WO2022102988A1 - Method for preparing antarctic lichen amandinea sp. extract, and composition containing amandinea sp. extract - Google Patents

Method for preparing antarctic lichen amandinea sp. extract, and composition containing amandinea sp. extract Download PDF

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WO2022102988A1
WO2022102988A1 PCT/KR2021/014012 KR2021014012W WO2022102988A1 WO 2022102988 A1 WO2022102988 A1 WO 2022102988A1 KR 2021014012 W KR2021014012 W KR 2021014012W WO 2022102988 A1 WO2022102988 A1 WO 2022102988A1
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extract
amandinea
inhibition
expression
inflammatory
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French (fr)
Korean (ko)
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임정한
김일찬
한세종
홍주미
김경희
김정은
민슬기
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한국해양과학기술원
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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
    • A61K36/09—Lichens
    • 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
    • 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
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • 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
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/208—Fungi extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material

Definitions

  • the present invention relates to a method for preparing an Antarctic lichen Amandinea sp. extract and to a composition for preventing or treating inflammatory diseases comprising the extract as an active ingredient.
  • the body has a defense mechanism called the immune system to protect itself from infection and damage.
  • the inflammatory response is the primary response of the body's immune system.
  • Several cells and molecules are implicated to effectively minimize damage or infection during the inflammatory response (Chen, L., et al. (2016). Oncotarget, 9(6) 7204-7218.).
  • Cytokines are generally known to be involved in the regulation of immune responses. Cytokines can be classified as pro- and anti-inflammatory cytokines, and soluble inhibitors of pro-inflammatory cytokines.
  • Pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin-1 (IL-1), IL-12 and interferon- ⁇ (IFN- ⁇ ) inflammatory cytokines) promote inflammation, whereas anti-inflammatory cytokines such as IL-1 receptor antagonists, IL-4, IL-6, IL-10, IL-11, and IL-13, inhibit the production of pro-inflammatory cytokines. Inhibits or exerts a number of other inhibitory effects in the inflammatory response (Turner, MD., et al. (2014). Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1843(11), 2563-2582.).
  • NF- ⁇ B pathway is considered as a prototype proinflammatory signaling pathway activated by proinflammatory cytokines, and the role of NF- ⁇ B has been extensively studied (Lawrence, T. (2009). Cold Spring Harbor Laboratory). Press, 1(6), 1-10.).
  • the murine macrophage cell line RAW 264.7 induced by LPS is commonly used in in vitro studies to screen for anti-inflammatory activity from native compounds.
  • LPS stimulation of RAW 264.7 cells increased levels of proinflammatory cytokines and inflammatory mediators were observed (Doyle, SL., O'Neill, LA. (2006). Biochemical pharmacology, 72(9), 1102-1113.; Kim, HK., et al. (1999) Biochemical pharmacology, 58(5), 759-765.).
  • the zebrafish (Danio rerio) has many advantages as an in vivo animal model.
  • Zebrafish have been widely used in various fields such as biology, embryology, molecular biology, immunology, and toxic drug discovery research, and the in vivo zebrafish anti-inflammatory test model is recognized as the best tool for anti-inflammatory assays (Liao, YF). (2011). Journal of Food & Drug Analysis, 19(2).; Lee, SH., et al. (2013). Carbohydrate polymers, 92(1), 84-89.).
  • lichens are complex organisms derived from the symbiotic relationship of two different organisms, fungi and algae. Because of their symbiotic existence, lichens are well adapted to extreme living conditions and can continue to grow. They are widely distributed in extreme environments such as desert areas, coastal areas, forest areas and polar regions. Antarctica is mostly covered by ice, and the ice-free aboveground area comprises less than 0.5% of ground cover. Even in these extreme conditions, 62 species of lichens inhabit the Barton Peninsula of King George Island in Antarctica. Lichens are known to produce several secondary metabolites with excellent pharmacological effects (Muller, K. (2001).
  • An object of the present invention is to provide a method for preparing a novel natural extract having excellent anti-inflammatory activity.
  • An object of the present invention is to provide a novel natural extract having excellent anti-inflammatory activity.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising the extract.
  • Another object of the present invention is to provide a food for preventing or improving inflammatory diseases comprising the extract.
  • Another object of the present invention is to provide a method for preventing or treating an inflammatory disease comprising administering the extract or the pharmaceutical composition.
  • Another object of the present invention is to provide an anti-inflammatory use of the extract.
  • Another object of the present invention is to provide a use of the extract for the prevention or treatment of inflammatory diseases.
  • Another object of the present invention is to provide the use of the extract in the manufacture of a medicament for the treatment of an inflammatory disease.
  • the present invention comprises the steps of (a) drying and pulverizing the Amandinea sample; (b) extracting the powdered amandinea with a polar organic solvent; and (c) removing the solvent from the extracted extract to obtain an Amandinea extract.
  • the present invention also provides an extract of Amandinea (Amandinea sp.) prepared by the above method.
  • the present invention also provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the extract as an active ingredient.
  • the present invention also provides a food for preventing or improving inflammatory diseases comprising the extract.
  • the present invention also provides a method for preventing or treating an inflammatory disease comprising administering the extract or the pharmaceutical composition.
  • the present invention also provides for the anti-inflammatory use of said extract.
  • the present invention also provides the use of said extract for the prevention or treatment of inflammatory diseases.
  • the present invention also provides for the use of said extract in the manufacture of a medicament for the treatment of an inflammatory disease.
  • the present invention also provides the use of said extract in the manufacture of a functional food for the prevention or amelioration of inflammatory diseases.
  • FIG. 1A shows the natural habitat of the Antarctic lichen, Amandinea sp.
  • Figure 1B shows the effect of Amandinea extract on cell viability
  • Figure 1C shows the effect of Amandinea extract on NO production.
  • Figure 4 depicts the measurement of protein p-1 ⁇ B- ⁇ (A) and p65 (B) expression in the NF- ⁇ B signaling pathway in LPS-stimulated Raw 264.7 cells using Western blotting. These data represent the mean ⁇ SEM of 3 replicates.
  • LPS stimulation group (#) NS, p>0.05; *, p ⁇ 0.05; **, p ⁇ 0.01; and ***, p ⁇ 0.001.
  • Figure 5 depicts the effect of amandinea extract on survival (A), morphological changes (B), heart rate (C), and body length (D) of zebrafish embryos. *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • FIG. 6 depicts the measurement of ROS accumulation in LPS-stimulated zebrafish larvae using DCF-DA.
  • Figure 6A shows the level of ROS production measured by fluorescence microscopy
  • Figure 6B shows the fluorescence intensity of ROS level in each zebrafish quantified using the Image J program. *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • NS 7 shows inflammatory genes IL-6 (A), IL-1 ⁇ (B), IL-10 (C), TNF- ⁇ (D), iNOS (E), inflammatory genes in zebrafish larvae after tail cutting using qPCR. and measurement of the relative expression of COX-2 (F).
  • NS p>0.05; *, p ⁇ 0.05; **, p ⁇ 0.01; and ***, p ⁇ 0.001.
  • lichen samples collected in Antarctica were completely dried and powdered. After the powdered lichen was extracted with methanol, the methanol was evaporated. Then, the dried extract was dissolved in DMSO to obtain an Amandinea extract.
  • Amandinea (Amandinea sp.) extract was produced in RAW264.7 macrophages in vitro and in zebrafish in vivo, pro-inflammatory cytokines such as IL-6, IL-1 ⁇ , IL-10 and TNF- ⁇ .
  • cytokines pro-inflammatory cytokines
  • inflammatory mediators such as inducible nitric oxide enzyme (iNOS) and cyclooxygenase-2 (COX-2)
  • cytosolic p-I ⁇ B- ⁇ By suppressing the expression of nuclear factor p65 and the generation of reactive oxygen species (ROS), it was confirmed to have a preventive or therapeutic effect on inflammatory diseases.
  • iNOS inducible nitric oxide enzyme
  • COX-2 cyclooxygenase-2
  • the present invention in one aspect (a) drying and pulverizing the Amandinea sample; (b) extracting the powdered amandinea with a polar organic solvent; and (c) removing the solvent from the extracted extract to obtain an Amandinea extract.
  • the polar organic solvent in step (b) may be characterized in that the extract is selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, and ethyl acetate, and preferably, the solvent is methanol. .
  • lichen refers to a symbiotic complex of fungi and algae or/and cyanobacteria, and is classified into about 14,000 species.
  • the term "extract" refers to a substance having anti-inflammatory activity isolated from the Amandinea sp.
  • the extract is used in the sense of including not only the extract, but also its dry powder or all forms formulated using the same.
  • the extract may be extracted using polar, non-polar, water, an organic solvent, or a mixed solvent thereof, preferably using a polar organic solvent.
  • the extracted liquid may be used in liquid form or may be used after concentration and/or drying.
  • the organic solvent is an anhydrous or hydrous lower alcohol (methanol, ethanol, isopropanol, butanol, etc.) having 1 to 4 carbon atoms, ethylene, acetone, hexane, ether, chloroform, dichloromethane, carbon tetrachloride and benzene, ethyl acetate, butyl acetate, N, N-dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof can be used, and the active ingredient of the extract is not destroyed or minimized at room temperature.
  • extraction may be performed by heating.
  • the degree of extraction and loss of the active ingredient of the extract may be different, so an appropriate organic solvent should be selected and used.
  • the extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction.
  • Filtration is a process of removing suspended solid particles from the extract, and may use cotton, nylon, etc. to filter out particles, ultrafiltration, cryofiltration, centrifugation, etc., but is not limited thereto.
  • a separation process by various chromatography chromatography according to size, charge, hydrophobicity or affinity may be further included.
  • Drying the filtrate includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying, and the like. In some cases, a process of pulverizing the final dried extract may be added.
  • the Amandinea extract may be obtained by removing the solvent by evaporating the solvent using a high-speed vacuum concentrator in the extracted extract in step (c), but is not limited thereto.
  • the present invention relates to an extract of Amandinea (Amandinea sp.) prepared by the above method.
  • the extract may be characterized as having one or more of the following characteristics:
  • NO nitric oxide
  • nitric oxide is a substance whose amount is increased by nitric oxide synthase when inducing an intracellular inflammatory reaction, and is a molecule that is an indicator of the inflammatory response.
  • NOS nervous system nitric oxide synthase
  • the synthesized nitric oxide increases the production of cGMP in brain cells, thereby performing a function of storing information recognized from the outside for a long time.
  • NO is a free radical and is known to be involved in physiological and pathological processes.
  • NO is synthesized by L-Arginine oxidation by nitric oxide synthase (Atkan, et al., 75: 639-653, 2004).
  • the extract inhibits the expression of IL-6, IL-1 ⁇ , IL-10 and TNF- ⁇ (Examples 3 and 7).
  • Excessive production of pro-inflammatory cytokines such as TNF- ⁇ and IL contributes to the pathogenesis of inflammatory diseases.
  • iNOS and COX-2 are inflammatory mediators, iNOS is an inflammatory molecule that produces NO, and COX-2 produces PGE2. Excessive increased activity of iNOS and COX-2 may be the etiology of various inflammatory diseases.
  • COX-2 is an enzyme involved in producing prostaglandin, a protein related to an inflammatory response, and an increase in intracellular COX-2 expression level can be an indicator indicating that an inflammatory response is in progress. there is.
  • the extract inactivates the NF- ⁇ B (nuclear factor kappa B) pathway and suppresses the expression of cytosolic p-I ⁇ B- ⁇ and nuclear factor p65 (Example 4).
  • NF- ⁇ B is a transcription factor that plays an important role in immune and inflammatory responses, and activated NF- ⁇ B induces the expression of various inflammatory factors.
  • NF- ⁇ B is known as a transcription factor that regulates more than 150 genes including IL-6, TNF- ⁇ , iNOS, COX-2 and IL-1 ⁇ .
  • I ⁇ B- ⁇ plays an important role in NF- ⁇ B activation mediated by LPS, TNF or IL-1. Activation of I ⁇ B- ⁇ occurs by LPS-induced phosphorylation, leading to nuclear translocation of p65.
  • Example 4 the treatment of the Amandinea extract had the effect of reducing the levels of p-I ⁇ B ⁇ and p-65, which inhibits NF- ⁇ B signaling, and thus TNF- ⁇ , IL-6 and NO It indicates that the expression of cytokines and mediators is reduced.
  • ROS levels are closely associated with inflammation, and high ROS production due to oxidative stress causes cell or tissue damage. ROS accumulation in activated neutrophils and macrophages induces the release of proinflammatory cytokines and mediators.
  • anti-inflammatory refers to an action that inhibits or reduces inflammation
  • anti-inflammatory is a protective reaction that occurs in the body when a living tissue is damaged, which is a cause of inflammatory disease.
  • anti-inflammatory refers to the efficacy of alleviating and preventing inflammation.
  • the anti-inflammatory activity is NO (nitric oxide) inhibitory effect, PGE2 inhibitory effect, NF- ⁇ B pathway inhibitory effect, Nrf2 / HO-1 induction effect, initial inflammatory factors IL-1 ⁇ , IL-6, TNF- ⁇ , etc. It may have anti-inflammatory activity due to the expression inhibitory effect, but is not limited thereto.
  • the term "inflammation” is one of the defensive responses of biological tissues to certain stimuli, and is a biological defense mechanism that seeks to recover to the original state by removing injuries caused by various harmful stimuli.
  • Inflammation stimuli include infection or chemical and physical stimuli, and the inflammatory process can be divided into acute and chronic inflammation.
