KR20220156119A - Cosmetic composition comprising extract of white clover for enhancing skin barrier - Google Patents

Abstract

The present invention relates to a cosmetic composition for strengthening a skin barrier containing a white clover extract as an active ingredient. By increasing expression of aquaporin-3, ochluddin, and claudin-1 which are major factors for strengthening the skin barrier, the white extract maintains skin health. In addition, the present invention relates to a cosmetic composition for improving skin winkles and moisturizing skin containing a white clover extract as an active ingredient. When using the white clover extract, the white clover extract is usefully used as a cosmetic for improving the wrinkles and moisturizing the skin by providing a fibroblast proliferation effect, a collagen biosynthesis-promoting effect, an MMP-1 production inhibiting effect, an elastase inhibitory activity effect, a complex effect of preventing and improving the wrinkles, and an effect of improving skin moisturizing due to an increase of an amount of skin moisture.

Description

Cosmetic composition for enhancing skin barrier containing extract of white clover as an active ingredient

The present invention relates to a cosmetic composition for strengthening the skin barrier, improving skin wrinkles, and moisturizing the skin by containing, as an active ingredient, a white clover extract containing phytosphingosine, phytoceramide, and an extract containing a large amount of sphingolipids.

The skin performs a variety of essential functions for the survival of the human body. The barrier function to maintain homeostasis inside the human body in response to environmental changes, the sensory function to recognize external changes, and the temperature control function belong to the most representative functions of the skin, all of which are internal functions of the human body in response to changes in the external environment. plays a role in maintaining homeostasis. Among various functions of the skin, in particular, the barrier function of the skin is mainly represented by the stratum corneum located in the outermost layer of the skin. However, according to recent research results, it has been reported that the stratum corneum affects not only the simple barrier function but also the function, role, and structure of the inner living cell layer, that is, the epidermal layer or the dermal layer, and its importance is continuously increasing. . As the importance of the stratum corneum is emphasized, research on this has been actively conducted, and in particular, studies on the structure and function of keratinocytes present in the stratum corneum are being actively conducted. There are two types of pores associated with water homeostasis in the epidermis: Aquaporins (AQP) and Tight Junction (TJ). Transport of water and solvents is carried out transcellularly through the cell via aquaporins (AQPs) and paracellularly through the extracellular space via tight junctions. Tight junction and tight junction-proteins act as a normal barrier to prevent TEWL in healthy skin and are upregulated when the epidermis is damaged or damaged, acting as a structural system. As such, Aquaporins (AQP), a skin factor for strengthening the skin barrier function and recovering the damaged barrier, is an integral membrane protein that acts as a water transporter, and there are 13 types (0-12) in mammals, and in addition to water, glycerol etc. can transport Among them, AQP1, 3, and 5 are expressed in the skin, and aquaporin 3 (AQP3) is mainly expressed in the basal layer of the epidermis. In atopic dermatitis and psoriasis, AQP3 expression is more strongly expressed than in normal tissues from the basal layer to the spinous layer. Judging from the increased expression of AQP3 in atopic dermatitis and psoriasis, it plays an important role in water transport and moisturizing, which promotes transdermal water loss and is related to the induction of dry skin. In addition, the tight junction (TJ) exists in the intercellular space of the granular cell layer of the epidermis, and is located on the outermost part of the above intercellular junction (ICJ) structures, that is, on the uppermost part of the epidermis, from the external environment to the internal It plays a beneficial role in protecting organs and maintaining homeostasis of the human body. These TJ structures include TJ-associated proteins such as occludin (OC), claudin, and junctional adhesion molecules. These bind to the actin-cytoskeleton and cytosolic regulatory proteins via plaque proteins (zona occludens-1 & cingulin) present in the cytoplasm, and are involved in adhesion between cells and around the cell. It has a biological barrier function that regulates the movement of water-containing solutes and water through space. Among TJs, occludin is a representative protein component of TJ discovered the earliest, and its expression level is known to have a high correlation with the biological function of TJ.

