KR102202554B1 - Skin whitening cosmetic composition with the extract of Saururus chinensis leaf, the extract of Grape seed, the extract of Portulaca oleracea and rice fermented product - Google Patents

Abstract

The present invention relates to a cosmetic composition made of a natural material-derived material. More specifically, the present invention provides a cosmetic composition which contains a complex of a Saururus chinensis leaf extract, a grape seed extract, a Portulaca oleracea extract, and a hard-boiled rice fermented product, thereby having no side effects and excellent skin whitening effects. The mixture is mixed with 2 to 3 parts by weight of grape seeds, and 6 to 7 parts by weight of Portulaca oleracea with respect to 1 part by weight of Saururus chinensis leaves.

Description

Skin whitening cosmetic composition with the extract of Saururus chinensis leaf, the extract of Grape seed, the extract of Portulaca oleracea and rice fermented product}

The present invention relates to a cosmetic composition for skin whitening comprising the extract of Sambaekcho leaf, the extract of grape seed, the extract of machi-suki and the fermented godubap.

In the cosmetic field, one of the areas of interest to East Asian women, especially Korean, Japanese, and Chinese women, is skin whitening. The most important factor in skin whitening is the skin melanin pigment. Melanin (melanin) is a black pigment found in animals, plants, microorganisms, etc., and is not essential for growth or development, but is a substance that enhances viability and competitiveness for a specific environment. Melanin in animals is related to skin disease and malignant melanoma, and in addition to DOPA melanin produced from tyrosine by tyrosinase, DHN melanin, GDHB melanin, catechol melanin ) Have been reported [ Bell, AA and Weeler MH, Ann. Rev. Phytophathol. 24, 411, 1986 ].

When melanin is overproduced, it is continuously released out of the skin, and spots are generated in local areas, or the skin becomes black. Therefore, various whitening methods to prevent excessive deposition of melanin on the skin and to reduce the pigment content in the skin are attracting attention. The main methods are UV protection, rapid bleaching of melanin pigments, blocking the step of biosynthetic melanin being loaded onto melanosomes and shearing into keratinocytes, inhibiting tyrosinase activity, which is the step of determining the rate of melanin biosynthesis. Method, a method of blocking signal transduction that induces tyrosinase activity and biosynthesis from the cell membrane of melanocytes, and a method of administering a substance that is specifically toxic to melanogenic cells [ Son KH, Heo MY. Int J Cosmeti Sci. 35: 9-18. 2013 ].

Arbutin, kojic acid, hydroquinone, vitamin C, etc., as cannonball substances that inhibit tyrosinase, which is known as an important factor in melanin synthesis, are known to date. It is known that it has an inhibitory effect on the production of melanosomes due to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) polymerase activity in addition to the synase activity inhibition function. However, controversy about the stability and safety of these substances and side effects continues.

Therefore, research on materials derived from natural substances is on the rise in order to overcome the problems of existing known materials that suppress melanin production and search for a material having a safer and superior effect.

Republic of Korea Patent Publication No. 10-2018-0048408 (published on May 10, 2018) discloses a cosmetic composition for skin whitening and a method for manufacturing the same, which contains three baekchos having improved skin whitening effect by using a traditional fermentation technique. Republic of Korea Patent Registration No. 10-1744579 (registered on January 1, 2017) discloses a cosmetic composition for skin whitening containing a mixed extract of water lily, chamomile flower, moringa, and machi.

An object of the present invention is to develop and provide a cosmetic composition having excellent skin whitening ability by utilizing a material derived from a natural product.

The present invention is a cosmetic for skin whitening comprising a mixed extract of a mixture of Saururus chinensis leaves, Grape seed, and Portulaca oleracea , and a fermented product obtained by inoculating yeast into rice and fermenting it. The composition is provided.

In the present invention, the mixture is preferably mixed in an amount of 2 to 3 parts by weight of grape seeds and 6 to 7 parts by weight of machihyun, based on 1 part by weight of the leaves of Sambaekcho.

In the present invention, the mixed extract and the fermented product are preferably mixed in an amount of 5 to 30 parts by weight of the fermented product to 70 to 95 parts by weight of the mixed extract on a dry weight basis.

In addition, the present invention for skin whitening, characterized in that it comprises a fermented product fermented by inoculating yeast into a sambaekcho ( Saururus chinensis ) leaf extract, grape seed (Grape seed) extract, portulaca oleracea extract, and godubap (rice) It provides a cosmetic composition.

