KR20100131598A - Skin composition for external application containing oryza sativa callus and preparing the same - Google Patents

Abstract

PURPOSE: An external use skin composition having rice plant callus extract is provided to ensure skin whitening, anti-oxidation, and anti-inflammation. CONSTITUTION: An external use skin composition for improving skin condition contains 0.1-10 weight% of rice plant callus extract as an active ingredient. A method for manufacturing the external use skin composition comprises: a step of inducing callus from rice plant seed and culturing; and a step of preparing the composition having the callus. The induction of callus is performed by culturing from rice plant seeds in a medium containing epigallocatechingallate.

Description

Skin composition for external application containing rice callus extract and method for preparing the same {Skin Composition for External Application Containing Oryza Sativa Callus and Preparing the Same}

The present invention relates to an external preparation composition for skin containing a rice callus extract and a method for producing the same.

Modern man is always threatened from the diseases associated with it by various internal and external stresses, and the skin is not free from such stresses. Various studies have been attempted to keep the skin healthy from such a skin stress, and in recent years, interest in cosmetics using natural extracts obtained from natural substances having little skin irritation is increasing.

Plants have long been a source of various natural substances that help human well-being. Most of these substances are secondary metabolites and are used for pharmaceuticals, food additives, cosmetic raw materials, fragrances, and pigments. Despite the synthesis of many new compounds with the development of organic synthesis, plants are still recognized as the source of new substances. Considering that there are more than a thousand new substances with new chemical structures found in plants every year, many of them are bioactive, which makes them an important ingredient.

 Many chemicals produced by plants are classified as primary metabolites because they are essential for cellular function. Primary plant metabolites are universally present in plant populations and constitute the majority of the chemicals produced by plants, while other compounds are classified as secondary metabolites and because of their many uses, both scientific research and commercial use are the focus of both. It is.

In some cases secondary metabolites are produced by plants, for example in response to stress induced by microbial infection, UV radiation, mechanical damage and also by treatment with organic and inorganic materials. The variety of secondary metabolites produced under such stress conditions is broad and includes alkaloids, terpenoids, prebonoids, carotenoids, phenolic compounds and glycosides. However, not all secondary metabolites are produced in large quantities by all plants, and the kinds of metabolites depend largely on the type of plant.

Plants have an excellent totipotency in which the entire individual is formed even when only a part of the leaves, stems, and roots constituting the plant are transplanted. Plant cells are callus derived from plant tissues. Callus is an amorphous tissue or cell mass that has lost its ability to cause normal organogenesis or tissue differentiation.Auxin or Cytokinins Occurs when cultured in a medium containing or wounding the plant, or treated with the auxin of plants.

Callus can be derived from any part of the plant such as leaves, stems, roots, rhizomes, and fruits through plant cell culture, especially tissue culture, and is characterized by being continuously produced by subculture. The basic principle of the cultivation of plant cells takes advantage of the biological fact that every plant cell has the ability to build the whole plant from which the cell is derived. This pluripotency is comparable to the pluripotency of embryonic stem cells in animals. Therefore, it can be accepted that plant cells have a positive effect on the protection and activation of skin stem cells.

On the other hand, callus is an oily tissue in which a wound cell recovers division ability and prevents and enlarges when a wound is caused to a plant, and a special mass of tissue generated by culturing the tissue cut from the plant in an auxin-containing medium or the like. In recent years, interest in the use of such callus has been increasing and is being studied in various ways. Further, as a public patent related to callus, there is a 10-2008-0038068 or the like.

 On the other hand, up to now, the method of producing rice extract used as a cosmetic raw material (Korea Patent Registration No. 0135251, Korean Patent Publication No. 2003-0068845) or the study on the tissue culture of rice, but the callus extract by inducing callus from rice seeds There was no example of manufacturing a cosmetic composition using or inducing a useful substance to the rice callus.

Therefore, the inventors of the present invention, while trying to manufacture a skin external preparation composition having a skin improvement ability, confirmed that the rice callus has a tyrosinase inhibitory activity and has a skin whitening effect, an antioxidant effect and an anti-inflammatory effect, and completed the present invention.

