KR102023571B1 - Cosmetic compositions for skin whitening comprising Tenebrio molitor extracts as an active ingredient - Google Patents

Abstract

The present invention relates to a skin-whitening cosmetic composition comprising, as active ingredients, one or more extracts from among Tenebrio Molitor extracts, Phyllostachys nigra leaf extracts, and Camellia sinensis leaf extracts. According to the present invention, the skin-whitening cosmetic composition suppresses melanin formation through a mechanism that inhibits the expression of tyrosinase, MITF, TRP-1, and TRP-2, thereby exhibiting skin-whitening effects. In addition, the composition of the present invention comprising the same as an active ingredient may be beneficially used as a material in functional cosmetics for skin whitening without causing skin irritation.

Description

Cosmetic compositions for skin whitening comprising Tenebrio molitor extracts as an active ingredient}

The present invention relates to a functional cosmetic composition comprising a brown gourd extract extracted from a brown gourd ( Tenebrio Molitor ) as an active ingredient, more specifically brown gourd extract; And Bamboo Leaves ( Phyllostachys) It relates to a functional cosmetic composition having a skin whitening function comprising as an active ingredient; at least one extract of nigra Leaf extract and Camellia sinensis Leaf extract.

When exposed to ultraviolet rays (UV), the skin produces reactive oxygen species (ROS), and excess free radicals oxidize, causing cell membranes, DNA, and lipids, proteins, Reacts with polysaccharides and nucleic acids to damage cells and cause skin aging. As the components for inhibiting such active oxygen, butylated hydroxy anisol (BHA) and butylated hydroxy toluene (BHT) are known.

In addition, UV is known to promote melanin production. Melanin is a major factor in determining the color of the skin. The melanin is secreted by the melanocortin 1 receptor (MC1R) and the pituitary gland in the melanocytes of the epidermis as the skin is stimulated by UV rays. The combination of -melanocyte stimulating hormone (α-MSH) is formed by increased expression of microphthalmia-associated transcription factor (MITF) by the cyclic adenosine monophosphate (cAMP) / protein kinase A (PKA) pathway. In addition, the hydrolysis process in which the precursor tyrosine protein is converted to 3,4-dihydroxyphenylalanin (L-DOPA) by an enzyme called tyrosinase, and the oxidation process converting L-DOPA to phenylanine-3,4-quinone (DOPA quinone) It is formed through. The resulting melanin is deposited in the skin and develops into "mushrooms" and "freckles". To prevent this, whitening cosmetics containing various cosmetic compositions are used.

Brown mealworm ( Tenebrio molitor (mealworm) is a colony of Coleoptera and is distributed all over the world including Korea. Patents related to cosmetic compositions using insects include cosmetic compositions for skin whitening containing cricket extract (Korean Patent No. 10-1429679), and cosmetic pack compositions containing sericin protein extracted from silkworm cocoons (Korean Patent No. 10- 0542266), but studies on the use of brown gourd extract as the main ingredient of cosmetics have been insignificant.

The inventors of the present invention, while searching for a natural material as a new material for producing cosmetics with excellent whitening effect without side effects on the living body, and tested the function by focusing on brown edible extract, brown edible extract has a melanin production inhibitory effect It was found that, when used in combination with the bamboo leaf extract or green tea extract was found to increase the effect, confirming the applicability as a cosmetic natural material for skin whitening to complete the present invention.

Korea Patent Registration No. 10-1429679 Republic of Korea Patent No. 10-0542266

Therefore, an object of the present invention is to provide a cosmetic composition for skin whitening having no skin side effects and having skin whitening effect.

In order to achieve the object of the present invention as described above, the present invention is brown rice wine ( Tenebrio Molitor ) extract; And Bamboo Leaves ( Phyllostachys) It provides a cosmetic composition for skin whitening containing at least one extract of nigra Leaf) extract and green tea ( Camellia sinensis Leaf) extract as an active ingredient.

The skin composition for skin whitening containing one or more extracts of brown edible extract and bamboo leaf extract and green tea extract according to the present invention has a mechanism of inhibiting the expression of tyrosinase, MITF, TRP-1 and TRP-2. It has a whitening effect by inhibiting the production of melanin, and the composition of the present invention comprising it as an active ingredient may be usefully used as a material of functional cosmetics for skin whitening does not cause skin irritation.

Hereinafter, the present invention will be described in detail.

