KR101499453B1 - Skin whitening composition comprising complex herbal extract - Google Patents

Abstract

The present invention relates to a composition containing an extract of Buchungcho extract as an active ingredient. Specifically, the Buchungchwa extract of the present invention has an antioxidative effect; High cell viability; Whitening activity; Wrinkle-improving effect, etc., and can be usefully used as a composition for treating and preventing whitening and skin aging.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for treating and preventing skin whitening and skin aging,

The present invention relates to a composition for treatment and prevention of skin whitening and skin aging, which comprises a Buchungchos extract as an active ingredient.

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

[Literature 2] Aroca, P., et al. 1993. Melanin biosynthesis patterns of following hormonal stimulation. J. Biol Chem 268, 25650-25655

[Literature 3] Jimenez-Cervantes C., et al. 1994. A new enzymatic function in the melanogenic pathway. J. Biol Chem 269, 17993-18001

[4] Paval, S. 1993. Dynamics of melanogenesis intermediates. J. Invest. Dermatology 100, 162-165

[Literature 5] Chin, J. E., et al. 2005. Effects of Houttuynia cordata extracts on tyrosinase gene expression. J. Korean Soc Food Sci Nutr 34, 1284-1288

[Literature 6] Lee, P. J. 2009. Inhibitory effect of muscat bailey a seed extract on melanin production in α-MSH stimulated B16 cell. J. Kor Plant Res 22, 477-482

[7] Kim HK, Kim YE, Do JR, Lee YC, Lee BY. (1995) Antioxidative activity and hysiological activity of some Korean medicinal plants. J. Food Sci . Technol . 27, 80-86

[8] Tsuji N, Moriwaki S, Suzuki Y, Takema Y and Imokawa G. (2001) The role of elastases secreted by fibroblasts in wrinkle formation: implication through selective inhibition of elastase activity. Photochem . Photobiol . 74 (2), 283-290

[Literature 9] Roth GJ, Siok CJ and Ozols J. (1980) Structural characteristics of prostaglandin synthetase from sheep vesicular gland. J. Biol . Chem . 255 (4), 1301-1304

[Literature 10] A physiology active minuteness chemical technique development road map. (2002) Ministry of Commerce , Industry and Energy . 229-322

[11] Jeroma SP, Gabrielle L and Raul F. (1998) Identification of collagen fibrils in scleroderma skin. J. Invest . Dermatol . 90 (1), 48-54

[Document 12] Oh KN. (2007) Protective effects of apigenin and luteolin on ultraviolet A-induced matrix metaaloproteinase expression in human keratinocyte. M. D. Thesis. Chosun university

[Document 13] Carmichael J, DeGraff WG, Gazdar AF, Minna JD and Mitchell JB. (1987) Evaluation of a tetrazolium based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res . 47 (4), 936-942

[14] Prota G. (1980) Recent advances in the chemistry of melanogenesis in mammals. J. Invest Dermatol. 75 (1), 122-127

[Literature 15] Pavel S and Muskiet FA. (1983) Eumelanin (precursor) metabolites as markers for pigmented malignant melanoma, a preliminary report. Cancer Detect Prev . 6, 311-316

[16] Lee, HC, Bu, HJ, Jung DS, Lee, SJ, Riu, KZ. (2001) Screening of the tyrosinase inhibitory and hyaluronidase inhibitory activities and radical scavenging effects using plants in Cheju. Kor . J. Pharmacogn . 32 (3), 175-180

[Literature 17] Ipseong et al. 1, Encyclopedia of Contemplation, Young Lim, pp. 277 ~ 278, 1998).

Suh, SS and Shin, JS: Studies on phytosterols, Yakhak HoeChi , 13 , 144-146 (1969)

[19] Wallace, J. W .: Biosynthesic studies on flavones and C-glycosylflavones: B-ring oxidation patterns, Phytochemistry, 14, 1765-1768 (1975)

[20] Harborne, JB: The natural distribution in angiosperms of anthocyanins acylated with aliphatic dicarboxylic acids, Phytochemistry, 25 (8), 1887-1894 (1981) .: Biological evaluation of Korean medicinal plants. II., Kor . J. Pharm ., 21 , 177-183 (1977)

[Literature 22] Woo Sik Sik, Eunbang Lee: Detection of physiological activities of Korean herbal medicines by extracted long-term specimens (Ⅱ), Journal of Pharmacognosy, 10 (1), 27-30 (1979)

[Literature 23] Jang, Myung-joo and Ji Hyung-joon: Studies on the pharmacokinetics and toxicity of Korean herbal medicines (3rd), Journal of Pharmacognosy, 13 (2), 55-61 (1982)

[24] Kim, CJ, Cho, SK, Shin, MS, Cho, H., Ro, DS, Park, JS and Yook, CS: Hypoglycemic activity of medicinal plants, Arch . Pharm . Res ., 13 (4), 371-373 (1990)

[Literature 25] Blois MS. (1958) Antioxidant determination by the use of a stable free radical. 26, 1199-1120

[26] Carmichael, J., WG DeGraff, AF Gazdar, JD Minna, and JB Mitchell. 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res . 47 , 936-942.

[Document 27] Cannell RJP, Kellan SJ, Owsians AM and Walker JM. (1988) Results of a large scale screen of microalgae for the production of protease inhibitors. Planta Media . 54 (1), 10-14.

