KR20130134976A - A cosmetic composition for whitening skin containing cynanchum atratum extracts - Google Patents

Abstract

The present invention relates to a cosmetic composition for whitening skin containing 4-Hydroxyacetophenone which is a compound purified from Cynanchum atratum extraact. According to the present invention, a naturally derived composition for whitening skin which protects the skin from ultraviolet rays and inhibits tyrosinase activity to handle the melanzation of the skin due to melanin and handles various oxidation factors causing skin aging and restores skin repairing effect reduced by external stimuli or stress, and facilitates absorrption into the skin is provided.

Description

A Cosmetic Composition for Whitening Skin Containing Cynanchum atratum Extracts}

The present invention relates to a skin whitening cosmetic composition containing a white rice extract, and more particularly, to protect the skin from ultraviolet rays containing 4-hydroxyacetophenone, a compound purified from the white rice extract, and to skin blackening due to melanin. In order to cope, it inhibits tyrosinase activity, copes with various oxidative factors that cause skin aging, and smoothes skin repair function that is degraded by damage factors such as external stimulus and various stresses, and it is easy to absorb skin. It relates to a derived skin lightening composition.

In modern society, the living conditions have been greatly improved due to industrialization. However, since it is frequently exposed to various harmful factors such as destruction of the ozone layer caused by air pollution, water pollution caused by waste water, various stresses, etc., Cosmetics applying high plant ingredients are attracting attention as a new concept that will lead the world cosmetics development in the 21st century.

In addition, trends in the cosmetics market are also reflected in 'Eco' and 'Cosmeceutical', which have scientific elements in 'Natural', 'Anti-aging' and 'Healthy and Bright skin' The flow is changing. Therefore, functional products developed with natural plant materials that are 'naturalism and environment friendly' that are growing steadily every year are expected to continue to grow.

As the standard of living of modern people has improved, consumption patterns have been changed in consideration of economic efficiency, convenience, and practicality in consumption propensity. Therefore, in selecting cosmetics, it is necessary to get rid of unconditional preference for famous brands and expensive cosmetics, The trend is to change the perception of cosmetics, which is not burdensome, and consumers are paying more attention to multifunctional cosmetics with more than two functions than single functional products. This multifunctionality is tailored to the needs of consumers who want to save time with simple treatment in busy modern societies and to have a certain and powerful effect.

However, as far as the products developed so far focus on the rapid trend change of the cosmetics market, it is very rare that products considering consumers' various skin types and living environment are fully taken into consideration. Also, And a product capable of manifesting functionalities by coping with various mechanisms for various functions required for the skin has not yet been developed so that the related products are desperately required to be developed.

Therefore, in the present invention, by applying a technology different from the previously developed products to overcome the problems and drawbacks as described above, to develop cosmetics that are more functional and safer skin, products that can realize the efficacy to consumers To develop

Accordingly, the technical problem to be achieved by the present invention is to protect the skin from ultraviolet rays, to inhibit the tyrosinase activity in order to cope with the skin blackening caused by melanin, to cope with various oxidative factors that cause skin aging and external stimulation or various It is to provide a skin-derived composition naturally derived from the skin can be smoothly reduced by the damage factors such as stress and easy to absorb the skin.

In order to achieve the above object, the present invention provides a skin whitening cosmetic composition containing a white rice extract.

The cosmetic composition of the present invention is 4-hydroxyacetophenone (4-Hydroxyacetophenone), which is a compound of the following Chemical Formula 1, purified from the compound purified from the white rice extract.

[Formula 1]

In the present invention, in order to protect the skin from ultraviolet rays and cope with the skin blackening caused by melanin, the skin such as inhibition of tyrosinase activity, reduction of melanin produced and generation of melanin formation information transmitting substances, and promotion of rapid release of melanin present in the stratum corneum It is based on developing a product that can realize the efficacy through the design of a product that copes with various mechanisms for expressing the whitening effect.

In the present invention, the skin repair function can be smoothly performed due to coping with various oxidizing factors causing skin aging and damage factors such as external stimuli or various stresses, thereby maintaining the skin healthy, It is featured to make full use of control function.

The skin whitening cosmetic composition of the present invention is easy to absorb the skin, and utilizes formulating techniques and materials such as efficient encapsulation, liposomeization, nanotechnology, etc. to enhance the efficacy and persistence, thereby facilitating the absorption of the active ingredient, thereby improving the utilization rate to the skin. There is a characteristic that can be maximized.

