KR20220099259A - Skin whitening composition comprising Safflower seed oil hydrolyzate and Saururus chinensis extract - Google Patents

Abstract

The present invention relates to a cosmetic composition containing, as an active ingredient, a mixture obtained by mixing a hydrolysate of Carthamus tinctorius L. seed oil and an extract of Saururus chinensis (Lour.) Baill. in a specific weight part proportion. The cosmetic composition has a synergistic effect due to the complex action of two mechanisms, such as decreased levels of tyrosinase proteins and inhibition of tyrosinase production caused by the inhibition of microphthalmia-associated transcription factor (MITF) protein production due to decreased levels of MITF mRNA expression, thereby exhibiting an excellent skin whitening effect.

Description

Skin whitening composition comprising Safflower seed oil hydrolyzate and Saururus chinensis extract as active ingredients

The present invention relates to a composition for skin whitening containing a mixture of safflower seed oil hydrolyzate and safflower seed extract as an active ingredient, and more particularly, to a composition having excellent skin whitening effect by including safflower seed oil hydrolyzate and safflower seed oil extract in a specific weight part ratio.

A person's skin color is largely determined by the amount of melanin, hemoglobin, and carotene, and melanin plays the most important role among them. Melanin is a pigment present in the epidermal layer of the skin, and is produced in melanocytes present in the epidermal layer of the skin. It plays a role in protecting the skin from harmful factors. However, when melanin is excessively generated by excessive exposure to ultraviolet rays or various environmental factors, there is a problem in that it causes pigmentation such as freckles, age spots, and blemishes and skin troubles.

Looking at the synthesis process of melanin in detail, an enzyme called tyrosinase present in the endoplasmic reticulum called melanosome in melanocytes uses tyrosine as a substrate to produce dopaquinone (DOPAauinone) and , dopaquinone undergoes an auto-oxidation reaction to become dopachrome (DOPAchirome). Again, dopachrome produces melanin by an enzymatic reaction. The generated melanin is transferred to keratinocytes through melanosomes, and from there, it is lost along with the dead skin cells through the keratinization process for about 28 days.

Among them, tyrosinase is an enzyme that determines the production of melanin in the initial stage of melanin synthesis and is expressed by a transcription factor called Microphtalmia-associated transcription factor (MITF). MITF plays an important role in the expression of enzymes TRP1 and TRP2 required for melanin formation as well as tyrosinase. The enzymes can convert L-tyrosine into two types of melanin, such as eumelanin and pheomelanin, in the organelle of melanocytes called 'melanosome', and the synthesized melanin is produced by surrounding keratinocytes. to determine the skin color or protect the skin from UV rays.

Conventionally, a substance having inhibitory activity against tyrosinase, such as hydroquinon, ascorbic acid, kojic acid, and glutathione, is mixed with cosmetics such as ointments and essences to whiten the skin. The effect was obtained or the skin hyperpigmentation such as freckles and freckles was improved.

However, hydroquinone exhibits a predetermined whitening effect, but has severe skin irritation, so the blending amount must be limited to a very small amount. In addition, thiol-based compounds such as glutathione and cysteine not only have a characteristic unpleasant odor, but also have a problem in transdermal absorption, and their glycosides and derivatives are also highly polar, making it difficult to use as a cosmetic formulation ingredient. On the other hand, the placenta extract has no skin irritation, but has a problem of insufficient whitening effect, and has recently been banned due to the risk of inducing mad cow disease.

The present inventors have devised to solve the problems of the prior art, and when it contains as an active ingredient a mixture obtained by mixing a hydrolyzate of safflower seed oil obtained from nature and an extract by weight in a specific weight part ratio, tyrosinase (Tyrosinase) protein It has been confirmed through experiments that an excellent skin whitening effect can be obtained through a synergistic action by a complex action of two mechanisms, such as inhibiting the level of MITF-M protein and inhibiting the production of tyrosinase. invention was completed.

Accordingly, it is an object of the present invention to provide a cosmetic composition for skin whitening, which contains a mixture of safflower seed oil hydrolyzate and extract of ginseng oil as an active ingredient.