In the present invention, the Sambaekcho leaf extract, grape seed extract, and portulaca oleracea extract are preferably used for skin whitening in 2 to 3 parts by weight of grape seed extract, and 6 to 7 parts by weight of portulaca oleracea extract based on 1 part by weight of the Sambaekcho leaf extract based on dry weight. It is better to be mixed with the cosmetic composition.

In the present invention, the Sambaekcho leaf extract, grape seed extract, portulaca oleracea extract, and fermented product are preferably 70 to 95 parts by weight of the sum of the Sambaekcho leaf extract, grape seed extract, and portulaca oleracea extract in a ratio of 5 to 30 parts by weight of the fermented product. Good to be mixed.

Meanwhile, in the cosmetic composition for whitening of the present invention, the extract is preferably extracted using an aqueous ethanol solution as an extraction solvent.

Meanwhile, in the cosmetic composition for whitening of the present invention, the fermented product is preferably a filtrate obtained through filtration after fermentation.

In the case of Sambaekcho leaves and grape seeds, although they are materials showing excellent whitening efficacy, there is a problem that their use is limited due to cytotoxicity. However, in the present invention, it was confirmed that the cytotoxicity was reduced by adding the fermented machisuki extract and the fermented godubap to these ingredients.Through this, it was confirmed that the whitening effect without side effects was observed. It was possible to develop a cosmetic composition containing as a composite.

1 is a graph showing the results of a toxicity test using melanoma cells (B16-F1) of three baekcho leaves and grape seeds.
Figure 2 is a graph of the results of the toxicity test using melanoma cells (B16-F1) of Preparation Examples 1 to 9.
3 is a graph of the results of a toxicity test using melanoma cells (B16-F1) of Preparation Examples 10 to 15.
4 is a graph showing the results of an experiment on the inhibitory effect of melanin biosynthesis using melanoma cells (B16-F1).
5 is a graph showing the results of an experiment for measuring the expression levels of Tyrosinase, TRP-1, and TRP-2 mRNA related to melanogenesis.
6 is a graph showing the results of an experiment for measuring the expression levels of Rab27a, MyosinVa, and Melanophilin mRNA related to melanosome transport.

The present invention is a cosmetic for skin whitening comprising a mixed extract of a mixture of Saururus chinensis leaves, Grape seed, and Portulaca oleracea , and a fermented product obtained by inoculating yeast into rice and fermenting it. The composition is provided.

In addition, a cosmetic composition for skin whitening, characterized in that it comprises a fermented product obtained by inoculating yeast on rice and inoculating sauerkraut ( Saururus chinensis ) leaf extract, grape seed extract, portulaca oleracea extract, and fermentation. to provide.

Saururus chinensis is a perennial herb that grows in some areas of Jeju Island and Jirisan Mountain, and grows in a humid valley where the air is well ventilated, the air humidity is high, and the shade is half shade. According to the herbal literature, it is recorded that Sambaekcho is effective in treating various adult diseases. Sambaekcho is known to have an excellent effect in anti-aging because tannins, etc., purify blood, strengthen capillaries, and smooth blood flow.

Grape seeds are high in polyphenols and have excellent antioxidant efficacy. Representatively, proanthocyanin has the effect of preventing cardiovascular disease, preventing senile dementia, anti-diabetic effect, and preventing colon cancer, and can increase immunity with strong antioxidant activity. In addition, resveratrol is a substance produced by grapes to protect itself from fungi, and it acts as a powerful antioxidant to prevent cell damage and aging. Resveratrol is contained in the order of grains of grapes, grape skins, and grape seeds, and it helps lower blood cholesterol.

Portulaca oleracea is also known as purslane, Jangmyeongchae, confectionery, and five elements. It is wild all over the country and is distributed all over the world. The roots are white, and the stems are reddish brown, with many branches spreading to the side and growing. Machihyeon has antipyretic, detoxifying, and hemostatic effects, and antibacterial effects have also been reported.

Rice is a peeled kernel of rice, a plant belonging to the rice family. Godubap refers to the rice that has been cooked very well and carefully cooked, and it is prepared by washing the rice well and soaking it overnight, draining it for 1 to 2 hours, and steaming it in a steamer or a steamer. In the present invention, a fermented product obtained by inoculating yeast and fermenting the prepared godubap is used.

The present invention was to develop a cosmetic composition for skin whitening by using the herbal medicines having the above efficacy, but according to the experiment of the present invention, a complex of Sambaekcho leaf extract, grape seed extract, portulaca oleracea extract and fermented Godubap yeast It was confirmed that the composition of the present invention including the present invention exhibits excellent skin whitening effect.