[Document 28] WE and Heindrich HG. (1963) Zur quantitativen bestimmung der collagenase. Hoppe-Seyler's. Physiol . Chem . 333: 149-151

[29] Kim MJ, Kim JY, Choi SW, Hong JT and Yoon KS. (2004) Anti-wrinkle effect of safflower ( Cathamus tinctorius ) seed extract. J. Soc . Cosmet . Scientisis Korea . 30 (1), 15-22

[Literature 30] Shim JS, Choi EJ, Lee CW, Kim HS and Hwang JK. (2009) Matrix metalloproteinase-1 inhibitory activity of Kaempferia pandurata Roxb. J of food . 12 (3), 601-607

[31] Yagi A, Kanbara T and Morinobu N. (1986) The effect of tyrosinase inhibition for aloe. Planta Media . 3981, 517-519

[32] Hosoi J. Abe E, Suda T and Kuroki T. (1985) Regulation of melanin synthesis of B16 mouse melanoma cells by 1 alpha, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res . 45 (4), 1474-1478

[Literature 33] Choi BW, Lee BH, Kang KJ, Lee ES and Lee NH. (1998) Screening of the tyrosinase inhibitors from marine algae and medicinal plants. Kor . J. Pharmacogn . 29 (3), 237-242

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.). Pigmentation of the skin is known to regulate the initial reaction from DOPA to DOPA quinone with tyrosinase enzyme, L-tyrosine, such as DHICA oxidase (TRP-1), and DOPA (3,4-dihydroxyphenyla-lanine) in melanin pigment biosynthesis (Aroca, P., et al 1993. Melanin biosynthesis patterns of the following hormonal stimulation: J. Biol Chem 268, 25650-25655 .; Jimenez-Cervantes C., et al 1994. A new enzymatic function in the melanogenic pathway. J. Biol Chem 269, 17993-18001; Paval, S. 1993. Dynamics of melanogenesis intermediates, J. Invest. Dermatology 100, 162-165.). On the basis of this, studies on the search for natural products that inhibit the activity of tyrosinase enzyme and affect the inhibition of melanin biosynthesis are actively conducted. As a result, the tyrosinase gene expression inhibitory effect of horsetail extract (Chin, JE, et al., 2005. Effects of Houttuynia cordata extracts on tyrosinase gene expression, J. Korean Soc Food Sci Nutr 34, 1284-1288) Inhibitory effect of α-MSH-stimulated melanin on the production of melanin in B16 cells (Lee, PJ 2009. Inhibitory effect of muscat bailey a seed extract on melanin production in α-MSH stimulated B16 cell. J. Kor Plant Res. 22, 477-482.) Are being actively studied. Other known antioxidative and whitening raw materials are Arbutin, Kojic acid, Ascorbic acid, etc. Plant extracts such as bark, mackerel and licorice are widely known.

DPPH is a chemically stabilized water-soluble substance with free radicals, which is reduced by ascorbic acid, tocopherol, and polyhydroxy aromatic compounds, resulting in discoloration of deep purple color, which is widely used to search for antioxidants from various natural materials . Reactive oxygen species (ROS) are largely eliminated by the body's defense system, but if not eliminated, they react with biomolecules rapidly to cause protein denaturation, lipid peroxidation and DNA damage of the biological membrane, Lipid peroxides promoted new radical reactions and acted as a cause of various diseases. (1995) Antioxidative activity and hysiological activity of some Korean medicinal plants J. Food (Kim HK, Kim YE, Do JR, Lee YC, Lee BY. Sci . Technol . 27, 80-86)

Elastase is an enzyme that degrades elastin, an important substrate for maintaining skin elasticity in the dermis. Elastase is also a nonspecific hydrolytic enzyme capable of degrading collagen, another important substrate protein. Thus, the elastase inhibitor has the function of improving the wrinkles of the skin, and ursolic acid has been used as the elastase inhibitor. Elastase is known to be the main enzyme of wrinkle formation by decomposing elastin, the insoluble elastic fiber protein of animal connective tissues, and breaking the network structure of the dermis early in the dermis (Tsuji N, Moriwaki S, Imokawa G. (2001) the role of elastases secreted by fibroblasts in wrinkle formation:... implication through selective inhibition of elastase activity Photochem Photobiol 74 (2), 283-290) ln dermal tissues of the skin associated with collagen and elasticity of the skin elastin forms a network structure. As the network structure is broken, that is, the elastin is decomposed by elastase, the skin is sagged and wrinkles are generated, so that the endogenous skin aging occurs. Therefore, skin aging can be suppressed by lowering the activity of elastase, elastin degrading enzyme, which is one of the major causes of skin aging. Currently, studies to prevent skin aging have been actively conducted, and up to now, there have been many studies on α-1-proteinase inhibitor, mucus proteinase inhibitor, α-2-macri globulin, inter-α-trypsin, bowman-birk inhibitor, verapamil, beta lactam, chondroitin sulfates , deoxycycline, heparin, and other elastase inhibitors have been reported. (Roth GJ, Siok CJ and Ozols J. (1980) Structural characteristics of prostaglandin synthetase from sheep vesicular gland. J. Biol. Chem . 255 (4), 1301-1304)

Collagen, a major component of the extracellular matrix, is a major substrate protein produced by the fibroblasts of the skin. It is also an important protein that accounts for about 30% of the total biomass protein weight, and has a solid triple helix structure. Collagen forms most of the organic matter in the skin, tendons, bones and teeth, especially in the bones and dermis of the skin. The main functions of collagen are known to be mechanical rigidity of skin, resistance of connective tissues and binding force of tissues, support of cell adhesion, induction of cell division and differentiation. Such collagen is reduced by aging and photoaging by ultraviolet irradiation, which is known to be closely related to wrinkling of the skin. In addition, collagen is not affected by proteases such as trypsin but is degraded by collagenase (A physiology active minuteness chemical technique development road map. (2002) Ministry of Commerce , Industry and Energy . 229-322)