Accordingly, it is an object of the present invention to provide a skin external preparation composition for improving skin containing rice callus extract as an active ingredient.

Still another object of the present invention is to provide a method for preparing a skin external composition for skin improvement containing the rice callus extract as an active ingredient.

In order to achieve the above object, the present invention provides a skin external composition for skin improvement containing rice callus extract as an active ingredient.

The present invention also comprises the steps of (a) inducing callus from the rice seed culture; And (b) provides a method for producing a skin external composition for skin improvement containing rice callus comprising the step of preparing a composition containing the callus.

The rice callus-containing skin external preparation composition according to the present invention activates skin cells due to skin cell growth and proliferation, has a moisturizing effect, and has an excellent effect on skin regeneration and skin soothing.

The present invention relates to a skin external preparation composition for improving skin containing a rice callus extract as an active ingredient, and a method for preparing the same. Specifically, the present invention induces callus (Callus) from rice seeds using tissue culture technology, and the skin A topical composition, for example, relates to a use as a cosmetic raw material and a method for producing the same.

The present invention relates to a skin external preparation composition for improving skin containing rice callus extract as an active ingredient in one aspect. Specifically, the rice callus extract may contain from 0.01 to 30% by weight, preferably 0.1 to 10% by weight relative to the total weight of the composition. If it is contained in less than 0.1% by weight, it is difficult to exhibit the desired skin improvement effect, when containing more than 10% by weight there is a disadvantage that shows a problem in the formulation.

In the present invention, "extract" means an extract obtained by powdering the rice callus itself or rice callus, or by hot water extraction, ethanol extraction, or the like.

In the present invention, the meaning of "contained as an active ingredient" means that the composition for external application for skin can contain a skin improving effect, for example, can contain and improve the proliferative activity of skin cells. do. In addition, it means that it contains an effective amount that can exhibit a moisturizing effect, skin soothing effect and the like as the external preparation for skin.

In the present invention, rice (Oryza sativa) is a monocotyledonous plant, with a plant and a year old, 50-100 cm high, forming a lively abandonment, the fruit matures in September-October, and is a crop that has been used for food for many years. . The rice from these rice consists of rice bran, rice pear, and rice bran, of which rice bran contains a large amount of fat-soluble components, which are rich in peptides, glue seeds, and various minerals. It consists of cellulose, glue seed, starch, etc., and the rice flakes contain unsaturated fatty acids and vitamin groups such as oleic acid, linoleic acid, filtmic acid and lecithin.

 In the present invention, callus refers to a special tissue or a mass of cells generated when a tissue cut out of a plant is cultured in an auxin-containing medium, a certain kind of plant is wounded, or an upper mouth is treated with auxin. Normally, callus is an amorphous tissue or cell mass that has lost its ability to cause normal organogenesis or tissue differentiation, and is mostly composed of flow cells. In a broad sense, it may also include plant tumor tissue caused by infections such as agrobacterium.

When callus, which is a mass of undifferentiated cells through tissue culture, is formed, that is, the cells in a plant are directed immediately after proliferation, and are assembled regularly into specific tissues and organs. However, when various plant growth hormones are given from the outside, the control that has been maintained until then is released, and cells are interpreted to proliferate arbitrarily. When the callus obtained by this dedifferentiation is put in a liquid medium having the same composition and shaken for culture, the cells are separated and continue to proliferate as a suspended state. The callus or cultured cells are subdivided and proliferated permanently by passage in fresh medium, which is essentially different from the differentiated cells in which complete plants age and die. Callus or cultured cells, which have been passaged for several generations, are applied to a solid medium from which various plant growth hormones have been removed. In other words, the plant is restored in one somatic cell that makes up the plant. Plant tissue cells isolated from the parent plants have the potential to be regenerated into complete plants when cultured in an appropriate culture environment. This morphogenic ability is of great value for the practical use of cosmetic raw materials, as well as for academic importance such as plant development.