Wherein the 4-hydroxyacetophenone is a solvent fraction of a white rice (CA) extract; Cynanchum , a plant belonging to the Asclepiadaceae atratum ), and then extracted with MeOH three times at room temperature for three days, followed by solvent fractionation with n- hexane, methylene chloride (MC), ethylacetate (EtOAc) and H ₂ O, as shown below. It is characterized by having the same ¹ H and ¹³ C-NMR data characteristics.

In the cosmetic composition for skin whitening of the present invention, the 4-hydroxyacetophenone is preferably included in an amount of 0.001-50% by weight based on the total weight of the cosmetic composition, and more preferably 0.01-20% by weight. And even more preferably 0.1-10% by weight. When the content of 4-hydroxyacetophenone is less than a certain level, no remarkable effect can be expected. When the content of the 4-hydroxyacetophenone is more than a certain level, safety of the cosmetic composition or preparation of the cosmetic composition is difficult.

The formulation of the cosmetic composition for skin whitening of the present invention as described above is not particularly limited and may be appropriately selected as desired. For example, the cosmetic composition of the present invention may be formulated into ointment for external use, softening longevity, nutritional lotion, nutritional cream, massage cream, essence, pack, emulsion, oil gel and the like. In addition to the 4-hydroxyacetophenone of the present invention, the external skin ointment may be prepared to contain 50 to 97% by weight of vaseline and 0.1 to 5% by weight of polyoxyethylene oleyl-ether phosphate, 1 to 10% by weight of polyhydric alcohols (propylene glycol, glycerin, etc.) and 0.05 to 2% by weight of a surfactant (polyethylene oleyl ether, polyoxyethylene hydrogenated castor oil, etc.) in addition to 4-hydroxyacetophenone have. In addition to the 4-hydroxyacetophenone of the present invention, the nutritional lotion and nutritional cream may contain 5 to 20% by weight of oils (squalane, petrolatum, octyldodecanol, etc.) and wax components (such as cetanol, stearyl alcohol, To 15% by weight, and the essence may be prepared by adding 5-30% by weight of polyhydric alcohols such as glycerin, propylene glycol, etc., in addition to the 4-hydroxyacetophenone of the present invention. In addition to the 4-hydroxyacetophenone of the present invention, the massage cream is prepared by containing 30 to 70% by weight of an oil such as liquid paraffin, petrolatum, isononylisononanoate and the like, and the pack contains, in addition to the 4-hydroxyacetophenone of the present invention, In addition to the 4-hydroxyacetophenone of the present invention, pigments such as kaolin, talc, zinc oxide and titanium dioxide may be added to a peel off pack or a general emulsifiable cosmetic containing 5 to 20% by weight of polyvinyl alcohol, And may be prepared in a wash-off pack containing wt.%.

In addition, the skin whitening cosmetic composition of the present invention contains ingredients commonly used in cosmetic compositions in addition to the 4-hydroxyacetophenone as an active ingredient, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances Conventional adjuvants and carriers may be included, but are not limited thereto.

When the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide May be used, but is not limited thereto.

When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, and in particular, in the case of a spray, additionally chlorofluorohydro Propellant such as carbon, propane / butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. Fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

According to the present invention, in order to protect the skin from ultraviolet rays, to cope with the skin blackening caused by melanin, it inhibits tyrosinase activity, copes with various oxidative factors that cause skin aging, and damage factors such as external stimulation or various stresses. It is possible to smooth the reduced skin repair function by providing a natural skin-derived skin composition is easy to absorb the skin.

1 is a schematic representation of the process of separating and purifying 4-hydroxyacetophenone from the white rice extract of the present invention.
Figure 2 shows the ¹³ C-NMR spectrum (125 MHz, CD ₃ OD) of 4-hydroxyacetophenone.
Figure 3 shows the ¹ H-NMR spectrum (500 MHz, CD ₃ OD) of 4-hydroxyacetophenone.
4 shows the results of analysis using a calibration curve of 4-hydroxyacetophenone.
5 shows the result of analysis using the calibration curve of 4-hydroxyacetophenone.
Figure 6 shows the HPLS chromatogram results of 4-hydroxyacetophen.
7 shows the comparison of the whitening effect of the white rice extract and 4-hydroxyacetophenone.
Figure 8 shows the effect of 4-hydroxyacetophenone on tyrosinase enzyme activity.
FIG. 9 shows the effect of 4-hydroxyacetophenone on tyrosinase protein expression.
Fig. 10 shows the effect of 4-hydroxyacetophenone on tyrosinase gene expression.
Fig. 11 shows the effect of 4-hydroxyacetophenone on MITF protein expression.
Fig. 12 shows the effect of 4-hydroxyacetophenone on MITF gene expression.
Figure 13 shows the effect of 4-hydroxyacetophenone on CREB phosphorylation.
Fig. 14 shows the effect of 4-hydroxyacetophenone on protein kinase A enzyme activity.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  1: white rice ( Cynanchum atratum Separation and Chemical Structure of 4-Hydroxyacetophenone from Extracts