Most of the collagen is present in the dermis of the skin and accounts for about 70 to 80% of the dry weight of the skin. It plays a role of supporting the skin occupying most of the extracellular matrix. However, degradation of collagen is accelerated by extrinsic factors such as decrease of collagen biosynthesis due to internal factors such as decrease of cell activity due to natural aging, increase of stress due to various harmful environments, and increase of active oxygen species by sunlight And the skin substrate is destroyed to form wrinkles. In addition, collagen plays an important role in wound healing and can quickly restore scarring without promoting the synthesis of collagen in damaged epithelium. The most prominent collagen synthesis promoters that have been identified so far include asiaticoside, asiatic acid, and madecasic acid, the major components of centella asiatica. (Jeroma SP, Gabrielle L and Raul F. (1998) Identification of collagen fibrils in scleroderma skin J. Invest . Dermatol . 90 (1), 48-54)

Among several kinds of MMPs produced in the body, MMP-1 is a collagen-specific protease that inhibits the activity of MMP-1 to reduce collagen degradation, thereby maintaining skin elasticity and preventing wrinkle formation It is known. (Oh KN. (2007) Protective effects of apigenin and luteolin on ultraviolet A-induced matrix metaaloproteinase expression in human keratinocyte M. D. Thesis.

MTT detection method is widely used as a cytotoxicity and cell proliferation detection method because many samples can be easily read using an ELISA reader using a 96-well plate, and the dehydrogenase activity of mitochondria in cell metabolism (1987), a yellow-soluble MTT tetrazolium is reduced to a purple, water-insoluble MTT formazan. (Carmichael J, DeGraff WG, and Gazdar AF, of chemosensitivity testing. Cancer Res . 47 (4), 936-942.)

Melanin, a key determinant of skin tone, is biosynthesized in melanosomes in pigmented cells called melanocytes in the epidermal basal layer. The starting material for the synthesis of melanin is tyrosine, an amino acid. Tyrosine is oxidized by tyrosinase in melanocytes to L-3,4-dihydroxyl phenylalanine (DOPA) and DOPA quinone. It is known that DOPA quinone then becomes DOPA chrome, 5,6-dihydroxyindole, and indole-5,6-quinone, and then melanin is formed by polymerization with indole-5,6-quinone. In addition, tyrosinase plays an important role in the formation of melanin in the skin. Tyrosine hydoxylase, which oxidizes tyrosine in melanosome to form DOPA, acts as a DOPA oxidase that oxidizes DOPA to form DOPA quinone. (Prota G. (1980) Recent advances in the chemistry of melanogenesis in mammals J. Invest Dermatol . 75 (1), 122-127.)

Melanin, which is the most important factor in determining human skin color, not only inhibits skin photoaging and sunburn keratosis but also causes partial hyperpigmentation of spots, freckles, and black spots. L-ascorbic acid and its derivatives and kojic acid, arbutin, lactic acid, glucosamine, and tunicamycin have been developed to inhibit melanogenesis. There is a problem with stability and only a very limited amount is used. (Pavel S and Muskiet FA. (1983) Eumelanin (precursor) metabolites as markers for pigmented malignant melanoma, a preliminary report. Cancer Detect Prev . 6, 311-316.).

Skin blackening is due to the fact that melanocytes present in the skin increase the production of melanin in response to external conditions such as UV exposure. In order to confirm the whitening effect of raw materials and substances, the inhibition of enzyme activity of tyrosinase, which is a melanin-producing enzyme, has been mainly used. However, in recent years, in addition to suppression of enzyme activity, whitening agents have been shown to increase tyrosinase and related enzyme expression in melanocyte There have been many reports on the mechanism studies that disturb (. Lee NH, Yang HC, Bu HJ, Jung DS, Lee SJ, Riu KZ (2001) Screening of the tyrosinase inhibition and hyaluronidase inhibition activities and radical scavenging effects using plants in Cheju. Kor. J. Pharmacogn. 32 (3) , 175-180.).

Spirodela polyrrhiza ) is a perennial herbaceous plant of the genus Sennaeus . It is a small grass floating on the surface of the water. It is also known as a fowlpowder, and its components such as potassium oxide, potassium chloride and fluorine are known to be effective for arteriosclerosis and hematopoiesis. (1) (1). (2).

Studies on the constituents of 浮萍 草 (Puchipcheon) showed that all the grasses (outposts) of the frog were treated with sterols such as campesterol, β-sitosterol and stigmasterol (4. Suh, SS and Shin, JS: Studies on phytosterols, Yakhak HoeChi, 13, 144-146 (1969) and, flavone-O-glycoside as apigenin-7-O-β- D-glucoside, cynaroside (luteolin-7-O-β-D-glucoside), hypolaetin-8-O (8-hydroxylase-8-O-β-D-glucoside) and flavone-C-glycoside as vitexin (apigenin-8-C-β-D-glucoside), orientin B-ring oxidation patterns, Phytochemistry , 14 , 1765-1768 (1975) and anthocyanin (6. Harborne, JW: Biosynthesic studies on flavones and C-glycosylflavones: JB: The natural distribution in angiosperms of anthocyanins acylated with aliphatic dicarboxylic acids, Phytochemistry , 25 (8), 1887-1894 (1981).

