KR20200046984A - Antiinflammatory compostion having components extracted from lichen - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing a Cup lichen extract component selected at least one from a Myelochroa entotheiochroa extract or a Heterodermia hypoleuca extract as an active component. The Cup lichen extract according to the present invention can be utilized in a pharmaceutical composition for treating or preventing inflammatory diseases, or a food composition and/or a cosmetic composition for alleviating inflammation.

Description

Anti-inflammatory composition containing extract component derived from lichens {ANTIINFLAMMATORY COMPOSTION HAVING COMPONENTS EXTRACTED FROM LICHEN}

This patent application relates to the results of the research conducted by the Korea Forest Service on the development of forest bioresource materials (organization: Sunchon National University; Project No .: 2017024A00-1720-BA01).

The present invention relates to an anti-inflammatory composition derived from natural products, and more particularly, to an anti-inflammatory or anti-inflammatory composition comprising an extract component derived from lichen as an active ingredient.

The Nagoya Protocol, which took effect internationally to implement the Biodiversity Convention, came into force in 2017 in Korea. Accordingly, the 「Act on Access to Genetic Resources and Sharing Profits」 is being implemented in Korea. According to the Nagoya Protocol, the interests of countries that make medicines and cosmetics from other countries' biological resources must be shared with the countries that provided them, and the permission of the countries to use them must be obtained. Considering these points, there is a need to research and develop various physiological functions of biological resources inhabited in Korea where there is relatively insufficient biological resources.

On the other hand, lichens symbiotic with fungi (fungi) and algae in complex. The fungus constituting the lichen is one type of Ascomycetes or Basidiomycotes, and the algae is one of blue-green algae or green algae. Due to its unique symbiotic lifestyle, lichens adapt even in extreme environments where ordinary fungi or algae cannot survive alone. Globally, lichens inhabit all parts of the world, from the equator to the polar regions up to 6,000 m above sea level, and in most parts of the world, such as the Antarctic, desert, and volcanic areas where common land plants are difficult to live.

Lichens break down the hard surfaces of rocks, creating a ground for moss to enter, followed by moss, plants that can live in barren acidic soils such as ferns, pines, and rhododendrons. Lichens live on inorganic nutrients in the air and are sensitive to air pollution, so they cannot live when exposed to heavy metals contained in rain and dust. Due to these characteristics, lichens have attracted attention as an indicator species of air pollution.

The lichens can be divided into sessile limbs that stick to the surface like blotch, leafy lichens that look like leaves and have distinct distinction on both sides.

In Korea, China, and Japan, lichens are used as tea and food ingredients, and the secondary metabolites produced by lichens are very diverse, and are unique materials that are hard to find in other living organisms. Recently, studies on the physiological activity of lichens, such as anti-cancer and antioxidant, have been conducted.

For example, in Korean Patent Publication No. 10-2017-0082762 , the lichen extract of Everniastrum vexans , or atlanorin isolated therefrom or a pharmaceutically acceptable salt thereof is effective. Disclosed is a pharmaceutical composition for treating or preventing lung cancer as an ingredient.

Domestically and internationally, lichens are rather unfamiliar to other biomes. Due to its low awareness in Korea, it is relatively less researched than other microorganisms, plants, and mushrooms, and natural material exploration and development are also insufficient. To date, it is known that more than 700 kinds of lichens inhabit the country. It is necessary to research and develop various physiological activities of lichens living in Korea, which is a poor country of biological resources, as a biological resource that can replace biological resources of other countries that are not easy to access and use by the fermentation of the Nagoya Protocol.

An object of the present invention is to provide an anti-inflammatory composition containing an extract component obtained from natural materials.

According to one aspect, the present invention provides an anti-inflammatory composition comprising, as an active ingredient, a lichen extract selected from at least one of an extract of a tangle of yellow plum blossom ( Myelochroa entotheiochroa ) or an extract of white back- grimmage ( Heterodermia hypoleuca ) do.

In an exemplary embodiment, the lichen extract may be a C ₁ ~ C ₅ alcohol extract, for example, a methanol extract.

For example, the active ingredient having anti-inflammatory activity may include a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof.

In Formula 1, R ₁ to R ₃ are each independently a C ₁ to C ₅ alkyl group.

In another exemplary embodiment, the active ingredient may also include a compound represented by Formula 1 or a food-technically acceptable salt thereof.

In another exemplary embodiment, the active ingredient may include a compound represented by Formula 1 or a cosmetically acceptable salt thereof.

For example, in Formula 1, R ₁ to R ₃ are each a methyl group, that is, the active ingredient may include attraric acid or a pharmaceutically, food engineering and / or cosmetically acceptable salt thereof. have.

In another aspect, the present invention provides a pharmaceutical composition for treating or preventing an inflammatory disease comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a food composition for alleviating inflammation comprising a compound represented by Formula 1 or a food-engineerable salt thereof as an active ingredient.

