KR101443840B1 - Cosmetic cmposition for skin-whitening - Google Patents

Abstract

The present invention relates to the use of Galega < RTI ID = 0.0 > officinalis extract or an alkaloid isolated from Galle officinalis as an active ingredient. The extracts and alkaloids effectively inhibit melanin production by selectively inhibiting mRNA expression of tyrosinase.

Description

[0001] Cosmetic composition for skin-whitening [0002]

The present invention relates to a skin whitening cosmetic composition, and more particularly, to a skin whitening cosmetic composition comprising Galega officinalis extract or a specific alkaloid isolated from Galle officinalis ( Galega officinalis ) as an active ingredient.

Melanin is a dark brown pigment that exists in the eyes, skin, and hair. It absorbs more than a certain amount of ultraviolet rays from the body and blocks the infiltration of ultraviolet rays, thereby protecting the body and maintaining a positive temperature. However, excessive exposure to ultraviolet rays can lead to excessive secretion of melanin to prevent penetration of the skin, and the skin color changes to brown. The production of melanin is known to be caused by a combination of tyrosinase and other enzymes and hormones.

Malignant melanoma is a tumor made by malignant alteration of melanocytes that produce melanin pigment. It is known that melanocytes may be present in any part of the body regardless of the site, but it is most frequently found in the skin and is known to have high malignancy among the tumors that can occur in the skin. In Asian countries, the incidence of melanoma is higher than that in western countries, but the frequency of melanoma is increasing year by year. Genetic factors and exposure to ultraviolet light are thought to be responsible for the development of malignant melanoma.

The present inventors carried out searches on various natural products in order to develop whitening materials derived from natural products. The present inventors have found that Galega officinalis extract and certain alkaloid compounds isolated therefrom effectively inhibit melanin production of the B16F1 cell line, which is one of the malignant melanomas.

Thus, the present invention is based on the discovery that Galega The present invention also provides cosmetic compositions for skin whitening comprising the above-mentioned alkaloid compounds as an active ingredient.

In accordance with one aspect of the present invention, the Galega officinalis ) or a compound of the following formula (1) as an active ingredient:

≪ Formula 1 >

In one embodiment, a skin whitening cosmetic composition comprising the compound of formula (1) as an active ingredient is provided.

In another embodiment, the Gallega operational leases during the day (Galega officinalis ) as an active ingredient is provided.

The extract can be obtained by an extraction method comprising extracting Galega officinalis with a C ₁ -C ₄ alcohol or a mixed solvent of C ₁ -C ₄ alcohol and water. The extraction is carried out by drying the leaves of Galega officinalis and extracting the obtained dried leaves with a C ₁ -C ₄ alcohol or a mixed solvent of C ₁ -C ₄ alcohol and water, .

According to the present invention, Galega ( Galega) officinalis extract and galactose isolated from Galega officinalis (i.e., the compound of formula (I)) selectively inhibit tyrosinase activity, thereby inhibiting melanin production. Thus, Galega officinalis extract and the compound of formula (I) can be usefully used in skin whitening cosmetics.

Figure 1 shows the results of the search and evaluation of extracts having anti-melanin producing activity. FIG. 1 (A) is a photograph of a cell pellet image of a malignant melanoma cell line, B16F1 cells treated with 7 kinds of extracts and cultured for 72 hours. Figure 1B shows the result of measuring the melanin content. C of Figure 1 is a graphical representation of Galega < RTI ID = 0.0 > officinalis ) at a concentration of 10 μg / ml and 20 μg / ml, and cultured the cells for 72 hours. The obtained cell pellet images were taken with a digital camera. D in Figure 1 is the Galega < RTI ID = 0.0 > officinalis ) treated with the extract of the present invention. In Figure 1, E is the Galega < RTI ID = 0.0 > The results of analysis of mRNA expression of tyrosinase, MITF, and MCIR by real-time PCR when treated with an extract of C. officinalis .
Fig. 2 shows the results of evaluating the anti-melanin producing activity of the compound of formula (1) (2- (3-methylbut-2-enyl) guanidine). FIG. 2A shows the result of photographing the obtained cell pellet image with a digital camera when the extract of Galale officinalis ( Galega officinalis ) and the compound of Chemical Formula 1 were treated, FIG. 2B shows the result of measuring the melanin content And FIG. 2C shows the result of analysis of tyrosinase mRNA expression by real-time PCR. Fig. 2D shows the results of immunohistochemical staining of B16F1 cells with Galega officinalis ) and the compound of Chemical Formula (1). In Figure 2, E indicates that Galega < RTI ID = 0.0 > officinalis ) and a compound of formula (I); And immunoblotting analysis of cell lysates obtained from cells irradiated with ultraviolet light of 5 mJ / cm ² for 6 hours as a control group.

The present invention relates to the use of Galega < RTI ID = 0.0 > officinalis ) or a compound of the following formula (1) as an active ingredient:

≪ Formula 1 >

In one embodiment, the present invention provides a cosmetic composition for skin whitening comprising the compound of Formula 1 as an active ingredient. The compound of Chemical Formula 1 is a known substance and its chemical name is 2- (3-methylbut-2-enyl) guanidine (CAS Registry Number: 543-83-9) .

