KR20150102449A - Composition comprising an extract of crude drug for treating and improving skin-aging or skin trouble - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for external use of the skin or a cosmetic composition containing extracts of Glycyrrhiza uralensis Fischer, Coptis chinensis, or Forsythia suspensa Vah as an active components for treating and alleviating skin aging or dermatitis.

Description

TECHNICAL FIELD The present invention relates to a composition for treating and improving skin aging and dermatitis,

The present invention relates to a dermatological pharmaceutical composition and a cosmetic composition for treating or ameliorating skin aging or dermatitis containing licorice, chrysanthemum or licorice extract as an active ingredient.

[1] Voegeli, R. 1996. Elastase and typing determination on human skin surface. Cosmetic & Toiletries. 111, 51-58

[2] Kim HH, Cho S, Lee S, Kim KH, Cho KH, Eun HC, Chung JH, Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo. J Lipid Res . 47 (5) , pp.921-30, 2006;

[3] Medina A, Ghaffari A, Kilani RT, Ghahary A., The role of stratifin in fibroblast-keratinocyte interaction. Mol Cell Biochem . 305 (1-2) , pp. 255-64, Review, 2007.

[4] Wang XJ, Han G, Owens P, Siddiqui Y, Li AG., Role of TGF beta-mediated inflammation in cutaneous wound healing. J Investig Dermatol Symp Proc . 11 (1) , pp. 112-7, Review, 2006;

[5] Traidl-Hoffmann C, MI, Ring J, Behrendt H., Impact of desloratadine and loratadine on the crosstalk between human keratinocytes and leukocytes: Implications for anti-inflammatory activity of antihistamines. Int Arch Allergy Immunol . 140 (4) , pp.315-20, 2006

[6] Cannon JG, Tatro JB, Reichlin S, Dinarello CA., Alpha melanocyte stimulating hormone inhibits immunostimulatory and inflammatory actions of interleukin 1. J Immunol . 137 (7) , pp.2232-6, 1986

[7] Yamamoto T, Eckes B, Mauch C, Hartmann K, Krieg T., Monocyte chemoattractant protein-1 enhancer gene expression and synthesis of matrix metalloproteinase-1 in human fibroblasts by an autocrine IL-1 alpha loop. J Immunol . 164 (12) , pp.6174-9, 2000;

[Patent Literature 8] Dai G, Freudenberger T, Zipper P, Melchior A, Grether-Beck S, Rabausch B, de Groot J, Twarock S, Hanenberg H, Homey B, Krutmann J, Reifenberger J, Fischer JW., Chronic ultraviolet B Hyaluronic acid synthase is the down-regulation of hyaluronic acid synthases. Am J Pathol . 171 (5) , pp.1451-61, 2007

[Literature 9] Information Supervision, Shin Min-gyo, Dae-hae Encyclopedia, Young Lim, pp684-687, 1998

[Reference 10] Metaphorical analysis of information and communication, Yeonglim, p491-493, 1998

[Literature 11] Information on the Interview with Professor Lee, Young Lim, p974-975, 1998

[Literature 12] Blois MS. Antioxidant determination by the use of a stable free radical. Nature . 1958; 26: 1199-1120

[Document 13] Amariwicz, R., Naczk, M., Shahidi, F. (2000). Antioxidant activity of various fractions of non-tannic phenolics of canola hulls. J. of Agric Food Chem. 48: 2755-2759 [Literature 14] Roberta, R. E., Pellegrini, N., Anna, P., Ananth, P., Yang, M., Catherine, R. E., (1999). Antioxidant activity was improved by ABTS radical cation decolorization assay. J. of Free Radical Biology & Medicine. 26: 1231-1237

[Document 15] Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur . J. Biochem . 1974; 47: 469-474

[Literature 16] Pryor WA. Oxy-radicals and related species: their formation, lifetimes, and reactions. Annu . Rev. Physiol . 1986; 48: 657-667

[Literature 17] Greenstock CL. Radiation and aging: free radical damage, biological response and possible antioxidant intervention. Med . Hypotheses . 1993; 41 (5): 473-482

[Literature 18] Marcocci L., Maguire J. J., Droylefaix M. T. and Packer L.: The Nitric Oxide Scavenging Properties of Ginkgo Biloba Extract EGb 761. Biochemical and Biophysical Research Communications. 201, 15, 748-755, 1994

[Literature 19] Sreejayan, N. and Rao, M. NA. : Nitric oxide scavenging by curcuminoids. Journal of Pharmacy and Pharmacology, 49, 105-107, 1997

[20] Han HK, Choi SS, Shin JC <Chung HS (2007) A study on single oral dose toxicity of highly anthocyanin-pigmented rice varieties. J. Kor. Soc. Food Sci. Nutr. 36: 527-533

[21] Kim SJ, Lee EJ, Song YS, Jeong HJ, Le KM, Kim HR, Chae HJ, Shin TY, Kim YK, Hong SH, Kim HM. Cheongyol saseuptang inhibits production of TNF-alpha, IL-6 and IL-8 as well as NF-kappa B activation in human mast cells. J Ethnopharmacol. 2005; 97: 83-8

[22] Kim HM, Park YA, Lee EJ, Shin TY. Inhibition of immediate-type allergic reation by Rosa davurica Pall. In a murine model. J Ethnophamacol. 1999; 67: 53-60

Modern people promote skin aging phenomena such as spots, freckles and skin pigmentation by inducing various skin troubles due to various internal and external factors such as ultraviolet rays and stress (Voegeli, R. 1996. Elastase and typing determination on human skin surface. Cosmetic & Toiletries. 111, 51-58.).

The increase of ultraviolet rays and the increase of various oxidizing substances due to the destruction of the environment can cause skin damage and deterioration of the total performance of proteins. Such damage or deterioration of the skin cells causes a decrease in skin elasticity, an increase in skin wrinkles, and a lethal phenomenon in skin cosmetics such as stain and freckle formation. Skin elasticity is maintained by collagen, elastin, hyaluronic acid, and fibroblast, which synthesizes collagen (1). Fibroblast function is regulated not only by various growth factors but also by cytokines secreted from skin cells such as keratinocytes and melanocytes (2-3). TGF-β, TβRII, and SGad3 of keratinocytes liberated under normal conditions increase the expression of procollagen, fibrillin-1, and tropoelastin from dermal fibroblasts, resulting in collagen production , And suppression of collagen degradation by inhibiting the expression of MMPs (Kim HH, Cho S, Lee S, Kim KH, Cho KH, Eun HC and Chung JH, Photoprotective and anti-skin-aging effects of eicosapentaenoic acid .. acid in human skin in vivo J Lipid Res 47 (5), pp.921-30, 2006;. Medina A, Ghaffari A, Kilani RT, Ghahary A., The role of stratifin in fibroblast-keratinocyte interaction Mol Cell Biochem . 305 (1-2) , pp. 255-64, Review, 2007.).

In addition to TGF-beta (transforming growth factor), tumor necrosis factor (TNF-alpha), PGE ₂ (prostaglandin E ₂ ), α-MSH (melanocyte stimulating hormone), GSF (granulocyte stimulating factor), IL-1 (interleukin-1), IL-6, IL-8 and IL-10 XJ, Han G, Owens P, Siddiqui Y, Li AG., Role of TGF beta-mediated inflammation in cutaneous wound healing. J Investig Dermatol Symp Proc . 11 (1) , pp. 112-7, Review, 2006; Traidl-Hoffmann C, MI, Ring J, Behrendt H., Impact of desloratadine and loratadine on the crosstalk between human keratinocytes and leukocytes: Implications for anti-inflammatory activity of antihistamines. Int Arch Allergy Immunol. 140 (4) , pp.315-20, 2006.). Both α-MSH and PGE ₂ stimulate melanocytes to promote melanogenesis, which enhances the antioxidant action of skin cells and prevents skin cells from being damaged by ultraviolet rays (Cannon JG, Tatro JB, Reichlin S, Dinarello CA., Alpha melanocyte stimulating hormone inhibits immunostimulatory and inflammatory actions of interleukin 1. J Immunol . 137 (7) , pp.2232-6, 1986.).

