KR101417300B1 - A cosmetic composition comprising rice stem cell extract with high temperature and high pressure treatment for a whitening activity - Google Patents

Abstract

The present invention relates to a whitening cosmetic composition containing Rice stem cell high-temperature high-pressure extract as an active ingredient. The rice embryonic stem cell high temperature and high pressure extract according to the present invention has excellent inhibitory activity against tyrosinase which is involved in melanin biosynthesis and inhibits melanin production and does not cause skin irritation, so that it can be usefully used for a whitening cosmetic composition.

Description

[0001] The present invention relates to a cosmetic composition containing a high-temperature and high-pressure extract of a rice stem cell,

The present invention relates to a cosmetic composition containing a rice embryonic stem cell high temperature and high pressure extract having a whitening activity.

In recent years, a healthy and relaxed life has been demanded, and with the desire to be beautiful, transparent and clear skin has become a dream for everyone. As evidenced by this, the preference for whitening cosmetics has expanded from summer to all seasons, leading to the development of more whitening lines. Especially, since the whitening market is concentrated in Asia such as China, research and development of functional whitening materials are mainly performed in Japan and Korea.

The skin color of a person is determined by the amount of melanin, carotene and hemoglobin, and melanin is the most crucial factor. Melanin is synthesized in the melanocyte, which is a pigmentary cell in the epidermal basal layer, and transformed into keratinocyte, which shows skin color. It disappears with keratinization along with the keratinization of the epidermis. In addition, melanin prevents ultraviolet rays from absorbing or scattering ultraviolet rays from the skin to prevent cells or tissues inside the skin from being damaged by ultraviolet rays. However, melanin is abnormally excessive due to external environmental changes such as exposure to ultraviolet rays, If the melanin is not completely eliminated due to lack of production or keratinization, pigmentation may occur, which may cause blackening of the skin, spots, and freckles. Such skin disorders can be a cosmetic problem for the patient and can cause serious psychological problems. In addition, unsightly facial lesions can have a profound effect on emotions and health and cause a reduction in social functioning and productivity in work and school.

Melanin synthesis in melanocytes is described in detail. An enzyme called tyrosinase acts on tyrosine as a substrate to generate dopaquinone and spontaneous reaction from dopaquinone And enzyme reaction to produce melanin in the copolymer black pigment. Therefore, in order to prevent pigmentation of the skin, it is general and easy to reduce the production of melanin by inhibiting some reactions during the melanin production process.

Since the 1980s, skin cancer researchers have studied the biosynthetic pathway of melanin, which has led to whitening cosmetics and drug development. In particular, kojic acid, arbutin, ascorbic acid, etc. have been developed and utilized in the development of a tyrosinase inhibitor, which is known as a melanin biosynthesis rate-limiting enzyme. However, COZSAN has been discontinued due to the problem of carcinogenicity to thyroid cancer, and arbutin has been reported to have weak activity. In addition, ascorbic acid is easily oxidized in a solution to be browned and lose its whitening activity. Therefore, it is troublesome to use ascorbic acid in its derivation without being able to use it alone.

In addition, many studies have been carried out to find whitening active ingredients in natural products, especially plants. Among them, many plant extracts and herbal medicine extracts such as alfalfa, licorice, peony, cinnamon, gossam, It has been found that they have an inhibitory activity against azithromycin. However, these also have many problems in using cosmetics or medicines at an effective concentration or more in terms of safety and possibility of discoloration, and there is a limit in that whitening effect is not clear. In particular, it has been pointed out that most of the plant extracts exhibit inhibitory activity of tyrosinase activity, which is effective only at a high concentration, and that the inhibitory effect is hardly exhibited at a low concentration.

The conventional whitening cosmetic composition has a problem that the stability of the product is poor and the skin whitening effect is unclear and the stability to the skin is also inferior. In particular, the conventional whitening cosmetic composition has a problem that the whitening effect is rapidly deteriorated with time there was.

Therefore, it is necessary to develop more effective whitening agents that are more effective than the whitening agents so far. Currently, most of cosmetic raw materials (80% ~ 90%) are imported from foreign countries.

Therefore, there has been continued research on natural raw materials which are excellent in stability in cosmetic compositions and excellent in whitening effect without stimulation to the skin, in particular, natural raw materials which are easily available in the domestic market.

Stem cells are cells that have the ability to differentiate into all types of cells that, if necessary, are not differentiated into specific cells and constitute a tissue or organ. The inherent characteristics of stem cells are called self-renewal and pluripotency, and stemness is a collective term for these characteristics.

In recent years, with the development of plant tissue culture technology using totipotency of plants, interest in functional secondary metabolites derived from plant stem cells has been amplified. These secondary metabolites are known to have a variety of physiological activities, and extracts derived from plant stem cells are attracting attention as a new raw material for high-function cosmetics. Using this technology, it is expected that it will be possible to develop and apply new components which have not been used so far, since they can economically produce high-performance active materials which are present in nature but were difficult to obtain by chemical synthesis.

Rice ( Oryza Sativa L.) is an annual herb and has many kinds of rice, but it is classified into rice, paddy rice, and rice paddy depending on the viscosity of the rice grain. The chromosome number is n = 12, 2n = 24 and belongs to the AA genome. Plant Classification Rice is one of the seed plants (an organ which becomes a seed after being formed in a fox), which is surrounded by a grasshopper. It also belongs to the monocotyledonous plant with one cotyledon in the seed. Rice roots consist of seed roots, mid-hypocotyl roots and crown roots, which absorb water and nutrients through root hair.

Rice grows and divides in the main stem and the primary tiller branch (the branches come out from the node in the ground of the plant). The stem is made between the nodes and nodes. The leaves are divided into leaf sheath and leaf sheath, with leaf veins and leaf ears in between. The flower contains 6 stamens and 1 pistil, and pollinator.

Rice contains about 75% starch, about 8% gray matter, and 0.5-1% fat. In addition, it contains a small amount of vitamin B, but the content of vitamin depends on the type of rice and the cultivation area. The fat part may include ester cholesterol and free cholesterol, brassicasterol, stigmasterol, sitosterol, monoglyceride, diglyceride, triglyceride, phospholipid, Contains free fatty acids.

In addition, rice is an edible crop identified as not producing toxic metabolites as in some crops such as tobacco (Tyagi AK and Mohanty A, Plant Sci . 2000; 158 (1-2): 1-18).

On the other hand, Korean Patent Laid-Open Publication No. 2010-0131598 entitled " Cosmetic Composition Containing Rice Callus Extract " describes that a composition prepared by inducing callus from rice seeds and incorporating it into a composition to be powdered has antioxidative, anti-inflammatory and whitening activity . However, as disclosed in the above-mentioned patent, it is difficult to obtain a sufficient extracting effect on a useful ingredient by merely pulverizing the callus itself and dissolving in water. In order to cultivate rice callus itself, It is disadvantageous in terms of time and economic cost since it must be cultured by inducing callus.