In another aspect, the present invention provides a cosmetic composition for alleviating inflammation comprising a compound represented by Formula 1 or a cosmetically acceptable salt thereof as an active ingredient.

The present invention proposes an anti-inflammatory composition containing as an active ingredient an extract obtained from at least one lichen selected from two lichens. In one example, the extraction component may include a compound represented by Chemical Formula 1 and salts that are pharmaceutically, food engineering or cosmetically acceptable.

Since the active ingredient having anti-inflammatory activity according to the present invention is obtained from natural materials, there is no toxicity or side effect to the human body. By using the extract component of lichen obtained according to the present invention as an active ingredient, it can be utilized in medicines for the treatment and / or prevention of inflammatory diseases, or food and / or cosmetics to relieve inflammation.

In addition, by utilizing the active ingredients that can be obtained from the lichens inhabited in Korea, it is possible to secure abundance of domestic biological resources. It is expected that it will be able to replace biological resources from other countries that are becoming difficult to use due to the entry into force of the Nagoya Protocol.

1 is a diagram schematically illustrating a process of extracting and purifying an active ingredient having anti-inflammatory efficacy in a tangle of yellowed plum ( Myelochroa entotheiochroa ; MM) according to an exemplary embodiment of the present invention. MeOH means methanol, EA means ethyl acetate, Fr means fraction, and LF means layer fraction.
Figure 2a shows a high performance liquid chromatography (HPLC) chromatogram of the extract obtained from the tangle of yellow plum blossoms (MM) according to an exemplary embodiment of the present invention.
Figure 2b shows the results of high performance liquid chromatography-molecular spectrometry (HPLC-MS) of an extract obtained from a tattered yellow plum blossom (MM) according to an exemplary embodiment of the present invention. It is a graph.
3 is a diagram schematically showing a process of extracting and purifying an active ingredient having anti-inflammatory efficacy in a white back- grimmage ( Heterodermia hypoleuca ; HH) according to an exemplary embodiment of the present invention. MeOH means methanol, EA means ethyl acetate, Fr means fraction, and LF means layer fraction.
Figure 4a shows the HPLC chromatogram of the extract obtained from the white back-grimmage (HH) according to an exemplary embodiment of the present invention.
Figure 4b is a graph showing the HPLC-MS results of the extract obtained from the white back-grimmage (HH) according to an exemplary embodiment of the present invention.
5 and 5b, after treating the active ingredient extracted from the lichen to the macrophage cell line stimulated with lipopolysaccharide (lipopolysaccharide; LPS) according to an exemplary embodiment of the present invention, the toxicity of the active ingredient to the macrophage cell line was measured. It is a graph showing the result and a graph showing the result of measuring the amount of nitrogen oxide (NO) remaining in the medium.
6A to 6C are graphs showing the results of measuring the amount of cytokine and chemokine associated with the inflammatory response after treating the active ingredient extracted from lichen in a macrophage cell line stimulated with lipid polysaccharide according to an exemplary embodiment of the present invention, respectively. to be.
7A to 7G are each treated with an active ingredient extracted from lichen in a macrophage cell line stimulated with lipid polysaccharides according to an exemplary embodiment of the present invention, followed by inducible nitric oxide synthase (iNOS) by concentration. It is a graph showing the results of measuring the amount of oil using flow cytometry.

The present inventors have completed the present invention by studying the physiological activity of lichens inhabited in Korea, and identifying that the extract component obtained from lichens has anti-inflammatory activity. Hereinafter, the present invention will be described with reference to the accompanying drawings, if necessary.

[Extraction ingredients and extraction method of lichen]

Inflammation is an immune response (Medzhitov, R., Origin and physiological roles of inflammation. Nature 2008 , 454, 428-435) caused by various factors, such as infection by a pathogen or tissue damage. In relation to inflammation, macrophage and phagocytes play an important role in the host's immune response, elimination of pathogenic factors in intrinsic immunity and adaptive immunity, a specific response. Induction, etc. (Hawiger J., Innate Immunity and Inflammation: A Transcriptional Paradigm.Immunologic Research . 2001 , 23, 99-109).

Macrophages play a major role in the immune system and are used to recognize and eliminate harmful stimuli such as damaged cells, pathogens, or LPS. All inflammatory cytokines IL-6, IL-1β, IL-12 and chemokine It is a kind of GM-CSF and prostaglandin E2, iNOS, NO production (Tan, NH, Zhou, J. Plant cyclopeptides. Chem. Rev. 2006 , 106, 840-895.).