IL-1 and TNF-α (tumor necrosis factor) act on fibroblasts to inhibit procollagen expression and inhibit MMP-1 (martrix metalloproteinase-1), MMP-2, MMP-3, hyaluronidase And the collagen degradation is accelerated (Yamamoto T, Eckes B, Mauch C, Hartmann K, Krieg T., Monocyte chemoattractant protein-1 enhancer gene expression and synthesis of matrix metalloproteinase-1 in human fibroblasts by an autocrine IL .. -1 alpha loop J Immunol 164 (12), pp.6174-9, 2000; Dai G, Freudenberger T, Zipper P, Melchior A, Grether-Beck S, Rabausch B, de Groot J, Twarock S, Hanenberg H , Homey B, Krutmann J, Reifenberger J, Fischer JW., Chronic ultraviolet B irradiation causes loss of hyaluronic acid from mouse dermis because of down-regulation of hyaluronic acid synthases. Am J Pathol. 171 (5) , pp.1451-61 , 2007.).

In addition, even if fibroblast is directly injured by UV, the collagen-related gene is suppressed and the expression of MMPs related to degradation is promoted and the wrinkles are increased. Thus, increasing the proliferation and collagen synthesis of dermal fibroblasts, or inhibiting MMPs, may be a means of inhibiting the formation of wrinkles.

Glycyrrhizae Radix is a perennial herbaceous plant belonging to the family Leguminosae and grows in the northern regions of Siberia, Mongolia and Inner Mongolia and Gansu in China. Its main components are triterpene saponins, Glycyrrhetic acid, flavonoids, isoflavonoids, cumestan derivatives, hydroxycoumarins, steroids, volatile oil, and the like. , Detoxification action, hepatitis, urticaria, dermatitis, eczema, Jinhae and geomung, muscle relaxation, diuretic action, and anti-inflammatory action. Licorice is a root of licorice belonging to Leguminosae (Glycyrrhiza uralensis) and the relative plants and its components are glycyrrhizin, liquiritigenin, isoliquiritigenin, (Neoisoliquiritigenein), and the detoxification effect, the Jinhae genotyping effect, the antiallergic action, and the gastric secretion inhibitory action are known (Lee, Seong-min, Shin, Min-gyo).

Coptidis chinensis Radix is a dried perennial rootstock of perennial herbaceous perennial herb and belongs to berberine, coptisine, palmatine, and jaterorrhizine. , Hari, vomiting, insecticide, disinfection, etc. (The information dictionary of the information and the survey, Yeonglim, p491-493, 1998).

It is a dried fruit of perennial herb Forsythia koreana which belongs to water bureaucrats. It contains ingredients such as forsythol, matairesinoside, oleanolic acid, alkaloid and the like It is known that it is known as the "Information and Communication Sourcing Contest", Younglim Corporation, p974-975, 1998).

Therefore, the present inventors have conducted extensive studies on the electron donating ability measurement, the ABTS radical cation-scavenging activity assay, the SOD dismutase-like assay, the nitric oxide antioxidant activity tests such as the radical scavenging activity method (Experimental Example 1); An antibacterial experiment (Experimental Example 2) for various strains including the skin uptake bacteria; iNOS, COX-2 expression, iNOS increased by LPS, Western blot assay for COX-2 protein iNOS mRNA expression level, and real time PCR (Experiment 3); Cell viability measurement test by MTT assay (Experimental Example 4); Anti-inflammatory, antimicrobial, and anti-allergic activity through the experiment of measuring anti-allergic activity (Example 5) by the test for suppressing the secretion of histamine, the cytokine measurement experiment of TNF-α and IL-8 Skin aging, dermatitis and the like, which are effective for the prevention of skin aging and skin irritation. The present invention has been completed based on this finding.

In order to accomplish the above object, the present invention provides a dermatological pharmaceutical composition for the treatment and improvement of skin aging or dermatitis, which comprises licorice, chrysanthemum or almond extract as an active ingredient.

The present invention provides a cosmetic composition for the treatment and improvement of skin aging or dermatitis containing licorice, chrysanthemum or licorice extract as an active ingredient.

The extracts as defined herein may be prepared by mixing water, a spirit, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water and a lower alcohol having 1 to 4 carbon atoms, more preferably 50 to 90 % Ethanol mixed solvent.

The dermatological pharmaceutical composition includes a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta agent or a cataplasma formulation.

The cosmetic composition may further comprise at least one selected from the group consisting of hair tonic, hair conditioner, hair essence, hair lotion, hair nourishing lotion, hair shampoo, hair rinse, hair treatment, Hair dye, hair gel, hair glaze, hair dresser, hair lacquer, hair moisturizer, hair dyes, hair dyes, hair dyes, hair dyes, hair dyes, Risers, hair mousses, or hair sprays.

Hereinafter, the method for obtaining the herbal medicine extract of the present invention will be described in detail.

The herbal extract of the present invention is obtained by washing and finely dividing Licorice, Rhodophilus or Salviae respectively and adding purified water of about 1 to 100 times (w / w), preferably 10 to 20 times (w / w) 1 time, including added water, alcohol, C ₁ to C ₄ lower alcohol or a mixed solvent thereof, preferably water and a lower alcohol mixed solvent, and more preferably, 50 to 90% ethanol, a mixed solvent of 1 to 4 carbon atoms For 1 to 10 times, preferably 2 to 5 times at a temperature of 10 to 120 DEG C, preferably 20 to 60 DEG C for 1 to 72 hours, preferably 2 to 24 hours, by cold extraction, hot water extraction, Extraction with a cold extraction method, preferably freeze extraction method, followed by lyophilization and concentration to obtain the extract of licorice, Chinese persimmon or allium herb medicine of the present invention.

The present invention provides an external skin pharmaceutical composition and a cosmetic composition having the skin aging or dermatitis treatment and improvement effect containing the herbal medicine extract obtained by the above production method and the above production method as an effective ingredient.

The inventors of the present invention conducted studies on the electron donating ability measurement, ABTS radical cation-scavenging activity inhibition activity assay, SOD dismutase-like activity assay, assay) and Nitric oxide radical scavenging activity (Experimental Example 1); An antibacterial experiment (Experimental Example 2) for various strains including the skin uptake bacteria; iNOS, COX-2 expression, iNOS increased by LPS, Western blot assay for COX-2 protein iNOS mRNA expression level, and real time PCR (Experiment 3); Cell viability measurement test by MTT assay (Experimental Example 4); Anti-inflammatory, antimicrobial, and anti-allergic activity through the experiment of measuring anti-allergic activity (Example 5) by the test for suppressing the secretion of histamine, the cytokine measurement experiment of TNF-α and IL-8 Skin aging, dermatitis and the like, which are effective for the prevention of skin irritation.

The extract is characterized by containing 0.0001 to 20% by weight, preferably 0.01 to 10% by weight based on the total weight of the composition.

The dermatological pharmaceutical composition of the present invention contains the above extract in an amount of 0.1 to 50% by weight based on the total weight of the composition.

The dermatological pharmaceutical composition containing the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The pharmaceutical composition containing the extract according to the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, external preparations such as syrups and aerosols, and sterilized injection solutions according to a conventional method, , Preferably a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta or a cataplasma. Examples of carriers, excipients and diluents that may be contained in the composition containing the extract include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition for improving whitening and wrinkles containing the extract of the present invention can be applied to external preparations for skin as external preparations for skin, such as cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta or cataplasma Of the present invention can be used as a pharmaceutical composition in the form of external preparation for skin, but the present invention is not limited thereto.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg per day, 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.