Accordingly, the present inventors have made efforts to provide a new whitening agent that exhibits a whitening function but has no side effect, and has successfully established a rice embryo stem cell capable of mass culture and has found that the extract extracted under high temperature and high pressure does not cause skin irritation, Melanin production by effectively inhibiting the production of melanin.

Korean Patent Publication No. 2010-0131598 Korean Patent No. 0542605 Korean Patent No. 0897159

Tyagi AK and Mohanty A, Rice transformation for crop improvement and functional genomics, Plant Sci. 2000; 158 (1-2): 1-18 Sant Saran Bhojwani and M. K. Razdan, Plant Tissue Culture: Theory and Practice, 1996, Elsevier Murashige T and Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 1962; 15 (3): 473-497 Chu et al., Establishment of an efficient medium for rice cultivation, through comparative experiments on the nitrogen sources, Sci. Sinica. 1975; 18: 659-668 JA Thompson, Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose, Plant Sci. 1986; 47 (2): 123-133

Accordingly, an object of the present invention is to provide a cosmetic composition comprising a high-temperature and high-pressure extract of rice embryonic stem cells excellent in whitening effect.

In order to achieve the above object, the present invention provides a cosmetic composition for whitening comprising a rice stem cell high-temperature high-pressure extract as an active ingredient.

The term "rice stem cell" is defined in this invention, rice (Oryza sativa L.), preferably from one or more tissues selected from the group consisting of leaves, stems, seeds and roots, and more preferably from undifferentiated seeds. The term " rice embryonic stem cell " in the present invention means a suspension cell derived from an undifferentiated cell mass (i.e., callus) derived from the plant tissue of the rice, particularly a stabilized micro-suspended cell. The term " stabilized microsuspended cells " in the present invention is characterized in that the cells do not exhibit the property of agglomerating to grow in suspension culture, and the diameter thereof is kept at 1 mm or less. For example, the rice stem cells of rice (Oryza sativa L.) calli (accession number KCTC 11760BP).

The term " high temperature and high pressure " as defined in the present invention means a temperature of 100 to 135 ° C, preferably 100 to 125 ° C and a pressure of 1.2 to 2.0 kgf / cm 2, preferably 1.2 to 1.5 kgf / Cm < 2 >, more preferably 1.5 kgf / cm < 2 >.

The present invention also provides a method for producing a rice stem cell high-temperature high-pressure extract, which comprises the following steps:

A large-scale culture step of mass-culturing rice stem cells;

The mass cultured rice stem cells are suspended in a solvent and incubated at a temperature of 100 to 135 ° C, preferably 100 to 125 ° C, and at a temperature of 1.2 kgf / cm 2 to 2.0 kgf / cm 2, preferably 1.2 kgf / pressure processing step of performing high-temperature high-pressure processing under a pressure of 1.5 kgf / cm2, more preferably 1.5 kgf / cm2.

Hereinafter, the present invention will be described in detail.

In the present invention, when preparing the rice embryonic stem cells, scutellum-derived calli obtained by germinating the surface-sterilized and washed rice seeds after removing the husks are inoculated, and the callus is inoculated / subcultured in the suspension culture medium. It can be prepared as a suspension cell. However, the present invention is not limited thereto, and may be obtained by applying Sant Saran Bhojwani and M. K. Razdan, Plant Tissue Culture: Theory and Practice, 1996, Elsevier, which is known to those skilled in the art.

For example, the rice embryonic stem cells of the present invention can be produced by modifying the cell line production method described in Korean Patent Registration Nos. 0542605 and 0897159 as follows:

One) Callus  Establish

Here, the callus can be obtained according to the following production example, but is not limited thereto.

Rice ( Oryza Sativa L.) seed husk is removed and surface sterilization treatment is performed using a functional ethanol and a NaOCl solution, followed by washing with sterilized distilled water, and it is applied to a callus induction solid medium, preferably a Chu (N6) agar medium. 20 to 30 < 0 > C, preferably 25 [deg.] C for 10 to 20 hours and 4 to 14 hours of dark condition; Preferably, after culturing for 5 to 20 days, preferably 7 to 14 days, under the condition of 16 hours of light and 8 hours of darkness, the callus derived from the germinated seeds is transferred to a new Chu (N6) agar medium, To 0.5 to 2 cm, preferably to about 1 cm, so as to obtain callus derived from rice. The callus derived from the rice prepared as described above was deposited on September 7, 2010 under the deposit number KCTC 11760BP by the Korean Society for Research in Microbiology and Culture (KCTC).

2) Rice stem cells  Produce

The process for introducing the rice-derived callus into a fine suspension cell for liquid culture is described below, but is not limited thereto.

As the medium for suspension cell culture, any culture medium for plant cell culture known in the technical field of the present invention can be used, and nutrients and other factors necessary for maintaining the growth potential of plant cells, such as carbon source, nitrogen source, Vitamins and the like. Specifically, MS (Murashige T and Skoog F, Physiol . 　 Plant . 1962; 15 (3): 473-497 ) medium, Chu (N6) (Chu et al., Sci . Sinica . 1975; 18: 659-668) medium, Amino Acid (JA Thompson, Plant Sci . 1986; 47 (2): 123-133) can be used, and if necessary, various additives such as plant growth regulators can be added, or a part of the components can be removed. Examples of the plant growth regulator that can be included in the culture medium for suspension cells of the present invention include 2,4-Dichlorophenoxyacetic acid (2,4-D), kinetin, gibberellic acid (GA3) But is not limited thereto.

Chu (N6) medium or AA (amino acid) medium, preferably containing 30 g / L sucrose, 2 mg / L 2,4-D and 0.2 mg / L kinetin, And callus-derived calli were inoculated at 25 ° C. to 30 ° C. (preferably 28 ° C.), 90 to 150 rpm (preferably 110 rpm), shaking incubation in dark condition, Cells are cultured. The rice suspension cells which were 2 to 3 months old suspension-cultured in the above-described manner were cultured in the same manner as in Example 1, except that 30 g / L of sucrose, 2.0 mg / L of 2,4-D, 0.2 mg / L of kinetin and 0.1 mg / L of gibberellic acid Lt; RTI ID = 0.0 > AA < / RTI > Alternatively, it may be transferred to an AA medium containing 30 g / L of chondroitin sulfate, 0.2 mg / l of kinetin and 0.1 mg / L of gibberellic acid, and subcultured in another example. At this time, it is preferable to subculture only the micro-suspended cells isolated from the cell aggregate. It is preferable that the cells are not agglomerated due to agglomeration among the cells in the suspension culture, and that the diameter of the micro- It is particularly preferable to subculture only the cells.

