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WO2025110757A1 - Composition for skin whitening - Google Patents

Composition for skin whitening Download PDF

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Publication number
WO2025110757A1
WO2025110757A1 PCT/KR2024/018525 KR2024018525W WO2025110757A1 WO 2025110757 A1 WO2025110757 A1 WO 2025110757A1 KR 2024018525 W KR2024018525 W KR 2024018525W WO 2025110757 A1 WO2025110757 A1 WO 2025110757A1
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WIPO (PCT)
Prior art keywords
peptide
test
manufacturer
catenin
present
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French (fr)
Korean (ko)
Inventor
한장희
김민서
유효정
김정회
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Han Do Sook
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Han Do Sook
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Priority claimed from KR1020230163186A external-priority patent/KR102892487B1/en
Application filed by Han Do Sook filed Critical Han Do Sook
Publication of WO2025110757A1 publication Critical patent/WO2025110757A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients

Definitions

  • the present invention relates to a cosmetic composition for skin whitening comprising a specific peptide.
  • the peptide acts as an antagonist for ⁇ -catenin, thereby effectively inhibiting ⁇ -catenin signaling as well as inhibiting the expression of tyrosinase and melanin synthesis.
  • WNT signaling is involved in cell proliferation and migration, cell differentiation, and various diseases including cancer. WNT also plays an important role in signaling that promotes melanin synthesis. When the WNT signal is activated, ⁇ -catenin accumulates in the cytoplasm and, when it reaches a certain concentration, binds to Tcf/Lef transcription factors in the nucleus to induce gene expression such as MITF, thereby inducing the expression of tyrosinase, which plays an important role in melanin formation (Wisurumuni Arachchilage Hasitha Maduranga Karunarathne, et al., Int J Mol Sci. 2020 Jan 2;21(1):312. et al.).
  • Inhibition of WNT/ ⁇ -catenin signaling can inhibit the activity of tyrosinase, which plays an important 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, a substance that can act as an antagonist for ⁇ -catenin can act as a functional cosmetic substance for skin whitening.
  • 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).
  • MITF microphthalmia-associated transcription factor
  • the present inventors have conducted various studies to develop an active substance based on small peptides that can act as an antagonist for ⁇ -catenin. As a result, they discovered that a specific peptide, i.e., a 3-mer peptide composed of Pro-Ala-Ile, acts as an antagonist for ⁇ -catenin, effectively inhibits ⁇ -catenin signaling, and can be usefully applied to skin whitening by inhibiting the expression of tyrosinase and melanin synthesis.
  • a specific peptide i.e., a 3-mer peptide composed of Pro-Ala-Ile
  • the present invention aims to provide a skin whitening cosmetic composition comprising the specific peptide.
  • a cosmetic composition for skin whitening comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof.
  • the present invention has revealed that the peptide according to the present invention (i.e., the peptide composed of Pro-Ala-Ile) acts as an antagonist for ⁇ -catenin. That is, the present invention has revealed that the peptide according to the present invention not only inhibits melanin synthesis by binding to ⁇ -catenin and inhibiting WNT signaling, but also promotes autophagy to exhibit whitening effects. 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.
  • the peptide according to the present invention i.e., the peptide composed of Pro-Ala-Ile acts as an antagonist for ⁇ -catenin. That is, the present invention has revealed that the peptide according to the present invention not only inhibits melanin synthesis by binding to ⁇ -catenin and inhibiting WNT signaling, but also promotes auto
  • Figure 1 shows the results of measuring the signal change of the peptide (VE-Bcatanin)-AMC of the present invention composed of Pro-Ala-Ile by treatment with ⁇ -MSH and ⁇ -catenin siRNA.
  • Figure 2 shows the results of analyzing the change in ⁇ -catenin/TCF interaction by ⁇ -MSH and peptide treatment of the present invention.
  • Figure 3 shows the results of analyzing the change in tyrosinase expression level by ⁇ -MSH and peptide treatment of the present invention.
  • Figure 4 shows the results of analyzing the change in the amount of melanin due to treatment with ⁇ -MSH and the peptide of the present invention.
  • Figure 5 shows the results of analyzing the change in LC3B expression level due to peptide treatment of the present invention.
  • Figure 6 shows the results of analyzing the change in melanin expression level due to peptide treatment of the present invention in a 3D human skin model.
  • 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.
  • the peptide of the chemical formula 1 may also be expressed as "Pro-Ala-Ile".
  • the amino acids constituting the peptide of the chemical formula 1 may independently be in the form of L-amino acid or D-amino acid.
  • Pharmaceutically acceptable salts of the peptide derivative of the chemical formula 1 include, but are not limited to, acid addition salts, for example.
  • the cosmetic composition of the present invention may be in the form of a functional cosmetic composition containing the above-described peptide as an effective ingredient.
  • the cosmetic composition may be manufactured in various forms according to a conventional cosmetic manufacturing method.
  • the cosmetic composition may be manufactured in the form of a cosmetic product, toner, cream, lotion, etc. containing the peptide, and may be used by being diluted with a conventional cleansing solution, astringent, or moisturizing solution.
  • the cosmetic composition may include conventional excipients such as a stabilizer, a solubilizer, vitamin, pigment, and fragrance commonly used in the field of cosmetic compositions.
  • the content of the peptide may be contained in an amount effective to achieve a skin whitening effect, for example, in a content of 1 x 10 -5 to 1 x 10 -2 wt% based on the total weight of the composition, and preferably in a content of about 1 x 10 -4 to 1 x 10 -3 wt%.
  • the peptide composed 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).
  • 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.
  • Test Example 1 Evaluation of ⁇ -catenin binding
  • ⁇ -catenin translocates into the nucleus. It was confirmed through immunofluorescence that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) binds to ⁇ -catenin and translocates into the nucleus by ⁇ -MSH stimulation.
  • 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.
  • 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.
  • DMEM Dulbecco's Modified Eagle Medium
  • Composition 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO
  • siRNA + Opti-MEM ® medium and transfection reagent + Opti-MEM ® medium were mixed slowly in a 1:1 ratio and reacted at room temperature for 5 minutes.
  • phalloidin-rhodamine (1:2000) was reacted at room temperature for 40 minutes, and then washed three times with washing solution (PBS).
  • the fluorescence signal (AMC) detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).
  • the degree of nuclear translocation of the peptide-AMC of the present invention was compared and analyzed in the ⁇ -MSH and ⁇ -catenin siRNA treatment groups based on the control siRNA negative control group.
  • ⁇ -catenin was spread throughout the cytoplasm and showed a weak FITC (green fluorescence) signal by the antibody, but in the ⁇ -MSH treatment group, it was observed that ⁇ -catenin accumulated after moving into the nucleus and the FITC signal increased.
  • the peptide-AMC (blue fluorescence) of the present invention bound to ⁇ -catenin that had moved into the nucleus in the ⁇ -MSH treatment group and showed an AMC (blue fluorescence) signal in the nucleus, and when the expression of ⁇ -catenin was inhibited by treatment with siRNA, the AMC signal was reduced (Fig. 1).
  • Test Example 2 Evaluation of ⁇ -catenin signaling inhibition efficacy
  • 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.
  • 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.
  • DMEM Dulbecco's Modified Eagle Medium
  • Composition 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO
  • Reactions A, B, and C were sequentially treated and reacted at 37°C for 60, 30, and 90 minutes, respectively, and finally washed twice with TBS (0.01 M Tris, 0.15 M NaCl) and mounted using a mounting solution containing DAPI (nuclear stain).
  • the PLA fluorescence signal was quantitatively analyzed and evaluated using NIS-Elements BR3.1.
  • the luminescence signal due to the interaction of ⁇ -catenin/TCF in the ⁇ -MSH and test substance treatment groups was compared and analyzed based on the negative control group.
  • Inhibition of WNT/ ⁇ -catenin signaling can inhibit the activity of tyrosinase, which plays an important role in melanin synthesis, and reduce melanin synthesis.
  • the level of tyrosinase expression is observed through Western blot analysis, and the change in the total amount of intracellular and free melanin is confirmed using melanin contents assay to confirm whether the peptide of the present invention has a whitening effect.
  • 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.
  • Cell management The cell line was frozen, thawed, and inoculated into a 100 cm2 animal cell culture dish containing culture medium. The cells were cultured in an incubator (5% CO2 , 37°C), and subcultured with new culture medium every 2–3 days.
  • DMEM Dulbecco's Modified Eagle Medium
  • Composition 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO
  • BSA Bovine serum albumin
  • BIO-TEK BIO-TEK
  • EL808 BIO-TEK, EL808
  • 1X106 cells were seeded into each well of a 6-well culture plate. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was performed when the cell confluency was 30% or higher.
  • Electrophoresis was performed by loading 20 ⁇ g of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.
  • the PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.
  • a blocking solution 3% BSA, 0.05% Tween 20, TBS
  • 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 ⁇ -Actin used as a loading control, which was captured using a Western blot imaging system.
  • 1X106 cells were seeded into each well of a 6-well culture plate. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was performed when the cell confluency was 30% or higher.
  • 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.
  • the total amount of melanin inside and outside the cells was quantitatively analyzed and evaluated using absorbance values measured using a microplate reader.
  • the total melanin production of cells increased in the ⁇ -MSH treatment group, but decreased in a concentration-dependent manner in the test substance treatment group (Fig. 4).
  • the test substance significantly reduced tyrosinase activity and total melanin production of cells in a concentration-dependent manner. Therefore, it is determined that the peptide of the present invention (peptide composed of Pro-Ala-Ile) has a skin whitening effect.
  • Autophagy is an activity that obtains energy by decomposing damaged proteins or unnecessary organelles within the cell. Since melanin delivered to keratinocytes can also be decomposed by activating the autophagy function, the expression of LC3B, a representative autophagy marker, was confirmed through Western blot analysis to determine whether the test substance induces autophagy by inhibiting WNT/ ⁇ -catenin signaling.
  • 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.
  • 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.
  • DMEM Dulbecco's Modified Eagle Medium
  • Composition 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO
  • BSA Bovine serum albumin
  • Electrophoresis was performed by loading 20 ⁇ g of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.
  • the PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.
  • a blocking solution 3% BSA, 0.05% Tween 20, TBS
  • 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 ⁇ -Actin used as a loading control, which was captured using a Western blot imaging system.
  • the test substance As a result of evaluating the autophagy-inducing efficacy of the test substance, it was confirmed that the test substance promoted the expression of LC3B in a concentration-dependent manner. Therefore, it is judged that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) has the efficacy of promoting autophagy.
  • Test Example 5 Evaluation of Whitening Efficacy in a 3D Human Skin Model
  • test substance exhibited whitening efficacy by inhibiting ⁇ -catenin at the cellular level. It was confirmed through Fontana Masson Staining whether the test substance also exhibited whitening efficacy in a 3D human skin model (Neoderm-ED) similar to human skin.
  • 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.
  • the positive control substance (L-ascorbic acid) was dissolved in distilled water to prepare a concentration of 1 mg/ml.
  • Composition 10% Fetal bovine serum / Storage conditions: Refrigerated storage / Manufacturer: TEGO SCIENCE
  • VILVER VILVER
  • Neoderm-ME After receiving Neoderm-ME, a dedicated medium was added and cultured for 24 hours.
  • UVB was irradiated at an intensity of 0.06 J/cm 2 using a UV irradiator.
  • Neoderm-ME was separated from the insert well using a blade to create a paraffin block.
  • 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.
  • Neoderm-ME After receiving Neoderm-ME, a dedicated medium was added and cultured for 24 hours.
  • UVB was irradiated at an intensity of 0.06 J/cm 2 using a UV irradiator.
  • Neoderm-ME was separated from the edge of the insert by cutting it using a blade.
  • Neoderm-ME tissue was placed in a 1.5 ml test tube and PBS was completely removed.
  • test substance 200 ⁇ l was transferred to a 96-well plate, and the absorbance (405 nm) was measured using a microplate reader.
  • 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.
  • the test substance significantly reduced the total melanin production of the skin model with an efficacy equal to or greater than that of the positive control group. Therefore, it is judged that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) has a whitening effect not only at the cell level but also in the skin model.