A solvent fraction of white rice (CA) extract; 3 kg of Cynanchum atratum , a plant belonging to Asclepiadaceae, was purchased from Kyungdong market, and then extracted with 15 L of MeOH three times at room temperature for 3 days. The MeOH extract (75 g) was solvent fractionated with n- hexane, methylene chloride (MC), ethylacetate (EtOAc) and H ₂ O.

Column fractionation for the active fraction (MC); Activation analysis In association with the research team, the activity of the whitening activity was detected and it was converted to the MC layer. Thus, the MC fraction (25 g) was separated into 10 fractions (CAC-1 to CAC-10) by silica gel column chromatography using MC-MeOH solvent.

Column fractionation for white rice CAC-3; CAC-3 fractions were separated by silica gel column chromatography with MPLC using MC-MeOH as a solvent and separated into six small fractions (CAC-3-1 to CAC-3-6). 4-hydroxyacetophenone (CAC-3 -3) was isolated and purified.

In the NMR spectrum of 4-hydroxyacetophenone, one methyl group was observed in ¹ H-NMR, and the proton due to two 1,4-substituted benzenes at δ 7.88 and 6.83 respectively and one methyl at δ 2.51 Three protons were observed. In the ¹³ C-NMR spectrum, a total of eight carbon signals were confirmed. One ketone was detected at δ 198.07, and six carbons were observed due to one benzene at δ 162.70, 130.72, 130.72, 128.70, 114.85 and 114.85 , And carbon at one of the methyl groups was identified at δ 24.85. The formula of 4-hydroxyacetophenone is shown below. Its ¹ H and ¹³ C-NMR data are shown in the following Table 1 and Figs. 2 and 3.

Formula 1

Position
4-Hydroxyacetophenone δ _C 隆_H ( J in Hz) One 128.70 - 2 130.72 7.88 d (8.5) 3 114.85 6.83 d (8.5) 4 162.70 - 5 114.85 6.83 d (8.5) 6 130.72 7.88 d (8.5) 7 198.07 - 8 24.85 2.51 s

Quantitative analysis of surface elements

 The content of 4-hydroxyacetophenone isolated from white rice was analyzed by HPLC. The HPLC used in the quantitative analysis was a 2696 Waters HPLC system with a 2996 PDA detector. The content was analyzed by the following analytical method using a calibration curve.

Prepare standard product - Take 500 μg of 4-hydroxyacetophenone as standard, dissolve in 500 μl of HPLC for MeOH, and dilute it to 500, 250, 100, 20 or 10 μg / ml with stock solution . 10 μl of the standard solution was analyzed by HPLC to obtain an area of the chromatogram, and a calibration curve was prepared using the area and concentration of the standard solution as parameters (FIG. 4). The r ² value of the standard product (4-hydroxyacetophenone) was 0.9999, and the respective values were obtained (FIG. 5).

Preparation and content analysis of the sample solution - The white rice extract was completely dried in a vacuum oven, exactly 10 mg, and completely dissolved in 1 ml of MeOH for HPLC. The filtrate was filtered through a 0.45 μm membrane filter and used as the sample solution. The content of 4-hydroxyacetophenone was determined from regression linear equation and the content (%) was calculated by repeating 3 times (FIG. 6) on the sample (Table 3).

Analysis condition

A) Detector: Ultraviolet absorptiometer (measuring wavelength T: 254 nm)

B) Column: YMC J'sphere ODS-H80 (150 X 4.6 mm)

C) Mobile _{phase: ACN (Acetonitrile): H 2} O

Time (min) H ₂ O ACN 0 80% 20% 40 0 % 100%

D) Flow rate: 1 ml / min

E) injection volume: 10 μl

Test Example  1: white rice extract and 4- Hydroxyacetophenone Whitening effect

Mouse melanoma B16 cells were plated at 2.5 × 10 ³ cells / well in a 96-well culture plate and cultured in a 37 ° C incubator with 5% CO ₂ for 24 hours. Twenty-four hours later, the medium of each well was removed and replaced with Dulbecco's modified Eagle's medium (DMEM) containing fresh 10% fetal bovine serum (FBS), followed by serial dilution of 4-hydroxyacetophenone and alpha-melanocyte stimulating hormone MSH), respectively. After 72 hours, the amount of melanin released into the medium was measured using a microplate reader at 405 nm. To use synthetic melanin as a standard, serial dilutions were made, and the cells were dispensed into a 96-well culture plate and absorbance was measured at 405 nm to create a standard curve. Experimental results were repeated at least 3 times. The effect on melanogenesis was expressed as a percentage of control compared to the control, with the control group treated with alpha-MSH alone as 100%.