The composition containing the extract of the present invention can be variously used for skin aging, cosmetic and cleansing agents for treating and improving dermatitis, and the like. Examples of products to which the present composition can be added include skin lotions, skin softeners, skin toners, astringents, lotions, milky lotions, moisturizing lotions, nutritional lotions, massage creams, nutritional creams, , Cosmetics such as essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, cleanser, soap, treatment, essence and cosmetic pack.

The cosmetic composition of the present invention comprises a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, polymeric polysaccharides, sphingolipids and seaweed extracts.

The water-soluble vitamin is not particularly limited as long as it can be compounded in cosmetics. Preferably, vitamin B, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, And their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium ascorbate-2-phosphate etc.) can also be added to water-soluble vitamins . The water-soluble vitamin can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzymatic method, or a chemical synthesis method.

Usable vitamins include vitamins such as vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol) Alpha-tocopherol vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl laurate, and the derivatives thereof (palmitic acid ascorbin, stearic acid ascorbic acid, dipalmitic acid ascorbin, dl-alpha tocopherol acetate, Ether, etc.) are also included in the usable vitamins used in the present invention. Usability Vitamins can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzyme or a chemical synthesis method.

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. The polymeric peptide can be obtained by a conventional method such as purification from a culture broth of a microorganism, an enzymatic method, or a chemical synthesis method, or it can be purified from natural products such as ducks such as pigs and cows and silk fiber of silkworms.

The polymeric polysaccharide may be any compound as long as it can be incorporated in cosmetics, and examples thereof include hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.). For example, chondroitin sulfate or a salt thereof can be usually purified from mammals or fish.

Sphingo lipids may be any as long as they can be incorporated into cosmetics, and preferable examples thereof include ceramides, phytosphingosine and sphingoglycolipids. Sphingoid lipids can be purified from ordinary mammals, fish, shellfish, yeast or plants by conventional methods or can be obtained by chemical synthesis.

The seaweed extract may be any of those which can be compounded in cosmetics. Preferably, the seaweed extract is selected from the group consisting of algae extract, red pepper extract, green algae extract and the like. Also, the algae extract may be colored guanine, arginic acid, Potassium alginate and the like are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained from seaweed by a conventional method.

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

Examples of the compounding ingredients that may be added include organic solvents such as a preservative component, a moisturizer, an emollient, a surfactant, an organic and inorganic pigment, an organic powder, an ultraviolet absorbent, a preservative, a bactericide, an antioxidant, a plant extract, a pH adjuster, A blood circulation accelerator, a cold agent, an antiperspirant agent, and purified water.

Examples of the oil retaining component include ester-based oil retaining, hydrocarbon-based oil retaining, silicone-based oil retaining, fluoric oil retaining, animal retention and plant retention.

Examples of ester-based fats include glyceryl tri-2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isostearyl isostearate, Butyl isopropyl myristate, isopropyl myristate, isopropyl myristate, isopropyl myristate, isopropyl myristate, isopropyl myristate, butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isosilyl myristate, isostearic acid isostearyl, isostearyl palmitate, octyldodecyl myristate, Trimethylol propane, triisostearic acid trimethylol propane, tetra 2-ethylhexanoic acid pentaerythritol tetra (2-ethylhexanoate) , Decyl caprylate, decyl laurate, hexyl laurate, myristate decyl, myristyl myristate, myristine monoethyl stearate, stearyl stearate, decyl oleate, ricinoleic acid tri , Isostearyl stearate, isostearyl stearate, isodecyl stearate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, isopropyl stearate, isopropyl stearate, isopropyl stearate, -Hexyl stearate, stearyl ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprate, di (capryl, capric acid) propylene glycol, Propyleneglycol propionate, propyleneglycol propionate, dicaproic acid neopentyl glycol, dioctanoic acid neopentyl glycol, tricarboxylic acid glyceryl, triunsaturated glyceryl, triisopalmitic acid glyceryl, triisostearic acid glyceryl, neopentanoic acid octyldodecyl Octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, octanoic acid octanoate, Octyldecyl lactate, octyldecyl lactate, octyldecyl lactate, polyglycerin oleic acid ester, polyglycerin isostearic acid ester, triisocetyl citrate, triisobutyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, But are not limited to, ethyl, acetyltriethyl citrate, acetyltributyl citrate, trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di-2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, But are not limited to, dioctyl sebacate, stearic acid cholesteryl, isostearic acid cholesteryl, hydroxystearic acid cholesteryl, oleic acid cholesteryl, oleic acid dihydrocholesteryl, isostearic acid pitostearyl, Stearoyl hydroxystearic acid isostearyl, 12-stearoyl stearyl hydroxystearate, 12-stearo And monohydroxystearic acid and esters such as sostearyl.

Examples of the hydrocarbon hydrocarbon-based fats include hydrocarbon fats and oils such as squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, floating isoparaffin, polybutene, microcrystalline wax and vaseline.

Examples of silicone based oils include polymethyl silicone, methylphenyl silicone, methyl cyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane-methylcetyloxysiloxane copolymer, dimethylsiloxane-methylstarchoxysiloxane copolymer, alkyl Modified silicone oils, and amino-modified silicone oils.

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

Examples of animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, new flower oil, soybean oil, corn oil, rape oil, apricot kernel oil, palm kernel oil, palm oil, castor oil, , Corn oil, palm oil, palm oil, cucumber nut oil, wheat germ oil, rice germ oil, shea butter, coltsfoot colostrum, marker daisy nut oil, mead home oil, egg oil, , Canned wax, carnauba wax, liquid lanolin, hardened castor oil, and the like.

Examples of the moisturizing agent include water-soluble low-molecular moisturizing agents, oil-soluble molecular moisturizing agents, water-soluble polymers, and oil-soluble polymers.

Examples of the water-soluble low-molecular moisturizing agent include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B Glycol (polymerization degree n = 2 or more), polyglycerin B (polymerization degree n = 2 or more), lactic acid, lactic acid salt and the like.

Examples of the lipid-soluble low-molecular moisturizing agent include cholesterol and cholesterol ester.

Examples of the water-soluble polymer include carboxyvinyl polymer, polyaspartic acid, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, dextrin, etc. .

Examples of the oil-soluble polymer include polyvinylpyrrolidone / eicosene copolymer, polyvinylpyrrolidone / hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymer silicone.

Examples of the emollients include long chain acyl glutamic acid cholesteryl ester, hydroxystearic acid cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid and lanolin fatty acid cholesteryl ester.

Examples of the surfactant include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

Examples of the nonionic surfactant include self emulsifying monostearate glycerin, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbit fatty acid ester, POE (Polyoxyethylene / polyoxypropylene) copolymer, POE.POP alkyl ether, polyether-modified silicone, polyether-modified silicone, polyoxyethylene-polyoxypropylene (POE) Alkanolamides, alkylamine oxides, hydrogenated soybean phospholipids, and the like.

Examples of the anionic surfactant include fatty acid soap, alpha-acylsulfonate, alkylsulfonate, alkylallylsulfonate, alkylnaphthalenesulfonate, alkylsulfate, POE alkylether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl phosphate, alkylamide phosphate , Alkyloyl taurine salts, N-acyl amino acid salts, POE alkyl ether carboxylates, alkylsulfosuccinates, sodium alkylsulfoacetates, acylated hydrolyzed collagen peptide salts, and perfluoroalkyl phosphate esters .

Examples of the cationic surfactant include alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, behenyl trimethyl ammonium chloride, Benzalkonium, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, quaternary ammonium salts of lanolin derivatives, and the like.