In vivo, nitric oxide (NO) is involved in inflammatory reactions such as removing pathogens by acting as cytotoxic substances, such as inhibiting the metabolism of pathogens or destroying DNA. Nitrogen monoxide is an 'endothelium-derived relaxing factor (EDRF)', which is bio-synthesized from arginine and oxygen atoms by various nitric oxide synthases (NOS), but also human immunity It is also produced by immune phagocytes, such as macrophages, as part of the reaction.

However, nitric oxide synthase is largely used to mediate an immune response, such as 'constitute nitric oxide synthase (cNOS)', which continuously synthesizes nitrogen monoxide regardless of the reaction of a living body. It can be largely classified as 'inducible nitric oxide synthase (iNOS)' which is activated and synthesizes nitrogen monoxide.

cNOS is constantly expressed to produce nitrogen monoxide that mediates physiological function at low levels, while iNOS is a lipopolysaccharide (LPS) or interferon-gamma (IFN-γ) or tumor necrosis factor (TNF). It is known to induce an inflammatory reaction by nitrogen monoxide by rapidly activating it in immune cells that have met with cytokines or bacterial toxins such as Liang, YC, Huang, YT, Tsai, SH, Lin -Shiau, SY, Chen, CF and Lin, JK, Suppression of inducible cyclooxygenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages, Carcinogenesis 1999 , 1945-1952; Sang-Sup Jew, Ok-Nam Bae and Jin- Ho Chung, Anti-inflammatory Effects of Asiaticoside on Inducible Nitric Oxide Synthase and Cyclooxygenase-2 in RAW 264.7 Cell Line, J. Toxicol. Pub. Health. 2003 , 19 (1) 33-37).

In particular, excessive inflammatory reactions can contribute to the pathogenesis of chronic diseases such as arthritis, asthma, arteriosclerosis, brain damage and serious tissue damage (Libby, P., Ridker, PM, Maseri, A., Inflammation and atherosclerosis, Circulation 2002, 105, 1135-1143; Manzi , S., Wasko, MC, Inflammation-mediated rheumatic diseases and atherosclerosis, Ann Rheum Dis 2000, 59, 321-325;... Tak, PP, Firestein, GS, NF- _B: A key role in inflammatory diseases, J. Clin. Investig. 2001 , 107, 7-11.).

Thus anti-inflammatory agent serves to reduce the damage caused by inflammatory factors that regulate excessively generated in inflammatory mediators (Kundu, JK; Surh, YJ Inflammation:... Gearing the journey to cancer Mutat Res 2008, 659, 15-30) . However, chemically synthesized anti-inflammatory agents may have side effects or toxicity to the human body, and accordingly, interest in anti-inflammatory active ingredients obtained from natural materials is increasing.

In order to obtain natural ingredients having anti-inflammatory activity, the present invention adopts lichens that live in Korea. The active ingredient having anti-inflammatory activity is at least one lichen extract selected from the extract of Myelochroa entotheiochroa or Heterodermia hypoleuca . Hereinafter, in the present specification, an extract of a tattered yellow plum tree branch and an extract of a white back gripper are collectively referred to as a lichen extract or lichen extract component.

The extraction solvent for obtaining the lichen extract is 50% to 100% of methanol, ethanol, such as C ₁ to C ₅ alcohol, ethyl acetate, a polar solvent, and an organic solvent such as hexane, chloroform or dichloromethane, a non-solvent. It may be a mixed solvent, preferably 50% to 100% of methanol, C ₁ ~ C ₅ alcohol, such as ethanol, more preferably 70% to 100% of methanol or ethanol.

If necessary, the lichen extract of the present invention may be a cold immersion extraction method, a warm immersion extraction method, or a heat extraction method, and a conventional extraction device, an ultrasonic pulverization extractor, or a fractionator may be used. As an example, a method of obtaining an extract of lichen using an extraction solvent is pulverized to a suitable size among dried lichens, placed in an extraction container, and an appropriate amount of an extraction solvent is added and heated to obtain an extract. In order to increase the extraction efficiency of lichens, the above process can be repeated several times or more.

The extract can be filtered and concentrated under reduced pressure to produce a crude extract of lichen. The obtained lichen extract can be stored in a deep freezer until use. In addition, the lichen extract may be to completely remove moisture through concentration and lyophilization of the obtained extract, and the lichen extract that completely removes the moisture is used in powder form or the powder is dissolved in distilled water or a conventional solvent. Can be used.

If necessary, the extract obtained using an organic solvent can be fractionated using a solvent. Specifically, the extract extracted with the solvent may be further subjected to a fractionation process with a solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof. Preferably, the fractionated solvent is an aqueous methanol solution, water, ethyl acetate, and chloroform, and more preferably, an aqueous methanol solution and ethyl acetate. Particularly, the fraction by ethyl acetate was found to have the best anti-inflammatory activity.