Even when cultured in a culture medium containing 2,4-D in the step of producing rice stem cells, the culture is carried out in a culture medium containing no 2,4-D in the mass culture stage, so that the possibility of toxicity due to the residue of 2,4-D There are advantages to be solved.

3) Mass culture

The stabilized microsuspension cells can be mass-cultured using any bioreactor for culturing known plant cells known to those skilled in the art, such as a stirred tank reactor, an airlift reactor, a fluidized reactor, etc. In the present invention, A bioreactor suitable for plant cell culture was added, including a gas sparger, a hollow-paddle impeller and a sampling port sized to harvest plant cells. ), But the present invention is not limited thereto. The culture method may be any of a batch method, a continuous method, and a feed method.

The culture conditions include a culture temperature of 25 to 30 캜 (preferably 28 캜), a stirring speed of 50 to 150 rpm (preferably 90 to 110 rpm), a dissolved oxygen (DO) level of 15% (Preferably 20% or more). In order to maintain it, controlling the aeration rate and stirring speed is preferable for the formation of a suitable culture environment.

The bioreactor culture alternates between the dark condition and the fluorescent condition, and it is preferable to recover the cells after 5 days (preferably 7 to 8 days) after the culture. Prepared rice stem cells can be used immediately for extraction, or can be stored frozen or frozen until they are removed.

Using such a rice stem cell culture method, it is possible to mass-produce rice stem cells having a certain quality in a controlled environment. In addition, in order to facilitate mass production, plant cell cultures using micro-suspended cells derived from callus do not use any animal components, and since plant cells are not infected with human viruses, they are advantageous over animal cell-derived components in terms of safety have.

In addition, when a high-temperature and high-pressure extract of rice embryonic stem cells is obtained using a deposited callus-derived callus (e.g., Accession No. KCTC 11760BP) prepared as described in 1) above, the step of obtaining callus derived from rice is omitted And can be advantageous in terms of quality control.

The Rice Stem Cell High Temperature and High Pressure Extract of the Present Invention The Rice Stem Cell High Temperature and High Pressure Extract can be obtained by subjecting the rice stem cell itself or the rice stem cells, which have been pulverized by freezing with liquid nitrogen, to water, buffer, C1 to C4 lower alcohols, polyhydric alcohols, The process comprises treating an extraction solvent selected from a solvent, preferably a buffer (for example, a phosphate buffer), water, a C1-C4 lower alcohol anhydride or their functional alcohols, glycerin, ethylene glycol, propylene glycol, butylene glycol and mixtures thereof And the extract is subjected to an extraction method such as a high-temperature high-pressure extraction method and extracted, followed by filtration to remove the cell suspension, and the filtrate can be obtained.

The rice embryonic stem cell high temperature and high pressure extract of the present invention can be obtained by suspending rice stem cells in an extraction solvent selected from a phosphate buffer solution, water, ethanol and a mixture thereof, extracting it by high temperature and high pressure treatment, and then lyophilizing it.

However, the present invention is not limited thereto. For example, the rice stem cells may be suspended in an extraction solvent selected from the group consisting of water, buffers, C1 to C4 lower alcohols, polyhydric alcohols and mixtures thereof, Cm 2 to 1.5 kgf / cm 2, more preferably from 1.5 kgf / cm 2 to 1.5 kgf / cm 2, at a temperature of about 100 ° C to 135 ° C (preferably 100 ° C to 125 ° C) / Cm < 2 >) for about 20 minutes to 30 minutes, followed by pulverization and extraction. Here, the extraction conditions are very preferable because they can achieve excellent melanin formation inhibitory effect at a temperature of 100 ° C to 125 ° C and a pressure of 1.5 kgf / cm 2.

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

In addition, the extract of the present invention includes not only the extract obtained by the high-temperature and high-pressure extraction described above, but also the extract obtained through ordinary purification process. For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation using various chromatographies (manufactured for separation according to size, charge, hydrophobicity or affinity) And the fractions obtained by the method are also included in the high-temperature high-pressure extract of the present invention. Herein, fractions obtained by further isolating rice stem cell high-temperature and high-pressure extracts by high performance liquid chromatography (HPLC) were prepared by dissolving the high-temperature and high-pressure extracts of rice embryonic stem cells in water and then fractionating the fractions with sequential use of hexane and ethyl acetate. The fraction and the ethyl acetate fraction were further separated by HPLC, but the extract of the present invention is not limited thereto.

The present invention provides a cosmetic composition comprising the rice embryonic stem cell high temperature high pressure extract obtained as described above as an active ingredient.

The extract of the composition of the present invention is preferably contained in an amount of 0.001 to 10% by weight based on the total weight of the composition. If the amount of the extract is less than 0.001% by weight, the effect of whitening is insufficient due to insufficient useful ingredients. If the amount of the extract is more than 10% by weight, the cosmetic formulation may become unstable.

In addition, the rice embryonic stem cell high temperature and high pressure extract of the present invention Inhibits the activity of tyrosinase in vivo and exhibits an excellent inhibitory effect on melanin production, so that it can be usefully used for a whitening cosmetic composition.

The composition of the present invention may contain at least one selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymeric polysaccharides, sphingolipids and seaweed extracts.

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

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

The polymeric peptide may be any compound as long as it can be compounded in cosmetics, and examples thereof include collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. The polymeric peptide can be obtained by a conventional method such as purification from a culture broth of a microorganism, an enzymatic method, or a chemical synthesis method, or it can be purified from natural products such as ducks such as pigs and cows and silk fiber of silkworms.

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

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

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

The cosmetic composition of the present invention, in addition to the above-mentioned components, may be blended with other components, which are usually added to cosmetics as required.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Examples of the anionic surfactant include fatty acid soap, alpha-acylsulfonate, alkylsulfonate, alkylarylsulfonate, alkylnaphthalenesulfonate, alkylsulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl ginseng salt, Alkylsulfosuccinic acid salts, acylated hydrolyzed collagen peptide salts, and perfluoroalkyl phosphoric acid esters, and the like can be mentioned. have.

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

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

Examples of the organic and inorganic pigments include inorganic pigments such as silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, Bengala, clay, bentonite, titanium mica, titanium oxide, bismuth chloride, zirconium oxide, magnesium oxide, Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, chromium oxide, chromium oxide, chromium hydroxide, But are not limited to, polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluororesin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, Silk powder, cellulose, CI Pigment Yellow, CI Pigment Orange, and composite pigments of inorganic pigments and organic pigments thereof.