Examples of the amphoteric surfactant include carboxybetaine type, amide betaine type, sulfobetaine type, hydroxysulfobetaine type, amidosulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type and amide amine type Amphoteric surfactants and the like.

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, But are not limited to, polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluororesin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, Silk powder, cellulose, CI Pigment Yellow, CI Pigment Orange, and composite pigments of inorganic pigments and organic pigments thereof.

As the organic powder, metallic soap such as calcium stearate; Metal salts of alkyl phosphates such as sodium zinc cetylate, zinc laurylate and calcium lauryl laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc and N-lauroylglycine calcium; Amidosulfonic acid multivalent 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-lauroylglycylglycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid, alpha-aminoaurauric acid, and the like; Polyethylene, polypropylene, nylon, polymethylmethacrylate, polystyrene, divinylbenzene-styrene copolymer, ethylene tetrafluoride, and the like.

Examples of ultraviolet absorbers include paraaminobenzoic acid, ethyl parnamobenzoate, amyl paranobenzoate, octyl paranobenzoate, ethyleneglycol salicylate, phenyl salicylate, benzyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, benzyl cinnamate , Octyl methoxycinnamate, dioctyl methoxycinnamate, mono-2-ethylhexane glyceryl dipyrromethoxycinnamate, isopropyl paratumoxycinnamate, diisopropyl-diisopropyl cinnamate ester mixture, Carninoic acid, ethyl urocanoate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and salts thereof, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone disulfonate, dihydroxybenzophenone , Tetrahydroxybenzophenone, 4- tert -butyl-4'-methoxydibenzoylmethane, 2,4,6-trianylino- p- (carbo-2'-ethylhexyl-1'- , 3,5-triazine, 2- (2- 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, gallic acid propyl, and eicosorbic acid.

Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogenphosphate and the like.

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

In addition, any of the above-mentioned components may be blended within the range not to impair the objects and effects of the present invention, but it is preferably 0.01 to 5% by weight based on the total weight, More preferably from 0.01 to 3% by weight.

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

The ingredients contained in the cosmetic composition of the present invention may contain, in addition to the above-mentioned extracts, the components conventionally used in cosmetic compositions, such as stabilizers, solubilizers, vitamins, pigments and fragrances, And a carrier.

The functional cosmetic composition of the present invention can be prepared in any formulations conventionally produced in the art, and examples thereof include emulsions, creams, lotions, packs, foundations, lotions, essences, and hair cosmetics.

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 .

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 as a carrier component. In the case of a spray, in particular, / 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 an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

As described above, the present invention provides a method for measuring an electron donating ability, an ABTS radical cation-scavenging activity inhibitory activity test, a SOD dismutase-like activity assay ) And Nitric oxide radical inhibition activity (Experimental Example 1); An antibacterial experiment (Experimental Example 2) for various strains including the skin uptake bacteria; iNOS, COX-2 expression, iNOS increased by LPS, Western blot assay for COX-2 protein iNOS mRNA expression level, and real time PCR (Experiment 3); Cell viability measurement test by MTT assay (Experimental Example 4); Anti-inflammatory, antimicrobial, and anti-allergic activity through the experiment of measuring anti-allergic activity (Example 5) by the test for suppressing the secretion of histamine, the cytokine measurement experiment of TNF-α and IL-8 Skin aging, dermatitis and the like, and can be used as a cosmetic composition.

FIG. 1 is a graph showing the effect on the electron donating ability of a sample,
2 shows the effect of the sample on the ABTS radical scavenging ability,
Figure 3 is a graph showing the effect of SOD-like activity of a sample,
FIG. 4 is a graph showing the influence of the sample on NO elimination performance,
FIG. 5 is a graph showing the antimicrobial activity (A: 0.5% B: 1% C: 2% D: 3%) of a sample,
FIG. 6 is a graph showing the effect of licorice samples on iNOS and COX-2 protein expression,
FIG. 7 is a graph showing the effect on the iNOS and COX-2 protein expression rates of the ligated samples,
8 is a graph showing the influence of the chromium samples on iNOS and COX-2 protein expression,
FIG. 9 is a graph showing the effect of licorice samples on iNOS and COX-2 mRNA expression rates,
FIG. 10 is a graph showing the effect on the expression rate of iNOS and COX-2 mRNA in a ligated sample,
FIG. 11 is a graph showing the influence of iNOS and COX-2 mRNA expression ratios of the chrome samples,
12 is a graph showing cell viability of licorice samples,
13 is a graph showing the cell viability of the ligated sample,
FIG. 14 is a graph showing the cell viability of a chrome sample,
Fig. 15 is a graph showing the effect of licorice samples on the histamine releasing ability,
16 is a graph showing the effect on the histamine releasing ability of the ligated sample,
FIG. 17 is a graph showing the effect of the zinc sample on the histamine releasing ability,
18 is a graph showing the effect of the licorice sample on TNF-a production ability,
FIG. 19 is a graph showing the effect of the crosslinked sample on the TNF-a production ability,
20 is a graph showing the effect of the chrome sample on TNF-a production ability,
21 is a graph showing the effect of the licorice sample on the IL-8 production ability,
FIG. 22 is a graph showing the effect on the IL-8 production ability of the ligated sample,
FIG. 23 is a graph showing the effect of the chrome sample on the IL-8 production ability,
Fig. 24 is a microscope photograph of lotion agent,
25 is a diagram showing the pH measurement and the viscosity measurement result of the lotion agent.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1. Materials and Sample Extraction

500 g of licorice, licorice and lichen used in this experiment were purchased from Omni Herb (www.omnoherb.com), and 70% ethanol was added at 10 times the weight of the sample and immersed at room temperature for 24 hours to separate the supernatant and precipitate, The recovering process was repeated three times. (Hereinafter, referred to as "GU", "FK" and "CC", respectively) were obtained by concentrating the dried liquorice, licorice, .

Reference Example 1. Statistics Processing

This study was repeated 3 times or more independently, and all the results were expressed as mean ± SD. Statistical significance was determined by Student's t-test and statistically significant when p value was less than 0.05 .

Experimental Example 1. Measurement of Antioxidative Activity

In order to measure the antioxidative activity of the samples obtained in the above Examples, the following literature methods were applied to conduct experiments.

1-1. Measurement of electron donating ability

In order to confirm the electron donating ability of the sample of the above example, the following experiment was carried out by modifying the Blois method described in literature [Blois MS. Antioxidant determination by the use of a stable free radical. Nature . 1958; 26: 1199-1120].

50 μl of 0.2 mM of 1,1-diphenyl-2-picrylhydrazyl was added to 100 μl of each sample solution. After stirring for 30 minutes, absorbance was measured at 517 nm.

1,1-diphenyl-2-picrylhydrazyl (DPPH) is a stable free radical. If the sample has antioxidant activity, DPPH will reduce the DPPH reduction by eliminating the free radicals involved in lipid oxidation, The higher the amount of purple DPPH is, the more violet the purple is lost, and the lower the UV value, the lower the value is [Amariwicz, R., Naczk, M., Shahidi, F. (2000). Antioxidant activity of various fractions of non-tannic phenolics of canola hulls. J. of Agric Food Chem. 48: 2755-2759.].

As a result, Eletron donating ability of three samples was 59.4%, 69.4% and 73.0% at the concentration of 100 ㎍ / ㎖, respectively, as shown in FIG. 1. At the same concentration, chrysanthemum was the most effective.

1-2. Measurement of ABTS radical cation-scavenging inhibitory activity

In order to test the antioxidative effect of the sample of the above example by ABTS radical cation decolorization, Roberta et al. [Roberta, RE, Pellegrini, N., Anna, P., Ananth, P., Yang, M., Catherine, RE, (1999). Antioxidant activity was improved by ABTS radical cation decolorization assay. J. of Free Radical Biology & Medicine. 26: 1231-1237].