In a study of bioactive浮萍草(bupyeongcho) are 1977 Woo, etc. (7.. Woo, WS, Lee , EB and Chang, I M .: Biological evaluation of Korean medicinal plants. Ⅱ, Kor. J. Pharm. , 21 , 177-183 (1977), the antitumor activity against sarcoma 180, leukemia SN 36 and Ehrlich ascites carcinoma, the cytotoxic action against HeLa cell, the effect of Staphylococcus aureus and Escherichia coli . In 1979, the MeOH extract of Fried Rice showed no direct shrinkage or anti-acetylcholine action on the ileal section of the rat, and no direct contraction or anti-oxytocin action on the uterine segment. woowonsik, yieunbang: extraction Journal of biologically active search (ⅱ), domestic herbal herbal medicine due to the long-term sample, 10 (1), 27-30 in Chapter (1979) .1982 years, etc. (9 jangilmu, jihyeongjun: Pharmacology of Korean herbal (3), Journal of the Korean Pharmacopoeia, 13 (2), 55-61 (1982). In this experiment, the foliar EtOH extract showed weak anti-mitotic activity against neuroglioma 9ASK but no cytotoxic effect, and the extract of CHCl ₃ fraction had anticancer activity against leukemia P388 (1990), Ro et al. (10) Kim, CJ, Cho, SK, Shin, MS, Cho, H., Ro, DS, Park, JS and Yook, CS: Hypoglycemic activity of medicinal plants, Arch Pharm . Res ., 13 (4), 371-373 (1990) have reported that water extract of frogs does not have anti-glycemic effect in streptozotocin-induced diabetic mice.

However, none of the above documents mentions or discloses the therapeutic effect on skin aging, such as whitening, wrinkle improvement, etc. of Puccio extract.

Accordingly, the present inventors have found that the antioxidative effects of the Puchoncho extracts such as DPPH free radical scavenging activity, ABTS radical scavenging activity inhibiting activity, and xanthine oxidase inhibiting activity; High cell viability; Tyrosinase inhibitory activity, Cellular tyrosinase Inhibitory activity Melanin inhibitory activity, whitening activity such as inhibition of tyrosinase protein expression associated with whitening; It was confirmed that it is useful as a composition for treating and preventing whitening and skin aging by confirming the effect of inhibiting elastase, MMP-1 inhibitory activity and pro-collagenase inhibitory activity Thereby completing the present invention.

In order to achieve the above object, the present invention provides an external dermatological pharmaceutical composition for treating and preventing whitening and skin aging, which comprises an extract of Buchungcho extract as an active ingredient.

The present invention also provides a cosmetic composition for improving and preventing whitening and aging of skin, which contains an extract of Buchungcho as an active ingredient.

Extracts as defined herein include crude extracts, or fractionated extracts.

As used herein, the term " crude extract " refers to water, ethanol, methanol, propanol, butanol, acetone, ethyl acetate, hexane, butylene glycol, propylene glycol, hydrolyzed butylene glycol, Or more, preferably water or a mixed solvent of water and ethanol, most preferably 60% to 80% ethanol-soluble extract.

The fraction extracts defined herein include nonpolar solvent soluble fraction extracts and polar solvent soluble fraction extracts.

The nonpolar solvent soluble fraction extract as defined herein is prepared by suspending the crude extract in water followed by treatment with a nonpolar solvent selected from hexane, methylene chloride, dichloromethane, chloroform or ethyl acetate, preferably hexane, methylene chloride fraction, or ethyl acetate , More preferably a fraction extract which is soluble in a non-polar solvent obtained by fractionation with a methylene chloride nonpolar organic solvent; The polar solvent-soluble fraction extract includes a fraction extract obtained by removing the nonpolar solvent-soluble tablet and allowing the remaining crude extract to be fractionated with butanol or water.

The extracts defined herein include crude extracts or non-polar solvent soluble extracts and can be prepared by the following process.

For example, the crude extract of the present invention may contain water, alcohol, water, and water containing about 1 to 200 times (w / w) of the total weight of the sample, preferably 10 to 100 times (w / 4 or a mixed solvent thereof, preferably water or a mixture of water and ethanol, more preferably water or a mixed solvent of 50-99% water and ethanol, for 2 hours to 1 week, preferably 4 hours to 1 week, The extraction method such as cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction, preferably reflux cooling extraction method, at room temperature for 10 to 150 ° C, preferably 20 to 100 ° C, Followed by a first step of obtaining an extract.

For example, the non-polar solvent and polar solvent-soluble extract of the present invention may be used in an amount of about 0.0005 to 50 times, preferably 0.05 to 5 times (v / v) of the crude extract, preferably 60 to 90% w%) of water, followed by a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate and butanol to obtain nonpolar solvent-soluble extraction tablets (n-hexane, water; And a polar solvent-soluble extracted and purified product soluble in polar solvents such as butanol and water.

As defined herein, "skin aging" includes wrinkles, stains, muscle wounds, skin damage due to ultraviolet light, skin cancer, and preferably wrinkles or stains.

The suppuryecho extract comprises 0.1 to 50% by weight based on the total weight of the skin pharmaceutical composition.

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.

In addition, the cosmetic composition includes formulations of lotion, skin, lotion, nutrition lotion, nutritional cream, massage cream, essence, and pack.

Hereinafter, the present invention will be described in detail.

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

Butanol, acetone, ethyl acetate, hexane, butylene glycol, propylene glycol, propylene glycol, propylene glycol, propylene glycol, butylene glycol, , Water, and ethanol, preferably water, at a temperature of about 10 캜 to 100 캜, preferably at a temperature of 60 캜 to 90 캜 A first step of subjecting the mixture to an extraction method such as hot water extraction, reflux cooling extraction, immersion extraction or pressure extraction for about 1 to 5 hours, preferably by reflux cooling extraction; The extract of the present invention can be obtained by centrifugation, filtration under reduced pressure, and concentration and lyophilization for 1 to 5 times.