In the present invention, after mixing three baekcho leaves, grape seeds and machihyeon, the extraction solvent is added thereto, and then extracted and used. However, as another embodiment, it is also possible to mix and use the extract of the extract of the extract of the extract of the extract of the leaf of the cedarium, the extract of the extract of the seed of the grape, and the extract of the extract of the machi-hyeon, respectively extracted by adding an extraction solvent to each of the three baekcho leaves, grape seeds, and machihyeon. During extraction, as an extraction solvent, purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, and hexane solvents may be used alone or a mixed solvent in which two or more solvents thereof are mixed may be used. However, preferably, it is good to use the one extracted by using an aqueous ethanol solution, more preferably a 50% aqueous ethanol solution. In addition to the extraction solvent, other extraction methods may use methods well known in the art.

In the cosmetic composition of the present invention, the fermented product refers to a fermented product obtained by inoculating yeast on rice and fermenting it, and preferably, a filtrate obtained by filtering is used. Godubap can be prepared by steaming after soaking rice grains, and a fermented godubap can be prepared by mixing godubap and yeast. In this case, filtration may be performed using a conventional method well known in the art.

On the other hand, skin whitening, characterized in that it comprises a fermented product fermented by inoculating yeast into rice and a mixed extract of a mixture of Saururus chinensis leaves, grape seed, and portulaca oleracea of the present invention. In the cosmetic composition for, the three baekcho leaf extract, grape seed extract, and machihyeon are preferably mixed with 2 to 3 parts by weight of grape seeds and 6 to 7 parts by weight of machihyun based on 1 part by weight of the three baekcho leaves. In addition, the mixed extract and the fermented product are preferably mixed in an amount of 5 to 30 parts by weight of the fermented product in 70 to 95 parts by weight of the mixed extract on a dry weight basis. In addition, it is preferable to contain the composite including the mixed extract and the fermented product in an amount of 1 to 20% by weight based on the total weight of the cosmetic composition.

On the other hand, it is characterized in that it comprises a fermented product fermented by inoculating yeast into another embodiment of the present invention, Saururus chinensis leaf extract, grape seed extract, portulaca oleracea extract, and godubap (rice). In the cosmetic composition for skin whitening, the Sambaekcho leaf extract, grape seed extract, and portulaca oleracea extract are, preferably, based on dry weight, based on 1 part by weight of the Sambaekcho leaf extract, 2 to 3 parts by weight of grape seed extract, 6 to 7 parts by weight of machihyun extract It is better to be mixed with the cosmetic composition for skin whitening. In addition, the Sambaekcho leaf extract, grape seed extract, portulaca oleracea extract, and fermented product are preferably mixed in a ratio of 5 to 30 parts by weight of the fermented product to 70 to 95 parts by weight of a combination of the Sambaekcho leaf extract, grape seed extract, and portulaca oleracea extract. In addition, it is preferable to contain the composite including the mixed extract and the fermented product in an amount of 1 to 20% by weight based on the total weight of the cosmetic composition.

On the other hand, the cosmetic composition of the present invention, for example, solution, suspension, emulsion, paste, lotion, gel, water-soluble liquid, cream, essence, surfactant-containing cleansing, oil, oil in water (O/W) type and oil Any one basic cosmetic formulation selected from heavy water (W/O) type; skin; Lotion; Eye cream; Soothing gel; Ointment; Formulation for mask pack; Formulation for body wash; Peeling gel; Water-in-water and water-in-oil makeup base; foundation; Skin cover; Any one color cosmetic formulation selected from lipstick, lip gloss, face powder, two-way cake, eye sado, cheek color, and eyebrow pencil; Formulation for scalp; It may be any one selected from among.

In addition, the cosmetic composition of the present invention may contain auxiliary agents commonly used in the cosmetic field, such as hydrophilic or lipophilic activators, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants, dyes, and the like. The amounts of these various adjuvants are those commonly used in the art, such as 0.001 to 30% by weight based on the total weight of the composition. However, in any case, the adjuvant and its ratio will be selected so as not to adversely affect the desirable properties of the cosmetic composition according to the present invention.

On the other hand, in the cosmetic composition of the present invention, when the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silaka as carrier components , Talc or zinc oxide may be used.

In addition, in the cosmetic composition of the present invention, when the formulation is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of soribitan may be used.

In addition, in the cosmetic composition of the present invention, when the formulation is a suspension, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester as carrier components. The same suspending agent, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, and the like may be used.

On the other hand, in the cosmetic composition of the present invention, when the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally Propellants such as chlorofluorohydrocarbon, propane/butane or dimethyl ether.

