KR20120139163A - A cosmetic composition comprising rice stem cell extract for an anti-aging activity - Google Patents

Abstract

PURPOSE: A cosmetic composition containing rice plant stem cell extract is provided to proliferate epidermis and dermis of skin and to enhance skin elasticity and anti-wrinkling effect. CONSTITUTION: A cosmetic composition for anti-aging contains rice plant stem cell extract as an active ingredient. The rice plant stem cells are undifferentiated cells derived from Oryza sativa L. leaves, stems, seeds, and roots. The rice plant stem cells are prepared using Oryza sativa L.-derived callus(deposit number KCTC 11760BP). The extract is prepared from rice plant stem cells using phosphate buffer solution, water, ethanol, or a mixture thereof. [Reference numerals] (AA) Left : control; (BB) Right : rice plant stem cell extract 30μg/ml

Description

A cosmetic composition comprising rice stem cell extract for an anti-aging activity}

The present invention relates to a cosmetic composition containing a rice stem cell extract having anti-aging activity.

Skin is the body's primary protective layer that protects organs in the body from temperature and humidity changes, ultraviolet rays, pollutants, and other external stimuli, and plays an important role in maintaining body homeostasis. The skin is composed of the uppermost epidermis and the dermis located in the lower epidermis. The epidermis is the middle-sized squamous epithelium, which is mainly occupied by keratinocytes, from the inside to the basal layer, spinous layer, and granular layer It is divided into four layers in the order of the horny layer. In other words, the keratinocytes divide in the basal layer and then move to the surface of the skin to differentiate into various stages.

This important skin, like other organs of the human body, gradually ages with age, and as a result, various changes occur. The basement membrane is the epidermis and dermis boundary structure known to be thinner with age (Vazquez F. et al. Changes of the basement membrane and type IV collagen in human skin during aging.Maturitas . 1996, 25 (3): 209-215), resulting in vulnerable trauma.

It is also well known that the skin is a continuously dividing tissue that is regenerated about every 3-4 weeks, and the skin regeneration ability decreases with age. Accordingly, interest in stem cells that can activate surrounding cells by proliferating and secreting various cell activators through endless cell division has increased.

Stem cells are cells that remain undifferentiated into specific cells and, if necessary, have the ability to differentiate into all kinds of cells that make up a tissue or organ. These stem cell inherent characteristics are called self-renewal and pluripotency, and stemism is a general term for these characteristics.

Recently, with the development of plant cell culture technology using plant totipotency, interest in functional secondary metabolite derived from plant stem cells has been amplified. This secondary metabolite is known to have a variety of physiological activity, and as a new raw material of high-functional cosmetics, plant stem cell-derived extracts are attracting attention. This technology is expected to enable the development and application of new ingredients that have not been used until now because of the economical production of highly functional active substances that existed in nature but were difficult to obtain by chemical synthesis.

In particular, since the development of new functional materials and related technologies has not been enough as consumers' demand for functional cosmetics increases, cosmetics using plant stem cells capable of producing a myriad of new functional ingredients have various skin effects, especially It is expected to be very popular in terms of anti-aging efficacy.

Epidermal stem cells, which are typical of adult stem cells in the skin, are located at the epidermal and dermal borders of the skin, divide into two, one remains as epidermal stem cells, and the other continues to divide and go out into epidermal cells. Differentiate

When the border between the epidermis and the dermis becomes flat due to endogenous aging and photoaging, the area of the epidermis is reduced, and thus, a part of the epidermal stem cell is observed.

Wrinkles occur around the missing portion, which may be problematic in terms of cosmetics. The epidermal cells also affect fibroblasts, which are located underneath the dermal cells, which affect collagen production. The epidermal stem cells are known to inhibit the production of collagen fibers and elastic fibers in the dermal layer.

Collagen is a major matrix protein produced in the fibroblasts of the skin and is present in extracellular epilepsy and accounts for 30% of the total weight of biological proteins and has a spiral structure.

Its main function is known as the main ingredient that gives skin elasticity through functions such as mechanical firmness of skin, resistance of connective tissue and binding of tissue, and support of cell adhesion. However, such collagen is deteriorated in the function of fibroblasts by photoaging due to aging and ultraviolet irradiation, and the production amount thereof is reduced. In general, at 80 years of age, the skin thickness is reduced by 65% compared to 20 years, and this phenomenon is known to be closely related to the wrinkle formation of the skin.

Therefore, there has been a steady effort to develop a bioactive substance having an anti-wrinkle effect in order to prevent skin aging and maintain a healthier and more elastic skin. For example, in 1995, tretinoin (trans-retinoic acid) was approved by the US FDA as a treatment for the improvement of photo-aged skin. Wrinkle improvement cosmetics have begun to be released. Since then, female hormone-like substances and antioxidants extracted from various plants have been introduced into cosmetics as an anti-wrinkle ingredient. However, most of these cosmetic raw materials have various problems such as insufficient efficacy or cause skin side effects such as irritation and troubles.

Rice ( Oryza sativa L.) is an annual herb, and there are many kinds of rice, but it is divided into rice, field rice, and waxy rice according to the viscosity of rice grains. The number of chromosomes is n = 12, 2n = 24 and belongs to AA genome. According to the plant taxonomy, rice belongs to a pizza plant, which is surrounded by an eggplant (an organ that becomes a seed after being formed in the eggplant) among seed plants. It also belongs to a monocotyledonous plant (單 子葉 植物) with one cotyledon in the seed. Rice root is composed of seed root (種子 根), jungbaebaeeun (中 胚軸 根) and root root (冠 根) and absorbs water and nutrients through the root hairs.

