WO2018155933A1 - 6,7-dimethoxy-2,2-dimethyl-2h-chromene for increasing expression of aquaporin-3 and use thereof - Google Patents

Abstract

The present invention relates to a cosmetic composition for moisturizing the skin and a pharmaceutical composition for preventing or treating atopic dermatitis, each of the compositions comprising the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an effective ingredient. The compound increases expression of the mRNAs or proteins of aquaporin-3 (AQP3) and CLOCK, thereby being effective for moisturizing the skin and useful for treating or alleviating atopic dermatitis.

Description

6,7-dimethoxy-2,2-dimethyl-2H-chromen and its use to increase expression of aquaporin-3

The present invention relates to 6,7-dimethoxy-2,2-dimethyl-2 H -chromen and its use to increase the expression of aquaporin-3.

Human skin is largely divided into three layers: epidermis, dermis and subcutaneous fat tissue. The human skin is primarily used to protect the human body from physical or chemical stimuli caused by the external environment such as harmful microorganisms, physical damage and ultraviolet rays. Serves as a protective barrier. In addition, the skin not only serves to transport various physiologically active substances necessary for the human body, but also controls the evaporation of moisture corresponding to about 65% to 70% of the human body to the body.

The epidermal layer is divided from the outside in the order of the stratum corneum, the granule layer, the pole layer and the base layer. The cells of the stratum corneum act like bricks, and the intercellular lipids between the keratinocytes are composed of a lipid mixture such as ceramide, cholesterol or fatty acid to form an intercellular lamellae structure to form a skin barrier. The cells constituting the epidermal layer include various cells such as keratinocytes, langelhans cells, and merkel cells, and most of them are keratinocytes. Keratinocytes are cell proliferation activity in the basal layer, and through the granule layer to synthesize the cell components, including keratin, finally reaches the skin stratum corneum. The cells that reach the stratum corneum are keratins that are flat dead cells that kill millions of dead keratinocytes a day and replace them with new keratinocytes. The flattened keratinocytes are stratified, like layers of bricks, making the stratum corneum stronger and at the same time gaining the flexibility described by free movement and functioning as a barrier. The stratum corneum contains about 10% to 20% of water and exists at the outermost part of the human body, thereby suppressing evaporation of moisture to the outside of the body to maintain a proper amount of water in the skin, while blocking excess penetration of substances from the outside Perform a defensive function.

Factors that weaken the defense function of the stratum corneum, which influences the skin barrier function, may be classified into internal factors and external factors. Internal factors include disease, genetic defects, or aging. External factors include frequent use of soaps or detergents, ultraviolet radiation, long-term exposure to adverse temperature and humidity conditions, or poor eating habits. However, as the skin dries, the skin barrier structure is impaired and the water content of the stratum corneum is reduced. At this time, the homeostatic reaction to maintain the normal skin barrier function occurs in a chain, the proliferation of the epidermal cells and the stratum corneum is increased, the surface of the skin becomes rough and a lot of keratin. Therefore, in order to maintain skin health, it is important to prevent skin dryness, and for this purpose, it is necessary to use an appropriate skin moisturizer.

The most widely used moisturizer in the current cosmetics moisturizes the skin, prevents evaporation, or draws moisture from the air to the skin. Many moisturizers used in cosmetics are glycerin (glycerol), propylene glycol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, sorbitol ( sorbitol, sodium lactate, sodium 2-pyrrolidone-5-carboxylate, sodium hyaluronate (N-acetglucosamine and gluconic acid) It is a kind of sugar) and keeps moisture in the connective tissue under the epidermal layer, and forms a gel matrix in the tissue to make the skin lubricity and flexibility, and to prevent external pressure and bacterial infection.

Aquaporin is present in the cell membrane as a transporter that actively transports water molecules into the cell membrane in the cell membrane (J Biol Chem 1999; 274: 21631-21636, Curr.Opin.Struct.Biol. 2005; 15; (176-183). There are 13 known aquaporin proteins in mammals.Aquaporin-3 (AQP3) protein is a kind of aquaglyceroporin protein that selectively passes water molecules and glycerol. It is known to play an important role in hydration and wound healing (J Invest Dermatol 2002; 118: 678-685, J. Mol. Med. 2008; 96: 523-529).

Glycerol, which is present in the body, is a type of aliphatic trihydric alcohol that binds to fatty acids to make neutral fatty acids. Glycerol has three hydroxyl groups (-OH) (molecular formula: C ₃ H ₅ (OH) ₃ ; CH ₂ OH-CHOH-CH ₂ OH) which has a close nature with water, so it is hygroscopic and natural in vivo Moisturizing effect (J. Biol. Chem. 2002; 277: 46616-46621). In mice lacking the aquaporin-3 (AQP3) gene, stratum corneum and epidermis were markedly lacking in water and skin elasticity (J. Biol. Chem. 2002; 277; 17147-17153). In addition, application of glycerol to aquaporin-3 deficient mice restored skin moisturizing and elasticity (Proc. Natl. Acad. Sci. USA 2002; 100: 7360-7365). These results suggest that aquaporin-3 (AQP3) protein plays an important role in maintaining skin moisture by maintaining moisture inflow into the stratum corneum and glycerol concentration in the epidermal layer.

An object of the present invention is to provide a skin moisturizing cosmetic composition comprising a compound represented by the following formula (1) as an active ingredient.

[Formula 1]

Another object of the present invention to provide a health functional food composition for skin moisturizing comprising the compound represented by the formula (1) as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Still another object of the present invention is to provide a cosmetic composition for improving atopic dermatitis comprising the compound represented by Formula 1 as an active ingredient.

Still another object of the present invention is to provide a skin moisturizing cosmetic composition comprising a fraction obtained by sequentially fractionating water, hexane, and chloroform from an fluorinated alcohol extract.

Still another object of the present invention is to provide a health functional food composition for skin moisturizing, comprising a fraction obtained by sequentially fractionating water, hexane and chloroform from the fluorinated alcohol extract.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating atopic dermatitis, comprising as an active ingredient a fraction obtained by sequentially fractionating water, hexane, and chloroform from an fluorinated alcohol extract.

Still another object of the present invention is to provide a cosmetic composition for improving atopic dermatitis comprising fractions obtained by sequentially fractionating water, hexane, and chloroform from an fluorinated alcohol extract as an active ingredient.

Still another object of the present invention is to provide a method for preventing and treating atopic dermatitis, comprising administering a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to a subject in a pharmaceutically effective amount.

It is another object of the present invention to provide a method for preventing and treating atopic dermatitis, comprising administering to a subject a pharmaceutically effective amount of a fraction obtained by sequentially dividing water, hexane and chloroform from an alcoholic extract of fluoride. will be.

Still another object of the present invention is to provide a method for enhancing skin moisturizing, comprising applying topically to the individual an effective amount of a skin moisturizing cosmetic composition comprising the compound represented by Formula 1.