Melanin concentration was increased to 9 ㎍ / ㎖ in B16 cells without alpha-MSH stimulation, and melanin production was increased 10-fold by 50 ㎍ / ㎖ when alpha-MSH alone was administered alone. The effect of the sample on the melanin production was expressed as control% with the alpha-MSH alone group as a control. When the sample was treated with alpha-MSH and 0.3 M g / ml of extract, the concentration of melanin was 93% compared to the control, 83% at 1 μg / ml, 1% at 10 μg / ml at 3 μg / , The concentration of melanin was inhibited by 0.3%, and the IC ₅₀ value was 1.8 쨉 g / ml (Fig. 7-A). In the case of treatment with alpha-MSH and sample 4-hydroxyacetophenone (concentration 50 ㎍ / ㎖), 89% of melanin was produced in comparison with the control, 78% at 100 ㎍ / ㎖, 44% at 300 ㎍ / Ml to 18% concentration-dependent inhibition of melanin production, and the IC ₅₀ value was 183 / / ml (Fig. 7-B). The effect of arbutin, a whitening substance, on the production of melanin was also measured in parallel. When treated with alpha-MSH and arbutin (concentration 10 ㎍ / ㎖), melanin was produced in 89% of the control, 72% in the arbutin concentration of 30 ㎍ / ㎖, 47% in 100 ㎍ / %, And the IC ₅₀ value was 92 쨉 g / ml (Fig. 7-C).

Test Example 2: Measurement of effect on cell viability

Mouse melanoma B16 cells were plated at 2.5 × 10 ³ cells / well in a 96-well culture plate and cultured in a 37 ° C incubator with 5% CO ₂ for 24 hours. After 24 hours, the medium of each well was removed and replaced with DMEM media containing fresh 10% fetal bovine serum (FBS), and then treated with serially diluted 4-hydroxyacetophenone and alpha-MSH, respectively. After 72 hours, the cells were changed to DMEM containing fresh 10% FBS and stabilized for 2 hours. After 2 hours, 3- [4,5-dimethylthiazol-2 -yl] -2,5-diphenyltetrazolium bromide (MTT) in the culture medium is 37 ℃ 5% CO ₂ is supplied to a treatment with a concentration of 100 μg / ml per well And reacted for 2 hours. The MTT solution was removed, and 200 μl of dimethyl sulfoxide (DMSO) was added to dissolve the cells. The absorbance at 590 nm was measured using a microplate reader.

The white rice extract did not show cytotoxicity at the sample concentration of 0.3-3 / / ㎖, but showed 30% cytotoxicity at the sample concentration of 10 / / ㎖ (Fig. 7-A). 4-hydroxyacetophenone showed no cytotoxicity at the sample concentration of 50 ㎍ / ㎖-100 ㎍ / ㎖, but showed cytotoxicity at 39% at the sample concentration of 200 ㎍ / ㎖ and 58% at the concentration of 300 ㎍ / ㎖ (eg 7-B ). The whitening substance arbutin did not show cytotoxicity at the concentration (10 / / ㎖-300 ㎍ / ㎖) (Fig. 7-C).

Test Example 3: Measurement of effect on tyrosinase enzyme activity

Mushroom tyrosinase was used as an enzyme source and L-tyrosine as a substrate. Sodium phosphate buffer was used as an enzyme reaction buffer, and mushroom tyrosinase 500 L-tyrosine, diluted white rice extract and 4-hydroxyacetophenone were added. Absorbance was measured at 475 nm for 2 minutes using a spectrophotometer set at 37 ° C. The activity of the enzyme was defined as 1 unit of the amount of enzyme that converted 1 nM of L-Tyrosine to the dopachrome per minute.

Tyrosinase catalyzes the oxidation of L-tyrosine and L-DOPA, the early stages of melanin biosynthesis.