ABTS radical cation decolorization was measured by Roberta et al. That is, 5 mL of 7 mM ABTS and 88 μl of 140 mM K ₂ S ₂ O ₈ were mixed with ethanol at a ratio of about 1:88, and the ABTS solution was used so that the absorbance of the control was adjusted to 0.7 ± 0.002 at 734 nm. 50 uL of the sample solution and 1 mL of ABTS solution were mixed, shaken for 30 seconds, reacted for 2.5 minutes, and the absorbance at 734 nm was measured to calculate the radical scavenging activity.

The ABTS method is the most commonly used method among other indirect methods. The addition of a sample containing a phenolic compound oxidizes ABTS (2,2'-azinobis- (3-ethylben zothiazoline-6-sulfonate) ABTS ˙ ⁺ is produced and measured for its antioxidant activity by measuring its degradation. ABTS ˙ ⁺ shows strong absorption in the range of 600-750 nm, stable when there is no phenolic compound, but hydrogen donor such as phenolic compound And the unique cyan color is discolored and converted into a colorless ABTS.

The results of ABTS radical cation decolorization are shown in FIG. Licorice, licorice and Rhodiola showed 95.3%, 99.0% and 100% activity at 50 ㎍ / ㎖, respectively. At the same concentration, chrysanthemum was the most effective.

1-3. SOD disutase-like activity measurement

In order to test the antioxidant efficacy of the sample according to the SOD dismutase like assay, the method of Marklund disclosed in the literature was applied as follows. [Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur . J. Biochem . 1974; 47: 469-4741.

To 0.2 mL of each sample solution, 2.6 mL of Tris-HCl buffer (50 mM tris + 10 mM EDTA, pH 8.5) and 0.2 mL of 7.2 mM pyrogallol were added and reacted at 25 ° C for 10 minutes. Then, 0.1 mL of 1M HCl was added to stop the reaction, The amount of oxidized pyrogallol was measured at 420 nm. The SOD - like activity was expressed as the absorbance reduction ratio of the sample solution and the non - added sphere.

One of the antioxidant enzymes, superoxide dismutase (SOD), is an enzyme that produces hydrogen peroxide (2O ₂ ^- + 2H ⁺ → H ₂ O ₂ + O ₂ ) by reacting with a very harmful peroxide anion radical (O ₂ ^- ). It is present in all species that consume oxygen and is a representative active oxygen inhibitor that protects against reactive oxygen defects in vivo. H ₂ O ₂ produced by SOD is known to be a function that protects living organisms from oxygen injury by converting into harmless water molecules and oxygen molecules by peroxidase or catalase. (Pryor WA) Oxy-radicals and related species: their formation, lifetimes, and reactions. Annu. Rev. Physiol. 1986; 48: 657-667; Greenstock CL. Radiation and aging: free radical damage, biological response and possible antioxidant intervention Med. Hypotheses 1993; 41 (5): 473-482)

Thus, living organisms have been adapting to the new environment through the evolutionary process of building a series of oxidation defense systems to protect themselves from free radical toxicity. The antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione reductase (GR), vitamin C (ascorbic acid), vitamin E (tocopherol) And other antioxidants. SOD, a typical active oxygen inhibitor, has a disadvantage in that it can not be easily absorbed into the body or is easily inactivated at a temperature of 70 ° C and very unstable at pH 10 or higher. Therefore, it is necessary to study SOD - like active substances in the body which can overcome these drawbacks.

The results of measuring the SOD-like activity are shown in FIG. Licorice, allium, and radish showed high activity of 27.1%, 86.1% and 93.5% at the concentration of 100 ㎍ / ㎖, respectively. At the same concentration, chrysanthemum was the most effective.

1-4. Measurement of nitric oxide radical inhibitory activity

In order to test the antioxidant activity of the sample according to the nitric oxide radical inhibition method, Marcocci et al. [Marcocci L., Maguire J. J., Droylefaix M. T. and Packer L.: The Nitric Oxide Scavenging Properties of Ginkgo Biloba Extract EGb 761. Biochemical and Biophysical Research Communications. 201, 15, 748-755,1994).

Sodium nitroprusside solutions were prepared by dissolving 10 mM sodium nitroprusside (Lot # BCBH0082V / SIGMA-ALORICH) in 20 mM phosphate buffer (pH 7.4). 0.5 mL of sample and 0.5 mL of sodium nitroprusside solution were reacted at 25 ° C for 150 minutes. 1 mL of the reaction solution and griess reagent solution were reacted at 1: 1 and the absorbance was measured at 540 nm.

Nitric oxide is one of the Reactive Oxygen Species (ROS) that is highly cytotoxic and is known to cause inflammation, cancer and other pathological conditions. In addition, a large amount of NO causes an indirect effect such as nitrozation, nitration, and an oxidation reaction, resulting in a harmful effect. [Sreejayan, N. and Rao, M. NA. : Nitric oxide scavenging by curcuminoids. Journal of Pharmacy and Pharmacology, 49, 105-107, 1997].

As shown in FIG. 4, the activity of licorice, licorice, and radish was 21.9%, 61.1% and 17.4% at a concentration of 100 ㎍ / ㎖, respectively. Inflammatory effects were most excellent.

Experimental Example  2. Antimicrobial activity measurement

In order to measure the antibacterial activity of the sample obtained in the above examples, the following literature test was applied to the experiment.

Streptococcus mutans (KTCT 3300), Propionibacterium (KTCT 3300) including skin microorganisms such as Escherichia coli (KTCT 1039), Staphylococcus epidermis (KTCT 1917), Candida albicans (KTCT 7965) and Staphylococcus aureus acnes (KTCT 3314).

(1) Culture of bacteria

To investigate the improvement of skin acne, Staphylococcus epidermidis , Escherichia coli , Staphylococcus aureus , Candida albicans Streptococcus mutans was used as an antimicrobial agent for mouthwash. Liquid medium for preculture and main culture was nutrient broth (NB) for S. epidermidis and E. coli , trypticsoybroth (TSB) for S. aureus and C. albicans for YM broth (YMB) (TSA) for S. aureus and YM Agar (YMA) medium for C. albicans were used as the solid medium for S. epidermidis and E. coli , and nutrient agar (NA) for S. epidermidis and E. coli . In P. acnes , liquid and solid medium were used for Gifu anaerobic medium (GAM) medium and S. mutans was used for liquid and solid medium for brain heart infusion (BHI). P. acnes and S. mutans were cultured in 5% CO ₂ incubator at 37 ℃, and all other strains were cultured at 37 ℃ in BOD incubator.

(2) Paper disk diffusion measurement

The antimicrobial activity was measured by paper disk diffusion method. Each strain cultivated in a single colony on a plate medium was inoculated into 10 mL of the liquid medium for 0.1 to 3 mL for 3 to 6 hours, and then cultured in 10 mL of the liquid medium for 18 to 24 hours. The cells were inoculated at a rate of about 1 × 10 ⁷ cells / well and homogeneously plated with a sterilized cotton swab. A 0.5, 1. 2. 3% sample of sterilized filter paper discs (8 mm, Whatman, Japan) loaded on a solid plate medium was absorbed by concentration and incubated at 37 ° C for 18-24 hours to remove the clear zone ) Was measured.

The antimicrobial effects of licorice, licorice and licorice were examined in Fig. 5 and Table 1. Licorice extract is Streptococcus mutans , Propionibacterium acnes The clear zone of the total concentration was confirmed in the bacteria. The extracts of agaricus extract did not have antimicrobial activity in all bacteria. candida albicans , Streptcoccus mutans , and Propionibacterium acnes .