Another object of the present invention is to enhance skin moisturization, comprising topically applying to the individual an effective amount of a skin moisturizing cosmetic composition comprising a fraction obtained by sequentially fractionating water, hexane, and chloroform from an fluorinated alcohol extract. To provide a way.

In order to achieve the above object, the present invention provides a skin moisturizing cosmetic composition comprising a compound represented by the following formula (1) as an active ingredient

[Formula 1]

.

.

In one embodiment of the present invention, the compound represented by Formula 1 may be 6,7-dimethoxy-2,2-dimethyl- 2H -chromen.

In one embodiment of the present invention, the compound may be to increase the expression level of aquaporin-3 (aquaporin-3, AQP3) or the amount of mRNA or protein of CLOCK.

The present invention also provides a health functional food composition for skin moisturizing comprising the compound represented by the formula (1) as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for improving atopic dermatitis comprising a compound represented by the following formula (1) as an active ingredient.

In addition, the present invention provides a cosmetic composition for moisturizing skin comprising a fraction obtained by sequentially fractionating water, hexane and chloroform from the fluorinated alcohol extract.

In one embodiment of the present invention, the alcohol may be ethanol.

In one embodiment of the present invention, the fraction may be a hexane fraction.

In one embodiment of the present invention, the fraction may be to increase the expression level of aquaporin-3 (aquaporin-3, AQP3) or mRNA or protein of CLOCK.

In addition, the present invention provides a health functional food composition for skin moisturizing comprising a fraction obtained by sequentially fractionating water, hexane and chloroform from the fluorinated alcohol extract.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising a fraction obtained by sequentially distilling water, hexane and chloroform from the fluorinated alcohol extract as an active ingredient.

In another aspect, the present invention provides a cosmetic composition for improving atopic dermatitis comprising a fraction obtained by sequentially fractionating the alcohol extract of fluorinated water, hexane and chloroform as an active ingredient.

In addition, the present invention provides a method for preventing and treating atopic dermatitis comprising administering to a subject a pharmaceutically effective amount of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

The present invention also provides a method for preventing and treating atopic dermatitis, comprising administering to a subject a pharmaceutically effective amount of a fraction obtained by sequentially dividing water, hexane, and chloroform from an alcoholic extract of fluoride.

In addition, the present invention provides a method for enhancing skin moisturizing comprising topically applying to the individual an effective amount of a skin moisturizing cosmetic composition comprising a compound represented by the formula (1).

In addition, the present invention provides a method for enhancing skin moisturizing, comprising topically applying to the individual an effective amount of a skin moisturizing cosmetic composition comprising a fraction obtained by sequentially fractionating water, hexane and chloroform from an fluorinated alcohol extract. to provide.

Agerarin, a compound represented by Formula 1 according to the present invention, is effective for moisturizing the skin by increasing the expression level of aquaporin-3 (AQP3) and mRNA or protein of CLOCK, and is useful for treating or improving atopic dermatitis. Can be used.

Figure 1 shows the results of measuring the amount of aquaporin-3 (AQP3) after treatment with fluorinated-ethanol extract (AHE). (A) and (B) measured the expression level of AQP3 mRNA by RT-PCR, and (C) measured the expression level of AQP3 protein by immunoblot method.

Figure 2 shows the result of measuring the amount of aquaporin-3 (AQP3) after irradiation with ultraviolet light after treatment with fluorinated-ethanol extract (AHE). (A) is the amount of expression of AQP3 mRNA by RT-PCR, (B) is the amount of mRNA expression by QRT-PCR, (C) is the expression of AQP3 protein by immunoblot method The amount was measured, and (D) the expression of AQP3 protein was measured in cells by immunofluorescence.

Figure 3 shows the results of the process of fractionation of hexane and chloroform in fluorinated-ethanol extract (AHE) and the expression of aquaporin-3 (AQP3) for the fraction. (A) shows the process of fractionation into hexane and chloroform in the fluorinated ethanol extract, (B) is treated with hexane, chloroform and water fractions separated from the fluorinated-ethanol extract (AHE) and then irradiated with UV light The results of measurement of the expression of aquaporin-3 (AQP3) by QRT-PCR is shown, and (C) shows the expression of aquaporin-3 (AQP3) of fluorinated-ethanol extract (AHE) and hexane fractions in RT-PCR. It shows the result measured.

4 is a chromatogram analysis of the fluorinated hexane fraction, (A) shows a chromatogram of the fluorinated hexane fraction, (B) shows a three-dimensional chromatogram taken with a photodiode array detector 8.6 minutes peak material will be.

Figure 5 shows the results of measuring the amount of aquaporin-3 (AQP3) expression of agerarin (Agerarin), a compound isolated from the fluoride-hexane fraction (AHE-Hx). (A) and (C) are the results by RT-PCR, and (B) and (D) are the results by QRT-PCR.

Figure 6 shows the primary nuclear magnetic resonance spectra for the compound Agerarin (Agerarin) isolated from the fluorine-hexane fraction (AHE-Hx), (A) is the carbon-nuclear magnetic resonance spectroscopy, (B ) Shows hydrogen-nuclear magnetic resonance spectra, (C) shows a distortionless enhancement by polarization transfer (DEPT) spectrum, and (D) shows a heteronuclear multiple quantum coherence (HMQC) spectrum.

Figure 7 shows the secondary nuclear magnetic resonance spectra for the compound Agerarin (Agerarin) isolated from the fluoride-hexane fraction (AHE-Hx), (A) is TOCSY (Total correlated spectroscopy) spectrum, (B ) Shows the correlated spectroscopy (COSY) spectrum, (C) the heteronuclear multiplebonded connectivities (HMBC) spectrum, and (D) shows the carbon-hydrogen connected relationship from COSY and HMBC experiments (dotted line: COSY result, solid line: HMBC result). (E) shows a high resolution mass spectrometry spectrum.

FIG. 7E uses UPLC-TOFMS (ultra-performance liquid chromatography-hybrid quadrupole-time-of-flightmass spectrometry) equipped with Waters ACQUITY UPLC system (Waters, Milford, Mass.) To reconfirm the structure of Agerarin. The results of the high resolution mass spectrometry experiments are shown.

Figure 8 shows the result of measuring the expression level of CLOCK by the agerarin (Agerarin) which is a compound separated from the fluoride-hexane fraction (AHE-Hx). (A) shows the expression of CLOCK mRNA and aquaporin-3 (AQP3) mRNA by RT-PCR after injecting shRNA (small hairpin RNA) to the CLOCK gene (shCLOCK). The amount of CLOCK mRNA expression over time was measured by RT-PCR. (C) The amount of CLOCK mRNA expression over time was quantified by QRT-PCR, and (D) the amount of CLOCK protein expression over time. Is measured by the immunoblot method.