For skin whitening Cosmetics  Composition

The present invention brown meal ( Tenebrio Molitor ) extract; And

Bamboo Leaves ( Phyllostachys nigra Leaf) extract and green tea ( Camellia sinensis Leaf) extract of at least one extract; it is characterized by providing a cosmetic composition for skin whitening containing as an active ingredient.

The brown mealworm ( Tenebrio molitor ) of the present invention, also called a worm (mealworm), means a larva of brown mealworms belonging to the Coleoptera mealworm family, is distributed throughout the world, including Korea. It is mainly used as food for pets, and recently, brown gourd is being researched as a value of food resource in the future by using easy mass production and rich in nutrients such as protein. Reported studies include antifungal effects using antifungal proteins isolated from brown rice wine. It is known that eubobogam and herbaceous wood have effects on cirrhosis, liver cancer, and tetanus. However, research on pharmacological efficacy is inadequate and needs to be explored in various ways.

Thus, the inventors of the present invention, while seeking to find a material that is stable to the human body while showing excellent whitening effect, derived from natural materials, paying attention to brown gourd extract, at least one of brown gourd extract, bamboo leaf extract and green tea extract It was confirmed through experiments whether the complex mixed with the extract has this effect.

On the other hand, melanin is a pigment present in the skin and plays an important role in protecting the body from external stimuli such as ultraviolet rays. However, when it is over-produced and deposited, it can cause blemishes and freckles, which can cause skin appearance problems. Furthermore, it can promote skin aging and cause skin cancer, so it is used as a whitening product for substances that can suppress melanin overproduction. This is possible.

According to one embodiment of the present invention, the mixture of the brown gourd extract, bamboo leaf extract and green tea extract of the present invention inhibits tyrosinase concentration-dependently, and MITF (microphthalmia-associated) Inhibition of the expression of transcription factor (TRP-1), tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2) has been shown to have activity to inhibit melanin biosynthesis (see Experimental Examples 1 to 5).

Therefore, in the case of the composite of the brown gourd extract of the present invention, which inhibits tyrosinase activity and melanin biosynthesis, and one or more extracts of bamboo leaf extract and green tea extract, it may be used for preventing pigmentation of the skin or whitening effect of the skin. Could be.

The extract according to the present invention may be obtained by extracting from nature using extraction methods known in the art, the brown rice wine extract, bamboo leaf extract and green tea extract as defined in the present invention using an appropriate solvent, respectively It is extracted from the raw materials of, and includes, for example, both crude extracts, polar solvent soluble extracts or nonpolar solvent soluble extracts. Preferably it may be an extract extracted from brown rice wine, bamboo leaves or green tea.

As a suitable solvent for extracting the extract, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used, but is not limited thereto. For example, purified water and methanol Alcohols containing 1 to 4 carbon atoms, acetone, ether, benzene, chloroform, including ethanol, propanol, isopropanol, isopropanol, butanol, etc. , Various solvents such as ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. Preferably the extract may be acetone, ethanol, methanol or water extract, more preferably may be ethanol, methanol or water extract, particularly preferably the water extract. .

As the extraction method, any one of hot water extraction method, cold leaching extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be used. In addition, the desired extract may further be subjected to a conventional fractionation process, it may be purified using conventional purification methods. There is no limitation on the preparation method of the extract of the present invention, any known method may be used.

For example, the brown gourd, bamboo leaf or green tea extract included in the composition of the present invention may be used as it is while the primary extract extracted by the solvent extraction method is centrifuged, filtered, concentrated and freeze-dried and stored in a refrigerator compartment. In addition, the primary extract may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying. In addition, the primary extract is further purified using a variety of chromatography, such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. You can also get

Therefore, in the present invention, the extract is a concept including all the extracts, fractions and purified products obtained at each step of extraction, fractionation or purification, their dilutions, concentrates or dried products.

In addition, the present inventors are brown gourd extract; And a mixture of one or more extracts of bamboo leaf extracts and green tea extracts to prepare a complex and confirm their whitening activity, all of the complexes have a whitening effect, and a mixture of all three extracts It was experimentally demonstrated that this more excellent effect was shown (see Experimental Examples 1 to 5).

In the cosmetic composition according to the present invention, the composition may include 5 to 55 parts by weight of one or more extracts of bamboo leaf extract and green tea extract based on 50 parts by weight of brown gourd extract, and preferably 8 to 52 weight parts. It may be included.

