KR20130062112A - A topical composition comprising the extract of cyperus rotundus l as an active ingredient for preventing and treating inflammatory disease - Google Patents

Abstract

PURPOSE: A Cyperus rotundus extract is provided to suppress IL-1beta, IL-6, and TNF-alpha, to inhibit COX-2 and iNOS generation, and to be used as a pharmaceutical composition or a cosmetic composition for preventing and treating inflammatory diseases. CONSTITUTION: A pharmaceutical composition for preventing and treating inflammatory diseases contains 0.1-50 wt% of a Cyperus rotundus extract as an active ingredient. The pharmaceutical composition is manufactured in the form of a cream, a gel, a patch, a spray, an ointment, a lotion, a liniment, a paste, or a cataplasma. The extract is prepared by water including purified water, C1-C4 alcohol, or a mixture thereof. A cosmetic composition for preventing and treating inflammatory diseases contains the extract as an active ingredient.

Description

External topical composition comprising the extract of Cyperus rotundus L as an active ingredient for preventing and treating inflammatory disease

The present invention relates to a skin external pharmaceutical composition or cosmetic composition for preventing and treating inflammatory diseases containing Cyperus rotundus extract as an active ingredient.

[Reference 1] Cho YJ, An BJ. 2008. Anti-inflammatory effects from Cheongmoknosang in Lipopolysaccharide-stimulated raw cells. J Korean Soc Appl Biol Chem 51 (1): 44-48.

[Reference 2] McCord JM. Wong K, Stpkes SH, Petrone WF and English D. 1980. Superoxide and inflammation: protection of synorial fluid by superoxide dismutase. Science, 185: 529-531.

Document 3 Azad N, Rojanasakul Y, Vallyathan V. 2008. Inflammation and lung cancer: roles of reactive oxygen / nitrogen species. J Toxicol Environ Health B Crit Rev. 11 (10): 5-11.

Document 4 Posadas I, Terencio MC, Guillen I, Ferrandiz ML, Coloma J, Oaya M and Alcaraz MJ. 2000. Coregulation between cyclooxygenase-2 and inducoble nitric oxide synthase expression in the time course of murine inflammation. Naunyn-Schmiedeberg's Arch. Pharmacol. 361: 98-106.

Document 5 Moncada S, Palmer RM and Higgs EA. Nitric oxide: physiology, pathophy siology and pharmacology. Pharmacol. Rev. 43: 109-142.

Document 6 Yun HY, Dawson VL and Dawsom ™. 1996. Neurobiology of nitric oxide. Crit. Rev. Neurobiol. 10: 291-316.

[Document 7] Yoon WJ, Lee JA, Kim JY, Oh DJ, Jung YH, Lee WJ and Park SY. 2006.Anti-oxident Activities and Anti-inflammatory Effects on Artemisia scoparia . Kor. J. Pharmacogn. 37 (4): 235-240.

Yamamura T, Onishi J, Nishiyama T. 2002. Antimelanogenic activity of hydrocoumarins in cultured normal human melanocytes by stimulating intracellular glutathione synthesis. Arch. Dermatol. Res. 294 (8): 349-354.

Lin CB, Babiarz L, Liebel F, Roydon Price E, Kizoulis M, Gendi menico GJ, Fisher DE, Seiberg M. 2002. Modulation of microphthal mia-associated transcription factor gene expression alters skin pigmentation. J. Invest Dermatol. 119 (6): 1330-1340.

10. Ando S, Ando O, Suemoto Y, Mishima Y. 1993. Tyrosinase gene transcription and its control by melanogenic inhibitors. J Invest Dermatol. 100 (2 Suppl): 150S-155S.

Imokawa G, Mishima Y. 1982. Loss of melanogenic properties in tyrosinase induced by glucosylation inhibitors within malignant melanoma cells. Cancer Res. 42 (5): 1994-2002.

Reference 12 et al. Hyangjedae Dictionary, Yeonglimsa Temple, 1034-1035. 1998.

13 Michikawa M, Lim KT, McLarnon JG, Kim SU. 1994. Oxygen radical-induced neurotoxicity in spinal cord neuron cultures. J. Neurosci Res. 37: 62-70.

14 Carmichael J, Defraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium based semiautomated colorimetric assay: assessment of chemosensitivuty tesing. Cancer Res. 47: 936-942.

