KR101557064B1 - Oryza Sativa L. extracts for Skin Whitening - Google Patents

Abstract

The present invention provides an extract of Oryza Sativa L. ethanol having whitening activity. The shinto black rice ethanol extract of the present invention inhibits melanin synthesis directly by inhibiting gene expression of enzymes involved in melanin synthesis and thus can provide a reliable whitening effect and can be used as a whitening cosmetic composition or a health supplement.

Description

{Oryza Sativa L. extracts for Skin Whitening}

The present invention relates to a whitening cosmetic composition, and more particularly, to a whitening cosmetic composition comprising an extract of Shinto black rice as an active ingredient.

Skin pigmentation is caused by stimuli such as ultraviolet rays and inflammation, and it is also known to be caused by factors such as genetic factors, hormones, foods, and chemicals. When the skin is exposed to external stimuli such as ultraviolet rays, a pigment called melanin present in the skin is synthesized to protect the skin from external stimuli. The biosynthesis of melanin occurs in a special form of melanocyte subcellular organ called melanosome, in which an enzyme called tyrosinase exists and plays an important role in the production of melanin. Tyrosine is converted to dopa by the action of tyrosinase and converted to dopachrome through dopaquinone through a series of oxidation processes to finally form melanin, will be. Although melanin is a necessary ingredient to protect the skin, when it is over-synthesized, it causes pigmentation, which may cause skin irritation and freckles, which may cause skin appearance problems, and even skin aging or skin cancer. Therefore, people began to search for ingredients that could inhibit the overproduction of melanin, and antioxidants have begun to appear as a solution. Antioxidant is a component that prevents the oxidation of active oxygen in the human body, and it is known that it not only prevents aging and cancer, but also has a skin whitening effect by suppressing melanin synthesis.

One example of an antioxidant-containing substance is rice, which contains antioxidants such as tocopherol (vitamin E), orozanol, tocotrienol, ferulic acid, phytate, isovitaxin and phenolic compounds. It is also known that rice has excellent effect of cleansing the skin as much as it was cleansed with rice flour which rice flour from the Joseon Dynasty washed rice. Especially, among rice varieties, colored rice contains antioxidant antioxidant, which can be expected to have a strong antioxidant effect. Black rice is one of the representative color pigments among them. Vitamin B and E in black rice contain more than four times of ordinary rice, which reduces female beauty and blood sugar. Oryzafuran, which is contained in black rice, has antioxidant ability much stronger than vitamin C or E , The black rice has been attracting attention as a well-being food.

It is said that according to the ancient farmer of China's ancient farmer Jemun magic records, black rice was cultivated in 286 ~ 534 AD, the latitude period. Recently, since 1980, we have been actively studying breeding, product development, ingredient investigation, and cultivation technology, and now it is creating new demand steadily. In Korea, since 1997, we have cultivated a new varieties of rice varieties called 'Shinto black rice' by artificially crossing the black rice nectar and black rice nectar Milyang 153. Shinto black rice is a new varieties of rice cultivated in Jeonbuk No.3, a high quality black rice with 2 years of productivity test and 3 years of regional adaptation test. It is small in height and stable against fallen down, and it is possible to grow stable. It is expected to be worthy of use because it has a relatively high production volume of 509kg / 10a of brown rice. Especially, Shinto black rice has the highest anthocyanin content of 149mg / 100g and the highest antioxidant activity among color pigments.

In the patent, Korean Patent Publication No. 2004-0033342 discloses a prior art using antioxidative and whitening function of black rice. The above patent relates to a cosmetic composition for whitening skin which contains an extract of black bean, black rice and black sesame as an active ingredient. It relates to a cosmetic composition for whitening which is superior in skin whitening function and has little skin irritation when each extract is used alone . On the other hand, as another prior art, there is Korean Patent No. KR 0611428 relating to a skin whitening cosmetic composition containing an extract of black rice as an active ingredient. This patent discloses a cosmetic composition which is excellent in inhibition of free radical scavenging action and melanin biosynthesis inhibition by containing a black rice extract, effective for suppressing aging and effective in whitening, and having excellent stability in formulation. However, according to the conventional technology related to general black rice extract, considering the fact that the black rice of various varieties has been improved, it is insufficient to prove the efficacy of the new black rice. Therefore, it is required to improve the whitening - related technology by deeply studying varieties of black rice cultivars which are improved variously, and the varieties having remarkably excellent whitening function.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Compounds that have been used as a composition for whitening cosmetics to prevent pigmentation have not solved most problems such as skin irritation, cytotoxicity, stability of product, and deterioration of whitening effect with time. Also, as the demand for cosmetics made of natural materials increases, research on natural substances having whitening effect needs to be continuously carried out, and it is necessary to secure the production amount of raw materials meeting the increasing demand.