On the other hand, in the cosmetic composition of the present invention, when the formulation is a surfactant-containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

In addition, in the cosmetic composition of the present invention, in the case of a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation or soap, it may be applied to the skin and then wiped off, removed, or washed with water. As an example, the soap is liquid soap, powder soap, solid soap and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel and cleansing pack, and the surfactant-free cleansing formulation is a cleansing cream, Cleansing lotion, cleansing water, and cleansing gel, but are not limited thereto.

In addition, the cosmetic composition of the present invention can be used in conjunction with other cosmetic compositions other than the present invention. In addition, the cosmetic composition according to the present invention may be used according to a conventional method of use, and the number of times of use thereof may be changed according to a user's skin condition or taste.

Hereinafter, the present invention will be described in more detail in the following Preparation Examples, Examples and Experimental Examples. However, the scope of the present invention is not limited to the following manufacturing examples, examples, and experimental examples, and includes all modifications of the equivalent technical idea.

[Experimental Example 1: Evaluation of cytotoxicity using melanoma cells (B16-F1)]

In order to measure the toxicity of three baekcho leaves and grape seeds, an experiment was performed by treating a sample in melanoma (B16-F1) cells of a commercially purchased mouse. DMEM (Dulbecco's modified Eagle's medium) medium containing 10% bovine serum and melanoma (B16-F1) cells were dispensed at a concentration of 1×10 ⁵ cells/well in a 6 well plate, and 37℃ 5% CO ₂ condition for 24 hours Cultured in. At this time, the sample to be used in the experiment was extracted by adding about 6 times the amount of 50% (v/v) ethanol to each of Sambaekcho leaves and grape seeds for 3 days at room temperature, and the extract was filtered with 0.45㎛ and concentrated under reduced pressure. .

After 24 hours, the samples (50% ethanol extract of Sambaekcho leaves and 50% ethanol extract of grape seeds) were diluted by concentration in a new DMEM medium containing α-Melanocyte stimulate hormone (α-MSH) and 10% bovine serum and cultured for 72 hours. do. After 72 hours of incubation, all of the medium was removed, and 2.5 mg/ml of MTT soluble solution was replaced with a 10-fold diluted medium, and cultured in a 4 hour incubator. After 4 hours, the supernatant was removed, and MTT formazan crystals produced by adding 2 ml dimethhyl sulfoxide (DMSO) solution were measured for absorbance at 570 nm.

The control group was treated with DMSO (dimethhyl sulfoxide) used as a dissolving solvent, and the α-MSH treated group was treated with α-MSH and dimethhyl sulfoxide (DMSO) used as a dissolving solvent. Arbutin (SK Bioland) was used as a positive control. The toxicity test results are shown in Table 1 and FIG. 1 below.

Sample Conc.(㎍/㎖) Cell viability (%) Control 97.21 α-MSH treatment group 100.00 α-MSH +
Sambaekcho Leaf Extract 0.1 96.52 0.25 93.83 0.5 74.12 One 67.62 2.5 58.53 5 52.83 10 48.99 α-MSH +
Grape seed extract One 91.55 10 85.39 25 77.76 50 57.54 100 44.82 250 40.12 α-MSH + Arbutin 100 88.17

According to the above experiment, cytotoxicity in melanoma was confirmed. The extract of Sambaekcho leaf showed cytotoxicity from 0.5 µg/ml and 25 µg/ml of grape seed extract.

[Experimental Example 2: Preparation of mixed extract for alleviating cytotoxicity]

In this experimental example, it was attempted to alleviate the cytotoxicity of Sambaekcho and grape seeds by selecting the machi-hyun as a substance for alleviating the cytotoxicity of leaves and grape seeds. After mixing a total of 100g of leaves, grape seeds, and machisuki at the mixing ratio (weight ratio) shown in Table 2 below, 600g of 50% (v/v) ethanol was added, extracted for 3 days at room temperature, filtered to 0.45㎛, and the filtrate was concentrated under reduced pressure. Then, vacuum-dried powder was prepared.

Sambaekcho Leaf Grape seed Machi Prefecture Manufacturing Example 1 5 3 2 Manufacturing Example 2 3 2 One Manufacturing Example 3 2 One One Manufacturing Example 4 One 2 2 Manufacturing Example 5 One 2 One Manufacturing Example 6 One 4 5 Manufacturing Example 7 One 3 6 Manufacturing Example 8 One 2 7 Manufacturing Example 9 One One One

In order to measure the toxicity of the obtained powder, an experiment was performed by treating a sample in melanoma (B16-F1) cells of a commercially purchased mouse. DMEM (Dulbecco's modified Eagle's medium) medium containing 10% bovine serum and melanoma (B16-F1) cells were dispensed at a concentration of 1×10 ⁵ cells/well in a 6 well plate, and 37℃, 5% CO ₂ for 24 hours. Incubated under conditions.