15 Hwang EY, Kim DH, Kim HJ, Hwang JY, Park TS, Lee IS, Son JH. 2011.Antioxidant Activities and Nitric Oxide Production of Medicine Plants in Gyeongsangbukdo ( Carthamus tinctorius seed, Cyperus rotundus , Schizonepeta tenuifolia , Polygonatum odoratum var. pluriflorum , Paeonia lactiflora ) J. Appl. Biol. Cheml. 54: 171-177.

16. Zhang X, Song Y, Xiong H, Ci X, Li H, Yu L, Zhang L, Deng X. 2009. Inhibitory effects of ivermectin on nitric oxide and prostaglandin E2 production in LPS-stimulated RAW 264.7 macrophages International Immunopharmacology . 9: 354359.

In modern society, researches are actively conducted to explore various substances that can treat or prevent diseases according to the desire to live healthier. Among them, it is focused on researches to explore various physiological activities using natural products, to isolate and purify the active ingredients to identify the biochemical functions of the components and to scientifically verify the efficacy (1). Oxidative stress plays an important role in many physiological pathological phenomena, including reactive oxygen species (ROS), oxygen ( ¹ O ₂ ), superoxide (O _2- ), hydroxy radical (HO), hydrogen peroxide (H2O2), and these reactive oxygen species are involved in the inflammatory response (2). Inflammation occurs through a complex process regulated by the production of cytokine and various inflammatory factors. Prolonged inflammatory response due to repetitive tissue damage or regeneration leads to inflammatory diseases caused by excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in inflammatory cells ( 3). During the inflammatory process, high levels of inflammatory cytokines, nitric oxide (NO) and prostaglandin E ₂ (PGE ₂ ) are produced by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (4). NO is synthesized by three types of NOS: neuronal NOS (nNOS), endothelial NOS (eNOS) and iNOS. nNOS and eNOS are always present in cells, but iNOS is expressed only when exposed to interferon-γ, lipopolysacchatide (LPS) and various proinflammatory cytokines (5-6).

Nitric oxide (NO), which is one of free radicals and has recently been known to play an important role in inducing inflammation, has various physiological functions such as defense, signal transduction, neurotoxicity, and vasodilation (7). In general, the formation of NO plays an important role in killing bacteria or eliminating tumors, but excessive formation of NO by pathological causes causes inflammation, tissue damage, genetic variation and nerve damage. iNOS does not normally exist intracellularly, but when induced, it generates a large amount of NO for a long time, and NO produced by iNOS in inflammatory state not only promotes inflammatory reactions such as vascular permeability and edema but also promotes biosynthesis of inflammatory mediators. In addition, it is known to intensify inflammation by promoting biosynthesis of inflammatory mediators. NO has been involved in the development of inflammation and cancer (8-9), and iNOS expression remains in NF-kB activity, an important mechanism by which LPS or cytokine is overproduced by macrophage (10).

Inhibition of prostagladin synthesis depends on the mechanism of action of a number of anti-inflammatory drugs, due to the production and inhibition of COX-2. With an enzyme to the telephone Arachidonic acid to PGE ₂ it is classified as COX-1 and COX-2. COX-1 acted on normal biological functions such as platelet formation, gastric wall protection, and renal function maintenance. The mechanism of action of many anti-inflammatory drugs has been shown to inhibit prostaglandin synthesis, which is due to the production and deactivation of COX-2 (11).

Hyangbuja is a perennial herb belonging to Cyperaceae and grows in southern Korea and Jeju Island. Its components include pinene, campene, 1,8-cineol, limonene, etc. It contains an essential oil component and is known for analgesic and antibacterial activity (12).

Therefore, in this study, the anti-inflammatory effect was examined using raw cells, macrophage cell lines, by selecting medicinal plants in Gyeongbuk as part of the physiological activity screening study. Completed.

In order to achieve the above object, the present invention provides a skin external pharmaceutical composition for the treatment and prevention of inflammatory diseases containing the extract of Hyangbuja as an active ingredient.

In another aspect, the present invention provides a cosmetic composition for the prevention and improvement of inflammatory diseases containing the extract of Hyangbuja as an active ingredient.

The hyangbuja extract is characterized in that it comprises 0.1 to 50% by weight relative to the total weight of the skin external pharmaceutical composition.

The pharmaceutical composition comprises a cream, gel, patch, spray, ointment, warning, lotion, linen, pasta or cataplasma formulation.

In addition, the cosmetic composition includes a lotion, skin, lotion, nutrition lotion, nutrition cream, massage cream, essence, the formulation of the pack.

Hyangbuja, as defined herein, is preferably characterized in that it is a Korean-rich hyangbuja, more preferably a Gyeongbuk cultivated hyangbuja.

Extracts as defined herein include water, including purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably water or extracts soluble in 50% to 90% ethanol.