Rice divides and develops in the main stem and the primary branch (the branch comes out of the node in the ground of the plant). The stem consists of nodes and nodes. The leaf is divided into leaf body and leaf shop, with leaf tongue and leaf ear in between. The flower contains 6 stamens and 1 pistil and has a pollen gutter.

Rice contains about 75% starch, about 8% gray matter, and about 0.5% to 1% fat. In addition, it contains a small amount of vitamin B. The vitamin content varies depending on the type of rice and the plantation. Fatty parts include ester and free cholesterol, brassicasterol, stigmasterol, sitosterol, monoglyceride, diglyceride, triglyceride, phospholipid, Contains free fatty acids.

Republic of Korea Patent Publication No. 2006-0065770 'Pharmaceutical composition comprising rice extract' in the rice extract inhibits the activity of elastase (elastase), maintain the elasticity of the blood vessels, prevent blood loss, blood flow control and blood pressure Although it is notified about the medicines that can be used for diseases caused by vascular protection and disorders of blood circulation, the contents of cosmetic compositions containing rice stem cell extracts having an anti-aging effect are published or known elsewhere. none.

Accordingly, the present inventors have made diligent research to search for substances that are helpful in improving skin wrinkles and improving skin elasticity. As a result, rice stem cell extracts are effective in improving skin wrinkles and elasticity without causing skin irritation. The present invention was completed by confirming that.

Therefore, the technical problem to be achieved by the present invention is to provide a cosmetic composition comprising a plant stem cell extract excellent in the anti-aging effect of the skin by the wrinkle relief / improvement effect and skin elasticity improving effect.

The present invention provides a cosmetic composition for anti-aging comprising a rice stem cell extract (Rice stem cell extract) as an active ingredient.

Rice stem cells of the present invention, rice ( Oryza sativa L. ) means undifferentiated cells derived from plant tissues, preferably from tissues selected from the group consisting of seeds, leaves, stems and roots, more preferably from seeds, in the present invention the surface after peeling Scutellum-derived callus with germinated sterile and washed seeds can be obtained, and the callus can be prepared as microsuspension cells derived by inoculation / passage in suspension culture. However, the present invention is not limited thereto, and may be obtained by applying a plant tissue culture method known to those skilled in the art. For example, the rice stem cells of the present invention was prepared by modifying the cell line manufacturing method described in Korean Patent Nos. 0542605 and 0897159 as follows:

One) Callus  Establish

Here, the callus may be obtained as in the following Preparation Examples, but is not limited thereto.

Rice ( Oryza sativa L.) Seeds are removed and surface sterilized using hydrous ethanol and NaOCl solution, washed with sterile distilled water and washed in a callus-induced solid medium, preferably Chu (N6) agar. Bright conditions of 10 to 20 hours and dark conditions of 4 to 14 hours at 20 to 30 ° C, preferably 25 ° C; Preferably incubated for 5 to 20 days, preferably 7 to 14 days under 16 hours of light conditions and 8 hours of dark conditions, and then take blastocyst-derived callus from the germinated seeds and transfer to a new Chu (N6) agar medium and diameter Rice-derived callus can be obtained by culturing until it grows to about 0.5-2 cm, preferably about 1 cm. The rice-derived callus prepared as described above in the present invention was deposited on September 7, 2010 at the Korea Colleciton for Type Culture (KCTC) and was assigned the accession number KCTC 11760BP.

2) fine suspension  By culture Rice Stem Cells  Produce

The process of inducing the rice-derived callus to microsuspension cells for liquid culture is described below, but is not limited thereto.

As a medium for culturing microsuspension cells, MS (Murashige & Skoog, 1962) medium, Chu (N6) (Chu et al., 1975) medium, Amino Acid (Thompson, 1986) medium, and the like may be used. Examples of plant growth regulators that may be used include, but are not limited to, 2,4-Dichlorophenoxyacetic acid (2,4-D), kinetin, gibberellic acid (GA3), and the like. Preferably, Chu (N6) medium or AA (amino acid) medium containing 30 g / L sucrose, 2 mg / L 2,4-D, and 0.2 mg / L kinetin may be used. Inoculate and subculture at intervals of two weeks with shaking incubation at 25 to 30 ° C. (preferably 28 ° C.) 90 to 150 rpm, (preferably 110 rpm), dark conditions. At this time, it is preferable to select and passage only the fine suspended cells separated from the cell aggregate. Rice suspension cells 2 to 3 months old, which were passaged and suspended in the above manner, include sucrose 30g / L, 2,4-D 2.0mg / L, kinetin 0.2mg / l and gibberellic acid 0.1mg / L Transfer to AA medium for subculture. Stabilized suspension cells may be cultured without 2,4-D in culture to eliminate the possibility of toxicity due to the retention of plant hormones.

3) Bulk Cultivation

The stabilized suspension cells can be mass cultured using any bioreactor for plant cell culture known to those skilled in the art, such as a stirred tank reactor, an airlift reactor, a fluidized reactor, and the like. Bioreactor modified for plant cell culture by adding gas sparger, hollow-paddle impeller and sampling port sized for plant cell collection Cultured in, but the present invention is not limited thereto. In addition, the culture method may be any method such as batch, continuous or fed-batch.