Figure 9 shows the results of measuring the expression of CLOCK and aquaporin-3 (AQP3) mRNA by agerarin (Agerarin) after inhibiting CLOCK gene expression. (A) and (C) measured the expression levels of CLOCK mRNA and aquaporin-3 (AQP3) mRNA by RT-PCR, respectively, and (B) and (D) respectively measured CLOCK mRNA and QRT-PCR. It is a result quantitatively showing the expression level of aquaporin-3 (AQP3) mRNA.

FIG. 10 is a diagram illustrating that agerarin has an effect on skin moisturizing. Agerarin increases CLOCK expression and CLOCK may promote aquaporin-3 (AQP3) gene expression. It is a summary.

The present invention provides a skin moisturizing cosmetic composition comprising a compound represented by the following formula (1) as an active ingredient.

The present invention also provides a health functional food composition for skin moisturizing comprising the compound represented by the following formula (1) as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for improving atopic dermatitis comprising a compound represented by the following formula (1) as an active ingredient.

[Formula 1]

Formula 1 "azelaic Lin (Agerarin)" as, 6,7-dimethoxy-2,2-dimethyl -2 H -chromene - of (6,7-Dimethoxy-2,2-dimethyl -2 H-chromen) It has a chemical name and can be represented by the following formula (1). The acerarine has a molecular formula of C ₁₃ H ₁₆ O ₃ , and a molecular weight of 220.1099.

In the cosmetic composition according to the present invention, the cosmetic composition may include the compound of Formula 1 in an amount of 0.0001 to 30% by weight based on the total weight of the composition, but includes an effective amount that can provide a desirable skin moisturizing effect If so, it is not limited thereto.

Cosmetic composition according to the present invention is an external skin ointment, cream, supple cosmetics, nourishing cosmetics, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, Group consisting of lotion, milk lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, nourishing essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser It may have a formulation selected from, but is not limited thereto. The composition of each of these formulations may contain a variety of bases and additives necessary for the formulation of the formulation and are suitable, and the types and amounts of these components can be readily selected by those skilled in the art.

When the formulation of the present invention is a paste, cream or gel, animal carriers, vegetable fibers, waxes, paraffins, starches, tracantes, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide, etc. may be used as carrier components. Can be.

When 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, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the dosage form of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can 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, isethionate, an imidazolinium derivative, methyltaurate, sarcosinate, 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.

Formulations of the present invention include fluorescent substances, fungicides, hydrogenic agents, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, water soluble vitamins, oil soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and And excipients, including plant extracts.

The water-soluble vitamins may be any compound as long as they 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. And salts thereof (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, etc.) can also be used in the present invention. Included in 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 formulated in cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol) and the like. Derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherolvitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl) Ethyl ether) and the like 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 polymer peptide may be any compound as long as it can be incorporated into cosmetics. Preferably, collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin and the like can be given. 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 can be normally purified from a mammal or fish.

The sphingolipid may be any compound as long as it can be incorporated 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.

In addition to the said essential component, you may mix | blend the cosmetics of this invention with the other components normally mix | blended with cosmetics as needed. Other ingredients 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.

Examples of the ester fats and oils include glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristin, butyl mystinate, isopropyl palmitate, ethyl stearate, octyl palmitate, and isocetyl isostearate and stearic acid. Butyl acid, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl acid, isocetyl acid, isostyl acid mystearyl, isostyl palmitate, octylate acid octylate dodecyl, isostearic acid isetyl, diethyl sebacate, adidi Diisopropyl phosphate, isoalkyl neopentane, tri (capryl, capric acid) glyceryl, trimethyl ethyl trimethylolpropane, trimethyl stearate trimethylolpropane, tetra 2-ethylhexanopentane Tol, cetyl caprylate, lauric acid decyl, hexyl laurate, myristic acid decyl, myristic acid myristyl, myristic acid cetyl, stearyl stearate, decyl oleate, ricinole Sancetyl, isostearyl laurate, isotridecyl myristin, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyl dodecyl oleate, octyl dodecyl linoleate, isopropyl isopropyl acid , 2-ethylhexanoic acid cetostearyl, 2-ethylhexanoic acid stearyl, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicapric acid, di (capryl, capric acid) propylene glycol , Propylene glycol dicaprylic acid, neopentyl glycol dicapric acid, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl tritridecyl, glyceryl triisopalmitate, glyceryl triisostearate, neopentanoic acid Octyldodecyl, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearic acid isosate Aryl, octyldecyl isostearate, polyglycerol oleic acid ester, polyglycerol isostearic acid ester, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauric lactate, myritic lactate, cetyl lactate, octyl lactate, Triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malic acid, 2-ethylhexyl hydroxystearate, diethyl 2-ethylhexyl succinate, diisobutyl adipicate, diisocebaic acid Propyl, dioctyl sebacate, cholesteryl stearate, cholesteryl isostearic acid, cholesteryl hydroxystearate, cholesteryl oleate, oleic acid dihydrocholesteryl, isosterate physteryl, oleate Stearyl, 12-stealoylhydroxystearate isocetyl, 12-stealoylhydroxystearate stearyl, 12 Ester type | system | groups, such as stearoyl hydroxy stearic acid isostearyl, etc. are mentioned.

Examples of the hydrocarbon-based oils and fats include hydrocarbon oils such as squalene, liquid paraffin, alpha-olefin oligomers, isoparaffins, ceresin, paraffins, liquid isoparaffins, polybutenes, microcrystalline waxes, and vaseline.

Examples of the silicone-based oils and fats include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane and methylcetyloxysiloxane copolymer, dimethylsiloxane and methylsteoxysiloxane copolymer, Alkyl modified silicone oil, amino modified silicone oil, etc. are mentioned.

Perfluoro polyether etc. are mentioned as said fluorine-based fat or oil.

Examples of the 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, grapes. Seed oil, Cottonseed oil, Palm oil, Cuckoo nut oil, Wheat germ oil, Rice germ oil, Shea butter, Walnut colostrum, Marker demia nut oil, Meadow home oil, Egg yolk oil, Uji, Horse oil, Mink oil, Orange rape oil, Jojoba Animal or vegetable fats and oils, such as oil, candler wax, carnava wax, liquid lanolin, hardened castor oil, etc. are mentioned.

As said moisturizer, a water-soluble low molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, a fat-soluble polymer, etc. are mentioned.

Examples of the water-soluble low molecular moisturizer 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), poly Propylene 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 polymers 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 mentioned.

Examples of the fat-soluble polymers include polyvinylpyrrolidone and eicosene copolymers, polyvinylpyrrolidone and hexadecene copolymers, nitrocellulose, dextrin fatty acid esters, polymer silicones, and the like. Examples of the emollient include long-chain acyl glutamic acid cholesteryl esters, hydroxystearic acid cholesterol, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl esters, and the like.

As said surfactant, a nonionic surfactant, anionic surfactant, cationic surfactant, an amphoteric surfactant, etc. are mentioned.

