ng/mL) 또는 PBS (대조군)를 처리하고, 농도별 귤핵 추출물(50, 100 또는 200 μg/mL)을 처리하여 4시간 동안 배양한 후, ICAM-1 및 VCAM-1의 발현량을 상기 실시예 3과 동일한 방법으로 웨스턴 블롯 분석하였다. 이때, 1차 항체로는 anti-rabbit-ICAM-1(sc-7891, Santa Cruz), anti-rabbit-VCAM-1(sc-8304, Santa Cruz) 및 anti-mouse-β-actin(ab6276, Abcam)을 사용하였으며, 2차 항체로는 Donkey anti-Rabbit IgG, HRP(Invitrogen) 및 Donkey anti-Mouse IgG, HRP (Invitrogen)를 사용하였다.Specifically, to produce the endotoxin-treated vascular endothelial cell model, human umbilical artery endothelial cells (HUAECs) were seeded on a 60 mm plate at a density of 4X10 5 cells/ml and pre-cultured for 16 hours. , treated with endotoxin (LPS, 50 ng/mL) or PBS (control), treated with tangerine core extract at different concentrations (50, 100, or 200 μg/mL) and cultured for 4 hours, then ICAM-1 and VCAM- The expression level of 1 was analyzed by Western blot in the same manner as in Example 3 above. At this time, the primary antibodies used were anti-rabbit-ICAM-1 (sc-7891, Santa Cruz), anti-rabbit-VCAM-1 (sc-8304, Santa Cruz), and anti-mouse-β-actin (ab6276, Abcam). ) was used, and as secondary antibodies, Donkey anti-Rabbit IgG, HRP (Invitrogen) and Donkey anti-Mouse IgG, HRP (Invitrogen) were used.
그 결과, 내독소 50 ng/mL를 처리한 내독소 처리 혈관 내피세포 모델에서 대조군 모델에 비해 ICAM-1 및 VCAM-1의 발현 수준이 현저히 증가하였으나, 귤핵 추출물을 처리한 경우 ICAM-1 및 VCAM-1의 발현 수준이 귤핵 추출물 농도 의존적으로 유의하게 감소함을 확인하였다(도 8). As a result, the expression levels of ICAM-1 and VCAM-1 were significantly increased in the endotoxin-treated vascular endothelial cell model treated with 50 ng/mL of endotoxin compared to the control model, but when treated with tangerine core extract, ICAM-1 and VCAM It was confirmed that the expression level of -1 was significantly decreased in a concentration-dependent manner of the tangerine core extract (FIG. 8).
실시예 9. 귤핵 추출물의 내독소 주입 마우스모델에서 항염증 효과 비교Example 9. Comparison of anti-inflammatory effects of tangerine core extract in endotoxin injection mouse model
귤핵 추출물을 100 mg/kg로 마우스에 경구 투여한 후 내독소가 처리된 마우스의 혈장 내 염증성 사이토카인의 발현량을 분석하였다.After orally administering 100 mg/kg of tangerine core extract to mice, the expression levels of inflammatory cytokines in the plasma of mice treated with endotoxin were analyzed.
구체적으로, 마우스는 C57BL6/J 계통의 8주령 마우스를 사용하였다. 귤핵 추출물질은 100 mg/kg로 단회 경구투여 하였으며, 대조군에는 DMSO를 사용하였다. 투여 30분 후 5 mg/kg의 LPS를 복강내로 투여하였으며, 대조군에는 인산완충액을 복강내로 투여하였다. 투여 후 24시간 뒤에 마우스의 안와부 정맥에서 모세관채혈튜브를 이용해 채혈하였고, 혈액 채취 후 얼음에 30분간 보관 후 3000rpm에서 15분간 원심분리하여 혈장을 분리하였다. 분리한 혈장에서 111종의 사이토카인 변화를 동시에 관찰할 수 있는 cytokine array를 실시하였다. 이때 사용한 키트는 Proteome Profiler Mouse XL Cytokine Array (R&D Systems, ARY028)를 사용하였으며, 제조회사가 제시한 방법대로 시행하였다. Specifically, 8-week-old mice of the C57BL6/J strain were used. Tangerine core extract was administered orally once at 100 mg/kg, and DMSO was used in the control group. 30 minutes after administration, 5 mg/kg of LPS was administered intraperitoneally, and phosphate buffer solution was administered intraperitoneally to the control group. 24 hours after administration, blood was collected from the orbital vein of the mouse using a capillary blood collection tube. After blood collection, it was stored on ice for 30 minutes and centrifuged at 3000 rpm for 15 minutes to separate plasma. A cytokine array was performed to simultaneously observe changes in 111 types of cytokines in separated plasma. The kit used at this time was Proteome Profiler Mouse XL Cytokine Array (R&D Systems, ARY028), and was performed according to the method suggested by the manufacturer.
그 결과, 발현이 통계적으로 유의미하게 변한 상위 6개의 혈장 단백질 Serpin E1/PAI-1, IGFBP-1, CXCL1/KC, CXCL9/MIG, P-Selectin/CD62P, CD14를 동정하였다. As a result, the top six plasma proteins whose expression changed statistically significantly were identified: Serpin E1/PAI-1, IGFBP-1, CXCL1/KC, CXCL9/MIG, P-Selectin/CD62P, and CD14.