In another embodiment, the invention relates to the use of a Galega < RTI ID = 0.0 > officinalis ) as an active ingredient.

The extract can be obtained by an extraction method comprising extracting Galega officinalis with a C ₁ -C ₄ alcohol or a mixed solvent of C ₁ -C ₄ alcohol and water. Preferably, the extraction can be carried out using methanol. The extraction is carried out by drying the leaves of Galega officinalis and extracting the obtained dried leaves with a C ₁ -C ₄ alcohol or a mixed solvent of C ₁ -C ₄ alcohol and water, preferably methanol .

The amount of the extraction solvent to be used is not particularly limited and may be, for example, about 1 to 10 times by volume, preferably about 1 to 5 times by volume based on 1 weight of dry leaves of Galle officinalis ( Galega officinalis ) . The extraction may be carried out by an extraction method such as cold extraction, hot water extraction, ultrasonic extraction, and reflux cooling extraction for about 1.5 to 48 hours, preferably about 2 to 24 hours. Preferably, the extraction can be performed by ultrasonic extraction at room temperature (about 25 ° C), and the extraction process can be performed once or plural times, preferably about 3 times. The obtained extract can be obtained in a liquid or powder form, if necessary, by a conventional method, for example, at a temperature of about 30 to 70 캜, by concentration under reduced pressure or drying under reduced pressure.

The cosmetic composition according to the present invention can be prepared in various forms according to a method conventionally used in the field of cosmetic composition. For example, the cosmetic composition of the present invention may be in the form of a conventional cosmetic composition such as a cream, a pack, a lotion, an essence, a lotion, a foundation and a makeup base. Can be prepared according to a conventional method. The Galega < RTI ID = 0.0 > The amount of the extract of officinalis and / or the compound of the formula (I) may be, for example, in the range of 0.001 to 20% by weight, preferably 0.01 to 15% by weight, and most preferably 0.1 to 10% by weight per unit cosmetic. Of course, the content may vary depending on the degree of skin whitening activity desired.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of extract

The leaves were harvested from Galega officinalis and dried for about 7 days. 700 ml of anhydrous methanol was added to about 300 g of dried leaves, and ultrasonic extraction was performed for 2 hours. The ultrasound extraction procedure was performed twice more. The obtained extract was dried under reduced pressure to obtain 33.7 g of an extract.

Test Example 1. Anti-melanin-producing extracts

A preliminary test was conducted on various extracts from Korean Plant Extract Bank (data not shown), and seven kinds of extracts described as having a relatively inhibitory activity on melanin production were selected to inhibit melanin production Respectively.

B16F1 cells (Korean Cell Line Bank, Daejeon), a malignant melanoma cell line, were divided into 6-well plates at a concentration of 1 × 10 ⁵ cells and cultured for 24 hours. At this time, the medium was DMEM medium containing 10% (v / v) fetal bovine serum and 0.1% gentamycin, and cultured in a 5% CO ₂ incubator at 37 ° C. The seven extracts obtained as a result of the preliminary test were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 μg / ml, and cultured in a 5% CO ₂ incubator at 37 ° C. for 72 hours. After completion of the incubation, the cell pellet image was photographed with a digital camera (FIG. 1A). Melanin was extracted from the cell pellet and the melanin content was measured according to the method described in Biol Pharm Bull 2008; 31: 606-610 (Fig. 1B). From the results of Figs. 1A and 1B, it was confirmed that the first extract (i.e., A extract) was superior to the other extracts in inhibiting the melanin formation, and the extract was confirmed to be Galega officinalis extract .

The cell pellet image obtained by dissolving the extract prepared in Example 1 at different concentrations, that is, 10 μg / ml and 20 μg / ml in DMSO, and culturing the cells for 72 hours in the same manner as described above, The results are shown in Fig. 1C.

From the B16F1 cells (FL) obtained by treating the untreated B16F1 cells (no treatment, NT) and the concentration of 20 占 퐂 / ml, J Invest Dematol. 1992: 98 (4): 481-487. The results of measurement of tyrosinase activity in each cell lysate using L-DOPA as a substrate are shown in Fig. 1 Of D. As can be seen from Fig. 1D, tyrosinase activity was significantly decreased in the cells treated with the extract.