The pharmaceutical composition containing the extract of Hyangbuja can be used in 0.1 to 50% by weight based on the total weight.

Inflammatory diseases as defined herein preferably include dermatitis diseases such as atopy, eczema, allergy, rhinitis, more preferably atopy, eczema, allergy, rhinitis.

Hereinafter, the present invention will be described in detail.

The flavor extract of the present invention can be obtained as follows.

After washing the perfume of the present invention, the volume of about 1 to 30 times the volume, preferably 5 to 15 times the dry weight of the dried perfume, dried 1 hour to 30 hours, preferably 5 to 25 hours before drying. Polar solvents of C₁ to C₄ lower alcohols such as water, ethanol and methanol, or a mixed solvent thereof, preferably 50% to 90% ethanol, at about 0 to 100 ° C, preferably at about 10 ° C to 50 ° C. The filtrate obtained by extracting the filtrate obtained by extracting 1 to 30 days, preferably 5 to 15 days, 1 to 10 times, preferably 2 to 5 times under reduced pressure, concentration and lyophilization may be obtained to obtain a flavor extract.

The present invention provides a skin external pharmaceutical composition or cosmetic composition for the prevention and treatment of inflammatory diseases containing the extract of Hyangbuja obtained as an active ingredient.

The external skin pharmaceutical composition containing the fragrant extract of the present invention may be used as a pharmaceutical composition in the form of an external skin preparation of creams, gels, patches, sprays, ointments, warnings, lotions, liniments, pasta or cataplasma. But it is not limited thereto.

The preferred dosage of the fragrant extract of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. However, for the preferred effect, the perfume extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Hyangbuja extract of the present invention is treated with lipopolysaccharide (LPS) to induce an inflammatory response and then interleukin ((interleukin) -1β, IL-1β, IL-6, IL-8) by the immune response, tumor necrosis factor ((tumor necrosis factor) -α, TNF-α), NO inhibitory activity and COX-2, showing the effect of inhibiting the production of iNOS excellently confirmed that it is useful for the treatment and prevention of inflammatory diseases.

In addition, the flavor extract of the present invention can be used in a variety of cosmetics and facial cleansing and the like having a skin inflammation improvement effect.

Products to which the composition can be added include, for example, cosmetics such as lotion, skin, lotion, nourishing lotion, nourishing cream, massage cream, essence, pack, etc., and cleansing, face wash, soap, treatment, essence, etc. have.

Cosmetics of the present invention comprises a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract.

The water-soluble vitamins may be any compound that can be incorporated into cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like. Their salts (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, etc.) may also be used in the water-soluble vitamins that can be used in the present invention. Included. The water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microorganism culture, enzyme or chemical synthesis.

The oil-soluble vitamin may be any compound that can be incorporated into cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), and the like. And derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherolvitamin E, dl-pantothenyl alcohol, D-pantothenyl alcohol, pantotenylethyl Ethers, etc.) are also included in the oil-soluble vitamins used in the present invention. Oil-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification of microorganism culture, enzyme or chemical synthesis.

The polymeric peptide may be any compound as long as it can be compounded in cosmetics, and examples thereof include collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. Polymeric peptides can be purified and obtained by conventional methods such as purification from microbial cultures, enzymatic methods or chemical synthesis methods, or can be purified and used from natural products such as dermis and pig silk such as pigs and cattle.

The polymer polysaccharide may be any compound as long as it can be blended into cosmetics. Preferably, hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.) may be mentioned. For example, chondroitin sulfate or its salt, etc. can be normally purified from a mammal or fish.

The sphingolipid may be any compound as long as it can be blended into cosmetics. Preferably, ceramide, phytosphingosine, sphingosaccharide lipid, etc. may be mentioned. Sphingo lipids can usually be purified from mammals, fish, shellfish, yeasts or plants by conventional methods or obtained by chemical synthesis.

The seaweed extract may be any compound as long as it can be blended into cosmetics. Preferably, the seaweed extract may include brown algae extract, red algae extract, green algae extract, and the like. Also, calginine, arginic acid, sodium arginate, Potassium arginate and the like are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purification from seaweed by conventional methods.

The cosmetic of the present invention may be blended with other essential ingredients, if necessary, in combination with the essential ingredients.

Other components that may be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, Blood circulation accelerators, cooling agents, restriction agents, purified water and the like.

Examples of the fat or oil component include ester fats, hydrocarbon fats, silicone fats, fluorine fats, animal fats, and vegetable fats and oils.