Accordingly, the present invention provides an extract of a black bean black rice having high antioxidative activity and high yield, especially in anthocyanin content among black rice having antioxidative and whitening activity. The extant black rice ethanol extract of the present invention inhibits melanin biosynthesis and has a whitening activity, so that it can be used as a whitening cosmetic composition or a health supplement.

The present invention provides an extract of Oryza Sativa L. ethanol having whitening activity.

The inventors of the present invention have sought to elucidate the whitening function of Shinto black rice, which has the highest anthocyanin content among the colored pigments and has the most excellent antioxidant activity. As a result, the extract of Shinto black rice ethanol having the above-mentioned merits was completed. The ethanol extract of Shinto black rice ethanol inhibited the activity of tyrosinase, which is a major enzyme causing skin pigment deposition due to melanin biosynthesis, and TRP-1 and TRP-2 enzymes It was confirmed that the inhibition of gene expression leads to a whitening effect.

According to one aspect of the present invention, there is provided an Oryza sativa L. ethanol extract having whitening activity.

According to a preferred embodiment of the present invention, the Shinto black rice can be characterized as a rice variety developed by artificial interbreeding of black rice paddy rice and Milyang rice No. 153. In the present invention, it is very important to use the shinto black rice as an extraction raw material. The anthocyanin content of Shinto black rice was the highest among colored pigments at 149 mg / 100 g, and the radical scavenging rates of DPPH and ABTS were 54.5% and 62.3%, respectively. In addition, Shinto black rice is small in height and stable to fallen and stable cultivation is possible. It is a high quality black rice with high quality of 509 kg / 10 a. As a result, it is very important to utilize the shinto black rice, which has a strong competitive edge in the supply and function of raw materials, against the steadily growing antioxidant product demand.

According to a preferred embodiment of the present invention, the ethanol is preferably 70-90% ethanol, more preferably 75-85% ethanol, most preferably 80% ethanol can do.

According to a preferred embodiment of the present invention, the extract of Shinto black rice ethanol may inhibit the melanin synthesis process. In the present invention, it is an important feature that the extant black rice ethanol extract inhibits melanin synthesis. This is because, when dark brown melanin is formed in skin cells, the skin looks dark or causes pigmentation, so that inhibition of melanin synthesis can be a key to whitening function.

According to a preferred embodiment of the present invention, the Shinto black rice ethanol extract may be characterized by inhibiting gene expression of tyrosinase, TRP-1, and TRP-2, enzymes involved in melanin synthesis. In the present invention, inhibition of the gene expression of enzymes involved in melanin synthesis may be an important feature of the extract of Shinto black rice ethanol. Especially, inhibition of the activity of tyrosinase among enzymes is a very important feature. Tyrosinase is directly involved in melanin synthesis because it is an enzyme that converts tyrosine in melanocytes to dopa and then oxidizes it to dopa quinone. When dopaquinone is formed by the activity of tyrosinase, dopaquinone is continuously polymerized to form melanin. Thus, it is possible to prevent formation of dopaquinone by inhibiting tyrosinase and finally to inhibit melanin synthesis. Therefore, it may be very important in the present invention to inhibit gene expression of enzymes such as tyrosinase and TRP-1 and TRP-2.

According to a preferred embodiment of the present invention, the extract of Shinto black rice ethanol may inhibit protein synthesis of tyrosinase transcription factors MITF and EPK. In the present invention, the inhibition of protein synthesis of MITF and EPK by the extract of Shinto black rice ethanol may be important since it essentially acts to inhibit the activity of tyrosinase as a transcription factor.

According to another aspect of the present invention, there is provided a whitening cosmetic composition comprising the above extract of Shinto black rice ethanol as an active ingredient.