  • Acute inflammation is a short-term reaction within a few days, and plasma components or blood cells are involved in the removal of foreign substances by opening the microcirculation system.
  • Chronic inflammation has a long duration, and tissue proliferation is seen.
  • the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising the Amandinea sp. extract as an active ingredient.
  • the anti-inflammatory pharmaceutical composition can be used for the prevention, treatment or improvement of inflammatory diseases by having anti-inflammatory activity.
  • the pharmaceutical composition may be characterized as having one or more of the following characteristics:
  • the pharmaceutical composition may be characterized in that it contains the extract at a concentration of 10 ⁇ g/mL to 80 ⁇ g/mL, and preferably contains the extract at a concentration of 25 ⁇ g/mL to 50 ⁇ g/mL.
  • the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and degenerative disease It may be characterized in that it is selected from the group consisting of neuroinflammation, but is not limited thereto.
  • inflammatory disease is a generic term for diseases in which inflammation is the main lesion.
  • the pharmaceutical composition may be characterized in that it further comprises a pharmaceutically acceptable carrier, excipient or diluent.
  • Carriers, excipients and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.
  • a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.
  • the pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods. Suitable formulations include tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, oral formulations such as aerosols, external preparations, suppositories, and sterile injection solutions, However, the present invention is not limited thereto.
  • the pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, when the dosage form is a tablet, a coated tablet, a dragee, and a hard capsule, lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid or a pharmaceutically acceptable salt thereof may be included.
  • a pharmaceutically inert organic or inorganic carrier when the dosage form is a tablet, a coated tablet, a dragee, and a hard capsule, lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid or a pharmaceutically acceptable salt thereof may be included.
  • the formulation when the formulation is a soft capsule, it may contain vegetable oils, waxes, fats, semi-solid and liquid polyols.
  • water, polyol, glycerol, and vegetable oil may be included.
  • the "pharmaceutically acceptable salt” refers to a formulation of a compound that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and properties of the compound.
  • the pharmaceutical salt is an acid that forms a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc., tartaric acid, formic acid, citric acid , organic carbonic acids such as acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc., sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.
  • acid addition salts formed with phonic acid and the like are included.
  • pharmaceutically acceptable salts of carboxylic acids include metal salts or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, and guanidine, dicyclohexylamine, N organic salts such as -methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine, and the like.
  • Acids such as oxalic acid are not pharmaceutically acceptable, but can be used in the preparation of useful salts as intermediates for obtaining pharmaceutically acceptable salts.
  • the pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizing agent, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant, and the like, in addition to the carrier described above.
  • the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • composition of the present invention may be used together with a pharmaceutical composition for treatment of inflammatory diseases, such as immunotherapy, chemotherapy and radiation therapy, and other inflammatory diseases, which can be recognized by those skilled in the art as being effective in the prevention or treatment of inflammatory diseases. .
  • inflammatory diseases such as immunotherapy, chemotherapy and radiation therapy, and other inflammatory diseases, which can be recognized by those skilled in the art as being effective in the prevention or treatment of inflammatory diseases.
  • the pharmaceutical composition of the present invention may be administered to an individual by various routes.
  • the mode of administration may be, for example, by subcutaneous, intravenous, intramuscular or intrauterine intrathecal or intracerebrovascular injection.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.
  • the present invention relates to a food for preventing or improving inflammatory diseases comprising the Amandinea (Amandinea sp.) extract.
  • the food product may be characterized as having one or more of the following characteristics:
  • the food may be characterized in that it contains the extract at a concentration of 10 ⁇ g/mL to 80 ⁇ g/mL, and preferably contains the extract at a concentration of 25 ⁇ g/mL to 50 ⁇ g/mL.
  • the term "food” refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, There are vitamin complexes, health functional foods, and health foods, and includes all foods in the ordinary sense.
  • the functional food is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently exhibit bioregulatory functions and has high medical effects.
  • function (sex) means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body.
  • the food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.
  • the formulation of the food may be manufactured without limitation as long as it is a formulation recognized as a food, and the health functional food according to the present invention may be in the form of powder, granule, tablet, capsule or beverage.
  • the health food means food having an active health maintenance or promotion effect compared to general food
  • health supplement food means food for the purpose of health supplementation.
  • the terms health functional food, health food, and dietary supplement are used interchangeably.
  • the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.
  • the composition may include additional ingredients that are commonly used in food to improve odor, taste, vision, and the like.
  • vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included.
  • it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr).
  • it may include amino acids such as lysine, tryptophan, cysteine, and valine.
  • the composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butylhydroxy Toluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG, etc.), sweeteners (dulcin, cyclimate, saccharin, sodium, etc.) ), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, food additives such as improving agents ) may be included.
  • the additive may be selected according to the type of
  • It may further include a food supplementary additive together with the Amandinea extract of the present invention, and may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.
  • the mixed amount of the active ingredient may be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment).
  • the terms “improvement”, “prevention” and “treatment” should be interpreted in the broadest sense, and “improvement” means any action that temporarily/continuously relieves a disease or one or more clinical symptoms.
  • "Prevention” means preventing the progression of one or more of the clinical symptoms of a disease in a patient who may be exposed to or susceptible to the disease but has not yet experienced or revealed symptoms of the disease.
  • Treatment means any action that arrests or reduces the development of a disease or one or more clinical symptoms thereof.
  • the present invention relates to a method for preventing or treating an inflammatory disease comprising administering the Amandinea sp. extract or the pharmaceutical composition.
  • the present invention relates to the anti-inflammatory use of the extract of Amandinea (Amandinea sp.).
  • the present invention relates to the use of the Amandinea (Amandinea sp.) extract for the prevention or treatment of inflammatory diseases.
  • the present invention relates to the use of the extract of Amandinea (Amandinea sp.) in the manufacture of a medicament for the treatment of an inflammatory disease.
  • the present invention relates to the use of the Amandinea (Amandinea sp.) extract in the manufacture of a functional food for the prevention or improvement of inflammatory diseases.
  • Amandinea Amandinea sp.
  • Example 1-1 Collection of lichen and preparation of extract
  • Lichen samples were collected at 62°12'57.82"S, 58°56'01.87"W on Ardley Island, Antarctica. Detailed information on sample identification and collection areas is shown in Table 1. Voucher specimens were stored at the Korea Polar Research Institute. The lichen samples were thoroughly dried and powdered using pestle and mortar. Powdered lichens were extracted with methanol (1 g/10 mL) in the dark at 25 °C for 2 weeks. Methanol in the extract was evaporated using a speed vacuum concentrator. The dried extract was then dissolved in DMSO to a final concentration of 20 mg/mL.
  • Example 1-2 Cell culture and treatment
  • Murine macrophage Raw 264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (PS) at 37 °C in 5% CO 2 /95% air. The medium was replaced with DMEM containing 5% FBS and 1% PS before treatment.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • PS penicillin streptomycin
  • Example 1-5 Measurement of IL-6 and TNF- ⁇
  • IL-6 DY406-05, R&D Systems Inc, USA
  • TNF- ⁇ DY410-05, R&D Systems Inc, USA
  • ELISA Sandwich-enzyme-linked immunosorbent assay kits
  • Example 1-6 Quantitative real-time PCR analysis (IL-6, TNF- ⁇ , iNOS, COX-2, IL-1 ⁇ , IL-10)
  • RNA was isolated using easy-BLUE TM Total RNA Extraction Kit (17061, Intron biology) according to the manufacturer's instructions.
  • RNA (1 ⁇ g) was reverse transcribed using M-MLV reverse transcriptase (RT001S, Sigma-Aldrich), and then real-time PCR amplification was performed with TOPrealTM qPCR 2X PreMIX (RT500, Enzynomics) using Rotor gene 6500 (Corbett research). did.
  • the target gene primer sequences are listed in Table 2. Expression data were analyzed using the comparative cycle threshold (C t ) method and normalized to the level of ⁇ -actin expression.
  • Proteins were extracted using RIPA buffer (Sigma), protease inhibitor cocktail purification (Roche) and phosphatase inhibitor cocktail purification (RACHe) or nuclear extraction kit (ab113474, Abcam).
  • the extracted proteins (20 ⁇ g) were separated by SDS-PAGE, transferred to PVDF membranes, washed with 5% skim milk in TBST buffer for 1 h, and incubated with primary antibody overnight at 4 °C.
  • Primary antibodies are iNOS (ADI-905-431, Enzo), COX-2 (SC-166475, Santa Cruz Biotechnology), p-I ⁇ B ⁇ (#2859, Cell signaling), p65 (#3033, Cell signaling), PCNA ( SC-25280, Santa Cruz Biotechnology) and GAPDH (SC-25778, Santa Cruz Biotechnology). Secondary antibodies were anti-mouse (HAF007, DuoSet) and anti-rabbit (SC-2537-CM, Santa Cruz Biotechnology). Blots were analyzed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher).
  • ECL enhanced chemiluminescence
  • Examples 1-8 Origin and maintenance of parental zebrafish
  • zebrafish were obtained from a merchant (Ansan aquarium, Korea) and maintained at 28 °C ⁇ 0.5 with a 14:10 h light:dark cycle. Prior to mating, zebrafish were separated into two groups: females and males. They reacted to light on contact in the morning and laid eggs within 30 minutes.
  • Viability was measured daily up to 120 hpf. Zebrafish were anesthetized with 0.25 mg/mL tricaine at 48 hpf, and heart rate and body length were analyzed using a microscope.
  • Example 1-10 Measurement of ROS level in zebrafish
  • DCF-DA 2',7'-dichlorodihydrofluorescein diacetate
  • Example 2 Effect of Amandinea sp. extract on cell viability and NO production
  • Lichens were collected from sources near King Sejong Antarctic Station in 2017 (Fig. 1A).
  • the lichen species was identified as Amandinea sp. by PCR (data not shown).
  • the extract inhibited the expression levels of pro-inflammatory cytokines IL-6 and TNF- ⁇ , and decreased the levels of inflammatory mediators iNOS and COX-2 in LPS-stimulated Raw 264.7 cells. These were determined by ELISA, qPCR, and Western blotting ( FIGS. 2 and 3 ). COX-2 protein levels were not significant, but the data show that the extract reduced LPS-stimulated cytokine production.
  • the present inventors confirmed the inhibitory effect of Amandinea extract on the overproduction of ROS in LPS-induced zebrafish larvae.
  • LPS-induced ROS accumulation was tested via DCF-DA staining.
  • DCF-DA staining in LPS-induced ROS generation produced clear fluorescence images.
  • the extract reduced ROS production in vivo in a dose-dependent manner ( FIG. 6 ).
  • Example 7 Effect of Amandinea extract on pro-inflammatory cytokine production in tail amputation-induced inflammation of zebrafish
  • the extract inhibited the expression levels of inflammatory cytokines IL-6, IL-1 ⁇ , IL-10, and TNF- ⁇ in a zebrafish tail-transection model, and the levels of inflammatory mediators iNOS and COX-2 decreased.
  • the mRNA expression of IL-1 ⁇ and COX-2 was significantly reduced compared to the expression of the dexamethasone-treated positive control group (FIG. 7).
  • Amandinea (Amandinea sp.) extract inhibits the production of pro-inflammatory cytokines and inflammatory mediators in RAW264.7 cells in vitro and zebrafish in vivo, and inactivates the NF- ⁇ B pathway, so that It can be usefully used for prophylactic or therapeutic purposes.

Abstract

The present invention relates to a method for preparing an Antarctic lichen Amandine sp. extract, and a pharmaceutical composition for preventing or treating inflammatory diseases, containing the extract as an active ingredient. An Amandinea sp. extract according to the present invention inhibits proinflammatory cytokine and inflammatory mediator production and inactivates the NF-κB pathway, in RAW264.7 cells in vitro and in zebrafish in vivo, and thus can be effectively used for preventing or treating inflammatory diseases.

Description

남극 지의류 아만디네아 추출물의 제조방법 및 아만디네아 추출물을 포함하는 조성물Method for producing Amandinea extract of Antarctic lichen and composition comprising Amandinea extract
본 발명은 남극 지의류(lichen) 아만디네아(Amandinea sp.) 추출물의 제조방법 및 상기 추출물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a method for preparing an Antarctic lichen Amandinea sp. extract and to a composition for preventing or treating inflammatory diseases comprising the extract as an active ingredient.