In one embodiment of the present invention, the composition may be one containing 5 to 55 parts by weight of bamboo leaf extract or green tea extract based on 50 parts by weight of brown gourd extract, preferably 45 to 55 weight of bamboo leaf extract or green tea extract It may be included, and more preferably may contain 48 to 52 parts by weight of bamboo leaf extract or green tea extract.

In one embodiment of the present invention, the composition may be one containing 15 to 45 parts by weight of bamboo leaf extract and 5 to 35 parts by weight of green tea extract, based on 50 parts by weight of brown gourd extract, preferably from 23 to bamboo leaf extract 37 parts by weight and 13 to 27 parts by weight of green tea extract may be included, and more preferably 25 to 35 parts by weight of bamboo leaf extract and 15 to 25 parts by weight of green tea extract, and more preferably bamboo leaves It may be to include 28 to 32 parts by weight of the extract and 18 to 22 parts by weight of green tea extract.

In addition, the cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, and as a product to which the cosmetic composition of the present invention may be added, for example, convergent cosmetics, soft cosmetics, nutrient cosmetics , Cosmetics such as various creams, essences, packs, foundations, and cleansing, face wash, soap, treatments, essences and the like.

Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, stick, makeup base, foundation, press powder, loose powder, eye shadow and the like, but is not limited thereto.

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

< Production Example  1> Brown meal  Preparation of Extract

Brown mealworm ( Tenebrio Molitor and mealworm were purchased from Sworm, Gyeonggi, Korea, and water was added in an amount of 10 times the weight of the sample, followed by extraction with a stirrer (250 rpm) at room temperature for 24 hours to separate the supernatant. This was repeated three times, and the separated supernatant was filtered, concentrated and lyophilized to prepare a brown gourd extract. The prepared brown gourd extract was used as a sample of the experiment while being stored in the refrigerating chamber (see Fig. 1).

< Production Example  2> Preparation of Bamboo Leaf Extract

Dried Bamboo Leaves ( Phyllostachys nigra Leaf) was purchased commercially and water was added in an amount of 10 times the weight of the sample, followed by extraction for 24 hours using a stirrer (250 rpm) at room temperature to separate the supernatant. This was repeated three times, and the separated supernatant was filtered, concentrated and lyophilized to prepare a bamboo leaf extract. The prepared bamboo leaf extract was used as a test sample while being stored in the refrigerator.

< Production Example  3> Preparation of Green Tea Extract

Dried green tea ( Camellia sinensis Leaf) was purchased on the market, water was added in an amount of 10 times the sample weight, and the supernatant was separated by extraction for 24 hours using a stirrer (250 rpm) at room temperature. This was repeated three times, and the separated supernatant was filtered, concentrated, and lyophilized to prepare a green tea extract. The prepared green tea extract was used as a sample of the experiment while storing in the refrigerator.

< Example  And Comparative example > Brown meal Of Bamboo, Bamboo Leaf and Green Tea Extracts

To prepare the composite of Examples 1 to 12 and Comparative Examples 1 to 4 by mixing the brown gourd extract, bamboo leaf extract and green tea extract of Preparation Examples 1 to 3 by weight.

Brown mealworm extract Bamboo Leaf Extract Green tea extract Example 1 50 20 20 Example 2 50 25 20 Example 3 50 30 20 Example 4 50 35 20 Example 5 50 40 20 Example 6 50 30 10 Example 7 50 30 15 Example 8 50 30 20 Example 9 50 30 25 Example 10 50 30 30 Example 11 50 50 - Example 12 50 - 50 Comparative Example 1 100 - - Comparative Example 2 - 100 - Comparative Example 3 - - 100 Comparative Example 4 - 50 50

< Preparation > Preparation of Experimental Materials

Mushroom tyrosinase, L-3,4-dihydroxy phenyl-alanine (L-DOPA), etc., reagents used for measuring the whitening effect, are sigma chemical Co. Ltd. (St. Louis, Mo., USA) and used. Antibodies such as cAMP kit (Cayman, Ann Arbor, MI, USA), MITF, Tyrosinase, TRP-1, TRP-2 were purchased from santa cruz (Biotech, CA, USA).