In addition, the technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned are clearly to those of ordinary skill in the art to which the present invention belongs from the description below. can be understood

In the present specification, it provides a cosmetic composition for skin whitening, which contains a mixture of safflower seed oil hydrolyzate and extract of safflower oil as an active ingredient.

The mixture may be a mixture of a hydrolyzate of safflower seed oil and an extract of safflower seed oil in a ratio of 2: 0.5 to 1.5 parts by weight.

The safflower seed oil hydrolyzate is hydrolyzed by treating safflower seed oil with at least one alkali metal hydroxide selected from the group consisting of sodium hydroxide (NaOH), calcium hydroxide (Ca(OH) ₂ ), lithium hydroxide (LiOH) and potassium hydroxide (KOH). It may be decomposed or hydrolyzed by adding an ester hydrolase to safflower seed oil.

The extract of trifolieae is one or more solvents selected from the group consisting of water, ethanol, methanol, propanol, butanol, acetone, ethyl acetate, hexane, butylene glycol, propylene glycol, hydrous butylene glycol, hydrous propylene glycol, and hydrous glycerin. may have been extracted.

The mixture may be included in an amount of 0.001 to 50% by weight based on the total weight of the composition.

The composition may have one or more formulations selected from the group consisting of softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray and powder.

The cosmetic composition for skin whitening prepared according to the present invention has a complex action of two mechanisms, such as inhibition of tyrosinase protein level and inhibition of MITF protein production by inhibition of MITF mRNA expression. It shows excellent skin whitening effect as a synergistic effect by

1 is a result of measuring the content of linoleic acid in a hydrolyzate of safflower seed oil prepared according to an embodiment of the present invention.
Figure 2 shows the results of performing a stability test according to the accelerated experiment of the hydrolyzate of safflower seed oil and the extract mixture of safflower seed oil prepared according to an embodiment of the present invention.
Figure 3 shows the cell viability test results of safflower seed oil hydrolyzate and extract (30% ethanol extract, 70% ethanol extract) prepared according to an embodiment of the present invention.
Figure 4 shows the results of measuring the melanin biosynthesis inhibitory effect by UVB irradiation with respect to an embodiment of the present invention.
Figure 5 shows the results of measuring the melanin biosynthesis inhibitory effect by α-MSH stimulation with respect to an embodiment of the present invention.
6 is a measurement of IC ₅₀ concentration of a sample for melanin biosynthesis generated by UV stimulation in Pam212 and B16F0 co-culture conditions for an embodiment of the present invention.
7 is a measurement of the IC ₅₀ concentration of the sample for melanin biosynthesis generated by α-MSH stimulation in the B16F0 cell line with respect to an embodiment of the present invention.
Figure 8 shows the cell viability test results of the hydrolyzate of safflower seed oil and the extract mixture of safflower seed oil according to an embodiment and a comparative example of the present invention.
Figure 9 is a result of measuring the effect of the sample on the melanin biosynthesis produced by UV stimulation in Pam212 and B16F0 co-culture conditions of a mixture of safflower seed oil hydrolyzate and trifolium extract according to an embodiment and a comparative example of the present invention.
10 is a result of measuring the effect of the sample on the melanin biosynthesis produced by α-MSH stimulation in the B16F0 cell line of a mixture of safflower seed oil hydrolyzate and Tribaekcho extract according to an embodiment and a comparative example of the present invention.
11 is a result of measuring the protein level inhibitory effect of the melanin synthesis marker MITF-M of the hydrolyzate of safflower seed oil and the extract of trifolieae according to an embodiment of the present invention.
Figure 12 is a result of measuring the mRNA and protein level inhibitory effect of the melanin synthesis marker tyrosinase (Tyrosinase) of the hydrolyzate of safflower seed oil and the extract mixture of safflower seed oil according to an embodiment of the present invention.
Figure 13 schematically shows the mechanism of action of a mixture of safflower seed oil hydrolyzate and safflower oil extract according to an embodiment of the present invention.

Hereinafter, with reference to the accompanying drawings, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement them. However, the present invention may be implemented in various different forms and is not limited to the embodiments disclosed below. In addition, in order to clearly disclose the present invention in the drawings, parts irrelevant to the present invention are omitted, and the same or similar symbols in the drawings indicate the same or similar components.