Antimicrobial activity of Glycyrrhiza uralensis FISCH, Forsythia suspensa Vahl , Coptis chinensis Franch . on several microorganisms Sample
Strains Glycyrrhiza uralensis FISCH Forsythia suspensa Vahl Coptis chinensis Franch 1A 2B 3C 4D One 2 3 4 One 2 3 4 Escherichia coli - - - - - - - - - - - - Staphylococcus epidermis - - - - - - - - - - - - candida albicans - - - - - - - - 15 20 23 27 Staphylococcus aureus - - - - - - - - - - - - Streptcoccus mutans 10 11 13 14 - - - - 12 15 20 23 Propionibacterium acnes 11 13 14 16 - - - - 10 12 15 20 a: no inhibition, b: inhibition zone diameter (mm)

Experimental Example 3. Molecular biological anti-inflammatory experiment of licorice, licorice and licorice

3-1. Western blot assay

After cell seeding on a 100-mm tissue culture dish, DMEM medium (AYK 59509 / ThermoScienTIFIC) was replaced with EMEM medium (51200-038 / gibco brl) for 80% confluence and the cells were stabilized for a certain period of time. And LPS (1 / ml (Lot # 043M4033V / SIGMA) or H ₂ O ₂ 500. After the supernatant was removed, the cells were washed twice with PBS, harvested with a scrapper, The protein was harvested and the amount of protein was corrected by substituting the OD value into a standard curve prepared with BSA (bovine serum albumin), followed by staining with coomassie blue (CAT # Description / expedeon InstantBlue) And then used as a western sample.

After electrophoresis using SDS-polyacrylamide gel (# 161-0416 / BIO-RAD), remove the gel and immerse in transfer buffer for 10 ~ 15 minutes. Put 2 sponge in transfer buffer and put 1 filter paper on it. Leave. Immerse the 3 mm paper and nitrocellulose filter in the buffer. Between the two sponge and 2mm paper, the nitrocellulose paper and the gel are closely adhered, and the electrode is inserted and transferred.

After transfer for more than 1 hour at 190mA, immerse in ponceau S (Cat # 161-0156 / BIO-RAD) for 2 minutes and check the band. After washing twice with PBS, remove the background with blocking buffer (Lot # T0713614S). After washing twice, the primary antibody is added at a ratio of 1: 1,000, followed by dilution with a secondary antibody (mouse-Lot.L0312 / SantaCruzBiotechnology, rabbit-Lot.H2912, goat-Lot.F1212) at a ratio of 1: 1,000. Secondary antibodies were attached and washed several times with PBS tween (0000298339 / LONZA). After transferring to a film using ECL kit (Amersham Pharmacia, England), the amount of iNOS and COX-2 protein expression was confirmed.

In order to confirm the inhibition mechanism of NO production by licorice, chrysanthemum and licorice ethanol extract, western blot was performed to measure the amount of protein expression, and the results are shown in FIGS.

As a result of iNOS and COX-2 expression of licorice and licorice, the amount of iNOS protein expression increased by LPS was reduced at 100 μg / ml. According to the band density ratio, iNOS protein production was reduced by 20% and 30% at 100 μg / ml for licorice and aconite, respectively. The amount of COX-2 protein was decreased only at 100 μg / ml in both licorice and licorice. As a result of iNOS and COX-2 expression of Huanglian, the expression level of iNOS and COX-2 protein increased by LPS was decreased in a concentration-dependent manner. iNOS, and COX-2 at 100 μg / ml, respectively. These results suggest that licorice, allium, and hwangryeong extract inhibit iNOS and COX-2 protein, and that they can be used as an anti-inflammatory cosmetic material by highly inhibiting iNOS and COX-2 protein.

3-2. Real time PCR

(1) RNA isolation Raw 264.7 cells (40071 / Dulbecco) was treated with 1 ml of TRIzol-reagent (MFCD00213058 / SIGMA) to isolate total RNA. 100 μl of phenol and 100 μl of chloroform / isoamylalcohol (24: 1) were added to the separated RNA, and the supernatant was separated by repeating centrifugation twice. RNA was precipitated with 0.5 ml isopropylalcohol, washed with 70% ethanol and air-dried. RNA was dissolved in RNAase-free water, and RNase-free DNase was added and stored at -70 ° C.

(2) Real-time PCR Transcriptor First strand cDNA synthesis kit (Roche, Germany) was used and 1 μg of total RNA was reacted with anchored-oligo (dT) Primer (Lot B456520 / Roche Diagnostics GmbH) The reaction was terminated by heating with the transcriptor reverse transcriptase, protector RNase inhibitor, and Deoxynucleotide Mix (Lot B456520 / Roche Diagnostics GmbH) for 5 min at 55 ° C for 30 min at 85 ° C. COX-2, iNOS, β -actin was amplified by real-time PCR using 2 μl cDNA, 5 'and 3' primer using SYBR Green I (Roche, Germany) and thermal cycler (D04BB-6DF36-0B299041969 / Roche Diagnostics GmbH) Respectively. The products generated by PCR were electrophoresed on 1.2% agarose gel (Lot 151204 / Mbiotech) to identify specific bands. RNA was extracted and analyzed by real-time PCR after 24 hours of LPS and sample treatment in order to confirm the changes in gene expression of the immune-related factors upon administration of the sample. The fluorescence material used in the real-time PCR is SYBR green I, which is a fluorescent substance that specifically binds to double strand DNA, SYBR green I, and uses this characteristic to detect fluorescence . In the denaturation process, when a single strand is made, it is not bonded, but when it becomes a double strand with a primer in the annealing process, it combines to emit light. The results of the experiment were compared with those of the LPS - treated group as a calibrator, and their values were normalized to quantify the gene expression change quantitatively. As shown in Figs. 9 to 11, iNOS and COX-2 mRNA expression of licorice and allium phloem showed a dose-dependent decrease in the expression of iNOS mRNA, which was increased by LPS, and expression of COX-2 mRNA in licorice and allium The results were reduced in a concentration dependent manner. These results are consistent with the results of western blot iNOS and COX-2 protein expression, indicating that the three samples inhibit iNOS and COX-2 gene expression at the mRNA stage, It suggests.

Experimental Example  4. Measurement of cell viability

In order to measure the cell viability of the samples obtained in the above examples, the MTT assay method of the following literature was applied (Han HK, Choi SS, Shin JC <Chung HS highly-developed anthocyanin-pigmented rice varieties, J. Kor. Soc., Food Sci. Nutr., 36: 527-533).

4-1. Cell culture

HMC-1 cells (human mast cell) were cultured in 100 U / ml penicillin, 100 mg / ml streptomycin 1%, and 10% inactivated fetal bovine serum 5% CO ₂ at 37 ° C using Iscove's Modified Dulbecco's Media (IMDM) medium (SH30228.01 / Thermo SCIENTIFIC) Lt; / RTI &gt;

4-2. Experimental course

To determine the cell viability, 0.2 mL of each well was inoculated into a 96-well plate at the rate of 5 × 10 ⁵ cells / ml. After inoculation, the cells were incubated in a 5% CO ₂ incubator at 37 ° C for 24 h, After replacement, SRA-B3 was treated by concentration and cultured for 24 h. 0.02 mL of a solution of MTT (3-4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide prepared at a concentration of 5 mg / mL was added thereto, followed by culturing for 4 hours. 0.15 mL of DMSO: Ethanol (1: 1) solution was added to each well, stirred for 30 minutes, and absorbance was measured at 550 nm with an ELISA reader.

As a result, the cytotoxicity was 100.85 ± 8.42%, 97.13% at the treatment concentrations of 0.1, 0.5 and 1 mg / ml of licorice on the basis of the cell survival rate when HMC-1 was cultured with PMA + A23187 ± 7.38% and 82.16 ± 3.06%, respectively, indicating no cytotoxicity. (See Fig. 12)

Cytotoxicity was 109.48 ± 9.49%, 100.61 ± 4.87% at treatment concentrations of 0.1, 0.5 and 1 mg / ml, respectively, based on the cell survival rate when HMC-1 was cultured with PMA + A23187 95.13. + -. 6.43% and was not cytotoxic (see FIG. 13).