In addition, in order to protect the skin in terms of other mechanisms, it is necessary to suppress aging of the skin. Skin aging is divided into intrinsic aging caused by physiological aging and extrinsic aging caused by continuous exposure to ultraviolet rays. Intrinsic aging is caused by a decrease in the function and cell number of fibroblasts with age, resulting in a decrease in the amount of synthesis of extracellular matrix protein fibers such as collagen, elastin, and fibrillin, and a decrease in elasticity due to loosening of the structure and loss of moisture in skin cells. The structure of the stratum corneum changes. Extrinsic aging occurs when external factors such as ultraviolet rays generate reactive oxygen species (ROS) to activate various signal transduction systems, thereby increasing inflammatory responses and actions by activating activator protein-1 (AP-1). Aging occurs because lipids, proteins, nucleic acids, and enzymes are damaged. All aging occurs due to structural changes in the extracellular matrix present in the dermal layer of the skin. A representative symptom of the component is the occurrence of fine lines and wrinkles caused by a decrease in elasticity, which can be said to be caused by a significant decrease in collagen, a skin elasticity component protein, among collagen in skin dermal tissue. In addition, sebum components and natural moisturizing factors exist in the stratum corneum of the skin to keep the skin always moist. When you get older or in a dry season, you cannot maintain enough moisture in the stratum corneum with only these natural moisturizing factors, so you can supply moisture with cosmetics. should do In particular, moisturizers prevent transepidermal water loss in the damaged epidermal layer, supply moisture to soften rough skin, and increase skin flexibility by filling irregular spaces created by exfoliated corneocytes. it is necessary to do

White clover ( Trifolium repense L.) is a single to perennial legume herb, straight-rooted, with short roots and very few stumps. It can be produced in the abnormal part of the protuberance and propagates as an independent object in the root node. In general, each leaf has a 'V'-shaped white pattern in the middle of the leaf. Because the flowers are particularly white, they are commonly called white clover. This white clover is used as a medicinal herb as well as a health food, and it is a very valuable mountain herb that can be used for food as well as leaves and flowers (Korean resource plants, 1996, 248P, Kim Tae-jeong). The flowers are good to eat fried, and you can enjoy a refreshing taste when you make a salad with the leaves. In addition, white clover, which is an excellent ingredient for healthy food, is also used for green juice and raw food. In addition, there is a record that white clover was used to relieve pain such as toothache in Western folk medicine. Raw leaves are effective in hemostasis and inflammation relief, so if you have purulent abrasions or burns, you can apply them as an emergency treatment. Such white clover is best collected in spring, and when collected in summer, it is tough to cook and eat, but it can continue to be used for green juice. White clover, a natural health food, is effective in pulmonary tuberculosis, asthma, cold, jaundice, diuresis, and fever, and is also effective in hemostasis, relieving inflammation, and burns. In addition, there is a report that it is effective for breast cancer, and in this regard, Korean Patent Publication No. 10-2001-0111912 includes a complex of isoflavone and tamoxifen, which are concentrated forms derived from soybeans or clover, to prevent or minimize the occurrence or proliferation of breast cancer. Or a composition for suppressing it is described. On the other hand, unlike white clover, red clover ( Trifolium pratense ) with red petals is larger than normal white clover and has different scientific names, and is in the spotlight for its individual characteristics.

Accordingly, the present inventors, while studying various physiological activities of white clover, confirmed that the white clover extract has functions of strengthening the skin barrier, improving skin wrinkles, and moisturizing the skin, thereby completing the present invention.

Republic of Korea Patent Registration No. 10-0545043 (Title of Invention: Cosmetic composition containing a particle-type agent containing white clover extract, Applicant: Nadry Cosmetics Co., Ltd., Registration date: January 13, 2006) Republic of Korea Patent Registration No. 10-1756812 (Title of Invention: Cosmetic composition for skin moisturizing including fermented extract and oxygenated water, Applicant: KOIS Co., Ltd. Registration date: July 5, 2017) Republic of Korea Patent Publication No. 10-2001-0111912 (Title of Invention: Composition and Method for Prevention or Treatment of Breast Cancer, Applicant: Sole LLC, Publication Date: December 20, 20101)

Plant Resources in Korea, 1996, 248P, Taejeong Kim

An object of the present invention is to provide a cosmetic composition for strengthening the skin barrier, improving skin wrinkles, and moisturizing the skin by containing a white clover extract containing an extract containing phytosphingosine, phytoceramide and a large amount of sphingolipid as an active ingredient is in

The present invention relates to a cosmetic composition for strengthening the skin barrier containing a white clover extract.

The white clover extract may be extracted using a mixed solution of chloroform and C1-C4 alcohol as a solvent.

The cosmetic composition has a function of improving skin wrinkles or moisturizing the skin.

The cosmetic composition is preferably characterized in that it has a fibroblast proliferation effect, collagen production effect, MMP-1 inhibitory effect, elastase activity inhibitory effect, water loss inhibitory effect, or water content increasing effect.

The cosmetic composition has an effect of increasing gene expression of aquaporin-3, occludin or claudin-1.

Hereinafter, the present invention will be described in detail.

The white clover extract may be extracted using a mixed solution of chloroform and C1-C4 alcohol as a solvent, and the C1-C4 alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and isobutanol.

The mixed solution of chloroform and C1-C4 alcohol used in the preparation of the white clover extract may use 1 to 40 times the volume (1 to 40 liters based on 1 kg) based on the weight of white clover used, and preferably 5 to 40 times the volume can be used The extraction condition of the white clover extract may be 1 minute to 48 hours at 20 to 100 ° C. The above process may be repeated from 1 to 4 times.