After 24 hours, the sample is diluted by concentration in a new DMEM medium containing α-Melanocyte stimulate hormone (α-MSH) and 10% bovine serum and incubated for 72 hours. After 72 hours of incubation, all of the medium is removed, and 2.5 mg/mL of MTT soluble solution is replaced with a 10-fold diluted medium and cultured in an incubator for 4 hours. After 4 hours, the supernatant was removed, and MTT formazan crystals produced by adding 2 ml dimethhyl sulfoxide (DMSO) solution were measured for absorbance at 570 nm.

The control group was treated with DMSO (dimethhyl sulfoxide) used as a dissolving solvent, and the α-MSH treated group was treated with α-MSH and dimethhyl sulfoxide (DMSO) used as a dissolving solvent. Arbutin (SK Bioland) was used as a positive control. The toxicity test results are shown in Table 3 and FIG. 2 below.

Sample Conc.(㎍/㎖) Cell viability (%) Control 98.17 α-MSH treatment group 100.00 Manufacturing Example 1 50 46.76 100 38.93 Manufacturing Example 2 50 44.7 100 34.79 Manufacturing Example 3 50 45.27 100 35.46 Manufacturing Example 4 50 67.62 100 55.02 Manufacturing Example 5 50 63.51 100 52.41 Manufacturing Example 6 50 82.69 100 78.15 Manufacturing Example 7 50 85.12 100 83.48 Manufacturing Example 8 50 97.21 100 96.84 Manufacturing Example 9 50 56.11 100 47.52 α-MSH + Arbutin 100 88.14

As a result of the experiment, it was found that the cytotoxicity was alleviated by the addition of the mixed extract of Macho vulgaris, and in particular, in Preparation Examples 7 and 8, the cytotoxic mitigation effect was most excellent.

[Experimental Example 3: Preparation of a complex product of three baekcho leaves, grape seeds, portulaca oleracea extract and fermented godubap filtrate]

In this experimental example, it was attempted to confirm the toxicity mitigation effect by adding the fermented filtrate of godubap to Preparation Examples 7 and 8, which had excellent cytotoxicity evaluation results among the mixed extracts of Sambaekcho leaves, grape seeds, and machisalis prepared above.

Three times purified water was added to well-soaked rice grains, soaked for 3 hours, and then treated with 150°C steam for 30 minutes to prepare godubap. After cooling the godubap, godubap and nuruk (Songhakgokja Co., Ltd., Soyulgok) were mixed at a ratio of 1:1 and left for at least 1 hour. After that, purified water equivalent to 3 times the weight of the mixture is added, aged at 40°C for 1 day, heated to 90°C, and heated and sterilized for 1 hour. After the completion of the manufacturing process, the filtered solution was concentrated under reduced pressure with 0.45 μm to prepare a fermented godubap filtrate. The mixed extract powder of Preparation Example 7 and Preparation Example 8 and the fermented filtrate powder of Godubap were blended as shown in Table 4 below.

division Manufacturing Example 7 Godubap fermentation filtrate Manufacturing Example 10 95 5 Manufacturing Example 11 90 10 Manufacturing Example 12 70 30 division Manufacturing Example 8 Godubap fermentation filtrate Manufacturing Example 13 95 5 Manufacturing Example 14 90 10 Manufacturing Example 15 70 30

In order to measure the cytotoxicity of Preparation Examples 10 to 15, an experiment was performed by treating a sample with melanoma (B16-F1) cells of a commercially purchased mouse. DMEM (Dulbecco's modified Eagle's medium) medium containing 10% bovine serum and melanoma (B16-F1) cells were dispensed at a concentration of 1×10 ⁵ cells/well in a 6 well plate, and 37℃, 5% CO ₂ for 24 hours. Incubated under conditions.

After 24 hours, the sample is diluted by concentration in a new DMEM medium containing α-Melanocyte stimulate hormone (α-MSH) and 10% bovine serum and incubated for 72 hours. After 72 hours of incubation, all of the medium was removed, and 2.5 mg/ml of MTT soluble solution was replaced with a 10-fold diluted medium, and cultured in a 4 hour incubator. After 4 hours, the supernatant was removed, and MTT formazan crystals produced by adding 2 ml dimethhyl sulfoxide (DMSO) solution were measured for absorbance at 570 nm.