신체는 감염 및 손상으로부터 자신을 보호하기 위해 면역계로 불리는 방어 메카니즘을 갖는다. 염증반응은 신체의 면역계의 일차 반응이다. 염증반응 동안 손상 또는 감염을 효과적으로 최소화하기 위해 여러 세포 및 분자가 관련된다(Chen, L., et al. (2018). Oncotarget, 9(6) 7204-7218.). 사이토카인은 일반적으로 면역반응의 조절에 관여하는 것으로 알려져 있다. 사이토카인은 전(pro-)염증성 및 항(anti-)염증성 사이토카인, 및 전염증성 사이토카인의 가용성 억제제로서 분류될 수 있다. 인터루킨-6(IL-6), 종양괴사인자-α(TNF-α), 인터루킨-1(IL-1), IL-12 및 인터페론-γ(IFN-γ)와 같은 전염증성 사이토카인(pro-inflammatory cytokine)은 염증을 촉진하는 반면, IL-1 수용체 길항제, IL-4, IL-6, IL-10, IL-11, 및 IL-13과 같은 항염증성 사이토카인은 전염증성 사이토카인의 생성을 억제하거나 염증반응에서 다수의 다른 억제효과를 발휘한다(Turner, MD., et al. (2014). Biochimica et Biophysica Acta (BBA)-Molecular Cell Research,　1843(11), 2563-2582.). 또한, NF-κB경로는 전염증성 사이토카인에 의해 활성화되는 프로토타입 전염증 신호전달 경로로서 간주되고, NF-κB의 역할은 광범위하게 연구되고 있다(Lawrence, T. (2009). Cold Spring Harbor Laboratory Press,　1(6), 1-10.). The body has a defense mechanism called the immune system to protect itself from infection and damage. The inflammatory response is the primary response of the body's immune system. Several cells and molecules are implicated to effectively minimize damage or infection during the inflammatory response (Chen, L., et al. (2018). Oncotarget, 9(6) 7204-7218.). Cytokines are generally known to be involved in the regulation of immune responses. Cytokines can be classified as pro- and anti-inflammatory cytokines, and soluble inhibitors of pro-inflammatory cytokines. Pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-12 and interferon-γ (IFN-γ) inflammatory cytokines) promote inflammation, whereas anti-inflammatory cytokines such as IL-1 receptor antagonists, IL-4, IL-6, IL-10, IL-11, and IL-13, inhibit the production of pro-inflammatory cytokines. Inhibits or exerts a number of other inhibitory effects in the inflammatory response (Turner, MD., et al. (2014). Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1843(11), 2563-2582.). In addition, the NF-κB pathway is considered as a prototype proinflammatory signaling pathway activated by proinflammatory cytokines, and the role of NF-κB has been extensively studied (Lawrence, T. (2009). Cold Spring Harbor Laboratory). Press, 1(6), 1-10.).
LPS에 의해 유도된 뮤린(murine) 대식세포 세포주 RAW 264.7는 천연 화합물로부터 항염증 활성을 스크리닝하는 시험관 내 연구에 통상적으로 사용된다. RAW 264.7 세포의 LPS 자극 시, 전염증성 사이토카인 및 염증성 매개체 수준의 증가가 관찰되었다(Doyle, SL., O'Neill, LA. (2006). Biochemical pharmacology,　72(9), 1102-1113.; Kim, HK., et al. (1999). Biochemical pharmacology,　58(5), 759-765.). 또한, 제브라피쉬(Danio rerio)는 생체 내 동물모델로서 많은 이점을 갖는다. 제브라피쉬는 생물학, 배아학, 분자생물학, 면역학 및 독성약물 발견 연구와 같은 다양한 분야에서 널리 사용되어 왔으며, 생체 내 제브라피쉬 항염증 시험 모델은 항염증 검정을 위한 최상의 도구로서 인식된다(Liao, YF., et al. (2011). Journal of Food & Drug Analysis,　19(2).; Lee, SH., et al. (2013). Carbohydrate polymers,　92(1), 84-89.).The murine macrophage cell line RAW 264.7 induced by LPS is commonly used in in vitro studies to screen for anti-inflammatory activity from native compounds. Upon LPS stimulation of RAW 264.7 cells, increased levels of proinflammatory cytokines and inflammatory mediators were observed (Doyle, SL., O'Neill, LA. (2006). Biochemical pharmacology, 72(9), 1102-1113.; Kim, HK., et al. (1999) Biochemical pharmacology, 58(5), 759-765.). In addition, the zebrafish (Danio rerio) has many advantages as an in vivo animal model. Zebrafish have been widely used in various fields such as biology, embryology, molecular biology, immunology, and toxic drug discovery research, and the in vivo zebrafish anti-inflammatory test model is recognized as the best tool for anti-inflammatory assays (Liao, YF). (2011). Journal of Food & Drug Analysis, 19(2).; Lee, SH., et al. (2013). Carbohydrate polymers, 92(1), 84-89.).
한편, 지의류(lichen)는 2개의 상이한 유기체인 진균 및 조류의 공생 관계로부터 유래되는 복합 유기체이다. 이들의 공생 존재로 인해, 지의류는 극단적인 생활 조건에 잘 적응할 수 있고 계속해서 성장할 수 있다. 이들은 사막지역, 연안지역, 임야지역 및 극지방과 같은 극단적인 환경에서 광범위하게 분포된다. 남극은 대부분 얼음으로 덮히고, 얼음이 없는 지상 영역은 지피식물(ground cover)의 0.5% 미만으로 구성된다. 이러한 극단적인 조건에서도, 62종의 지의류는 남극대륙에서 King George Island의 Barton Peninsula에 서식한다. 지의류는 우수한 약리학적 효과를 갖는 여러 2차 대사산물을 생산하는 것으로 알려져 있으며(Muller, K. (2001). Applied Microbiology and Biotechnology,　56(1-2), 9-16.), 다양한 종의 지의류로부터의 추출물은 항산화, 항박테리아, 및 항증식 활성을 갖는 것으로 보고되어 있다(Tanas, S., et al. (2010). Journal of natural medicines,　64(1), 42.; Mitrovic, T., et al. (2011). International journal of molecular sciences,　12(8), 5428-5448.). 그러나, 남극 지의류 아만디네아(Amandinea sp.)에 대한 연구는 미흡한 실정이며, 아만디네아 추출물의 항염증 효과는 보고된 바 없다. On the other hand, lichens are complex organisms derived from the symbiotic relationship of two different organisms, fungi and algae. Because of their symbiotic existence, lichens are well adapted to extreme living conditions and can continue to grow. They are widely distributed in extreme environments such as desert areas, coastal areas, forest areas and polar regions. Antarctica is mostly covered by ice, and the ice-free aboveground area comprises less than 0.5% of ground cover. Even in these extreme conditions, 62 species of lichens inhabit the Barton Peninsula of King George Island in Antarctica. Lichens are known to produce several secondary metabolites with excellent pharmacological effects (Muller, K. (2001). Applied Microbiology and Biotechnology, 　56(1-2), 9-16.), various species of lichens It has been reported that extracts from et al. (2011), International journal of molecular sciences, 12(8), 5428-5448.). However, studies on the Antarctic lichen Amandinea sp. are insufficient, and the anti-inflammatory effect of Amandinea extract has not been reported.
이에, 본 발명자들은 항염증 활성이 우수한 남극 지의류(lichen) 추출물을 스크리닝하고자 예의 노력한 결과, 아만디네아(Amandinea sp.) 추출물이 RAW264.7 대식세포 및 제프라피쉬에서 LPS에 의해 유도되는 염증반응에 대한 항염증 효과를 갖는 것을 확인하고, 본 발명을 완성하였다.Accordingly, the present inventors made diligent efforts to screen extracts of Antarctic lichen with excellent anti-inflammatory activity. As a result, Amandinea sp. inflammatory response induced by LPS in RAW264.7 macrophages and zebrafish It was confirmed that it has an anti-inflammatory effect on, and completed the present invention.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술을 형성하는 정보를 포함하지 않을 수 있다.The information described in the background section is only for improving the understanding of the background of the present invention, and it does not include information forming prior art known to those of ordinary skill in the art to which the present invention pertains. it may not be
발명의 요약Summary of the invention
본 발명의 목적은 항염증 활성이 우수한 신규한 천연 추출물의 제조방법을 제공하는 데 있다.An object of the present invention is to provide a method for preparing a novel natural extract having excellent anti-inflammatory activity.
본 발명의 목적은 항염증 활성이 우수한 신규한 천연 추출물을 제공하는 데 있다.An object of the present invention is to provide a novel natural extract having excellent anti-inflammatory activity.
본 발명의 다른 목적은 상기 추출물을 포함하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공하는 데 있다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising the extract.
본 발명의 또 다른 목적은 상기 추출물을 포함하는 염증성 질환의 예방 또는 개선용 식품을 제공하는 데 있다.Another object of the present invention is to provide a food for preventing or improving inflammatory diseases comprising the extract.
본 발명의 또 다른 목적은 상기 추출물 또는 상기 약학 조성물을 투여하는 단계를 포함하는 염증성 질환의 예방 또는 치료방법을 제공하는 데 있다.Another object of the present invention is to provide a method for preventing or treating an inflammatory disease comprising administering the extract or the pharmaceutical composition.
본 발명의 또 다른 목적은 상기 추출물의 항염증의 용도를 제공하는 데 있다.Another object of the present invention is to provide an anti-inflammatory use of the extract.
본 발명의 또 다른 목적은 염증성 질환의 예방 또는 치료를 위한 상기 추출물의 용도를 제공하는 데 있다.Another object of the present invention is to provide a use of the extract for the prevention or treatment of inflammatory diseases.
본 발명의 또 다른 목적은 염증성 질환의 치료를 위한 약제의 제조에 있어 상기 추출물의 사용을 제공하는 데 있다.Another object of the present invention is to provide the use of the extract in the manufacture of a medicament for the treatment of an inflammatory disease.
상기 목적을 달성하기 위하여, 본 발명은 (a) 아만디네아 샘플을 건조하여 분말화시키는 단계; (b) 상기 분말화된 아만디네아를 극성 유기용매로 추출하는 단계; 및 (c) 상기 추출된 추출액에서 용매를 제거하여 아만디네아 추출물을 수득하는 단계를 포함하는 아만디네아(Amandinea sp.) 추출물의 제조방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of (a) drying and pulverizing the Amandinea sample; (b) extracting the powdered amandinea with a polar organic solvent; and (c) removing the solvent from the extracted extract to obtain an Amandinea extract.
본 발명은 또한 상기 방법으로 제조된 아만디네아(Amandinea sp.) 추출물을 제공한다.The present invention also provides an extract of Amandinea (Amandinea sp.) prepared by the above method.
본 발명은 또한 상기 추출물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the extract as an active ingredient.
본 발명은 또한 상기 추출물을 포함하는 염증성 질환의 예방 또는 개선용 식품을 제공한다.The present invention also provides a food for preventing or improving inflammatory diseases comprising the extract.
본 발명은 또한 상기 추출물 또는 상기 약학 조성물을 투여하는 단계를 포함하는 염증성 질환의 예방 또는 치료방법을 제공한다.The present invention also provides a method for preventing or treating an inflammatory disease comprising administering the extract or the pharmaceutical composition.
본 발명은 또한 상기 추출물의 항염증의 용도를 제공한다.The present invention also provides for the anti-inflammatory use of said extract.
본 발명은 또한 염증성 질환의 예방 또는 치료를 위한 상기 추출물의 용도를 제공한다.The present invention also provides the use of said extract for the prevention or treatment of inflammatory diseases.
본 발명은 또한 염증성 질환의 치료를 위한 약제의 제조에 있어 상기 추출물의 사용을 제공한다.The present invention also provides for the use of said extract in the manufacture of a medicament for the treatment of an inflammatory disease.
본 발명은 또한 염증성 질환의 예방 또는 개선을 위한 기능성 식품의 제조에 있어 상기 추출물의 사용을 제공한다.The present invention also provides the use of said extract in the manufacture of a functional food for the prevention or amelioration of inflammatory diseases.
도 1A는 남극 지의류인 아만디네아(Amandinea sp.)의 천연 서식지를 나타낸다. 도 1B는 아만디네아 추출물이 세포 생존율에 미치는 효과를 나타내고, 도 1C는 아만디네아 추출물이 NO 생산에 미치는 효과를 나타낸다. 이들 데이터는 3회 반복의 평균±SEM을 나타낸다. LPS 자극군(#)에 대하여, *p < 0.05, **p < 0.01, 및 ***p < 0.001이다.1A shows the natural habitat of the Antarctic lichen, Amandinea sp. Figure 1B shows the effect of Amandinea extract on cell viability, Figure 1C shows the effect of Amandinea extract on NO production. These data represent the mean±SEM of 3 replicates. For the LPS stimulation group (#), *p < 0.05, **p < 0.01, and ***p < 0.001.
도 2는 ELISA 및 qPCR을 사용하여 LPS-자극된 Raw 264.7 세포에서의 염증성 유전자 발현의 측정을 도시한다. IL-6 (A) 및 TNF-α (C)의 단백질 수준을 도시하며, IL-6 (B) 및 TNF-α (D)의 상대적인 mRNA발현을 도시한다. 이들 데이터는 3회 반복의 평균±SEM을 나타낸다. LPS 자극군(#)에 대하여, *p < 0.05, **p < 0.01, 및 ***p < 0.001 이다.2 depicts the measurement of inflammatory gene expression in LPS-stimulated Raw 264.7 cells using ELISA and qPCR. Protein levels of IL-6 (A) and TNF-α (C) are shown, and relative mRNA expression of IL-6 (B) and TNF-α (D) is shown. These data represent the mean±SEM of 3 replicates. For the LPS stimulation group (#), *p < 0.05, **p < 0.01, and ***p < 0.001.