The white clover extract is obtained by a known method used for separation and extraction of plant components, such as extraction with an organic solvent (alcohol, ether, acetone, etc.), partitioning of hexane and water, and column chromatography according to a conventional method, alone or It may be used after fractionation or purification using a suitably combined method.

The chromatography is silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography chromatography), thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

As the formulation of the cosmetic composition, It can be prepared in any formulation commonly manufactured in the art, and can be used in essence, lotion, emulsion, pack, hand cream, foot cream, lip balm, lipstick, eye shadow, eye liner, eyebrow pencil, blush, highlighter, general Lotion, toner, cream, serum, beauty soap, softening lotion, medicated lotion, body cleanser, cleansing foam, cleansing lotion, gel, cleansing oil, cleansing cream, shampoo, conditioner, hair treatment, hair lotion, cleansing tissue and cleansing water You can provide what you choose from.

The formulation of the cosmetic composition preferably contains 0.1 to 10% by weight of the white clover extract.

The cosmetic composition of the present invention may further include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extracts.

Any water-soluble vitamin can be used as long as it can be formulated into cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, etc. salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium salt of ascorbic acid-2-phosphate, etc.) included Water-soluble vitamins can be obtained by conventional methods such as a microbial transformation method, a purification method from a microbial culture, an enzymatic method, or a chemical synthesis method.

Any useful vitamin may be used as long as it can be formulated into cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (DL-alpha tocopherol, D-alpha tocopherol, D-alpha tocopherol), etc. are mentioned. , their derivatives (ascorbine palmitate, ascorbine stearate, ascorbine dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherol nicotinate, vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenylethyl ether, etc.) and the like are also included in the useful vitamins used in the present invention. The useful vitamins can be obtained by conventional methods such as microbial transformation, purification from microbial culture, enzymatic or chemical synthesis.

Any polymer peptide may be used as long as it can be blended into the cosmetic composition, but preferably collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin and the like are mentioned. Polymer peptides can be purified and obtained by conventional methods such as a purification method from a culture medium of microorganisms, an enzyme method, or a chemical synthesis method, or can be used after being purified from natural products such as pig or cow dermis or silkworm silk fibers.

Any polymeric polysaccharide may be used as long as it can be incorporated into the cosmetic composition, but hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt or the like) is preferable. For example, chondroitin sulfate or a salt thereof can be used after being purified from mammals or fish.

Any sphingolipid can be used as long as it can be incorporated into the cosmetic composition, but ceramide, phytosphingosine, sphingoglycolipid and the like are preferable. Sphingolipids can be purified by conventional methods or obtained by chemical synthesis from mammals, fish, shellfish, yeast, or plants.

Any seaweed extract can be used as long as it can be formulated into a cosmetic composition, but brown algae extract, red algae extract, green algae extract, etc. are preferable, and calrageenan, arginic acid, and sodium alginate purified from these seaweed extracts , potassium alginate, etc. are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purifying from seaweed by a conventional method.

The cosmetic composition of the present invention may also contain other ingredients that are commonly used in cosmetics.

Other ingredients that may be added include fats and oils, humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, A blood circulation accelerator, a cooling agent, an antiperspirant, purified water, etc. are mentioned.

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

Examples of ester oils include glyceryl tri-2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, and stearic acid. Butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, adipine Diisopropyl acid, isoalkyl neopentanoate, tri(capryl, capric acid) glyceryl, trimethylolpropane tri2-ethylhexanoate, trimethylolpropane triisostearate, pentaelislitol tetra2-ethylhexanoate , Cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinooleate, isostea laurate Lil, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, 2-ethylhexanoate Aryl, 2-ethyl stearyl hexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprate, di(caprylic, capric acid) propylene glycol, propylene glycol dicaprylate, dicap Neopentyl glycol prinate, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl triundecylate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate , Octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerin oleate, polyglycerin isostearate, Triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyltriethyl citrate, acetyltributyl citrate, trioctyl citrate, dimalate Isostearyl, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, Cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phyteryl isostearate, phyteryl oleate, 12-stealoyl and esters such as isocetyl hydroxystearate, stearyl 12-stealoylhydroxystearate, and isostearyl 12-stealoylhydroxystearate.

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

Examples of silicone oils include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane/methylcetyloxysiloxane copolymer, dimethylsiloxane/methylstealoxysiloxane copolymer, alkyl Modified silicone oil, amino modified silicone oil, etc. are mentioned.

Examples of fluorine-based fats and oils include perfluoropolyether and the like.

Animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, birdflower oil, soybean oil, corn oil, rapeseed oil, algae oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil , cottonseed oil, palm oil, kukui nut oil, wheat germ oil, rice germ oil, shea butter, moonshine colostrum, marker damian nut oil, meadowsweet oil, egg yolk oil, beef tallow, horse oil, mink oil, orange raffia oil, jojoba oil , animal or plant fats and oils such as candelilla wax, carnaba wax, liquid lanolin, and hydrogenated castor oil.

Examples of the moisturizer include water-soluble low-molecular moisturizers, fat-soluble molecular moisturizers, water-soluble polymers, and oil-soluble polymers.

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

Cholesterol, cholesterol ester, etc. are mentioned as a fat-soluble low-molecular moisturizer.

Examples of water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water-soluble chitin, chitosan, dextrin, and the like. can

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

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

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

Nonionic surfactants include self-emulsifying glycerin monostearate, propylene glycol fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, sorbitan fatty acid esters, POE (polyoxyethylene) sorbitan fatty acid esters, POE sorbitan fatty acid esters, POE Glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POE·POP (polyoxyethylene·polyoxypropylene) copolymer, POE·POP alkyl ether, polyether modified silicone, lauric acid alkanolamides, alkylamine oxides, hydrogenated soybean phospholipids, and the like.

Anionic surfactants include fatty acid soaps, alpha-acyl sulfonates, alkyl sulfonates, alkyl allyl sulfonates, alkylnaphthalene sulfonates, alkyl sulfates, POE alkyl ether sulfates, alkylamide sulfates, alkyl phosphates, POE alkyl phosphates, and alkylamides. Phosphates, alkyloylalkyl taurine salts, N-acylamino acid salts, POE alkyl ether carboxylates, alkyl sulfosuccinates, sodium alkyl sulfoacetates, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphate esters, and the like. have.

As the cationic surfactant, alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, behenyltrimethylammonium bromide, chloride Benzalkonium, stearic acid diethylaminoethylamide, stearic acid dimethylaminopropylamide, lanolin derivative quaternary ammonium salt, etc. are mentioned.

Amphoteric surfactants include carboxybetaine type, amidebetaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type, amideamine type, etc. An amphoteric surfactant etc. are mentioned.

As organic and inorganic pigments, silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium-coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide , inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine and complexes thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenolic resin, fluororesin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene/styrene copolymer, organic pigments such as silk powder, cellulose, CI pigment yellow and CI pigment orange, and composite pigments of these inorganic pigments and organic pigments.

Examples of the organic powder include metal soaps such as calcium stearate; Alkyl phosphate metal salts, such as zinc sodium cetylrate, zinc laurylate, and calcium laurylate; polyvalent metal salts of acyl amino acids such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroyl glycine calcium; polyvalent metal salts of amide sulfonic acids such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoylizine, N-alpha-paritoylolnithine, N-alpha-lauroylarginine, N-alpha-hardened beef fatty acid acylarginine, etc. -acyl basic amino acids; N-acyl polypeptides such as N-lauroyl glycyl glycine; alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene/styrene copolymer, tetrafluoroethylene, and the like.

Examples of UV absorbers include para-aminobenzoic acid, ethyl para-aminobenzoate, amyl para-aminobenzoate, octyl para-aminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, and benzyl cinnamate. , paramethoxycinnamic acid-2-ethoxyethyl, paramethoxycinnamic acid octyl, diparamethoxycinnamic acid mono-2-ethylhexane glyceryl, paramethoxycinnamic acid isopropyl, diisopropyl-diisopropylcinnamic acid ester mixture, uro Cannic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and its salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone sodium disulfonate, dihydroxybenzophenone , tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2'-ethylhexyl-1'-oxy)-1 ,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc. are mentioned.

Bactericides include hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zincophylthione, benzalkonium chloride, photosensitizer So No. 301, mononitroguacol sodium, undecyrenic acid, etc. are mentioned.

Examples of antioxidants include butylhydroxyanisole, propyl gallic acid, and elisorbic acid.

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

Examples of alcohol include higher alcohols such as cetyl alcohol.

In addition, the blending components that may be added are not limited to these, and any of the above components can be blended within a range not impairing the objects and effects of the present invention, but is preferably 0.01 to 5% by weight, more preferably 0.01 to 5% by weight relative to the total weight. may be formulated at 0.01 - 3% by weight.

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

Ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetics in addition to the extract as an active ingredient, for example, conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and fragrances, and contains a carrier.

When the cosmetic composition formulation of the present invention is a paste, cream or gel, animal fibers, vegetable fibers, wax, paraffin, starch, tracanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. can be used

When the cosmetic composition formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluorohydrocarbon , propellants such as propane/butane or dimethyl ether.