Specifically, the cosmetic composition for skin whitening according to an embodiment of the present invention may contain a mixture of a hydrolyzate of safflower seed oil and an extract of safflower oil as an active ingredient.

Safflower (Carthamus tinctorious L.) is an annual plant belonging to the Asteraceae family, and its origin is in the mountainous regions of Afghanistan or Ethiopia, and is cultivated in China and Tibet. The medicinal parts of safflower include flowers and seeds, which inhibit platelet coagulation and delay bleeding time, as well as lower plasma cholesterol and triglycerides.

Safflower seeds contain phenolic compounds such as serotonin, lignans and flavonoids, which have strong antioxidant and anti-hyperlipidemic effects. By doing so, it is possible to prevent osteoporosis in menopausal women and to form new bone in case of bone loss.

In particular, safflower seed oil contains a large amount of unsaturated fatty acids including linoleic acid and tocopherol, so it is used as a new material with antioxidant, antithrombotic and antihypertensive properties.

Meanwhile, in the present invention, a hydrolyzate of safflower seed oil is used, and the hydrolyzate of safflower oil according to an embodiment of the present invention is sodium hydroxide (NaOH), calcium hydroxide (Ca(OH) ₂ ), lithium hydroxide (LiOH) and It may be hydrolyzed by treatment with one or more alkali metal hydroxides selected from the group consisting of potassium hydroxide (KOH), or hydrolyzed by adding an ester hydrolase to safflower seed oil, and the ester hydrolase is lipase ( Lipase), for example, lipozyme or lipopna may be used.

For example, the hydrolyzate of safflower seed oil may be obtained by adding safflower seed oil to an aqueous sodium hydroxide solution and performing a hydrolysis reaction for about 50 to 70 minutes while stirring in a constant temperature water bath within a temperature range of about 80 to 100 ° C.

Saururus chinensis Baill (三白草; Saururus chinensis Baill ) is a perennial herb in the family Trifoliumaceae, distributed in Korea, China, and Japan, and is a plant that grows wild in wetlands and beaches. The forage contains essential oils such as quercetin, quercitrin, isoquercitrin, rutin, and water-soluble tannin. It has antibacterial, anti-mutagenic, and carcinogenic properties, inhibits the growth of cancer cells, lowers blood pressure, and strengthens capillaries, and quercitrin is known to have anti-inflammatory, analgesic and antioxidant effects. In addition, three hundred seconds are used for beriberi, jaundice, ointment, oyster mushroom, swelling, hydrocephalus, redness, acute and chronic urethritis, prostatitis, cystitis, gonorrhea, dysentery, aftereffects and muscle pain caused by bruises, and inflammation of the skeleton and bone marrow. It is known to be effective in treating pain caused by

On the other hand, the extract according to an embodiment of the present invention is composed of water, ethanol, methanol, propanol, butanol, acetone, ethyl acetate, hexane, butylene glycol, propylene glycol, hydrous butylene glycol, hydrous propylene glycol and hydrous glycerin. It may be extracted with one or more solvents selected from the group, specifically, obtained by extraction with 30% ethanol aqueous solution at room temperature to 70% ethanol aqueous solution for one week, and more specifically, one week with 30% ethanol aqueous solution at room temperature It may be obtained by extraction.

On the other hand, the cosmetic composition for skin whitening according to an embodiment of the present invention contains a mixture of hydrolyzate safflower seed oil and extract of safflower seed oil as an active ingredient, wherein the mixture contains hydrolyzate of safflower oil and extract of safflower seed oil: 0.5 to 1.5 weight It may be mixed by booby ratio. On the other hand, when mixing the hydrolyzate of safflower seed oil and the extract of safflower seed oil within the weight part ratio range, the mechanism of inhibiting the tyrosinase protein level of the hydrolyzate of safflower oil and the inhibition of MITF protein production according to the inhibition of MITF mRNA expression of the extract It is possible to optimize the skin whitening effect due to the synergistic action of the tyrosinase production inhibitory mechanism.

In addition, in the cosmetic composition for skin whitening according to an embodiment of the present invention, the mixture may be included in an amount of 0.001 to 50% by weight based on the total weight of the composition.