The control group was treated with DMSO (dimethhyl sulfoxide) used as a dissolving solvent, and the α-MSH treated group was treated with α-MSH and dimethhyl sulfoxide (DMSO) used as a dissolving solvent. Arbutin (SK Bioland) was used as a positive control. The toxicity test results are shown in Table 5 and FIG. 3 below.

Sample Conc.(㎍/㎖) Cell viability (%) Control 98.47 α-MSH treatment group 100.00 Manufacturing Example 10 50 98.78 100 97.77 Manufacturing Example 11 50 90.11 100 89.17 Manufacturing Example 12 50 89.12 100 87.02 Manufacturing Example 13 50 99.78 100 98.62 Manufacturing Example 14 50 90.18 100 89.94 Manufacturing Example 15 50 91.31 100 89.64 α-MSH + Arbutin 100 89.41

As shown in FIG. 3, it was confirmed that the cytotoxicity was alleviated when the fermented godubap filtrate was added to the mixed extract of Sambaekcho leaves, grape seeds, and machihyeon. In particular, it was found that the cytotoxicity was most excellently alleviated in Preparation Example 10 and Preparation Example 13.

[Experimental Example 4: Measurement of melanin biosynthesis inhibitory effect using melanoma (B16-F1) cells]

In this experiment, it was intended to measure the whitening efficacy of Preparation Examples 10 and 13, which were found to have the best alleviation of cytotoxicity in the above Examples.

The experiment was carried out by treating the sample with melanoma (B16-F1) cells of commercially purchased mice. DMEM (Dulbecco's modified Eagle's medium) medium containing 10% bovine serum and melanoma (B16-F1) cells were dispensed at a concentration of 1×10 ⁵ cells/well in a 6 well plate, and 37℃, 5% CO ₂ for 24 hours. Incubated under conditions.

After 24 hours, the sample is diluted by concentration in a new DMEM medium containing α-Melanocyte stimulate hormone (α-MSH) and 10% bovine serum and incubated for 72 hours. After 72 hours of incubation, cells from which all of the medium was removed were washed with 2 ml of PBS (phosphate buffer, saline) and treated with 0.5 ml of 1N NaOH to collect the cells into a 1.5 ml tube to obtain intracellular melanin. The collected cells were transferred to a 96 well plate, and the absorbance was measured at 450 nm to determine the amount of melanin per protein.

Arbutin (SK Bioland) was used as a positive control. The melanin biosynthesis inhibition test results are shown in Table 6 and FIG. 4 below.

Sample Conc.(㎍/㎖) Melanin formation per cell (%) Control 33.91 α-MSH treatment group 100.00 α-MSH + Preparation Example 10 5 85.46 10 82.85 25 80.74 50 79.89 100 75.31 α-MSH + Preparation Example 13 5 83.31 10 80.96 25 77.05 50 66.81 100 56.56 α-MSH + Arbutin 100 52.10

As shown in Table 6 and FIG. 3, it was found that the composites of Preparation Example 10 and Preparation 13 of the present invention inhibited melanin production per unit cell in a concentration-dependent manner.

[Experimental Example 5: Investigation of the expression level of tyrosinase, TRP-1, TRP-2 mRNA related to melanogenesis]

In this experiment, the melanogenesis-related whitening effect of the composite of Preparation Example 13, which had excellent melanin production inhibitory effect in Experimental Example 4, was measured.

The experiment was carried out by treating the sample with melanoma (B16-F1) cells of commercially purchased mice. DMEM (Dulbecco's modified Eagle's medium) medium containing 10% bovine serum and melanoma (B16-F1) cells were dispensed at a concentration of 2×10 ⁵ cells/well in a 6 well plate, and 37℃, 5% CO ₂ for 24 hours. Incubated under conditions.

After 24 hours, the samples were diluted by concentration in a new DMEM medium containing α-Melanocyte stimulate hormone (α-MSH) and 10% bovine serum, and cultured for 24 hours. After culturing for 24 hours, the cells from which all the medium was removed were washed with 2 ml of PBS (phosphate buffer, saline), and the cells were collected with TRIzol (invitrogen, USA) to isolate RNA. The isolated RNA was quantified using a Qubit fluoremeter with RNA BR Assay kit, and then cDNA (complementary DNA) was synthesized to perform real-time PCR. For cDNA synthesis, a cDNA synthesis Kit (High Capacity RNA to-cDNA Kit, Applied Biosystems, USA) was used, and an experiment was performed according to the method of the Kit. Real-Time PCR: After amplifying the gene using 7500 Fast with Power SYBR Green PCR master mix (Applied Biosystems, USA), the amplified product was quantitatively analyzed. Tyrosinase, TRP-1, TRP-2, and GAPDH primers used in PCR were synthesized and used by Cosmo Genetec (Korea), and the primer sequences are shown in Table 7 below.