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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

피부 미백용 조성물 Composition for skin whitening

본 발명은 특정 펩타이드를 포함하는 피부 미백용 화장료 조성물에 관한 것이다. 상기 펩타이드는 β-카테닌에 대한 길항제(antagonist)로 작용함으로써, β-카테닌 신호전달을 효과적으로 억제할 뿐만 아니라, 티로시나아제의 발현 및 멜라닌 합성을 억제한다.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 inhibiting the expression of tyrosinase and melanin synthesis.

WNT 신호전달은 세포의 증식과 이동, 세포 분화 및 암을 포함한 다양한 질병에 관여한다. 멜라닌 합성을 촉진하도록 하는 신호전달에도 WNT가 중요한 역할을 하며, WNT신호가 활성화 되면 세포질 내의 β-카테닌이 축적되고 일정 농도가 되면 핵 내에서 Tcf/Lef 전사인자에 결합하여 MITF 등의 유전자 발현을 유도하고 이에 따라 멜라닌 형성에 중요한 역할을 하는 티로시나아제의 발현을 유도한다(Wisurumuni Arachchilage Hasitha Maduranga Karunarathne, et al., Int J Mol Sci. 2020 Jan 2;21(1):312. 등).WNT signaling is involved in cell proliferation and migration, cell differentiation, and various diseases including cancer. WNT also plays an important role in signaling that promotes melanin synthesis. When the WNT signal is activated, β-catenin accumulates in the cytoplasm and, when it reaches a certain concentration, binds to Tcf/Lef transcription factors in the nucleus to induce gene expression such as MITF, thereby inducing the expression of tyrosinase, which plays an important role in melanin formation (Wisurumuni Arachchilage Hasitha Maduranga Karunarathne, et al., Int J Mol Sci. 2020 Jan 2;21(1):312. et al.).

WNT/β-카테닌 신호전달을 억제하면 멜라닌 합성에 중요한 역할을 하는 티로시나아제의 활성을 저해하고 멜라닌 합성을 감소시킬 수 있다(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 등). 따라서, β-카테닌에 대한 길항제로 작용할 수 있는 물질은 피부 미백을 위한 기능성 화장료 물질로서 작용할 수 있다. Inhibition of WNT/β-catenin signaling can inhibit the activity of tyrosinase, which plays an important 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, a substance that can act as an antagonist for β-catenin can act as a functional cosmetic substance for skin whitening.

본 발명자들은 작은안구증-연관 전사인자(microphthalmia-associated transcription factor: MITF)로부터 유래된 펩타이드가 MITF 표적분자의 전사를 조절함으로써 멜라닌 색소의 형성을 억제하고; 타이로시나아제의 합성 및 활성을 억제함으로써, 피부 미백 활성을 갖는다는 것을 개시한 바 있다(대한민국 특허등록 제10-1457371호).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).

본 발명자들은 β-카테닌에 대한 길항제로 작용할 수 있는 물질로서 작은 펩타이드(small peptides)를 기반으로 한 활성물질을 개발하기 위하여 다양한 연구를 수행하였다. 그 결과, 특정 펩타이드, 즉 Pro-Ala-Ile으로 구성된 3-mer의 펩타이드가 β-카테닌에 대한 길항제(antagonist)로 작용함으로써, β-카테닌 신호전달을 효과적으로 억제할 뿐만 아니라 티로시나아제의 발현 및 멜라닌 합성을 억제함으로써, 피부 미백에 유용하게 적용될 수 있다는 것을 발견하였다.The present inventors have conducted various studies to develop an active substance based on small peptides that can act as an antagonist for β-catenin. As a result, they discovered that a specific peptide, i.e., a 3-mer peptide composed of Pro-Ala-Ile, acts as an antagonist for β-catenin, effectively inhibits β-catenin signaling, and can be usefully applied to skin whitening by inhibiting the expression of tyrosinase and melanin synthesis.

따라서, 본 발명은 상기 특정 펩타이드를 포함하는 피부 미백용 화장료 조성물을 제공하는 것을 목적으로 한다.Accordingly, the present invention aims to provide a skin whitening cosmetic composition comprising the specific peptide.

본 발명의 일 태양에 따라, 하기 화학식 1의 펩타이드 또는 이의 약학적으로 허용가능한 염을 포함하는, 피부 미백용 화장료 조성물이 제공된다.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.

<화학식 1><Chemical Formula 1>

Figure PCTKR2024018525-appb-img-000001
Figure PCTKR2024018525-appb-img-000001

본 발명에 따른 펩타이드(즉, Pro-Ala-Ile으로 구성된 펩타이드)가 β-카테닌에 대한 길항제(antagonist)로 작용한다는 것이 본 발명에 의해 밝혀졌다. 즉, 본 발명에 따른 펩타이드는 β-카테닌에 결합하여 WNT 신호전달을 억제함으로써 멜라닌 합성을 억제할 뿐만 아니라 자가포식 작용을 촉진시켜 미백효능을나타내는 것이 본 발명에 의해 밝혀졌다. 본 발명에 따른 펩타이드가 3D 사람 피부 모델에서 효과적으로 멜라닌 합성을 억제한다는 것이 또한 본 발명에 의해 밝혀졌다. 따라서, 본 발명에 따른 펩타이드는 피부 미백용 화장료 조성물에 유용하게 적용될 수 있다.The present invention has revealed that the peptide according to the present invention (i.e., the 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 to exhibit whitening effects. 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.