On the other hand, the active ingredient contained in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions, in addition to a mixture of safflower seed oil hydrolyzate and safflower extract, for example, antioxidants, stabilizers, solubilizers, vitamins , and conventional containing agents such as pigments and fragrances.

In addition, the cosmetic composition containing the mixture of the hydrolyzate of safflower seed oil and the extract of safflower seed oil of the present invention as an active ingredient can be prepared in any conventionally prepared formulation, for example, a solution, a suspension, an emulsion, a paste, a gel, It may have one or more formulations selected from the group consisting of cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray.

Example

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited only to the following examples and experimental examples, and includes modifications of technical ideas equivalent thereto.

production example

Manufacture of safflower seed oil hydrolyzate (hereinafter referred to as SFH)

After putting 20 g of safflower seed oil in a 500 ml beaker, an aqueous solution in which 20 g of NaOH was dissolved in 36 g of purified water was added. The hydrolysis reaction was carried out for 60 minutes while stirring the mixed solution in a constant temperature water bath maintained at 90°C. During the hydrolysis reaction, an aqueous solution in which 100 g of NaCl was dissolved in 300 g of purified water was cooled in a refrigerator, and then the solution in which the hydrolysis reaction was completed was slowly added to the brine solution. The resulting solid was filtered under reduced pressure and dried again under reduced pressure at room temperature to prepare 16.4 g of a hydrolyzate of safflower seed oil.

Preparation of extract (hereinafter referred to as "SC")

Three hundred seconds ( Saururus chinensis ) was prepared, and extracted for a week at room temperature using each of an ethanol 30% aqueous solution and an ethanol 70% aqueous solution. After completion of the extraction, the extracted solution was obtained after filtration using a filter paper, and concentrated under reduced pressure and dried to obtain a target extract of Trichomaceae (obtained as a 30% aqueous solution of ethanol (SC30) 4.52 g, as a 70% aqueous solution of ethanol) (SC70) 3.95 g) were obtained, respectively.

Example 1: Preparation of a mixture of safflower seed oil hydrolyzate (SFH) and trifolieae extract (SC)

After completely dissolving 2.34 g of the prepared trifolieae extract (SC30) in 110 g of a 30% aqueous 1,3-butylene glycol solution, 4.68 g of the prepared safflower seed oil hydrolyzate was added thereto and dissolved while stirring at 50°C. After filtration using a 1.0 μm membrane filter and a 0.45 μm membrane filter sequentially, 100 g of a mixture of safflower seed oil hydrolyzate and ginseng extract from which insoluble matter and microorganisms were completely removed was prepared.

[Experiment 1: Measurement of Linoleic Acid Content]

In order to measure the content of linoleic acid contained in the hydrolyzate of safflower seed oil (SFH) prepared according to Preparation Example, analysis was performed under the following conditions, and the standard material was supelco 37 component FAME Mix (purity 100%). The equipment used was a gas chromatograph (GC) Agilent 7890A, and the analysis conditions and results were as follows.

Detector FID Column SPTM-2560 (100m x 0.25mm x 0.20μm) Injection Temp. 225℃ Injection Volume 1.0μl Detector Temp. 285℃ Oven Temp. 100°C (4min.), 208°C (3°C/min, 5min), 244°C (2°C/min, 15min) Carrier Gas He Column Flow 0.75 mL/min. Split Ratio 200:1

sample safflower seed oil Safflower seed oil NaOH hydrolyzate Safflower Seed Oil Lipase Hydrolyzate Linoleic acid content (%) 1.3 59.88 57.24

Referring to Table 2 and the results of FIG. 1, in the case of the hydrolyzate of safflower seed oil prepared according to the preparation example of the present invention, the linoleic acid content was about 59.88%, and it was confirmed that it exhibited a significantly higher value than other oils. there was.

[Experiment 2: Stability test by accelerated experiment]

A mixture of safflower seed oil hydrolyzate (SFH) and trifolieae extract (SC30) obtained according to Example 1 in an incubator at room temperature (25 ° C) and 45 ° C. at 0 month, 1 month, and 2 month intervals, respectively. Changes in color and pH of the solution As a result of measuring with the naked eye and a pH meter, no color change was observed by visual observation, and it was also measured that there was almost no change in pH with time (see FIG. 2).