The inventors of the present invention found that the extract of Buchungcho extract of the present invention has an antioxidative effect such as DPPH free radical scavenging activity, ABTS radical scavenging activity inhibiting activity, and xanthine oxidase inhibiting activity; High cell viability; Tyrosinase inhibitory activity, Cellular tyrosinase Inhibitory activity Melanin inhibitory activity, whitening activity such as inhibition of tyrosinase protein expression associated with whitening; It is useful as a composition for the treatment and prevention of whitening and skin aging by confirming the wrinkle-reducing effect through the Elastase inhibitory activity, the MMP-1 inhibitory activity and the pro-collagenase inhibitory activity experiment Respectively.

In addition, the Puccio extract of the present invention has no toxicity and side effects, and has proved to be a non-irritant sample in the skin patch test, so that it can be safely used even in long-term use.

The dermatological pharmaceutical composition containing the Buchungcho extract of the present invention may be manufactured and used as a pharmaceutical composition in the form of a cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta or cataplasma But is not limited thereto.

The preferred dosage of the Bucurrhoeae extract of the present invention varies depending on the condition and body weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, in order to obtain the desired effect, it is preferable that the Puchoncho extract of the present invention is 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 Pucci extract of the present invention can be used variously in cosmetics and cleansers having a whitening effect.

Examples of products to which the present composition can be added include cosmetics such as lotion, skin, lotion, nutrition lotion, nutritional cream, massage cream, essence, pack, cleansing, cleanser, soap, have.

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) , Derivatives thereof (such as palmitic acid ascorbin, stearic acid ascorbic acid, dipalmitic acid ascorbin, dl-alpha tocopherol acetic acid, dl-alpha tocopherol nicotinic acid vitamin E, dl-pantothenyl alcohol, D-pantothenyl alcohol, 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, alkylarylsulfonate, alkylnaphthalenesulfonate, alkylsulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl ginseng salt, Alkylsulfosuccinic acid salts, acylated hydrolyzed collagen peptide salts, and perfluoroalkyl phosphoric acid esters, and the like can be mentioned. have.

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 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, Preferably 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, as an active ingredient, the components commonly used in cosmetic compositions in addition to the above-mentioned Puchonchia extract, and may contain conventional ingredients such as stabilizers, solubilizers, vitamins, Adjuvants and carriers.

The cosmetic composition of the present invention can be prepared into any formulation 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.

The suppuryecho extract of the present invention has an antioxidative effect such as DPPH free radical scavenging activity, ABTS radical cation scavenging activity inhibiting activity, xanthine oxidase inhibiting activity; High cell viability; Tyrosinase inhibitory activity, Cellular tyrosinase Inhibitory activity Melanin inhibitory activity, whitening activity such as inhibition of tyrosinase protein expression associated with whitening; It is useful as a composition for the treatment and prevention of whitening and skin aging by confirming the wrinkle-reducing effect through experiments such as Elastase inhibitory activity, MMP-1 inhibitory activity and pro-collagenase inhibitory activity Can be used.

1 is a graph showing the DPPH radical scavenging activity of a sample;
2 is a graph showing the ABTS cation radical scavenging activity of a sample;
3 is a graph showing the survival rate of the fibroblasts of the sample;
4 is a graph showing the survival rate of melanoma cells of a sample;
5 is a graph showing the elastase inhibitory activity of the sample;
6 is a graph showing collagenase inhibitory activity of a sample;
7 is a graph showing the pro-collagen inhibitory activity of the sample;
8 is a graph showing the MMP-1 inhibitory activity of the sample;
9 is a graph showing the tyrosinase inhibitory activity of the sample;
10 is a graph showing the influence of the sample on melanin biosynthesis;
11 is a graph showing the intracellular tyrosinase inhibitory activity of Melanoma cells of a sample.

Below, The present invention will be described in detail by 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. Preparation of Puchipcheon extract

1-1. Puchipcheon water extract

Bupyeongcho was purchased from Human Hub Co., Ltd. In the case of the hot-water extract, 100 g of the sample was added with 10-fold amount of distilled water, and the mixture was refluxed for cooling at 85 ° C for 9 hours to separate the supernatant and precipitate. The extract was centrifuged at 12,000 rpm for 10 minutes. 1 filter paper. Each extract was concentrated and lyophilized to obtain 13.9 g of a water extract (hereinafter referred to as SW).

1-2. Puchoncho Ethanol Extract

Bupyeongcho was purchased from Human Hub Co., Ltd. The supernatant and precipitate were separated by centrifugation at 85 ° C for 12 hours. The extracts were centrifuged at 12,000 rpm for 10 minutes. 1 filter paper. Each extract was concentrated and lyophilized to obtain 12 g of 70% ethanol extract (hereinafter referred to as SE).

Example 2. Preparation of Pu-Pyungcho fraction extract

The supernatant fraction of the supernatant was fractionated by 1: 1 (v / v) and concentrated under reduced pressure to obtain hexane fractions. Ethyl acetate layer, butanol layer and water layer were sequentially added through the same procedure to obtain respective fractions. Each of the extracts and the solvent fractions were concentrated and lyophilized to give 94.05 g (hereinafter referred to as SE-H) of hexane fraction, 68.80 g (hereinafter referred to as SE-E) of the ethyl acetate fraction and 50.26 g , And 205.5 g of water fraction (hereinafter referred to as SE-W) were used as samples in this experiment while being stored in a refrigerated room in powder form.