도 1은 α-MSH 및 β-카테닌 siRNA 처리 의한 Pro-Ala-Ile으로 구성된 본 발명의 펩타이드(VE-Bcatanin)-AMC 신호 변화를 측정한 결과이다.Figure 1 shows the results of measuring the signal change of the peptide (VE-Bcatanin)-AMC of the present invention composed of Pro-Ala-Ile by treatment with α-MSH and β-catenin siRNA.

도 2는 α-MSH 및 본 발명의 펩타이드 처리에 의한 β-카테닌/TCF 상호작용 변화를 분석한 결과이다.Figure 2 shows the results of analyzing the change in β-catenin/TCF interaction by α-MSH and peptide treatment of the present invention.

도 3은 α-MSH 및 본 발명의 펩타이드 처리에 의한 티로시나아제 발현량 변화를 분석한 결과이다.Figure 3 shows the results of analyzing the change in tyrosinase expression level by α-MSH and peptide treatment of the present invention.

도 4는 α-MSH 및 본 발명의 펩타이드 처리에 의한 멜라닌 양의 변화를 분석한 결과이다.Figure 4 shows the results of analyzing the change in the amount of melanin due to treatment with α-MSH and the peptide of the present invention.

도 5는 본 발명의 펩타이드 처리에 의한 LC3B 발현량 변화를 분석한 결과이다.Figure 5 shows the results of analyzing the change in LC3B expression level due to peptide treatment of the present invention.

도 6은 3D 사람피부모델에서 본 발명의 펩타이드 처리에 의한 멜라닌 발현량 변화를 분석한 결과이다.Figure 6 shows the results of analyzing the change in melanin expression level due to peptide treatment of the present invention in a 3D human skin model.

본 발명은 하기 화학식 1의 펩타이드 또는 이의 약학적으로 허용가능한 염을 포함하는, 피부 미백용 화장료 조성물을 제공한다.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.

<화학식 1><Chemical Formula 1>

Figure PCTKR2024018525-appb-img-000002
Figure PCTKR2024018525-appb-img-000002

본 발명의 화장료 조성물에 있어서, 상기 화학식 1의 펩타이드는 "Pro-Ala-Ile"으로도 표기될 수 있다. 상기 화학식 1의 펩타이드를 구성하는 아미노산은 독립적으로 L-아미노산 또는 D-아미노산의 형태일 수 있다. 상기 화학식 1의 펩타이드 유도체의 약학적으로 허용가능한 염은 예를 들어 산부가염 등을 포함하나, 이에 제한되는 것은 아니다.In the cosmetic composition of the present invention, the peptide of the chemical formula 1 may also be expressed as "Pro-Ala-Ile". The amino acids constituting the peptide of the chemical formula 1 may independently be in the form of L-amino acid or D-amino acid. Pharmaceutically acceptable salts of the peptide derivative of the chemical formula 1 include, but are not limited to, acid addition salts, for example.

본 발명의 화장료 조성물은 상기한 펩타이드를 유효성분으로 포함하는 기능성 화장료 조성물 형태일 수 있다. 상기 화장료 조성물은 통상의 화장료 제조방법에 따라, 다양한 형태로 제조될 수 있다. 예를 들어, 상기 화장료 조성물은 상기 펩타이드를 함유하는 향장 제품, 화장수, 크림, 로오숀 등의 형태로 제조될 수 있으며, 이는 통상의 클렌징액, 수렴액 및 보습액으로 희석하여 사용될 수 있다. 또한, 상기 화장료 조성물은 화장료 조성물 분야에서 통상적으로 사용되는 안정화제, 용해화제, 비타민, 안료, 및 향료와 같은 통상적인 보조제를 포함할 수 있다. 상기 화장료 조성물에 있어서, 상기 펩타이드의 함량은 피부 미백 효과를 달성하기에 유효한 양, 예를 들면 조성물 총 중량에 대하여 1 x 10-5 ∼ 1 x 10-2 중량%의 함량으로 함유될 수 있고, 바람직하게는 약 1 x 10-4 ∼ 1 x 10-3 중량%의 함량으로 함유될 수 있다.The cosmetic composition of the present invention may be in the form of a functional cosmetic composition containing the above-described peptide as an effective 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, and may be used by being diluted with a conventional cleansing solution, astringent, or moisturizing solution. In addition, the cosmetic composition may include conventional excipients such as a stabilizer, a solubilizer, vitamin, pigment, and fragrance commonly used in the field of cosmetic compositions. In the cosmetic composition, the content of the peptide may be contained in an amount effective to achieve a skin whitening effect, for example, in a content of 1 x 10 -5 to 1 x 10 -2 wt% based on the total weight of the composition, and preferably in a content of about 1 x 10 -4 to 1 x 10 -3 wt%.

이하, 본 발명을 실시예 및 시험예를 통하여 더욱 상세히 설명한다. 그러나, 이들 실시예 및 시험예는 본 발명을 예시하기 위한 것으로, 본 발명이 이들 실시예 및 시험예에 한정되는 것은 아니다.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.

실시예 1. 펩타이드의 합성Example 1. Synthesis of peptides

Pro-Ala-Ile으로 구성된 펩타이드는 자동화합성기(PeptrEx-R48, 펩트론사, 대전, 대한민국)를 이용하여 FMOC 고체-상 방법(FMOC solid-phase method)으로 합성하였다. 합성된 펩타이드는 C18 분석용 RP 컬럼(Shiseido capcell pak)을 사용한 역상 고속액체크로마토그래피(reverse-phase HPLC) (Prominence LC-20AB, Shimadzu사, 일본)로 정제 및 분석하였으며, 질량분석기(HP 1100 Series LC/MSD, Hewlett-Packard사, Roseville, 미국)를 이용하여 동정하였다. The peptide composed 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).

실시예 2. 펩타이드를 포함하는 조성물의 제조Example 2. Preparation of a composition containing a peptide

실시예 1에서 제조한 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)를 3차 증류수 용해시켜 1000 ppm 농도가 되도록 제조하였다. 얻어진 펩타이드 용액을 하기 시험예에서 사용하였다.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.

시험예 1: β-카테닌 결합 평가Test Example 1: Evaluation of β-catenin binding

α-MSH로 B16F10 세포에 자극을 주면 β-카테닌이 핵안으로 이동한다. 본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)가 β-카테닌에 결합하여 α-MSH 자극에 의해 핵으로 이동하는지를 면역형광염색법(Immunofluorescence)을 통해 확인하였다.When B16F10 cells are stimulated with α-MSH, β-catenin translocates into the nucleus. It was confirmed through immunofluorescence that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) binds to β-catenin and translocates into the nucleus by α-MSH stimulation.

(1) 시험재료(1) Test material

- 시험물질 준비- Test substance preparation

본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)를 3차 증류수에 용해시켜 1000 ppm의 농도가 되도록 제조하였다.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.

- 시험계- Test system

1) 세포주: 마우스 멜라노사이트(mouse melanocyte, B16F10, ATCC)1) Cell line: Mouse melanocyte (mouse melanocyte, B16F10, ATCC)

2) 세포관리: 세포주는 동결 보존한 것을 해동시켜 배양액이 담긴 100 ㎠ 동물세포 배양접시에 접종한 후 배양기(5% CO2, 37℃)에서 배양하고, 매 2~3일 마다 새로운 배양액으로 계대배양하였다.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.