Examples of the nonionic surfactant include self-emulsifying monoglycerate, 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 and POP (polyoxyethylene and polyoxypropylene) copolymer, POE and POP alkyl ether, polyether modified silicone, laurin Acid alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc. are mentioned.

Examples of the anionic surfactant include fatty acid soap, alpha-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, and POE alkyl. Amide phosphate, alkyloylalkyltaurine salt, N-acylamino acid salt, POE alkyl ether carboxylate salt, alkyl sulfosuccinate salt, sodium alkyl sulfo acetate, acyl hydrolyzed collagen peptide salt, perfluoroalkyl phosphate ester, etc. Can be.

Examples of the cationic surfactant include alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyl trimethylammonium chloride, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzyl ammonium bromide, behenyltrimethylammonium, Benzalkonium chloride, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, quaternary ammonium salts of lanolin derivatives, and the like. Examples of amphoteric surfactants include carboxybetaine type, amidebetaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type and amideamine type. An amphoteric surfactant etc. are mentioned.

Examples of the organic and inorganic pigments include silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengal, clay, bentonite, titanium film mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, and oxide Inorganic pigments such as aluminum, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine, chromium oxide, chromium hydroxide, coloramine and composites thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene and 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 said organic powder, Metal soaps, such as a calcium stearate; Alkyl phosphate metal salts such as sodium cetylinate, 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; N-epsilon-lauroyl-L-lysine, N-epsilon-palmitolyzine, N-alpha-paratoylol nitin, N-alpha-lauroyl arginine, N-alpha-cured fatty acid acyl arginine -Acyl basic amino acid; N-acyl polypeptides, such as N-lauroyl glycyl glycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene and styrene copolymer, ethylene tetrafluoride and the like.

As said ultraviolet absorber, paraamino benzoic acid, ethyl paraamino benzoate, amyl paraamino benzoate, octyl paraamino benzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylic acid, and cinnamic acid Benzyl, paramethoxy cinnamic acid-2-ethoxyethyl, paramethoxy cinnamic acid octyl, diparamethoxy cinnamic acid mono-2-ethylhexaneglyceryl, paramethoxy cinnamic acid isopropyl, diisopropyl and diisopropyl cinnamic acid ester mixture , Urocanoic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone disulfonate, dihydroxy Benzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p- (carbo-2'-ethylhexyl-1'-oxy) -1,3,5-triazine, 2- ( 2-hydroxy-5-methylphenyl) benzotriazole, etc. are mentioned.

Examples of the fungicides include hinokithiol, trichloric acid, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcinol, isopropylmethylphenol, azulene, salicylic acid, zinphylthione, benzalkonium chloride, Photosensitizer No. 301, mononitrourecosodium sodium, undecylenic acid, etc. are mentioned.

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 fmarate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, and the like.

Higher alcohols, such as cetyl alcohol, are mentioned as said alcohol.

In addition, the compounding component which may be added other than this is not limited to this, Moreover, any of the above components can be mix | blended within the range which does not impair the objective and effect of this invention.

When the cosmetic composition is formulated as a medicine or cosmetic, it may include a known excipient applicable to the skin to act as a carrier for the active ingredient. When formulating into a pharmaceutical, reference may be made to the contents disclosed in [Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA], and when formulating into a cosmetic, [International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995, may be referred to. Such documents are incorporated as part of this specification.

The pharmaceutical composition according to the present invention may use the compound of Formula 1 as an active ingredient as it is or in the form of a pharmaceutically acceptable salt. The salt is not particularly limited as long as it is pharmaceutically acceptable. For example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid , Benzene sulfonic acid, toluene sulfonic acid, naphthalene sulfonic acid and the like can be used. In addition to acid addition salts, base addition salts such as sodium hydroxide, potassium hydroxide, triethylamine, tert-butylamine can also be used.

In the formulation of the pharmaceutical composition according to the present invention, the content of the active ingredient can vary widely depending on the formulation, according to conventional methods.

The pharmaceutical composition of the present invention may further comprise an adjuvant. The adjuvant may be used without any limitation as long as it is known in the art, but may further include the Freund's complete adjuvant or incomplete adjuvant to increase its effect.

The pharmaceutical composition according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical art. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. .

When formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient in the active ingredient, for example starch, calcium carbonate, sucrose, lactose, gelatin It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the individual. Obviously, the concentration of the active ingredient included in the pharmaceutical composition may be variously selected according to the object, and preferably, the pharmaceutical composition is included in a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, the pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg / ml, the human body may be toxic.

In addition to the extract as an active ingredient, the health functional food composition of the present invention may contain various flavors or natural carbohydrates and the like as additional ingredients, as in general food compositions.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The aforementioned flavoring agents can advantageously be used natural flavoring agents (tautin), stevia extracts (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice creams, alcoholic beverages, vitamin complexes, and health supplements. There is this.

In addition to the extract as an active ingredient, the functional food composition is a flavor, such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and its Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the food composition of the present invention may contain a fruit flesh for producing natural fruit juice and fruit juice beverage and vegetable beverage.

The health functional food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like. In the present invention, the "health functional food composition" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act. Ingestion is intended to obtain useful effects for health purposes such as nutrient control or physiological action. The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards. Examples of the items listed in the 'Food Additive Revolution' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high color pigment and guar gum; Mixed preparations, such as a sodium L- glutamate preparation, an addition of an alkali, a preservative preparation, and a tar coloring agent, etc. are mentioned. For example, the health functional food in the form of tablets may be granulated in a conventional manner by mixing the active ingredient of the present invention with excipients, binders, disintegrants and other additives, and then compression-molded with a lubricant or the like, or The mixture can be compression molded directly. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary. Hard capsules among the health functional foods in the form of capsules may be prepared by filling a conventional hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and the soft capsule agent may include the active ingredient of the present invention as an additive such as an excipient. It can be prepared by mixing the mixture with a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary. The health functional food in the form of a cyclic form can be prepared by molding a mixture of the active ingredient and the excipient, the binder, the disintegrant, etc. of the present invention by a known method, and can be avoided with sucrose or other epidermis, if necessary, Alternatively, the surface may be coated with a material such as starch or talc. The health functional food in the form of granules may be prepared by granulating a mixture of an excipient, a binder, a disintegrant, and the like of the active ingredient of the present invention in a known manner, and may contain a flavoring agent, a coagulant, etc. as necessary. Can be.

In addition, the present invention provides a cosmetic composition for moisturizing skin comprising a fraction obtained by sequentially fractionating water, hexane and chloroform from the fluorinated alcohol extract.

In addition, the present invention provides a health functional food composition for skin moisturizing comprising a fraction obtained by sequentially fractionating water, hexane and chloroform from the fluorinated alcohol extract.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising a fraction obtained by sequentially distilling water, hexane and chloroform from the fluorinated alcohol extract as an active ingredient.