도 3은 qPCR 및 웨스턴 블롯팅을 사용하여 LPS-자극된 Raw 264.7 세포에서의 염증성 유전자 발현의 측정을 도시한다. iNOS(A) 및 COX-2(C)의 상대적인 mRNA발현을 도시하며, iNOS (B) 및 COX-2 (D)의 단백질 수준을 도시한다. 이들 데이터는 3회 반복의 평균±SEM을 나타낸다. LPS 자극군(#)에 대하여, NS, p>0.05; *, p < 0.05; **, p < 0.01; 및 ***, p < 0.001 이다.3 depicts the measurement of inflammatory gene expression in LPS-stimulated Raw 264.7 cells using qPCR and Western blotting. Relative mRNA expression of iNOS (A) and COX-2 (C) is shown, and protein levels of iNOS (B) and COX-2 (D) are shown. These data represent the mean±SEM of 3 replicates. For the LPS stimulation group (#), NS, p>0.05; *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
도 4는 웨스턴 블롯팅을 사용하여 LPS-자극된 Raw 264.7 세포에서 NF-κB 신호전달 경로에서의 단백질 p-lκB-α (A) 및 p65 (B) 발현의 측정을 도시한다. 이들 데이터는 3회 반복의 평균±SEM을 나타낸다. LPS 자극군(#)에 대하여, NS, p>0.05; *, p < 0.05; **, p < 0.01; 및 ***, p < 0.001 이다.Figure 4 depicts the measurement of protein p-1κB-α (A) and p65 (B) expression in the NF-κB signaling pathway in LPS-stimulated Raw 264.7 cells using Western blotting. These data represent the mean±SEM of 3 replicates. For the LPS stimulation group (#), NS, p>0.05; *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
도 5는 생존율(A), 형태학적 변화(B), 심장 박동수(C), 및 제브라피쉬 배아의 신체 길이(D)에 대한 아만디네아 추출물의 효과를 도시한다. *p < 0.05, **p < 0.01, 및 ***p < 0.001. Figure 5 depicts the effect of amandinea extract on survival (A), morphological changes (B), heart rate (C), and body length (D) of zebrafish embryos. *p < 0.05, **p < 0.01, and ***p < 0.001.
도 6은 DCF-DA를 사용하여 LPS-자극된 제브라피쉬 유충에서 ROS 축적의 측정을 도시한다. 도 6A는 형광 현미경에 의해 측정된 ROS 생성의 수준을 도시하고, 도 6B는 이미지 J 프로그램을 사용하여 정량화된 각각의 제브라피쉬에서의 ROS 수준의 형광 강도를 도시한다. *p < 0.05, **p < 0.01, 및 ***p < 0.001.6 depicts the measurement of ROS accumulation in LPS-stimulated zebrafish larvae using DCF-DA. Figure 6A shows the level of ROS production measured by fluorescence microscopy, and Figure 6B shows the fluorescence intensity of ROS level in each zebrafish quantified using the Image J program. *p < 0.05, **p < 0.01, and ***p < 0.001.
도 7은 qPCR을 사용하여 꼬리 절단 후 제브라피쉬 유충에서 염증성 유전자인 IL-6 (A), IL-1β (B), IL-10 (C), TNF-α (D), iNOS (E), 및 COX-2 (F)의 상대적인 발현의 측정을 도시한다. 꼬리 절단군(#)에 대하여, NS, p>0.05; *, p < 0.05; **, p < 0.01; 및 ***, p < 0.001 이다.7 shows inflammatory genes IL-6 (A), IL-1β (B), IL-10 (C), TNF-α (D), iNOS (E), inflammatory genes in zebrafish larvae after tail cutting using qPCR. and measurement of the relative expression of COX-2 (F). For the tail amputation group (#), NS, p>0.05; *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.
본 발명의 일 실시예에서, 남극에서 수집한 지의류 샘플을 완전히 건조하여, 분말화시켰다. 분말화된 지의류를 메탄올로 추출한 후, 메탄올을 증발시켰다. 이어서, 건조된 추출물을 DMSO에 용해시켜 아만디네아 추출물을 수득하였다. 또한, 아만디네아(Amandinea sp.) 추출물은 시험관 내 RAW264.7 대식세포 및 생체 내 제브라피쉬에서 NO의 생성, IL-6, IL-1β, IL-10 및 TNF-α 등과 같은 전염증성 사이토카인(pro-inflammatory cytokine)의 발현, 유도성 산화질소 효소(iNOS) 및 시클로옥시게나아제-2(COX-2) 등과 같은 염증성 매개체(inflammatory mediators)의 발현, 세포질(cytosolic) p-IκB-α 및 핵 인자 p65의 발현 및 활성산소종(reactive oxygen species, ROS)의 생성을 억제시킴으로써, 염증성 질환의 예방 또는 치료 효과를 갖는 것을 확인하였다. In one embodiment of the present invention, lichen samples collected in Antarctica were completely dried and powdered. After the powdered lichen was extracted with methanol, the methanol was evaporated. Then, the dried extract was dissolved in DMSO to obtain an Amandinea extract. In addition, Amandinea (Amandinea sp.) extract was produced in RAW264.7 macrophages in vitro and in zebrafish in vivo, pro-inflammatory cytokines such as IL-6, IL-1β, IL-10 and TNF-α. Expression of pro-inflammatory cytokines, expression of inflammatory mediators such as inducible nitric oxide enzyme (iNOS) and cyclooxygenase-2 (COX-2), cytosolic p-IκB-α and By suppressing the expression of nuclear factor p65 and the generation of reactive oxygen species (ROS), it was confirmed to have a preventive or therapeutic effect on inflammatory diseases.
따라서, 본 발명은 일 관점에서 (a) 아만디네아 샘플을 건조하여 분말화시키는 단계; (b) 상기 분말화된 아만디네아를 극성 유기용매로 추출하는 단계; 및 (c) 상기 추출된 추출액에서 용매를 제거하여 아만디네아 추출물을 수득하는 단계를 포함하는 아만디네아(Amandinea sp.) 추출물의 제조방법에 관한 것이다.Accordingly, the present invention in one aspect (a) drying and pulverizing the Amandinea sample; (b) extracting the powdered amandinea with a polar organic solvent; and (c) removing the solvent from the extracted extract to obtain an Amandinea extract.
본 발명에 있어서, 상기 (b) 단계의 극성 유기용매는 추출물은 메탄올, 에탄올, 이소프로판올, 부탄올, 아세톤, 에틸아세테이트로 구성된 군에서 선택되는 것을 특징으로 할 수 있으며, 바람직하게는 상기 용매는 메탄올이다.In the present invention, the polar organic solvent in step (b) may be characterized in that the extract is selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, and ethyl acetate, and preferably, the solvent is methanol. .
본 발명에서 용어, "지의류"는 균류와 조류 또는/및 시아노박테리아(cyanobacteria)의 공생 복합체를 의미하며, 약 14,000 종으로 분류된다.As used herein, the term "lichen" refers to a symbiotic complex of fungi and algae or/and cyanobacteria, and is classified into about 14,000 species.
본 발명에서 용어 "추출물"은 상기 아만디네아(Amandinea sp.)으로부터 분리된 항염증 활성을 가지는 물질을 말한다. 또한 본 발명에서 추출물은 추출액뿐만 아니라 이의 건조 분말 또는 이를 이용하여 제형화된 모든 형태를 포함하는 의미로 사용된다. 본 발명에서 추출물은 극성, 비극성, 물, 유기용매 또는 이의 혼합 용매를 사용하여 추출할 수 있으며, 바람직하게는 극성 유기용매를 사용하여 추출할 수 있다. 추출한 액은 액체 형태로 사용하거나 또는 농축 및/또는 건조하여 사용할 수 있다. 상기 유기용매는 탄소수 1 내지 4의 무수 또는 함수 저급알코올(메탄올, 에탄올, 이소프로판올, 부탄올 등), 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 디클로로메탄, 사염화탄소 및 벤젠, 에틸아세테이트, 부틸아세테이트, N, N-디메틸 포름아미드(DMF), 디메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합용매 등을 사용할 수 있으며, 추출물의 유효 성분이 파괴되지 않거나 최소화된 조건에서 실온 또는 가온하여 추출할 수 있다. 추출하는 유기용매에 따라 추출물의 유효성분의 추출정도와 손실정도가 차이가 날 수 있으므로, 알맞은 유기용매를 선택하여 사용하도록 한다. 추출 방법은 특별히 제한되지 않고, 예를 들어 냉침 추출, 초음파 추출, 환류 냉각 추출 등이 있다. 여과는 추출액으로부터 부유하는 고체 입자를 제거하는 과정으로, 면, 나일론 등을 이용하여 입자를 걸러 내거나 한외여과, 냉동여과법, 원심분리법 등을 사용할 수 있으나, 이에 제한되지 않는다. 또한, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 크로마토그래피)에 의한 분리 과정을 추가로 포함할 수 있다. 여액을 건조하는 단계는 동결건조, 진공건조, 열풍건조, 분무건조, 감압건조, 포말건조, 고주파건조, 적외선건조 등을 포함하나 이에 제한되지 않는다. 경우에 따라, 최종 건조된 추출물을 분쇄하는 공정을 추가할 수 있다.In the present invention, the term "extract" refers to a substance having anti-inflammatory activity isolated from the Amandinea sp. In addition, in the present invention, the extract is used in the sense of including not only the extract, but also its dry powder or all forms formulated using the same. In the present invention, the extract may be extracted using polar, non-polar, water, an organic solvent, or a mixed solvent thereof, preferably using a polar organic solvent. The extracted liquid may be used in liquid form or may be used after concentration and/or drying. The organic solvent is an anhydrous or hydrous lower alcohol (methanol, ethanol, isopropanol, butanol, etc.) having 1 to 4 carbon atoms, ethylene, acetone, hexane, ether, chloroform, dichloromethane, carbon tetrachloride and benzene, ethyl acetate, butyl acetate, N, N-dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof can be used, and the active ingredient of the extract is not destroyed or minimized at room temperature. Alternatively, extraction may be performed by heating. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the extract may be different, so an appropriate organic solvent should be selected and used. The extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction. Filtration is a process of removing suspended solid particles from the extract, and may use cotton, nylon, etc. to filter out particles, ultrafiltration, cryofiltration, centrifugation, etc., but is not limited thereto. In addition, a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity) may be further included. Drying the filtrate includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying, and the like. In some cases, a process of pulverizing the final dried extract may be added.
본 발명에 있어서, 상기 (c) 단계에서 상기 추출된 추출액에서 고속 진공 농축기를 사용하여 용매를 증발시킴으로써 용매를 제거하여 아만디네아 추출물을 수득할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the Amandinea extract may be obtained by removing the solvent by evaporating the solvent using a high-speed vacuum concentrator in the extracted extract in step (c), but is not limited thereto.
본 발명은 다른 관점에서, 상기 방법으로 제조된 아만디네아(Amandinea sp.) 추출물에 관한 것이다.In another aspect, the present invention relates to an extract of Amandinea (Amandinea sp.) prepared by the above method.
본 발명에 있어서, 상기 추출물은 하나 이상의 하기 특징을 가지는 것을 특징으로 할 수 있다:According to the present invention, the extract may be characterized as having one or more of the following characteristics:
1) 산화질소(NO)의 생성 억제;1) inhibition of nitric oxide (NO) production;
2) IL-6, IL-1β, IL-10 및 TNF-α의 발현 억제;2) inhibition of expression of IL-6, IL-1β, IL-10 and TNF-α;
3) 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제;3) inhibition of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2);
4) NF-κB(nuclear factor kappa B)의 불활성화;4) inactivation of NF-κB (nuclear factor kappa B);
5) 세포질(cytosolic) p-IκB-α 및 핵 인자 p65의 발현 억제; 및5) inhibition of expression of cytosolic p-IκB-α and nuclear factor p65; and
6) 활성산소종(reactive oxygen species, ROS)의 생성 억제.6) Inhibition of generation of reactive oxygen species (ROS).
본 발명에 있어서, 상기 추출물은 산화질소(NO)의 생성을 억제하는 것을 확인하였다(실시예 2). NO는 다양한 면역 세포에서 생성되는 세포 내 매개체인 작은 분자이며, 염증 증상의 생리학적 및 병리학적 상태에 중추적인 역할을 한다.In the present invention, it was confirmed that the extract inhibits the production of nitric oxide (NO) (Example 2). NO is a small molecule that is an intracellular mediator produced by various immune cells and plays a pivotal role in the physiological and pathological conditions of inflammatory conditions.
본 발명에서 용어 "산화질소(NO)"는 세포 내 염증반응 유발시 산화질소 합성효소에 의해 생성량이 증가하는 물질로, 염증반응의 지표가 되는 분자이다. 신경계에서 산화질소는 신경세포에 존재하는 신경계 산화질소 합성효소(NOS)에 의하여 합성된다. 상기 합성된 산화질소는 뇌세포에서 cGMP의 생성을 증가시키고, 이로 인하여 외부로부터 인지한 정보를 오랫동안 저장하는 기능을 수행한다. NO는 자유 라디칼로 생리학적, 병리학적 과정에 관련된 것으로 알려져 있다. NO는 산화질소 합성효소에 의해 엘-아르기닌(L-Arginine) 산화에 의해 합성된다(Atkan, et al., 75: 639-653, 2004). In the present invention, the term “nitric oxide (NO)” is a substance whose amount is increased by nitric oxide synthase when inducing an intracellular inflammatory reaction, and is a molecule that is an indicator of the inflammatory response. In the nervous system, nitric oxide is synthesized by the nervous system nitric oxide synthase (NOS) present in nerve cells. The synthesized nitric oxide increases the production of cGMP in brain cells, thereby performing a function of storing information recognized from the outside for a long time. NO is a free radical and is known to be involved in physiological and pathological processes. NO is synthesized by L-Arginine oxidation by nitric oxide synthase (Atkan, et al., 75: 639-653, 2004).
본 발명에 있어서, 상기 추출물은 IL-6, IL-1β, IL-10 및 TNF-α의 발현을 억제하는 것을 확인하였다(실시예 3 및 7). TNF-α, IL 등과 같은 전염증성 사이토카인의 과잉 생성은 염증질환의 발병에 기여한다.In the present invention, it was confirmed that the extract inhibits the expression of IL-6, IL-1β, IL-10 and TNF-α (Examples 3 and 7). Excessive production of pro-inflammatory cytokines such as TNF-α and IL contributes to the pathogenesis of inflammatory diseases.