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

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

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

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

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

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

In addition, any of the above components may be blended within the range not to impair the objects and effects of the present invention, but it is preferably 0.01 to 50% by weight, More preferably from 5 to 20% by weight.

The ingredients contained in the cosmetic composition of the present invention may further contain ingredients commonly used in cosmetic compositions in addition to the above compounds, for example, conventional adjuvants such as stabilizers, solubilizers, vitamins, Carrier.

In addition, the cosmetic composition of the present invention can be prepared in any formulations conventionally produced in the art, and examples thereof include pastes, creams, gels, powders, sprays, solutions, emulsions and suspensions. More specifically, the present invention relates to a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutritional cream, a moisturizing cream, a hand cream, a foundation, , Cleansing lotion, cleansing cream, body lotion and body cleanser.

The preparation method of these formulations can be produced according to a conventional method of producing a 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 .

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

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

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

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyltaurate, sarcosinates, fatty acid amides Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

The skin whitening cosmetic composition containing the Rice Stem Cell High Temperature and High Pressure Extract of the present invention has an excellent inhibitory activity against tyrosinase involved in melanin biosynthesis and inhibits melanin production, and therefore, whitening for prevention and improvement of pigmentation, And can be usefully used as a cosmetic for cosmetics.

FIG. 1 shows the effect of the extract on the melanin content and cell viability of B16F10 cells according to the change of extraction temperature during the extraction of the high-temperature and high-pressure extracts of rice embryonic stem cells of the present invention (first to third methods of Example 1) Graph.
FIG. 2 is a graph showing the effect of the solvent-extracted fraction of the high-temperature high-pressure extract of rice embryonic stem cells of the present invention (Example 2, Method 2) on the melanin content and cell viability of B16F10 cells.
Fig. 3 is an HPLC chromatogram showing the isolation of a whitening active substance from the high-temperature high-pressure extract of rice embryonic stem cells of the present invention (second method of Example 1).
4 is an HPLC chromatogram showing separation of the whitening active substance from the ethyl acetate fraction of the high-temperature and high-pressure extract of rice embryonic stem cells of the present invention.
FIG. 5 is a graph showing the relationship between the melanin content and the cell survival rate in B16F10 cells of the rice embryonic stem cell high-temperature high-pressure extract of the present invention (the second method of Example 1), the ethyl acetate fraction of the high- [Sample 1: Rice Stem Cell High Temperature & High Pressure Extract 200 ppm; Sample 2: HPLC fractionation of Rice Stem Cell High Temperature & High Pressure Extract 200 ppm; Sample 3: Ethyl acetate fraction of the rice embryonic stem cell high temperature and high pressure extract 200 ppm; And Sample 4: HPLC fraction 200 ppm separated from the ethyl acetate fraction of the rice embryonic stem cell high temperature and high pressure extract]

Hereinafter, the present invention will be described more specifically with reference to the following Reference Examples, Examples, Formulation Examples and Test Examples. However, the description of the following reference examples, formulation examples, examples, and test examples is for illustrative purposes only and is not intended to limit or limit the scope of the present invention.

[ Reference Example  One] Of rice stem cells  Ready

A rice-derived callus (accession number: KCTC 11760BP) was cultivated in a medium containing 30 g / L sucrose, 2 mg / L 2,4-D, 0.2 mg / L kinetin, AA (amino acid, Duchefa, Netherlands) and incubated at 28 ° C and 110 rpm in shaking culture at 2 weeks intervals. Three-month-old rice suspension cells cultured as described above were cultured in an AA medium containing 30 g / L of sucrose, 2.0 mg / L 2,4-D, 0.2 mg / L kinetin and 0.1 mg / L gibberellic acid And transferred. At this time, only the micro-suspended cells isolated from the cell aggregate were selected and subcultured. In order to eliminate the possibility of toxicity due to residues of 2,4-D, micro-suspended cells having a diameter stabilized at about 1 mm or less were cultured in the medium except 2,4-D in the medium.

The stabilized microsuspension cells can be prepared by adding a disk type gas sparger, a hollow-paddle impeller and a sampling port sized to harvest plant cells, Were cultivated batch-wise in a 7 L capacity bioreactor (Applikon, The Netherlands) modified for culture. The working volume was 3.2 L and the initial agitation rate and the aeration rate were 80 rpm and 0.2 vvm, respectively. AA medium (2.3 L) was sterilized by autoclaving in a bioreactor at 121 ° C for 25 minutes, and then 300 ml of a 10-fold concentrated (10x) amino acid solution filtered with a 0.22-μm filter (Millipore, USA) And injected into the AA medium. The flask-cultured cells were then inoculated into the bioreactor in a 10% FCWV (fresh cell weight volume). At this time, a tube welder (Wave Biotech, Switzerland) was used to sterilize the tubing between the inoculation bottle and the bioreactor vessel.

The incubation temperature was 28 ℃ and 90-110 rpm. In order to maintain the dissolved oxygen (DO) level at 20% or more during the incubation period, the growth rate and stirring speed were controlled. Air and oxygen were mixed and the rate of expansion was adjusted to intermittently enter between 0.5 L / min for air and between 0.1 ml / min and 100 ml / min for oxygen.

Bioreactor cultures were alternated between dark and fluorescent conditions, and cells were harvested at 7 days of culture. The recovered cells were suspended again in PBS and washed 2 to 3 times to remove the excess medium. After the last washing, the washing solution was completely removed by filtering. The rice stem cells thus prepared were directly used for extraction or frozen or frozen until the time of extraction.

[ Reference Example  2] mouse melanoma ( melanoma ) Preparation of cells

Mouse melanoma cells (B16-F10 cell, ATCC CRL-6475) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 0.1% penicillin-streptomycin (100 unit / ml, penicillin- streptomycin) (Dulbecco ' s Modified Eagle ' s Medium, GIBCO) medium and incubated at 37 < 0 > C in a 5% CO ₂ incubator maintained at a saturated humidity. The medium was changed twice a week. Cells that reached 70 ~ 80% confluency were kept in a subculture after trypsinization using 0.25% trypsin (GIBCO, 15050-065).

[ Example  One] Rice stem cells  Preparation of high-temperature high-pressure extract

First Method - 60 g of the rice stem cell prepared in Reference Example 1 was suspended in 300 ml of water and then put into a high-temperature and high-pressure treatment apparatus (MLS-3750, SANYO, JAPAN) and extracted at a pressure of 1.5 kgf / , And the extract was filtered with Watman paper to remove cell suspension to obtain a filtrate. The filtrate was lyophilized and placed in a container and kept at room temperature until use.