Experimental Example 1. Identification

1-1. Electronic Donor  ( electron donating ability ) Measure

In order to confirm the electron donating ability of the sample of the above example, the Blois method described in the literature was modified and the following experiment was conducted. (Blois MS. (1958) Antioxidant determination by the use of a stable free radical. 26, 1199-1120)

1 ml of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 ml of each sample solution. After stirring for 30 minutes, absorbance was measured at 517 nm. The electron donating ability was expressed by the absorbance reduction rate of the sample solution addition group and the no addition group. The DPPH radical scavenging activity was expressed by the concentration value of the sample required to reduce the absorbance of the control without addition of the sample to ½ according to the following equation (1).

As a result, DPPH radical scavenging activity of Buchungcho ethanol, hot water extract and ethanol extract fraction was shown in FIG. The supernatant of ethanol extracts showed better scavenging activity than that of the hot - water extracts, indicating that the ethanol extract showed a scavenging activity of 36.1% at 100 μg / ml concentration and a 24.7% scavenging activity at the concentration of 100 μg / ml of hot - water extract. The solvent fraction of Buchung-checho ethanol extract showed 44.2% and 42.6% cleavage in the EtOAC fraction and BuOH fraction at 100 μg / ml, respectively.

1-2. ABTS Radical  Cation Scatters  Measurement of inhibitory activity

In order to test the antioxidant activity of the sample of the present invention by the radical cation decolorization (ABTS) radical cation decolorization method, the following method was applied as described below (Roterta, R., P. Nicoletta, P. Anna, P. Ananth, Y. Min and RE Catherine . 1999. Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization Assay. Radic. Biol. Med. 26, 1231-1237.)

2.45 mM potassium persulfate was mixed with 7 mM 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Wako Chemical Co., Japan) and allowed to stand at room temperature for 24 hours. ⁺ , And diluted with phosphate buffered saline (PBS, pH 7.4). 50 μl of the sample was added to 100 μl of the diluted solution, and the solution was allowed to stand for 5 minutes, and the absorbance was measured. The ABTS radical scavenging activity was expressed as the concentration of the sample required to reduce the absorbance of the control without addition of the sample to 1/2 as shown in Equation 2 below.

The ABTS radical cation scavenging activity of ABTS + radicals is due to the reaction of 2,2-azino-bis (3-ethyl-benthiazoline-6-sulfonic acid) diammonium salt (ABTS) with potassium persulfate. Both antioxidants and chain breaking antioxidants can be measured.

The results of the measurement of ABTS radical scavenging ability of Bucheoncho were shown in Fig. In the case of Bukul Pyongcho ethanol extract, 36.7% at 10 μg / ml showed better ABTS radical scavenging ability than hot water extract. The fraction of the solvent fraction of Buchungchon ethanol extract showed 44.2% scavenging activity at the concentration of 10 μg / ml of EtOAC fraction and showed the best ABTS radical scavenging ability among the fractions.

Experimental Example  2. MTT assay Cells by Survival rate  Measure

Cell viability was measured according to a method such as Carmichael (Carmichael, J., DeGraff WG, Gazdar AF, Minna JD, Mitchell JB and 1987. Evaluation of a tetrazolium based semiautomated colorimetric assay:.. Assessment of chemosen- sitivity testing Cancer Res . 47 , 936-942.).

2-1. Cell culture

B16F10 melanoma cells were obtained from Michikawa [Michikawa, M., KT Lim, JG McLarnon and SU Kim. 1994. Oxygen radical-induced neurotoxicity in spinal cord neuron cultures. J. Neurosci Res. 37, 62-70. The culture broth was incubated with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (DMEM) supplemented with Dulbecco's modified eagle's medium penicillin / streptomycin) (100 U / ml) was added and incubated at 37 ° C in a 5% CO ₂ incubator.

2-2. Cell viability measurement

Each cell line [melanoma (B16F10), fibroblast (CCD-986sk)] was dispensed in a 96-well plate at a concentration of 0.6 to 8 × 10 ³ cells / well in an amount of 0.18 ml, and 0.02 ml was added to each sample. And cultured in a 5% CO ₂ incubator for 24 hours. In the control group, the same amount of distilled water as that of the sample was added and the cells were cultured under the same conditions. After adding 0.02 ml of MTT solution (5 mg / ml) for 4 hours, the culture medium was removed. 0.15 ml of DMSO: ethanol (1: 1) was added to each well and reacted at room temperature for 30 minutes. Absorbance was measured at 550 nm.

As a result of measuring the cell survival rate of CCD986sk cells, supernatant ethanol, hot water extract and solvent fraction showed cell survival rate of 80% or more at a concentration of 25 μg / ml (FIGS. 3 and 4). Based on the cell viability results, Puccio ethanol, hot-water extract and solvent fractions were tested for procollagen biosynthesis and MMP-1 activity at a concentration of 25 μg / ml.

EXPERIMENTAL EXAMPLE 3 Experiment of wrinkle-improving effect

3-1. Measurement of Elastase Inhibitory Activity

(Cannell RJP, Kellan SJ, Owsians AM and Walker JM. (1988) Results of a (a) Test for the Elastase Inhibitory Activity of the Samples of the Examples large scale screen of microalgae for the production of protease inhibitors. Planta Media . 54 (1), 10-14.)

N-succinyl- (L-Ala) 3-Aster glehni-nitroanilide was used as a substrate and the amount of Aster glehni-nitroanilide produced from the substrate at 37 ° C for 20 minutes was measured at 405 nm. In other words, prepare each test solution to a constant concentration, take 0.5 mL each in a test tube, add 0.5 mL of porcine pancreas elastase (2.5 U / mL, Sigma Chemical Co) dissolved in 50 mM tris-HCl buffer (pH 8.6) N-succinyl- (L-Ala) 3-Aster glehni-nitroanilide (0.5 mg / mL) dissolved in 50 mM tris-HCl buffer (pH 8.6) Elastase inhibitory activity was represented by the absorbance decreasing rate of the addition solution of the sample solution and the non-addition solution as shown in the following equation (3).