Cytotoxicity was 94.43 ± 2.77%, 86.78 ± 4.60% at 0.1, 0.5 and 1 mg / ml, respectively, on the basis of 100% cell survival rate when HMC-1 was cultured with PMA + A23187 84.13 ± 5.08% and was not cytotoxic. (See Fig. 14)

Experimental Example  5 Anti-allergy  Experiment

In order to measure the anti-allergic activity through the inhibitory activity against the secretory activity of histamine and cytokine of the samples obtained in the above examples, the following literature test was applied.

In order to induce contact dermatitis, the back part of the rats was depilated and left to stand for 24 hours to heal the micro wounds of the skin. The DNCB solution used in this experiment was diluted to 0.5% and 1% in a 3: 1 mixture of acetone and olive oil. 150 [mu] l of 1% DNCB solution was applied on the back, and immunity was induced for 4 days. After 3 weeks, 150 [mu] l of 0.5% DNCB solution was applied on the back to induce contact dermatitis. The herbal extracts were dissolved in PBS and administered orally at a dose of 400 mg / kg for 2 weeks. The visual change of skin condition of dermatitis induced mice was visualized.

On the end of the experiment, the blood was collected from the heart with a syringe and centrifuged at 5,000 rpm at 4 ° C for 3 minutes to separate the serum. Serum IgE levels in Balb / c mice and TNF-α, IL-6 and IL-8 in HMC-1 cells stimulated with PMA + A231867 were measured using an enzyme-linked immunosorbent assay. Dilute the monoclonal antibody against each cytokine in PBS (pH 7.4) to a 96-well plate, and coat each 100-well plate on a 96-well plate. The plate is allowed to stand at 4 ° C for 12 hours. The plate is washed with PBS containing 0.05% tween and blocked with PBS containing 1% BSA, 5% sucrose and 0.05% NaN3 for 1 hour. After washing several times, serum is added and incubated at 37 ° C for 2 hours. The wells are washed again, and biotin-conjugated secondary antibody is added and left for another 2 hours. After washing the wells, avidin peroxidase was added and incubated at 37 ° C for 30 min. After washing the wells again, the substrate ABTS solution was added and the color reaction was measured at 405 nm using an ELISA reader.

5-1. Cell culture

HMC-1 cells, a human mast cell, were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin 1% and 10% inactivated fetal bovine serum (FBS) medium 5% CO ₂ at 37 ℃ using (SH30228.01 / SCIENTIFIC Thermo) Lt; / RTI &gt;

5-2. Sample Preparation

HMC-1 cells (3 × 10 ⁵ cells / ml) were plated in 24-well plates for 1 hour in CO ₂ After stabilization in the bacterial incubator, the phlyls, alum and licorice at concentrations of 0.1, 0.5 and 1 mg / ml were pretreated for 1 hour. After treatment with PMA (MFCD00036736 / SIGMA) (50 nM) + A23187 (MFCD00151202 / SIGMA) (1 μM), the cells were cultured for 8 hours. The supernatant was removed to determine the amount of cytokine change. And then PMA (50 nM) + A23187 (1 μM) was treated, cultured for 12 hours, and the supernatant was counted for histamine release.

5-3. Hiscamine Secretory function  Inhibition experiment

Histamine quantitation The amount of histamine secreted in activated HMC-1 was measured using a histamine assay kit (IBL-America, Inc., Minneapolis, MN, USA).

Briefly, 100 μl of each sample and 100 μl of Histamine-Alkaline Phosphate conjugate (IBL-America, Inc., Minneapolis, MN, USA) are added to a 96-well plate and left at room temperature for 40 minutes. After washing the plate three times with PBS containing 0.05% tween, 100 μl of p-Nitrophenyl Phosphate substrate (MFCD00007319 / SIGMA) is added and left at room temperature for 20 minutes. After 20 minutes, 50 μl of a stop solution containing 3N NaOH was added, and the color reaction was measured at 405 nm using an ELISA reader (Power Wave XS / BIO-TEK).

As shown in FIG. 15, HMC-1 cells were pretreated with various concentrations of licorice (0.1-1 mg / ml) and stimulated with PMA + A23187 for 12 hours. The amount of histamine released from the cells was measured using a Histamine assay kit. As a result of the experiment, it was confirmed that the amount of Histamine increased by PMA + A23187 was dose-dependently inhibited by licorice. Especially, high concentration of licorice showed a suppression rate of about 22.16% (0.51 ± 0.005 pg / ml, P <0.05).

In addition, as shown in FIG. 16, HMC-1 cells were pretreated with various concentrations of bridges (0.1-1 mg / ml) and stimulated with PMA + A23187 for 12 hours. The amount of histamine released from the cells was measured using a Histamine assay kit. As a result, it was confirmed that the amount of Histamine increased by PMA + A23187 was dose - dependently inhibited by ligand. In particular, the high-dose rate control showed a 28.53% inhibition rate (0.47 ± 0.003 pg / ml, P <0.05).

Also, as shown in FIG. 17, histamine is well known as a major mediator involved in various allergic reactions, and it causes itching. The effects of Rhodiola on Histamine secretion were investigated. HMC-1 cells were stimulated with PMA + A23187 for 12 hours after pretreatment with various concentrations of phthalocyanine (0.1-1 mg / ml). The amount of histamine released from the cells was measured using a Histamine assay kit. As a result of the experiment, it was confirmed that the amount of Histamine increased by PMA + A23187 was inhibited in a concentration dependent manner by the phloem strain. In particular, the high concentration of thrips showed a suppression rate of about 24.89% (0.49 ± 0.00005 pg / ml, P <0.05).

4-4. Cytokine measurement: TNF -α and IL -8 measurement

The HMC-1 cells were diluted in PBS (pH 7.4) with a monoclonal antibody (IBL-America, Inc., Minneapolis, MN, USA) And then allowed to stand at 4 DEG C for 12 hours. The plate is washed with PBS containing 0.05% tween and blocked with PBS (0000298339 / LONZA) containing 1% BSA, 5% sucrose, 0.05% NaN ₃ for 1 hour. After washing several times, the sample is added and left at 37 ° C for 2 hours. The wells were washed again and incubated with biotin-conjugated secondary antibody (IBL-America, Inc., Minneapolis, MN, USA) for 2 hours. After washing the wells, avidin peroxidase (MFCD00130587 / SIGMA) was added and the wells were washed again at 37 ° C. for 30 minutes. Substrate ABTS solution (SIGMA A1888, SIGMA P5592) was added. (Power Wave XS / BIO-TEK) at 405 nm.

As shown in FIG. 18, HMC-1 cells were pretreated with various concentrations of (0.1-1 mg / ml) licorice and stimulated with PMA + A231867. The concentration of TNF-α secreted from the cells was measured by ELISA. Experiment result. The production of TNF-α was increased by PMA + A231867 (6.47 ± 0.04 ng / ml, P <0.05) compared with the control, but was inhibited by the licorice concentration-dependent. In particular, the high concentration (1 mg / ml) of licorice inhibited about 43.4% (3.67 ± 0.03 ng / ml, P <0.05).

In addition, as shown in Fig. 19, HMC-1 cells were stimulated with PMA + A231867 after pretreatment with various concentrations of (0.1-1 mg / ml). The concentration of TNF-α secreted from the cells was measured by ELISA. Experiment result. The production of TNF-α was increased by PMA + A231867 (6.47 ± 0.04 ng / ml, P <0.05). Especially, the inhibition effect of high concentration (1 mg / ml) was about 37.7% (4.03 ± 0.05 ng / ml, P <0.05).