As ester fats and oils, glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl mystinate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, and stearic acid Butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl acid isocetyl, isostyl acid isostearyl, isostaryl palmitate, octylate acid octyldodecyl, isostearic acid isetyl, diethyl sebacate, adipine Acid isopropyl, isoalkyl neopentane, tri (capryl, capric acid) glyceryl, trimethyl ethyl trimethylol propane, triisostearic acid trimethylol propane, tetra 2-ethylhexanoic penta erythritol Cetyl caprylate, lauric acid decyl, hexyl laurate, decyl myristin, myristin acid myristyl, myritic acid cetyl, stearyl stearate, decyl oleate, rininooleic acid , Isostearyl laurate, isotridecyl myristin, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecate oleate, octylate decyl oleate, octyl dodecyl linoleate, isopropyl isopropyl acid, 2 -Cetostearyl ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicapric acid, propylene glycol dicacapate, capric acid Propylene glycol, dicapric acid neopentyl glycol, dioctanoate neopentyl glycol, tricaprylic acid glyceryl, triundecyl glyceryl, triisopalmitinate glyceryl, triisostearate glyceryl, neopentane dodecyl Isostearyl octanoate, octyl isononate, hexyl decyl neodecanoate, octyl dodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, Sothete octylate, polyglycerol oleate, polyglycerine isostearate, triisocetyl citrate, triisoalkyl citrate, triisoctyl citrate, lauryl lactate, myritic lactate, octyl lactate, octyl lactate, tricitric acid Ethyl, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malic acid, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipicate, diisopropyl sebacinate, Dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, physteryl isostearate, phytic oleate, 12-Steloylhydroxystearate isocetyl, 12-Steloylhydroxystearate stearyl, 12-stealo Esters such as monohydroxystearic acid isostearyl; and the like.

Hydrocarbon-based fats and oils, such as squalene, a liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, a liquid isoparaffin, polybutene, microcrystal wax, and a vaseline, etc. are mentioned as a hydrocarbon-type fats and oils.

Examples of the silicone-based oils and fats include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane, methylcetyloxysiloxane copolymer, dimethylsiloxane and methylsteoxysiloxane copolymer, and alkyl. Modified silicone oil, amino modified silicone oil and the like.

Examples of the fluorine-based oil include perfluoropolyether and the like.

Animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, soybean oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil. , Cottonseed oil, Palm oil, Cucumber nut oil, Wheat germ oil, Rice germ oil, Shea butter, Walnut colostrum oil, Marker demia nut oil, Meadow home oil, Egg yolk oil, Uji, Horse oil, Mink oil, Orange rape oil, Jojoba oil And animal or plant fats and oils such as candeler wax, carnava wax, liquid lanolin and hardened castor oil.

Examples of the moisturizing agent include a water-soluble low molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

Water-soluble low molecular humectants include serine, glutamine, sorbitol, mannitol, pyrrolidone-sodium carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or more), polypropylene Glycol (polymerization degree n = 2 or more), polyglycerol B (polymerization degree n = 2 or more), lactic acid, lactic acid salt, etc. are mentioned.

Examples of the fat-soluble low molecular humectants include cholesterol and cholesterol esters.

Examples of the water-soluble polymer include carboxyvinyl polymer, polyasparaginate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water soluble chitin, chitosan, and dextrin. Can be.

Examples of the fat-soluble polymers include polyvinylpyrrolidone-eicosene copolymers, polyvinylpyrrolidone-hexadecene copolymers, nitrocellulose, dextrin fatty acid esters, polymer silicones, and the like.

Examples of the emollient include long-chain acyl glutamic acid cholesteryl esters, hydroxy stearic acid cholesterol, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl esters, and the like.

As surfactant, nonionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, etc. are mentioned.

As the nonionic surfactant, self-emulsifying glycerin monostearate, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE Glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE castor oil, POE / POP (polyoxyethylene / polyoxypropylene) copolymer, POE / POP alkyl ether, polyether modified silicone, lauric acid Alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc. are mentioned.

Anionic surfactants include fatty acid soaps, alpha-acyl sulfonates, alkyl sulfonates, alkyl allyl sulfonates, alkyl naphthalene sulfonates, alkyl sulfates, POE alkyl ether sulfates, alkylamide sulfates, alkyl phosphates, POE alkylphosphates, and alkylamides. Phosphates, alkyloylalkyltaurine salts, N-acylamino acid salts, POE alkyl ether carboxylate salts, alkyl sulfosuccinate salts, sodium alkyl sulfo acetates, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphate esters, and the like. have.