The fraction obtained by performing the fractionation process can be filtered or concentrated or dried to remove the solvent, and then filtered, concentrated and dried. Specifically, the filtration may use a filter paper or a reduced pressure filter, the concentration may be concentrated under reduced pressure using a reduced pressure concentrator, for example, a rotary evaporator, and the drying may be performed, for example, by lyophilization. In an alternative embodiment, the lichen extract obtained above may be fractionated using column chromatography.

In another alternative embodiment, the lichen fraction can be subjected to high performance liquid chromatography (HPLC) to purify certain active ingredients. According to an exemplary embodiment of the present invention, the active ingredient having anti-inflammatory activity is a compound represented by Formula 1 below.

[Formula 1]

In Formula 1, R ₁ to R ₃ are each independently a C ₁ to C ₅ alkyl group.

In one example, the compound having anti-inflammatory activity may be atraric acid (AA) in which R ₁ to R ₃ are all methyl groups in Formula 1.

In the present invention, the lichen extract is a concept including all extracts, fractions and purified products obtained at each stage of extraction, fractionation or purification, and their dilutions, concentrates or dried products.

According to an exemplary embodiment of the present invention, the lichen extract obtained according to the present invention has no cytotoxicity (FIG. 5A) and reduces the production of nitrogen oxides produced by the induced inflammatory response (FIG. 5B). In particular, it inhibits the expression of various cytokines or chemokines associated with the inflammatory response (see FIGS. 6A-6C) and inhibits the expression of iNOS (see FIGS. 7A-7G).

[Anti-inflammatory composition]

The present invention relates to an anti-inflammatory composition comprising the above-described lichen extract, or a compound represented by Formula 1 or a pharmaceutical / food engineering / cosmetic acceptable salt thereof as an active ingredient. Pharmaceutically / food engineering / cosmetically acceptable salts of the compound represented by the formula (1) include acid addition salts or basic salts formed by free acids.

Acids used in pharmaceutical / food engineering / cosmetic engineering acceptable acid addition salts include inorganic and organic acids. Inorganic acids include, but are not limited to, hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid and / or phosphorous acid. Organic acids include aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids.

Pharmaceutical / food engineering / cosmetic engineering non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulphite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride , Bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, Succinate, suberate, sebacate, fumarate, maleate, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, and hydrate Hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonay , Chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate , Naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

For example, pharmaceutical / food engineering / cosmetic engineering acceptable organic acids include acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, benzoic acid and ascorbic acid. , Methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid and camphoric acid. On the other hand, the pharmaceutically / food engineering / cosmetic engineering acceptable base includes sodium hydroxide, potassium hydroxide, triethylamine and tert-butylamine, but is not limited thereto.

In one exemplary embodiment, the lichen extract, or a compound represented by Formula 1, or a pharmaceutical / food engineering / cosmetic acceptable salt thereof (hereinafter, collectively referred to as lichen extract, etc.) is one of the anti-inflammatory compositions. It may be added at a concentration of 1000 μM, preferably 10 to 500 μM, but the present invention is not limited thereto.

In one exemplary embodiment, the present invention provides a pharmaceutical composition for treating or preventing inflammation containing lichen extract or the like as an active ingredient and, if necessary, a pharmaceutically acceptable carrier.

For example, the pharmaceutical composition for treating or preventing an inflammatory disease may include a lichen extract or the like as an active ingredient alone, and additional ingredients, pharmaceutically acceptable or nutritionally, depending on the formulation, method of use, and purpose of use. It may further include acceptable carriers, excipients, diluents or sub-components.

Pharmaceutical compositions comprising the lichen extract according to the present invention as an active ingredient, respectively, according to a conventional method, powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, external preparations, suppositories and sterilization It can be formulated and used in the form of an injection solution. For example, the lichen extract of the present invention may be administered in various formulations, oral or parenteral, during clinical administration. When formulated, fillers, bulking agents, binders, wetting agents, surfactants, anti-agglomerates, lubricants, which are usually used , Wetting agents, fragrances, emulsifiers or preservatives, and may further include diluents or excipients, and may be used either orally or parenterally.

For example, carriers, excipients, and diluents that can be included in pharmaceutical compositions including lichen extracts, etc. include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene It may be one or more selected from the group consisting of glycol, liquid paraffin, and physiological saline, but is not limited thereto, and may be used as a common carrier, excipient, or diluent.

As an example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient such as a lichen extract of the present invention, such as starch, calcium carbonate ), Sucrose (lactose), gelatin, etc. are prepared by mixing. In addition, lubricants such as magnesium styrene talc are used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances and preservatives, in addition to water and liquid paraffin, which are commonly used as diluents. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, and glycerogelatin can be used. The pharmaceutical composition of the present invention may be administered by subcutaneous injection, intravenous injection or intramuscular injection when parenterally administered.