Primer Sense Antisense Tyrosinase 5'TATAAACTCAGTGTTTCCCTTTATCACAA-3' 5'-TCTATGATGACATGAAGTGGCAAA-3' TRP-1 5'-GTTCAATGGCCAGGTCAGGA-3' 5'-CAACGCAGCCACTACAGCA-3' TRP-2 5'-TGG CTC ACT CCT TCC TGA ATG-3' 5'-CAA ACA CAG GGT CGT TGG CT-3' GAPDH 5'-GGCATCTTGGGCTACACTGAG-3' 5'-GGAAGAGTGGGAGTTGCTGTTG-3'

The control group was treated with DMSO (dimethhyl sulfoxide) used as a dissolving solvent, and the α-MSH treatment group was treated with DMSO (dimethhyl sulfoxide) used as a dissolving solvent. Arbutin (SK Bioland) was used as a positive control. The experimental results are shown in Table 8 and FIG. 5 below.

Sample Name Conc. (㎍/mL) Tyrosinase
expression rate(%) Stdev. TRP-1 expression rate(%) Stdev. TRP-2 expression rate(%) Stdev. Control 64.34 0.52 64.34 4.45 81.74 0.82 α-MSH treatment group 100.00 0.00 100.00 0.00 100.00 0.00 α-MSH +
Manufacturing Example 13 25 89.48 0.49 85.88 1.69 82.79 2.02 50 86.53 0.29 82.77 1.75 80.90 0.69 100 73.99 1.40 67.06 0.21 64.30 0.42 α-MSH +
Arbutin 100 75.85 1.54 78.44 1.76 75.92 2.69

As shown in Table 8 and FIG. 5, it was confirmed that the increased expression of Tyrosinase, TRP-1 and TRP-2 genes due to α-MSH during Preparation Example 13 treatment was significantly suppressed in a concentration-dependent manner, and 100 μg/of each gene It was confirmed that the inhibitory effect of melanin production was superior to Arbutin used as a positive control at a concentration of ml.

[Experimental Example 6: Investigation of the expression levels of Rab27a, MyosinVa, and Melanophilin mRNA related to melanosome transport]

In this experiment, it was to confirm the whitening effect related to the transport of melanosomes of the composite of Preparation Example 13.

The experiment was carried out by treating the sample with melanoma (B16-F1) cells of commercially purchased mice. DMEM (Dulbecco's modified Eagle's medium) medium containing 10% bovine serum and melanoma (B16-F1) cells were dispensed at a concentration of 1.5×10 ⁵ cells/well in a 6 well plate, and 37℃, 5% CO ₂ for 24 hours. Incubated under conditions.

After 24 hours, the sample is diluted by concentration in a new DMEM medium containing α-Melanocyte stimulate hormone (α-MSH) and 10% bovine serum and incubated for 18 hours. After 18 hours of culture, the cells from which all the medium was removed were washed with 2 ml of PBS (phosphate buffer, saline), and the cells were collected with TRIzol (invitrogen, USA) to isolate RNA. The isolated RNA was quantified using a Qubit fluoremeter with RNA BR Assay kit, and then cDNA (complementary DNA) was synthesized to perform real-time PCR. For cDNA synthesis, a cDNA synthesis Kit (High Capacity RNA to-cDNA Kit, Applied Biosystems, USA) was used, and an experiment was performed according to the method of the Kit. Real-Time PCR: After amplifying the gene using 7500 Fast with Power SYBR Green PCR master mix (Applied Biosystems, USA), the amplified product was quantitatively analyzed. Rab27a, MyosinVa, Melanophilin, and GAPDH primers used in PCR were synthesized and used by Cosmogenetec (Korea), and the primer sequence is shown in Table 9 below.

Primer Sense Antisense Rab27a 5'-ATGTCGGATGGAGATTACGA-3' 5'-CCATAACTGCAGGTGGATTC-3' MyosinVa 5'-AGTGCAGCAGCTAAGAGCAT-3' 5'-ATTCTTGCACGTTTGCTTTC-3' Melanophilin 5'-AGCCCCTCAACAGCAAAAA-3' 5'-TTCCTCAAAGTCCACATCTCG-3' GAPDH 5'-GGCATCTTGGGCTACACTGAG-3' 5'-GGAAGAGTGGGAGTTGCTGTTG-3'

The control group was treated with DMSO (dimethhyl sulfoxide) used as the dissolving solvent, and the α-MSH treatment group was treated with DMSO (dimethhyl sulfoxide) used as the dissolving solvent. The experimental results are shown in Table 10 and FIG. 6 below.