As cationic surfactant, alkyl trimethylammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium bromide, behenyl trimethyl ammonium chloride, chloride Benzalkonium, diethylaminoethyl stearate, dimethylaminopropyl stearate, lanolin derivatives, quaternary ammonium salts, and the like.

Examples of amphoteric surfactants include the carboxybetaine type, the amide betain type, the sulfobetain type, the hydroxysulfobetain type, the amide sulfobetain type, the phosphobetaine type, the aminocarboxylate type, the imidazoline derivative type, and the amideamine type. An amphoteric surfactant etc. are mentioned.

Examples of the organic and inorganic pigments include inorganic pigments such as silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, Bengala, clay, bentonite, titanium mica, titanium oxide, bismuth chloride, zirconium oxide, magnesium oxide, Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, chromium oxide, chromium oxide, chromium hydroxide, Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene, styrene copolymer, Organic pigments such as silk powder, cellulose, CI pigment yellow, CI pigment orange, and composite pigments of these inorganic pigments and organic pigments;

As the organic powder, metallic soap such as calcium stearate; Alkyl phosphate metal salts such as zinc sodium cetyl acid, zinc lauryl acid and calcium laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroylglycine calcium; Amide sulfonic acid polyvalent metal salts, such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; Such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoylidene, N-alpha-paratyylnitine, N-alpha-lauroyl arginine, Acyl basic amino acids; N-acylpolypeptides, such as N-lauroyl glycyl glycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid, alpha-aminoaurauric acid, and the like; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride and the like.

Examples of the ultraviolet absorber include paraaminobenzoic acid, ethyl paraaminobenzoate, amyl paraaminobenzoic acid, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylic acid and benzyl cinnamic acid. , Paramethoxy cinnamic acid-2-ethoxyethyl, paramethoxy cinnamic acid octyl, diparamethoxy cinnamic acid mono-2-ethylhexaneglyceryl, paramethoxy cinnamic acid isopropyl, diisopropyl diisopropyl cinnamic acid ester mixture, wuro Canonic acid, ethyl urokanoate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone sodium sulfonate, dihydroxybenzophenone , Tetrahydroxybenzophenone, 4- tert -butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino- p- (carbo-2'-ethylhexyl-1'-oxy) -1 , 3,5-triazine, 2- (2-hi And the like can be mentioned hydroxy-5-methylphenyl) benzotriazole.

Examples of the disinfectant include hinokitiol, trichloroacid, trichlorohydroxydiphenyl ether, crohexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zinc filitione, benzalkonium chloride, No. 301, mononitro and eicol sodium, and undecylenic acid.

Examples of the antioxidant include butylhydroxyanisole, propyl gallic acid, and erythorbic acid.

Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, fmaric acid, sodium pmarate, succinic acid, sodium succinate, sodium hydroxide, sodium dihydrogen phosphate, and the like.

Examples of the alcohol include higher alcohols such as cetyl alcohol.

Moreover, the compounding component which may be added other than this is not limited to this, Moreover, Although all said components can be mix | blended within the range which does not impair the objective and effect of this invention, Preferably it is 0.01-5% weight with respect to gross weight, More Preferably it is compounded at 0.01-3% weight.

The cosmetic of the present invention may take the form of a solution, an emulsion, a viscous mixture or the like.

Components included in the cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the perfume extract as an active ingredient, for example, conventional stabilizers, solubilizers, vitamins, pigments and fragrances Adjuvants and carriers.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, and includes, for example, milky lotion, cream, lotion, pack, foundation, lotion, essence, hair cosmetic, and the like.

Specifically, the cosmetic composition of the present invention can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, Packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

Hyangbuja extract of the present invention is treated with lipopolysaccharide (LPS) to induce an inflammatory response and then interleukin ((interleukin) -1β, IL-1β, IL-6, IL-8) by the immune response, tumor necrosis factor ((tumor necrosis factor) -α, TNF-α), NO inhibitory activity and COX-2, iNOS is shown to be an excellent inhibitory effect can be used as a skin external pharmaceutical composition or cosmetic composition for the treatment and prevention of inflammatory diseases have.