Forms for parenteral administration include toothpaste, mouthwash, and topical administration (creams, ointments, dressing solutions, sprays, other coatings, etc.). As an example of the formulation of the topical administration agent, an active ingredient such as lichen extract may be impregnated into a carrier such as gauze made of natural or synthetic fibers. In the case of the cream or ointment, periodontal disease or gingival disease may be suitable for application directly to or around the affected area. In the case of the above-mentioned spraying agent, it can be prepared by a conventional spraying agent manufacturing method except that it contains an extract of lichen as an active ingredient. It can be prevented or treated. In the case of the dressing solution, it can be prepared by a conventional method of preparing a dressing solution, except that it contains an extract of lichen as an active ingredient, and used for dressing at an inflammatory disease site or dressing of other bacterial infection sites to reduce inflammatory diseases. It can be prevented or treated.

In addition, the formulation of the pharmaceutical composition for the prevention or treatment of inflammatory diseases of the present invention may be in a preferred form depending on the method of use, and in particular, it can be provided in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated by adopting a known method. Examples of specific formulations include warning agents, granules, lotions, linen agents, limonades, powders, syrups, eye ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, fluid extracts, emulsions, Suspensions, premise, acupuncture, eye drops, tablets, suppositories, injections, spirits, capsules, creams, pills, soft or hard gelatin capsules, and the like.

Furthermore, pharmaceutical compositions for the prevention or treatment of inflammatory diseases of the present invention may be prepared using appropriate methods known in the art or disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. It can be preferably formulated using.

If necessary, pharmaceutical compositions for the prevention or treatment of inflammatory diseases, in addition to the above active ingredients, nutrients, vitamins, electrolytes, flavoring agents, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, It may further contain a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.

The lichen extract of the present invention can be administered to various mammals such as rats, mice, livestock, and humans by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine epidural or intracerebroventricular injection.

In another alternative embodiment, the present invention provides a food composition for alleviating inflammation, which contains an extract of lichen and the like as an active ingredient. Food in the present specification means a natural product or a processed product containing one or more nutrients, and includes food, food additives, health functional foods, beverages, and beverage additives. The food composition is not particularly limited to other ingredients besides lichen extracts, and may contain various flavoring agents or food supplement additive ingredients such as natural carbohydrates as additional ingredients.

Examples of natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; As well as conventional sugars such as polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors / sweeteners such as taumatin and stevia extract (eg rebaudioside A, glycyrrhizine) as flavors or sweeteners other than those described above; Synthetic flavor / sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the food composition according to the present invention.

In addition, food compositions include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, It may contain organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, carbonic acid used in alcoholic beverages, and the like. In addition, the food composition according to the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportion of these additives is not so important, but it is generally selected from 0 to about 20 parts by weight, preferably 0.01 to 1.0 parts by weight per 100 parts by weight of the lichen extract according to the present invention.

In another exemplary embodiment, the present invention provides a cosmetic composition for alleviating inflammation, which contains an extract of lichen and the like as an active ingredient. The cosmetic composition for alleviation of inflammation may include ingredients that can be used in the cosmetic field, in addition to including, as an active ingredient, a lichen extract that can be prepared according to the present invention.

Cosmetic compositions include cosmetically and / or dermatologically acceptable media. In one example, the cosmetic composition is an aqueous, aqueous / alcoholic or oily solution, or an aqueous, aqueous / alcoholic or oily gel, or a solid or dough anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase (O / W) or vice versa (W / O), suspension, microemulsion, microcapsules, microgranules or ionic (liposomes) and / or nonionic vesicle dispersants.

Ethanol, glycerin, butylene glycol, propylene glycol, glycereth-26, methylgluceth-20, isocetyl myristate, cetyl octanoate, isocetyl octanoate, octyldodecyl miri as a solvent for preparing a cosmetic composition One or more selected from states, octyldodecanol, isostearyl isostearate, and neopentiglycol dicaprate may be used.

In addition, the cosmetic composition may include at least one component selected from the group consisting of water-soluble vitamins, fat-soluble vitamins, polymer peptides, polymer polysaccharides, and sphingoli lipids.

Water-soluble vitamins include vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, and vitamin H, and salts thereof (thiamine hydrochloride, ascorbic acid) Sodium salt, etc.) and derivatives (ascorbic acid-2-phosphate sodium salt, ascorbic acid-2-phosphate magnesium salt, etc.) are also included in the water-soluble vitamins that can be used in cosmetic compositions.

Fat-soluble vitamins include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (alpha-tocopherol), and derivatives thereof (ascorbyl palmitate, ascorbate stearate, acetic acid-alphatocopherol, nicotinic acid- Alpha-tocopherol, pantonyl alcohol, etc.) are also included in fat-soluble vitamins that can be used in cosmetic compositions.

