Composition for skin whitening

Abstract

The present invention provides a cosmetic composition for skin whitening, comprising a specific peptide. The peptide acts as an antagonist for β-catenin, thereby effectively inhibiting β-catenin signaling as well as inhibiting tyrosinase expression and melanin synthesis. As a result, the peptide can be effectively used for skin whitening.

Description

skin whitening composition

[0001] The present invention relates to a cosmetic composition for skin whitening comprising a specific peptide. The peptide acts as an antagonist for β-catenin, thereby effectively inhibiting β-catenin signaling as well as tyrosinase expression and melanin synthesis.

[0002] WNT signaling is involved in cell proliferation and migration, cell differentiation, and various diseases including cancer. WNT also plays a crucial role in signaling that promotes melanin synthesis. When WNT signaling is activated, β-catenin accumulates in the cytoplasm, and when a certain concentration is reached, it binds to the Tcf / Lef transcription factor in the nucleus, inducing the expression of genes such as MITF, thereby inducing the expression of tyrosinase, which plays a key role in melanin formation (Wisurumuni Arachchilage Hasitha Maduranga Karunarathne, et al., Int J Mol Sci. 2020 Jan 2;21(1):312. et al.).

[0003] Inhibition of WNT / β-catenin signaling can inhibit the activity of tyrosinase, which plays a key role in melanin synthesis, and reduce melanin synthesis (Chao Niu, et al., Molecules. 2017 Aug 4;22(8):1303. doi: 10.3390; Dao-Pei Zou, et al., Genes Dis. 2020 Jun 15;8(5):677-688. doi: 10.1016, etc.). Therefore, substances that can act as antagonists of β-catenin can serve as functional cosmetic substances for skin whitening.

[0004] The present inventors have disclosed that a peptide derived from microphthalmia-associated transcription factor (MITF) inhibits the formation of melanin pigment by regulating the transcription of MITF target molecules and has skin whitening activity by inhibiting the synthesis and activity of tyrosinase (Korean Patent Registration No. 10-1457371).

[0005] The present inventors conducted various studies to develop active substances based on small peptides that can act as β-catenin antagonists. As a result, we discovered that a specific peptide, a 3-mer peptide composed of Pro-Ala-Ile, acts as a β-catenin antagonist, effectively inhibiting β-catenin signaling and suppressing tyrosinase expression and melanin synthesis, making it useful for skin whitening.

[0006] Accordingly, the present invention aims to provide a skin whitening cosmetic composition comprising the specific peptide.

[0007] According to one aspect of the present invention, a cosmetic composition for skin whitening is provided, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0008] <Chemical Formula 1>

[0009]

[0010] The present invention has revealed that the peptide according to the present invention (i.e., a peptide composed of Pro-Ala-Ile) acts as an antagonist for β-catenin. That is, the present invention has revealed that the peptide according to the present invention not only inhibits melanin synthesis by binding to β-catenin and inhibiting WNT signaling, but also promotes autophagy, thereby exhibiting a whitening effect. The present invention has also revealed that the peptide according to the present invention effectively inhibits melanin synthesis in a 3D human skin model. Therefore, the peptide according to the present invention can be usefully applied to a cosmetic composition for skin whitening.

[0011] Figure 1 shows the results of measuring the change in the signal of the peptide (VE-Bcatanin)-AMC of the present invention composed of Pro-Ala-Ile by treatment with α-MSH and β-catenin siRNA.

[0012] Figure 2 shows the results of analyzing changes in β-catenin / TCF interaction by α-MSH and peptide treatment of the present invention.

[0013] Figure 3 shows the results of analyzing changes in tyrosinase expression levels due to treatment with α-MSH and the peptide of the present invention.

[0014] Figure 4 shows the results of analyzing changes in the amount of melanin due to treatment with α-MSH and the peptide of the present invention.

[0015] Figure 5 shows the results of analyzing changes in LC3B expression levels due to peptide treatment of the present invention.

[0016] Figure 6 shows the results of analyzing changes in melanin expression levels due to peptide treatment of the present invention in a 3D human skin model.

[0017] The present invention provides a cosmetic composition for skin whitening, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0018] <Chemical Formula 1>

[0019]

[0020] In the cosmetic composition of the present invention, the peptide of Chemical Formula 1 may also be expressed as "Pro-Ala-Ile." The amino acids constituting the peptide of Chemical Formula 1 may independently be in the form of L-amino acids or D-amino acids. Pharmaceutically acceptable salts of the peptide derivative of Chemical Formula 1 include, but are not limited to, acid addition salts.