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

When the cosmetic composition formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the cosmetic composition formulation of the present invention is a surfactant-containing cleanser, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

The present invention relates to a cosmetic composition for strengthening the skin barrier containing a white clover extract as an active ingredient. The white extract maintains a healthy state of the skin by increasing the expression of aquaporin-3, occludin, and claudin-1, which are major factors for strengthening the skin barrier.

In addition, the present invention relates to a cosmetic composition for improving skin wrinkles and moisturizing the skin, containing an extract of white clover as an active ingredient. The use of white clover extract shows fibroblast proliferation effect, collagen biosynthesis promotion effect, MMP-1 production inhibition effect, elastase inhibitory effect, and increase in skin moisture content, etc., showing complex wrinkle prevention and improvement effects and skin moisturizing improvement effects Therefore, it can be usefully used as a cosmetic for improving wrinkles and moisturizing the skin.

1 is a photograph showing an example of a process for preparing a white clover extract.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the invention to those skilled in the art, so that the disclosure herein will be thorough and complete.

Preparation Example 1: Preparation of white clover extract

White clover leaves and stems were washed with purified water, dried, and white clover was pulverized using a mixer. Next, a chloroform:methanol (2:1, v/v) solution corresponding to 10 times the weight of the pulverized white clover was added as an extraction solvent, followed by extraction and filtration using a paper filter. This extract was concentrated under reduced pressure to obtain a solid phase (see FIG. 1).

Example 1: Analysis of Phytosphingosine and Phytoceramides and Related Sphingolipids in White Clover Extracts

Weigh exactly 10 mg of the white clover extract, add 800 μl of chloroform, 400 μl of methanol, and 300 μl of distilled water thereto, extract for 30 minutes, transfer the chloroform layer to a new tube through centrifugation, and concentrate using a vacuum concentrator. It was redissolved in 1000 μl of methanol and analyzed for phytosphingosine, phytoceramide and related sphingolipids using LC-MS/MS. The analysis results were quantified as pmol or nmol or mmol of phytosphingosine, phytoceramide and related sphingolipids measured per gram of extract.

lipid types content Phytoceramide 0.74 mmol/g Extract Phytosphingosine 0.24 nmol/g Extract Phytosphingosine 1-phosphate 124.27 pmol/g Extract Sphingomyeline 0.19 mmol/g Extract Ceramide 0.18 nmol/g Extract Sphingosine 185.41 pmol/g Extract Sphinganine 24.41 pmol/g Extract Sphingosine 1-phosphate 18.63 pmol/g Extract Sphinganine 1-phosphate 7.56 pmol/g Extract

As can be seen from the results of Table 1, it can be confirmed that the white clover extract contains excessive amounts of phytosphingosine, phytoceramide and related sphingolipids.

Example 2: Skin cell proliferation activity test

After culturing human normal fibroblasts (ATCC) in a 96-well plate using DMEM medium containing 10% (v/v) fetal bovine serum (FBS), the medium was replaced with FBS-free medium and human normal fibroblasts were used as a control. After treatment with ethanol, white clover extract was administered to the cell culture medium at 50 to 500 μg/mL and cultured for 24 hours. Thereafter, 10 μl of CCK-8 reagent (Dojindo) was added and incubated for 3 hours, and absorbance was measured at 450 nm. At this time, the effect of the test drug on human fibroblast cell proliferation was expressed as a growth factor in the absorbance of the control group treated with ethanol.

The measured value was substituted into "cell proliferation effect (%) = absorbance of the experimental group / absorbance of the control group x 100" to calculate the cell proliferation effect, and the results are shown in Table 2 below.

White clover extract (μg/mL) Cell proliferation effect (%) control group 100.00±6.32 50 118.34±9.52 100 134.68±10.27 200 170.51±11.24 300 172.36±8.74 500 195.23±15.47

As can be seen from the results of Table 2, the white clover extract of the present invention generally induces the proliferation of normal human fibroblasts in a concentration-dependent manner. In particular, the experimental group treated with 500 μg/mL of the white clover extract showed It can be understood that the proliferative effect of 200-fold fibroblasts is exhibited.

Example 3: Collagen synthesis enhancing effect

Human normal fibroblasts (ATCC) were cultured in a 3 x 10 ⁴ cell/well 96-well plate in DMEM medium containing 10% (v/v) fetal bovine serum (FBS) until the cells reached 80-90% growth. After that, 50 to 500 μg/mL of white clover extract was administered to the cell culture medium and cultured for 36 hours. After culturing, the amount of newly synthesized collagen was measured by collecting the supernatant of each well and measuring the amount of procollagen type IC-peptide (PICP) using a kit (TAKARA, Japan). The amount of PICP was expressed in terms of ng/10 ⁵ cells.