3) 배지: DMEM(Dulbecco's Modified Eagle Medium)3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

조성: 10% 소태아혈청(Fetal bovine Serum), 1% 항생제 / 보관조건: 냉장보관 / 제조회사: GIBCOComposition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

- 시험부재료- Test materials

1) 면역형광염색법(Immuno- Fluorescence) 1) Immuno-Fluorescence

1-1) α-MSH1-1) α-MSH

보관 조건: -20℃ 냉동보관 / 제조 회사: SIGMA, M4135-1MG Storage conditions: -20℃ frozen storage / Manufacturer: SIGMA, M4135-1MG

1-2) 본 발명의 펩타이드-AMC1-2) Peptide-AMC of the present invention

보관 조건: -20℃ 냉동보관 / 제조 회사: 펩트론 Storage conditions: -20℃ frozen storage / Manufacturer: Peptron

1-3) 팔로이딘-로다민(Phalloidin-Rhodamin)1-3) Phalloidin-Rhodamin

보관 조건: -20℃ 냉동보관 / 제조 회사: : INVITROGEN, R415 Storage conditions: -20℃ frozen storage / Manufacturer: : INVITROGEN, R415

1-4) 항-β-카테닌(S33/S37/T41) 항체1-4) Anti-β-catenin (S33/S37/T41) antibodies

보관 조건: -20℃ 냉동보관 / 제조 회사: CELL SIGNALING, 4270 Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 4270

1-5) 항-래빗 IgG-FITC 항체1-5) Anti-rabbit IgG-FITC antibody

보관 조건: 4℃ 냉장보관 / 제조 회사: INVITROGENStorage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN

2) siRNA 형질 주입(siRNA transfection) 2) siRNA transfection

2-1) β-카테닌 siRNA(h)2-1) β-catenin siRNA(h)

보관 조건: -20℃ 냉동보관 / 제조 회사: SANTA CRUZ BIOTECHNOLOGY, sc-29209 Storage Conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-29209

2-2) Lipofectamine™ RNAiMAX Transfection Reagent2-2) Lipofectamine™ RNAiMAX Transfection Reagent

보관 조건: 4℃ 냉장보관 / 제조 회사: INVITROGEN, 13778075 Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

2-3) Opti-MEM® Medium2-3) Opti-MEM ® Medium

보관 조건: 4℃ 냉장보관 / 제조 회사: GIBCO, 31985062Storage Conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

(2) 시험방법(2) Test method

- 시험군의 구성- Composition of the test group

Figure PCTKR2024018525-appb-img-000003
Figure PCTKR2024018525-appb-img-000003

- 시험 과정- Exam process

1) 24-웰 배양 플레이트에 12 mm 현미경용 원형 커버글라스를 넣어 2.5X104 개의 세포를 각 웰에 분주한 후, 24시간 배양 후 세포의 단층 배양 상태를 확인하고 세포의 콘플루언시가 50% 이상일 때 시험을 진행하였다.1) 2.5 x 10 4 cells were seeded into each well of a 24-well culture plate with a 12 mm microscope circular cover glass. After culturing for 24 hours, the monolayer culture status of the cells was checked and the test was performed when the confluency of the cells was 50% or higher.

2) siRNA 처리를 위해 전용 배지(DMEM+10% FBS)로 배지를 교체하였다.2) The medium was replaced with a dedicated medium (DMEM + 10% FBS) for siRNA treatment.

3) siRNA + Opti-MEM® 배지와 transfection reagent + Opti-MEM® 배지를 1:1 비율로 만들어 천천히 섞어준 뒤, 5 분간 상온에서 반응시켜 주었다.3) siRNA + Opti-MEM ® medium and transfection reagent + Opti-MEM ® medium were mixed slowly in a 1:1 ratio and reacted at room temperature for 5 minutes.

4) 3)용액을 배양한 세포에 넣어 주고 48시간 동안 처리하여 시험을 진행하였다.4) 3) The solution was added to cultured cells and treated for 48 hours to conduct the test.

5) 본 발명의 펩타이드-AMC(1:100)을 16시간 동안 4℃에서 반응시킨 후 세척용액(PBS)으로 3회 세척하였다.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).

6) 세포골격을 염색하기 위해 팔로이딘-로다민(1:2000)을 상온에서 40분 반응시킨 후 세척용액(PBS)으로 3회 세척하였다.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).

7) β-카테닌 항체(1:100)를 처리하여 40분 동안 상온에서 반응시킨 후 세척용액(PBS)으로 3회 세척하였다.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).

8) 2차 항체(1:1000)을 처리하여 40분 동안 상온에서 반응시킨 후 세척용액(PBS)으로 3회 세척하였다.8) After treating with secondary antibody (1:1000), reacting at room temperature for 40 minutes, washing three times with washing solution (PBS).

9) 마운팅 용액을 사용하여 마운팅하였다.9) Mounting was performed using a mounting solution.

10) 세포에서 검출된 형광 신호(AMC)는 디지털 형광 이미징 시스템(LOGOS BIOSYSTEMS, CS20002)을 이용하여 관찰, 촬영하였다.10) The fluorescence signal (AMC) detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

- 결과 관찰 및 판정- Observe and judge the results

Control siRNA 음성 대조군을 기준으로 하여 α-MSH 및 β-카테닌 siRNA 처리군에서의 본 발명의 펩타이드-AMC의 핵이동 정도를 비교 분석하였다.The degree of nuclear translocation 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.

(3) 시험결과(3) Test results

음성대조군에서 β-카테닌는 세포질에 퍼져 있어 항체에 의해 약한 FITC (초록색 형광) 신호를 나타냈으나, α-MSH 처리군에서는 핵 안으로 이동한 후 β-카테닌이 축적되어 FITC 신호가 증가하는 것이 관찰되었다. 본 발명의 펩타이드-AMC (파란색 형광)은 α-MSH 처리군에서 핵 안으로 이동한 β-카테닌에 결합하여 핵 안에서 AMC (파란색 형광) 신호를 나타냈고, siRNA를 처리하여 β-카테닌의 발현을 억제시켰을 때는 AMC 신호가 감소되었다(도 1).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 inhibited by treatment with siRNA, the AMC signal was reduced (Fig. 1).

(4) 결론(4) Conclusion

음성 대조군과 비교하였을 때 α-MSH처리군에서 본 발명의 펩타이드-AMC가 핵으로 이동하는 것이 관찰되었고, β-카테닌 siRNA 처리군에서는 핵 안의 본 발명의 펩타이드-AMC 신호가 감소됨이 관찰되었다. 따라서 본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)는 β-카테닌에 특이적으로 결합하는 것으로 판단된다.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 decrease in the β-catenin siRNA treatment group. Therefore, it is judged that the peptide of the present invention (peptide composed of Pro-Ala-Ile) specifically binds to β-catenin.

시험예 2: β-카테닌 신호전달 억제 효능 평가Test Example 2: Evaluation of β-catenin signaling inhibition efficacy

본 발명의 펩타이드의 β-카테닌 길항제로서의 가능성을 검증하기 위하여 WNT(또는 α-MSH) 하위신호전달(downstream signaling) 경로인 β-카테닌/TCF의 상호작용의 변화를 in situ PLA를 통해 확인하였다.To verify the potential of the peptide of the present invention as a β-catenin antagonist, the change in the interaction of β-catenin/TCF, a downstream signaling pathway of WNT (or α-MSH), was confirmed through in situ PLA.

(1) 시험재료(1) Test material

- 시험물질 준비- Test substance preparation

본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)를 3차 증류수에 용해시켜 1000 ppm의 농도가 되도록 제조하였다.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.

- 시험계- Test system

1) 세포주: 마우스 멜라노사이트(mouse melanocyte, B16F10, ATCC)1) Cell line: Mouse melanocyte (mouse melanocyte, B16F10, ATCC)

2) 세포관리: 세포주는 동결 보존한 것을 해동시켜 배양액이 담긴 100 ㎠ 동물세포 배양접시에 접종한 후 배양기(5% CO2, 37℃)에서 배양하고, 매 2~3일 마다 새로운 배양액으로 계대배양하였다.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.