According to a preferred embodiment of the present invention, the amount of the extract of Shinto black rice ethanol is preferably 0.00001-30% by weight, more preferably 0.0001-10% by weight, and most preferably 0.001-0.5% by weight based on the total weight of the whitening cosmetic composition %. ≪ / RTI > In the composition of the present invention, the most preferred amount of the extract of Shinto black rice ethanol is 0.001-0.5% by weight. When the content of the extract of Shinto black rice ethanol is less than 0.001% by weight, there is a problem that the whitening effect is not large. The stability of the product may be somewhat problematic.

According to a preferred embodiment of the present invention, the whitening cosmetic composition may contain components other than the above-described Shinoto black rice ethanol extract. At least one auxiliary component selected from the group consisting of glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate and allantoin, Further comprising at least one component selected from the group consisting of butylene glycol, citric acid, panthenol and allantoin, wherein the at least one ingredient is selected from the group consisting of glycerin, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, squalane and sodium citrate, Most preferably glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate and allantoin.

According to a preferred embodiment of the present invention, since the cosmetic composition of the present invention is basically applied to the skin, it can be provided with reference to the cosmetic composition of the related art, for example, a solution, a suspension, an emulsion, a paste, But are not limited to, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, 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.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 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 suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

According to one aspect of the present invention, there is provided a functional health supplement containing the extract of Shineto black rice ethanol as an active ingredient. The extract of Shinto black rice ethanol according to the present invention has antioxidant properties due to various antioxidants such as tocopherol (vitamin E), orozanol, tocotrienol, ferulic acid, phytic acid, isovistaticin, phenolic compound and anthocyanin, Can be produced as a health supplement.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an extract of Oryza sativa L. ethanol having whitening activity;

(b) The shinto black rice used as a raw material in the present invention has a high production yield and can be extracted in a large amount and is easy to use;

(c) The Shinto black rice used as a raw material in the present invention has an especially high antioxidative effect in all rice varieties because the anthocyanin content is particularly high;

(d) The extract of Shinto black rice ethanol according to the present invention has an antioxidative effect basically, thus protecting skin from active oxygen and preventing aging;

(e) The Shinto black rice ethanol extract of the present invention can inhibit the melanin synthesis directly by inhibiting gene expression of tyrosinase among enzymes involved in melanin synthesis, thereby bringing about a definite whitening effect;

(f) The extract of Shinto black rice ethanol according to the present invention can be used not only as a cosmetic composition having a whitening function but also as a health functional supplement; And

(g) It can be safely used as a product which is harmless to the human body and has a low side effect by utilizing natural materials.

FIG. 1 is a graph showing cytotoxicity of skin cell B16 / F1 treated with an extract of Shinoto black rice ethanol.
2 is a graph showing the activity of mushroom tyrosinase according to the concentration of ethanol extract of Shinto black rice.
3 is a graph showing the melanin content of skin cell B16 / F1 treated with the extract of Shinoto black rice ethanol.
Fig. 4 is a graph showing tyrosinase activity of skin cell B16 / F1 treated with the extract of Shinanto black rice ethanol.
FIG. 5 is a graph showing the degree of gene expression of melanin synthesis related enzymes in skin cell B16 / F1 treated with the extract of Shinto black rice ethanol.
FIG. 6 is a graph showing the amounts of protein synthesis of melanin synthesis related enzymes and enzyme transcription factors in skin cell B16 / F1 treated with the extract of Shinoto black rice ethanol.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

Example

In the present invention, the inhibitory effect of tyrosinase activity was confirmed by comparing the treatment result of Shinanto black rice ethanol extract composition with the control group, and the whitening effect was confirmed by quantifying melanin production amount in melanocytes. In addition, the efficacy of black bean black rice was verified by comparing the whitening efficacy by treatment with albutin at the same concentration, using the arbutin known to have a whitening effect as a positive control.

Example . Shinto black rice  Preparation of ethanol extract composition

Oryza sativa L.) was pulverized in a pulverizer, followed by stirring at room temperature for 48 hours with 700 ml of 80% ethanol. Then, the ethanol component was removed using a vacuum concentrator, and the remaining water was lyophilized for two days using lyophilization to finally produce a dark red black rice ethanol extract composition. The 80% ethanol extract composition thus obtained showed a yield of 1.38% as compared with the initial ground black rice meal.