As a result of this experiment, elastase inhibitory activity related to wrinkle formation was measured as shown in FIG. In the case of Bukul Pyongcho ethanol, the inhibitory effect of elastase was higher than that of the hot water extract (7.2%) at the concentration of 1,000 μg / ml of 27.3%. The fraction of EtOAC fraction was the best at 28.5% at the concentration of 1,000 μg / ml.

2-2. Collagenase  Measurement of inhibitory activity

In order to test the collagenase inhibitory activity of the samples obtained in the examples, the following experiment was carried out by applying the method described in the literature (WE and Heindrich HG. (1963) Zur quantitativen bestimmung der collagenase. Hoppe- Seyler's. Physiol . Chem . 333: 149-151.)

Specifically, 0.25 ml of a substrate solution prepared by adding 4 mM CaCl ₂ to 0.1 M tris-HCl buffer (pH 7.5) and dissolving 4-phenylazo benzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg 0.1 ml of collagenase (0.2 mg / ml) was added to the mixture. After incubation at room temperature for 20 minutes, 0.5 ml of 6% citric acid was added to stop the reaction, and 1.5 ml of ethyl acetate was added thereto. Were measured. The collagenase inhibitory activity was expressed by the absorbance decreasing rate of the addition solution of the sample solution and the non-addition solution as shown in the following equation (4).

The collagenase inhibitory activity of hydrothermal, ethanol extracts and solvent fractions of Bucheoncho was measured (Fig. 6). As a result of the measurement, the ethanol extract showed a collagenase inhibitory activity of 83.8% and the hot water extract of 77.2% at a concentration of 1,000 μg / ml. In addition, the EtOAC fraction showed the highest inhibition of collagenase activity by 84.3% at a concentration of 1,000 μg / ml.

2-3. Pro-collagen biosynthesis assay

In order to test the effect of the samples obtained in the examples on the pro-collagen biosynthesis, the following methods were applied to the methods described in the literature (Kim MJ, Kim JY, Choi SW, Hong JT and Yoon KS. (2004) Anti-wrinkle effect of safflower ( Cathamus tinctorius ) seed extract. J. Soc . Cosmet . Scientisis Korea. 30 (1), 15-22.).

Procollagen Type-I C-Peptide (PIP) EIA kit (# MK101) was purchased from TAKARA Bio Inc. to measure the amount of pro-collagen type I synthesis when CCD-986sk cells (ATCC) (Shiga, Japan). The cells were inoculated on a 96-well plate at a density of 5 × 10 ⁴ cells / well in a 12-well plate, and then stabilized for 24 hours. Thereafter, the cultured medium was removed, and the sample extracts were treated at different concentrations, followed by incubation for 48 hours in a CO ₂ incubator. The supernatant from each well was recovered and added to each well of procollagen Type-I C-Peptide (PIP) EIA kit, and then the total amount of procollagen type I was measured according to the manufacturer's method.

The amount of collagen biosynthesis by procollagen type Ⅰ C-peptide (PICP) enzyme immunoassay for fibroblasts was measured and is shown in FIG. The amount of procollagen biosynthesis was 45.1% and 37.2% at the concentration of 25 μg / ml for Puchoncho ethanol and hot water extract, respectively. Bucolic acid fraction showed the activity of Procollagen biosynthesis in the order of EtOAC (57.2%)> BuOH (41.5%)> water (42.8%)> hexane (32.3%).

2-4. Measurement of inhibitory activity of MMP-1

To test the MMP-1 inhibitory activity of the samples obtained in the Examples, the method described in the literature was applied as follows (Shim JS, Choi EJ, Lee CW, Kim HS and Hwang JK. (2009) Matrix metalloproteinase-1 inhibitory activity of Kaempferia pandurata Roxb. J of food 12 (3), 601-607.).

Cells were inoculated on a 12-well plate at a concentration of 5 × 10 ⁴ cells / well, and samples were added to each well and cultured in a CO ₂ incubator for 48 hours. At this time, TNF-α was added at a concentration of 10 ng / mL to increase the activity of MMP-1. Cell cultures were harvested and counted using the matrix metalloproteinase-1 biotrack activity assay kit (D & D system, DMP100).

The results of measurement of MMP-1 inhibitory activity of Buchungcho were shown in FIG. As a result, the ethanol extract of Buchungcho showed 17.5% at 25 μg / ml, which is more effective than the water extract. The EtOAC fraction showed an inhibition rate of 31.7% as compared with the control, and the MMP-1 inhibitory activity was the highest among the fractions.

Experimental Example 4. Whitening activity test

4-1. Tyrosinase inhibitory activity

In order to test the tyrosinase inhibitory activity of the samples obtained in the examples, the following experiment was conducted by applying the method of Yagi et al. (Yagi A, Kanbara T and Morinobu N. (1986)) to the effect of tyrosinase inhibition aloe, Planta Media , 3981, 517-519.)

To the reaction mixture, 0.2 mL of mushroom tyrosinase (110 U / mL) was added to a mixture of 0.2 mL of the substrate solution in which 10 mM L-DOPA was dissolved in 0.5 mL of 0.175 M sodium phosphate buffer (pH 6.8) and 0.1 mL of the sample solution, DOPA chrome produced in the reaction solution was measured at 475 nm for 2 minutes. The tyrosinase inhibitory activity was expressed by the absorbance decreasing rate of the addition solution of the sample solution and the non-addition solution as shown in the following equation (5).