As the culture conditions, the culture temperature of 25 to 30 ℃ (preferably 28 ℃), the stirring speed of 50 to 150rpm (preferably 90 to 110rpm), dissolved oxygen (DO) level during the culture period is more than 15% ( Preferably 20% or more), and controlling by controlling the aeration rate and the stirring speed to maintain is preferable for the appropriate culture environment composition.

Bioreactor culture alternates between dark and fluorescent conditions, it is recommended to recover the cells after 5 days (preferably 7-8 days). Rice stem cells prepared by removing excess medium, washing, and completely removing the washing solution may be used for extraction immediately or may be stored frozen or frozen until extraction.

Rice stem cell extract of the present invention is one or more extracts selected from the group consisting of water, buffers, C1 to C4 lower alcohols, polyhydric alcohols and mixtures thereof in rice stem cells themselves or powdered rice stem cells frozen by liquid nitrogen Treatment with a solvent, preferably one or more extracting solvents selected from buffers (e.g. phosphate buffer), water, C1-C4 lower anhydrous alcohols or hydrous alcohols thereof, glycerin, ethylene glycol, propylene glycol and butylene glycol, This may be added to extraction methods such as heat extraction, reflux cooling extraction, stirring extraction, ultrasonic extraction, high temperature and high pressure extraction, extraction and filtration to obtain a filtrate.

The rice stem cell extract of the present invention is preferably extracted by lyophilizing and extracting rice stem cells with an extraction solvent selected from phosphate buffer, water, ethanol or a mixture thereof, the extraction is stirred extraction method, ultrasonic extraction method, thermal extraction method And high temperature and high pressure extraction.

However, the present invention is not limited thereto, and examples thereof include manufacturing processes as follows:

i) The rice stem cells are frozen, preferably frozen with LN ₂ and triturated at a temperature of about 1 ° C. to 10 ° C. (preferably about 4 ° C.), followed by water, buffer, C1 to C4 lower alcohol, polyvalent Stirring for about 60 to 360 minutes (preferably 120 to 180 minutes) in one or more extraction solvents (preferably phosphate buffered saline (PBS)) selected from the group consisting of alcohols, or mixtures thereof Extraction process; or

ii) the rice stem cells are suspended in one or more extracting solvents selected from the group consisting of water, buffers, C1 to C4 lower alcohols, polyhydric alcohols, or mixtures thereof, preferably water or hydrous ethanol, about 4 to 30 For about 60 minutes to 360 minutes (preferably 120 minutes to 180 minutes) or at about 100 ° C. to 130 ° C. (preferably between 121 ° C. and 125 ° C.) at a temperature of < RTI ID = 0.0 > The temperature and the pressure of about 1.0 MPa to 2.0 MPa (preferably 1.2 MPa to 1.5 MPa) were applied for about 20 to 30 minutes, crushed and stirred for about 30 to 120 minutes (preferably 60 to 90 minutes) Extraction process.

In the method for obtaining the filtrate, after the filtration, a concentration process may be further performed if necessary.

The present invention provides a cosmetic composition containing the rice stem cell extract obtained by the above manufacturing process as an active ingredient.

In the composition of the present invention, the extract is preferably contained in 0.001 to 10% by weight based on the total weight of the composition. If the extract is less than 0.001% by weight, the elasticity improvement and anti-wrinkle effect due to collagen synthesis is weak due to the lack of useful ingredients, and when the extract exceeds 10% by weight, the cosmetic formulation may become unstable or cause irritation to the user's skin. There is.

In addition, the composition of the present invention exhibits skin elasticity improvement and anti-wrinkle effect through epidermal and dermal cell proliferation, epidermal stem cell proliferation and colony forming effect, and collagen synthesis enhancement effect, which is useful as an anti-aging cosmetic composition. Can be. Accordingly, the anti-aging in the composition of the present invention is due to the effect of improving wrinkles and skin elasticity based on the synthesis of collagen type I or the improvement of wrinkles and skin elasticity based on the growth of epidermal stem cells (WPE-stem cell) ( Lifting effect).

The composition of the present invention may comprise one or more of the components selected from the group consisting of water soluble vitamins, oil soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract.

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

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

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

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

The sphingolipid may be any compound that can be blended into cosmetics. Preferably, ceramide, phytosphingosine, sphingosaccharide lipid, and the like can be given. Sphingo lipids can usually be purified from mammals, fish, shellfish, yeasts or plants by conventional methods or obtained by chemical synthesis.

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

The cosmetic composition of the present invention may be blended with the above components and with other components which are usually formulated into cosmetics as needed.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In addition, the compounding component which may be added other than this is not limited to this, Moreover, Although all said components can be mix | blended within the range which does not impair the objective and effect of this invention, Preferably it is 0.01-50 weight% with respect to the composition total weight, More preferably, it is mix | blended in 5-20 weight%.

The components included in the cosmetic composition of the present invention may further include components conventionally used in the cosmetic composition in addition to the compound, and include, for example, conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments and flavorings, and And a carrier.

In addition, the composition of the present invention may be prepared in any formulation commonly prepared in the industry, for example, paste, cream, gel, powder, spray, solution, emulsion, suspension, etc. have. Specifically, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing foam It may be a formulation selected from, cleansing lotion, cleansing cream, body lotion and body cleanser.

The preparation method of these formulations can be manufactured according to the manufacturing method of a normal cosmetic formulation.

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

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

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

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

When the formulation of the present invention is 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.

Rice stem cell extract of the present invention, the effect of increasing the mechanical tightness of the skin and the resistance of connective tissue and the adhesion of tissues by increasing the epidermal and dermal cells of the skin and promoting the synthesis of collagen to improve wrinkles and elasticity of the skin There is.