In another aspect, the present invention provides a cosmetic composition for improving atopic dermatitis comprising a fraction obtained by sequentially fractionating the alcohol extract of fluorinated water, hexane and chloroform as an active ingredient.

The cosmetic composition, nutraceutical composition or pharmaceutical composition is omitted in order to avoid the excessive complexity of the present specification by the description of the overlapping description of the composition of the present invention described above.

Extracts of Ageratum houstonianum may be those obtained by extraction and separation from nature using extraction and separation methods known in the art. "Extract" as defined in the present invention is extracted from fluorination using a suitable solvent and includes, for example, both crude extracts, polar solvent soluble extracts or nonpolar solvent soluble extracts. As a suitable solvent for extracting the extract from the fluoride, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used, but is not limited thereto. For example, purified water and methanol alcohols having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform, including methanol, ethanol, propanol, isopropanol, butanol, and the like (chloroform), ethyl acetate (ethyl acetate), methylene chloride (methylene chloride), hexane (hexane) and various solvents such as cyclohexane (cyclohexane) may be used alone or in combination. As the extraction method, any one of hot water extraction method, cold leaching extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be used. In addition, the desired extract may further be subjected to a conventional fractionation process, it may be purified using conventional purification methods.

There is no limitation on the preparation method of the extract of the present invention, any known method may be used. For example, the extract included in the composition of the present invention may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying, which are extracted by the hot water extraction or solvent extraction. The primary extract may be further purified using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, and the like. You can also get Therefore, in the present invention, the extract is a concept including all the extracts, fractions and purified products obtained at each step of extraction, fractionation or purification, their dilutions, concentrates or dried products.

In addition, the present invention provides a method for preventing and treating atopic dermatitis, comprising administering to a subject a pharmaceutically effective amount of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

The present invention also provides a method for preventing and treating atopic dermatitis, comprising administering to a subject a pharmaceutically effective amount of a fraction obtained by sequentially dividing water, hexane, and chloroform from an alcoholic extract of fluoride.

The pharmaceutical composition of the present invention is administered in a therapeutically effective amount or in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, drug Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.

In addition, the present invention provides a method for enhancing skin moisturizing comprising topically applying to the individual an effective amount of a skin moisturizing cosmetic composition comprising a compound represented by the formula (1).

In addition, the present invention provides a method for enhancing skin moisturizing, comprising topically applying to the individual an effective amount of a skin moisturizing cosmetic composition comprising a fraction obtained by sequentially fractionating water, hexane and chloroform from an fluorinated alcohol extract. to provide.

As used herein, the term "effective amount" means an amount sufficient to enhance the moisturization of the skin at a reasonable benefit / risk ratio applicable to cosmetics, the effective dose level is the type of individual and the degree of dry skin, the degree of skin damage by drying, Age, sex, activity of the active ingredient, sensitivity to the active ingredient, the time of application, the duration of the application, factors including other active ingredients used at the same time and other factors well known in the cosmetic field.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1 Experimental Method

1.1. Preparation of Fluoride Extracts and Fractions

Ageratum houstonianum plants were dried to obtain a dry weight of 1,248 g, and soaked for 3 days with the same amount of ethanol. After filtration, the solvent was distilled off under reduced pressure distillation to obtain 150.9 g of ethanol extract (AHE; Ageratum houstonianum Ethanol Extract). The yield was 12.09%.

120 g of fluorinated-ethanol extract (AHE) was subjected to liquid-liquid separation depending on polarity. The same amount of hexane and water was added to separate the layers to obtain a hexane layer (17.63 g, 14.69%) and a chloroform layer (6.7 g, 5.58%). Separation of the fluorinated extract according to the polarity was performed as shown in Figure 3A.

1.2. Component Separation Using Liquid Chromatography and Molecular Structure Analysis Using Nuclear Magnetic Resonance Spectroscopy

In order to separate the active material of the hexane layer, preparative liquid chromatography was performed using prep-HPLC, Agilent 1100 Series and LunaC18 column (10 × 250mm, 5㎛). The mobile phase was acetonitrile / methanol / Water (v / v 30:50:20) at 30 ℃, the moving speed was 3.0 mL / min. The column injection sample volume was 50 μL and was detected at 230 nm using a potodiode array detector. All samples used for preparative liquid chromatography were dissolved in isopropanol.

In order to identify the structure of Agerarin, an active substance, a sample was dissolved in CDCl _{3 and placed} in a 2.5-mm nuclear magnetic resonance spectrometer to carry out nuclear magnetic resonance spectroscopy. The instrument used was run at room temperature with a Bruker AVANCE 400 spectrometer system (9.4 T; Bruker, Karlsruhe, Germany). Detailed experimental methods were performed following the method published in the paper (Magn Reson Chem 51: 364).

1.3. Culture of skin keratinocytes

Human keratinocytes (HaCaT) were purchased from the American Type CultureCollection (ATCC) and contained 10% FBS (Fetal Bovine Serum, Invitrogen Life Technologies) and Antibiotic-Antimycotic solution (Invitrogen Life Technologies) cultures with DMEM (Invitrogen Life Technologies) Cultured in a 37%, 5% CO ₂ incubator while passaged to a seed density of 1 x 10 ⁶ in a 100-mm cell culture dish once every two days using.

1.4. Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted using TRIzol RNA Isolation Reagents (QIAGEN). The double-stranded cDNA using reverse transcription was synthesized by using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's method. PCR was performed with 0.0125 μg double helix cDNA. Primer base sequences for gene amplification are shown in Table 1 below. As a control group, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), a housekeeping gene, was used. PCR denatured DNA at 95 ° C., 5 min, then 94 ° C., 1 min; 65 ° C., 2 minutes; And 30 cycles of 70 DEG C for 1 minute of reaction. PCR final product was electrophoresed on 1% agarose gel and confirmed by staining with EtBr (ethidium bromide).

TABLE 1

1.5. Quantitative Real-time polymerase chain reaction (QRT-PCR)

Quantitative Real-time polymerase chain reaction (QRT-PCR) is a quantitative experimental method based on polymerase chain reaction. That is, by measuring the amplification degree and the expression amount of the target gene at the same time it is a method that can measure the exact mRNA amount. After cDNA was synthesized in the same manner as in Example 1.4, PCR was performed using TaqMan-iQ supermixkit (Bio-Rad). TaqMan probe sequence for measuring aquaporin-3 (AQP3) mRNA was 5'-6-FAM-CCCTTCACGATCCACCCTTTCA-BHQ-1-3 '(SEQ ID NO: 7), and the TaqMan probe sequence used for control GAPDH mRNA was determined. The experiment was carried out as 5'-Yakima YellowTM-CGTCGCCAGCCGAGCCACATCGC-BHQ-1-3 '(SEQ ID NO: 8).