3) 배지: DMEM(Dulbecco's Modified Eagle Medium)3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

조성: 10% 소태아혈청(Fetal bovine Serum), 1% 항생제 / 보관조건: 냉장보관 / 제조회사: GIBCOComposition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

- 시험부재료- Test materials

1) α-MSH1) α-MSH

보관 조건: -20℃ 냉동보관 / 제조 회사: SIGMA, M4135-1MGStorage conditions: -20℃ frozen storage / Manufacturer: SIGMA, M4135-1MG

2) 항-β-카테닌(S33/S37/T41) 항체(1차 항체)2) Anti-β-catenin (S33/S37/T41) antibody (primary antibody)

보관 조건: -20℃ 냉동보관 / 제조 회사: CELL SIGNALING, 4270Storage Conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 4270

3) 항-TCF-4(D-4) 항체(1차 항체)3) Anti-TCF-4 (D-4) antibody (primary antibody)

보관 조건: -20℃ 냉동보관 / 제조 회사: SANTA CRUZ BIOTECHNOLOGY, SC-166699Storage Conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, SC-166699

4) NaveniFlex 100RM4) NaveniFlex 100RM

보관 조건: -20℃ 냉동보관 / 제조 회사: NaveniFlex, NV C-NF MR.100Storage Conditions: -20℃ frozen storage / Manufacturer: NaveniFlex, NV C-NF MR.100

5) Prolong™ diamond antifade mountant with DAPI5) Prolong™ diamond antifade mountant with DAPI

보관 조건: -20℃ 냉동보관 / 제조 회사: INVITROGEN, P36962Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

6) 디지털 형광 이미징 시스템(Digital Fluorescence Imaging System)6) Digital Fluorescence Imaging System

제조 회사: LOGOS BIOSYSTEMS, CS20002Manufacturer: LOGOS BIOSYSTEMS, CS20002

(2) 시험방법(2) Test method

- 시험군의 구성- Composition of the test group

Figure PCTKR2024018525-appb-img-000004
Figure PCTKR2024018525-appb-img-000004

- 시험 과정- Exam process

1) 24-웰 배양 플레이트에 12 mm 현미경용 원형 커버글라스를 넣어 2.5X104 개의 세포를 각 웰에 분주한 후, 24시간 배양 후 세포의 단층 배양 상태를 확인하고 세포의 콘플루언시가 50% 이상일 때 시험을 진행하였다.1) 2.5 x 10 4 cells were seeded into each well of a 24-well culture plate with a 12 mm microscope circular cover glass. After culturing for 24 hours, the monolayer culture status of the cells was checked and the test was performed when the confluency of the cells was 50% or higher.

2) 음성 대조, α-MSH 및 시험물질을 각 처리군의 농도에 맞게 1시간 동안 처리하였다.2) The negative control, α-MSH, and test substances were treated for 1 hour at the appropriate concentration for each treatment group.

3) 4% 파라포름알데히드를 이용하여 세포를 고정한 시킨 후, 0.1% Triton X-100으로 세포를 천공하여 세포 항체의 투과성을 높이는 전처리를 진행하였다.3) After fixing the cells using 4% paraformaldehyde, pretreatment was performed to increase the permeability of cell antibodies by perforating the cells with 0.1% Triton X-100.

4) 이후 시험은 In Situ PLA Kit(NaveniFlex 100RM)를 사용하여 진행하였고 시험은 제조사 지침에 따라 진행하였다.4) Subsequent tests were conducted using the In Situ PLA Kit (NaveniFlex 100RM) and the tests were conducted according to the manufacturer’s instructions.

5) PBS로 1회 세척 후, 블로킹 용액으로 37℃에서 30 분 동안 블로킹하였다.5) After washing once with PBS, blocking was performed with blocking solution at 37°C for 30 minutes.

6) 확인을 위한 2개의 항체를 항체 희석액에 10μg/mL 로 희석하여 4℃에서 16시간 반응 시키고 TTBS(0.01 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4)로 3회 세척하였다.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).

7) PLA 프로브를 넣어 37℃에서 1시간 동안 반응시킨 후, TTBS로 3회 세척하였다.7) The PLA probe was added and reacted at 37°C for 1 hour, then washed three times with TTBS.

8) Reaction A, B, C 를 차례로 처리하여 각각 60분, 30분, 90분 동안 37℃에서 반응시킨 후 마지막으로 TBS(0.01 M Tris, 0.15 M NaCl)로 2회 세척하고 DAPI(핵염색)가 포함된 마운팅 용액을 사용하여 마운팅하였다.8) Reactions A, B, and C were sequentially treated and reacted at 37°C for 60, 30, and 90 minutes, respectively, and finally washed twice with TBS (0.01 M Tris, 0.15 M NaCl) and mounted using a mounting solution containing DAPI (nuclear stain).

9) 세포에서 검출된 PLA신호는 디지털 형광 이미징 시스템(LOGOS BIOSYSTEMS, CS20002)을 이용하여 관찰, 촬영하였다.9) The PLA signal detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

- 결과 관찰 및 판정- Observe and judge the results

NIS-Elements BR3.1을 통해 PLA 형광 신호를 정량 분석하여 평가하였다. 음성 대조군을 기준으로 하여 α-MSH 및 시험물질 처리군에서의 β-카테닌/TCF의 상호작용에 의한 발광 신호를 비교 분석하였다.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 based on the negative control group.

(3) 시험결과(3) Test results

음성 대조군과 비교하였을 때 α-MSH 처리군에서 증가한 β-카테닌/TCF의 상호작용이 시험물질 처리군에서 농도의존적으로 억제됨이 관찰되었다(도 2).Compared to the negative control group, the interaction of β-catenin/TCF, which was increased in the α-MSH treatment group, was observed to be suppressed in a concentration-dependent manner in the test substance treatment group (Fig. 2).

(4) 결 론(4) Conclusion

시험물질이 α-MSH자극에 의한 β-카테닌 활성화를 억제하는 것을 확인하였다. 따라서 본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)는 β-카테닌 길항제로 작용가능한 것으로 판단된다.It was confirmed that the test substance inhibited β-catenin activation by α-MSH stimulation. Therefore, it is judged that the peptide of the present invention (peptide composed of Pro-Ala-Ile) can act as a β-catenin antagonist.

시험예 3: 피부 미백 효능 평가Test Example 3: Skin Whitening Efficacy Evaluation

WNT/β-카테닌 신호전달을 억제하면 멜라닌 합성에 중요한 역할을 하는 티로시나아제(Tyrosinase)의 활성을 저해하고 멜라닌 합성을 감소시킬 수 있다. 웨스턴 블랏 분석를 통해 티로시나아제 발현 정도를 관찰하고 멜라닌 함량 분석(Melanin contents assay)를 이용해 세포내 및 유리된 멜라닌 총량의 변화를 확인하여 본 발명의 펩타이드가 미백 효능이 있는지 확인하는 것이다.Inhibition of WNT/β-catenin signaling can inhibit the activity of tyrosinase, which plays an important role in melanin synthesis, and reduce melanin synthesis. The level of tyrosinase expression is observed through Western blot analysis, and the change in the total amount of intracellular and free melanin is confirmed using melanin contents assay to confirm whether the peptide of the present invention has a whitening effect.

(1) 시험재료(1) Test material

- 시험물질 준비- Test substance preparation

본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)를 3차 증류수에 용해시켜 1000 ppm의 농도가 되도록 제조하였다.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.

- 시험계- Test system

1) 세포주: 마우스 멜라노사이트(mouse melanocyte, B16F10, ATCC)1) Cell line: Mouse melanocyte (mouse melanocyte, B16F10, ATCC)

2) 세포관리: 세포주는 동결 보존한 것을 해동시켜 배양액이 담긴 100 ㎠ 동물세포 배양접시에 접종한 후, 배양기(5% CO2, 37℃)에서 배양하고, 매 2~3일 마다 새로운 배양액으로 계대배양하였다.2) Cell management: The cell line was frozen, thawed, and inoculated into a 100 cm2 animal cell culture dish containing culture medium. The cells were cultured in an incubator (5% CO2 , 37°C), and subcultured with new culture medium every 2–3 days.

3) 배지: DMEM(Dulbecco's Modified Eagle Medium)3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

조성: 10% 소태아혈청(Fetal bovine Serum), 1% 항생제 / 보관조건: 냉장보관 / 제조회사: GIBCOComposition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

- 시험부재료- Test materials

1) 웨스턴 블랏 분석 1) Western blot analysis

1-1) α-MSH1-1) α-MSH

보관 조건: -20℃ 냉동보관 / 제조 회사: SIGMA, M4135-1 Storage conditions: -20℃ frozen storage / Manufacturer: SIGMA, M4135-1

1-2) 항-티로시나아제(C-19) 항체1-2) Anti-tyrosinase (C-19) antibody

보관 조건: 4℃ 냉장보관 / 제조 회사: SANTACRUZ, SC-7833 Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTACRUZ, SC-7833

1-3) 항-β-actin 항체1-3) Anti-β-actin antibody

보관 조건: 4℃ 냉장보관 / 제조 회사: CEL SIGNALING, 4967S Storage Conditions: Refrigerated at 4℃ / Manufacturer: CEL SIGNALING, 4967S

1-4) NP40 세포 용해 완충액1-4) NP40 cell lysis buffer

보관 조건: -20℃ 냉동보관 / 제조 회사: INVITROGEN, FNN0021 Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

1-5) 소혈청 알부민(Bovine serum albumin, BSA)1-5) Bovine serum albumin (BSA)

보관 조건: 4℃ 냉장보관 / 제조 회사: CELLCONIC, FNN0021 Storage Conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

1-6) 단백질 분석 염색 시약(Protein assay dye reagent concentrate)1-6) Protein assay dye reagent concentrate