1 is an HPLC result graph of the rice stem cell extract of the present invention, the upper portion shows the HPLC fingerprint in the 190 ~ 400nm range, the lower portion shows the HPLC fingerprint at 254nm.
Figure 2 is a photograph of the colony forming assay test results of human epidermal stem cells (WPE-stem cell, keratinocyte progenitor cell, ATCC CRL-2887) of the rice stem cell extract of the present invention, Figure 2A is a micrograph taken at 40 times the ratio 2B is a photograph of a plate stained with 0.2% crystal violet.

Hereinafter, the present invention will be described in more detail with reference to Examples, Examples, Formulation Examples and Test Examples. However, the following description of the reference examples, formulation examples, examples and test examples are intended to illustrate the invention by way of example only, and are not intended to limit or limit the scope of the invention to the contents described therein.

[ Reference Example  One] Rice stem cells  Ready

Rice-derived callus (Accession No .: KCTC 11760BP) was prepared using AA (amino acid) medium containing 30 g / L sucrose, 2 mg / L 2,4-D and 0.2 mg / L kinetin, which are medium for culturing microsuspension cells. Inoculated at 28 ° C., 110 rpm, and cultured with shaking at 2 weeks intervals under dark conditions. At this time, only microsuspension cells isolated from cell aggregates were selected and passaged. Three-month old rice suspension cells that were passaged and suspended in culture as described above were AA medium containing sucrose 30g / L, 2,4-D 2.0mg / L, kinetin 0.2mg / l, and gibberellic acid 0.1mg / L. Subcultured. In order to eliminate the possibility of toxicity due to the retention of plant hormones, microsuspension cells stabilized to about 1 mm or less in diameter were cultured without 2,4-D.

The stabilized suspension cells, plant cell culture by adding a disk type gas sparger, hollow-paddle impeller and a sampling port made in a size suitable for plant cell collection Batch culture was carried out in a 7 L dose bioreactor (Applikon, The Netherlands) modified to suit. The working volume was 3.2 L, and the initial agitation rate and the aeration rate were 80 rpm and 0.2 vvm, respectively. The AA medium (2.3L) was sterilized by autoclaving for 25 minutes in a bioreactor at 121 ° C., and then 300 ml of 10-fold concentrated (10 ×) amino acid solution was sterilized and filtered with a 0.22-μm filter (Millipore, USA). It was injected into the AA medium. The flask cultured cells were then seeded in the bioreactor with 10% fresh cell weight volume (FCWV). At this time, a tube welder (Wave Biotech, Switzerland) was used to connect tubing between the inoculation bottle and the bioreactor vessel in a sterile state.

As the culture conditions, the culture temperature was 28 ℃, 90-110rpm, was controlled by adjusting the aeration rate and agitation rate to maintain the dissolved oxygen (DO) level 20% or more during the culture period. Air and oxygen were mixed and added, and the aeration rate was adjusted to intermittently enter between 0.5L / min for air and 0.1ml / min and 100ml / min for oxygen.

Bioreactor culture alternated between dark and fluorescent conditions, cells were recovered on day 7 of culture. The recovered cells were resuspended in PBS and washed 2-3 times to remove excess medium. After the last wash the filter was completely removed. Thus prepared rice stem cells were immediately used for extraction, or frozen or frozen until extraction.

[ Reference Example  2] Preparation of Human Skin Cells

2-1. Preparation of Human Fibroblasts

Human fibroblast cells (ATCC 2076) contained 5 cells in Iscove's modified Dulbeco's medium (IMDM) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. × 10 was cultured to ensure that the ⁵ cells / dish. The culture vessel was used 100mm cell culture dish and cultured in a single layer in a 5% CO ₂ incubator maintained at 37 ℃, saturated humidity in 10-12 ㎖ medium. The medium was changed twice a week, and the cells that reached confluency were maintained by subculture after trypsinization using 0.25% trypsin-EDTA.

2-2. Preparation of Human Epidermal Cells

Human epidermal cells (HaCaT cells, sold from Dongguk University) were used with DMEM (Dulbacco's Modified Eagle's Medium, GIBCO) medium supplemented with 10% FBS and 1% penicillin-streptomycin. Subcultured and maintained under the same conditions.

2-3. Preparation of Human Epidermal Stem Cells

Human epidermal stem cells (WPE-stem cell, keratinocyte progenitor cell, ATCC CRL-2887) were added Keratinocyte Serum Free Medium (K -SFM) cell number 1ⅹ10 ^5- The culture was performed at 1.5 × 10 ⁵ cells / cm 2. Culture vessels were 2.5 ㎍ / ㎠ Mouse Collagen Type IV and 2.5 ㎍ / ㎠ A 75T flask pre-coated with a human fibronectin mixture was used and cultured in a single layer in a 5% CO ₂ incubator maintained at 37 ° C. and saturated humidity in 15-20 ml of medium. The medium is changed twice a week, and the cells that reach confluency are trypsinized with 0.05% Trypsin-EDTA diluted in D-PBS at a 1: 1 ratio, and then trypsin-EDTA with 0.1% Trypsin inhibitor. Activation was maintained by subculture.

[ Example  One] Rice Stem Cells  Preparation of extract

Method 1—60 g of the rice stem cells prepared in Reference Example 1 was frozen with liquid nitrogen, ground in a mortar and then pulverized in 300 ml of phosphate buffered saline (PBS) at room temperature on a stirrer (magnetic stirrer, Corning, USA). After stirring for 24 hours, the prepared extract was filtered through Whatman paper to remove cell suspensions, and the filtrate was obtained. The filtrates were lyophilized and stored in a container at room temperature until use.