Second Method - 60 g of the rice stem cell prepared in Reference Example 1 was suspended in 300 ml of water and placed in a high-temperature and high-pressure treatment apparatus (MLS-3750, SANYO, JAPAN) and extracted at 125 캜 for 30 minutes under a pressure of 1.5 kgf / , And the extract was filtered with Watman paper to remove cell suspension to obtain a filtrate. The filtrate was lyophilized and placed in a container and kept at room temperature until use.

(Method 3) 60 g of the rice stem cell prepared in Reference Example 1 was suspended in 300 ml of water and then put into a high-temperature and high-pressure treatment apparatus (MLS-3750, SANYO, JAPAN) and extracted at a pressure of 1.5 kgf / , And the extract was filtered with Watman paper to remove cell suspension to obtain a filtrate. The filtrate was lyophilized and placed in a container and kept at room temperature until use.

[ Example  2] Rice stem cells  Of high-temperature high-pressure extract Solvent fraction  Produce

2-1. Hexane  Preparation of soluble extracts

25 g of the extract obtained in the second method of Example 1 was added to 250 ml of distilled water and extracted with stirring at room temperature for 12 hours. Thereafter, the process of evaporating water using a rotary vacuum evaporator (N1110, EYELA, JAPAN) was repeated three times . 24 g of the water extract thus obtained was dissolved in 250 ml of distilled water and then mixed with 250 ml of hexane in a separating funnel. The hexane insoluble layer (water layer) and the hexane soluble layer were fractionated to obtain 250 ml of the hexane insoluble extract and 250 ml of the hexane soluble extract Respectively.

2-2. Preparation of Ethyl Acetate Soluble Extracts

250 ml of the hexane-insoluble extract obtained in 2-1 above was mixed with 250 ml of ethyl acetate, and then the ethyl acetate-soluble layer and the insoluble layer were fractionated to obtain 250 ml of the ethyl acetate-insoluble extract and 175 mg of the ethyl acetate-soluble extract.

The hexane-soluble extract, ethyl acetate-soluble extract and ethyl acetate-insoluble extract were concentrated under reduced pressure and lyophilized to obtain hexane fraction (7 g), ethyl acetate fraction (175 mg) and water fraction (10.5 g).

[ Comparative Example ] Other Rice stem cells  Preparation of extract

Fourth Method - 60 g of the rice stem cell prepared in Reference Example 1 was suspended in 300 ml of water and then put into a high-temperature and high-pressure treatment apparatus (MLS-3750, SANYO, JAPAN) and extracted at a pressure of 1.5 kgf / , And the extract was filtered with Watman paper to remove cell suspension to obtain a filtrate. The filtrate was lyophilized and placed in a container and kept at room temperature until use.

Fifth Method - 60 g of the rice stem cell prepared in Reference Example 1 was frozen in liquid nitrogen, ground in a mortar and then placed in 300 ml of phosphate buffered saline (PBS) on a magnetic stirrer (Corning, USA) After stirring for 24 hours at room temperature, the prepared extract was filtered with Watman Paper to remove cell suspension, and a filtrate was obtained. The filtrate was lyophilized and placed in a container and kept at room temperature until use.

Sixth Method - 60 g of the rice stem cell prepared in Reference Example 1 was frozen with liquid nitrogen, ground in a mortar, suspended in 300 ml of water, treated with a sonicator (Sonic & Materials, Model Vibra Cell) for 10 seconds, For 24 hours. Then, the prepared extract was filtered with Watman Paper to remove cell suspension, and a filtrate was obtained. The filtrate was lyophilized and stored in a container at room temperature until use.

Seventh method - 60 g of the rice stem cell prepared in Reference Example 1 was frozen with liquid nitrogen, ground in a mortar, suspended in 300 ml of water, treated with a sonicator (Model Vibra Cell) for 10 seconds, The extract was filtered through Watman paper to remove cell suspension, and a filtrate was obtained. The filtrate was lyophilized and stored in a container at room temperature until use.

Method 8-60 g of the rice stem cells prepared in Reference Example 1 were frozen in liquid nitrogen, ground in a mortar at 4 ° C, suspended in 300 ml of 80% (v / v)% ethanol and sonicated for 10 seconds After 24 hours of stirring at room temperature for 24 hours, the prepared extract was filtered with Watman paper to remove cell suspension, and a filtrate was obtained. The filtrate was lyophilized and stored at room temperature until use.

Method 9 - The callus derived in accordance with Example 1 (1) of Korean Patent Laid-Open Publication No. 2010-0131598 was finely ground on liquid nitrogen and dissolved in purified water and used in the following test examples.

[ Test Example  One] Rice stem cells  Measurement of Melanogenesis Inhibitory Activity of B16F10 Cell Lines from High Temperature and High Pressure Extracts

1 ml of the melanoma cells prepared in Reference Example 2 (2 x 10 ⁴ cells / ml) was dispensed in a 12-well plate containing the culture medium, and then cultured in a 37 ° C and 5% CO ₂ incubator for 24 hours. Then, the culture medium was changed, and the rice embryonic stem cell high-temperature and high-pressure extracts of Example 1 were dissolved in water and cultured for 48 hours at 200 ppm (concentration in terms of total volume of the culture medium) at 200 ppm. Negative control group was treated with 1X PBS (phosphate buffered saline, Gibco, 21600-010), and positive control group was treated with 200ppm (concentration of arbutin) in the same manner. The extract of Comparative Example was also dissolved in water and treated at the concentration of 200 ppm (concentration relative to the total volume of the culture medium) and cultured in the same manner. After the culture, the culture supernatant was removed, and 1 ml of PBS was added to each well. After washing twice, 0.2 ml of 0.25% trypsin (Gibco, 15050-065) was treated to recover the cells. The recovered cells were placed in a 1.5 ml tube and centrifuged at 5000 rpm for 5 minutes. 100 μl of 1 N sodium hydroxide (NaOH) solution containing 10% DMSO was added to the cell pellet and suspended, . The intracellular melanin dissolved by the above reaction was measured at 405 nm using a 96-well plate reader. The melanin content was expressed as a percentage of the absorbance as compared with the negative control, as shown in the following formula (1), and the results are shown in Table 1 and FIG.

[Equation 1]

Melanin content (%) = absorbance of the sample treated group (OD405? Value) / negative control absorbance (OD405? Value) 100

[Table 1]

As shown in Table 1, the melanin production inhibitory effect was observed at a concentration of 200 μg / ml in the rice embryonic stem cell high-temperature high-pressure extract of the present invention. In particular, melanin production inhibitory effect was observed at an extraction temperature of 100 ° C. to 125 ° C. I was particularly impressed with the excellent. Further, even when the rice callus was extracted with water at room temperature as in Comparative Example 9, or even under high pressure conditions (1.5 kgf / cm 2) as in Comparative Example 4, even when the rice stem cell was extracted with water at an extraction temperature of less than 100 ° C % Inhibition effect on the melanin production.