[Experiment 3: Cell viability measurement experiment]

cell culture

Pam212 cells are mouse keratinocytes, and B16F0 cells are mouse melanoma cell lines, which are adhered to the culture dish and proliferate. 6 × 10 ⁵ Pam212 cells and 3 × 10 ⁵ B16F0 cells were diluted with DMEM medium containing 10 ml of 10% FBS in a 100 mm culture dish and subcultured every 48 hours before use.

Cell viability measurement

The MTT method was used to measure the viability of cells by a colorimetric method using absorbance. Pam212 cells at 5 × 10 ³ cells/well in a 96-well culture plate and B16F0 cells at 2.5 × 10 ³ cells/well in a 96-well culture plate. After 24 hours, remove the medium from the 96-well culture plate. Fresh DMEM 10% FBS medium and serially diluted samples were treated respectively. After 72 hours, the medium of the 96-well culture plate was removed and the MTT reagent was reacted with the medium at a concentration of 500 ug/ml for 1 hour. After removing the MTT solution, 200 ul of DMSO was added to dissolve the formazan crystal, and the absorbance was measured at a wavelength of 590 nm.

Specifically, in order to examine the cytotoxicity of each sample against the mouse melanoma B16F0 cell line and the mouse keratinocyte cell line Pam212, the yellow water-soluble substrate tetrazolium was converted to a blue-violet insoluble formazan by mitochondrial dehydrogenase action in living cells. The maximum concentration that does not exhibit cytotoxicity was confirmed by using the MTT method using the function of reducing to . In order to confirm the presence or absence of toxicity to B16F0 and Pam212 cells, each sample was incubated for 72 hours, and then cell viability was measured using the MTT method. After treatment with each concentration of SC30, SC70 and SFH according to Preparation Example, MTT cell viability was measured in B16F0 and Pam212 cells. As a result, when the survival rate of the control group was set to 100%, the concentration without toxicity was confirmed as the maximum concentration that can be treated. As a result, as shown in FIG. 3, it was confirmed that 100 ug/ml in the case of SC30, 10 ug/ml in the case of SC70, and 100 ug/ml in the case of SFH can be set as the maximum concentration.

[Experiment 4: Melanin amount measurement experiment]

melanin assay by UVB irradiation (Co-cullture system)

After growing B16F0 3.5 × 10 ⁴ cells/well in a 6-well plate and Pam212 1 × 10 ⁴ cells/well in a 6-well plate insert for 24 hours, the medium was removed and enough PBS was added to lightly cover the cells. Using a UV-crosslinker, it was irradiated with an intensity of 2 mJ/cm ² that does not affect the cell growth rate. After removing the PBS, the medium containing the sample was added and cultured for 72 hours, and melanin was measured by measuring the absorbance at 405 nm.

Specifically, to measure the effect of the sample on the melanin biosynthesis generated by UV stimulation in Pam212 and B16F0 co-culture conditions, melanin biosynthesis was induced by UV stimulation in keratinocytes and melanocytes co-culture conditions similar to skin conditions. After SC30 and SC70 extract was confirmed the effect on melanin biosynthesis. After stimulation with UV 2mJ/cm ² in B16F0 and Pam212 co-culture conditions, each sample was treated at a concentration that was not toxic to cells for 72 hours, and the amount of melanin synthesized and released into the medium at an absorbance of 405 nm was measured. As a result of the experiment, SC30 and SC70 inhibited melanin biosynthesis (see FIG. 4).

melanin assay by α-MSH stimulation

After dispensing at 2.5 × 10 ³ cells/well of B16F0 in a 96-well plate, the cells are grown for 24 hours. After adding the medium containing 100 nM α-MSH and the sample, it was cultured for 72 hours, and the amount of melanin was calculated by measuring the absorbance at 405 nm.

Specifically, in order to measure the effect of the sample on the biosynthesis of melanin produced by α-MSH stimulation in the B16F0 cell line, each concentration of the sample was treated in B16F0 cells for 72 hours, and the absorbance was synthesized in the cells at 405 nm and converted into a medium. The amount of released melanin was measured. As a result of the experiment, it was confirmed that the samples (SC30, SC70, SFH) prepared according to Preparation Example inhibit melanin biosynthesis by α-MSH stimulation (see FIG. 5 ).