Method 2-60 g of the rice stem cells prepared in Reference Example 1 were frozen with liquid nitrogen, ground in a mortar, suspended in 300 ml of water, and then treated with a sonicator (Sonic & Materials, Model Vibra Cell) for 10 seconds, followed by room temperature. After extracting by stirring for 24 hours at, the prepared extract was filtered with Whatman paper to remove the cell suspension, and the filtrate was obtained, all the filtrate was lyophilized and stored in a container at room temperature until use.

Method 3—60 g of the rice stem cells prepared in Reference Example 1 was frozen with liquid nitrogen, ground in a mortar, suspended in 300 ml of water, and treated with sonicator (Sonic & Materials, Model Vibra Cell) for 10 seconds. After extracting by stirring at 24 ° C. for 24 hours, the prepared extract was filtered through Whatman paper to remove cell suspensions, and the filtrate was obtained. All the filtrates were lyophilized and stored in a container at room temperature until use.

Method 4—60 g of the rice stem cells prepared in Reference Example 1 was frozen with liquid nitrogen, ground in a mortar and pestle at 4 ° C., and then suspended in 300 ml of 80 (v / v)% hydrous ethanol for 10 seconds with a sonicator. After extracting by stirring at room temperature for 24 hours, the prepared extract was filtered with Whatman paper to remove cell suspension, and the filtrate was obtained. All the filtrates were lyophilized and stored in a container until use.

Method 5—60 g of rice stem cells prepared in Reference Example 1 were suspended in 300 ml of water, extracted at 125 ° C. for 30 minutes at a pressure of 1.5 MPa, and then the extract was filtered with Whatman paper to remove cell suspension and to obtain a filtrate. The filtrates were all lyophilized and placed in a container and stored at room temperature until use.

[ Example  2] of Rice Stem Cell Extract HPLC Fingerprint  Confirm

By identifying the relative ratios of the major peaks by recording the peaks of the components contained in the prepared rice stem cell extract, the HPLC fingerprint of the rice stem cell extract was confirmed as a method for consistently preparing the rice stem cell extract.

8.8 mg of rice stem cell extract prepared by the fifth method of Example 1 was taken and placed in a 50 ml volumetric flask. 40 mL of 50% (v / v) MeCN (acetonitrile) solution was added to dissolve the solution, and 50% (v / v) MeCN was poured into the 50 ml mark. HPLC fingerprints were confirmed, and the results are shown in FIG. 1.

Mobile phase: H ₂ OMeCN (30:70 (v / v))

Flow rate: 1.0 ml / min

Column: Kromasil 100-5NH2. 250 * 4.6 mm (Akzo Nobel)

Wavelength: 254nm

- Temperature: 30 ℃

Injection volume: 20 μl

Instrument used: HPLC (LC-10AT, SHIMADZU),

            Photodiode detector (SPD-M10A, SHIMADZU),

            Injector (SPD-M10AVP, SHIMADZU),

            Pump (LC-10AT, SHIMADZU),

            Controller (SPD-10AVA, SHIMADZU),

Software (SHIMADZU CLASS-VP)

[ Formulation Example  One] Example  Flexible lotion containing extract of 1 skin Manufacturing

1 kg of a flexible lotion (skin) containing the rice stem cell extract of Example 1 was prepared in the composition ratio shown in Table 1 below.

[Table 1]

<Manufacturing method>

Components 2, 3, 4, and 8 were sequentially added to component 11 in Table 1, stirred, and dissolved at room temperature. Component 5 was dissolved by heating at about 60 ° C., and then component 10 was added and stirred. Finally, the components 1, 6, 7 and 9 were added and sufficiently stirred, then passed through a microfluidizer, and aged to prepare a flexible skin lotion (skin) of the present invention.

[ Comparative formulation example  One]

Except for component 1 was prepared in the same manner as in Formulation Example 1.

[ Formulation Example  2] Example  Production of a nutritious lotion (lotion) containing an extract of 1

To the composition ratio shown in Table 2 below, 1kg of nutrient cosmetic water (lotion) containing the rice stem cell extract of Example 1 was prepared.

[Table 2]

<Manufacturing method>

Nonionic amphiphilic lipids were prepared by homogenizing components 2, 3, 4 and 5 in Table 2. The nonionic amphiphilic lipids and components 1, 6, 7 and 13 are mixed and homogenized at room temperature and passed through a microfluidizer, and then component 8 is slowly added at room temperature to homogenize and then passed again through a microfluidizer. I was. Ingredients 9, 10, 11, and 12 were added, dispersed, stabilized, and aged to prepare nutrient lotion (lotion).

[ Comparative formulation example  2]

Except for component 1 was prepared in the same manner as in Formulation Example 2.

[ Formulation Example  3] Example  Preparation of nutrition cream containing extract of 1

To the composition ratio shown in Table 3 below, 1kg of nourishing cream containing rice stem cell extract of Example 1 was prepared.

[Table 3]

<Manufacturing method>

Nonionic amphiphilic lipids were prepared by homogenizing components 2, 3, 4 and 5 in Table 3. The nonionic amphiphilic lipids and components 1, 6, 7 and 13 are mixed and homogenized at room temperature to pass through a microfluidizer, and then component 8 is slowly added at room temperature to homogenize and then passed back to the microfluidizer again. I was. And ingredients 9, 10, 11 and 12 were added to the dispersion, stabilized and aged to prepare a nourishing cream.