1.6. Immunoblotting

Antibodies against glyceraldehyde phosphate dehydrogenase (GAPDH) and aquaporin-3 (AQP3) proteins were purchased from BOSTER (Pleasanton, Calif.). Cultured HaCaT cells were harvested and harvested at 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 µg / μl Leupeptin, 1 mM PMphenyl (phenylmethylsulfonyl fluoride After lysing the cells with the buffer solution containing), only the cell lysate was obtained by high-speed centrifugation. The amount of protein in the cell lysate was analyzed using Pierce BCA Protein Assay Reagent (Thermo Scientific, Rockford, IL, USA). Samples prepared to contain the same amount of protein (10 ~ 20 ㎍) was subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamidegel) electrophoresis to separate the proteins present in the cells. After transferring the protein separated by electrophoresis to a polystyrene membrane and reacting with aquaporin-3 (AQP3) and GAPDH primary antibody for 5 hours, the secondary antibody recognizing the primary antibody (Cell Signaling Technology) ) Was reacted for 1 hour. Protein changes were analyzed using an Enhanced Chemiluminescence System (Amersham Pharmacia Biotechnology).

1.7. Immunofluorescence microscopy

HaCaT cells were cultured in cover glass, treated with fluorinated extracts for 24 hours, and then fixed with the addition of 4% formaldehyde (formaldehyde). 0.1% Triton X-100 solution containing 2% bovine serum albumin was added to allow the antibody to pass through the cell membrane. The primary antibody binding to the aquaporin-3 (AQP3) protein was reacted for 90 minutes and for 30 minutes with the secondary antibody (Invitrogen, Carlsbad, CA, USA) to which the red phosphor Alexa-Fluor 555 was bound. The cover glass was washed with PBS buffer for 10 minutes and added using reagent Hoechst 33258 (Invitrogen) to selectively stain DNA with blue fluorescence, and then observed using an EVOS FL fluorescence microscope (Life Technologies, Carlsbad, CA, USA).

1.8. Inhibition of CLOCK gene expression using shRNA (small hairpin RNA)

HaCaT cells were cultured in a 60-mm cell culture dish and inoculated with lentiviral particles (Sigma) expressing shRNA for the CLOCK gene at a 50-80% seed density. After 24 hours, incubated for 10-14 days in a culture medium containing 2 μg / ml of puromycin, colonies that inhibited CLOCK gene expression were selectively used for the experiment.

Example 2 Effect of Increased Expression of Aquaporin-3 (AQP3) by Fluorinated-Ethanol Extract (AHE)

In the prior patents of the present inventors, aquaporin-3 (AQP3) by fluorinated-methanol extract It was confirmed that the expression level of the gene is increased (Korean Patent Publication No. 10-2014-0070192). In the present invention, it was confirmed that the expression level of aquaporin-3 (AQP3) gene by fluorinated-ethanol extract (AHE) was increased.

Specifically, HaCaT cells were treated with fluorinated-ethanol extracts (AHE) at 0, 5, 10, and 20 μg / ml concentrations for 24 hours, and total RNA was isolated to perform RT-PCR. As a result, it was confirmed that fluorinated-ethanol extract (AHE) increased the mRNA amount of aquaporin-3 (AQP3) in a concentration-dependent manner (FIG. 1A).

In addition, in order to analyze time-dependent reactivity, cells were harvested with the group treated without treatment (0 h) after 6, 12, or 24 hours of fluorinated-ethanol extract (AHE) at a concentration of 20 µg / ml. Afterwards, total RNA was isolated and RT-PCR was performed. As a result, when treated with fluorinated-ethanol extract (AHE), it was confirmed that the mRNA amount of aquaporin-3 (AQP3) began to increase within 6 hours after the treatment and continued to increase until 24 hours after the treatment (FIG. 1B). .

In addition, the present inventors performed an immunoblot experiment to confirm whether the expression level of aquaporin-3 (AQP3) protein by fluorinated-ethanol extract (AHE) is also changed. As a result, the expression of aquaporin-3 (AQP3) protein was higher in HaCaT cells than in the group without treatment (0 h) when treated with 20 μg / ml fluorinated-ethanol extract (AHE) for 12 and 24 hours. It was confirmed that the amount increased (Fig. 1C).

Therefore, it was confirmed that fluorinated-ethanol extract (AHE) increased the expression level of aquaporin-3 (AQP3) gene and protein in human keratinocytes.

Example 3 Recovery Effect of Aquaporin-3 (AQP3) Expression Reduced by Ultraviolet Irradiation of Fluorinated-Ethanol Extract (AHE)

Exposure to the skin of UV-B (UVB) reduces the expression of the Aquaporin-3 (AQP3) gene, which reduces skin moisturization and promotes skin aging (J. Cell. Physiol. 2008; 215: 506-516). We investigated whether fluorinated-ethanol extract (AHE) can affect aquaporin-3 (AQP3) gene expression, which is reduced by UVB irradiation. HaCaT cells were either irradiated with UVB (30 J / m ² ) alone, or pre-treatment with fluorinated-ethanol extract (AHE) at various concentrations (5, 10, 20 μg / ml) for 30 minutes. After irradiating UVB, cells were harvested 24 hours later. The expression level of aquaporin-3 (AQP3) mRNA was analyzed by RT-PCR and QRT-PCR. As a result, fluorinated-ethanol extract (AHE) recovered the aquaporin-3 (AQP3) mRNA expression level reduced by UVB in a concentration-dependent manner (Figs. 2A and 2B). In particular, the treatment of ethanol extract (AHE) with a concentration of 10 μg / ml or more was confirmed that the effect of expressing more aquaporin-3 (AQP3) mRNA than the control group not treated with UVB.

In addition, the present inventors were pre-treatment of fluorinated-ethanol extract (AHE) at a concentration of 20 μg / ml for 30 minutes, and then irradiated with UVB, and harvested cells after 24 hours to obtain aquaporin by immunoblot. The expression level of -3 (AQP3) protein was confirmed. As a result, it was confirmed that the aquaporin-3 (AQP3) protein reduced by UVB showed a similar protein expression level as the control group not treated with UVB (FIG. 3C).

The inventors analyzed the effects of aquaporin-3 (AQP3) protein production by fluorinated-ethanol extract (AHE) at the cellular level using fluorescence microscopy. Incubate HaCaT cells in cover glass and irradiate with 30 J / m ² UVB alone or pre-treatment with 20 μg / ml fluorinated-ethanol extract (AHE) 30 minutes before UVB irradiation. UVB was irradiated, and after 24 hours, aquaporin-3 (AQP3) protein distribution was analyzed by fluorescence microscopy. As a result, when the UVB was irradiated to the cells, the cells showing red fluorescence was reduced, red fluorescence appeared again in the cells treated with fluorinated-ethanol extract (AHE). As a result, it was confirmed that fluorinated-ethanol extract (AHE) recovered the aquaporin-3 (AQP3) protein expression level reduced by UVB.