1 is a diagram showing cell viability of macrophages (Raw264.7) of samples (individual values are expressed as mean ± SD (n ≧ 3));
2 is a diagram showing the effect on NO production in macrophages (Raw264.7) of samples (individual values are expressed as mean ± SD (n ≧ 3));
3 is a diagram showing the inhibitory effect on TNF-α production of samples (individual values are expressed as mean ± SD (n ≧ 3));
4 is a diagram showing the inhibitory effect on the IL- 1β production of the samples (individual values are expressed as mean ± SD (n ≧ 3));
5 shows PGE ₂ of the samples. A diagram showing the inhibitory effect on production (individual values expressed as mean ± SD (n ≧ 3));
6 shows IL- 8 of samples. A diagram showing the inhibitory effect on production (individual values expressed as mean ± SD (n ≧ 3));
7 is a diagram showing the inhibitory effect on iNOS expression in LPS-activated macrophages (Raw264.7) of samples (cells were cultured in 50 μg / ml containing medium for 24 hours and iNOS protein expression levels in individual samples) normalized to β-actin odor and individual values expressed as mean ± SD (n ≧ 3);
8 is a diagram showing the inhibitory effect on COX- 2 expression in LPS-activated macrophages (Raw264.7) of samples (cells were cultured in 50 μg / ml containing medium for 24 hours and COX-2 in individual samples). Protein expression levels were normalized to β-actin odor and individual values expressed as mean ± SD (n ≧ 3)).

Hereinafter, the present invention will be described in detail.

However, the following Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example 1. Sample Extraction

Cyperus rotundus (500 g), grown in Gyeongbuk, Korea, was extracted three times at room temperature with 70% ethanol and filtered through filter paper (Whatman No. 3, Maidstone, England). The filtrate was concentrated under reduced pressure (UT-). 1000, EYELA, Tokyo, Japan). The concentrated extract was freeze-dried with a freeze dryer (Labconco freeze dryer, 77500 model, Kansas City, MI, USA) and then obtained 37.05 g (yield 7.41%) of ethanol extract (hereinafter referred to as “CR-EtoH”). It was stored in a freezer and used as a sample of the following experimental example.

Reference Example 1. Experiment Preparation

1-1. Reagents and Instruments

Lipopolysaccharide (LPS) was commercially available from Sigma Chemical Co. (St. Louis, MO, USA). Raw 264.7 cells used for measuring cell viability were purchased from Korean Cell Line Bank (KCLB). Cell viability assay reagents were Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin / streptomycin, trypsin 250, 0.4% trypan blue stain, Gibco BRL Co. (Grand Island, USA) and Haemacytometer (Marienfeld, Germany) 3- [4,5-dimethyl thiazol-2-yl] -2,5-diphenyltetrazoliumbromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). INOS BD bioscience (Sanjose, CA, USA), the primary antibody used in the experiment, COX-2 cayman (Ann arbor, MI, USA), β-actin and anti-rabbit Ig-G horseradish peroxidase (HRP), the secondary antibody -conjantibody (Santa cruz, CA, USA). ELISA kits for Tumor necrosis factor (TNF) -α, IL-1β, IL-6 and IL-8 were purchased from Pierce endogen (Rockford, IL., USA). In addition, the band density was a gel doc (Amersham Pharmacia, England) instrument.

1-2. Cell culture

Murine macrophage cell line, RAW 264.7, purchased from the Korean Cell Line Bank (KCLB) was diluted with 0.25% trypsin solution in cultured cells according to the method of Michikawa et al. (13). Next, 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (100U / ml) were added to DuLbeco's modified eagle's medium (DMEM) medium, and then cultured in a 37 ° C. and 5% CO ₂ incubator.

Experimental Example  One. MTT assay Cells by Survival rate measurement

In order to confirm the cell viability of the sample obtained in the above example, the experiment was performed by applying the method disclosed in the literature (14).

RAW 264.7 cells were dispensed in 0.18ml to 1 × 10 ⁴ cells / well in a 96well plate. Samples were prepared by concentration, added with 0.02ml, and incubated in 37 ° C. and 5% CO ₂ incubator for 48 hours. In the control group, the same amount of distilled water as that of the sample was added and the cells were cultured under the same conditions. After incubation for 4 hours with the addition of 0.02ml of MTT solution prepared at 5mg / ml concentration, the culture solution was removed, and 0.15ml of DMSO: EtOH (1: 1) was added to each well for 30 minutes at room temperature, followed by ELISA reader. Absorbance was measured at 550 nm. The cytotoxicity measurement was expressed by the absorbance decreasing rate of the sample solution addition group and no addition group according to the following equation (1).

As a result of the above experiment, the cytotoxicity by the extract of Hyangbuja was confirmed by MTT assay, and the results are shown in FIG. 1. Hyangbuja extract showed a high survival rate of 100% at concentrations of 10, 100 and 250 ㎍ / ml, and showed no cytotoxicity compared to the control.