Manufacturing example  One. Shinto black rice  Preparation of Essence Containing Ethanol Extract

The composition of the essence and the prescription examples of the cosmetic as the formulation having the whitening effect containing the extract of Shinoto black rice ethanol of Example 1 are as follows.

ingredient Content (% by weight) Example 1 0.0025 glycerin 5.0 1,3-butylene glycol 2.0 Polyethylene glycol 2.0 Carbomer 1.0 Sodium hyaruronate 0.1 Glycine 3.0 Polyacrylamide 2.0 Hydroxyethyl cellulose 0.2 ethanol 3.0 Antioxidant 0.3 Triethanolamine 0.1 Polyoxyethylene hardened castor oil 1.0 EDTA 0.1 antiseptic Suitable amount Purified water Balance

Sodium hyaruronate and hydroxyethyl cellulose were dispersed in purified water in a propeller mixer at 2000 rpm to prepare a 1% solution. The carbomer was prepared by dispersing the purified water in a propeller mixer at 4000 rpm in a 2% solution. The raw material 1-12 was added to the water dissolution tank, homogenized at 2000 rpm, dispersed, and then heated to 75 캜. The warmed water phase was cooled to room temperature. The raw materials 13 to 15 were completely dissolved in a separate dissolution tank, and the mixture was added to the above-mentioned water dissolution tank and mixed by stirring. Example 1 was added thereto and thoroughly stirred and mixed to prepare an essence containing Example 1.

Manufacturing example  2. Shinto black rice  Manufacture of nutritional cream containing ethanol extract

The composition and prescription examples of the nutritive cream as a formulation having a whitening effect and containing the extract of Shinoto black rice ethanol of Example 1 are as follows.

ingredient Content (% by weight) Example 1 0.0025 1,3-butylene glycol 3.0 glycerin 3.0 Hydrogenated lecithin 1.0 Octyldodecanol 3.0 Trioctanoin 2.0 Stearic Acid 1.5 Cetostearyl alcohol 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 2.0 Dimethicone 3.0 Antioxidant 0.3 Xanthan gum 0.2 Triethanolamine 0.1 EDTA 0.1 antiseptic Suitable amount Purified water Balance

The raw material 1-8 was added to the water dissolution tank and homogenized at 2000 rpm with stirring, and then heated to 75 캜. The raw material 9-16 was added to the oily melting tank and dissolved by heating to 80 ° C. The oil phase dissolved in the water-dissolving tank was charged and emulsified (3000 rpm / 10 minutes) and then cooled to room temperature. Example 1 was added thereto and thoroughly stirred and mixed to prepare a nutritive cream containing Example 1.

Experimental Example  1. Mushroom Tyrosinase  ( mushroom tyrosinase ) Active experiment

Tyrosinase is a rate-limiting enzyme for the synthesis of melanin in the skin. If it inhibits the activity of the enzyme, it can regulate melanin production in skin cells. Therefore, in order to investigate the whitening effect of the extract composition of Shinto black rice ethanol, tyrosinase activity test was conducted. The tyrosinase activity test used mushroom tyrosinase and dopa as substrates. (50 μL), 0.03% (w / v) tyrosine solution (Sigma, USA) and 75 μL of 0.1 M phosphate buffer (pH 6.8, Sigma, USA) Mucin tyrosinase (Sigma, USA) (400 units / mL 0.1 M phosphate buffer) was added to the extracts of black rice, which were added at 0 mg / mL, 0.05 mg / mL, 0.1 mg / mL and 0.2 mg / And the absorbance was measured at 475 nm. The activity of mushroom tyrosinase according to the absorbance measurement is shown in the following table.

Example 1 Concentration (mg / ml) Activation of mushroom tyrosinase (%) 0 99.78 0.05 61.28 0.1 60.04 0.2 60.01

As a result of measuring the activity of mushroom tyrosinase, it was confirmed that the activity of the extract of Shinto black rice ethanol was inhibited in a concentration-dependent manner compared to the control group when treated with 0.05-0.2 mg / mL. Since tyrosinase is an important enzyme for the synthesis of melanin, it was judged that the extract of Shinto black rice ethanol could inhibit melanin synthesis directly by inhibiting tyrosinase enzyme.

Experimental Example  2. Melanocyte culture and MTT  analysis

B16-F1 mouse melanocytes (B16-F1 murine melanocytes, Korean Cell Line Bank, Seoul, Korea, 80007) were cultured in order to examine the effect of the ethanol extract of Shinto black rice produced in Example 1 directly on melanocytes of skin. ) Were cultured. Dulbecco's modified Eagle medium (DMEM) was supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, Germany), 100 U / mL penicillin (Thermo Scientific, Germany) and 0.1 mg / mL streptomycin (Thermo Scientific, Germany) Were added together and melanocytes were cultured at 37 ° C, 95% air, 5% CO ₂ .