[ Comparative formulation example  3]

Except for component 1 was prepared in the same manner as in Formulation Example 3.

[ Test Example Terminology

Hereinafter, "rice stem cell extract of Example 1" mentioned in Test Examples 1 to 5 means "rice stem cell extract prepared by the fifth method of Example 1" unless there is a special modifier.

[ Test Example １] Rice Stem Cells  Confirmation of Cytotoxicity of Extracts

Cytotoxicity of rice stem cells was measured by MTT assay based on metabolic reduction of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide]. In living cells, MTT is converted into MTT-formazan crystals by the dehydrogenase present in the mitochondria, and the amount of this can be used to measure the viability of living cells (Ref. ).

1-1. Cytotoxicity Measurements on Human Fibroblasts

Human fibroblasts prepared in Reference Example 2-1 were aliquoted at a concentration of 5 × 10 ⁴ cells / well in 24-multiwell plates and inoculated in IMDM medium to which 10% FBS was added and incubated for 24 hours. The cultured cells were replaced with a serum-free medium, and the rice stem cell extracts of Example 1 were dissolved in water and treated by concentration, and cultured for 24 hours. The control group was incubated in the same amount of water treatment. 100 μl of MTT solution (2.5 mg / ml) was added to the incubated cells, followed by reaction at 37 ° C. for 4 hours. The culture supernatant was removed, and 500 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the resulting MTT-formazan crystals. The absorbance was measured at 570 nm with an ELISA reader. Cell viability was expressed as a percentage of absorbance compared to the control group, as shown in Equation 1 below, and the results are shown in [Table 4].

[Equation 1]

[Table 4]

As shown in Table 4, the rice stem cell extract of the present invention was found to show little cytotoxicity to human fibroblasts up to a concentration of 100 ㎍ / ㎖.

1-2.Cytotoxicity Measurement on Human Epidermal Cells

Test Example 1 except that the human epidermal cells prepared in Reference Example 2-2 were inoculated in 48 × multiwell plates at a concentration of 2 × 10 ⁵ cells / well and inoculated in DMEM medium to which 10% FBS was added and cultured for 24 hours. The test was performed in the same manner as -1, and the cell viability was also calculated as shown in [Equation 1] and the results are shown in [Table 5]. As shown in [Table 5], the rice stem cell extract of the present invention was found to show little cytotoxicity to human epidermal cells up to a concentration of 100 ㎍ / ㎖.

[Table 5]

As shown in [Table 5], the rice stem cell extract of the present invention was found to show little cytotoxicity to human epidermal cells up to a concentration of 100 ㎍ / ㎖.

1-3. Cytotoxicity Measurements on Human Epidermal Stem Cells

WPE-Stem cells prepared in Reference Example 2-3 were dispensed at a concentration of 2 × 10 ⁴ cells / well in a 96-multiwell plate pre-coated with 2.5 μg / cm 2 Mouse Collagen Type IV and 2.5 μg / cm 2 Human Fibronectin mixture. It was incubated for 24 hours in K-SFM medium to which 0.05 mg / ml BPE and 5 ng / ml EGF were added. The cultured cells were replaced with a fresh medium, and the rice stem cell extracts of Example 1 were dissolved in water and treated by concentration to incubate for 24 hours. The control group was incubated with the same amount of water. 0.01 ml of MTT solution (2.5 mg / ml) was added to the incubated cells, followed by reaction at 37 ° C for 4 hours. The culture supernatant was removed, and 100 μl of DMSO was added to each well to dissolve MTT-formazan crystals, and the absorbance was measured at 570 nm with an ELISA reader. Cell viability was calculated by the above [Equation 1], the results are shown in [Table 6].

TABLE 6

As shown in Table 6, the rice stem cell extract of the present invention was found to show little cytotoxicity to human epidermal stem cells up to a concentration of 100 ㎍ / ㎖.

[ Test Example  2] Rice Stem Cells  Extract Skin cell  Confirmation of proliferation effect

Skin cell proliferation effect of rice stem cell extract was also measured by MTT assay.

2-1. Measurement of proliferative effect of human fibroblasts

Human fibroblasts prepared in Reference Example 2-1 were aliquoted at a concentration of 5 × 10 ⁴ cells / well in a 12-multiwell plate and incubated in IMDM medium with 10% FBS for 24 hours.

The cultured cells were replaced with serum-free medium, and the rice stem cell extracts of Example 1 were dissolved in water and treated by concentration to incubate for 72 hours. The control group was treated with the same amount of water and incubated for 72 hours. 100 μl of MTT solution (2.5 mg / ml) was added to the incubated cells, followed by reaction at 37 ° C. for 4 hours. After removing the culture supernatant and dissolving the resulting MTT-formazan crystals by adding 500 μl of DMSO to each well, the absorbance was measured at 570 nm with an ELISA reader. Cell growth rate (%) was expressed as a percentage of absorbance compared to the control, as shown in the following [Equation 2], the results are shown in [Table 7].

&Quot; (2) &quot;

[Table 7]

As shown in Table 7, the rice stem cell extract of the present invention was found to be more effective in the growth of fibroblasts in a concentration-dependent manner, especially at a concentration of 250 ㎍ / ㎖ 15% compared to the control It confirmed that it was shown.