The cell line used for measuring cell viability was obtained from the American type culture collection (Manassas, VA, USA) as a mouse melanoma cell (B16F10). Dulbecco's modified eagle medium (DMEM) and fetal bovine serum (FBS) for cell culture were purchased from LONZA Co., and 0.25% trypsin-EDTA and 0.4% trypan blue stain were used for gibco BRL Co. (Grand Island, NY, USA], 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromid (MTT) was obtained from sigma chemical Co. Ltd. (St. Louis, Mo., USA) and used.

Instruments used in the present invention are UV / VIS spectrophotometer (Hitachi, Japan), polarization microscope (Olympus BX 51, Japan), western imaging system (CAS-400SM, Davinch-K, Korea), rotary vacuum evaporator (Tokyo, Rikakikai Co Japan), centrifuge (Hitachi, Japan), freeze drier (Ilsin, Korea), microscope (Olympus, Japan), CO2 incubator (Hanbaek Scientific Co., Korea), pH meter (Metrohm. Switzerland), BOD Incubator (Hanbaek Co. , Korea), autoclave (Hanbaek Scientific Co., Korea), ELISA reader (Molecular Devices, USA) was used.

< Experimental Example  1> Brown meal Tyrosinase Inhibitory Activity of the Complex of Bamboo, Bamboo Leaf and Green Tea Extracts

One of the factors that affect skin color is melanin, which is biosynthesized in the melanocyte melanosomes in the basal layer of the epidermis. To synthesize this melanin, it starts with a kind of amino acid called tyrosine, which is oxidized to L-3,4-dihydroxylphenylalaine (DOPA) and DOPA quinone by tyrosinase in melanocytes. DOPA quinone becomes DOPA chrome, 5,6-dihydroxyindole, indole-5,6-quinone and it is known that melanin is produced by polymerization [Ko JS. Dermatology. Soomoonsa press. Seoul. Korea. 2000; 73., Prota G. Recent advances in the chemistry of melanogenesis in mammals. J Invest Dermatol. 1980; 75 (1): 122-7., Pavel S, Muskiet FA. Eumelanin (precursor) metabolites as markers for pigmented malignant melanoma: A preliminary report. Cancer Detect Prev. 1983; 6 (1-2): 311-6.]. Melanin is also known to be synthesized by oxidation by reactive oxygen species (Hachinohe M, Matsumoto H. Involvement of reactive oxygen species generated from melanin synthesis pathway in phytotoxicty of L-DOPA. J Chem Ecol. 2005; 31 (2): 237-46. Reactive oxygen species are one of the products that occur during the respiratory process in the body. When excessively produced, not only melanin synthesis but also oxidative stress causes cell damage and organ damage, resulting in various diseases such as cancer, diabetes, vascular disease, and arthritis. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: Role in inflammatory disease and progression to cancer. Biochem J. 1996; 313: 17-29.]. For this reason it is important to confirm the inhibitory activity of tyrosinase, in the present invention was measured tyrosinase inhibitory activity derived from the mushrooms of extracts and complexes of brown rice wine, bamboo leaves and green tea.

Tyrosinase inhibition activity was measured by Yagi et al. [Yagi A, Kanbara T, Morinobu N. The effect of tyrosinase inhibition for aloe. Planta Med. 1986; 3981: 517-9.]. The reaction zone was prepared by adding 0.2 mL of mushroom tyrosinase (110 U / mL) to a mixture of 0.2 mL of substrate solution dissolved in 10 mM L-DOPA in 0.5 mL of 0.175 M sodium phosphate buffer (pH 6.8) and 0.1 mL of sample solution. The reaction was carried out for a minute to measure the DOPA chrome produced in the reaction solution at 475 nm. At this time, butylated hydroxyl anisole (BHA) was used as a positive control. The tyrosinase inhibitory activity was expressed as the absorbance reduction rate of the addition and non-addition of the sample solution using the formula shown in Equation 1 below. The analytical values were expressed as mean ± S.D. Of three repeated experiments, and were evaluated as having a significant difference when the p value was less than 0.01 (p <0.01).