본 발명에 있어서, 상기 추출물은 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현을 억제하는 것을 확인하였다(실시예 3 및 7). iNOS 및 COX-2는 염증성 매개체로서, iNOS는 NO를 생성하는 염증성 분자이며, COX-2는 PGE2를 생성한다. iNOS 및 COX-2의 과도한 활성 증가는 다양한 염증질환의 병인일 수 있다.In the present invention, it was confirmed that the extract inhibits the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (Examples 3 and 7). iNOS and COX-2 are inflammatory mediators, iNOS is an inflammatory molecule that produces NO, and COX-2 produces PGE2. Excessive increased activity of iNOS and COX-2 may be the etiology of various inflammatory diseases.
본 발명에서 용어 "COX-2"는 염증반응과 관련된 단백질인 프로스타글라딘을 생성하는데 관여하는 효소로, 세포내 COX-2 발현 수준의 증가는 염증반응이 진행되고 있음을 나타내는 지표가 될 수 있다.In the present invention, the term "COX-2" is an enzyme involved in producing prostaglandin, a protein related to an inflammatory response, and an increase in intracellular COX-2 expression level can be an indicator indicating that an inflammatory response is in progress. there is.
본 발명에 있어서, 상기 추출물은 NF-κB(nuclear factor kappa B) 경로를 불활성화하고, 세포질(cytosolic) p-IκB-α 및 핵 인자 p65의 발현을 억제하는 것을 확인하였다(실시예 4). In the present invention, it was confirmed that the extract inactivates the NF-κB (nuclear factor kappa B) pathway and suppresses the expression of cytosolic p-IκB-α and nuclear factor p65 (Example 4).
NF-κB는 면역 및 염증반응에서 중요한 역할을 하는 전사인자이며, 활성화된 NF-κB는 다양한 염증인자의 발현을 유도한다. NF-κB는 IL-6, TNF-α, iNOS, COX-2 및 IL-1β를 포함하는 150개 이상의 유전자를 조절하는 전사인자로 알려져 있다. IκB-α는 LPS, TNF 또는 IL-1에 의해 매개되는 NF-κB 활성화에서 중요한 역할을 한다. IκB-α의 활성화는 LPS-유도된 인산화에 의해 발생하여, p65의 핵 전좌(translocation)를 유도한다. 실시예 4에서, 아만디네아 추출물의 처리는 p-IκBα 및 p-65 수준의 감소효과가 있었으며, 이는 추출물의 성분이 NF-κB 신호전달을 억제하여 TNF-α, IL-6 및 NO와 같은 사이토카인 및 매개체의 발현을 감소시켰음을 나타낸다.NF-κB is a transcription factor that plays an important role in immune and inflammatory responses, and activated NF-κB induces the expression of various inflammatory factors. NF-κB is known as a transcription factor that regulates more than 150 genes including IL-6, TNF-α, iNOS, COX-2 and IL-1β. IκB-α plays an important role in NF-κB activation mediated by LPS, TNF or IL-1. Activation of IκB-α occurs by LPS-induced phosphorylation, leading to nuclear translocation of p65. In Example 4, the treatment of the Amandinea extract had the effect of reducing the levels of p-IκBα and p-65, which inhibits NF-κB signaling, and thus TNF-α, IL-6 and NO It indicates that the expression of cytokines and mediators is reduced.
본 발명에 있어서, 제브라피쉬의 생체 내 실험을 통하여 상기 추출물은 활성산소종(reactive oxygen species, ROS)의 생성을 용량-의존적으로 억제하는 것을 확인하였다(실시예 6 및 도 6).In the present invention, it was confirmed that the extract dose-dependently inhibits the generation of reactive oxygen species (ROS) through in vivo experiments of zebrafish (Example 6 and FIG. 6).
ROS 수준은 염증과 밀접하게 연관되고, 산화적 스트레스로 인한 높은 ROS 생성은 세포 또는 조직 손상을 야기한다. 활성화된 호중구 및 대식세포에서의 ROS 축적은 전염증성 사이토카인 및 매개체의 방출을 유도한다.ROS levels are closely associated with inflammation, and high ROS production due to oxidative stress causes cell or tissue damage. ROS accumulation in activated neutrophils and macrophages induces the release of proinflammatory cytokines and mediators.
본 발명의 용어 "항염증"이란, 염증을 억제하거나 감소시키는 작용을 의미하며, 상기 용어 "항염증"은 생체 조직이 손상을 입었을 때에 체내에서 일어나는 방어적 반응으로, 염증성 질환을 유발하는 원인이다. 본 명세서에서 사용된 용어, "항염증"이란 염증을 가라앉히고 예방하는 효능을 의미한다. 상기 항-염증 활성은 NO (산화질소) 억제 효과, PGE2 억제 효과, NF-κB 경로 억제 효과, Nrf2/HO-1 유도 효과, 초기 염증성 인자인 IL-1β, IL-6, TNF-α 등의 발현 억제 효과에 의해 항-염증 활성을 가지는 것일 수 있으나, 이에 제한되지 않는다. As used herein, the term “anti-inflammatory” refers to an action that inhibits or reduces inflammation, and the term “anti-inflammatory” is a protective reaction that occurs in the body when a living tissue is damaged, which is a cause of inflammatory disease. . As used herein, the term "anti-inflammatory" refers to the efficacy of alleviating and preventing inflammation. The anti-inflammatory activity is NO (nitric oxide) inhibitory effect, PGE2 inhibitory effect, NF-κB pathway inhibitory effect, Nrf2 / HO-1 induction effect, initial inflammatory factors IL-1β, IL-6, TNF-α, etc. It may have anti-inflammatory activity due to the expression inhibitory effect, but is not limited thereto.
본 발명에서 용어 "염증"이란, 어떤 자극에 대한 생체조직의 방어반응의 하나로, 각종 유해한 자극에 의한 상해를 제거하여 원래의 상태로 회복하려는 생체방어 기전이다. 염증의 자극에는, 감염 혹은 화학적, 물리적 자극이 있으며, 염증의 과정은 급성과 만성 염증의 2가지로 나눌 수 있다. 급성염증은 수일 이내의 단기적인 반응이며, 혈장성분이나 혈구 등이 미소순환계를 게재하여 이물 제거에 관련한다. 만성염증은 지속시간이 길며, 조직의 증식 등이 보여진다.In the present invention, the term "inflammation" is one of the defensive responses of biological tissues to certain stimuli, and is a biological defense mechanism that seeks to recover to the original state by removing injuries caused by various harmful stimuli. Inflammation stimuli include infection or chemical and physical stimuli, and the inflammatory process can be divided into acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days, and plasma components or blood cells are involved in the removal of foreign substances by opening the microcirculation system. Chronic inflammation has a long duration, and tissue proliferation is seen.
본 발명은 다른 관점에서, 상기 아만디네아(Amandinea sp.) 추출물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학 조성물에 관한 것이다.In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising the Amandinea sp. extract as an active ingredient.
본 발명에 있어서, 항염증 약학 조성물은 항염증 활성을 가짐으로써 염증성 질환의 예방, 치료 또는 개선에 이용될 수 있다.In the present invention, the anti-inflammatory pharmaceutical composition can be used for the prevention, treatment or improvement of inflammatory diseases by having anti-inflammatory activity.
본 발명에 있어서, 상기 약학 조성물은 하나 이상의 하기 특징을 가지는 것을 특징으로 할 수 있다:According to the present invention, the pharmaceutical composition may be characterized as having one or more of the following characteristics:
1) 산화질소(NO)의 생성 억제;1) inhibition of nitric oxide (NO) production;
2) IL-6, IL-1β, IL-10 및 TNF-α의 발현 억제;2) inhibition of expression of IL-6, IL-1β, IL-10 and TNF-α;
3) 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제;3) inhibition of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2);
4) NF-κB(nuclear factor kappa B)의 불활성화;4) inactivation of NF-κB (nuclear factor kappa B);
5) 세포질(cytosolic) p-IκB-α 및 핵 인자 p65의 발현 억제; 및5) inhibition of expression of cytosolic p-IκB-α and nuclear factor p65; and
6) 활성산소종(reactive oxygen species, ROS)의 생성 억제.6) Inhibition of generation of reactive oxygen species (ROS).
본 발명에 있어서, 상기 약학 조성물은 상기 추출물을 10μg/mL 내지 80μg/mL의 농도로 포함하는 것을 특징으로 할 수 있으며, 바람직하게는 25μg/mL 내지 50μg/mL의 추출물을 포함한다.In the present invention, the pharmaceutical composition may be characterized in that it contains the extract at a concentration of 10 µg/mL to 80 µg/mL, and preferably contains the extract at a concentration of 25 µg/mL to 50 µg/mL.
본 발명에 있어서, 상기 염증성 질환은 관절염, 비염, 간염, 각막염, 위염, 장염, 신장염, 기관지염, 흉막염, 복막염, 척추염, 췌장염, 염증성 통증, 요도염, 방광염, 화상 염증, 피부염, 치주염, 치은염 및 퇴행성 신경염증으로 구성된 군에서 선택되는 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and degenerative disease It may be characterized in that it is selected from the group consisting of neuroinflammation, but is not limited thereto.
본 발명에 있어서 "염증성 질환"은 염증을 주 병변으로 하는 질병을 총칭하는 것이다.In the present invention, "inflammatory disease" is a generic term for diseases in which inflammation is the main lesion.
본 발명에 있어서, 상기 약학 조성물은 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 포함하는 것을 특징으로 할 수 있다.In the present invention, the pharmaceutical composition may be characterized in that it further comprises a pharmaceutically acceptable carrier, excipient or diluent.
상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제는 비제한적으로 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등이 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다.Carriers, excipients and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.
본 발명에 따른 약학 조성물은 통상의 방법에 따라 다양한 형태로 제형화하여 사용될 수 있다. 적합한 제형으로는 정제, 환제, 산제, 과립제, 당의정, 경질 또는 연질의 캡슐제, 용액제, 현탁제 또는 유화액제, 주사제, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액 등이 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods. Suitable formulations include tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, oral formulations such as aerosols, external preparations, suppositories, and sterile injection solutions, However, the present invention is not limited thereto.
본 발명에 따른 약학 조성물은 약학적으로 불활성인 유기 또는 무기 담체를 이용하여 적합한 제형으로 제조할 수 있다. 즉, 제형이 정제, 코팅된 정제, 당의정 및 경질 캡슐제인 경우 락토스, 수크로스, 전분 또는 그 유도체, 탈크, 칼슘 카보네이트, 젤라틴, 스테아르산 또는 그 약학적으로 허용가능한 염을 포함할 수 있다. 또한, 제형이 연질 캡슐제인 경우에는 식물성 오일, 왁스, 지방, 반고체 및 액체의 폴리올을 포함할 수 있다. 또한, 제형이 용액 또는 시럽 형태인 경우, 물, 폴리올, 글리세롤, 및 식물성 오일 등을 포함할 수 있다.The pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, when the dosage form is a tablet, a coated tablet, a dragee, and a hard capsule, lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid or a pharmaceutically acceptable salt thereof may be included. In addition, when the formulation is a soft capsule, it may contain vegetable oils, waxes, fats, semi-solid and liquid polyols. In addition, when the formulation is in the form of a solution or syrup, water, polyol, glycerol, and vegetable oil may be included.
상기 "약학적으로 허용가능한 염"이란, 화합물이 투여되는 유기체에 심각한 자극을 유발하지 않고 화합물의 생물학적 활성과 물성들을 손상시키지 않는 화합물의 제형을 의미한다. 상기 약학적 염은, 약학적으로 허용되는 음이온을 함유하는 무독성 산부가염을 형성하는 산, 예를 들어, 염산, 황산, 질산, 인산, 브롬화수소산, 요오드화수소산 등과 같은 무기산, 타타르산, 포름산, 시트르산, 아세트산, 트리클로로아세트산, 트리플로로아세트산, 글루콘산, 벤조산, 락트산, 푸마르산, 말레인산, 살리신산 등과 같은 유기 카본산, 메탄설폰산, 에탄술폰산, 벤젠설폰산, p-톨루엔설폰산 등과 같은 설폰산 등에 의해 형성된 산부가염이 포함된다. 예를 들어, 약학적으로 허용되는 카르복실산 염에는, 리튬, 나트륨, 칼륨, 칼슘, 마그네슘 등에 의해 형성된 금속염 또는 알칼리 토금속 염, 라이신, 아르지닌, 구아니딘 등의 아미노산 염, 디시클로헥실아민, N-메틸-D-글루카민, 트리스(히드록시메틸) 메틸아민, 디에탄올아민, 콜린 및 트리에틸아민 등과 같은 유기염 등이 포함된다. 옥살산 (oxalic)과 같은 산은 약학적으로 허용되는 것은 아니지만 약학적으로 허용되는 염을 얻기 위한 중간체로서, 유용한 염의 제조에 사용될 수 있다. The "pharmaceutically acceptable salt" refers to a formulation of a compound that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and properties of the compound. The pharmaceutical salt is an acid that forms a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc., tartaric acid, formic acid, citric acid , organic carbonic acids such as acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc., sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. acid addition salts formed with phonic acid and the like are included. For example, pharmaceutically acceptable salts of carboxylic acids include metal salts or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, and guanidine, dicyclohexylamine, N organic salts such as -methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine, and the like. Acids such as oxalic acid are not pharmaceutically acceptable, but can be used in the preparation of useful salts as intermediates for obtaining pharmaceutically acceptable salts.
본 발명에 따른 약학 조성물은 상기의 담체 외에도 보존제, 안정화제, 습윤제, 유화제, 용해제, 감미제, 착색제, 삼투압 조절제, 산화방지제 등을 더 포함할 수 있다.The pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizing agent, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant, and the like, in addition to the carrier described above.