In the present invention, the plant tissue for inducing callus may be any part of the seed or the plant. For example, germinated or sterilized seeds can be germinated to cut away parts of the plant, or to cut and sterilize parts of the plant. In this way callus is induced through sterile tissue culture methods. Preferably, the plant fragments are cultured in a tissue culture medium. The medium includes MS (Murashige & Skoog, 1962) medium, N6 (Chu et al. 1975) medium, B5 (Gamborg et al., 1968), NN (Nitsch and Nitsch, 1967) medium, and the basic medium of various plants Available at Plant growth regulators should be added to this basal medium. The most commonly used plant growth regulators are 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2), which are auxins. , 4,5-trichlorophenoxyacetic acid, Dicamba (2-methoxy-3,6-dichlorobenzoic acid), IAA (Indole-3-acetic acid), IBA (Indole-3-butyric acid), MCPA (2-methyl-4 -chlorophenoxyacetic acid), NAA (1-naphthylacetic acid), NOA (2-naphthyloxyacetic acid), Picloram (4-amino-2,5,6-trichloropicolinic acid) and Cytokinin BAP (6-benzylaminopurine) , 2iP (N ^6- (2-isopentyl) adenine), Kinetin (6-furfurylaminopurine), Thidiazuron (1-phenyl-3- (1,2,3-thiadiazol-5-yl) urea), Zeatin (4-hydroxy -3-methyl-trans-2-butenylaminopurine is used at a concentration suitable for the plant, taking into account the species, age, site, and ripening of the plant. In addition, secondary metabolites such as alkaloids, terpenoids, prebonoids, carotenoids, phenolic compounds, and glycosides can be added to plant types to enhance the various physiological activities of callus induced by growth regulators. Can be added accordingly.

A summary of the preferred callus induction and culture method is as follows. A part of the tissue of the plant is separated and sterilized, and then cultured in a medium containing growth regulators of auxin or cytokinin in the plant basic medium. After incubation for 2 to 3 weeks, when the cells of the tissue divide and the callus, which is a mass of cells, is separated, only the callus is separated and placed on a fresh medium, and then passaged by observing a constant time interval or callus state.

In another aspect, the present invention, (a) inducing callus from the rice seed cultured; And (b) relates to a method for producing a skin external composition for skin improvement containing rice callus extract comprising the step of preparing a composition containing rice callus.

In the step (a), specifically, the appropriate medium may be selected to induce callus by germinating rice seeds. If there is a medium generally used in the callus culture in the art, it can be used without limitation.

In the present invention, as a medium for inducing callus from rice seeds, 2N6 medium (N6 salt 3.95g / L, sucrose 30g / L, casamino acid 1g / L, 2,4-D 2mg / L, phytagel 2g / L, pH5.6-5.7) can be used. In addition to this, various media conventionally known as rice callus induction media such as N6 medium may also be applicable in the present invention.

When the callus is cultured in a medium containing useful substances, the callus absorbs the useful substances added to the medium and thus contains the useful substances in the callus. It can exhibit physiological activity. The introduction of the useful substance in the callus may introduce the useful substance into the cultured callus by additionally performing the step of densifying the induced callus in the medium containing the useful substance.

EGCG used as a useful substance in the present invention is epigallocatechinallate, which is the most abundant biologically active antioxidant component in green tea. It has been known as the main ingredient that contributes to the various pharmacological effects of green tea. 74% of catechins are dry ingredients, and 80-90% of the world's academic green tea research has received much attention recently to focus on this EGCG study, and the antimicrobial, skin regeneration, cholesterol lowering and anti-cancer effects have been revealed. The bactericidal effect is also known to be excellent.

In step (b), the callus obtained in step (a) may be powdered and included in the composition. In addition to this, it can be prepared by containing the extract obtained by hot water extraction method, ethanol extraction method and the like in an appropriate concentration.