Inhibition rate of tyrosinase by sample concentration (%) Brown meal: Bamboo leaves: Green tea 10 μg / mL 50 μg / mL 100 μg / mL 500 μg / mL Example 1 50:20:20 14.1 30.2 36.6 40.4 Example 2 50:25:20 17.0 38.9 47.3 63.9 Example 3 50:30:20 19.2 43.6 55.3 74.8 Example 4 50:35:20 17.8 39.5 48.5 66.4 Example 5 50:40:20 15.2 31.6 37.9 41.7 Example 6 50:30:10 14.3 31.2 36.2 40.9 Example 7 50:30:15 17.2 38.7 47.4 64.7 Example 8 50:30:20 19.2 43.6 55.3 74.8 Example 9 50:30:25 17.6 39.3 48.1 65.6 Example 10 50:30:30 14.9 30.7 36.5 41.5 Example 11 50: 50: 0 13.6 29.7 32.3 36.5 Example 12 50: 0: 50 13.7 29.2 31.9 35.1 Comparative Example 1 100: 0: 0 12.3 17.9 18.2 19.6 Comparative Example 2 0: 100: 0 7.6 12.2 14.8 17.3 Comparative Example 3 0: 0: 100 7.4 10.4 13.6 16.9 Comparative Example 4 0:50:50 10.9 18.5 20.6 24.7 Positive control group (BHA) - 12.1 47.2 81.3 91.4

As a result, the brown chopped extract, bamboo leaves and green tea of Comparative Examples 1 to 3 or Comparative Example 4 which does not include the brown chopped extract, respectively, treated with brown extract, bamboo leaf and green tea extract 1 The tyrosinase activity inhibition rate of the complexes mixed with more than one species was found to be high, and the tyrosinase inhibitory activity was better in the group treated with the complexes of Examples 1 to 10, in which all three extracts were mixed .

< Experimental Example  2> Brown meal Cytotoxicity of Melanoma cell (B16F10) of Complexes of Bamboo, Bamboo Leaf and Green Tea Extracts

The survival rate of melanoma cells by extracts or complexes of brown mealworms, bamboo leaves, and green teas was evaluated by Carmichael [Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res. 1987; 47 (4): 936-42.] Was measured by the MTT assay.

Specifically, 0.18 mL of the cell line B16F10 cells were dispensed in a 96 well plate at 5 × 10 ⁴ cells / well, and the sample was prepared by concentration (10, 50, 100 and 500 μg / mL), and then added 0.02 mL 37 It was incubated for 24 hours in a 5% CO₂incubator. After incubation for 4 hours by adding 0.02 mL of MTT solution prepared at a concentration of 5 mg / mL, the culture solution was removed, and 0.15 mL of DMSO was added to each well for 30 minutes at room temperature, and the absorbance was measured at 540 nm with an ELISA reader. Cytotoxicity measurement was expressed as the absorbance reduction rate of the sample solution addition group and no addition group using the formula shown in Equation 2 below, the analysis values were expressed as mean ± SD of three repeated experiments, p value is less than 0.01 There was a significant difference (p <0.01).

As a result, the survival rate of more than 95% at all 10, 50 and 100 μg / mL concentration was set as the experimental section.

< Experimental Example  3> Brown meal Inhibitory Activity of Tyrosinase in Melanoma Cell (B16F10)

After culturing B16F10 cells in each culture dish, the medium was removed and washed with PBS. 100 μL of lysis buffer (67 mM sodium phosphate buffer, 1% triton X-100, 0.1 mM phenylmethyl sulfonyfluoride) was added to each dish, and the cells were left on ice and ultra sonicated by collecting cells. After standing for 1 hour, the supernatant obtained by centrifugation at 13,200 rpm for 20 minutes was used as an enzyme solution. 120 μL of 8.0 mM L-DOPA dissolved in 67 mM phosphate buffer (pH 6.8) and 40 μL of sample solution (10, 50 and 100 μg / mL) were added to a 96 well plate, followed by 67 mM phosphate buffer (pH). 6.8) was added and 40 μL of the enzyme solution dissolved therein was incubated at 37 ° C. for 30 minutes, and the amount of DOPA chrome produced was measured at 490 nm. The analytical values are expressed as mean ± S.D. of three repeated experiments.