MTT assay (cytotoxicity assay) was first performed using cultured melanocytes. The analysis was carried out in a culture medium supplemented with MTT solution (Sigma, USA) after treatment of B16-F1 melanocytes with 8 concentrations of the Shinoto black rice extract composition for 72 hours. MTT solution was added and the mixture was reacted at 37 ° C for 3 hours to form formazan. Dimethyl sulfoxide (DMSO, Biobasic, Canada) was added to dissolve formazan and absorbance was measured at 570 nm. The cytotoxicity according to the absorbance analysis was different according to the concentration of the extract of Shintoko rice. The results are shown in Table 4 below.

Example 1 Concentration (mg / ml) Cytotoxicity (%) 0 100.00 0.05 100.00 0.1 78.90 0.2 72.10 0.5 10.01 One 9.20 2 9.30 3 9.20

Cytotoxicity analysis showed that the cytotoxicity was maintained at a concentration of 0.2 mg / ml or less, whereas the cytotoxicity was decreased from 0.5 mg / ml or more.

Experimental Example  3. Intracellular Melanin content  Measure

Experiments were conducted to measure the amount of melanin synthesized from melanocytes treated with Shinanto black rice ethanol extract for 72 hours. After the melanin cells cultured in Experimental Example 2 were centrifuged, the melanin in the cells was measured at 400 nm, which is the maximum absorbance of melanin. First, cells were washed with PBS buffer to determine melanin content, and cells were lysed by adding 20 mmol / L Tris-0.1%, Triton X-100 (pH 7.5) (Biobasic, Canada). The cell lysate was then centrifuged at 4 캜 for 5 minutes to obtain a pellet of 12000 xg. The resulting pellet was dissolved in 1 N NaOH at 60 ° C for 1 hour, and the absorbance at 400 nm was observed. The melanin content was measured by comparing this value with a standard curve using synthetic melanin.

Control group (no treatment) Arbutine (1 mM) Shinto black rice extract
High (0.1 mg / mL) Shinto black rice extract
Low (0.01 mg / mL) Melanin content (%) 100 46.23 80.12 78.64

Melanin content of melanocytes treated with 1 mM of arbutin (Arbutin, Sigma, USA) was also measured in order to investigate the inhibitory effect on melanin synthesis of Shinto black rice extract. Because albutin is commonly used as a chemical raw material having a function of inhibiting tyrosinase activity, it could be an index of whitening function of Shintoko black rice extract. The results showed that melanocytes treated with Shintoko rice extract had higher melanin synthesis than albutin but less melanin than control. In other words, it was concluded that Shinto black rice extract as a natural raw material has excellent whitening function.

Experimental Example  4. Melanocyte tyrosinase ( tyrosinase ) Active measurement

Experiments were conducted to see that the extract of Shinto black rice extract absorbed into cells inhibited tyrosinase activity. Melanocytes were added to a 6-well plate at a concentration of 1 × 105 cells / well, and melanocytes were cultured for 24 hours at 37 ° C. and 5% CO _2. 10 μg / ml, 100 μg / ml, respectively. After 72 hours of culture, the medium was removed, washed twice with PBS buffer, and 150 μl of 20 mmol / L Tris-0.1% Triton X-100 (pH 7.5) buffer (Biobasic, Canada) After dissolution, rapid freezing and thawing were repeated using liquid nitrogen and disrupted for 1 hour. 70 μl of the tyrosinase crude extract obtained by centrifugation at 12,000 g for 5 minutes and 140 μl of 0.1% L-DOPA (Sigma, USA) were reacted in a 37 ° C. incubator for 2 hours, and the absorbance at 475 nm Were measured.

Control group (no treatment) Arbutine (1 mM) Shinto black rice extract
High (0.1 mg / mL) Shinto black rice extract
Low (0.01 mg / mL) Tyrosinase activity (%) 100 32.14 77.45 63.90

When the protein was extracted from the cells and the activity of tyrosinase was measured using L-DOPA as a substrate, the enzymatic activity was significantly decreased when the extract composition of Shin-tao black rice ethanol was treated as shown in Table 6. It was confirmed that the tyrosinase inhibitory effect was particularly excellent at low concentrations.