보관 조건: 4℃ 냉장보관 / 제조 회사: BIO_RAD, #5000006 Storage Conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

1-7) 단백질 블랏팅을 위한 Immuno-bolt® PVDF 막1-7) Immuno-bolt ® PVDF membrane for protein blotting

보관 조건: 실온보관 / 제조 회사: BIO-RAD, #1620177 Storage Conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

1-8) WEST SAVE GOLD,1-8) WEST SAVE GOLD,

보관 조건: 4℃ 냉장보관 / 제조 회사: AB FRONTIER, LF-QC0103 Storage Conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

1-9) 다빈치 웨스턴 이미징 시스템(Davinch western Imaging System)1-9) DaVinci Western Imaging System

제조 회사: DAVINCH-K, CAS-400SMManufacturer: DAVINCH-K, CAS-400SM

2) 멜라닌 함량 분석2) Melanin content analysis

2-1) 1M NaOH2-1) 1M NaOH

보관 조건: 상온보관 / 제조 회사 : SIGMA Storage conditions: Room temperature storage / Manufacturer: SIGMA

2-2) 마이크로플레이트 리더(Microplate reader)2-2) Microplate reader

제조 회사: BIO-TEK, EL808Manufacturer: BIO-TEK, EL808

(2) 시험방법(2) Test method

(2-1) 웨스턴 블랏 분석(2-1) Western blot analysis

- 시험군의 구성- Composition of the test group

Figure PCTKR2024018525-appb-img-000005
Figure PCTKR2024018525-appb-img-000005

- 시험 과정- Exam process

1) 6-웰 배양 플레이트에 1X106 개의 세포를 각 웰에 분주하였다. 24시간 배양 후 세포의 단층 배양 상태를 확인하고 세포의 콘플루언시가 30% 이상일 때 시험을 진행하였다.1) 1X106 cells were seeded into each well of a 6-well culture plate. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was performed when the cell confluency was 30% or higher.

2) 음성 대조, α-MSH 및 시험 물질을 각 처리군의 농도에 맞게 72시간 동안 처리하였다.2) The negative control, α-MSH, and test substances were treated for 72 hours at the appropriate concentrations for each treatment group.

3) NP40 세포 용해 완충액을 이용하여 세포를 용해하여, 브래드포드 분석(Bradford assay) 방법을 이용한 정량을 통해 전기영동용 세포추출물을 제조하였다.3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

4) 소듐 도데실 설페이트-폴리아크릴아미드 겔에 정량한 세포추출물을 각 웰에 20 ㎍씩 로딩하여 전기영동을 진행하였다.4) Electrophoresis was performed by loading 20 ㎍ of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

5) SDS-PAGE에 전개된 단백질을 PVDF 막으로 이송하였다5) The proteins developed in SDS-PAGE were transferred to a PVDF membrane.

6) PVDF 막에 블로킹 용액(3% BSA, 0.05% Tween 20, TBS)을 처리하여 상온에서 1시간 동안 반응하였다.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.

7) 1차 항체를 2시간 동안 상온에서 반응시키고, 세척용액(0.05% Tween 20, TBS)으로 3회 세척하였다.7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

8) 2차 항체를 1시간 동안 상온에서 반응시키고, 세척용액으로 5회 세척하였다.8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

9) 항체 검출 키트(Antibody detection kit)를 이용하여 감광한 후 웨스턴블랏 이미징 시스템을 통해 확인하였다.9) After photosensitization using an antibody detection kit, the result was confirmed using a Western blot imaging system.

- 결과 관찰 및 판정- Observe and judge the results

웨스턴블랏 이미징 시스템을 이용해 촬영하여 로딩 콘트롤(loading control)로 사용한 β-Actin의 발현량을 기준으로 시험물질 처리에 의한 티로시나아제의 발현량을 ImageJ을 통해 정량 분석하여 평가하였다.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 used as a loading control, which was captured using a Western blot imaging system.

(2-2) 멜라닌 함량 분석(2-2) Melanin content analysis

- 시험군의 구성- Composition of the test group

Figure PCTKR2024018525-appb-img-000006
Figure PCTKR2024018525-appb-img-000006

- 시험 과정- Exam process

1) 6-웰 배양 플레이트에 1X106 개의 세포를 각 웰에 분주하였다. 24시간 배양 후 세포의 단층 배양 상태를 확인하고 세포의 콘플루언시가 30% 이상일 때 시험을 진행하였다.1) 1X106 cells were seeded into each well of a 6-well culture plate. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was performed when the cell confluency was 30% or higher.

2) 음성 대조, α-MSH 및 시험 물질을 각 처리군의 농도에 맞게 72시간 동안 처리하였다.2) The negative control, α-MSH, and test substances were treated for 72 hours at the appropriate concentrations for each treatment group.

3) 세포외의 멜라닌 양 측정을 위해 세포 배양액은 1.5 mL 시험관에 옮겨 원심분리한 후 상층액을 96-웰 플레이트에 옮겨 마이크로플레이트 리더를 이용하여 400 nm에서 흡광도를 측정하였다.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.

4) 세포내의 멜라닌 양 측정을 위해 배양한 세포는 PBS로 세척하고 각 웰 마다 1M NaOH를 넣어 60℃에서 용해한 후 세포 용해액을 마이크로플레이트 리더를 이용하여 400 nm에서 흡광도를 측정하였다.4) To measure the amount of intracellular melanin, cultured cells were washed with PBS and lysed at 60°C in 1 M NaOH added to each well. The absorbance of the cell lysate was measured at 400 nm using a microplate reader.

- 결과 관찰 및 판정- Observe and judge the results

마이크로플레이트 리더를 이용하여 측정한 흡광도 값으로 세포내외의 총 멜라닌 양을 정량분석 평가하였다.The total amount of melanin inside and outside the cells was quantitatively analyzed and evaluated using absorbance values measured using a microplate reader.

(3) 시험결과(3) Test results

(3-1) 웨스턴 블랏 분석(3-1) Western blot analysis

음성 대조군과 비교하였을 때 α-MSH 처리군에서 티로시나아제 발현이 증가하였고, 시험물질 처리군에서는 농도의존적으로 감소함이 관찰되었다(도 3).Compared to the negative control group, tyrosinase expression increased in the α-MSH treatment group, and a concentration-dependent decrease was observed in the test substance treatment group (Fig. 3).

(3-2) 멜라닌 함량 분석(3-2) Melanin content analysis

음성 대조군과 비교하였을 때 α-MSH 처리군에서 증가한 세포의 총 멜라닌 생성량이 시험물질 처리군에서는 농도의존적으로 감소함이 관찰되었다(도 4)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).

(4) 결론(4) Conclusion

시험물질의 미백효능을 평가한 결과, 시험물질이 농도의존적으로 티로시나아제 활성 및 세포의 총 멜라닌 생성량을 유의하게 감소시켰다. 따라서, 본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)는 피부 미백 효력이 있는 것으로 판단된다.As a result of evaluating the whitening effect of the test substance, the test substance significantly reduced tyrosinase activity and total melanin production of cells in a concentration-dependent manner. Therefore, it is determined that the peptide of the present invention (peptide composed of Pro-Ala-Ile) has a skin whitening effect.

시험예 4: 자가포식촉진 효능 평가Test Example 4: Evaluation of Autophagy Promotion Efficacy

자가포식(Autophagy)은 세포 내 손상된 단백질이나 불필요한 세포 소기관을 분해해 에너지를 얻는 활동이다. 피부각질세포에 전달된 멜라닌 역시 자가포식 기능을 활성화해 분해할 수 있으므로, 시험물질이 WNT/β-카테닌 신호를 억제하여 자가포식을 유도하는지 대표적인 자기포식 표지인 LC3B의 발현을 웨스턴 블랏 분석를 통해 확인하였다.Autophagy is an activity that obtains energy by decomposing damaged proteins or unnecessary organelles within the cell. Since melanin delivered to keratinocytes can also be decomposed by activating the autophagy function, the expression of LC3B, a representative autophagy marker, was confirmed through Western blot analysis to determine whether the test substance induces autophagy by inhibiting WNT/β-catenin signaling.

(1) 시험재료(1) Test material

- 시험물질 준비- Test substance preparation

본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)를 3차 증류수에 용해시켜 1000 ppm의 농도가 되도록 제조하였다.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.

- 시험계- Test system

1) 세포주: 사람각질세포(Human Keratinocyte, HaCaT, CLS)1) Cell line: Human Keratinocyte (HaCaT, CLS)

2) 세포관리: 세포주는 동결 보존한 것을 해동시켜 배양액이 담긴 100 ㎠ 동물세포 배양접시에 접종한 후 배양기(5% CO2, 37℃)에서 배양하고, 매 2~3일 마다 새로운 배양액으로 계대배양하였다.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.