Hereinafter, " Rice stem cell high-temperature high-pressure extract of Example 1 " referred to in Test Examples 2 to 5, Formulation Examples and Comparative Formulation Examples was referred to as " Rice stem prepared by Method 2 of Example 1 Cell high temperature high pressure extract ".

[ Formulation Example  One] Example  1 < / RTI > skin )

(Skins) containing the high-temperature high-pressure extract of Rice Stem Cells of Example 1 at a composition ratio shown in Table 2 below.

[Table 2]

<Manufacturing Method>

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

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

1 kg of a nutritional lotion (lotion) containing the high-temperature and high-pressure extract of Rice Stem Cells of Example 1 was prepared at the composition ratios shown in Table 3 below.

[Table 3]

<Manufacturing Method>

Components 2, 3, 4 and 5 of Table 3 were homogenized at room temperature to produce nonionic amphipathic lipids. The above nonionic amphipathic lipids were mixed with components 1, 6, 7 and 13, homogenized at room temperature and passed through a microfluidizer, followed by slow addition of component 8 at room temperature, homogenization, and then passed again through a microfluidizer . Then, ingredients 9, 10, 11 and 12 were added, dispersed, stabilized and aged to prepare a nutrition lotion (lotion).

[ Formulation Example  3] Example  1 &lt; / RTI &gt;

1 kg of nutritional cream containing the rice embryonic stem cell high-temperature high-pressure extract of Example 1 was prepared at the composition ratios shown in Table 4 below.

[Table 4]

<Manufacturing Method>

Components 2, 3, 4 and 5 of Table 4 were homogenized at room temperature to prepare nonionic amphipathic lipids. The nonionic amphipathic lipids were mixed with components 1, 6, 7 and 13, homogenized at room temperature and passed through a microfluidizer, followed by slow addition of component 8 at room temperature to homogenize, and then passed again through a microfluidizer . Then, ingredients 9, 10, 11 and 12 were added, dispersed, stabilized and aged to prepare nutritional cream.

[ Test Example  2] Rice stem cells  Cytotoxicity of high-temperature and high-pressure extracts

The cytotoxicity of the high-temperature and high-pressure extracts of rice embryonic stem cells was determined by MTT assay based on the metabolic reduction of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide]. Live cells are converted to MTT formazan crystals by the dehydrogenase present in the mitochondria, so that the viable cell viability can be determined by measuring the amount of the MTT formazan crystals (Reference: The EMBO Journal (2002) 21, pp2407-2417 et al.) .

The mouse melanoma cells prepared in Reference Example 2 (1 x 10 ⁴ cells / ml) were dispensed into a 96-multiwell plate at 200 μl / well, and inoculated on a culture medium and incubated for 24 hours. Then, the culture medium was changed, and the rice embryonic stem cell high-temperature high-pressure extract of Example 1 was dissolved in water and treated for 48 hours. Negative control group was treated with 1X PBS (phosphate buffered saline, Gibco, 21600-010), and positive control group was treated with 200ppm (concentration of arbutin) in the same manner. The extract of Comparative Example was also dissolved in water and treated at the concentration of 200 ppm (concentration relative to the total volume of the culture medium) and cultured in the same manner. 50 쨉 l of MTT solution (500 / / ml) was added to the cultured cells and reacted at 37 째 C for 4 hours to form formazan crystals. After removing the culture supernatant, 500 μl of DMSO was added to each well to dissolve the formazan crystal, and the absorbance was measured at 540 nm in a 96-well plate reader (Spectra Max M2, Molecular Device, US). Cell viability was expressed as a percentage of the absorbance as compared with the negative control, as shown in the following formula (2), and the results are shown in Table 5.

&Quot; (2) &quot;

Cell viability (%) = absorbance of the sample treated group (OD540? Value) / negative control absorbance (OD540? Value) 100

[Table 5]

As shown in the above Table 5, it was found that the rice embryonic stem cell high-temperature high-pressure extract of the present invention showed almost no cytotoxicity to mouse melanoma cells up to a concentration of 200 μg / ml. In addition, most of the extracts of the rice stem cell extracts of Examples and Comparative Examples except for the extracts obtained by the methods of Comparative Examples 5 and 9 showed almost no cytotoxicity.

[ Test Example  3] Rice stem cells  Of high-temperature high-pressure extract in vitro tyrosinase  Confirmation of inhibition

The inhibitory activity of tyrosinase, an enzyme that plays an important role in the production of melanin, is caused by a reaction with L-DOPA (L-3,4-dihydroxyphenylalanine), which is a substrate of mushroom tyrosinase as an enzyme source The absorbance of the produced L-dopaquinone was measured.

850 0.1 of 0.1 M phosphate buffer (pH 7.0), 50 쨉 l of the rice embryonic stem cell high-temperature high-pressure extract of Example 1 diluted by concentration, and 50 쨉 l of mushroom tyrosinase (250 U / ml) For 6 min. To this solution, 50 μl of a 0.06 mM L-DOPA solution was added and reacted at 37 ° C. for 1 minute. 0.1M phosphate buffer and kojic acid (200 ppm) were used as negative control and positive control, respectively. After the end of the experiment, the absorbance was measured at 475 nm using a 96 well plate reader.

The inhibition rate of tyrosinase activity was expressed as a percentage of the absorbance as compared with the negative control, as shown in the following formula (3), and the results are shown in Table 6.

&Quot; (3) &quot;

(%) = [1- (sample treated group absorbance (OD475? Value) / negative control absorbance (OD475? Value)}] × 100

[Table 6]

As described above, since the rice embryonic stem cell high-temperature high-pressure extract of the present invention does not inhibit mushroom tyrosinase activity at all treatment concentrations, unlike kojic acid, which is a positive control, the whitening activity is shown by a mechanism directly acting on tyrosinase I could confirm that.