Experimental Example  5. Measurement of gene expression of enzymes in melanocytes PCR )

(RT-PCR) was performed to investigate changes in gene expression of tyrosinase, which is involved in melanin synthesis in cells, and microphthalmia-associated transcription factor (MITF), a transcription factor of TRP-1, TRP-2 and tyrosinase enzyme. Law. Cells were added to a 6-well plate, which is a tissue cell culture container, at a density of 4 × 10 5 cells / well. The cells were cultured at 37 ° C. and 5% CO 2 for 24 hours. The samples were subjected to no- And treated with 10 μg / ml and 100 μg / ml ethanol extract of Shinto black rice. After 24 hours of culture, the medium was removed, and the total RNA prepared by adding TRIzol reagent (Invitrogen) was subjected to RT-PCR using Superscript II (Invitrogen), and cDNA was synthesized.

The PCR reaction mixture containing the above cDNA as a template was prepared by mixing the synthesized cDNA with 10 pmol forward primer and reverse primer, 10X PCR Buffer (2.5 mM dNTP Mix, m-biotech, Korea) and Taq DNA polymerase (Corebiosystem Co., . Here, the primer set used in the PCR reaction was as follows.

(a) Tyrosinase: upstream 5'-GGC CAG CTT TCA GGC AGA GGT-3 '

                 downstream 5'-TGG TGC TTC ATG GGC AAA ATC-3 '

(b) TRP-1: upstream 5'-GCT GCA GGA GCC TTC TTT CTC-3 '

            downstream 5'-AAG ACG CTG CAC TGC TGG TCT-3 ',

(c) TRP-2: upstream 5'-GGA TGA CCG TGA GCA ATG GCC-3 '

            downstream 5'-CGG TTG TGA CCA ATG GGT GCC-3 ',

(d) MITF: upstream 5'-GTA TGA ACA CGC ACT CTC TCGA-3 '

           dowm stream 5'-CTT CTG CGC TCA TAC TGC TC-3 '

The reaction was repeated 25 times at 94 ° C for 30 seconds of thermal denaturation, 30 seconds of primer annealing at 56 ° C, and 2 minutes of extension at 72 ° C. The obtained PCR products were electrophoresed using a 1% agarose gel and TAE (Tris-Acetate-EDTA) buffer according to a conventional method, and amplification bands were quantified using GelPro analysis software.

Control group (no treatment) Arbutine (1 mM) Shinto black rice extract
High (0.1 mg / mL) Shinto black rice extract
Low (0.01 mg / mL) Tyrosinase 1.0 0.8 0.79 0.72 TPR1 1.0 0.43 0.89 0.71 TPR2 1.0 0.41 0.74 0.78 MITF 1.0 0.21 0.23 0.29

As a result of the analysis, it was confirmed that the expression of tyrosinase, TRP1, RRP2, and MITF was decreased when the extract of Shintoko rice was treated as shown in Table 7 above. As a result of the above experiment, it was found that the extract composition of Shineto black rice ethanol was involved in the inhibition of melanin synthesis at gene expression level.

Experimental Example  6. Measurement of Protein Synthesis of Enzymes in Melanocytes Western blot )

Protein expression levels of tyrosinase, an enzyme involved in intracellular melanin synthesis, and microphthalmia-associated transcription factor (MITF), ERK, and p-ERK, TRP-1, TRP-2 and tarosinase enzyme transcripts were determined by Western blotting western blotting). Cells were added to a 16-well plate for tissue culture, and the cells were incubated at 37 ° C and 5% CO ₂ for 24 hours. To each cell sample, 0 μg / ml (no addition) , 10 μg / ml and 100 μg / ml, respectively. After incubation for 72 hours, the medium was removed, and the plate was washed twice with PBS buffer solution (4 ° C). Then, the cells were washed with a lipase solution (RIPA buffer, 10 mM Tris-HCl, pH 7.5, 1% NP-40, 0.1% sodium deoxycholate, 0.1 % SDS, 150 mM NaCl, 1 mM EDTA) was homogenized to obtain the total protein extract of cells. The extracted protein content was determined by the Bradford method using BSA (Bolvin Serum Albumin, Pierce, USA) as a standard solution. The protein extracts were separated on a 7.5% SDS gel and transferred to a nitrocellulose membrane (Schleicher & Schuell BioScience). Primary antibodies against mouse tyrosinase, TRP-1, TRP-2 and MITF (C-19), anti-TRP1 (G-17), anti-TRP2 (D-18), anti- Santa Cruz, Calif.) Was diluted 500-fold with buffer and incubated overnight at 4 ° C. They were then extensively washed and reacted with a goat peroxidase conjugated second antibody (Santacruz, USA). The washed membranes were developed using an ECL kit (Amersham-Pharmacia), and the developed proteins were quantitated using GelPro analysis software.