3) 배지: DMEM(Dulbecco's Modified Eagle Medium)3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

조성: 10% 소태아혈청(Fetal bovine Serum), 1% 항생제 / 보관조건: 냉장보관 / 제조회사: GIBCOComposition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

- 시험부재료- Test materials

1) 항-LC3B 항체1) Anti-LC3B antibody

보관 조건: -20℃ 냉동보관 / 제조 회사: CELL SIGNALING, 3868SStorage Conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 3868S

2) 고우트 항-래빗 IgG Fc-HRP2) Goat anti-rabbit IgG Fc-HRP

보관 조건: 4℃ 냉장보관 / 제조 회사: ABFRONTEIRStorage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

3) NP40 세포 용해 완충액3) NP40 cell lysis buffer

보관 조건: -20℃ 냉동보관 / 제조 회사: INVITROGEN, FNN0021Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

4) 소혈청 알부민(Bovine serum albumin, BSA)4) Bovine serum albumin (BSA)

보관 조건: 4℃ 냉장보관 / 제조 회사: CELLCONIC, FNN0021Storage Conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

5) 단백질 분석 염색 시약(Protein assay dye reagent concentrate)5) Protein assay dye reagent concentrate

보관 조건: 4℃ 냉장보관 / 제조 회사: BIO_RAD, #5000006Storage Conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

6) 단백질 블랏팅을 위한 Immuno-bolt® PVDF 막 6) Immuno-bolt ® PVDF membrane for protein blotting

보관 조건: 실온보관 / 제조 회사: BIO-RAD, #1620177Storage Conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

7) WEST SAVE GOLD,7) WEST SAVE GOLD,

보관 조건: 4℃ 냉장보관 / 제조 회사: AB FRONTIER, LF-QC0103Storage Conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

8) 다빈치 웨스턴 이미징 시스템(Davinch western Imaging System) 8) DaVinci Western Imaging System

제조 회사: DAVINCH-K, CAS-400SMManufacturer: DAVINCH-K, CAS-400SM

(2) 시험방법(2) Test method

- 시험군의 구성- Composition of the test group

Figure PCTKR2024018525-appb-img-000007
Figure PCTKR2024018525-appb-img-000007

- 시험 과정- Exam process

1) 6-웰 배양 플레이트에 5X106 개의 세포를 각 웰에 분주하였다. 24시간 배양 후 세포의 단층 배양 상태를 확인하고 세포의 콘플루언시가 80% 이상일 때 시험을 진행하였다.1) 5X106 cells were seeded into each well of a 6-well culture plate. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was performed when the cell confluency was 80% or higher.

2) 음성 대조 및 시험 물질을 각 처리군의 농도에 맞게 24시간 동안 처리하였다.2) The negative control and test substances were treated for 24 hours at the appropriate concentration for each treatment group.

3) NP40 세포 용해 완충액을 이용하여 세포를 용해하여, 브래드포드 분석(Bradford assay) 방법을 이용한 정량을 통해 전기영동용 세포추출물을 제조하였다.3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

4) 소듐 도데실 설페이트-폴리아크릴아미드 겔에 정량한 세포추출물을 각 웰에 20 ㎍씩 로딩하여 전기영동을 진행하였다.4) Electrophoresis was performed by loading 20 ㎍ of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

5) SDS-PAGE에 전개된 단백질을 PVDF 막으로 이송하였다5) The proteins developed in SDS-PAGE were transferred to a PVDF membrane.

6) PVDF 막에 블로킹 용액(3% BSA, 0.05% Tween 20, TBS)을 처리하여 상온에서 1시간 동안 반응하였다.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.

7) 1차 항체를 2시간 동안 상온에서 반응시키고, 세척용액(0.05% Tween 20, TBS)으로 3회 세척하였다.7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

8) 2차 항체를 1시간 동안 상온에서 반응시키고, 세척용액으로 5회 세척하였다.8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

9) 항체 검출 키트(Antibody detection kit)를 이용하여 감광한 후 웨스턴블랏 이미징 시스템을 통해 확인하였다.9) After photosensitization using an antibody detection kit, the result was confirmed using a Western blot imaging system.

- 결과 관찰 및 판정- Observe and judge the results

웨스턴블랏 이미징 시스템을 이용해 촬영하여 로딩 콘트롤(loading control)로 사용한 β-Actin의 발현량을 기준으로 시험물질 처리에 의한 LC3B의 발현량을 ImageJ을 통해 정량 분석하여 평가하였다.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 used as a loading control, which was captured using a Western blot imaging system.

(3) 시험결과(3) Test results

음성 대조군과 비교하였을 때 시험물질 처리군에서 자가포식 표지단백질인 LC3B의 발현량이 농도의존적으로 증가됨이 관찰되었다(도 5).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).

(4) 결론(4) Conclusion

시험물질의 자가포식작용유도 효능을 평가한 결과 시험물질이 농도의존적으로 LC3B의 발현을 촉진하는 것이 확인되었다. 따라서 본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드) 자가포식작용을 촉진하는 효능이 있는 것으로 판단된다.As a result of evaluating the autophagy-inducing efficacy of the test substance, it was confirmed that the test substance promoted the expression of LC3B in a concentration-dependent manner. Therefore, it is judged that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) has the efficacy of promoting autophagy.

시험예 5: 3D 사람피부모델에서 미백 효능 평가Test Example 5: Evaluation of Whitening Efficacy in a 3D Human Skin Model

상기 시험을 통해 세포단위에서 시험물질이 β-카테닌을 억제하여 미백효능을 보이는 것이 확인되었다. 시험물질이 사람 피부와 유사한 3D 사람피부모델(Neoderm-ED)에서도 미백 효능을 보이는지 폰타나 마손 염색(Fontana Massons Staining)을 통해 확인하였다.Through the above test, it was confirmed that the test substance exhibited whitening efficacy by inhibiting β-catenin at the cellular level. It was confirmed through Fontana Masson Staining whether the test substance also exhibited whitening efficacy in a 3D human skin model (Neoderm-ED) similar to human skin.

(1) 시험재료(1) Test material

- 시험물질 준비- Test substance preparation

본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)를 3차 증류수에 용해시켜 1000 ppm의 농도가 되도록 제조하였다.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.

- 대조물질- Control material

양성 대조물질(L-아스코르브산)을 증류수에 용해시켜 1 mg/ml의 농도가 되도록 제조하였다.The positive control substance (L-ascorbic acid) was dissolved in distilled water to prepare a concentration of 1 mg/ml.

- 시험계- Test system

1) 3D사람피부모델 : Neoderm-ME1) 3D human skin model: Neoderm-ME

2) 관리: 배양기(5% CO2, 37℃ )에서 배양하고, 수령 후 3일이내에 시험하였다.2) Management: Cultured in an incubator (5% CO2 , 37℃) and tested within 3 days of receipt.

3) 배지: Maintenance medium3) Badge: Maintenance medium

조성: 10% 소태아혈청(Fetal bovine Serum) / 보관조건: 냉장보관 / 제조회사: TEGO SCIENCEComposition: 10% Fetal bovine serum / Storage conditions: Refrigerated storage / Manufacturer: TEGO SCIENCE

- 시험부재료- Test materials

1) VLX-3W research radiometer 1) VLX-3W research radiometer

제조회사: VILVERManufacturer: VILVER

2) Solvable TM 용해 완충액2) Solvable TM dissolution buffer

보관 조건: 상온보관/ 제조회사: PerkinElmer, 6NE9100Storage Conditions: Room Temperature Storage/ Manufacturer: PerkinElmer, 6NE9100

3) 폰타나 마손 염색 키트(Fontana-Masson Stain kit)3) Fontana-Masson Stain kit

보관 조건: 4℃ 냉장보관 / 제조회사: ABCAM, ab150669Storage Conditions: Refrigerated at 4℃ / Manufacturer: ABCAM, ab150669

4) 현미경4) Microscope

제조 회사: OLYMPUS, BX53F2Manufacturer: OLYMPUS, BX53F2

(2) 시험방법(2) Test method

(2-1) 폰타나 마손 염색(2-1) Fontana Masson dyeing

- 시험군의 구성- Composition of the test group

Figure PCTKR2024018525-appb-img-000008
Figure PCTKR2024018525-appb-img-000008

- 시험 과정- Exam process

1) Neoderm-ME를 수령한 후 전용 배지를 첨가하여 24시간 동안 배양하였다.1) After receiving Neoderm-ME, a dedicated medium was added and cultured for 24 hours.