Polymeric peptides include gelatin, elastin, hydrolyzed elastin, keratin and the like. Examples of the polymer polysaccharide include hydroxycellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate, or salts thereof (sodium salt, etc.). Sphingo lipids include ceramide, phytosphingosine, and sphingosaccharide lipids.

In addition to the above ingredients, the cosmetic composition may be blended with other ingredients, which are usually formulated in cosmetics, as necessary. Ingredients that can be added include fats and oils, moisturizers, emollients (softeners), surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, fungicides, antioxidants, antioxidants, plant extracts, pH adjusters , Alcohols, foaming agents, fillers, ultraviolet absorbers, pigments, colorants, gelling agents or thickening agents, flavoring agents, blood circulation accelerators, cooling agents, limiting agents, purified water, and the like.

In addition, the compounding components that may be added are not limited thereto, and any of the above components may be compounded within a range not impairing the objects and effects of the present invention, but is preferably 0.01 to 5% by weight, preferably 0.01 to the total weight. To 3% by weight. In addition, the cosmetic composition of the present invention can be variously applied to cosmetics, face washes and shampoos for alleviating inflammation.

For example, as a product to which the cosmetic composition of the present invention can be added, there are, for example, cosmetics such as various creams, lotions, skins, and shampoos, rinses, cleansing agents, face washes, soaps, treatments, and cosmetics. Specifically, the cosmetic composition of the present invention is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, It can be formulated in a variety of formulations such as packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

Hereinafter, the present invention will be described through exemplary embodiments, but the present invention is not limited to the technical idea described in the following examples.

Example 1: Extraction and purification of tattered yellow plum blossoms

In the process shown in Fig. 1, active ingredients were extracted and purified from tattered yellow plum blossoms. Dissolve the methanol extract (1.4988g) of Myelochroa entotheiochroa (ME) pre-sold at the Korean Apparel Research Center with 50 mL of ethyl acetate (EA), add 50 mL of water (30% methanol), shake well and wait for the layer to separate. , When the layers were separated, only the EA layer was recovered, combined with the previously collected EA layer, and concentrated under reduced pressure to remove the solvent. The concentrated EA layer (291.7 mg) was dissolved in DMSO: Methanol 25 mg / mL. The dissolved sample was subjected to mild pressure liquid chromatography (MPLC) conditions. Specifically, acetonitrile (acetonitrile, ACN) aqueous solution (0.05% formic acid) (5%, 5%, 100%, 100%, 5%) with ACN (0.05% formic acid) -water (0.05% formic acid) mixed solution stepwise MPLC was performed using a gradient solvent system. Using a column, 3.5 mL (87.5 mg) was injected and separated into 5 fractions. Separation was carried out at a flow rate of 15 mL / min, and a third fraction having an anti-inflammatory effect was obtained from 40 minutes to 44.8 minutes, and the fraction (19.4 mg) was preparative reverse phase HPLC (semi-preparative reverse phase) using a C18 column. The active ingredient was separated and purified repeatedly by HPLC).

The HPLC used in this example is an Agilent Technologies 1260 series HPLC (Agilent Technologies, USA) system, and absorbance was measured at 365 nm. The column used was for preparative: Luna 5u (C18 100A 250Х10mm, Phenomenex Inc.), and the column temperature was fixed at 24 ° C. The solvent A of the mobile phase was 0.1% formic acid (v / v, in water), and B used 0.1% formic acid (v / v, in acetonitrile). The flow rate of the mobile phase was 1.0 mL / min, and the solvent gradient was set to 50% of the B solvent for 0 to 60 minutes. MS analysis was performed using an Agilent Technologies 6130 Quadrupole mass spectrometer (Agilent Technologies, USA), and an Agilent 1260 UV / visible detector on an Agilent Technologies 1260 series HPLC (Agilent Technologies, USA) system, CAPCELL C18 5 μm, 250 × 10 mm ( Shiseido Inc.) column was used and the column temperature was fixed at 24 ° C. The solvent A of the mobile phase was 0.1% formic acid (v / v, in water), and B used 0.1% formic acid (v / v, in methanol). The flow rate of the mobile phase was 1.0 mL / min, and the solvent gradient was maintained at 0 to 5 minutes B solvent 5%, 5 to 45 minutes B solvent 5 to 70%, 45 to 50 minutes B solvent 70 to 100%, 50 to 60 minutes. B solvent was 100%. The fraction (8.6 mg) obtained by the above HPLC method was collected and dried, and then used as a sample for ¹ H-NMR and ¹³ C-NMR analysis. The HPLC chromatogram is shown in Fig. 2A, and the MS analysis results are shown in Fig. 2B.