[ Test Example  4] Rice stem cells  Of high-temperature high-pressure extract in vivo tyrosinase  Confirmation of inhibition

1 ml of mouse melanoma cells prepared in Reference Example 2 (5 × 10 ⁵ cells / ml) was inoculated in a 100-mm cell culture dish containing the culture medium, and cultured in a 37 ° C. and 5% CO ₂ incubator for 24 hours Cultured and adhered to the plate. Thereafter, the medium was replaced with DMEM (containing 10% fetal bovine serum) supplemented with no antibiotic, and the high-temperature and high-pressure extracts of rice embryonic stem cells prepared in Example 1 were treated at different concentrations. As negative control and positive control, 0.1 M phosphate buffer and arbutin (200 ppm). After 3 days of culture, the medium was discarded and the cells were recovered with a scrapper. The recovered cells were placed in a 1.5 ml tube and centrifuged at 2000 rpm for 3 minutes. The obtained pellet was washed with PBS. 300 μl of 0.1 M potassium phosphate buffer (pH 6.8) containing 1% triton X-100 and 2 nM PMSF was added to the pellet, and the cells were disrupted by ultrasonic wave for 10 minutes and centrifuged at 10,000 × g for 10 minutes to collect the supernatant . The supernatant was used as an enzyme source for tyrosinase activity measurement and as a sample for protein quantification. The supernatant was quantitated using the BCA method (Bio-Rad protein kit (Hercules, CA, USA)) and 0.1 ml of the same amount of protein was mixed with 0.1 ml of 1.5 mM L-tyrosine. After incubating for 1 hour at 37 ° C in an incubator, absorbance was measured at 450 nm using a 96 well plate reader (SpectraMax M2, Molecular Device, USA).

The tyrosinase activity was expressed as a percentage of the absorbance as compared with the negative control, as shown in the following formula (4), and the results are shown in Table 7 below.

&Quot; (4) &quot;

Tyrosinase activity (%) = {sample treated group absorbance (OD450? Value) / negative control absorbance (OD450? Value)} 100

[Table 7]

As described above, it was confirmed that the rice embryonic stem cell high-temperature high-pressure extract of the present invention inhibits intracellular tyrosinase activity in a concentration-dependent manner at all experimental concentrations. In particular, the concentration of 200ppm or more excellent compared to the positive control arbutin in vivo tyrosinase activity.

The rice embryonic stem cell high-temperature high-pressure extract of the present invention can be prepared by subjecting the rice stem cell to an after-treatment with an extract (Comparative Example 5) prepared by suspending and stirring the rice embryonic stem cells in a solvent, It was confirmed that the extracts (Comparative Examples 6 to 8) and rice callus were finely ground and exhibited far superior intracellular tyrosinase activity than the extract (Comparative Example 9) dissolved in purified water.

Comparing these results with the results of Test Example 2, the rice embryonic stem cell high-temperature and high-pressure extracts of the present invention are different from the conventional whitening active ingredients in vivo tyrosinase-specific whitening activity.

[ Test Example  5] Human skin patch test ( Patch test )

To examine the degree of skin irritation on the cosmetic composition containing the high-temperature and high-pressure extract of Rice Stem Cell Preparations prepared in Example 1, a human skin patch test was conducted.

The subjects were selected as women without skin disease at the test site of 20-50 years old. The supernatant, which is the patch area, was wiped with 70% (v / v)% ethanol and dried. The softening length of Formulation Example 1, the nutrition lotion of Formulation Example 2, the nutritional cream of Formulation Example 3, 0.02 g each was added to the test site.

After 24 hours, the skin response was evaluated within 1 hour after chamber removal and the skin reaction was evaluated again the next day (after 48 hours). Skin reaction determinations were made according to the evaluation criteria shown in Table 8 below and the evaluation of the possibility of skin irritation was performed by calculating the average reaction rates calculated according to the following formula 5 and the results are shown in Table 9 Respectively.

&Quot; (5) &quot;

Average reaction rate (%) = {Σ (number of respondents after 24 hours × number of scores) + Σ (number of respondents after 48 hours × number of scores)} × 100 / (total number of tests × maximum number × total number of subjects)

[Table 8]

[Table 9]

As shown in Table 9, the cosmetic composition containing the high-temperature and high-pressure extract of Rice Stem Cells of the present invention did not induce skin irritation, and thus the safety of the Rice Stem Cell High Temperature and High Pressure Extracts of the present invention could be confirmed.

[ Test Example  6] Rice stem cells  High temperature high pressure extract and its Of the solvent fraction  Measurement of cytotoxicity and inhibition of melanin formation

In order to isolate the whitening active substance from the high-temperature and high-pressure extracts of rice embryonic stem cells of the present invention, it was determined whether any fraction of the solvent fractions of the high-temperature and high-pressure extracts of the rice stem cell high temperature and pressure extract prepared in Example 2 inhibited melanogenesis, And whether or not they have toxicity.

1 ml of the melanoma cells prepared in Reference Example 2 (2 x 10 ⁴ cells / ml) was inoculated on a 12-well plate containing the culture medium, and then incubated in a 37 ° C and 5% CO ₂ incubator for 24 hours. Thereafter, the culture medium was changed, and the high-temperature high-pressure extract of Rice Stem Cell Preparations prepared in Example 1 and the solvent fractions prepared in Example 2 were each treated with 200 ppm (concentration relative to the total volume of the culture medium), and the positive control group was arbutin 200 ppm (concentration for the entire volume of the culture medium) was treated. The negative control group was also treated with 1X PBS (Gibco, 21600-010). After the extract was treated and cultured for 2 days as described above, the medium was removed, and 1 ml of PBS was added thereto. The cells were washed twice, and the melanin content in the cells was checked in accordance with the procedure of Test Example 1, Was checked for cell viability according to the procedure of Test Example 2. [ Melanin content and cell viability were calculated as shown in Equations (1) and (2), and the results are shown in Table 10 and FIG.

[Table 10]

As described above, the ethyl acetate fraction of the solvent fraction of the rice embryonic stem cell high temperature and high pressure extract of the present invention showed the most excellent inhibitory effect on melanin formation.

These rice embryonic stem cell high-temperature high-pressure extracts and ethyl acetate fractions thereof were further fractionated as chromatographic samples. 10 μl of the sample was injected into a column (Prep Nova-Pak HR C18 Column, 6 μm, 7.8 × 300 mm), methanol: water = 10: 90 (v / v% (HPLC) (High Performance Liquid Chromatography, 1525 Binary HPLC Pump, Waters). The elution rate of the mobile phase solvent was 2 ml / min. And the UV detection wavelength was maintained at 210 nm. The temperature during the HPLC process was room temperature (25 캜).

The rice embryonic stem cell high-temperature high-pressure extract of the present invention and its ethylacetate fractions were each detected within 100 minutes of elution time, and each of them was separated into three fractions. In the case of the rice embryonic stem cell high-temperature high-pressure extract, fraction 6 of 10 minutes to 20 minutes, 6 mg of fraction 2 between 20 minutes and 30 minutes, 207 mg of fraction 2 between 20 minutes and 30 minutes, ; In the case of the ethyl acetate fraction, 16 mg of fraction 3 between 33 minutes, 112 minutes and 20 minutes to 20 minutes and 20 minutes to 30 minutes were obtained, respectively.