White clover extract (μg/mL) Amount of collagen synthesis (ng/10 ⁵ cell) control group 491.87±35.41 50 511.35±41.79 100 637.97±87.32 200 745.11±62.88 300 813.29±84.00 500 969.13±59.81

Referring to Table 3, when compared to the control group not treated with the white clover extract, it can be confirmed that the experimental group treated with the white clover extract exhibited a remarkably excellent collagen synthesis enhancement effect in fibroblasts.

Example 4: Collagenase inhibitory effect

Human normal fibroblasts (ATCC) were cultured in a 3 x 10 ⁴ cell/well 96-well plate in DMEM medium containing 10% (v/v) fetal bovine serum (FBS) until the cells reached 80-90% growth. After that, the white clover extract was administered to the cell culture medium at 50 to 500 μg/mL and cultured for 24 hours. The degree of collagenase activity of the cell culture medium was measured using a commercially available collagenase measuring instrument (Abcam, USA).

To this end, a color-inducing substance is added to induce color development at room temperature for 15 minutes, and 1M sulfuric acid is added to stop the color reaction. The absorbance of the yellow 96-well was measured at 405 nm using an absorbance meter, and the degree of synthesis of collagenase was calculated.

White clover extract (μg/mL) Collagenase expression (%) control group 100.00±7.55 50 97.23±6.72 100 87.39±5.79 200 80.17±8.71 300 73.99±7.12 500 65.28±8.44

As a result, referring to Table 4, the reaction absorbance of the cell culture medium not treated with the white clover extract was used as a control, and the expression level was shown using the synthetic ability of the control group as 100%. It was confirmed that the ability to inhibit the expression of collagenase in blast cells was increased.

Example 5: MMP-1 production inhibitory effect

Normal human fibroblasts (ATCC) were inoculated into 24-well microplates at 1 x 10 ⁶ cells per well, cultured for 24 hours in DMEM medium and 37°C, and then white clover extract was added at 50 to 500 μg/ml. After administration in mL, cultured in serum-free DMEM medium for 48 hours. After culturing, the supernatant of each well was collected and the amount (μg/ml) of newly synthesized MMP-1 was measured using an MMP-1 assay kit (Amersham, USA), and the inhibition rate of MMP-1 production was determined according to the following formula. It was calculated, and the results are shown in Table 4 below. At this time, TGF-β (10 mg/ml, Roche) was used as a positive control for the inhibition of MMP-1 production.

To this end, the measured value was substituted into "MMP-1 production inhibition rate (%) = [1-(amount of MMP-1 in the experimental group/amount of MMP-1 in the control group) x 100]" to calculate the effect of inhibiting MMP-1 production, , The results are shown in Table 5 below.

White clover extract (μg/mL) MMP-1 production inhibition rate (%) 50 23.64±1.96 100 35.41±2.57 200 47.75±3.99 300 56.24±5.41 500 64.12±4.21 TGF-β (1000 μg/mL) 73.21±6.83

Thus, as shown in the results of Table 4, it was confirmed that the white clover-treated experimental group had an increased inhibition rate of MMP-1 production in fibroblasts.

Example 6: Elastase control activity test

Test sample and elastase enzyme solution (1,000 units/mL, Sigma) were mixed in 2.5 wt% agar containing 0.1 wt% Elastin-Congo red (Sigma, USA) and reacted at room temperature for 10 minutes, injecting 10 μl of the solution After incubation at 37 ℃ for 24 hours. After completion of the culture, when a zona pellucida was formed around the Elastin-Congo red agar due to the activity of elastase, the diameter of the halo was measured to measure the effect of inhibiting elastase activity.

While only elastase was added dropwise to an agar medium containing elastin, in order to confirm the elastase inhibitory activity of the white clover extract, as shown in Table 6 below, a solution having a concentration of 50 to 500 μg/mL was used as a sample. It was added dropwise together with elastase to measure the diameter of the halo appearing on the medium to measure the elastase inhibitory activity, and retinol crystal was compared as a control example.

White Clover Extract Concentration (%) Halo diameter (cm) control group 1.8 0.01 1.6 0.05 1.5 0.1 1.2 0.5 1.1 Retinol crystals 0.8

As a result, it can be seen that the experimental group treated with the white clover extract exhibited an excellent elastase inhibitory effect with a significant decrease in halo diameter.

Example 7: Activity test of sphingosine kinase of white clover extract

After culturing human normal fibroblasts (ATCC) in a 6-well plate using DMEM medium containing 10% fetal bovine serum (FBS), the medium was replaced with FBS-free medium, and white clover extract dissolved in ethanol was added with 50 ~ 500 μg/mL After incubation for 24 hours, cells were collected and protein was quantified, and d17:1 sphingosine monophosphate produced from the administration of the substrate d17:1 sphingosine to 100 μg of protein was analyzed using LC-MS/MS. The analysis result was quantified by dividing the pmol of phytosphingosine monophosphate measured per mg protein of cells by dividing the reaction time for 30 minutes.