Subsequently, the purified sample was analyzed using an MR analyzer (JNM-ECZS series FT 400 NMR (JEOL, Japan) .Trimethyl silane: Cambridge isotope laboratory, Inc., USA (TMS) was used as a reference. Results, ¹ H NMR (methanol- d ₄ , 400 MHz); 6.25 (1H, s, H-6), 3.83 (3H, s, H-10), 2.31 (3H, s, H-8), 1.89 ( 3H, s, H-9), ¹³ C NMR (methanol- d ₄ , 100 MHz); 172.5 (C-1), 162.1 (C-3), 160.1 (C-5), 140.0 (C-7), Atraric acid was confirmed by 111.1 (C-6), 108.4 (C-4), 104.4 (C-2), 52.4 (C-10), 23.9 (C-8), and 8.1 (C-9).

Example 2: Extraction and purification of white back Grimage

In the process shown in Figure 3, the active ingredient was extracted and analyzed from the white back Grimage. After dissolving methanol extract (1.9337g) of Heterodermia hypoleuca (HH) pre-sold at the Korean Apparel Research Center with 50 mL of EA, add 50 mL of water (30% methanol), shake well and wait for the layer to separate. When separated, collect only the EA layer and shake it by adding 50 mL of EA again. When the layers were separated, only the EA layer was recovered, combined with the previously collected EA layer, and concentrated under reduced pressure to remove the solvent. MPLC, HPLC, MS and NMR analysis were performed according to the same procedure as in Example 1. The third fraction having anti-inflammatory effect in MPLC was obtained from 40 minutes to 44.8 minutes, and the fraction (70.1 mg) was analyzed according to the same procedure as in Example 1. The HPLC chromatogram is shown in Fig. 4A, and the MS analysis results are shown in Fig. 4B. In Example 1, it was confirmed that the same Atraric acid was finally separated and purified.

Example 3: Confirmation of the effect of pretreatment of atraric acid

With respect to the amount of nitrite induced by LPS, the pretreatment effect of purified Atraric acid (AA) in Example 1 and Example 2 was confirmed. RAW 264.7 macrophage is pre-sale from the Korea Cell Line Bank (KTCC, Seoul, Korea) and uses RPMI1640 medium containing 10% fetalbovine serum (FBS), 1% antibiotics, and 2-ME (2-mercaptoethanol, 50 mM). Then, the cells were cultured in an incubator controlled at 37 ° C and 4% CO ₂ .

To measure cytotoxicity, RAW 264.7 macrophage was dispensed into 96-well plates at 5X10 ⁴ cells / 100 µl per well, followed by CO ₂ incubation for 24 hours. LPS (lipopolysaccharide) 1 μg / mL and AA isolated were treated with 100 μl at concentrations of 1, 3, 10, 30, 100, and 300 μM / mL, respectively, and CO ₂ incubation was performed for 24 hours. Then, 100 µl of the culture supernatant was removed per condition, and 10 µl of Cell counting Kit-8 (CCK-8) was treated, followed by CO ₂ incubation for 3 hours, followed by absorbance at 450 nm using a micro plate reader (VERSA). Was measured. As a result, it was confirmed that there was no toxicity to RAW 264.7 macrophage at 1-300 μM / mL (FIG. 5A).

In addition, RAW 264.7 macrophage was activated with LPS to determine the production of NO. RAW 264.7 macrophage was dispensed in 96-well plates at ⁵ × 10 ⁴ cells / 100 µl per well and cultured for 24 hours. After 1 hour, 50 μl of 1 μg / mL of LPS was dispensed, and AA was treated with concentrations of 1, 3, 10, 30, 100, and 300 μM / mL, respectively, and CO ₂ incubation was performed for 24 hours. Then, 100 µl of the culture supernatant per condition was added to a 96-well plate, and the same amount of Griess reagent (0.1% N-1-naphtyl-ethylendiamine in H ₂ O: 1% sulfanilamide in 5% H ₃ PO ₄ = 1) : 1) was added and reacted for 10 minutes, and absorbance was measured at 550 nm with a Microplate reader. The concentration of NO was calculated by comparing NaNO ₂ (sodium nitrite) with a standard curve obtained by diluting twice from 64 μM to 0.5 μM. As a result, it was found that the concentration of LPS stimulated with LPS at 10-300 μM was shown to decrease the NO production of 264.7 macrophage, and the concentration from the next experiment was determined to be 10-300 μM (FIG. 5B).