In the experiment of ELISA kit, western blot was carried out using the 50 ㎍ / ml concentration.

Experimental Example  2. Hyangbuja  Extract Nitric Oxide  Production measurement

In order to confirm the effect on the NO production of the sample obtained in the above example, the experiment was performed as follows by applying the method disclosed in the literature (15).

NO concentration was measured by griess reagent (Sigma No. G4410, USA). Raw 264.7 cells were inoculated in a 12 well plate after adjusting to 5 x 10 ⁴ cells / ml using DMEM medium, 5% CO ₂ Incubated 24 hours before incubator. Cells were treated with 1 μg / ml LPS and 1 hour later, 1, 10, 100 μg / ml sample extract was incubated for 24 hours. After the supernatant of the culture was obtained and reacted with the griess reagent, the absorbance was measured at 540 nm with an ELISA reader to express the NO production rate as a percentage.

In order to measure the NO production of Raw 264.7 cells, the result of measuring the amount of NO produced by treating 70% ethanol extract of Hyangbuja, a medicinal crop of Gyeongbuk, with 10 and 50 ㎍ / ml, was measured in the cells.

As a result of the experiment, compared to the control group treated with LPS, the fragrance extract showed an effect of 83% at 10 ㎍ / ml concentration of NO, and it was confirmed that the fragrance extract exhibited an excellent effect of 40% at 50 ㎍ / ml concentration.

Experimental Example  3. Hyangbuja  Extract PGE ₂  Production measurement and Cytokine assay  ( TNF -α, IL -1β, IL -6, IL -8)

In order to confirm the effect on the production of pro-inflammatory factors of the sample obtained in the above example, the experiments were performed as follows by applying the method disclosed in the literature (16).

The amount of PGE _{2 in the} cell culture was measured by purchasing a commercial competitive enzyme immunoassay kit from R & D systems (Minneapolis, MN, USA).

Cells are pretreated with hyangbuja extract and treated with 100ng / ml LPS (Sigma No. L6636, USA). After 18 hours, cell cultures are obtained and used for PGE ₂ determination. 100 μl of the culture solution was loaded on a 96-well plate coated with goat anti-mouse, and 50 μl of primary antibody solution and 50 μl of PGE ₂ conjugate were added thereto, followed by incubation at 4 ° C. overnight. After washing four times with the wash buffer solution, the substrate solution was treated with 200 μl each reaction for 5 to 20 minutes, and then treated with 50 μl stop solution, and the absorbance at 450 nm was measured.

In cultured cells, TNF-α, IL-1β, IL-6, and IL-8 are measured using an enzyme-linked immunosorbent assay (ELISA) kit (R & D systems, Minneapolis, MN, USA). That is, anti-human TNF-α, IL-1β, IL-6, and IL-8 capture monoclonal antibodies were coated on 2 wells / ml on 96 well plates and reacted at 4 ° C. for 12 hours. In order to prevent nonspecific reaction after coating, a blocking solution composed of PBS (phosphate buffered saline) containing 10% FBS was added and reacted at 37 ° C. for 2 hours. After washing four times with washing buffer (PBS) containing 0.05% tween 20, the standard recombinant proteins TNF-α, IL-1β, IL-6, and IL-8 were serially diluted to an appropriate concentration. After the preparation, the culture supernatant is diluted and put into 100 씩 of each well and reacted at 37 ° C. for 2 hours. After washing 4 times with washing buffer, dilute biotinylated anti-human TNF-α, IL-1β, IL-6, IL-8 to 300 ng / ml using blocking solution, and then 100 μl each well. Put and react for 1 hour at 37 ℃. After washing four times with washing buffer again, each well was treated with a streptavidin-horseradish peroxidase conjugate enzyme at a concentration of 2.5 µg / ml, and then washed four times after reaction at 37 ° C. for 30 minutes. 50 μl of substrate solution was added to each well to induce color development for 20 minutes. Then, the reaction was stopped using 2 N sulfuric acid, and TNF-α, IL-1β, IL-6, and IL-8 proteins were used at 450 nm using an ELISA reader. Measure the amount.

The production of TNF-α, IL-1β, and IL-6 was measured to investigate whether the extract of Hyangbuja inhibits the formation of prostagladin E ₂ and pro-inflammatory cytokine produced by LPS in Raw 264.7 cells. . The results of measuring the expression of PGE ₂ and cytokines (TNF-α, IL-1β, IL-6) by treating 50 ㎍ / ml concentration of Hyangbuja extract, a medicinal crop of Gyeongbuk, were shown in FIGS. 3 to 6.