White clover extract (μg/mL) Sphk activity (pmol/mg protein/min) control group 3.25±0.29 50 3.38±0.33 100 3.75±0.39 200 4.12±0.45 300 4.68±0.26 500 5.59±0.49

As can be seen from the results of Table 7, treatment with the white clover extract can confirm the result of increasing the activity of sphingosine kinase.

Example 8: Intracellular phytosphingosine monophosphate and sphingosine monophosphate accumulation test of white clover extract

After culturing human normal fibroblasts (ATCC) in a 6-well plate using DMEM medium containing 10% fetal bovine serum (FBS), the medium was replaced with FBS-free medium, and white clover extract dissolved in ethanol was added with 50 ~ 500 μg/mL After incubation for 24 hours, the cells were collected, and after protein quantification, 800 μl of chloroform, 400 μl of methanol, and 300 μl of 0.1 M hydrochloric acid were added to 100 μg of protein, extracted for 30 minutes, and centrifuged to remove the chloroform layer into a new tube. After transferring and concentrating using a vacuum concentrator, it was redissolved in 100 μl of methanol and analyzed for phytosphingosine monophosphate using LC-MS/MS.

The assay results were quantified as pmol of phytosphingosine monophosphate measured per mg protein of cells.

white clover extract
(μg/mL) Phytosphingosine
(pmol/mg protein) Phytosphingosine 1-phosphate
(pmol/mg protein) control group 3.47±0.69 ND 50 517.24±42.19 68.12±3.41 100 873.54±59.25 79.23±5.17 200 1354.62±112.03 135.37±10.24 300 1529.73±132.00 169.77±11.83 500 2006.14±160.90 239.71±14.09

As can be seen from the results of Table 8, it was confirmed that the treatment of the white clover extract dissolved in ethanol significantly increased the amount of phytosphingosine monophosphate and sphingosine monophosphate within the cells than the control treatment.

Preparation Example 2: Preparation of cream containing white clover extract

For a clinical test on the skin wrinkle improvement effect, skin moisturizing improvement effect, and skin stability of the cosmetic composition containing the white clover extract of the present invention, a control sample and a test sample cream were prepared according to Table 9, respectively.

ingredient control sample ingredient test sample caprylic/
capric triglyceride 7.0 caprylic/
capric triglyceride 7.0 Triethylhexanoin 0.3 Triethylhexanoin 0.3 phytosterols 5.0 phytosterols 5.0 PEG-40 Stearate 0.02 PEG-40 Stearate 0.02 Cetearyl Olivate 0.03 Cetearyl Olivate 0.03 Sorbitan Olivate 1.00 Sorbitan Olivate 1.00 white clover extract 0.0 white clover extract 5.0 Purified water To 100 Purified water To 100

Example 9: Skin moisturizing improvement effect

The skin moisturizing improvement effect of the test sample and control sample cream of Preparation Example 2 was measured on 20 healthy women aged 35 to 50 years old. As a test substance application method, after washing twice a day in the morning and evening, apply it to the test area (forearm) and control area, visit on a weekly basis, and measure transdermal water loss per unit time (TEWL) with Tewameter TM-300 (Courage+Khazaka, Germany) , transepiderma water loss) was measured. TEWL was measured weekly using a Tewameter TM-300 after washing with a standard face wash, tapping lightly with a paper towel to remove moisture, and resting in constant temperature and humidity conditions (20-24, 40-60% RH) for 30 minutes. The measurement results are summarized in Table 10.

No. Moisture loss before use g/m ² /h Moisture loss after 2 weeks of using control sample cream g/m ² /h Moisture loss after 2 weeks of test sample cream use g/m ² /h One 10.9 7.2 5.1 2 8.3 6.7 4.5 3 8.2 7.4 5.2 4 7.4 6.7 5.2 5 9.1 8.9 4.9 6 8.6 7.6 5.2 7 7.0 6.4 5.0 8 11.4 9.6 4.8 9 9.6 9.1 6.0 10 8.3 9.0 4.8 11 10.0 8.5 6.0 12 10.9 9.2 5.3 13 10.5 9.8 5.3 14 12.0 10.1 4.8 15 9.9 8.4 5.0 16 6.5 6.9 5.1 17 9.4 8.0 5.6 18 9.4 7.6 5.8 19 9.2 7.2 5.3 20 7.4 5.2 5.8 Average 9.2 8.0 5.3 Standard Deviation 1.6 1.3 1.3

As a result, from Table 10, it was found that the test sample cream of Preparation Example 2 containing the clover extract had a very good skin moisturizing effect.