상기 실시예의 결과로부터, 귤핵 추출물은 체내에서 염증 반응 및 세포 부착 분자의 발현 증가를 억제시킴을 확인하였는바, 이를 미세먼지에 의해 유발된 질환의 예방, 개선 또는 치료에 적용할 수 있다.From the results of the above examples, it was confirmed that the tangerine core extract suppresses the inflammatory response and the increased expression of cell adhesion molecules in the body, and this can be applied to the prevention, improvement or treatment of diseases caused by fine dust.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (14)

  1. 귤핵(Citri tangerinae Semen) 추출물을 유효성분으로 포함하는, 미세먼지로 유발된 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating diseases caused by fine dust, comprising Citri tangerinae Semen extract as an active ingredient.
  2. 제1항에 있어서, 상기 귤핵 추출물은 물, 탄소수 1 내지 4의 알코올 및 이들의 혼합용매로 이루어지는 군으로부터 선택되는 어느 하나 이상의 용매로 추출된 것인, 조성물.The composition according to claim 1, wherein the tangerine core extract is extracted with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.
  3. 제1항에 있어서, 상기 질환은 염증성 질환, 심혈관계 질환, 대사성 질환, 신경계 질환, 호흡기계 질환 및 안질환인 것인, 조성물.The composition of claim 1, wherein the disease is an inflammatory disease, a cardiovascular disease, a metabolic disease, a neurological disease, a respiratory disease, and an eye disease.
  4. 제1항에 있어서, 상기 질환은 염증성 질환인 것인, 조성물.The composition of claim 1, wherein the disease is an inflammatory disease.
  5. 제1항에 있어서, 상기 조성물은 귤핵 추출물을 100 내지 200 μg/mL 농도로 포함하는 것인, 조성물.The composition of claim 1, wherein the composition includes tangerine core extract at a concentration of 100 to 200 μg/mL.
  6. 제1항에 있어서, 상기 조성물은 면역세포에서 일산화질소 생성 억제, IL(interleukin)-1β 발현 감소 및 TNF-α(tumor necrosis factor-α) 발현 감소로 이루어지는 군으로부터 선택되는 어느 하나 이상의 효과를 갖는 것인, 조성물.The method of claim 1, wherein the composition has one or more effects selected from the group consisting of inhibiting nitric oxide production in immune cells, reducing interleukin (IL)-1β expression, and reducing tumor necrosis factor-α (TNF-α) expression. A composition.
  7. 제1항에 있어서, 상기 조성물은 혈관 내피세포에서 세포 부착 분자의 발현 증가를 억제하는 것인, 조성물.The composition of claim 1, wherein the composition inhibits increased expression of cell adhesion molecules in vascular endothelial cells.
  8. 제7항에 있어서, 상기 세포 부착 분자는 ICAM-1(intercellular adhesion molecule-1) 또는 VCAM-1(vascular cell adhesion molecule-1) 중 선택되는 어느 하나 이상인 것인, 조성물.The composition of claim 7, wherein the cell adhesion molecule is at least one selected from intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1).
  9. 제1항의 약학적 조성물을 인간을 제외한 개체에 투여하는 단계를 포함하는, 미세먼지로 유발된 질환의 예방 또는 치료 방법.A method for preventing or treating diseases caused by fine dust, comprising administering the pharmaceutical composition of claim 1 to an entity other than a human.
  10. 제9항에 있어서, 상기 질환은 염증성 질환, 심혈관계 질환, 대사성 질환, 신경계 질환, 호흡기계 질환 및 안질환인 것인, 방법.The method of claim 9, wherein the disease is an inflammatory disease, a cardiovascular disease, a metabolic disease, a neurological disease, a respiratory system disease, and an eye disease.
  11. 제10항에 있어서, 상기 질환은 염증성 질환인 것인, 방법.The method of claim 10, wherein the disease is an inflammatory disease.
  12. 귤핵 추출물을 포함하는, 미세먼지 유발 질환의 예방 또는 개선용 식품 조성물.A food composition for preventing or improving fine dust-induced diseases, comprising a tangerine core extract.
  13. 귤핵 추출물을 포함하는, 미세먼지 유발 질환의 예방 또는 개선용 사료 조성물.A feed composition for preventing or improving fine dust-induced diseases, comprising a tangerine core extract.
  14. 귤핵 추출물을 포함하는, 미세먼지 유발 질환의 예방 또는 개선용 의약외품 조성물.A quasi-drug composition for preventing or improving fine dust-induced diseases, comprising a tangerine core extract.
PCT/KR2023/014337 2022-09-23 2023-09-21 Composition for prevention, amelioration or treatment of diseases caused by particulate matter comprising citri tangerinae semen extract and use thereof WO2024063550A1 (en)

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KR101973950B1 (en) * 2018-01-09 2019-04-30 (주)셀트리온 Composition comprising mixture of a citrus unshiu peel extract and chamaecyparis obtuse extract for alleviating skin inflammation or skin hyperpigmentation by yellow sand or fine dust
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