PGE _{2 with} LPS Inhibitory activity of hyangbuja extract was 26%, which showed excellent inhibitory activity, and cytokines (TNF-α, IL-1β, IL-6) showed excellent inhibitory effect, especially IL-1β. At 47%, the excellent inhibitory activity was observed.

Experimental Example  4. From external stimulation Intracellular  Measurement of pigmentation and inflammation-related protein expression

In order to confirm the effect on the expression of inflammation-related proteins from external stimulation of the sample obtained in the above example, the experiments were performed as follows by applying the method disclosed in the literature (16).

Inflammation-related proteins iNOS, COX-2 protein was measured. After culturing in a culture dish using each cell line, the sample was treated, washed with PBS after 24 hours, collected by centrifugation, and RIPA buffer (Sigma No. R0278, USA). Cell lysate is prepared. The amount of protein contained in the cell lysate is measured with a Bicinchoninic acid (BCA) kit (Thermo Scientific No. 23225, USA).

20 μg of protein was separated by SDS-PAGE containing 10% polyacrylamide (Sigma, USA) and subjected to 1 hour at 120 V electroblot (GE Healthcare EPS 301, USA) with PVDF membrane. Subsequently, the PVDF membrane is left in 5% skim milk (Becton and Dickinson Company No. 232100, USA) for 1 hour and then incubated overnight at 4 ° C. in primary antibody. After washing three times with TBST (Sigma, USA), the secondary antibody was reacted at room temperature for 1 hour, and then washed three times with TBST and reacted with ECL kit (Amersham Pharmacia # RPN2109D2, Biotech) for LAS 4000 chemiluinescence. Development and quantification were performed using a detection system (LAS 4000, Fuji, Tokyo, Japan).

Induction of NO production using LPS (1 ㎍ / ml) in Raw 264.7 cells resulted in low expression of COX-2 protein as shown in FIG. 8 when the Kyungbuk medicinal crop Hyangbuja extract was treated at 50 ㎍ / ml. Based on the above results, hyangbuja extract inhibits inflammation by inhibiting overexpression of inflammatory factors NO and PGE ₂ and synthase enzymes iNOS, COX-2 and inflammatory cytokines in LPS-induced macrophages. It is expected to be used as.

Examples of the formulation of the composition comprising the extract of the present invention will be described, but the present invention is not intended to limit it, but is intended to explain in detail only.

Hereinafter, the formulation of the present invention is exemplified as a cream, massage cream, lotion, skin lotion, essence, packs, cleansing foam formulation, but is not limited to the formulation comprising the cosmetic composition of the present invention.

Formulation Example  1. Cream composition

The oil and water phases are heated and mixed at 75 ° C., respectively, and then cooled to room temperature.

Formulation Example 2 Massage Cream Composition

     The oil phase and the water phase are each dissolved by heating at 75 ° C., and then cooled to room temperature.

Formulation Example  3. Lotion Composition

     The oil phase and the water phase are each heat mixed emulsified at 75 ° C., and then cooled to room temperature.

Formulation Example  4. Skin Lotion Composition

     The aqueous phase and the ethanol phase are each prepared, mixed and filtered.

Formulation Example  5. Essence Composition

     The aqueous phase and the ethanol phase are each prepared, mixed and filtered.

Formulation Example  6. Pack Composition

     The aqueous and ethanol phases are dispersed, dissolved and mixed, and then cooled to room temperature.

Formulation Example  7. Cleansing Foam  Composition

     The aqueous and oil phases are dispersed, dissolved and mixed, and then cooled and cooled to room temperature.

Claims (8)

Skin external pharmaceutical composition for the treatment and prevention of inflammatory diseases containing the extract of Hyangbuja. The method of claim 1,
The hyangbuja extract is a pharmaceutical composition, characterized in that it comprises 0.1 to 50% by weight relative to the total weight of the external skin pharmaceutical composition. The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition, characterized in that the cream, gel, patch, spray, ointment, warning, lotion, linen, pasta or cataplasma formulation. The method of claim 1,
The fragrance is a Korean pharmaceutical composition characterized in that the fragrance. The method of claim 1,
The extract is a pharmaceutical composition, characterized in that the extract is available in water, purified alcohol, lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The method of claim 1,
The inflammatory disease is a dermatological composition, characterized in that the disease. Cosmetic composition for the prevention and improvement of inflammatory diseases containing a flavor extract as an active ingredient. 8. The method of claim 7,
Wherein the cosmetic composition is a formulation of lotion, skin, lotion, nutrition lotion, nutritional cream, massage cream, essence, pack.

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