Example 10: Confirmation of gene expression of skin cell barrier enhancement related factors

In order to confirm the gene expression of aquaporin-3, occludin, and claudin-1, the white clover extract sample of Preparation Example 1 was added to human keratinocyte (CRL-2310, ATCC) by concentration and cultured for 24 hours, and Trizol was added to the cells. (Trizol, invitrogen, USA) 1mL was added and separated according to Invitrogen's RNA separation method. For cDNA synthesis, a cDNA synthesis kit (High Capacity RNA-to-cDNA Kit, Applied Biosystems, USA) was used, and for real-time PCR, a Real-time PCR Kit (Power SYBR Green PCR Master Mix, Applied Biosystems, CA, USA) was used. It was used, and primers were synthesized and used in an optimal state by a conventional method. Then, the gene was amplified using Applied Biosystems 7500 FAST Real-Time PCR System (Applied Biosystems, CA, USA), and the amplified product was quantitatively analyzed.

white clover extract
(μg/mL) Aquaporin-3 mRNA
Expression level (%) occludin mRNA
Expression level (%) claudin-1 mRNA
Expression level (%) Control group (untreated group) 100.0±1.2 100.0±3.2 100.0±2.4 100 132.0±0.3 132.4±2.4 162.2±3.1 500 273.4±0.5 253.2±3.3 295.8±1.3 Retinoic acid (1 μM) 153.2±0.4 143.1±2.3 126.3±2.2

Referring to Table 11, it can be seen that the white clover extract of Preparation Example 1 clearly exhibits an effect of enhancing the gene expression of aquaporin-3, occludin, and claudin-1.

In addition, as in the preparation of the white clover extract of Preparation Example 1, a red clover extract was prepared using the same solvent using red clover as a raw material, and a new extract was prepared using only chloroform as a solvent for the white clover extract to compare and confirm its activity was made The results thereof are shown in Table 12, representing the mRNA expression of claudin-1.

Claudin-1 mRNA expression level (%) Control group (untreated group) 100.0±2.5 Red clover extract 100 μg/mL
(Solvent: Same as Preparation Example 1) 134.2±4.1 White clover extract 100 μg/mL
(Solvent: propylene glycol) 128.4±3.9

Referring to Table 12, it can be seen that the mRNA activity of claudin-1 does not significantly increase in the extract obtained by extracting red clover extract or white clover with propylene glycol, which is another solvent, compared to the extract of Preparation Example 1.

Example 12: Confirmation of protein expression of factors related to skin cell barrier enhancement

In order to confirm the gene expression of aquaporin-3, occludin, and claudin-1, the white clover extract sample of Preparation Example 1 was added to human keratinocyte (CRL-2310, ATCC) by concentration, cultured for 24 hours, and cell lysate Cells were lysed.

Western blotting was performed to detect aquaporin-3, occludin, and claudin-1 proteins contained in the cell lysate. Signaling, USA) and anti-claudin-1 (4933S, Cell Signaling, USA) antibodies, and as a control, anti-β-Actin-Peroxidase antibody (clone AC-15, Sigma-Aldrich, Seoul, Korea) was used. Cell lysate was subjected to electrophoresis with 10% (w/v) SDS-PAGE, then proteins were transferred to PVDF and the PVDF membrane was reacted with the primary antibodies at 4° C. overnight. The HRP-conjugated secondary antibody was reacted at room temperature for 1 hour, washed with TBST, and reacted using an ECL reaction kit to confirm the band. The intensity of each band was confirmed based on the expression of β-action. The results for this are shown in Figure 2.

Referring to FIG. 2 , it can be seen that, like gene expression, the white clover extract of Preparation Example 1 of the present invention clearly exhibits an effect of enhancing protein expression of aquaporin-3, occludin, and claudin-1.

Claims (5)

A cosmetic composition for strengthening the skin barrier, comprising a white clover extract. According to claim 1,
The white clover extract is a cosmetic composition for strengthening the skin barrier, characterized in that extracted with a mixed solution of chloroform and C1 ~ C4 alcohol as a solvent. According to claim 1,
The cosmetic composition is a cosmetic composition for strengthening the skin barrier, characterized in that it has a skin wrinkle improvement or skin moisturizing function. According to claim 1,
The cosmetic composition is a cosmetic composition for strengthening the skin barrier, characterized in that it has a fibroblast proliferation effect, collagen production effect, MMP-1 inhibitory effect, elastase activity inhibitory effect, water loss inhibitory effect or water content increasing effect. According to claim 1,
The cosmetic composition is a cosmetic composition for strengthening the skin barrier, characterized in that it has an effect of increasing the gene expression of aquaporin-3, occludin or claudin-1.

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