2-2. Measurement of proliferative effect of human epidermal cells

Except for using the human epidermal cells prepared in Reference Example 2-2 was tested in the same manner as in Test Example 2-1, the cell proliferation rate was also calculated as shown in [Equation 2] and the results are shown in [Table 2 8].

[Table 8]

As shown in [Table 8], the rice stem cell extract of the present invention was found to have an effect on epidermal cell proliferation compared to the control group at a concentration of 1 µg / ml or more.

2-3. Measurement of proliferative effect on human epidermal stem cells

2-3-1. MTT assay

WPE-Stem cells prepared in Reference Example 2-3 were dispensed at a concentration of 1 × 10 ⁴ cells / well in 96-multiwell plates pre-coated with 2.5 μg / cm 2 Mouse Collagen Type IV and 2.5 μg / cm 2 Human Fibronectin mixture. It was incubated for 24 hours in K-SFM medium to which 0.05 mg / ml BPE and 5 ng / ml EGF were added. The cultured cells were replaced with a fresh medium, and the rice stem cell extracts of Example 1 were dissolved in water and treated by concentration to incubate for 72 hours. The control group was incubated for 72 hours with the same amount of water. The cultured cells were added with 0.02 ml of MTT solution (2.5 mg / ml) and reacted at 37 ° C. for 4 hours. After removing the culture supernatant and adding 100 μl of DMSO to each well, the resulting MTT-formazan crystals were dissolved, and the absorbance was measured at 570 nm with an ELISA reader. Cell proliferation was calculated as shown in [Equation 2] and the results are shown in [Table 9].

TABLE 9

As shown in [Table 9], the rice stem cell extract of the present invention increased the cell proliferation effect in a concentration-dependent manner, showed a significant proliferation effect of about 37% compared to the control at a concentration of 30 ㎍ / ㎖. In addition, it showed a cell proliferation effect of 20% or more up to the treatment concentration of 100 ㎍ / ㎖, it was found that the effect on the epidermal stem cell proliferation compared to the control.

2-3-2. Colony forming assay

Stem cells in vitro (in in vitro ) to form colonies in culture.

The colony is composed of stem cells, transient amplifying cells, and differentiated cells. The number of colonies formed here can be seen as a stem cell density value, because differentiated cells lose their ability to divide, and transiently rapidly dividing cells do not proliferate enough to form colonies. The number of colonies formed is called colony forming efficiency (CFE).

WPE-Stem cells prepared in Reference Example 2-3 were dispensed in a 100 mm cell culture dish at a concentration of 1 × 10 ⁴ cells / dish and in K-SFM medium containing 0.05 mg / ml BPE and 5 ng / ml EGF for 24 hours. Incubated. The medium of the cultured cells was replaced with a new medium, and the rice stem cell extract of Example 1 was dissolved in water and treated by concentration to incubate for 11 days. The control group was incubated with the same amount of water. After incubation was completed, the medium was removed, and 1 ml of 0.2% Crystal violet solution was added and stained at room temperature for 10 minutes. The results are shown in FIG. 2.

As shown in Figure 2, the number of colonies per cell and the number of cells in the colonies significantly increased when the rice stem cell extract treatment of the present invention.

In addition, after the staining, by counting the number of colonies (number of cells 50 or more) to calculate the colony-forming efficiency (% CFE) as shown in the following [Equation 3], the results [ Table 10].

&Quot; (3) &quot;

Colony Formation Rate (% CFE)

= (number of colonies formed at the end of growth period / number of cells inoculated at start of growth period) X100 (%)

[Table 10]

As shown in Table 10, the rice stem cell extract of the present invention exhibited an increase in colony formation of about 30-50% at all test concentrations. In addition, as shown in Figure 2, it can be seen that the treatment of the rice stem cell extract of the present invention has the effect of promoting the proliferation of cells and the colony formation accordingly.

This indicates that the rice stem cell extract of the present invention not only promotes the proliferation of epidermal stem cells, but also maintains the stem cell properties.

[ Test Example  3] Rice Stem Cells  Extract Collagen Type  Confirmation of I expression promoting effect

Human fibroblasts prepared in Reference Example 2-1 were seeded at a density of 1 × 10 ⁶ in a 60 mm culture dish (NUNC, USA) containing IMDM medium with 10% FBS. After 24 hours of incubation, the cells were washed with Han's balanced salt solution (HBSS) and replaced with DMEM medium without FBS. At 24 hours after the culture washed again with HBSS and treated at different concentrations dissolved rice stem cell extract in the water of Example 1 and replaced with IMDM medium FBS was not added and 37 ℃, 5% CO ₂ incubator (REVCO, USA) 24 Time incubation. The negative control group was cultured by treating the same amount of water, and the positive control group was cultured by treating Vit C, which is known to activate collagen formation (the Vit C activates collagen formation but is known to be unstable in cosmetic formulations).

In order to collect RNA of the cultured cells, the cultured cells were collected into 1 ml of TRLZOL (Invitrogen, USA), transferred to a 1.5 ml tube, 200 µl of Chloroform (Sigma, USA) was added, vortexed, and allowed to stand at room temperature for 5 minutes. After centrifugation (15,000 rpm, 4 ℃, 30 minutes), the supernatant was transferred to a new tube and mixed with the same amount of 2-propanol and allowed to stand at room temperature for 5 minutes. After centrifugation (12,000 rpm, 4 ° C, 20 minutes), 2-propanol was discarded and 1% of 75% (v / v) hydrous ethanol was added and centrifuged (10,000 rpm, 4 ° C, 10 minutes) and 75%. (v / v) hydrous ethanol was discarded and RNA pellets were dried at room temperature to remove residual ethanol. 40 μl of purified water treated with DEPC (diethyl pyrocarbonate) was added to the pellet, and then quantified at 260 nm.