[0021] The cosmetic composition of the present invention may be in the form of a functional cosmetic composition containing the above-described peptide as an active ingredient. The cosmetic composition may be manufactured in various forms according to a conventional cosmetic manufacturing method. For example, the cosmetic composition may be manufactured in the form of a cosmetic product, toner, cream, lotion, etc. containing the peptide, which may be diluted with a conventional cleansing solution, astringent solution, or moisturizing solution and used. In addition, the cosmetic composition may include conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions. In the cosmetic composition, the content of the peptide is an amount effective to achieve a skin whitening effect, for example, 1 x 10 based on the total weight of the composition. -5 ~ 1 x 10 -2 It may be contained in a content of weight %, preferably about 1 x 10 -4 ~ 1 x 10 -3 It can be contained in a content of weight%.

[0022] Hereinafter, the present invention will be described in more detail through examples and test examples. However, these examples and test examples are intended to illustrate the present invention, and the present invention is not limited to these examples and test examples.

[0023] Example 1. Synthesis of peptides

[0024] A peptide consisting of Pro-Ala-Ile was synthesized by the FMOC solid-phase method using an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, Korea). The synthesized peptide was purified and analyzed by reverse-phase HPLC (Prominence LC-20AB, Shimadzu, Japan) using a C18 analytical RP column (Shiseido capcell pak), and identified using mass spectrometry (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA).

[0025] Example 2. Preparation of a composition containing a peptide

[0026] The peptide (peptide composed of Pro-Ala-Ile) manufactured in Example 1 was dissolved in triple-distilled water to prepare a concentration of 1000 ppm. The obtained peptide solution was used in the following test examples.

[0027] Test Example 1: Evaluation of β-catenin binding

[0028] Stimulation of B16F10 cells with α-MSH causes β-catenin to translocate into the nucleus. Whether the peptide of the present invention (a peptide composed of Pro-Ala-Ile) binds to β-catenin and translocates into the nucleus upon α-MSH stimulation was confirmed through immunofluorescence.

[0029] (1) Test materials

[0030] - Preparation of test substances

[0031] The peptide of the present invention (peptide composed of Pro-Ala-Ile) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0032] - Test system

[0033] 1) Cell line: mouse melanocyte (mouse melanocyte, B16F10, ATCC)

[0034] 2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% CO2, 37°C), and subcultured with new culture medium every 2-3 days.

[0035] 3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0036] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0037] - Test materials

[0038] 1) Immunofluorescence

[0039] 1-1) α-MSH

[0040] Storage conditions: -20℃ frozen storage / Manufacturer: SIGMA, M4135-1MG

[0041] 1-2) Peptide-AMC of the present invention

[0042] Storage conditions: -20℃ frozen storage / Manufacturer: Peptron

[0043] 1-3) Phalloidin-Rhodamin

[0044] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, R415

[0045] 1-4) Anti-β-catenin (S33 / S37 / T41) antibody

[0046] Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 4270

[0047] 1-5) Anti-rabbit IgG-FITC antibody

[0048] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN

[0049] 2) siRNA transfection

[0050] 2-1) β-catenin siRNA(h)

[0051] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-29209

[0052] 2-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0053] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0054] 2-3) Opti-MEM ® Medium

[0055] Storage conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0056] (2) Test method

[0057] - Composition of the test group

[0058]

[0059] - Exam process

[0060] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4 After dispensing the cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the confluency of the cells was 50% or higher.

[0061] 2) The medium was replaced with a dedicated medium (DMEM + 10% FBS) for siRNA treatment.

[0062] 3) siRNA + Opti-MEM ® Media and transfection reagent + Opti-MEM ®The mixture was mixed in a 1:1 ratio and slowly stirred, then allowed to react at room temperature for 5 minutes.

[0063] 4) 3) The solution was added to cultured cells and treated for 48 hours to conduct the test.

[0064] 5) The peptide-AMC (1:100) of the present invention was reacted at 4°C for 16 hours and then washed three times with a washing solution (PBS).

[0065] 6) To stain the cytoskeleton, phalloidin-rhodamine (1:2000) was reacted at room temperature for 40 minutes, and then washed three times with washing solution (PBS).