본 발명에 따른 약학 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 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 발명에 따른 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
본 발명의 조성물은 염증성 질환의 예방 또는 치료에 효과가 있는 것으로 당업자에게 인식될 수 있는 면역요법, 화학요법 및 방사선요법 등과 같은 염증성 질환의 치료법 및 다른 염증성 질환의 치료용 약학 조성물 등과 함께 사용될 수 있다.The composition of the present invention may be used together with a pharmaceutical composition for treatment of inflammatory diseases, such as immunotherapy, chemotherapy and radiation therapy, and other inflammatory diseases, which can be recognized by those skilled in the art as being effective in the prevention or treatment of inflammatory diseases. .
본 발명의 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 방식은, 예를 들면, 피하, 정맥, 근육 또는 자궁 내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다. 본 발명의 약학 조성물은 치료할 질환, 투여 경로, 환자의 연령, 성별 및 체중 및 질환의 중등도 등의 여러 관련 인자와 함께, 활성성분인 약물의 종류에 따라 결정된다.The pharmaceutical composition of the present invention may be administered to an individual by various routes. The mode of administration may be, for example, by subcutaneous, intravenous, intramuscular or intrauterine intrathecal or intracerebrovascular injection. The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.
본 발명은 또 다른 관점에서, 상기 아만디네아(Amandinea sp.) 추출물을 포함하는 염증성 질환의 예방 또는 개선용 식품에 관한 것이다.In another aspect, the present invention relates to a food for preventing or improving inflammatory diseases comprising the Amandinea (Amandinea sp.) extract.
본 발명에 있어서, 상기 식품은 하나 이상의 하기 특징을 가지는 것을 특징으로 할 수 있다:According to the present invention, the food product may be characterized as having one or more of the following characteristics:
1) 산화질소(NO)의 생성 억제;1) inhibition of nitric oxide (NO) production;
2) IL-6, IL-1β, IL-10 및 TNF-α의 발현 억제;2) inhibition of expression of IL-6, IL-1β, IL-10 and TNF-α;
3) 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제;3) inhibition of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2);
4) NF-κB(nuclear factor kappa B)의 불활성화;4) inactivation of NF-κB (nuclear factor kappa B);
5) 세포질(cytosolic) p-IκB-α 및 핵 인자 p65의 발현 억제; 및5) inhibition of expression of cytosolic p-IκB-α and nuclear factor p65; and
6) 활성산소종(reactive oxygen species, ROS)의 생성 억제.6) Inhibition of generation of reactive oxygen species (ROS).
본 발명에 있어서, 상기 식품은 상기 추출물을 10μg/mL 내지 80μg/mL의 농도로 포함하는 것을 특징으로 할 수 있으며, 바람직하게는 25μg/mL 내지 50μg/mL의 추출물을 포함한다.In the present invention, the food may be characterized in that it contains the extract at a concentration of 10 µg/mL to 80 µg/mL, and preferably contains the extract at a concentration of 25 µg/mL to 50 µg/mL.
본 발명에서 용어 "식품"은 육류, 소시지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료, 비타민 복합제, 건강기능식품 및 건강식품 등이 있으며, 통상적인 의미에서의 식품을 모두 포함한다.In the present invention, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, There are vitamin complexes, health functional foods, and health foods, and includes all foods in the ordinary sense.
상기 건강기능(성) 식품(functional food)이란, 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미한다. 여기서 "기능(성)"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 식품은 당 업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당 업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 식품의 제형 또한 식품으로 인정되는 제형이면 제한 없이 제조될 수 있으며, 본 발명에 따른 건강기능식품은 분말, 과립, 정제, 캡슐 또는 음료의 형태일 수 있다.The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, "function (sex)" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be manufactured without limitation as long as it is a formulation recognized as a food, and the health functional food according to the present invention may be in the form of powder, granule, tablet, capsule or beverage.
상기 건강식품(health food)은 일반식품에 비해 적극적인 건강유지나 증진 효과를 가지는 식품을 의미하고, 건강보조식품(health supplement food)은 건강보조 목적의 식품을 의미한다. 경우에 따라, 건강 기능 식품, 건강 식품, 건강보조식품의 용어는 혼용된다.The health food means food having an active health maintenance or promotion effect compared to general food, and health supplement food means food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement are used interchangeably.
상기 식품조성물은 생리학적으로 허용 가능한 담체를 추가로 포함할 수 있는데, 담체의 종류는 특별히 제한되지 않으며 당해 기술 분야에서 통상적으로 사용되는 담체라면 어느 것이든 사용할 수 있다.The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.
또한, 상기 조성물은 식품에 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 추가 성분을 포함할 수 있다. 예들 들어, 비타민 A, C, D, E, B1, B2, B6, B12, 니아신(niacin), 비오틴(biotin), 폴레이트(folate), 판토텐산(panthotenic acid) 등을 포함할 수 있다. 또한, 아연(Zn), 철(Fe), 칼슘(Ca), 크롬(Cr), 마그네슘(Mg), 망간(Mn), 구리(Cu), 크륨(Cr) 등의 미네랄을 포함할 수 있다. 또한, 라이신, 트립토판, 시스테인, 발린 등의 아미노산을 포함할 수 있다.In addition, the composition may include additional ingredients that are commonly used in food to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. Also, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr). In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.
또한, 상기 조성물은 방부제(소르빈산 칼륨, 벤조산나트륨, 살리실산, 데히드로초산나트륨 등), 살균제(표백분과 고도 표백분, 차아염소산나트륨 등), 산화방지제(부틸히드록시아니졸(BHA), 부틸히드록시톨류엔(BHT) 등), 착색제(타르색소 등), 발색제(아질산 나트륨, 아초산 나트륨 등), 표백제(아황산나트륨), 조미료(MSG 등), 감미료(둘신, 사이클레메이트, 사카린, 나트륨 등), 향료(바닐린, 락톤류 등), 팽창제(명반, D-주석산수소칼륨 등), 강화제, 유화제, 증점제(호료), 피막제, 검기초제, 거품억제제, 용제, 개량제 등의 식품 첨가물(foodadditives)을 포함할 수 있다. 상기 첨가물은 식품의 종류에 따라 선별되고 적절한 양으로 사용될 수 있다.In addition, the composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butylhydroxy Toluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG, etc.), sweeteners (dulcin, cyclimate, saccharin, sodium, etc.) ), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, food additives such as improving agents ) may be included. The additive may be selected according to the type of food and used in an appropriate amount.
본 발명의 아만디네아 추출물과 함께 식품학적으로 허용가능한 식품 보조 첨가제를 더 포함할 수 있으며, 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.It may further include a food supplementary additive together with the Amandinea extract of the present invention, and may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment).
본 발명에서 용어 "개선", "예방" 및 "치료"는 최광의의 개념으로 해석되어야 하며, "개선"이란 질환 또는 하나 이상의 임상적 증상을 일시적/지속적으로 완화시키는 모든 행위를 의미한다. "예방"이란, 질환에 노출되거나 질환에 걸리기 쉬울 수 있으나 질환의 증상을 아직 경험하거나 드러내지 아니한 환자에게서 질환의 임상적 증상 중 하나 이상이 진행되지 아니하도록 하는 것을 의미한다. "치료"란, 질환 또는 이의 하나 이상의 임상적 증상의 발달을 저지 또는 감소시키는 모든 행위를 의미한다.In the present invention, the terms "improvement", "prevention" and "treatment" should be interpreted in the broadest sense, and "improvement" means any action that temporarily/continuously relieves a disease or one or more clinical symptoms. "Prevention" means preventing the progression of one or more of the clinical symptoms of a disease in a patient who may be exposed to or susceptible to the disease but has not yet experienced or revealed symptoms of the disease. "Treatment" means any action that arrests or reduces the development of a disease or one or more clinical symptoms thereof.
본 발명은 또 다른 관점에서, 상기 아만디네아(Amandinea sp.) 추출물 또는 상기 약학 조성물을 투여하는 단계를 포함하는 염증성 질환의 예방 또는 치료방법에 관한 것이다.In another aspect, the present invention relates to a method for preventing or treating an inflammatory disease comprising administering the Amandinea sp. extract or the pharmaceutical composition.
본 발명은 또 다른 관점에서, 상기 아만디네아(Amandinea sp.) 추출물의 항염증의 용도에 관한 것이다.In another aspect, the present invention relates to the anti-inflammatory use of the extract of Amandinea (Amandinea sp.).
본 발명은 또 다른 관점에서, 염증성 질환의 예방 또는 치료를 위한 상기 아만디네아(Amandinea sp.) 추출물의 용도에 관한 것이다.In another aspect, the present invention relates to the use of the Amandinea (Amandinea sp.) extract for the prevention or treatment of inflammatory diseases.
본 발명은 또 다른 관점에서, 염증성 질환의 치료를 위한 약제의 제조에 있어 상기 아만디네아(Amandinea sp.) 추출물의 사용에 관한 것이다.In another aspect, the present invention relates to the use of the extract of Amandinea (Amandinea sp.) in the manufacture of a medicament for the treatment of an inflammatory disease.
본 발명은 또 다른 관점에서, 염증성 질환의 예방 또는 개선을 위한 기능성 식품의 제조에 있어 상기 아만디네아(Amandinea sp.) 추출물의 사용에 관한 것이다.In another aspect, the present invention relates to the use of the Amandinea (Amandinea sp.) extract in the manufacture of a functional food for the prevention or improvement of inflammatory diseases.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예1: 물질 및 분석방법Example 1: Materials and Analytical Methods
실시예1-1: 지의류(lichen)의 수집 및 추출물의 제조Example 1-1: Collection of lichen and preparation of extract
지의류 샘플은 남극대륙의 Ardley island 상의 62°12'57.82"S, 58°56'01.87"W 에서 수집하였다. 샘플 동정 및 수집지역에 관한 상세한 정보는 표 1에 도시되어 있다. 확증표본(Voucher specimens)은 한국 극지연구소(Korea Polar Research Institute)에 보관되었다. 지의류 샘플을 완전히 건조하여, 막자(pestle) 및 모르타르(mortar)를 사용하여 분말화시켰다. 분말화된 지의류를 2주 동안 25°C의 어둠속에서 메탄올(1g/10mL)로 추출하였다. 추출물 중의 메탄올을 고속 진공 농축기(speed vacuum concentrator)를 사용하여 증발시켰다. 이어서, 건조된 추출물을 DMSO에 최종 농도 20 mg/mL로 용해시켰다.Lichen samples were collected at 62°12'57.82"S, 58°56'01.87"W on Ardley Island, Antarctica. Detailed information on sample identification and collection areas is shown in Table 1. Voucher specimens were stored at the Korea Polar Research Institute. The lichen samples were thoroughly dried and powdered using pestle and mortar. Powdered lichens were extracted with methanol (1 g/10 mL) in the dark at 25 °C for 2 weeks. Methanol in the extract was evaporated using a speed vacuum concentrator. The dried extract was then dissolved in DMSO to a final concentration of 20 mg/mL.
Figure PCTKR2021014012-appb-img-000001
Figure PCTKR2021014012-appb-img-000001
실시예1-2: 세포 배양 및 처리Example 1-2: Cell culture and treatment
뮤린 대식세포 Raw 264.7 세포를 5% CO2/95% 공기 중 37°C에서 10% 소태아혈청(FBS) 및 1% 페니실린 스트렙토마이신(PS)을 함유하는 Dulbecco's modified Eagle's medium(DMEM)에서 배양하고 배지를 5% FBS 및 1% PS를 함유하는 DMEM으로 교체한 후 처리하였다.Murine macrophage Raw 264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (PS) at 37 °C in 5% CO 2 /95% air. The medium was replaced with DMEM containing 5% FBS and 1% PS before treatment.
실시예1-3: 세포독성 분석Example 1-3: Cytotoxicity assay
The MTT [(3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] 분석을 사용하여 세포 생존율을 평가하였다. Raw 264.7 세포(96-웰 플레이트에서 2.5 X 104 cells/well)를 4시간 동안 배양한 후, 다양한 농도의 지의류 추출물(0, 10, 20, 40, 및 80 μg/mL)로 24시간 동안 처리하였다. 배양 후, 5 μM MTT 용액을 각 웰에 첨가하고 4시간 동안 배양하였다. 각 웰의 배지를 제거한 다음, 100 μL/well의 DMSO를 첨가하고 570 nm에서 흡광도를 측정하여 세포 생존율을 결정했다. 상대적인 세포 생존율(%)은 미처리 대조군 세포에 대한 백분율로서 계산하였다.Cell viability was evaluated using the MTT [(3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Raw 264.7 cells (2.5 X 10 4 cells/well in 96-well plates) were incubated for 4 hours and then treated with various concentrations of lichen extracts (0, 10, 20, 40, and 80 μg/mL) for 24 hours. did. After incubation, 5 μM MTT solution was added to each well and incubated for 4 hours. After removing the medium from each well, 100 μL/well of DMSO was added and the absorbance was measured at 570 nm to determine the cell viability. Relative cell viability (%) was calculated as a percentage of untreated control cells.