In order to investigate whether the whitening effect of 4-hydroxyacetophenone directly inhibited the enzyme activity of tyrosinase, the experiment was carried out using mushroom tyrosinase. The activity of the enzyme was defined as 1 unit of the amount of enzyme that converted 1 nM of L-Tyrosine to the dopachrome per minute. The enzyme activity of tyrosinase was not inhibited by treatment with 1-10 μg / ㎖ of white rice extract. In addition, treatment with 4-hydroxyacetophenone at a concentration of 100-400 ㎍ / ㎖ did not inhibit the enzyme activity of tyrosinase. Inhibition% was calculated using the control group treated with mushroom tyrosinase and L-Tyrosine as a control group, and the concentration of arbutin used as a positive control was 6% at 10 ㎍ / mL, 20% at 30 ㎍ / mL, and 100 ㎍ / mL 49%, showed an inhibitory effect of the 10 ㎍ / ㎖ 75% in, ₅₀ IC The value was 109 μg / ml (FIG. 8).

Test Example  4: Tyrosinase  Effect on protein expression

Mouse melanoma B16 cells were seeded in a 6-well culture plate at 3.5 × 10 ⁵ cells / well and cultured in a 37 ° C incubator with 5% CO ₂ for 24 hours. After 24 hours, the medium of each well was removed and replaced with fresh DMEM. Then, the diluted white rice extract, 4-hydroxyacetophenone, and alpha-MSH were treated 2 hours later. tyrosinase protein expression was 48 hours, MITF protein expression was 4 hours, and CREB phosphorylation was treated with alpha-MSH for 15 minutes. Cells were harvested using cold PBS, and cells were lysed using RIPA buffer. After electrophoresis, gels were transferred to polyvinylidene fluoride (PVDF) membrane and stained with TBS / T (Tris-buffered saline with tween-20) buffer containing 5% nonfat dry milk Shaking for 1 hour. Anti-tyrosinase, anti-MITF, anti-GAPDH, anti-phospho-CREB and anti-CREB primary antibodies were added and reacted overnight at 4 ° C and then washed three times with TBS / T buffer. Add HRP (Horseradish peroxidase) -conjugated secondary antibody diluted buffer and incubate at room temperature for 2 hours. After washing three times with TBS / T buffer, the PVDF membrane is reacted with ECL (enhanced chemiluminescence) solution and each band emitting chemiluminescence is identified using Chemi Doc ^TM XRS ⁺ machine (chemiluminescence imaging system).

Western blot analysis was performed to investigate the effect of tyrosinase on protein expression of melanin biosynthesis. B16 cells were treated with alpha-MSH and 4-hydroxyacetophenone for 48 hours. Expression of tyrosinase protein was increased when alpha-MSH was administered to B16 cells without alpha-MSH stimulation, compared to the control group. α-MSH-treated group was used as a control, and the expression pattern of tyrosinase protein changed by 4-hydroxyacetophenone treatment was observed. The concentration of tyrosinase protein was decreased in the group treated with 1-10 ㎍ / ㎖ of rice extract compared to the control group. 4-hydroxyacetophenone at a concentration of 100-200 / / ㎖ showed a concentration-dependent inhibition of tyrosinase protein expression compared to the control group (Fig. 9).

Test Example  5: Tyrosinase  Effect on gene expression

RT-PCR was performed to investigate the effect of tyrosinase on gene expression.

Mouse melanoma B16 cells were seeded in a 6-well culture plate at 3.5 × 10 ⁵ cells / well and cultured in a 37 ° C incubator with 5% CO ₂ for 24 hours. After 24 hours, the medium of each well was removed and replaced with fresh DMEM. Then, the diluted white rice extract, 4-hydroxyacetophenone, and alpha-MSH were treated 2 hours later. Tyrosinase expression was treated with alpha-MSH for 18 hours and MITF expression for 2 hours. Cells were harvested using cold PBS and RNA was isolated using TRIZOL reagent. The cDNA was synthesized by using 1 ug of RNA at 42 ° C for 60 minutes, and the polymerase chain reaction (PCR) was performed using the prepared cDNA as a template. PCR was performed at 94 ° C for 30 seconds, 54 ° C for 30 seconds, and 72 ° C for 60 seconds, tyrosianse for 27 cycles, MITF for 31 cycles, and housekeeping gene for beta-actin for 27 cycles. The resulting PCR products were loaded on 1% agarose gel and electrophoresed at 100V. Each primer sequence used in the experiment is as follows.