In addition, as shown in Fig. 20, TNF-a is a proinflammatory factor and is well known as a major mediator involved in the early allergic response. The effect of Rhodiola on TNF-α production was investigated. HMC-1 cells were stimulated with PMA + A231867 after pre-treatment with various concentrations of (0.1-1 mg / ml) The concentration of TNF-α secreted from the cells was measured by ELISA. Experiment result. The production of TNF-α was increased by PMA + A231867 (6.47 ± 0.04 ng / ml, P <0.05) compared to the control, In particular, the high concentration (1 mg / ml) of the rhizomes inhibited about 49.8% (3.25 ± 0.03 ng / ml, P <0.05).

In addition, as shown in Fig. 21, the cells were pretreated with various concentrations of (0.1-1 mg / ml) licorice and stimulated with PMA + A231867 for 8 hours. The amount of IL-8 secreted from the cells was measured by ELISA. Experiment result. The production of IL-8 was increased by PMA + A231867 (5.03 ± 0.12 ng / ml, P <0.05) compared to the control group, but was inhibited by the licorice in a concentration-dependent manner. Especially, high dose (1 mg / ml) licorice inhibited about 41.4% (2.95 ± 0.03 ng / ml, P <0.05).

In addition, as shown in FIG. 22, the cells were pre-treated with various concentrations of (0.1-1 mg / ml) and stimulated with PMA + A231867 for 8 hours. The amount of IL-8 secreted from the cells was measured by ELISA. Experiment result. The production of IL-8 was increased by PMA + A231867 (5.03 ± 0.12 ng / ml, P <0.05) compared to the control group, but was inhibited by the intercalator in a concentration-dependent manner. The inhibitory effect of high concentration (1 mg / ml) was about 19.1% (4.07 ± 0.07 ng / ml, P <0.05).

IL-8 is known to play an important role in the migration of neutrophils. [Kim, SJ, Lee EJ, Song YS, Jeong HJ, Le KM, Kim HR, Chae HJ, Shin TY, Kim YK, Hong SH, Kim HM. Cheongyol saseuptang inhibits production of TNF-alpha, IL-6 and IL-8 as well as NF-kappa B activation in human mast cells. J Ethnopharmacol. 2005; 97: 83-8; Kim, HM, Park YA, Lee EJ, Shin TY. Inhibition of immediate-type allergic reation by Rosa davurica Pall. In a murine model. J Ethnophamacol. 1999; 67: 53-60]

Cells were stimulated with PMA + A231867 for 8 h after pretreatment with various concentrations of (0.1-1 mg / ml) The amount of IL-8 secreted from the cells was measured by ELISA. As shown in FIG. 23, the production of IL-8 was increased (5.03 ± 0.12 ng / ml, P <0.05) by PMA + A231867 in comparison with the control group, In particular, the high concentration (1 mg / ml) of Rhodiola showed about 43.4% inhibition (2.84 ± 0.10 ng / ml, P <0.05).

Experimental Example  5. Lotion formulation  Manufacturing and Experiment

5-1. Manufacture of lotions

The raw materials used to make the lotion are shown in Table 2. To prepare lotion, water phase and oil phase were heated to 85 ℃ respectively and emulsified for 5 minutes at 2,500 rpm using a homo mixer. After cooling to 45 ° C, the extract was added and finally Silkem 1650 and Phenoxyethanol and DS-Cerix 5 were added and mixed at 1,700 rpm for 5 minutes using an agi-mixer.

Lotion composition table INCI Name Water Butylene Glycol Glycerin Carpylic Betaine Butylene Glycol Sorbitan Stearate Cetyl Alcohol Triethylhexanoin Squalane Tocopheryl Acetate Panthenol

5-2. Particle size measurement of lotion

In order to observe the lotion particles, the distribution of the inner particles was confirmed using a microscope (Eclipse 80i / 100X, Nicon, Japan). As a result of observing at a magnification of 400 times under a microscope, it was confirmed that the particle shape was varied, uniform distribution and no dispersion or separation of particles as shown in FIG. The minimum particle size was 3 μm, the maximum particle size was 9 μm, and the average particle size was 5 to 6 μm.

5-3. Lotion stability test ( pH , Viscosity)

The lotion was measured by repeatedly measuring the pH of the O / W type lotion containing the extract stored at 25 ° C three times using a pH meter (Hanna Co., Romania), and then the average was determined. Before measurement, the glass electrode was preliminarily immersed in basic buffer or distilled water for several hours, and the pH meter was connected to a power source for more than 10 minutes. The detector was washed with distilled water and gently wiped off.

Viscosity measurements were made using a Viscometer (Brookfield Co., USA). The O / W type lotion stored at 25 ° C was measured with a spindle No. 4 at 6 rpm for 1 minute. To reduce the effect of bubbles, defoaming was performed at the time of manufacturing and the average value was obtained by repeating 3 times. After 3 hours, the value was measured again for 3 weeks. In other words, the factors influencing the viscosity were minimized.

5-3. Experiment result

5-3-1. pH measurement

The pH of the lotion stored at room temperature (25 ° C), 0 ° C, 40 ° C, cycle chamber, sunlight (natural light) and artificial light (28 ° day) The pH value of the lotion stored in the broth was slightly lower than that of the other conditions, but there was no specific change in the observation period and the storage temperature.

5-3-2. Viscosity measurement

Cosmetics should be free of alteration, discoloration, alteration, microbial contamination, etc. due to external environmental factors. Viscosity measurement is important to understand the viscosity because the quality of the quality may change physically or chemically depending on the temperature of the sunlight or the surrounding environment, directly or indirectly. The viscosity of the lotion stored at room temperature (25 ° C.), 0 ° C., 40 ° C., cycle chamber, sunlight (natural light) and artificial light (artificial light) was measured for 28 days. As shown in FIG. 25, It was shown that the viscosity remained stable without significant change for 28 days under each temperature condition.

Examples of the preparation of the cosmetic composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.

Preparation Example 1. Preparation of a skin lotion (skin)

An example of production of a soft lotion (skin) in cosmetics containing licorice ethyl alcohol extract is shown below.

Manufacturing example  2. Manufacture of nutrition lotion (lotion)

An example of production of a nutritional lotion (lotion) among the cosmetics containing the ethyl alcohol extract of allium is shown below.

Manufacturing example  3. Manufacture of nutritional cream

An example of preparation of a nutritional cream among the cosmetics containing the ethyl alcohol extract is shown below.

Manufacturing example  4. Manufacture of Essence

An example of the preparation of the essence in cosmetics containing licorice ethyl alcohol extract is shown below.

Manufacturing example  5. Emulsion type  Manufacture of foundations

Table 5 shows an example of the production of an emulsified foundation among the cosmetic compositions containing the almond extract.

Manufacturing example  6. Preparation of hydrophilic ointment

Manufacturing example  7. Of rice pack  Produce

Claims (6)

A method for treating or ameliorating skin aging or dermatitis, which comprises an extract of licorice, chrysanthemum or licorice as an active ingredient. The dermatological pharmaceutical composition according to claim 1, wherein the extract is an extract which is soluble in water, alcohol, lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, including purified water. The dermatological pharmaceutical composition according to claim 1, wherein the dermatological pharmaceutical composition is a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta or a catarcoma. A cosmetic composition for the treatment and improvement of skin aging or dermatitis containing licorice, chrysanthemum or licorice extract as an active ingredient. [Claim 5] The cosmetic composition according to claim 4, wherein the extract is an extract that is soluble in water, alcohol, lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, including purified water. The cosmetic composition according to claim 4, wherein the cosmetic composition is at least one selected from the group consisting of hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair conditioner, hair treatment, Hair wax, hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair wave agent, hair bleaching agent, hair gel, hair glaze, hair dresser, hair Lacquer, hair moisturizer, hair mousse or hair spray.

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