Analyzing the above experimental results, it was confirmed that melanin production was increased by UV stimulation in the co-culture system or stimulation by α-MSH in B16F0, and SC30, SC70 and SFH according to the preparation example reduced it.

Sample IC for melanin biosynthesis produced by UV stimulation in Pam212 and B16F0 co-culture conditions ₅₀ concentration measurement

By selecting SC30, SC70, and SFH, which have excellent effect of inhibiting melanin production by UV stimulation, the sample concentration (IC ₅₀ ) when the melanin synthesis inhibition rate of the sample was 50% was confirmed. After stimulation with UV 2 mJ/cm ² in B16F0 and Pam212 co-culture conditions, each sample was treated with various concentrations that were not toxic to cells for 72 hours, and melanin synthesized in cells and released into the medium at absorbance 405 nm was measured. As a result, the 50% inhibition of melanin concentration (IC ₅₀ ) by UV stimulation was confirmed to be 5.9 ug/ml for SC30, 0.7 ug/ml for SC70, and 22 ug/ml for SFH (see FIG. 6).

Sample IC for melanin biosynthesis produced by α-MSH stimulation in B16F0 cell line ₅₀ density

By selecting SC30 and SC70 having excellent melanin inhibitory effect by α-MSH stimulation, the sample concentration (IC ₅₀ ) when the melanin synthesis inhibition rate of the sample was 50% was confirmed. The B16F0 cell line was stimulated with α-MSH, samples of various concentrations were treated for 72 hours, and the amount of melanin synthesized and released into the medium at an absorbance of 405 nm was measured. As a result, it was confirmed that the IC ₅₀ , which is the 50% inhibitory concentration of melanin by α-MSH stimulation, was 22 ug/ml in the case of SC30 and 2 ug/ml in the case of SC70 (see FIG. 7).

Comparative Examples 1-1, 1-2 and 1-3

A mixture was prepared in the same manner as in Example 1, except that SFH and SC70 were mixed in a ratio by weight of 2: 0.5 (Comparative Example 1-1), and mixed in a ratio by weight of 2: 0.1 (Comparative Example 1-2) ), SFH and SC30 were mixed in a weight part ratio of 2: 0.5 (Comparative Example 1-3).

[Experiment 5: Cell viability measurement experiment]

Cell viability measurement

The MTT method was used to measure the viability of cells by a colorimetric method using absorbance. After dispensing Pam212 cells at 5 × 10 ³ cells/well in a 96-well culture plate and B16F0 cells at 2.5 × 10 ³ cells/well in a 96-well culture plate, remove the medium from the 96-well culture plate after 24 hours. Fresh DMEM 10% FBS medium and serially diluted samples were treated respectively. After 72 hours, the medium from the 96-well culture plate was removed, and MTT reagent was reacted with the medium at a concentration of 500 ug/ml for 1 hour. After removing the MTT solution, 200 ul of DMSO was added to dissolve the formazan crystal, and the absorbance was measured at a wavelength of 590 nm.

Specifically, in order to examine the cytotoxicity of each sample against the mouse melanoma B16F0 cell line and the mouse keratinocyte cell line Pam212, the yellow water-soluble substrate tetrazolium was converted to a blue-violet insoluble formazan by mitochondrial dehydrogenase action in living cells. The highest concentration that does not show cytotoxicity was confirmed using the MTT method using the function of reducing In order to confirm the presence or absence of toxicity to B16F0 and Pam212 cells, each sample was incubated for 72 hours, and then cell viability was measured using the MTT method. Cell viability was measured using the MTT method after 72 hours of cell culture in the mixture of Example 1 and Comparative Examples 1-1 to 1-3, and as a result, when the viability of the control group was set to 100%, treatment without toxicity The maximum concentration possible was confirmed (see FIG. 8).