Example 3: Measurement of inflammatory cytokine / chemokine secretion

RAW 264.7 macrophage was dispensed into a 24-well plate to be 5 × 10 ⁵ cells / 1 mL per well and incubated for 24 hours. Thereafter, LPS was dispensed into 500 μl at a concentration of 1 μg / mL in the well, and incubated for 1 hour, and then treated with 500 μl at a concentration of 10, 30, 100, and 300 μM / mL, which had a NO inhibition effect, for 24 hours of CO ₂ incubation. Did. The cell culture supernatant was then collected. The cytokines IL-6, GM-CSF, and IL-1β contained in the collected supernatant were measured using an enzyme-linked immunosorbent assay (ELISA). Specifically, a primary antibody (2 µg / mL) was diluted in a plate-bottom micro-well in a coating buffer (0.1 M NaHCO ₃ , pH 8.2), dispensed at 50 µl / well, and overnight at 4 ° C, followed by washing solution ( 0.05% Tween 20 / PBS). The washed micro-well was blocked with PBS with 10% FBS added, and the supernatant collected previously was diluted at an appropriate ratio and then dispensed into each well to react at room temperature. Then, after reacting with 100 μl / well of biotin-attached secondary antibody (1 μg / mL) at room temperature for a period of time, 100 μl / well of avidin-peroxidase (2.5 μg / mL) was added. Finally, the substrate (2, 2'-azino-bis, 0.1M citritic acid, H ₂ O ₂ ) was added to develop color, and absorbance was measured at 405 nm using a micro-plate reader. The measured concentrations of IL-6, GM-CSF, and IL-1β were converted using a standard curve. As a result, the cytokine production of RAW 264.7 macrophage stimulated with LPS showed a concentration-dependent pattern in IL-6, IL-1β, and GM-CSF, a type of chemokine, and showed a large inhibition rate at 100-300 μM (FIGS. 6A to 6A). 6c). Therefore, the concentration in the next experiment was determined.

Example 4: Flow cytometry analysis

RAW 264.7 macrophage was dispensed into 24-well plates at 5 × 10 cells / mL per well and incubated for 24 hours. Subsequently, 500 μl of LPS was dispensed into the well at a concentration of 1 μg / mL, followed by CO2 incubation for 1 hour, followed by treatment with 500 μl at a concentration of 100, 300 μM / mL, which has a cytokine inhibition effect, and incubation for 24 hours. After collecting the cells and the culture solution, the centrifugation was performed, the supernatant was discarded, only the cells were obtained, and then blocked with anti-CD16 / 32 for 30 minutes. Then, fix and fix Fixation Buffer (BD, U.S.) for 20 minutes. Next, the cells were perforated for 5 min with Perm Buffer III (BD, U.S), and finally stained with PE-conjugated monoclonal antibodies iNOS, and then measured using flow cytometry (FACS CantoII). In this case, BD FACS Diva software was used for data analysis. As a result, the expression level of iNOS in mouse macrophages stimulated with LPS was reduced by A.A. (See FIGS. 7A to 7G).

In the above, the present invention has been described based on exemplary embodiments and examples of the present invention, but the present invention is not limited to the technical ideas described in the above embodiments and examples. Rather, those skilled in the art to which the present invention pertains can easily suggest various modifications and changes based on the above-described embodiments and examples. However, it is clear from the appended claims that all such modifications and variations fall within the scope of the present invention.

Claims (10)

An anti-inflammatory composition comprising as an active ingredient at least one lichen extract selected from among the extracts of a ragged yellow plum ( Myelochroa entotheiochroa ) or an extract of white back- grimmage ( Heterodermia hypoleuca ). The anti-inflammatory composition according to claim 1, wherein the lichen extract is a C ₁ -C ₅ alcohol extract. The anti-inflammatory composition according to claim 1, wherein the lichen extract is a methanol extract. The anti-inflammatory composition according to claim 1, wherein the active ingredient comprises a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof.
[Formula 1]

In Formula 1, R ₁ to R ₃ are each independently a C ₁ to C ₅ alkyl group. The anti-inflammatory composition according to claim 1, wherein the active ingredient comprises a compound represented by Formula 1 below or a food-engineerable salt thereof.
[Formula 1]

In Formula 1, R ₁ to R ₃ are each independently a C ₁ to C ₅ alkyl group. According to claim 1, The active ingredient is an anti-inflammatory composition comprising a compound represented by the formula (1) or a cosmetically acceptable salt thereof.
[Formula 1]

In Formula 1, R ₁ to R ₃ are each independently a C ₁ to C ₅ alkyl group. The anti-inflammatory composition according to any one of claims 4 to 6, wherein R ₁ to R ₃ in Formula 1 are each methyl groups.
A pharmaceutical composition for treating or preventing an inflammatory disease comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

In Formula 1, R ₁ to R ₃ are each independently a C ₁ to C ₅ alkyl group. A food composition for alleviating inflammation comprising a compound represented by the following Chemical Formula 1 or a food-engineerable salt thereof as an active ingredient.
[Formula 1]

In Formula 1, R ₁ to R ₃ are each independently a C ₁ to C ₅ alkyl group. A cosmetic composition for alleviating inflammation comprising a compound represented by Formula 1 or a cosmetically acceptable salt thereof as an active ingredient.
[Formula 1]

In Formula 1, R ₁ to R ₃ are each independently a C ₁ to C ₅ alkyl group.

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