실시예1-4: NO 측정Example 1-4: NO measurement
Griess 시약은 세포 배양 배지에서 아질산염(Nitrite) 생산을 검정하는 데 사용하였다. Raw 264.7 세포(96-well plate 에서 5 x 105 cells/well)를 24시간 동안 배양하였다. 배지를 제거하고 신선한 DMEM 배지 (5 % FBS)로 교체한 다음 37 °C에서 4시간 동안 배양했다. 세포를 다양한 농도의 지의류 추출물(0, 10, 20, 40, 80 μg/mL)로 1시간 동안 처리한 후, E.coli 지질다당류(LPS, serotype O111:B4)로 24시간 동안 처리했다. 세포 배양 배지(100 μL/well)를 96-웰 플레이트의 웰로 옮긴 다음 동일한 부피의 Griess 시약(1% sulfanilamide 및 0.1% N-1-naphylenediamine dihydrochloride in 2.5% phosphoric acid)을 첨가하고, NO 농도를 확인하기 위해 540 nm에서 흡광도를 측정하였다.Griess reagent was used to assay nitrite production in cell culture media. Raw 264.7 cells (5 x 10 5 cells/well in a 96-well plate) were cultured for 24 hours. The medium was removed and replaced with fresh DMEM medium (5% FBS) and then incubated at 37 °C for 4 h. Cells were treated with various concentrations of lichen extract (0, 10, 20, 40, 80 μg/mL) for 1 hour, and then treated with E. coli lipopolysaccharide (LPS, serotype O111:B4) for 24 hours. Transfer the cell culture medium (100 µL/well) to the wells of a 96-well plate, add an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-1-naphylenediamine dihydrochloride in 2.5% phosphoric acid), and check the NO concentration To measure the absorbance at 540 nm.
실시예1-5: IL-6 및 TNF-α의 측정Example 1-5: Measurement of IL-6 and TNF-α
Sandwich-enzyme-linked immunosorbent assay kits(ELISA)를 사용하여 세포로부터 분비된 IL-6(DY406-05, R&D Systems Inc, USA) 및 TNF-α(DY410-05, R&D Systems Inc, USA)의 농도를 측정하였다. Raw 264.7 세포(2.5 x 104 cells/mL)를 24시간 동안 배양하였다. 배지를 제거하고 새로운 DMEM 배지로 교체한 다음 37°C에서 4시간 동안 배양하였다. 세포를 다양한 농도의 지의류 추출물(0, 10, 20, 40, 80 μg/mL)로 1시간 동안 처리한 후, 0.5 μg/mL의 E.coli 지질다당류(LPS, serotype O111:B4)로 24시간 동안 처리했다. ELISA는 제조업체의 지침에 따라 수행하였고, 광학밀도는 마이크로플레이트 리더를 사용하여 450nm에서 즉시 측정하였다.The concentrations of IL-6 (DY406-05, R&D Systems Inc, USA) and TNF-α (DY410-05, R&D Systems Inc, USA) secreted from the cells were measured using Sandwich-enzyme-linked immunosorbent assay kits (ELISA). measured. Raw 264.7 cells (2.5 x 10 4 cells/mL) were cultured for 24 hours. The medium was removed and replaced with fresh DMEM medium, and then incubated at 37 °C for 4 h. Cells were treated with various concentrations of lichen extract (0, 10, 20, 40, 80 μg/mL) for 1 hour, and then treated with 0.5 μg/mL E. coli lipopolysaccharide (LPS, serotype O111:B4) for 24 hours. processed while ELISA was performed according to the manufacturer's instructions, and optical density was measured immediately at 450 nm using a microplate reader.
실시예1-6: 정량적 실시간 PCR 분석 (IL-6, TNF-α, iNOS, COX-2, IL-1β, IL-10)Example 1-6: Quantitative real-time PCR analysis (IL-6, TNF-α, iNOS, COX-2, IL-1β, IL-10)
총 RNA를 제조업체의 지침에 따라 easy-BLUETM Total RNA Extraction Kit (17061, Intron biology)를 사용하여 분리하였다. RNA (1 μg)를 M-MLV 역전사효소 (RT001S, Sigma-Aldrich)를 사용하여 역전사시킨 후, Rotor 유전자 6500 (Corbett research)을 사용하여 TOPrealTM qPCR 2X PreMIX (RT500, Enzynomics)로 실시간 PCR 증폭을 수행했다. 표적 유전자 프라이머 서열은 표 2에 기재되어 있다. 발현 데이터는 비교 사이클 역치(Ct) 방법을 사용하여 분석하였고, β-액틴 발현의 수준으로 표준화하였다.Total RNA was isolated using easy-BLUE TM Total RNA Extraction Kit (17061, Intron biology) according to the manufacturer's instructions. RNA (1 μg) was reverse transcribed using M-MLV reverse transcriptase (RT001S, Sigma-Aldrich), and then real-time PCR amplification was performed with TOPreal™ qPCR 2X PreMIX (RT500, Enzynomics) using Rotor gene 6500 (Corbett research). did. The target gene primer sequences are listed in Table 2. Expression data were analyzed using the comparative cycle threshold (C t ) method and normalized to the level of β-actin expression.
Figure PCTKR2021014012-appb-img-000002
Figure PCTKR2021014012-appb-img-000002
실시예1-7: 면역블로팅(Immunoblotting)Example 1-7: Immunoblotting
단백질을 RIPA 완충액(Sigma), 프로테아제 억제제 칵테일 정제(Roche) 및 포스파타제 억제제 칵테일 정제(Rhoche) 또는 핵 추출 키트 (ab113474, Abcam)를 사용하여 추출하였다. 추출된 단백질(20μg)을 SDS-PAGE에 의해 분리하고, PVDF 막으로 옮기고, TBST 완충액에서 5% 탈지유로 1시간 동안 세척하고, 1차 항체와 4°C에서 밤새 배양하였다. 1차 항체는 iNOS (ADI-905-431, Enzo), COX-2 (SC-166475, Santa Cruz Biotechnology), p-IκBα (#2859, Cell signaling), p65 (#3033, Cell signaling), PCNA (SC-25280, Santa Cruz Biotechnology) 및 GAPDH (SC-25778, Santa Cruz Biotechnology)이었다. 2차 항체는 항-마우스(HAF007, DuoSet) 및 항-래빗(SC-2537-CM, Santa Cruz Biotechnology)이였다. 블롯을 증강된 화학발광(ECL) 키트(Thermo Fisher)를 사용하여 분석하였다.Proteins were extracted using RIPA buffer (Sigma), protease inhibitor cocktail purification (Roche) and phosphatase inhibitor cocktail purification (Rhoche) or nuclear extraction kit (ab113474, Abcam). The extracted proteins (20 μg) were separated by SDS-PAGE, transferred to PVDF membranes, washed with 5% skim milk in TBST buffer for 1 h, and incubated with primary antibody overnight at 4 °C. Primary antibodies are iNOS (ADI-905-431, Enzo), COX-2 (SC-166475, Santa Cruz Biotechnology), p-IκBα (#2859, Cell signaling), p65 (#3033, Cell signaling), PCNA ( SC-25280, Santa Cruz Biotechnology) and GAPDH (SC-25778, Santa Cruz Biotechnology). Secondary antibodies were anti-mouse (HAF007, DuoSet) and anti-rabbit (SC-2537-CM, Santa Cruz Biotechnology). Blots were analyzed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher).
실시예1-8: 모(parental) 제브라피쉬의 기원 및 유지Examples 1-8: Origin and maintenance of parental zebrafish
성인 제브라피쉬를 상인(Ansan aquarium, Korea)으로부터 입수하고, 14:10h 명:암 주기로 28°C±0.5로 유지하였다. 짝을 이루기 전에, 제브라피쉬를 암컷 및 수컷의 2개의 그룹으로 분리하였다. 이들은 아침에 접촉하여 빛에 반응하여 30분 이내에 알을 낳았다.Adult zebrafish were obtained from a merchant (Ansan aquarium, Korea) and maintained at 28 °C ± 0.5 with a 14:10 h light:dark cycle. Prior to mating, zebrafish were separated into two groups: females and males. They reacted to light on contact in the morning and laid eggs within 30 minutes.
실시예1-9: 배아 독성의 측정Example 1-9: Determination of embryo toxicity
수정 후 6시간(hpf)에서, 제브라피쉬 배아(n = 10)를 웰 당 2 mL의 배아 배지를 함유하는 12-웰 플레이트로 옮겼다. 배아를 다양한 농도의 지의류 추출물(0, 1, 10, 100, 200 및 400 μg/mL)로 120 hpf 동안 처리하였다. 처리액 중의 최종 DMSO 농도는 1%였다.At 6 h post-fertilization (hpf), zebrafish embryos (n = 10) were transferred to 12-well plates containing 2 mL of embryo medium per well. Embryos were treated with various concentrations of lichen extracts (0, 1, 10, 100, 200 and 400 μg/mL) for 120 hpf. The final DMSO concentration in the treatment solution was 1%.
생존율은 120 hpf까지 매일 측정하였다. 제브라피쉬를 48 hpf에서 0.25 mg/mL tricaine으로 마취시키고, 심장 박동수 및 신체 길이를 현미경을 사용하여 분석하였다.Viability was measured daily up to 120 hpf. Zebrafish were anesthetized with 0.25 mg/mL tricaine at 48 hpf, and heart rate and body length were analyzed using a microscope.
실시예1-10: 제브라피쉬에서의 ROS 수준의 측정Example 1-10: Measurement of ROS level in zebrafish
제브라피쉬 유충에서의 ROS 생성은 인용문헌(Jeong, JW., et al. (2017). Molecular & Cellular Toxicology,　13(4), 405-417.)에 기재된 바와 같이, 산화-민감성 형광 프로브 염료인 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA, Sigma)를 사용하여 검출하였다. 3 hpf에서, 제브라피쉬 유충 (n = 10)을 12-웰 플레이트로 옮기고, 10 μg/mL LPS (지의류 추출물을 갖거나 갖지 않음)로 24시간 동안 처리하였다. 처리 후, 제브라피쉬 유충을 12-웰 플레이트로 옮기고 DCF-DA를 최종 농도 20 ㎍/mL로 배아 배지에 첨가하였다. 암실에서 28.5°C에서 1시간 동안 배양한 후, 제브라피쉬 유충을 배아 배지로 세척한 다음, 0.25mg/mL 트리카인(tricaine)으로 마취시켰다. ROS 생성을 형광 현미경으로 관찰하고, ImageJ 소프트웨어를 사용하여 정량화하였다.ROS production in zebrafish larvae is an oxidation-sensitive fluorescent probe dye, as described in Jeong, JW., et al. (2017). Molecular & Cellular Toxicology , 13 (4), 405-417. Detection was performed using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA, Sigma). At 3 hpf, zebrafish larvae (n = 10) were transferred to 12-well plates and treated with 10 μg/mL LPS (with or without lichen extract) for 24 h. After treatment, zebrafish larvae were transferred to 12-well plates and DCF-DA was added to the embryo medium to a final concentration of 20 μg/mL. After incubation for 1 h at 28.5 °C in the dark, zebrafish larvae were washed with embryonic medium and then anesthetized with 0.25 mg/mL tricaine. ROS generation was observed by fluorescence microscopy and quantified using ImageJ software.
실시예1-11: 꼬리 절단(Tail transection) 모델Examples 1-11: Tail transection model
염증반응에 대한 지의류 추출물의 효과는 인용문헌(Li, JJ., et al. (2018). Fish & shellfish immunology, 83, 205-212.)에 기재된 바와 같이 꼬리 절단(tail transection) 모델을 사용하여 결정하였다. 96 hpf에서, 제브라피쉬 유충을 0.25 mg/mL 트리카인(tricaine)으로 마취시키고 꼬리의 절단을 메스(scalpel)를 사용하여 수행하였다. 이어서, 제브라피쉬 유충을 배아 배지로 세척하고, 6-웰 플레이트(n=20/well)로 옮겨, 다양한 농도의 지의류 추출물(0, 25, 50μg/mL) 및 100μM 덱사메타손으로 24시간 동안 처리했다.The effect of lichen extract on the inflammatory response was evaluated using a tail transection model as described in Li, JJ., et al. (2018). Fish & shellfish immunology , 83, 205-212. decided. At 96 hpf, zebrafish larvae were anesthetized with 0.25 mg/mL tricaine and tail amputation was performed using a scalpel. Then, zebrafish larvae were washed with embryo medium, transferred to 6-well plates (n=20/well), and treated with various concentrations of lichen extract (0, 25, 50 μg/mL) and 100 μM dexamethasone for 24 hours.
실시예1-12: 통계적 분석Examples 1-12: Statistical Analysis
모든 실험을 3회 실시하여, 유사한 결과를 얻었다. 결과는 평균 ± 표준 오차로서 표현하고 t-검증(student's t-test)을 사용하여 분석하였다. 각 그래프의 바 상의 별표는 P-값(* P < 0.05, ** P < 0.01, ***P < 0.001)을 나타낸다.All experiments were performed three times, and similar results were obtained. Results were expressed as mean ± standard error and analyzed using student's t-test. Asterisks on the bars of each graph indicate P-values (* P < 0.05, ** P < 0.01, ***P < 0.001).
실시예 2: 세포 생존율 및 NO 생산에 대한 아만디네아(Amandinea sp.) 추출물의 효과Example 2: Effect of Amandinea sp. extract on cell viability and NO production
2017년에 King Sejong Antarctic Station 근처의 공급원으로부터 지의류를 수집하였다(도 1A). 지의류 종은 PCR(데이터 미표시)에 의해 아만디네아(Amandinea sp.)으로 동정되었다. Lichens were collected from sources near King Sejong Antarctic Station in 2017 (Fig. 1A). The lichen species was identified as Amandinea sp. by PCR (data not shown).