[0066] 7) After treating with β-catenin antibody (1:100), the cells were reacted at room temperature for 40 minutes and washed three times with washing solution (PBS).

[0067] 8) After treating with secondary antibody (1:1000), reacting at room temperature for 40 minutes, the cells were washed three times with washing solution (PBS).

[0068] 9) Mounting was performed using a mounting solution.

[0069] 10) The fluorescence signal (AMC) detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0070] - Observe and judge results

[0071] The degree of nuclear migration of the peptide-AMC of the present invention was compared and analyzed in the α-MSH and β-catenin siRNA treatment groups based on the control siRNA negative control group.

[0072] (3) Test results

[0073] In the negative control group, β-catenin was spread throughout the cytoplasm and showed a weak FITC (green fluorescence) signal by the antibody, but in the α-MSH treatment group, it was observed that β-catenin accumulated after moving into the nucleus, and the FITC signal increased. The peptide-AMC (blue fluorescence) of the present invention bound to β-catenin that had moved into the nucleus in the α-MSH treatment group and showed an AMC (blue fluorescence) signal in the nucleus, and when the expression of β-catenin was suppressed by treatment with siRNA, the AMC signal was reduced (Fig. 1).

[0074] (4) Conclusion

[0075] Compared to the negative control group, the peptide-AMC of the present invention was observed to move to the nucleus in the α-MSH treatment group, and the peptide-AMC signal of the present invention in the nucleus was observed to be reduced in the β-catenin siRNA treatment group. Therefore, it is believed that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) specifically binds to β-catenin.

[0076] Test Example 2: Evaluation of β-catenin signaling inhibition efficacy

[0077] In order to verify the potential of the peptide of the present invention as a β-catenin antagonist, changes in the interaction of β-catenin / TCF, a downstream signaling pathway of WNT (or α-MSH), were confirmed through in situ PLA.

[0078] (1) Test materials

[0079] - Preparation of test substances

[0080] The peptide of the present invention (peptide composed of Pro-Ala-Ile) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0081] - Test system

[0082] 1) Cell line: mouse melanocyte (mouse melanocyte, B16F10, ATCC)

[0083] 2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% CO2, 37°C), and subcultured with new culture medium every 2-3 days.

[0084] 3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0085] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0086] - Test materials

[0087] 1) α-MSH

[0088] Storage conditions: -20℃ frozen storage / Manufacturer: SIGMA, M4135-1MG

[0089] 2) Anti-β-catenin (S33 / S37 / T41) antibody (primary antibody)

[0090] Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 4270

[0091] 3) Anti-TCF-4 (D-4) antibody (primary antibody)

[0092] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-166699

[0093] 4) NaveniFlex 100RM

[0094] Storage conditions: -20℃ frozen storage / Manufacturer: NaveniFlex, NV C-NF MR.100

[0095] 5) Prolong™ diamond antifade mountant with DAPI

[0096] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

[0097] 6) Digital Fluorescence Imaging System

[0098] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0099] (2) Test method

[0100] - Composition of the test group

[0101]

[0102] - Exam process

[0103] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4 After dispensing the cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the confluency of the cells was 50% or higher.

[0104] 2) The negative control, α-MSH, and test substances were treated for 1 hour at the appropriate concentrations for each treatment group.

[0105] 3) After fixing the cells using 4% paraformaldehyde, pretreatment was performed to increase the permeability of the cell antibody by perforating the cells with 0.1% Triton X-100.

[0106] 4) Subsequent tests were conducted using the In Situ PLA Kit (NaveniFlex 100RM) and the tests were conducted according to the manufacturer's instructions.

[0107] 5) After washing once with PBS, blocking was performed with blocking solution at 37°C for 30 minutes.

[0108] 6) Two antibodies for confirmation were diluted to 10 μg / mL in antibody diluent, reacted at 4°C for 16 hours, and washed three times with TTBS (0.01 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4).

[0109] 7) After adding the PLA probe and reacting at 37°C for 1 hour, it was washed three times with TTBS.

[0110] 8) Reactions A, B, and C were sequentially processed and reacted at 37°C for 60, 30, and 90 minutes, respectively. Finally, the cells were washed twice with TBS (0.01 M Tris, 0.15 M NaCl) and mounted using a mounting solution containing DAPI (nuclear stain).