The rice stem cell extract and the high temperature and high pressure within the tyrosinase analysis the ethyl acetate fraction to each of the fractions 1 to 3 to the sample in Test Example 4 of the cells (in vivo tyrosinase assay). As a result, it was found that each fraction 2 contained a major whitening active substance (see separation of main substances at 25 minutes of retention time, see FIGS. 3 and 4). The rice embryonic stem cell high temperature high pressure extract (I) of the present invention, its HPLC fraction 2 (II); The ethyl acetate fraction (Ⅲ) and the HPLC fraction 2 (Ⅳ) of the rice embryonic stem cell high-temperature high-pressure extract were treated with melanoma cells as in Test Example 4, and the results are shown in Table 11 and FIG.

[Table 11]

As shown in Test Example 6, the rice embryonic stem cell high temperature and high pressure extract of the present invention, the ethyl acetate fraction thereof and the respective HPLC fractions thereof exhibit excellent whitening activity, and thus can be usefully used for a whitening cosmetic composition.

Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology KCTC11760BP 20100907

Claims (27)

A cosmetic composition for whitening comprising a high-temperature and high-pressure extract of rice stem cells as an active ingredient, Wherein said rice stem cell high-temperature high-pressure extract is extracted at a temperature of 100 ° C to 135 ° C and a pressure of 1.2 kgf / cm 2 to 2.0 kgf / cm 2. The method according to claim 1,
When the rice stem cell is rice ( Oryza wherein the cells are undifferentiated cells derived from one or more tissues selected from the group consisting of leaves, stems, seeds and roots of S. sativa L. The method according to claim 1,
When the rice stem cell is rice ( Oryza sativa L.) calli (Accession No. KCTC 11760BP). The method of claim 3,
Wherein said rice stem cell is a stabilized micro-suspended cell derived from said rice callus. The method of claim 4,
Wherein the stabilized microsuspension cells do not exhibit the property of agglomeration of cells in suspension culture, and the diameter thereof is maintained at 1 mm or less. The method according to claim 1,
Wherein said rice stem cell high-temperature high-pressure extract is obtained by extracting from rice stem cells with an extraction solvent selected from phosphate buffer, water, ethanol and a mixed solvent thereof. delete The method according to claim 1,
Wherein said rice stem cell high temperature and high pressure extract is extracted at a temperature of 100 ° C to 125 ° C and a pressure of 1.2 kgf / cm 2 to 1.5 kgf / cm 2. The method according to claim 1,
Wherein said rice stem cell high-temperature high-pressure extract is extracted at a temperature of 100 ° C to 125 ° C and a pressure of 1.5 kgf / cm 2. [Claim 7] The cosmetic composition according to claim 6, wherein the rice stem cell high temperature high pressure extract is a fraction obtained by further isolating the rice stem cell extract as an extraction solvent under high temperature and high pressure using high performance liquid chromatography (HPLC). [Claim 7] The method according to claim 6, wherein the rice embryonic stem cell high temperature and high pressure extract is an ethyl acetate fraction obtained by dissolving the rice stem cell extract as an extraction solvent under high temperature and high pressure and then fractionating the extract with water and sequentially using hexane and ethyl acetate Cosmetic composition. [Claim 12] The cosmetic composition according to claim 11, wherein the Rice stem cell high temperature high pressure extract is a fraction obtained by further separating the ethyl acetate fraction using high performance liquid chromatography (HPLC). [Claim 7] The cosmetic composition according to claim 6, wherein the Rice Stem Cell High Temperature and High Pressure Extract is obtained by Extraction from Rice Stem Cells with Water. The method according to claim 1,
Wherein said rice stem cell high-temperature high-pressure extract is contained in an amount of 0.001 to 10% by weight based on the total weight of the composition. The method according to claim 1,
The high-temperature and high-pressure extracts of the rice stem cells were obtained by intracellular tyrosinase ( in vivo tyrosinase activity, thereby inhibiting the production of melanin. The method according to any one of claims 1 to 6 and claims 8 to 15,
The cosmetic composition may be at least one selected from the group consisting of a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, a foundation, Foam, cleansing lotion, cleansing cream, body lotion and body cleanser. A large-scale culture step in which rice stem cells are mass-cultured;
The mass cultured rice embryonic stem cells are suspended in a solvent, and then subjected to a high-temperature high-pressure treatment step at a temperature of 100 ° C to 135 ° C and a high-temperature and high-pressure treatment under a pressure of 1.2 kgf / cm 2 to 2.0 kgf /
Lt; RTI ID = 0.0 &gt; high-pressure &lt; / RTI &gt; 18. The method of claim 17,
A large-scale culture step of mass-culturing rice stem cells;
The high-temperature and high-pressure treatment step in which the mass cultured rice stem cells are suspended in a solvent and subjected to a high-temperature high-pressure treatment under a temperature condition of 100 ° C to 125 ° C and a pressure of 1.2 kgf / cm 2 to 1.5 kgf /
Lt; RTI ID = 0.0 &gt; high-pressure &lt; / RTI &gt; 18. The method of claim 17,
A large-scale culture step of mass-culturing rice stem cells;
The mass cultured rice embryonic stem cells are suspended in a solvent, and then subjected to a high-temperature high-pressure treatment step at a temperature of 100 ° C to 125 ° C and a high-temperature and high-pressure treatment under a pressure of 1.5 kgf /
Lt; RTI ID = 0.0 &gt; high-pressure &lt; / RTI &gt; 18. The method of claim 17,
The rice embryonic stem cells are selected from the group consisting of rice A method for producing a high-temperature and high-pressure extract of rice embryonic stem cells, which is a stabilized microsuspension cell derived from a sativa L. derived callus (Accession No. KCTC 11760BP). The method of claim 20,
Wherein the stabilized microsuspension cells do not exhibit the property of agglomeration of cells in suspension culture, and the diameter thereof is maintained at 1 mm or less. 18. The method of claim 17,
Wherein the solvent is an extraction solvent selected from a phosphate buffer, water, ethanol and a mixed solvent thereof. 18. The method of claim 17,
Further comprising the step of applying the extract obtained in the high-temperature and high-pressure treatment step to high-performance liquid chromatography (HPLC) to obtain a fraction. 18. The method of claim 17,
Further comprising the step of dissolving the extract obtained in the step of high-temperature and high-pressure treatment in water, followed by further fractionation with hexane and ethyl acetate sequentially to obtain an ethyl acetate fraction. 27. The method of claim 24,
Further comprising fractionating the ethyl acetate fraction by high performance liquid chromatography (HPLC) to obtain a fraction. The method according to claim 22, wherein the solvent is water. 18. The method of claim 17,
Further comprising the step of concentrating the filtrate obtained by removing the suspended matter of the cells by filtration of the extract and concentrating the resulting filtrate.

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