- Tyrosinase primer seq. (size: 1,191 bp)

Sense primer: 5'-CATTTTTGATTTGAGTGTCT-3 '(SEQ ID NO: 1)

Antisense primer: 5'-TGTGGTAGTCGTCTTTGTCC-3 '(SEQ ID NO: 2)

- MITF primer seq. (size: 397 bp)

Sense primer: 5'-TACAGTCACTACCAGGTGCAG-3 '(SEQ ID NO: 3)

Antisense primer: 5'-CCATCAAGCCCAAAATTTCTT-3 '(SEQ ID NO: 4)

- beta-actin primer seq. (size: 349 bp)

Sense primer: 5'-TGGAATCCTGTGGCATCCATGAAAC-3 '(SEQ ID NO: 5)

Antisense primer: 5'-TAAAACGCAGCTCAGTAACAGTCCG-3 '(SEQ ID NO: 6)

B16 cells were treated with alpha-MSH and 4-hydroxyacetophenone for 18 hours. The expression of tyrosinase gene was increased when alpha-MSH was administered to B16 cells in the absence of alpha-MSH stimulation, compared to the control group. α-MSH-treated group was used as a control, and the expression pattern of tyrosinase, which was changed upon treatment with 4-hydroxyacetophenone, was examined. The expression of tyrosinase gene was suppressed in a concentration - dependent manner in the group treated with 1-10 μg / ㎖ of white rice extract compared to the control group. 4-hydroxyacetophenone at a concentration of 200 ㎍ / ㎖ inhibited tyrosinase protein expression compared to the control group. The PKA inhibitor, H-89, was used as a positive control, and tyrosinase protein expression was inhibited in the group treated with 10 μg / ml of H-89 compared to the control (FIG. 10).

Test Example  6: MITF  Effect on protein expression

Western blot analysis was performed to investigate the effect of MITF, a transcription factor controlling the expression of tyrosianse, on protein expression. B16 cells were treated with diluted white rice extract and treated for 2 hours with alpha-MSH for 4 hours. The expression of MITF protein was increased when alpha-MSH was administered to B16 cells without alpha-MSH stimulation, compared to the control group. The expression of MITF protein was examined in the control group of the alpha-MSH-treated group. MITF protein expression was inhibited in a dose - dependent manner in the group treated with 10 ㎍ / ㎖ of white rice extract. The PKA inhibitor, H-89, was used as a positive control and the MITF protein expression was suppressed in the group treated with 10 μg / ml of H-89 compared to the control group (FIG. 11).

Test Example  7: MITF  Effect on gene expression

RT-PCR was performed to investigate the effect of MITF on gene expression. B16 cells were treated with alpha-MSH and 4-hydroxyacetophenone for 2 hours. The expression of MITF gene was increased when alpha-MSH was administered to B16 cells without alpha-MSH stimulation, compared to the control group. α-MSH-treated group was used as a control, and the expression pattern of MITF, which was changed upon treatment with 4-hydroxyacetophenone, was examined. MITF gene expression was inhibited in a dose - dependent manner in the group treated with 10 ㎍ / ㎖ of white rice extract. 4-hydroxyacetophenone at a concentration of 200 ㎍ / ㎖ inhibited MITF protein expression compared to the control group. The PKA inhibitor, H-89, was used as a positive control, and MITF protein expression was suppressed in the group treated with 10 μg / ml of H-89 compared to the control (FIG. 12).

Test Example  8: CREB  Effect on phosphorylation

CREB is a transcription factor that is phosphorylated by PKA and binds to the CRE portion of the MITF promoter to control the expression of MITF. Western blot analysis was performed to determine the effect of CREB on phosphorylation. B16 cells were treated with 4-hydroxyacetophenone, which had been diluted series, and then treated with alpha-MSH for 2 hours. The phosphorylation of CREB was induced by treatment with alpha-MSH compared to the group supplemented with B16 cells without alpha-MSH stimulation. alpha-MSH-treated group was used as a control, and the degree of CREB phosphorylation changed by 4-hydroxyacetophenone treatment was observed. In the group treated with 3-10 ㎍ / ㎖ of extracts, the inhibition of CREB phosphorylation was dose - dependently inhibited compared to the control group. 4-hydroxyacetophenone at a concentration of 200 ㎍ / ㎖ inhibited CREB phosphorylation in a concentration-dependent manner compared to the control group. The PKA inhibitor, H-89, was used as a positive control, and the group treated with 10 μg / ml of H-89 inhibited CREB phosphorylation as compared to the control (FIG. 13).