In addition, mRNA expression of tyrosinase, MITF, and MCIR in B16F1 cells obtained by treating the untreated B16F1 cells with a concentration of 20 μg / ml was analyzed by real-time PCR as follows. Using Trizol reagent (Invitrogen), total RNA was extracted from the cells according to the manufacturer's instructions and then treated with DNase I. Trizol was removed by adding chloroform, and mRNA was precipitated using isopropanol. The precipitated mRNA was washed with 75% ethanol. The optical density was measured using a UV spectrometer at 260 nm to 280 nm to determine the amount and purity of RNA, and agarose gel electrophoresis was performed to confirm the integrity of the RNA. The primers used for the real-time PCR are shown in Table 1 below, and GAPDH was used as a housekeeping gene. All amplification procedures were performed using 1x SYBR supermix final concentrate, 500 nmol / l gene specific Primer, and 2 [mu] l of template. The initial denaturation was 5 min at 95 [deg.] C, 45 sec at 95 [deg.] C, 45 sec at 58 [ 45 cycles at 72 ° C for 45 seconds and final extension at 72 ° C for 5 minutes After amplification, standard and threshold values for each reaction were determined using a software package (Light cycler 480II Roche). , The amplification products were separated into 2% agarose gels and visualized by staining with ethidium bromide. The results of the real time PCR analysis are shown in FIG. 1E. The results of E in FIG. 1 indicate that the extract selectively inhibits the expression of tyrosinase, which plays an important role in the melanogenesis process, thereby suppressing the production of melanin have.

gene SEQ ID NO: order Tyrosinase
One Forward 5'-GTCGTCACCCTGAAAATCCTAACT-3 ' 2 Reverse 5'-CATCGCATAAAACCTGATGGC-3 ' MITF
3 Forward 5'-ACGACAACTCTGGATCTCAC-3 ' 4 Reverse 5'-CATCAGGATGTCTTCCAAGT-3 ' MCIR
5 Forward 5'-GCCACCCTTACTATCCTTCT-3 ' 6 Reverse 5'-GAGGAGGAAGAGGTTGAAGT-3 ' GAPDH
7 Forward 5'-CAGATGCCTGCTTCACCACCTTC-3 ' 8 Reverse 5'-TCTGGAAAGCTGTGGCGTGATGG-3 '

Test Example 2. Evaluation of anti-melanin producing activity of 2- (3-methylbut-2-enyl) guanidine

B16F1 cells were treated with 20 μg / ml of the extract prepared in Example 1 and 10, 100, and 1000 μM of 2- (3-methylbut-2-enyl) guanidine And cultured in the same manner as in Example 1 for 72 hours. The cell pellet image was photographed with a digital camera (A in FIG. 2) and the melanin content was measured (FIG. 2B) in the same manner as in Test Example 1, and mRNA expression of tyrosinase was measured by real- PCR) (Fig. 2C). From the results of Figs. 2A to 2C , it can be seen that the compound of the formula (1), which is an alkaloid isolated from Galega officinalis , effectively inhibits melanogenesis by selectively inhibiting tyrosinase activity .

In addition, B16F1 cells were treated by dissolving 5 mu g / ml, 10 mu g / ml of the extract prepared in Example 1 and 1 mM of the compound of formula 1 in DMSO and culturing for 2 days in the same manner as in Test Example 1, Cell growth measurement tests were performed using XTT assay kit (Roche, Cat # 11465015001) at day 1 and 2 (FIG. 2 D). From the results of FIG. 2 D, it can be seen that Galega officinalis extract and the compound of formula (1) did not significantly affect the growth of cells at the inhibitory concentration of melanin synthesis.

The cells were cultured for 72 hours in the same manner as in Test Example 1 by dissolving 5 mg / ml, 10 mg / ml, 20 mg / ml of the extract prepared in Example 1 and 1 mM of the compound of Formula 1 in DMSO ; And the control group was irradiated with 5 mJ / cm ² of ultraviolet rays for 6 hours to induce apoptosis. Cells were washed twice with cold phosphate-buffered saline and dissolved in 300 μl of tissue lysis buffer And centrifuged at 14,000 rpm for 10 minutes. About 20 μg of total protein was separated by SDS-PAGE and immunoblotting analysis was performed. That is, proteins were transferred to a PVDF membrane and blocked with 5% skim milk powder in Tris-buffered saline (TBS-T) containing 0.1% Tween-20 for 1 hour. (Cat. # SC-7150, Santa Cruz, CA, USA) and anti-ERK2 (cat. # SC-154, Santa Cruz, CA, USA) in TBS containing 1% )] For 1 hour at room temperature or 16 hours at 4 [deg.] C. The membranes were washed with TBS-T solution and reacted with HRP-conjugated secondary antibody (0.1 μg / ml; Jackson Immuno Research Laboratories, West Grove, PA) for 45 minutes. Immunoreactivity was detected using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). Immunoblotting analysis results for the Parp-1 protein (F: full-length protein, T: truncated protein) are shown in FIG. In Figure 2E, ERK2 was used as a control protein. From the immunoblot analysis results of E in Fig. 2, it was found that Galega officinalis extract and the compound of formula (I) all exhibited only cell death that could normally occur in the cells.

<110> CUSKIN. CO,. LTD <120> Cosmetic cmposition for skin-whitening <130> PN0602 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 gtcgtcaccc tgaaaatcct aact 24 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 catcgcataa aacctgatgg c 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 acgacaactc tggatctcac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 catcaggatg tcttccaagt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gccaccctta ctatccttct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gaggaggaag aggttgaagt 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 cagatgcctg cttcaccacc ttc 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 tctggaaagc tgtggcgtga tgg 23

Claims (6)

delete A cosmetic composition for skin whitening comprising a compound of the following formula (1) as an active ingredient:
&Lt; Formula 1 >

delete delete delete delete

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