Example 4 Analysis of Aquaporin-3 (AQP3) Expression Effects of Hexane and Chloroform Fractions Isolated from Fluorinated-Ethanol Extract (AHE)

In order to identify active ingredients that promote aquaporin-3 (AQP3) gene expression in fluorinated-ethanol extract (AHE), the hexane and chloroform fractionation layers in fluorinated-ethanol extract (AHE) were prepared as in Example 1.1. Isolated (FIG. 3A). The inventors confirmed through RT-PCR whether the fraction changed the amount of aquaporin-3 (AQP3) mRNA expression reduced by UVB. As a result, there was no difference in the chloroform fraction compared to the control group, but it was confirmed that the expression of aquaporin-3 (AQP3) mRNA decreased by UVB was significantly increased in the fluorinated-ethanol extract (AHE) and the hexane fraction ( 3B).

In addition, the inventors conducted an experiment to see if the fluorinated-hexane fraction (AHE-Hx) promoted aquaporin-3 (AQP3) gene expression. HaCaT cells were treated with 20 μg / ml fluorinated-hexane fractions (AHE-Hx) for 24 hours and analyzed for expression of aquaporin-3 (AQP3) mRNA via RT-PCR. As a result, it was confirmed that the fluorinated-hexane fraction (AHE-Hx) increased the expression level of aquaporin-3 (AQP3) mRNA similarly to the case of treatment with the fluorinated-ethanol extract (AHE) (FIG. 3C). .

Example 5 Assay for Active Material Isolated from Fluorinated-Hexane Fraction (AHE-Hx)

Separation of the active substance from the hexane fraction was carried out via preparative liquid chromatography experiment as in Example 1.2 above. As a result, the peak of the retention time of 8.6 minutes was the most quantitative to separate the material (FIG. 4A). To confirm whether the separated material was a single material, a three-dimensional chromatogram was performed with a photodiode array detector to confirm that it was a single material (FIG. 4B). The inventors have named the isolated single substance 'Agerarin'.

Example 6 Effect of Increased Expression of Aquaporin-3 (AQP3) by Agerarin

The inventors analyzed the aquaporin-3 (AQP3) gene expression changes for Agerarin of Example 5. HaCaT cells were treated with 20 μg / ml fluorinated-ethanol extract (AHE) or 20 μg / ml agerarin for 24 hours, after which the cells were harvested to separate total RNA and aquaporin via RT-PCR. MRNA expression level of -3 (AQP3) was measured. As a result, it was confirmed that the mRNA expression level of aquaporin-3 (AQP3) was increased when treated with fluorinated-ethanol extract (AHE) and agerarin (Agerarin) (FIG. 5A).

In addition, in order to quantitatively analyze aquaporin-3 (AQP3) mRNA expression by Agerarin via QRT-PCR, 20 μg / ml fluorinated-ethanol extract (AHE) or azera in HaCaT cells. The mRNA expression level of aquaporin-3 (AQP3) was compared for 24 hours of lean treatment. As a result, the mRNA expression level of aquaporin-3 (AQP3) was increased by 3.2 times compared to the control group when treated with fluorinated-ethanol extract (AHE), and compared with the control group when treated with agerarin. It was confirmed that it increased 7.2 times (FIG. 5B).

In addition, we analyzed whether Agerarin restores aquaporin-3 (AQP3) mRNA expression, which is reduced by UVB irradiation. HaCaT cells were irradiated with UVB (30J / m ² ) alone or UVB after 30 minutes of pre-treatment of fluorinated-ethanol extract (AHE) and Agerarin at a concentration of 20 μg / ml After 24 hours, cells were harvested to perform RT-PCR (FIG. 5C) and QRT-PCR (FIG. 5D). As a result, it was confirmed that agerarin restored the expression level of aquaporin-3 (AQP3) mRNA reduced by UVB more significantly than when treated with fluorinated-ethanol extract (AHE) (FIG. 5C). And 5D).

Example 7 Structural Identification of Agerarin

The present inventors analyzed the molecular structure by using a nuclear magnetic resonance spectroscopy as in Example 1.2. To identify the structure of Agerarin. A total of 12 peaks were observed in the carbon-nuclear magnetic resonance spectroscopy experiment spectrum (FIG. 6A), and seven peaks were observed in the hydrogen-nuclear magnetic resonance spectroscopy experiment spectrum (FIG. 6B). Agerarin was found to be composed of three quartet carbons, four doublet carbons, and five singlet carbons from a distortionless enhancement by polarization transfer (DEPT) experiment (FIG. 6C). The two carbon peaks observed at 56.1 and 56.7 ppm were determined to be methoxy groups because the heteronuclear multiple quantum coherence (HMQC) experiments revealed that they were bound to 3.80 and 3.78 ppm hydrogen, respectively (FIG. 6D). In addition, since the hydrogen peak at 1.38 ppm was directly bonded to 27.8 ppm carbon peak, it was determined to be a methyl group, and the area of the hydrogen peak was determined to have two methyl groups, so agerarin had two methyl groups. Was determined. Since the remaining seven carbon peaks and four hydrogen peaks are similar to the results of nuclear magnetic resonance spectroscopy of the chromene skeleton, Agerarin is considered to have a chromene skeleton and is described in the paper (Magn Reson Chem 50: 759). Comparison with the data confirmed the structure of Agerarin.

Agerarin contains 2 H- chromenone from the interpretation of secondary nuclear magnetic resonance experiments, TOCSY (total correlated spectroscopy, FIG. 7A), COSY (correlated spectroscopy, FIG. 7B), and heteronuclear multiplebonded connectivities (FIG. 7C). It was decided to. The result of determining the connection between carbon and hydrogen from the COSY and HMBC experiment results is shown in FIG. 7D. The carbon and hydrogen peaks obtained as a result of the nuclear magnetic resonance spectroscopy experiment are summarized as shown in Table 2.

TABLE 2

From the above results, azelaic Lin (Agerarin) is 6,7-dimethoxy-2,2-dimethyl- 2 H -chromene - have been identified as (6,7-dimethoxy-2,2-dimethyl -2 H-chromen), The structure is shown in the following formula (1).

[Formula 1]

The molecular formula of Agerarin was finally determined to be C ₁₃ H ₁₆ O ₃ and molecular weight 220.1099. To reconfirm the structure, high-resolution mass spectrometry was performed using ultra-performance liquid chromatography-hybrid quadrupole-time-of-flightmass spectrometry (UPLC-TOFMS) equipped with a Waters ACQUITY UPLC system (Waters, Milford, Mass.). As a result, the peak of [M + H] was observed at 221.5345 and the theoretical value was 221.1178, confirming that the structure of AG-H-1 is correct (FIG. 7E).