[0111] 9) The PLA signal detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0112] - Observe and judge results

[0113] The PLA fluorescence signal was quantitatively analyzed and evaluated using NIS-Elements BR3.1. The luminescence signal due to the interaction of β-catenin / TCF in the α-MSH and test substance treatment groups was compared and analyzed with the negative control group as the standard.

[0114] (3) Test results

[0115] Compared to the negative control group, it was observed that the increased β-catenin / TCF interaction in the α-MSH treatment group was suppressed in a concentration-dependent manner in the test substance treatment group (Fig. 2).

[0116] (4) Conclusion

[0117] It was confirmed that the test substance inhibits β-catenin activation induced by α-MSH stimulation. Therefore, it is judged that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) can act as a β-catenin antagonist.

[0118] Test Example 3: Skin Whitening Efficacy Evaluation

[0119] Inhibition of WNT / β-catenin signaling inhibits the activity of tyrosinase, which plays a crucial role in melanin synthesis, thereby reducing melanin synthesis. The level of tyrosinase expression was observed through Western blot analysis, and changes in the total amount of intracellular and free melanin were confirmed through a melanin contents assay to determine whether the peptide of the present invention has a whitening effect.

[0120] (1) Test materials

[0121] - Preparation of test substances

[0122] The peptide of the present invention (peptide composed of Pro-Ala-Ile) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0123] - Test system

[0124] 1) Cell line: mouse melanocyte (mouse melanocyte, B16F10, ATCC)

[0125] 2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% CO2, 37°C), and subcultured with new culture medium every 2 to 3 days.

[0126] 3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0127] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0128] - Test materials

[0129] 1) Western blot analysis

[0130] 1-1) α-MSH

[0131] Storage conditions: -20℃ frozen storage / Manufacturer: SIGMA, M4135-1

[0132] 1-2) Anti-tyrosinase (C-19) antibody

[0133] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTACRUZ, SC-7833

[0134] 1-3) Anti-β-actin antibody

[0135] Storage conditions: Refrigerated at 4℃ / Manufacturer: CEL SIGNALING, 4967S

[0136] 1-4) NP40 cell lysis buffer

[0137] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0138] 1-5) Bovine serum albumin (BSA)

[0139] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0140] 1-6) Protein assay dye reagent concentrate

[0141] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0142] 1-7) Immuno-bolt for protein blotting ® PVDF membrane

[0143] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0144] 1-8) WEST SAVE GOLD,

[0145] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0146] 1-9) DaVinci Western Imaging System

[0147] Manufacturer: DAVINCH-K, CAS-400SM

[0148] 2) Melanin content analysis

[0149] 2-1) 1M NaOH

[0150] Storage conditions: Store at room temperature / Manufacturer: SIGMA

[0151] 2-2) Microplate reader

[0152] Manufacturer: BIO-TEK, EL808

[0153] (2) Test method

[0154] (2-1) Western blot analysis

[0155] - Composition of the test group

[0156]

[0157] - Exam process

[0158] 1) 1X10 in a 6-well culture plate 6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 30% or higher.

[0159] 2) The negative control, α-MSH, and test substances were treated for 72 hours at the concentrations of each treatment group.

[0160] 3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0161] 4) Electrophoresis was performed by loading 20 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0162] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0163] 6) The PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.

[0164] 7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0165] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0166] 9) After exposure using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0167] - Observe and judge results

[0168] The expression level of tyrosinase due to treatment with the test substance was evaluated by quantitative analysis using ImageJ based on the expression level of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system.

[0169] (2-2) Melanin content analysis

[0170] - Composition of the test group

[0171]

[0172] - Exam process

[0173] 1) 1X10 in a 6-well culture plate 6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 30% or higher.

[0174] 2) The negative control, α-MSH, and test substances were treated for 72 hours at the concentrations of each treatment group.

[0175] 3) To measure the amount of extracellular melanin, the cell culture medium was transferred to a 1.5 mL test tube, centrifuged, and the supernatant was transferred to a 96-well plate, and the absorbance was measured at 400 nm using a microplate reader.

[0176] 4) To measure the amount of melanin in the cells, cultured cells were washed with PBS, lysed at 60°C by adding 1 M NaOH to each well, and the absorbance of the cell lysate was measured at 400 nm using a microplate reader.

[0177] - Observe and judge results

[0178] The total amount of melanin inside and outside the cells was quantitatively analyzed and evaluated using absorbance values ​​measured using a microplate reader.