Tyrosinase activity (%) by sample concentration Brown meal: Bamboo leaves: Green tea 10 μg / mL 50 μg / mL 100 μg / mL No additive group (Con) - 100.0 100.0 100.0 Example 1 50:20:20 92.8 69.9 56.5 Example 2 50:25:20 86.1 62.6 48.8 Example 3 50:30:20 82.9 57.5 44.8 Example 4 50:35:20 85.3 61.1 48.1 Example 5 50:40:20 92.2 67.5 55.6 Example 6 50:30:10 92.8 69.1 56.2 Example 7 50:30:15 85.9 62.0 48.4 Example 8 50:30:20 82.9 57.5 44.8 Example 9 50:30:25 85.5 61.8 47.9 Example 10 50:30:30 92.2 68.4 55.7 Example 11 50: 50: 0 93.5 71.4 63.8 Example 12 50: 0: 50 93.9 72.3 65.2 Comparative Example 1 100: 0: 0 96.6 83.6 78.2 Comparative Example 2 0: 100: 0 97.3 88.8 83.7 Comparative Example 3 0: 0: 100 97.6 88.7 84.6 Comparative Example 4 0:50:50 95.4 80.6 74.8

As a result of confirming the tyrosinase inhibitory activity in melanoma cells by the extracts or complexes of brown rice wine, bamboo leaves and green tea of Examples 1 to 12 and Comparative Examples 1 to 4 according to the above method, As shown, the tyrosinase inhibitory activity of the complexes of Examples 1 to 12 mixed with brown gourd extract and bamboo leaves and green tea compared to the extracts or the complexes of Comparative Examples 1 to 4 alone or one of the complexes was higher. In addition, it was shown that the complexes of Examples 1 to 10, in which all three species were mixed, compared to the complexes of Examples 11 and 12, which mixed two species, further reduced tyrosinase activity. Example 8> Example 9> Example 4> Example 7> Example 2> Example 5> Example 10> Example 6> Example 1 In the order of tyrosinase inhibitory activity was excellent.

< Experimental Example  4> Brown meal Inhibitory Activity of Melanin Biosynthesis in Melanoma Cell (B16F10)

Measurement of melanin biosynthesis inhibition in B16F10 cells is described by Hosoi et al. [Hosoi J, Abe E, Suda T, Kuroki T. Regulation of melanin synthesis of B16 mouse melanoma cells by 1 alpha, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res. 1985; 45 (4): 1474-8.]. B16F10 cells incubated in DMEM medium were dispensed to 1 × 10 cells / dish in a 100mm culture dish, and cultured for 24 hours, 2 mL of the samples were prepared by concentration, and washed 48 hours later with phosphate buffer (pH 7.4). Then, the cells were detached with 0.25 M trypsin-EDTA solution, and the harvested cells were treated with 1 mL of 5% TCA per 1 × 10 cell, centrifuged twice at 2,500 rpm, and the separated melanin was washed with phosphate buffer. Then, 1 mL of ether: ethanol (1: 3) was added thereto, followed by centrifugation twice, followed by washing with 1 mL of ether and drying. 1 mL of 1 N NaOH was added to the dried melanin and reacted at 80 ° C. for 1 hour, and then the absorbance was measured at 405 nm. Melanin biosynthesis inhibition was expressed as the absorbance decrease rate of the sample solution addition group and no addition group using the formula shown in Equation 3 below, and the analysis values were expressed as mean ± S.D. of three repeated experiments.

Melanin biosynthesis inhibition rate (%) by sample concentration Brown meal: Bamboo leaves: Green tea 10 μg / mL 50 μg / mL 100 μg / mL No additive group (Con) - 100.0 100.0 100.0 Example 1 50:20:20 3.1 30.8 36.4 Example 2 50:25:20 5.2 42.6 47.1 Example 3 50:30:20 7.2 45.7 50.2 Example 4 50:35:20 5.6 43.6 47.8 Example 5 50:40:20 3.7 32.1 37.3 Example 6 50:30:10 3.3 31.6 36.7 Example 7 50:30:15 5.5 42.8 47.1 Example 8 50:30:20 7.2 45.7 50.2 Example 9 50:30:25 5.7 43.2 47.0 Example 10 50:30:30 3.6 31.8 37.2 Example 11 50: 50: 0 3.4 28.6 33.1 Example 12 50: 0: 50 3.0 28.4 32.8 Comparative Example 1 100: 0: 0 0.8 14.2 18.2 Comparative Example 2 0: 100: 0 1.4 16.2 16.4 Comparative Example 3 0: 0: 100 1.1 15.3 15.7 Comparative Example 4 0:50:50 2.1 21.9 25.4