Example 8. Effect of Increased CLOCK Gene Expression by Agerarin

Aquaporin-3 (AQP3) is known to be expressed in a 24-hour cycle by the Circadian Locomotor Output Cycles Kaput (CLOCK) gene, a circadian clock regulation gene (Journal of Investigative Dermatology 2014; 134: 1636-1644). Indeed, Example 1.8 above. Inhibiting CLOCK gene expression by injecting shRNA (small hairpin RNA) to the CLOCK gene (shCLOCK), as compared with the control shRNA (shCont) -expressing cells, the amount of aquaporin-3 (AQP3) mRNA expression is significantly Confirmed inhibition (FIG. 8A).

From the above results, the CLOCK gene plays an important role in aquaporin-3 (AQP3) gene expression. In the present invention, an experiment was performed to determine whether agerarin changes the expression of the CLOCK gene. HaCaT cells were treated with Agerarin at a concentration of 20 μg / ml for 6, 12, or 24 hours, and then harvested with the untreated group (0 h). Was performed. As a result, it was confirmed that when treated with agerarin (Agerarin), the mRNA expression level of CLOCK began to increase within 6 hours after treatment and gradually decreased after 24 hours of treatment (FIG. 8B).

In addition, QRT-PCR was performed to quantitatively measure the amount of CLOCK mRNA increased by Agerarin. As a result, when Agerarin at 20 μg / ml was treated to HaCaT cells, the amount of CLOCK mRNA was 2.5 and 3.8 times higher after 6 and 12 hours, respectively, compared to the group without any treatment (0 h). After 24 hours, it was increased slightly and increased 1.8 times compared to the control (Fig. 8C).

The immunoblot was performed to confirm whether the expression of CLOCK protein by Agerarin also changes. As a result, when Hagerain cells were treated with Agerarin at a concentration of 20 μg / ml for 6, 12, or 24 hours, it was significantly increased after 6 and 12 hours as in the change of mRNA expression level, compared to the control group. After 24 hours, it increased compared to the control group, but showed a slightly decreased expression compared to 12 hours (FIG. 8D).

Therefore, agerarin (Agerarin) was confirmed to increase the expression level of the CLOCK gene and protein, a biological clock control gene in human keratinocytes.

Example 9.Aquaporin-3 (AQP3) Gene Expression Effect through Agerarin's CLOCK Gene

To determine whether CLOCK gene expression by Agerarin is directly related to aquaporin-3 (AQP3) gene expression, agararin-induced aqua in a cell with low CLOCK gene expression using shRNA. The effect of porin-3 (AQP3) gene expression was analyzed.

HaCaT cells expressing the shRNA (shCLOCK) and the control shRNA (shCont) of the CLOCK gene were treated with 20 μg / ml of agerarin for 6 hours and harvested together with untreated cells, and then total RNA. RT-PCR was performed by separation. As a result, the amount of mRNA expression of CLOCK was increased by agerarin in the control shRNA-expressing cells, but in the cells expressing shRNA (shCLOCK) of the CLOCK gene, even when treated with agerarin, CLOCK MRNA expression was not increased (Fig. 9A). Similar results were also confirmed in QRT-PCR (FIG. 9B).

To determine whether the increase in aquaporin-3 (AQP3) gene expression by agerarin is dependent on the CLOCK gene, a concentration of 20 μg / ml in cells expressing the control shRNA (shCont) and shRNA of the CLOCK gene (shCLOCK) are expressed. After 24 hours treatment with agerarin (Agerarin), the amount of aquaporin-3 (AQP3) mRNA was analyzed.

When the RT-PCR method was performed, the amount of aquaporin-3 (AQP3) mRNA was increased by agerarin in the control shRNA (shCont) -expressing cells, but the shRNA (shCLOCK) of the CLOCK gene was expressed. In the treatment of agerarin (Agerarin) did not increase the amount of aquaporin-3 (AQP3) mRNA (Fig. 9C). In addition, QRT-PCR was performed to quantitatively measure the expression level of aquaporin-3 (AQP3) mRNA. As a result, Agerarin treatment resulted in a 3.0-fold increase in aquaporin-3 (AQP3) mRNA in HaCaT cells expressing control shRNA (shCont), whereas in cells expressing shRNA (shCLOCK) for the CLOCK gene. Under no treatment, aquaporin-3 (AQP3) mRNA levels were reduced by 40% compared to control cells (0.6-fold), and treatment with agerarin increased only 1.2-fold compared to control cells.

Thus, agerarin (Agerarin) was confirmed to increase the expression of aquaporin-3 (AQP3) gene by the CLOCK gene (Fig. 10).

Claims (17)

Skin moisturizing cosmetic composition comprising a compound represented by the formula (1) as an active ingredient: [Formula 1]

. The method of claim 1, The compound represented by Chemical Formula 1 is 6,7-dimethoxy-2,2-dimethyl-2 H -chromen composition. The method of claim 1, The compound is characterized in that the aquaporin-3 (aquaporin-3, AQP3) or a composition characterized in that to increase the expression of mRNA or protein of CLOCK. A health functional food composition for skin moisturizing comprising the compound represented by Formula 1 as an active ingredient: [Formula 1]

. A pharmaceutical composition for preventing or treating atopic dermatitis comprising the compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Formula 1]

. A cosmetic composition for improving atopic dermatitis comprising a compound represented by the formula (1) as an active ingredient: [Formula 1]

. Fire screen (Ageratum houstonianum) of skin-friendly cosmetic composition comprising a fraction obtained is fractionated sequentially with water, hexane and chloroform from the alcohol extract. The method of claim 7, wherein The alcohol is a composition, characterized in that ethanol. The method of claim 7, wherein Wherein said fraction is a hexane fraction. The method of claim 7, wherein The fraction is aquaporin-3 (Aquaporin-3, AQP3) or composition characterized in that to increase the expression of mRNA or protein of CLOCK. A health functional food composition for skin moisturizing comprising fractions obtained by sequentially fractionating water, hexane and chloroform from an alcoholic extract of fluorinated. A pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising a fraction obtained by sequentially distilling water, hexane and chloroform from the fluorinated alcohol extract as an active ingredient. A cosmetic composition for improving atopic dermatitis, comprising a fraction obtained by sequentially distilling water, hexane and chloroform from an alcoholic extract of fluoride as an active ingredient. A method for preventing and treating atopic dermatitis, comprising administering to a subject a pharmaceutically effective amount of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. [Formula 1]

. A method for preventing and treating atopic dermatitis, comprising administering to a subject a pharmaceutically effective amount of a fraction obtained by sequentially fractionating water, hexane and chloroform from an alcoholic extract of fluorinated. Skin moisturizing enhancement method comprising the step of topically applying to the individual in an effective amount of a skin moisturizing cosmetic composition comprising a compound represented by the formula (1). [Formula 1]

. A method of enhancing skin moisturizing comprising topically applying to the individual an effective amount of a skin moisturizing cosmetic composition comprising a fraction obtained by sequentially fractionating water, hexane and chloroform from an alcoholic extract of fluorinated.

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