2) UVB 처리군의 경우 UV조사기를 이용하여 0.06 J/cm2 의 강도로 UVB를 조사하였다.2) For the UVB treatment group, UVB was irradiated at an intensity of 0.06 J/cm 2 using a UV irradiator.

3) 음성, 양성 대조 및 시험 물질을 각 처리군의 농도에 맞게 48시간 동안 처리하였다.3) The negative, positive control, and test substances were treated for 48 hours at the appropriate concentration for each treatment group.

4) Neoderm-ME는 블레이드(blade)를 이용하여 인서트 웰(insert well)로부터 분리하여 파라핀 블럭(Paraffin block)을 제작하였다.4) Neoderm-ME was separated from the insert well using a blade to create a paraffin block.

5) 4 ㎛ 두께로 섹션하여 슬라이드를 준비하였다.5) Slides were prepared by cutting sections at 4 ㎛ thickness.

6) 자일렌(Xylene)을 이용한 파라핀 세척 및 함수 과정(Et-OH 100% >95%>90%>80%>70%)을 진행하였다.6) Paraffin washing and water rehydration process using xylene (Et-OH 100% >95%>90%>80%>70%) was performed.

7) 이후 시험은 폰타나 마손 염색 키트를 사용하여 진행하였고 시험은 제조사 지침에 따라 진행하였다.7) Subsequent tests were conducted using the Fontana Masson staining kit and the tests were conducted according to the manufacturer's instructions.

7) 염화금 용액(Gold Chloride Solution)(0.2%)을 상온에서 30초간 반응시킨 후 증류수로 여러 번 세척하였다.7) After reacting with gold chloride solution (0.2%) at room temperature for 30 seconds, it was washed several times with distilled water.

8) 소듐 티오설페이트 용액(5%)를 상온에서 2분간 반응시킨 후 2분간 흐르는 물에 세척한 후 증류수로 2번 세척하였다.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.

9) Nuclear Fast Red Solution으로 상온에서 5분간 반응시킨 후 2분간 흐르는 물에 세척한 후 증류수로 2번 세척하였다.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.

10) 100% 에탈올에 3번 반응시켜 탈수 과정을 진행한 후 마운팅하였다.10) After dehydration by reacting three times with 100% ethanol, mounting was performed.

11) 조직에서 검출된 염색은 현미경을 이용하여 관찰, 촬영하였다.11) The staining detected in the tissue was observed and photographed using a microscope.

- 결과 관찰 및 판정- Observe and judge the results

음성 대조군을 기준으로 하여 양성 대조군 및 시험물질 처리군에서의 멜라닌 생성 정도를 비교 분석 하였다.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.

(2-2) 멜라닌 함량 분석(2-2) Melanin content analysis

- 시험군의 구성- Composition of the test group

Figure PCTKR2024018525-appb-img-000009
Figure PCTKR2024018525-appb-img-000009

- 시험 과정- Exam process

1) Neoderm-ME를 수령한 후 전용 배지를 첨가하여 24시간 동안 배양하였다.1) After receiving Neoderm-ME, a dedicated medium was added and cultured for 24 hours.

2) UVB 처리군의 경우 UV조사기를 이용하여 0.06 J/cm2 의 강도로 UVB를 조사하였다.2) For the UVB treatment group, UVB was irradiated at an intensity of 0.06 J/cm 2 using a UV irradiator.

3) 음성, 양성 대조 및 시험 물질을 각 처리군의 농도에 맞게 48시간 동안 처리하였다.3) The negative, positive control, and test substances were treated for 48 hours at the appropriate concentration for each treatment group.

4) 블레이드(blade)를 이용하여 Neoderm-ME를 인서트(insert)의 가장자리로부터 도려내듯이 분리하였다.4) Neoderm-ME was separated from the edge of the insert by cutting it using a blade.

5) 분리한 Neoderm-ME 조직을 1.5 ml 시험관에 담고 PBS를 완전히 제거하였다.5) The separated Neoderm-ME tissue was placed in a 1.5 ml test tube and PBS was completely removed.

6) Neoderm-ME 가 담긴 시험관에 용해 완충액을 360 μl씩 첨가하였다.6) 360 μl of dissolution buffer was added to each test tube containing Neoderm-ME.

7) 상온에서 5~10분간 반응시킨 후, 막이 Neoderm-ME 조직으로부터 분리되면 포셉으로 막을 제거하였다.7) After reacting at room temperature for 5 to 10 minutes, the membrane was separated from the Neoderm-ME tissue and removed with forceps.

8) 95℃ 히트 블록(heat block)에서 45분 동안 반응시켰다.8) The reaction was carried out in a 95℃ heat block for 45 minutes.

9) 13,000 rpm으로 실온에서 10분 동안 원심분리 한 후 상층액을 튜브에 옮겼다.9) After centrifugation at room temperature for 10 minutes at 13,000 rpm, the supernatant was transferred to a tube.

10) 96-웰 플레이트에 시험물질을 200 μl씩 옮겨 마이크로플레이트 리더를 이용하여 흡광도(405 nm)를 측정하였다.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.

- 결과 관찰 및 판정- Observe and judge the results

음성 대조군을 기준으로 하여 양성대조군 및 시험물질 처리군에서의 멜라닌 생성 정도를 비교 분석하였다.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.

(3) 시험결과(3) Test results

3D 사람피부모델에서 UVB에 의해 유도되는 멜라닌 생성에 대한 시험물질의 억제 효능을 평가한 결과, 음성 대조군과 비교하였을 때 UVB처리군에서 증가한 멜라닌량이 양성대조군 및 시험물질 처리군에서는 감소됨이 확인되었다(도 6).As a result of evaluating the inhibitory efficacy of the test substance on melanin production induced by UVB in a 3D human skin model, it was confirmed that the amount of melanin increased in the UVB treatment group compared to the negative control group was decreased in the positive control group and the test substance treatment group (Fig. 6).

(4) 결론(4) Conclusion

시험물질이 양성대조군과 동등 이상의 효능으로 피부모델의 총 멜라닌 생성량을 유의하게 감소시키는 것이 확인되었다. 따라서, 본 발명의 펩타이드(Pro-Ala-Ile으로 구성된 펩타이드)는 세포단위에서 뿐만 아니라 피부모델에서도 미백 효력이 있는 것으로 판단된다.It was confirmed that the test substance significantly reduced the total melanin production of the skin model with an efficacy equal to or greater than that of the positive control group. Therefore, it is judged that the peptide of the present invention (a peptide composed of Pro-Ala-Ile) has a whitening effect not only at the cell level but also in the skin model.

Claims (1)

하기 화학식 1의 펩타이드 또는 이의 약학적으로 허용가능한 염을 포함하는, 피부 미백용 화장료 조성물.A cosmetic composition for skin whitening, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof. <화학식 1><Chemical Formula 1>
Figure PCTKR2024018525-appb-img-000010
Figure PCTKR2024018525-appb-img-000010
PCT/KR2024/018525 2023-11-22 2024-11-21 Composition for skin whitening Pending WO2025110757A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002068601A2 (en) * 2001-02-28 2002-09-06 Skubitz Keith M Small peptides capable of modulating the function of cd66 (ceacam) family members
WO2016105375A1 (en) * 2014-12-23 2016-06-30 Avon Products, Inc. Peptides and their use in the treatment of skin
WO2016204841A1 (en) * 2015-06-17 2016-12-22 Avon Products, Inc. Peptides and their use in the treatment of skin
EP2510982B1 (en) * 2006-05-05 2017-04-26 Sederma Cosmetic compositions comprising at least one peptide with at least one immobilized aromatic cycle
WO2020142103A1 (en) * 2019-01-04 2020-07-09 Avon Products, Inc. Oxidized derivatives of gdf-11 fragments

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002068601A2 (en) * 2001-02-28 2002-09-06 Skubitz Keith M Small peptides capable of modulating the function of cd66 (ceacam) family members
EP2510982B1 (en) * 2006-05-05 2017-04-26 Sederma Cosmetic compositions comprising at least one peptide with at least one immobilized aromatic cycle
WO2016105375A1 (en) * 2014-12-23 2016-06-30 Avon Products, Inc. Peptides and their use in the treatment of skin
WO2016204841A1 (en) * 2015-06-17 2016-12-22 Avon Products, Inc. Peptides and their use in the treatment of skin
WO2020142103A1 (en) * 2019-01-04 2020-07-09 Avon Products, Inc. Oxidized derivatives of gdf-11 fragments

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