Specifically, in the rice callus extract skin external preparation composition according to the present invention, in addition to the aqueous phase component, the oil phase component, may include additives and the like. The additives include alcohols, colorants, fragrances and the like.

In addition, the topical skin preparation composition according to the invention can be prepared in various forms, for example emulsions, lotions, creams (water-in-oil, water-in-oil, multiphase), solutions, suspensions (anhydrous and aqueous), anhydrous products (Oil and glycol based), gels, masks, packs, powders and the like formulations.

The preparation method of the rice callus extract-containing skin external preparation composition according to the present invention is not limited to the above-described manufacturing method, and those skilled in the art to which the present invention pertains may also be seen as a method of partially modifying the manufacturing method. A rice callus extract-containing skin external preparation composition according to the invention can be prepared.

In addition, the topical skin composition according to the present invention may be a cosmetic composition or a pharmaceutical composition.

When the external skin composition is a pharmaceutical composition, it may further include a pharmaceutically acceptable carrier that is already known and used.

In the cosmetic composition, an acceptable carrier may be included in the cosmetic formulation. Herein, "acceptable carrier in cosmetic preparation" means a compound or composition already known and used that may be included in a cosmetic preparation or a compound or composition which will be developed in the future, and which has no toxicity, instability or irritation that can be adapted to the human body upon contact with the skin. Say nothing.

The carrier may be included in the topical skin composition of the present invention in an amount from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight of the composition. However, since the ratio depends on the formulation of the above-described skin external preparation composition of the present invention and its specific application site (face, neck, etc.) or its preferred application amount, the ratio may be viewed in any aspect. It should not be understood as limiting the scope of the invention.

Meanwhile, examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, sunscreen agents, colorants, fragrances, and the like. The alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, sunscreens, colorants, compounds / compositions that can be used as fragrances and the like are already known in the art, Appropriate materials / compositions may be selected and used.

As an embodiment of the present invention, the external preparation composition for skin according to the present invention may include glycerin, butylene glycol, propyleneglycol, polyoxyethylene hardened castor oil, ethanol, triethanolamine, etc., in addition to the above-mentioned seaweed extract, A trace amount may be included if necessary such as a medicine, a pigment, purified water, and the like.

In particular, the external preparation composition for the skin may be prepared in the form of general emulsion formulations and solubilized formulations, using a conventionally known manufacturing method, in addition to the manufacturing method specifically disclosed in the present invention.

When the cosmetic composition is prepared, cosmetics of the emulsion formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations include soft cosmetics. In addition, by containing a dermatologically acceptable medium or base, it can be prepared in the form of topical or systemic adjuvants commonly used in the field of dermatology.

In addition, formulations of suitable cosmetics include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase, for example. It may be provided in the form of a vesicle dispersant in the form of a cream, skin, lotion, powder, ointment, spray or concealed stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the topical skin composition of the present invention may further comprise fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ions Any commonly used in type or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic or dermatology field as other ingredients. In addition, the above components may be introduced in an amount generally used in the dermatology field.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Rice Callus According to the Present Invention

(1) Preparation of rice callus according to the present invention

Brown rice of mature rice seeds (Oryza sativa L.) was sterilized with 70% ethanol for 1 minute and 2% NaClO for 1 hour. After sterilization, the seeds were washed 5 times or more with sterile water, 2N6 (N6 salt 3.95g / L, sucrose 30g / L, casamino acid 1g / L, 2,4-D 2mg / L, phytagel 2g / L, pH5. 6 to 5.7) and incubated for about 3 weeks in a dark condition of 25 ℃ to induce callus. The induced callus was finely ground on liquid nitrogen, dissolved in purified water, and used in the following experimental example.

(2) Preparation of Rice Callus Induced in Medium Containing EGCG

After sterilization and washing in the same manner as in (1), 2N6 (N6 salt 3.95g / L, sucrose 30g / L, casamino acid 1g / L, 2,4-D 2mg / L, phytagel 2g / L) , pH5.6 ~ 5.7) EGCG 50mg / L was added to the medium and incubated for about 3 weeks in a dark condition of 25 ℃ to induce callus. The induced callus was finely ground on liquid nitrogen, dissolved in purified water, and used in the following experimental example.