[0179] (3) Test results

[0180] (3-1) Western blot analysis

[0181] Compared to the negative control group, tyrosinase expression was observed to increase in the α-MSH treatment group, and to decrease in a concentration-dependent manner in the test substance treatment group (Fig. 3).

[0182] (3-2) Melanin content analysis

[0183] Compared to the negative control group, the total melanin production of cells increased in the α-MSH treatment group, but decreased in a concentration-dependent manner in the test substance treatment group (Fig. 4).

[0184] (4) Conclusion

[0185] The whitening efficacy of the test substance was evaluated, and it was found that the test substance significantly reduced tyrosinase activity and total melanin production in cells in a concentration-dependent manner. Therefore, the peptide of the present invention (a peptide composed of Pro-Ala-Ile) is believed to have a skin whitening effect.

[0186] Test Example 4: Evaluation of Autophagy Promotion Efficacy

[0187] Autophagy is a cellular process that degrades damaged proteins and unnecessary organelles to generate energy. Since melanin delivered to keratinocytes can also be degraded by activating autophagy, we examined whether the test substance induced autophagy by inhibiting WNT / β-catenin signaling, using Western blot analysis to detect the expression of LC3B, a representative autophagy marker.

[0188] (1) Test materials

[0189] - Preparation of test substances

[0190] The peptide of the present invention (peptide composed of Pro-Ala-Ile) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0191] - Test system

[0192] 1) Cell line: Human Keratinocyte (HaCaT, CLS)

[0193] 2) Cell management: The cell line was frozen and thawed, inoculated into a 100 cm2 animal cell culture dish containing culture medium, and cultured in an incubator (5% CO2, 37°C), and subcultured with new culture medium every 2-3 days.

[0194] 3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0195] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0196] - Test materials

[0197] 1) Anti-LC3B antibody

[0198] Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 3868S

[0199] 2) Goat anti-rabbit IgG Fc-HRP

[0200] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0201] 3) NP40 cell lysis buffer

[0202] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0203] 4) Bovine serum albumin (BSA)

[0204] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0205] 5) Protein assay dye reagent concentrate

[0206] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0207] 6) Immuno-bolt for protein blotting ® PVDF membrane

[0208] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0209] 7) WEST SAVE GOLD,

[0210] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0211] 8) DaVinci Western Imaging System

[0212] Manufacturer: DAVINCH-K, CAS-400SM

[0213] (2) Test method

[0214] - Composition of the test group

[0215]

[0216] - Exam process

[0217] 1) 5X10 in a 6-well culture plate6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 80% or higher.

[0218] 2) The negative control and test substances were treated for 24 hours at the concentrations appropriate for each treatment group.

[0219] 3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0220] 4) Electrophoresis was performed by loading 20 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0221] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0222] 6) The PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.

[0223] 7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0224] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0225] 9) After exposure using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0226] - Observe and judge results

[0227] The expression level of LC3B due to treatment with the test substance was evaluated by quantitative analysis using ImageJ based on the expression level of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system.

[0228] (3) Test results

[0229] Compared to the negative control group, it was observed that the expression level of LC3B, an autophagy marker protein, increased in a concentration-dependent manner in the test substance treatment group (Fig. 5).

[0230] (4) Conclusion

[0231] As a result of evaluating the autophagy-inducing efficacy of the test substance, it was confirmed that the test substance promoted LC3B expression in a concentration-dependent manner. Therefore, it is determined that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) has the efficacy of promoting autophagy.

[0232] Test Example 5: Evaluation of Whitening Efficacy in a 3D Human Skin Model

[0233] The above test confirmed that the test substance exhibited whitening effects by inhibiting β-catenin at the cellular level. The test substance's whitening effects were also confirmed in a 3D human skin model (Neoderm-ED) similar to human skin using Fontana-Masson staining.

[0234] (1) Test materials

[0235] - Preparation of test substances

[0236] The peptide of the present invention (peptide composed of Pro-Ala-Ile) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0237] - Control substance

[0238] The positive control substance (L-ascorbic acid) was dissolved in distilled water to prepare a concentration of 1 mg / ml.

[0239] - Test system

[0240] 1) 3D human skin model: Neoderm-ME

[0241] 2) Management: Cultured in an incubator (5% CO2, 37℃) and tested within 3 days of receipt.