Sample Name Conc. (㎍/mL) Rab27a
expression rate(%) Stdev. MyosinVa expression rate(%) Stdev. Melanophilin expression rate (%) Stdev. Control 52.11 3.09 55.40 2.37 79.20 00.83 α-MSH treatment group 100.00 0.00 100.00 0.00 100.00 0.00 α-MSH +
Manufacturing Example 13 25 92.12 2.95 90.37 0.52 85.22 3.44 50 86.69 3.15 83.33 2.27 83.54 6.93 100 78.72 2.43 72.87 2.35 71.66 4.85

As shown in Table 10 and FIG. 6, it was confirmed that the gene expression of Rab27a, MyosinVa, and Melanophilin increased due to α-MSH during Preparation Example 13 treatment was inhibited in a concentration-dependent manner.

[Example 1: Flexible lotion]

ingredient Content (% by weight) Manufacturing Example 13 2 glycerin 5.0 1,3-butylene glycol 3.0 REG 1500 1.0 Allantoin 0.1 DL-panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Sodium hyaluronate 5.0 ethanol 10.0 Octyldodeces-16 0.2 Polysorbate 20 0.2 Uniside-U 13 0.01 incense a very small amount Distilled water Balance Sum 100

[Example 2: Nourishing Cream]

ingredient Content (% by weight) Manufacturing Example 13 2 Glycerin monostearate 1.5 Cetearyl alcohol 1.5 Stearic acid 1.0 Allan Polysorbate 60 1.5 Sorbitastearate 0.6 Isostearyl isosterate 5.0 Squalane 5.0 Mineral oil 35.0 Dimethicone 0.5 Hydroxyethyl cellulose 0.12 glycerin 6.0 Triethanolamine 0.7 Hyang Uniside-U13 0.02 incense a very small amount Distilled water Balance Sum 100

[Example 3: Massage cream]

ingredient Content (% by weight) Manufacturing Example 13 2 Propylene glycol 6 glycerin 4.0 Triethanolamine 0.5 Beeswax 2.0 Tocopheryl Acetate 0.1 Polysorbate 60 3.0 Sorbitansquioleate 2.5 Cetearyl alcohol 2.0 Floating paraffin 30.0 Carboxyvinyl polymer 0.5 incense a very small amount Distilled water Balance Sum 100

[Example 4: Pack]

ingredient Content (% by weight) Manufacturing Example 13 2 glycerin 7.0 1,3-butylene glycol 3.0 Squalene 3.0 Dimethicone 3.0 Sodium hyaluronate 0.1 Glycine 2.0 Polyacrylamide 5.0 Antioxidant 4 0.3 Triethanolamine 0.1 EDTA 0.1 antiseptic a very small amount Distilled water Balance Sum 100

Claims (8)

Including a mixed extract of a mixture of Saururus chinensis leaves, Grape seed, and Portulaca oleracea , and fermented product fermented by inoculating yeast in rice,
The mixture,
A cosmetic composition for skin whitening, characterized in that it is mixed with 2 to 3 parts by weight of grape seeds and 6 to 7 parts by weight of machi-hyun based on 1 part by weight of the leaves of Sambaekcho.
delete The method of claim 1,
The mixed extract and fermented product,
A cosmetic composition for skin whitening, characterized in that the fermented product is mixed in an amount of 5 to 30 parts by weight of the fermented product to 70 to 95 parts by weight of the mixed extract on a dry weight basis.
Saururus chinensis leaf extract, grape seed extract, portulaca oleracea extract, and fermented product fermented by inoculating yeast on rice,
The Sambaekcho leaf extract, grape seed extract, and machi-hyeon extract,
Based on dry weight, with respect to 1 part by weight of Sambaekcho leaf extract, 2 to 3 parts by weight of grape seed extract, 6 to 7 parts by weight of machihyeon extract cosmetic composition for skin whitening, characterized in that mixed.
delete The method of claim 4,
The Sambaekcho leaf extract, grape seed extract, portulaca oleracea extract and fermented product,
The cosmetic composition for skin whitening, characterized in that mixed in a ratio of 5 to 30 parts by weight of the fermented product to 70 to 95 parts by weight of the sum of the three baekcho leaf extract, grape seed extract, and machi-hyun extract.
The method of claim 1 or 4,
The extract,
A cosmetic composition for skin whitening, characterized in that it is extracted using an aqueous ethanol solution as an extraction solvent.
The method of claim 1 or 4,
The fermented product,
A cosmetic composition for skin whitening, characterized in that it is a filtrate obtained through filtration after fermentation.

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