9: to the test Protein kinase  A ( PKA Effect on Enzyme Activity

Recombinant human PKA was used as an enzyme source and kemptide was used as a substrate. PKA enzyme, a series of diluted white rice extract and 4-hydroxyacetophenone, kemptide, [γ- ³² P] ATP were added and reacted at 30 ° C for 15 minutes. The reaction solution is dropped on P81 phosphocellulose paper and air-dried. Add 40 ml of 0.75% phosphoric acid, wash 3 times, and wash once with acetone. scintillation cockail was added and radioactivity was measured using a scintillation counter.

The kinase assay was performed in cell-free system to determine whether PKA directly affects the enzyme activity. Recombinant human PKA was used as an enzyme source and kemptide was used as a substrate. The PKA enzyme, substrate, [γ- ³² P] ATP was added and reaction was carried out at 30 ° C. for 15 min. The degree of phosphorylation of the substrate was measured using a scintillation counter. PKA enzyme and substrate treated with kemptide were used as a control to calculate inhibition%. Rice extract showed the sample 1 ㎍ / ㎖ 21% In, 3 ㎍ / ㎖ 51% in the inhibition of 71% at 10 ㎍ / ㎖ effect, IC ₅₀ The value was 3 μg / ml. Inhibitory effects of 4-hydroxyacetophenone samples were 30% at 50 ㎍ / mL, 47% at 100 ㎍ / mL, 69% at 200 ㎍ / mL and 92% at 400 ㎍ / mL and IC ₅₀ The value was 113 μg / ml. The ATP-competitive inhibitor H-89, which acts on the catalytic subunit of PKA, was used as a positive control and showed an inhibitory effect of 93% at 10 μg / ml of H-89 (FIG. 14).

Formulation example

It is selected as skin, lotion, cream, and essence formulations which are the best formulations that have high functionality and compatibility with functional materials and cosmetic formulations, and which have the best functionality in terms of functionality, usability, stability and safety. .

Formulation example  One: skin  Produce

The skins were prepared in the compositions shown in Table 4 below. Specifically, a preservative and a humectant are placed in a dissolution zone for water, and the mixture is dissolved by heating at 45 to 50 ° C., and the soluble image is put into a dissolution zone for water, slowly solubilized using an Agi Mixer, Cool.

Formulation example  2: Lotion manufacturing

The skins were prepared in the compositions shown in Table 5 below. Specifically, the emulsifier, the oil, the antioxidant, and the preservative are heated and dissolved in the oily dissolution part at 80 to 82 DEG C, and some additives and solvent are added to the manufacturing part heated and dissolved at 78 to 80 DEG C and emulsified using a HOMO MIXER Cool to 40 캜, add the remaining additives, mix, and cool.

Formulation example  3: Cream production

The skins were prepared in the compositions shown in Table 6 below. Specifically, the emulsifier, the oil, the antioxidant, and the preservative are heated and dissolved in the oily dissolution part at 80 to 82 DEG C, and some additives and solvent are added to the manufacturing part heated and dissolved at 78 to 80 DEG C and emulsified using a HOMO MIXER Cool to 40 캜, add the remaining additives, mix, and cool.

Formulation Example 4: Preparation of Essence

The skins were prepared as shown in Table 7 below. Specifically, the emulsifier, the oil, the antioxidant, and the preservative are heated and dissolved in the oily dissolution part at 80 to 82 DEG C, and some additives and solvent are added to the manufacturing part heated and dissolved at 78 to 80 DEG C and emulsified using a HOMO MIXER Cool to 40 캜, add the remaining additives, mix, and cool.

Test Example  10: Evaluation of stability on cosmetics

Time Stability Test

Stability was measured by temperature (4 ℃, 25 ℃, 44 ℃) for a certain period after the application of functional material to skin and lotion formulation. After checking the stability, I could. Addition of the stabilizer in the formulation was further carried out. As a result of observing changes in the composition at various temperatures (4 ° C, 25 ° C and 44 ° C), stable formulations were obtained for a certain period of time. Tables 8 and 9 below show the results of observation of the condition of each skin and lotion cosmetic condition.

1) Appearance: Good-G, slight discoloration -SC, discoloration -C

1) Appearance: Good-G, slight discoloration -SC, discoloration -C

2) Viscosity: 4 Spindle 12 rpm at 25 ° C

Test Example  11: Evaluation of stability on cosmetics

Thirty patients were subjected to skin patch test (IQ chamber) using IQ chamber. Patients with psoriasis, eczema, other skin lesions, pregnant, lactating or contraceptive, and antihistamines were excluded from the study. The test site was wiped with 70% ethanol and dried. Then, 25 μl of each of Skin, Lotion, Cream, Essence formulations was dropped on the top of 30 prepared subjects and placed on the IQ chamber. And fixed. After applying the patch for 24 hours and removing the patch, the test area was marked with a marking pen. After 30 minutes, 24 hours, and 48 hours, the test site was observed.