As a result, the tyrosinase-inhibiting activity of mushroom-derived tyrosinase inhibitory activity, which can effectively inhibit melanin polymer biosynthesis in the skin, is shown in FIG. As a result, the inhibition rate of Bu Puiljia ethanol extract was 25.8% at the concentration of 1,000 μg / ml and 8.7% of the water extract. Among the solvent fractions, the EtOAC fraction showed the inhibition rate of 38.2% at the concentration of 1,000 μg / ml, showing the most excellent tyrosinase inhibitory effect.

4-2. Melanin ( melanin ) Biosynthesis inhibition rate

In order to test the melanin biosynthesis inhibitory activity of skin melanoma cells of the samples obtained in the examples, a method of measuring the inhibition rate of melanin biosynthesis in Melanoma cell (B16F10) disclosed in Hosoi et al. (Hosoi J. Abe E, Suda T and Kuroki T. (1985) Regulation of melanin synthesis of B16 mouse melanoma cells by 1 alpha, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res 45 (4), 1474-1478 .)

The melanoma cells cultured in the DMEM medium were divided into 2 × 10 ⁶ cells / dish in a 100 mm culture dish. After culturing for 24 hours, 2 ml of the sample was prepared by concentration, and after 48 hours, the cells were incubated with phosphate buffer (pH 7.4) And washed. The cells were then desalted with 0.25 M trypsin-EDTA solution, and the harvested cells were treated with 1 ml of 5% TCA per 1 × 10 ⁶ cells, centrifuged twice at 2,500 rpm, and the separated melanin was washed with phosphate buffer After that, 1 ml of ether: ethanol (1: 3) is added, centrifuged twice, and washed with 1 ml of ether. 1 ml of 1N NaOH was added to the dried melanin, reacted at 80 ° C for 1 hour, and the absorbance was measured at 405 nm by a spectrophotometer. The inhibition of melanin biosynthesis was expressed by the absorbance decreasing rate of the addition solution of the sample solution and the non-addition solution as shown in the following equation (6).

Melanin biosynthesis in the extract of melanoma cells was examined. The survival rate of melanoma cells by Puchoncho ethanol, hot water extract and fraction of solvent was examined. As a result, the survival rate of melanoma cells was 80% or more at 25 μg / .

4-3. Intracellular tyrosinase inhibitory activity

In order to test the cellular tyrosinase inhibitory activity of the samples obtained in the examples, Choi et al. (1995) and Choi et al. (1998) Screening of the tyrosinase inhibitors from marine algae and medicinal plants Kor . J. Pharmacogn . 29 (3), 237-242.)

B16F10 melanoma cells (ATCC) were inoculated in 6 × 5 × 10 ⁴ cells and cultured for 24 hours. Each sample was treated for 48 hours. After washing with PBS twice, lysis buffer (1% triton X-100, 0.1M sodium phosphate buffer, 50 mM PMSF, pH 6.8) was added to each well. Cells were disrupted on ice and centrifuged, and only the supernatant was collected and used as the enzyme solution. L-DOPA was dissolved in 0.1 M sodium phosphate buffer (pH 6.8) at a concentration of 2 mg / mL to prepare a substrate. The enzyme solution (40 μL) was added to 160 μL of the substrate and incubated at 37 ° C. for 1 hour. The amount of DOPA chrome produced was measured at 490 nm (1-absorbance of the sample / absorbance of the control) × 100.

As a result, the inhibitory activity against melanin biosynthesis of Puchoncho ethanol and hot water extract was measured as shown in FIG. The concentration of 25 μg / ml of Buchyeongcho ethanol and hot water extracts showed inhibitory activity of 9.6% and 0.8%, respectively. The fractions of EtOAC fraction showed 16.5% inhibition of melanin biosynthesis.

Hereinafter, formulations of cream, massage cream, lotion, skin lotion, essence, pack, and cleansing foam are exemplified as the formulation examples of the present invention, but the formulations including the cosmetic composition of the present invention are not limited thereto.

Formulation Example  1. Cream composition

The oil phase and water phase are heated to 75 ° C and cooled to room temperature.

Formulation Example  2. Massage Cream  Composition

The oil phase and water phase are mixed by heating at 75 DEG C and then cooled to room temperature.

Formulation Example  3. lotion composition

The oil phase and water phase are mixed and emulsified by heating at 75 ° C and then cooled to room temperature.

Formulation Example 4. Skin lotion composition

The water phase and the ethanol phase are respectively prepared and mixed and then filtered.

Formulation Example  5. Essence composition

The water phase and the ethanol phase are respectively prepared and mixed and then filtered.

Formulation Example  6. Pack composition

The water phase and the ethanol phase are dispersively dissolved and mixed, and then cooled to room temperature.

Formulation Example  7. Cleansing Foam  Composition

The water phase and the oil phase are dispersed and dissolved, mixed and sieved, and then cooled to room temperature.

Claims (8)

A skin pharmaceutical composition for treating and preventing whitening and skin aging comprising an ethyl acetate fraction of Puchoncho ethanol extract as an active ingredient. delete delete delete delete The dermatological pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment, a pasta or a cataplasma. A cosmetic composition for improving and preventing whitening and skin aging comprising an ethyl acetate fraction of an ethanol extract of Buchung - The cosmetic composition according to claim 7, wherein the cosmetic composition is a formulation of lotion, skin, lotion, nutrition lotion, nutritional cream, massage cream, essence, pack.

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