RT-PCR was used as a total RNA of 1 ㎍, All-in-one RT-PCR kit from RexGene Biotech (Korea). Collagen Type I and Actin primers were used in Bioneer (Korea). Synthesis was used, and the sequence and reverse transcription conditions of the primers are described in Tables 11a and 11b. RT-PCR products were quantified using an image analysis system (BIO-RAD, USA) by electrophoresis on 2% agarose gel, the results are shown in Table 12 below.

[Table 11a]

Table 11b

[Table 12]

As shown in [Table 12], the rice stem cell extract of the present invention, when treated at a concentration of 1, 10, 25, 50 ㎍ / ㎖, the expression level of Collagen Type I compared to the control group did not process the sample 294.39, 417.20, 371.93, 288.07% increased the effect was found. In addition, the rice stem cell extract of the present invention was shown to promote the expression of Collagen Type I as compared to the treatment group of Vit C, a positive control group. Thus, it was confirmed that the rice stem cell extract of the present invention can contribute to the wrinkle improvement, elasticity improvement effect by collagen synthesis.

[ Test Example  4] human patch test ( Patch test )

In order to determine the degree of skin irritation for the cosmetic composition comprising the rice stem cell extract prepared in Example 1, a human patch test was performed.

The subjects were selected as a woman without skin disease at the test site of 20-50 years old. After wiping the upper part of the patch area with 70 (v / v)% hydrated ethanol and drying it, using a fin chamber, the flexible cosmetic water of Formulation Example 1, the nutrition lotion of Formulation Example 2, and the nutrition cream of Formulation Example 3 were used. 0.02g each was added and attached to the test site.

After 24 hours, the skin reaction was evaluated within 1 hour after the chamber was removed, and the skin reaction was evaluated the next day (after 48 hours). The skin response was determined by the evaluation criteria of the following [Table 13], and the evaluation of the possibility of skin irritation was performed by calculating the average response rate calculated according to [Equation 4] below, and the results are shown in [Table 14]. Indicated.

&Quot; (4) &quot;

Average response rate (%) = [Σ (Number of response subjects after 24 hours × score) + Σ (Number of response subjects after 48 hours × score)] × 100 / (Total number of tests × maximum score × total number of subjects)

[Table 13]

[Table 14]

As shown in Table 14, the cosmetic composition containing the rice stem cell extract of the present invention did not cause skin irritation, thereby confirming the safety of the rice stem cell extract of the present invention.

[ Test Example  5] Rice Stem Cells  Evaluation of Skin Wrinkle and Elasticity Improvement Effect of Extracts

In order to confirm the effect of improving the skin wrinkles and elasticity according to the skin coating of the nourishing cream of Formulation Example 3 and Comparative Formulation Example 3 of the present invention was carried out the following test.

The subjects were 60 women aged 20-55 years, and 25% of the owners of normal, oily, dry and combination skin were composed based on skin condition.

To the same person, apply the nourishing cream of Formulation Example 3 on the left side of the face and the nourishing cream of Comparative Formulation 3 on the right side of the face. It was measured visually, and the results are shown in the following [Table 15].

TABLE 15

As shown in [Table 15], the formulation containing the rice stem cell extract (Formulation Example 3) was applied, compared to the site where the formulation without the rice stem cell extract of the present invention (Comparative Example 3) was applied. The effect of improving skin wrinkles and elasticity at one site was high. As such, it could be confirmed that the rice stem cell extract of the present invention has an anti-aging effect according to skin wrinkles and elasticity improvement.

Korean Collection for Type Cultures (KCTC) KCTC11760BP 20100907

Claims (11)

An anti-aging cosmetic composition containing rice stem cell extract as an active ingredient. According to claim 1, wherein the rice stem cells are rice ( Oryza sativa L.) A cosmetic composition which is an undifferentiated cell derived from a tissue selected from the group consisting of leaves, stems, seeds and roots. According to claim 1, wherein the rice stem cells are rice ( Oryza sativa L.) A cosmetic composition prepared using callus derived (Accession No. KCTC 11760BP). The cosmetic composition according to claim 1, wherein the rice stem cell extract is obtained by extracting from rice stem cells with an extraction solvent selected from phosphate buffer, water, ethanol and a mixed solvent thereof. The cosmetic composition according to claim 4, wherein the extraction is performed by a method selected from among agitation extraction, ultrasonic extraction, thermal extraction, and high temperature and high pressure extraction. The cosmetic composition according to claim 5, wherein the extraction is a high temperature and high pressure extraction method. According to claim 6, wherein the high temperature and high pressure extraction method is a temperature of 100 to 130 ℃ Cosmetic composition to extract at a pressure of 1.0MPa to 2.0MPa. The cosmetic composition of claim 1, wherein the rice stem cell extract is contained in an amount of 0.001 to 10% by weight based on the total weight of the composition. The cosmetic composition according to claim 1, wherein the anti-aging is caused by wrinkle improvement and skin elasticity improvement effects based on the synthesis of Collagen type I. According to claim 1, wherein the anti-aging cosmetic composition is due to the effect of improving wrinkles and skin elasticity based on epidermal stem cell proliferation. The cosmetic composition according to any one of claims 1 to 10, wherein the cosmetic composition is a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand Cosmetic composition is a formulation selected from cream, foundation, essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser.

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