The judgment was made after 30 minutes, 24 hours, and 48 hours after the removal of the patch, and the skin reaction was classified according to the criterion of the International Contact Dermatitis Research Group (ICDRG) shown in Table 10 below. The mean score was calculated according to the formula for calculating the average skin reactivity shown in Table 11 below, and the presence or absence of stimulation was determined according to the skin patch test result.

※ Criteria for Skin Response

1) Negative (-): No irritation

2) Doubtful or slight reaction and erythema (±): Mild irritation Mild or barely detectable mild erythema

3) Erythema + Induration (+): Erythema + Induration + Vesicle (++): Erythema + Induration + Bullee (+++): Severe erythema, : Strong irritation Severe erythema and alveoli, scab formation

Formula (1) below is a formula for calculating the mean skin response (mean score).

i = subject number 30 minutes after patch removal

j = subject number after 24 hours after patch removal

k = number of subjects 48 hours after patch removal

Score i, j, k = score after 30 minutes, 24 hours, and 48 hours after removal of the patch, scores for each ICDRG score, erythema and edema

The information of the subjects participating in the human body test is summarized in Table 12 below.

The ages of the subjects participating in the human body test are subdivided as shown in Table 13 below. The average age of participants was 40.4 years. Gender classification was 26 women and 4 men.

Skin, Lotion, Cream and Essence were dripped into the IQ chamber of 25 ㎕ each at the top of 30 subjects and then skin patch test was performed. After 24 hours and after 48 hours, the skin reaction at the test site was evaluated according to the criteria of the International Contact Dermatitis Research Group (ICDRG) Were classified according to the skin patch test results, and are summarized in Tables 14 to 17 below.

Position
4-Hydroxyacetophenone δ _C 隆_H ( J in Hz) One 128.70 - 2 130.72 7.88 d (8.5) 3 114.85 6.83 d (8.5) 4 162.70 - 5 114.85 6.83 d (8.5) 6 130.72 7.88 d (8.5) 7 198.07 - 8 24.85 2.51 s

According to the present invention, in order to protect the skin from ultraviolet rays, to cope with the skin blackening caused by melanin, it inhibits tyrosinase activity, copes with various oxidative factors that cause skin aging, and damage factors such as external stimulation or various stresses. It is possible to smooth the reduced skin repair function by providing a natural skin-derived skin composition is easy to absorb the skin.

<110> Cosmecca Korea Co., Ltd. <120> A Cosmetic Composition for Whitening Skin Containing White Rice          Extracts <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tyrosinase sense primer <400> 1 catttttgat ttgagtgtct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tyrosinase antisense primer <400> 2 tgtggtagtc gtctttgtcc 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MITF sense primer <400> 3 tacagtcact accaggtgca g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MITF antisense primer <400> 4 ccatcaagcc caaaatttct t 21 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> beta-actin sense primer <400> 5 tggaatcctg tggcatccat gaaac 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> beta-actin antisense primer <400> 6 taaaacgcag ctcagtaaca gtccg 25

Claims (7)

Skin whitening cosmetic composition containing a white rice extract. The method of claim 1,
Cosmetic composition, characterized in that the white rice extract contains 4-hydroxyacetophenone (4-Hydroxyacetophenone) which is a compound of the formula (1).
Formula 1

3. The method of claim 2,
Wherein the 4-hydroxyacetophenone is a solvent fraction of a white rice (CA) extract; Cynanchum , a plant belonging to the Asclepiadaceae atratum ), which is obtained by distilling MeOH three times at room temperature for three days and then fractionating the solvent with n- hexane, methylene chloride (MC), ethylacetate (EtOAc) and H ₂ O. 3. The method of claim 2,
The 4-hydroxyacetophenone has a ¹ H and ¹³ C-NMR data characteristics as shown in Table 18, wherein the cosmetic composition. 3. The method of claim 2,
Cosmetic composition, characterized in that the 4-hydroxyacetophenone is included in an amount of 0.001-50% by weight relative to the total weight of the cosmetic composition. 6. The method according to any one of claims 1 to 5,
Cosmetic composition, characterized in that the composition has a tyrosinase activity inhibitory activity. 6. The method according to any one of claims 1 to 5,
The skin whitening is a cosmetic composition, characterized in that the skin wrinkle improvement, skin elasticity enhancement, skin strengthening, skin shine or skin spot suppression.

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