Amount of protein synthesis (%) Control group (no treatment) Shinto black rice extract
High (0.1 mg / mL) Shinto black rice extract
Low (0.01 mg / mL) Tyrosinase 100 113.5 106.4 TPR1 100 62.7 50.7 TPR2 100 58.2 59.3 MITF 100 50.6 51.8 EPK 100 87.4 72.4 p-EPK 100 410.3 243.2

As shown in Table 8, the protein synthesis of p-ERK (Phosphorylated-ERK) did not decrease but the protein synthesis amounts of TRP-1 and TRP-2 decreased significantly , MITF, and tyrosinase also significantly inhibited protein synthesis. Thus, it was confirmed that the extract composition of Shinto black rice ethanol inhibited melanin synthesis and whitening function by inhibiting protein synthesis of tyrosinase and its transcription factors.

Room  7. Skin whitening effect test

In order to confirm whether the proven whitening effect in the above experimental example actually aids in skin whitening of human beings, a human body application test was conducted on the nutritional cream of Preparation Example 2 above. The skin whitening test using Roboskin analyzer equipment periodically evaluated the improvement of pigmentation and skin tone (brightness) while applying samples or products to the subject's face. The test consisted of 10 subjects (5 control subjects, 5 test subjects), all of whom were female and whose mean age was 34 years. The average value was obtained by measuring 5 times in total for 1 week, 2 weeks, 4 weeks and 6 weeks before use. All the tests were conducted in a constant temperature and humidity chamber of 20-24 ° C and a humidity of 45-55% For at least 1 hour.

Skin Whitening Effect Test of Nutritive Cream Containing Ethanol extract of Shinto black rice Before use 1 week 2 weeks 4 weeks 6 weeks Control Test section Control Test section Control Test section Control Test section Control Test section Pigment
Calm (large) 35.2 32.1 32.1 30.1 30.1 29.2 28.9 25.5 27.8 21.5 brightness 65.4 67.0 66.2 68.2 67.2 72.5 68.8 76.0 69.1 78.9

As a result of measurement of skin whitening effect according to Production Example 2, it was confirmed that the pigmentation tendency decreased from the 2nd week and the brightness of the skin became brighter. Thus, it was found that the cosmetic composition containing the extract of Shintoko rice helps skin whitening I could.

Claims (11)

Which comprises an extract of Oryza sativa L. ethanol as an active ingredient and has an inhibitory effect on the activity of melanocyte tyrosinase in the range of 0.01 to 0.1 mg / mL and 22.55 to 36.1% A cosmetic composition for whitening.
The method according to claim 1,
The present invention relates to a cosmetic composition for the whitening of rice varieties, which is developed by artificial crossing of Shinto black rice with Black Nambara and Milyang 153.
The method according to claim 1,
Wherein the ethanol is 75-85% ethanol.
The method according to claim 1,
Wherein the extant black rice ethanol extract inhibits the melanin synthesis process.
The method according to claim 1,
Wherein said Shineto black rice ethanol extract inhibits gene expression of tyrosinase, TRP-1, and TRP-2, enzymes involved in melanin synthesis.
The method according to claim 1,
Wherein said extract of Shinto black rice ethanol inhibits protein synthesis of tyrosinase transcription factors MITF and ERK.
delete The method according to claim 1,
Wherein the amount of the extract of Shinto black rice ethanol is 0.00001-30% by weight based on the total weight of the whitening cosmetic composition.
The method according to claim 1,
Wherein the whitening cosmetic composition further comprises at least one auxiliary ingredient selected from the group consisting of glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate and allantoin , Whitening cosmetic composition.
The method according to claim 1,
Wherein the whitening cosmetic composition is in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray By weight, based on the total weight of the cosmetic composition.
Wherein the content of the extract of Shinto black rice ethanol is 0.01 to 0.1 mg / mL, and the activity inhibitory effect of melanocyte tyrosinase is 22.55 to 36.1%.

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