LPS의 존재 하에 RAW 264.7 세포 상에서 아만디네아 추출물의 독성을 측정하기 위해, MTT 분석을 수행하여 세포 생존율을 측정하였다. 추출물은 LPS-자극된 RAW264.7 세포에 대해 세포독성 효과를 갖지 않는 것으로 밝혀졌다. 이들은 10, 20, 40 및 80 μg/ml의 농도에서 생존율에 대해 유의한 효과를 나타내지 않았다(도 1B). 이어서, NO 검정을 사용하여 LPS-자극된 Raw 264.7 세포에서 NO 생산에 대한 추출물의 효과를 평가하였다. 결과는 추출물이 용량-의존적 방식으로 NO 생산이 감소시킴을 나타내었다(도 1C). 이러한 결과에 기초하여, 본 발명자들은 모든 후속 실험에서 10, 20, 40 및 80 μg/ml의 추출물 농도를 사용하였다.To determine the toxicity of Amandinea extract on RAW 264.7 cells in the presence of LPS, MTT assay was performed to determine cell viability. The extract was found to have no cytotoxic effect on LPS-stimulated RAW264.7 cells. They showed no significant effect on viability at concentrations of 10, 20, 40 and 80 μg/ml ( FIG. 1B ). The NO assay was then used to evaluate the effect of extracts on NO production in LPS-stimulated Raw 264.7 cells. The results indicated that the extract reduced NO production in a dose-dependent manner ( FIG. 1C ). Based on these results, we used extract concentrations of 10, 20, 40 and 80 μg/ml in all subsequent experiments.
실시예 3: 전염증성 사이토카인 생산에 대한 아만디네아 추출물의 효과Example 3: Effect of Amandinea Extract on Pro-inflammatory Cytokine Production
NO 생산 결과에 기초하여, LPS-유도된 염증성 사이토카인 생산에 대한 추출물의 효과를 추가로 조사하였다. 추출물은 전염증성 사이토카인 IL-6 및 TNF-α의 발현 수준을 억제하였고, LPS-자극된 Raw 264.7 세포에서 염증성 매개체인 iNOS 및 COX-2의 수준을 감소시켰다. 이들은 ELISA, qPCR, 및 웨스턴 블롯팅에 의해 측정하였다(도 2 및 3). COX-2 단백질 수준은 유의미하지 않았지만, 데이터는 추출물이 LPS-자극된 사이토카인 생산을 감소시킴을 보여준다.Based on the NO production results, the effect of the extract on LPS-induced inflammatory cytokine production was further investigated. The extract inhibited the expression levels of pro-inflammatory cytokines IL-6 and TNF-α, and decreased the levels of inflammatory mediators iNOS and COX-2 in LPS-stimulated Raw 264.7 cells. These were determined by ELISA, qPCR, and Western blotting ( FIGS. 2 and 3 ). COX-2 protein levels were not significant, but the data show that the extract reduced LPS-stimulated cytokine production.
실시예 4: NF-κB 신호전달에 대한 아만디네아 추출물의 효과Example 4: Effect of Amandinea extract on NF-κB signaling
본 발명자들은 NF-κB 신호전달에 대한 추출물의 효과를 시험하였다. 웨스턴 블롯팅에 의해 검출된 바와 같이 세포질 p-IκB-α 및 핵 p65의 수준이 감소되었음을 확인하였다(도 4). 이들 결과는 추출물이 세포질 p-IκB-α 수준을 감소시킴으로써 핵 p65 수준을 억제한다는 것을 암시한다.We tested the effect of extracts on NF-κB signaling. It was confirmed that the levels of cytoplasmic p-IκB-α and nuclear p65 were decreased as detected by Western blotting ( FIG. 4 ). These results suggest that the extract inhibits nuclear p65 levels by decreasing cytoplasmic p-IκB-α levels.
실시예 5: 제브라피쉬에서 생존율, 심장 박동수 및 신체 길이에 대한 아만디네아 추출물의 효과Example 5: Effect of Amandinea Extract on Survival Rate, Heart Rate and Body Length in Zebrafish
제브라피쉬 추출물에서 아만디네아 추출물의 독성을 결정하기 위해, 생존율, 심장 박동수 및 신체 길이를 관찰하였다. 생존율은 200 및 400 μg/mL의 지의류 추출물로의 처리에서 상당히 감소되었다(도 5A). 이들 결과에 기초하여, 본 발명자들은 25 및 50 μg/mL의 추출물 농도를 사용하여 심장 박동수 및 신체 길이를 실험하였다. 추출물의 처리 시 대조군과 비교하여 심장 박동수 및 신체 길이에서의 유의한 변화는 검출되지 않았다. 따라서, 아만디네아 추출물은 100 μg/mL 이하에서 독성이 존재하지 않음을 알 수 있다.To determine the toxicity of Amandinea extract in zebrafish extract, survival rate, heart rate and body length were observed. Viability was significantly reduced with treatment with 200 and 400 μg/mL lichen extract ( FIG. 5A ). Based on these results, we tested heart rate and body length using extract concentrations of 25 and 50 μg/mL. No significant changes in heart rate and body length were detected upon treatment of the extract compared to the control group. Therefore, it can be seen that the Amandinea extract does not have toxicity at 100 μg/mL or less.
실시예 6: LPS-처리된 제브라피쉬에서의 ROS 생산에 대한 아만디네아 추출물의 효과Example 6: Effect of Amandinea extract on ROS production in LPS-treated zebrafish
본 발명자들은 LPS-유도된 제브라피쉬 유충에서 ROS의 과생산에 대한 아만디네아 추출물의 억제효과를 확인하였다. LPS-유도된 ROS 축적을 DCF-DA 염색을 통해 실험하였다. LPS-유도된 ROS 생성에서 DCF-DA 염색은 투명한 형광 이미지를 생성하였다. 추출물은 용량-의존성 방식으로 생체 내 ROS 생성을 감소시켰다(도 6).The present inventors confirmed the inhibitory effect of Amandinea extract on the overproduction of ROS in LPS-induced zebrafish larvae. LPS-induced ROS accumulation was tested via DCF-DA staining. DCF-DA staining in LPS-induced ROS generation produced clear fluorescence images. The extract reduced ROS production in vivo in a dose-dependent manner ( FIG. 6 ).
실시예 7: 제브라피쉬의 꼬리 절단-유도된 염증에서 전염증성 사이토카인 생성에 대한 아만디네아 추출물의 효과Example 7: Effect of Amandinea extract on pro-inflammatory cytokine production in tail amputation-induced inflammation of zebrafish
추출물은 제브라피쉬 꼬리-절단(tail-transection) 모델에서 염증성 사이토카인 IL-6, IL-1β, IL-10, 및 TNF-α의 발현 수준을 억제하였고, 염증성 매개체인 iNOS 및 COX-2의 수준을 감소시켰다. IL-1β 및 COX-2의 mRNA 발현은 덱사메타손이 처리된 양성대조군의 발현에 비해 상당히 감소되었다(도 7).The extract inhibited the expression levels of inflammatory cytokines IL-6, IL-1β, IL-10, and TNF-α in a zebrafish tail-transection model, and the levels of inflammatory mediators iNOS and COX-2 decreased. The mRNA expression of IL-1β and COX-2 was significantly reduced compared to the expression of the dexamethasone-treated positive control group (FIG. 7).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.
본 발명에 따르면 아만디네아(Amandinea sp.) 추출물은 시험관 내 RAW264.7 세포 및 생체 내 제브라피쉬에서 전염증성 사이토카인 및 염증성 매개체의 생성을 억제하고 NF-κB 경로를 불활성화 시키므로, 염증성 질환의 예방 또는 치료 용도로 유용하게 사용될 수 있다.According to the present invention, Amandinea (Amandinea sp.) extract inhibits the production of pro-inflammatory cytokines and inflammatory mediators in RAW264.7 cells in vitro and zebrafish in vivo, and inactivates the NF-κB pathway, so that It can be usefully used for prophylactic or therapeutic purposes.
전자파일 첨부하였음.An electronic file is attached.

Claims (12)

  1. 다음 단계를 포함하는 아만디네아(Amandinea sp.) 추출물의 제조방법:A method for preparing an extract of Amandinea sp. comprising the steps of:
    (a) 아만디네아 샘플을 건조하여 분말화시키는 단계;(a) drying and pulverizing the Amandinea sample;
    (b) 상기 분말화된 아만디네아를 극성 유기용매로 추출하는 단계; 및(b) extracting the powdered amandinea with a polar organic solvent; and
    (c) 상기 추출된 추출액에서 용매를 제거하여 아만디네아 추출물을 수득하는 단계.(c) removing the solvent from the extracted extract to obtain an Amandinea extract.
  2. 제1항에 있어서, 상기 (b) 단계의 극성 유기용매는 추출물은 메탄올, 에탄올, 이소프로판올, 부탄올, 아세톤, 에틸아세테이트로 구성된 군에서 선택되는 것을 특징으로 하는 추출물의 제조방법.The method of claim 1, wherein the polar organic solvent in step (b) is selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, and ethyl acetate.
  3. 제1항 또는 제2항의 방법으로 제조된 아만디네아(Amandinea sp.) 추출물.Claims 1 or 2 Amandinea (Amandinea sp.) extract prepared by the method.
  4. 제3항에 있어서, 하나 이상의 하기 특징을 가지는 것을 특징으로 하는 추출물:4. The extract according to claim 3, characterized in that it has one or more of the following characteristics:
    1) 산화질소(NO)의 생성 억제;1) inhibition of nitric oxide (NO) production;
    2) IL-6, IL-1β, IL-10 및 TNF-α의 발현 억제;2) inhibition of expression of IL-6, IL-1β, IL-10 and TNF-α;
    3) 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제;3) inhibition of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2);
    4) NF-κB(nuclear factor kappa B)의 불활성화;4) inactivation of NF-κB (nuclear factor kappa B);
    5) 세포질(cytosolic) p-IκB-α 및 핵 인자 p65의 발현 억제; 및5) inhibition of expression of cytosolic p-IκB-α and nuclear factor p65; and
    6) 활성산소종(reactive oxygen species, ROS)의 생성 억제.6) Inhibition of generation of reactive oxygen species (ROS).
  5. 아만디네아(Amandinea sp.) 추출물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising Amandinea sp. extract as an active ingredient.
  6. 제5항에 있어서, 하나 이상의 하기 특징을 가지는 것을 특징으로 하는 약학 조성물:6. The pharmaceutical composition according to claim 5, characterized in that it has one or more of the following characteristics:
    1) 산화질소(NO)의 생성 억제;1) inhibition of nitric oxide (NO) production;
    2) IL-6, IL-1β, IL-10 및 TNF-α의 발현 억제;2) inhibition of expression of IL-6, IL-1β, IL-10 and TNF-α;
    3) 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제;3) inhibition of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2);
    4) NF-κB(nuclear factor kappa B)의 불활성화;4) inactivation of NF-κB (nuclear factor kappa B);
    5) 세포질(cytosolic) p-IκB-α 및 핵 인자 p65의 발현 억제; 및5) inhibition of expression of cytosolic p-IκB-α and nuclear factor p65; and
    6) 활성산소종(reactive oxygen species, ROS)의 생성 억제.6) Inhibition of generation of reactive oxygen species (ROS).
  7. 제5항에 있어서, 상기 추출물을 10μg/mL 내지 80μg/mL의 농도로 포함하는 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 5, wherein the extract is contained at a concentration of 10 μg/mL to 80 μg/mL.
  8. 제5항에 있어서, 상기 염증성 질환은 관절염, 비염, 간염, 각막염, 위염, 장염, 신장염, 기관지염, 흉막염, 복막염, 척추염, 췌장염, 염증성 통증, 요도염, 방광염, 화상 염증, 피부염, 치주염, 치은염 및 퇴행성 신경염증으로 구성된 군에서 선택되는 것을 특징으로 하는 약학 조성물.According to claim 5, wherein the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and Pharmaceutical composition, characterized in that selected from the group consisting of degenerative neuroinflammation.
  9. 제5항에 있어서, 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 포함하는 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 5, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
  10. 아만디네아(Amandinea sp.) 추출물을 포함하는 염증성 질환의 예방 또는 개선용 식품.Food for preventing or improving inflammatory diseases, including Amandinea sp. extract.
  11. 제10항에 있어서, 하나 이상의 하기 특징을 가지는 것을 특징으로 하는 식품:11. The food product according to claim 10, characterized in that it has one or more of the following characteristics:
    1) 산화질소(NO)의 생성 억제;1) inhibition of nitric oxide (NO) production;
    2) IL-6, IL-1β, IL-10 및 TNF-α의 발현 억제;2) inhibition of expression of IL-6, IL-1β, IL-10 and TNF-α;
    3) 유도성 산화질소 효소(inducible nitric oxide synthase; iNOS) 및 시클로옥시게나아제-2(COX-2)의 발현 억제;3) inhibition of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2);
    4) NF-κB(nuclear factor kappa B)의 불활성화;4) inactivation of NF-κB (nuclear factor kappa B);
    5) 세포질(cytosolic) p-IκB-α 및 핵 인자 p65의 발현 억제; 및5) inhibition of expression of cytosolic p-IκB-α and nuclear factor p65; and
    6) 활성산소종(reactive oxygen species, ROS)의 생성 억제.6) Inhibition of generation of reactive oxygen species (ROS).
  12. 제10항에 있어서, 상기 추출물을 10μg/mL 내지 80μg/mL의 농도로 포함하는 것을 특징으로 하는 식품.The food according to claim 10, wherein the extract is contained at a concentration of 10 μg/mL to 80 μg/mL.
PCT/KR2021/014012 2020-11-11 2021-10-12 Method for preparing antarctic lichen amandinea sp. extract, and composition containing amandinea sp. extract WO2022102988A1 (en)

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