[Experiment 6: Measurement of the effect on melanin biosynthesis]

Effects of samples on melanin biosynthesis produced by UV stimulation in Pam212 and B16F0 co-culture conditions of the mixtures of Examples and Comparative Examples

Based on the mixture of Example 1 and Comparative Examples 1-1 to 1-3, after stimulation with UV 2 mJ/cm ² in B16F0 and Pam212 co-culture conditions, the effect of inhibiting melanin synthesis by the mixture was confirmed. As a result, the IC ₅₀ , which is the 50% inhibitory concentration of melanin by UV stimulation, showed SFH 5ug/ml, SC70 1.3 ug/ml, and ratio 2: 0.1 in the case of a mixture (Comparative Example 1-1) of SFH and SC70 ratio of 2: 0.1 In the case of a phosphorus mixture (Comparative Example 1-2), 11.7 ug/ml of SFH and 0.6 ug/ml of SC70 were shown. In the case of a mixture (Example 1) having a SFH and SC30 ratio of 2: 1, IC ₅₀ is SFH 6.5 ug/ml, SC70 0.6 ug/ml, and in the case of a mixture (Comparative Example 1-3) having a ratio of 2: 0.5, IC ₅₀ showed SFH 12.3 ug/ml and SC70 3.1 ug/ml (see FIG. 9).

Effect of sample on melanin biosynthesis produced by α-MSH stimulation in B16F0 cell line of the mixture of Examples and Comparative Examples

Based on the mixture of Example 1 and Comparative Examples 1-1 to 1-3, it was confirmed how it affects melanin biosynthesis produced in α-MSH in the B16F0 cell line. Samples were treated at each concentration in B16F0 cells for 72 hours, and the amount of melanin synthesized in the cells and released into the medium at an absorbance of 405 nm was measured. As a result of the experiment, the IC ₅₀ , which is the 50% inhibitory concentration of melanin by α-MSH stimulation, showed 5.9 ug/ml of SFH and 1.5 ug/ml of SC70 in the case of the mixture of Comparative Example 1-1, and SFH in the case of the mixture of Comparative Example 1-2. 19.3 ug/ml, and 1 ug/ml SC70. For the mixture of Example 1, IC ₅₀ SFH 18.5 ug/ml, SC70 9.3 ug/ml, and for the mixture of Comparative Example 1-3, IC ₅₀ SFH 19.6 ug/ml, SC70 4.9 ug/ml ( see Fig. 10).

Referring to the above experimental results, it can be confirmed that the melanin IC ₅₀ value is lower when using a mixture by a combination of SFH and SC70 and a combination of SFH and SC30 compared to when SFH, SC70, and SC30 are used alone, Accordingly, it can be confirmed that the SC70/SFH or SC30/SFH mixture exhibits a better whitening effect than the single agent.

In particular, in the case of a mixture containing SFH at a concentration of 30 ug/ml and SC30 at a concentration of 15 ug/ml (mixed in a ratio by weight of 2: 1), as in the mixture according to Example 1, within a range that does not exhibit cytotoxicity, UV Alternatively, it could be confirmed that melanin production by α-MSH stimulation could be inhibited by more than 90% (see Table 3 below).

[Experiment 7: Western Blot]

Confirmation of protein level of melanin synthesis marker MITF-M

To confirm the protein level of the melanin synthesis marker MITF-M of the mixture according to Example 1, quantitative analysis was performed through Western blot. In the case of SFH alone treatment, the amount of MITF-M protein increased by α-MSH stimulation could not be suppressed, but it was confirmed that the amount of MITF-M protein was suppressed when SC30 alone or the mixture of Example 1 was treated (Fig. 11).

Confirmation of mRNA and protein levels of the melanin synthesis marker Tyrosinase

Whether the mixture of Example 1 affects mRNA and protein levels of Tyrosinase, the most important protein for melanin biosynthesis, was quantitatively analyzed through RT-PCR and Western blot. As a result, when SFH alone was treated, the amount of tyrosinase protein increased by αMSH stimulation was suppressed, but the mRNA level was not suppressed. However, SC30 alone and the mixture of Example 1 both inhibited both Tyrosinase mRNA and protein levels. These results indicate that SFH alone is involved in tyrosinase protein inhibition, and SC30 inhibits melanin synthesis by inhibiting MITF-M protein production by inhibiting MITF mRNA expression and by inhibiting tyrosinase production. In particular, in the case of the mixture of Example 1, two mechanisms such as a mechanism for directly inhibiting the tyrosinase protein level and a mechanism for inhibiting the production of tyrosinase by inhibition of MITF protein production according to the inhibition of MITF mRNA expression It shows an excellent skin whitening effect as a synergistic effect by a complex action (see Fig. 12).