[0242] 3) Badge: Maintenance medium

[0243] Composition: 10% fetal bovine serum / Storage conditions: Refrigerated storage / Manufacturer: TEGO SCIENCE

[0244] - Test materials

[0245] 1) VLX-3W research radiometer

[0246] Manufacturer: VILVER

[0247] 2) Solvable TM dissolution buffer

[0248] Storage conditions: Store at room temperature / Manufacturer: PerkinElmer, 6NE9100

[0249] 3) Fontana-Masson Stain kit

[0250] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABCAM, ab150669

[0251] 4) Microscope

[0252] Manufacturer: OLYMPUS, BX53F2

[0253] (2) Test method

[0254] (2-1) Fontana Masson dyeing

[0255] - Composition of the test group

[0256]

[0257] - Exam process

[0258] 1) After receiving Neoderm-ME, a dedicated medium was added and cultured for 24 hours.

[0259] 2) For the UVB treatment group, 0.06 J / cm was applied using a UV irradiator. 2 UVB was irradiated at an intensity of .

[0260] 3) The negative, positive control, and test substances were treated for 48 hours at the appropriate concentration for each treatment group.

[0261] 4) Neoderm-ME was separated from the insert well using a blade to create a paraffin block.

[0262] 5) Slides were prepared by cutting sections at 4 ㎛ thickness.

[0263] 6) Paraffin washing and water dehydration process using xylene (Et-OH 100% >95%>90%>80%>70%) was performed.

[0264] 7) Subsequent tests were conducted using the Fontana Masson staining kit and the tests were conducted according to the manufacturer's instructions.

[0265] 7) After reacting with gold chloride solution (0.2%) at room temperature for 30 seconds, it was washed several times with distilled water.

[0266] 8) After reacting with sodium thiosulfate solution (5%) at room temperature for 2 minutes, it was washed in running water for 2 minutes and then washed twice with distilled water.

[0267] 9) After reacting with Nuclear Fast Red Solution at room temperature for 5 minutes, wash in running water for 2 minutes and then wash twice with distilled water.

[0268] 10) After dehydration by reacting three times with 100% ethanol, mounting was performed.

[0269] 11) The staining detected in the tissue was observed and photographed using a microscope.

[0270] - Observe and judge results

[0271] The degree of melanin production in the positive control group and test substance treatment group was compared and analyzed based on the negative control group.

[0272] (2-2) Melanin content analysis

[0273] - Composition of the test group

[0274]

[0275] - Exam process

[0276] 1) After receiving Neoderm-ME, a dedicated medium was added and cultured for 24 hours.

[0277] 2) For the UVB treatment group, 0.06 J / cm was applied using a UV irradiator. 2 UVB was irradiated at an intensity of .

[0278] 3) The negative, positive control, and test substances were treated for 48 hours at the appropriate concentration for each treatment group.

[0279] 4) Neoderm-ME was separated from the edge of the insert using a blade.

[0280] 5) The separated Neoderm-ME tissue was placed in a 1.5 ml test tube and PBS was completely removed.

[0281] 6) 360 μl of dissolution buffer was added to each test tube containing Neoderm-ME.

[0282] 7) After reacting at room temperature for 5 to 10 minutes, the membrane was separated from the Neoderm-ME tissue and removed with forceps.

[0283] 8) The reaction was carried out for 45 minutes in a 95℃ heat block.

[0284] 9) After centrifugation at 13,000 rpm for 10 minutes at room temperature, the supernatant was transferred to a tube.

[0285] 10) 200 μl of the test substance was transferred to a 96-well plate and the absorbance (405 nm) was measured using a microplate reader.

[0286] - Observe and judge results

[0287] The degree of melanin production in the positive control group and test substance treatment group was compared and analyzed based on the negative control group.

[0288] (3) Test results

[0289] As a result of evaluating the inhibitory efficacy of the test substance on UVB-induced melanin production in a 3D human skin model, it was confirmed that the amount of melanin increased in the UVB-treated group compared to the negative control group was reduced in the positive control group and the test substance-treated group (Fig. 6).

[0290] (4) Conclusion

[0291] The test substance was confirmed to significantly reduce total melanin production in the skin model with an efficacy equal to or greater than that of the positive control group. Therefore, the peptide of the present invention (a peptide composed of Pro-Ala-Ile) is believed to have a whitening effect not only at the cellular level but also in the skin model.

Claims

1. A cosmetic composition for skin whitening, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof. <Chemical formula 1>

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