In order to measure the melanin biosynthesis inhibitory activity in melanoma cells by extracts or complexes of brown rice wine, bamboo leaves and green tea of Examples 1 to 12 and Comparative Examples 1 to 4, the results were confirmed by concentration, as shown in Table 4 above. Likewise, the melanin biosynthesis inhibitory activity of the complexes of Examples 1 to 12, which were mixed with brown extracts and one or more extracts of bamboo leaf green tea, compared to the extracts of Comparative Examples 1 to 4 or the complex containing no brown meals, as in the results of tyrosinase inhibition activity Found to be high. In addition, it was found that the melanin content was further reduced when the complexes of Examples 1 to 10, which were mixed with all three species, were mixed, compared to the complexes of Examples 11 and 12, which were mixed with two species. Example 3 Example 8> Example 4> Example 2> Example 7> Example 9> Example 5> Example 10> Example 6> Example 1 The effect of reducing melanin biosynthesis was excellent in this order. appear.

< Experimental Example  5> Brown meal Inhibitory Effects of Tyrosinase, MITF, TRP-1, and TRP-2 Expression on Western Blot of Complex of Bamboo, Leaf and Green Tea Extracts

Melanin production occurs mainly by oxidative reactions. TRP-1 oxidizes 5,6-dihydroxy indole-2-carboxylic acid (DHICA) produced by TRP-2 to produce indole-2-carboxylic acid (IQCA). . Microphthalmia-associated transcription factor (MITF) is a factor that regulates the expression of tyrosinase. Therefore, inhibition of MITF inhibits tysinase expression and consequently inhibits melanin pigment production.

The complex of Example 3 (= Example 8) brown mouse, bamboo leaf and green tea extract to the expression of tyrosinase, MITF, TRP-1, TRP-2 in the mouse melanoma cell (B16F10) After treatment at a concentration of 10, 25, 50, 100 μg / mL and recovered after 48 hours, the expression of the proteins was confirmed by western blot.

Specifically, the cells were washed twice with cold PBS, dissolved by adding complate mini 1 tab to 10 mL of RIPA buffer, and centrifuged at 12,000 rpm for 15 minutes at 4 ° C. The supernatant obtained by centrifugation was quantified by bradford assay and 25 μL of protein was separated by electrophoresis on 10% SDS-PAGE. The separated proteins were transferred to PVDF membrane using a semi dry transfer cell (Hofer, USA) and incubated in blocking buffer (5% skim milk in TBST) for 30 minutes at room temperature. After diluting the primary antibody and reacting for 3 hours, the mixture was washed three times with TBST at 10 minute intervals and the membrane was reacted for 60 minutes by diluting each secondary antibody polymerized with HRP at 1: 1,000. After washing three times, the band was identified and quantified using a davinch-k western imaging system. The analytical values were expressed as mean ± S.D. of three repeated experiments, and it was evaluated that there was a significant difference when the p value was less than 0.01 or 0.05 depending on the analysis target (p <0.01 or 0.05). Β-actin was used as house keeping gene.

Sample amount (%) of protein expression by concentration Con 10 μg / mL 25 μg / mL 50 μg / mL 100 μg / mL Tyrosinase / β-actin ratio 100.0 85.6 71.3 66.8 58.4 MITF / β-actin ratio 100.0 99.8 98.2 95.4 87.5 TRP-1 / β-actin ratio 100.0 58.5 30.7 14.6 7.1 TRP-2 / β-actin ratio 100.0 78.4 41.6 24.3 13.8

As a result, as shown in Table 5, all of tyrosinase, MITF, TRP-1 and TRP-2 showed a tendency to decrease the expression depending on the concentration, brown meal, bamboo leaf and Example 3 (= Example 8) It was confirmed that the complex of green tea extracts may inhibit the expression of the factors and have an inhibitory effect on tyrosinase activity and melanin biosynthesis.

Therefore, based on these results, the present inventors confirmed that the composite of brown rice meal, bamboo leaf and green tea extract of the present invention can be used as a cosmetic composition having a whitening activity.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (3)

Based on 50 parts by weight of Tenebrio Molitor extract;
25-35 parts by weight of Phyllostachys nigra Leaf extract; And
15-25 parts by weight of green tea ( Camellia sinensis Leaf) extract;
Cosmetic composition for skin whitening containing as an active ingredient. The method of claim 1,
The extract is a skin whitening cosmetic composition, characterized in that the ethanol (ethanol), methanol (methanol) or water (water) extract. delete