In summary, as can be seen from the above experiment, when UV stimulates epidermal keratinocytes, keratinocytes secrete α-MSH, ET-1, and SCF, which are paracrine factors that can stimulate the melanin synthesis signaling system. . These proteins bind to respective receptors in melanocytes to increase MITF mRNA expression, and the increased MITF-M binds to a promoter of tyrosinase production and increases tyrosinase protein production, thereby promoting skin melanin synthesis. On the other hand, when SFH is applied alone, it directly inhibits the tyrosinase protein level, and when SC30 is applied alone, both MITF-M protein and Tyrosinase mRNA are inhibited. After all, the mixture of Example 1 suppresses the Tyrosinase protein level due to the combined effect of the two agents, and inhibits melanin synthesis by inhibiting both the mRNA of Tyrosinase and the amount of MITF-M protein, which is an important transcription factor for Tyrosinase production. It can be seen that an excellent whitening effect is exhibited (see FIG. 13).

[Experiment 8: Clinical trial for skin brightness improvement effect]

For 12 healthy adult women (average age 51.25), the cream formulation containing the mixture of Example 1 was applied to the face area, and the skin brightness ( L*) and the average of the individual increase rate (%) for the L* value were respectively measured and shown in Tables 4 and 5 below.

2 weeks after application 4 weeks after application Personal increase rate of L* value (%) 0.099 0.295

Looking at the results in Table 4, the L* value showed an increase rate of 0.099% and 0.285%, respectively, after 2 weeks and 4 weeks after application compared to before product application. On the other hand, as a result of analyzing the proportion of study subjects with increased L* value, it was confirmed that 75.000% and 100.000% of study subjects showed an increase in L* value after 2 weeks of application and 4 weeks after application (Table below) 5).

2 weeks after application 4 weeks after application Proportion of study subjects with increased L* value (%) 75.000 100.000

As described above, in the case of a mixture (SS-Complex) containing safflower seed oil hydrolyzate (SFH) and trifolieae extract (SC30) in a specific weight part ratio according to the present invention, the mechanism of inhibiting the tyrosinase protein level and MITF protein production and It can be confirmed that the mechanism of inhibiting tyrosinase mRNA can be simultaneously performed, effectively inhibiting melanin production by two pathways, thereby exhibiting a strong synergistic effect related to skin whitening.

In the foregoing, specific embodiments of the present invention have been described and illustrated, but it is common knowledge in the art that the present invention is not limited to the described embodiments, and that various modifications and variations can be made without departing from the spirit and scope of the present invention. It is self-evident to those who have Accordingly, such modifications or variations should not be individually understood from the technical spirit or point of view of the present invention, and modified embodiments should be said to belong to the claims of the present invention.

Claims (6)

A cosmetic composition for skin whitening, comprising a mixture of safflower seed oil hydrolyzate and safflower extract as an active ingredient. The method of claim 1,
The mixture is a mixture of safflower seed oil hydrolyzate and safflower seed extract in a ratio of 2: 0.5 to 1.5 parts by weight, a cosmetic composition for skin whitening. The method of claim 1,
The safflower seed oil hydrolyzate is hydrolyzed by treating safflower seed oil with at least one alkali metal hydroxide selected from the group consisting of sodium hydroxide (NaOH), calcium hydroxide (Ca(OH) ₂ ), lithium hydroxide (LiOH) and potassium hydroxide (KOH). decomposed or
A cosmetic composition for skin whitening, which is hydrolyzed by adding an ester hydrolytic enzyme to safflower seed oil. The method of claim 1,
The trifolium extract is water, ethanol, methanol, propanol, butanol, acetone, ethyl acetate, hexane, butylene glycol, propylene glycol, water-containing butylene glycol, water-containing propylene glycol, and water-containing one or more solvents selected from the group consisting of glycerin. Extracted, cosmetic composition for skin whitening. The method of claim 1,
The mixture is included in an amount of 0.001 to 50% by weight based on the total weight of the composition, a cosmetic composition for skin whitening. The method of claim 1,
The composition is one or more formulations selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, and sprays. Having, a cosmetic composition for skin whitening.

Images