Experimental Example 1 Safety Test of a Composition Containing Rice Callus According to the Present Invention

Fifty subjects (mean age 35 years, age 20 to 50 years) were subjected to a skin patch test on the upper arm using a fin chamber. However, Psoriasis, Eczema, and other skin lesion holders, and those who are pregnant, nursing or contraceptives, and antihistamines were excluded from this study.

Wipe the upper arm area of the test site with primary distilled water and dry it for 50 subjects, and then, 0.5 wt%, 1 wt%, 5 wt% of the rice callus extract according to Example 1 in purified water (negative control) and purified water. Composition containing 0.5 wt%, 1 wt%, 5 wt% of rice callus extract derived from the medium to which EGCG was added, and 10 wt% of sodium lauryl sulfate (SLS) in purified water. (Positive control group) was added dropwise to the pin chamber by 25 μl each, and fixed on the upper arm. In general, when 10% SLS touches the skin, it swells red and is frequently used as a positive control group in skin irritation experiments.

The patch was applied to the skin for 48 hours, and after removing the patch, the test area was marked with a marking pen, and after 4 hours, the skin condition was visually read and the results are shown in Table 1 below. Stimulus degree was calculated according to the following equation.

[Equation 1]

TABLE 1

 sample Number of test subjects  Judgment result  Stimulus  ++  +  + -  - 10% SLS 50 5 10 25 10 1.20  EGCG no addition  Contains 0.5% of rice callus extract 50 0 3 One 46 0.14 Contains 1% of rice callus extract 50 One 0 2 47 0.1 Contains 5% of rice callus extract 50 0 3 2 45 0.16 EGCG addition Contains 0.5% of rice callus extract 50 0 2 4 44 0.16 Contains 1% of rice callus extract 50 0 One 2 47 0.08 Contains 5% of rice callus extract 50 0 One 3 46 0.1 * Criteria *
-: No erythema or unusual phenomenon
+-: Slightly reddish
+: Significantly redder than the surroundings
++: reddening and swelling worse than the surroundings

As shown in Table 1, the test results of 50 subjects showed that the rice callus induced by the addition of rice callus extract and EGCG to the medium has no safety problem. The result of experimenting with purified water itself was shown as 0 stimulus, which is not separately indicated in Table 1 above.

Experimental Example 2: Test of Tyrosinase Inhibition Effect of Rice Callus Extract According to the Present Invention

To confirm the whitening effect of the composition prepared in Example 1, the degree of inhibition of the function of the tyrosinase (Tyrosinase), an enzyme that promotes the oxidation process of a tyrosine (Tyrosine) substance to help melanin production in vivo saw. Specifically, a method of measuring the degree of inhibition of the formation of a black polymer called melanin by oxidizing tyrosine by inhibiting the function of tyrosinase (Pomerantz SH, J. Biochem., 24: 161-168, 1966) is applied. Determined.

The composition of the present invention was put in 96 well-plates so as to be 0.5%, 1%, and 5%, respectively, 220 μl of 100 mM sodium phosphate buffer (pH 6.8) and 40 μl of 1.5 mM L-tyrosine solution were added, followed by mushroom tyrosinase. 20 μl of (1,500 units / ml, Sigma) was added and reacted at 37 ° C. for 20 minutes, and then 0.2 ml of the solution was transferred to a 96 well plate and absorbed at 490 nm using a microplate reader. The inhibition rate for tyrosinase was measured by measuring. The degree of inhibition of tyrosinase function was expressed as a percentage based on the absorbance intensity of the control using pure water.

