WO2022102988A1 - Procédé de préparation d'extrait de lichen amandinea sp. d'antarctique et composition contenant de l'extrait d'amandinea sp. - Google Patents

Procédé de préparation d'extrait de lichen amandinea sp. d'antarctique et composition contenant de l'extrait d'amandinea sp. 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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PCT/KR2021/014012
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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.

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Abstract

La présente invention se rapporte à un procédé de préparation d'un extrait de lichen Amandine sp. d'antarctique, et à une composition pharmaceutique destinée à prévenir ou à traiter des maladies inflammatoires, contenant l'extrait en tant que principe actif. Un extrait d'Amandinea sp. selon la présente invention inhibe la production de cytokine pro-inflammatoire et de médiateur inflammatoire et inactive la voie du NF-κB, dans les cellules RAW264.7 in vitro et chez le poisson zèbre in vivo, et peut ainsi être efficacement utilisé pour prévenir ou traiter des maladies inflammatoires.
PCT/KR2021/014012 2020-11-11 2021-10-12 Procédé de préparation d'extrait de lichen amandinea sp. d'antarctique et composition contenant de l'extrait d'amandinea sp. WO2022102988A1 (fr)

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KR20110132938A (ko) * 2010-06-03 2011-12-09 한국해양연구원 라말린을 함유하는 염증질환 또는 면역질환의 예방 또는 치료용 약학 조성물
US20190358327A1 (en) * 2016-12-23 2019-11-28 Klox Technologies Inc. Biophotonic compositions comprising lichen extract and their use to treat skin disorders
US20200029575A1 (en) * 2018-07-24 2020-01-30 Frances Trail Lichen compounds that inhibit mycotoxin production
KR20200046984A (ko) * 2018-10-26 2020-05-07 순천대학교 산학협력단 지의류 유래의 추출 성분을 포함하는 항 염증 조성물

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KR20110132938A (ko) * 2010-06-03 2011-12-09 한국해양연구원 라말린을 함유하는 염증질환 또는 면역질환의 예방 또는 치료용 약학 조성물
US20190358327A1 (en) * 2016-12-23 2019-11-28 Klox Technologies Inc. Biophotonic compositions comprising lichen extract and their use to treat skin disorders
US20200029575A1 (en) * 2018-07-24 2020-01-30 Frances Trail Lichen compounds that inhibit mycotoxin production
KR20200046984A (ko) * 2018-10-26 2020-05-07 순천대학교 산학협력단 지의류 유래의 추출 성분을 포함하는 항 염증 조성물

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