[Equation 2]

% Inhibition = 1-[(Experimental absorbance-Blank absorbance) / control absorbance] X 100

As shown in Figure 1, compared to the control using purified water, containing 0.5% of the rice callus extract showed 5% oxidation inhibitory activity (tyrosinase inhibitory activity), 1% containing 18% of the antioxidant activity was inhibited In case of containing 5%, oxidation inhibition was 32%. In addition, 0.5% of rice callus extract derived from EGCG-containing medium showed 6% inhibition of oxidation, and 1% contained 25% of inhibition. Showed an inhibition of oxidation of 40%. Therefore, the composition according to the present invention was also able to inhibit the oxidation reaction mediated by enzymes, and thus could confirm the applicability as a whitening material.

Experimental Example 3: Antioxidant Test Using DPPH

Compound 1,1-diphenyl-2-picryl hydrazyl (DPPH, Sigma D9132-1G, USA) generates free radicals in ethanol, the amount of free radicals produced by mixing with the composition according to the invention in a proportion By checking whether the degree of decrease (free radical scavenging activity test) was checked whether the antioxidant effect. The free radical scavenging activity was modified from the method of Kim et al. picryl hydrazyl, Sigma D9132-1G, USA) reagent was used.

150 μl of 0.2 mM DPPH solution (ethanol in the case of Blanc) was prepared to contain various extracts at various concentrations, and 150 μl of these mixtures were added and mixed, and the absorbance was measured at 517 nm after being allowed to stand at room temperature for 30 minutes. The control group was set to the case using purified water. After absorbance measurement for the experimental group and the control group, the degree of antioxidant capacity was expressed as a percentage based on the absorbance intensity of the control group using pure water.

Using the following Equation 3 to obtain the free radical scavenging effect is shown in Table 3 below.

&Quot; (3) "

Free radical scavenging effect (%) = 1- [(Experimental absorbance-Blank absorbance) / control absorbance] X 100

As shown in Figure 2, compared to the control using purified water, 5% of the rice callus extract, 21% decrease when the DPPH free radicals of the reaction system contains 32% and 0.5% when containing 1% In case of containing 5% of rice callus extract derived from EGCG-added medium, the DPPH free radicals of the reaction system were reduced by 62%, 1% by 60% and 0.5% by 23%. there was.

Experimental Example 4: iNOS Activity Inhibition Test

To investigate the inhibitory effects of artificially induced inflammation using macrophage raw264.7 cells, 10% FBS-DMEM (Fetal Bovine Serum, Fetal Bovine Serum, Dulbecco's Modified Eagle Medium, GIBCO) containing no iNOS G) 1 × 10 ⁵ cells were suspended in the medium and inoculated in a 24 well plate.

After one day, the composition prepared in Example 1 was treated by concentration, followed by 18 hours of incubation, and then treated with 1 μg / ml of Lipopolysaccharide (Sigma) for 24 hours. Thereafter, the supernatant was collected, 100 µl each was added to a 96 well plate, and the same amount of Griess reagent (Sigma) was added thereto. A calibration curve was prepared using sodium nitrite as a standard, and the amount of nitric oxide produced in each culture was determined. The yield of% of each sample was determined using the amount of Nitric Oxide produced in the lipopolysaccharide-treated group as 100%.

TABLE 2

As shown in Table 2, the activity of iNOS was inhibited by rice callus extract (extract), rice callus extract (extract) induced in the medium to which EGCG was added.

Figure 1 shows the tyrosinase inhibitory test results of the composition containing the rice callus extract according to the present invention.

Figure 2 is a graph showing the results of testing the DPPH antioxidant effect of the rice callus extract containing composition according to the present invention.

Claims (5)

Skin external preparation composition containing rice callus extract as an active ingredient. According to claim 1, wherein the rice callus extract is a skin external composition for skin improvement, characterized in that containing 0.1 to 10% by weight based on the total weight of the composition. Method for preparing a skin external preparation composition for improving skin containing rice callus extract comprising the following steps: (a) inducing and incubating callus from rice seeds; And (b) preparing a composition containing the callus. The method of claim 3, wherein the callus is powdered and contained in the composition. The method according to claim 3, wherein in step (a), the callus is induced from rice seeds in a medium containing epigallocatechinallate and cultured.

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