Retinoic acid receptor activating peptide and cosmetic composition comprising same

Abstract

The present invention relates to a peptide activating a retinoic acid receptor and a cosmetic composition using same.

Description

Retinoic acid receptor activating peptide and cosmetic composition containing the same

[0001] The present invention relates to a peptide that activates a retinoic acid receptor and a cosmetic composition using the same.

[0002] The retinoic acid receptor (RAR) is required for retinoic acid, the active form of vitamin A, to function. It binds to specific genes and controls their transcription. Furthermore, the RA receptor is known to be involved in melanin production, wrinkle formation, and anti-aging (skin regeneration).

[0003] Accordingly, a substance is being developed to treat related diseases by controlling the action of the retinoic acid receptor, as described in the following patent document.

[0004] <Patent Document>

[0005] Patent Publication No. 10-1990-07000601 (published on August 16, 1990) "Retinoic Acid Receptor Composition and Preparation Method Thereof"

[0006] However, existing substances that control the action of retinoic acid receptors are either not effective enough or are mostly chemically synthesized, which can cause side effects when used.

[0007] The present invention has been devised to solve the above problems.

[0008] The purpose of the present invention is to provide a peptide that activates a retinoic acid receptor and a cosmetic composition using the same, which can be used for wrinkle improvement, skin whitening, and skin regeneration (anti-aging).

[0009] In order to achieve the above-mentioned purpose, the present invention is implemented by an embodiment having the following configuration.

[0010] According to one embodiment of the present invention, the peptide according to the present invention is characterized in that it consists of an amino acid sequence of SEQ ID NO: 1 or 2.

[0011] According to another embodiment of the present invention, the peptide according to the present invention is characterized by activating a retinoic acid receptor.

[0012] According to another embodiment of the present invention, the peptide according to the present invention is characterized in that it is used for skin whitening, wrinkle improvement or skin regeneration.

[0013] According to another embodiment of the present invention, the cosmetic composition according to the present invention is characterized in that it comprises a peptide consisting of an amino acid sequence of SEQ ID NO: 1 or 2.

[0014] According to another embodiment of the present invention, the peptide according to the present invention is characterized in that it comprises a peptide consisting of an amino acid sequence of SEQ ID NO: 1 or 2.

[0015] According to another embodiment of the present invention, a cosmetic composition for skin whitening according to the present invention is characterized in that it comprises a peptide consisting of an amino acid sequence.

[0016] According to another embodiment of the present invention, a cosmetic composition for improving skin wrinkles according to the present invention is characterized in that it comprises a peptide consisting of an amino acid sequence of SEQ ID NO: 1 or 2.

[0017] The present invention can obtain the following effects through the above-described examples.

[0018] The present invention has the effect of promoting wrinkle improvement, whitening, and regeneration (anti-aging) of the skin by activating a retinoic acid receptor using a peptide consisting of an amino acid sequence of sequence number 1 or 2.

[0019] Figure 1 is a graph showing the effect of a peptide according to one embodiment of the present invention on the physical binding of RAR and RXR.

[0020] Figure 2 is a graph showing the effect of a peptide according to one embodiment of the present invention on the physical binding of P53 and POLII.

[0021] Figure 3 is a graph showing the effect of a peptide according to one embodiment of the present invention on inducing intracellular collagen synthesis.

[0022] Hereinafter, the retinoic acid receptor activating peptide according to the present invention and the cosmetic composition containing the same will be described in detail with reference to the attached drawings. Unless otherwise defined, all terms in this specification have the same general meaning as those terms understood by a person skilled in the art to which the present invention pertains, and if there is a conflict with the meaning of a term used in this specification, the definitions used in this specification will apply. In addition, detailed descriptions of well-known functions and structures that may unnecessarily obscure the gist of the present invention will be omitted. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components can be included, but rather that other components can be included, unless specifically stated otherwise.

[0023]

[0024] One embodiment of the present invention relates to a peptide that activates a retinoic acid receptor, wherein the peptide is characterized by having an amino acid sequence of SEQ ID NO: 1 (Ile-Gln-Glu-Met) or 2 (Thr-Val-Glu-Phe). The peptide can improve the condition of the skin by activating the retinoic acid receptor. Specifically, the peptide can promote wrinkle improvement, skin whitening, and skin regeneration (anti-aging). The peptide can be used in cosmetic compositions, therapeutic compositions, etc.

[0025]

[0026] Another embodiment of the present invention relates to a cosmetic composition comprising a peptide comprising an amino acid sequence of SEQ ID NO: 1 or 2, wherein the cosmetic composition can promote wrinkle improvement, skin whitening, and skin regeneration (anti-aging). Various known ingredients that can provide formulation stability and additional skin condition improvement effects can be added to the cosmetic composition.

[0027]

[0028] Hereinafter, the present invention will be described in more detail through examples. However, these examples are provided solely to illustrate the present invention in more detail and are not intended to limit the scope of the present invention.

[0029]

[0030] <Example 1> Synthesis of peptides

[0031]

[0032] Peptides of sequence numbers 1 to 2 listed in Table 1 below were synthesized by the FMOC solid-phase method using an automated synthesizer (PeptrEx-R48, Peptron, Korea). The synthesized peptides were purified and analyzed by reverse-phase high-performance liquid chromatography (Prominence LC-20AB, Shimadzu, Japan) using a C18 analytical RP column (Shiseido capcell pak), and identified using a mass spectrometer (HP 1100 Series LC / MSD, Hewlett-Packard, USA).

[0033]

[0034]

[0035] <Example 2> Confirmation that the peptide of Example 1 activates the retinoic acid receptor and increases the physical binding between RAR and RXR.

[0036]

[0037] 1. Retinol regulates transcription of target DNA by inducing binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR). To confirm whether the peptide of Example 1 activates binding of RXR / RAR, the effect on physical binding of RAR and RXR was evaluated using the in situ PLA (proximity ligation assay) method.

[0038] 2. Specifically, 4.5 × 10 in each well of a 24-well microplate 4 HaCaT cells (human keratinocytes) of dogs were seeded in DMEM serum medium and cultured for 24 hours in a 37℃, 5% CO2 incubator, and then treated with each peptide of Example 1 and incubated for 30 minutes under the same conditions. The concentration of each peptide in the medium was 1 and 10 μM, and untreated cells were used as a control group. Afterwards, the cells in each well were washed with PBS and fixed by treating with 2% formaldehyde for 15 minutes, and then treated with 0.1% TritonX-100 for 5 minutes to increase antibody permeability into the cells. Anti-RAR monoclonal antibody (Santa Cruz, USA) and anti-RXR polyclonal antibody (Santa Cruz, USA) were added, and PLA probes were added using an in situ PLA kit (Sigma-Aldrich), followed by hybridization, ligation, amplification, and mounting steps.

[0039] 3. Afterwards, the luminescence signals (PLA signals) detected in each cell were measured using a fluorescence microscope (logosbio / CelenaS, Korea) to quantify the physical interaction between RAR and RXR antibodies, and the results are shown in Figure 1.

[0040] 4. Referring to Figure 1, it can be confirmed that when Raragin A to B of Example 1 are treated, the physical binding between RAR and RXR is increased compared to the untreated control group. In other words, Raragin A to B activate RAR, thereby increasing the physical binding between RAR and RXR, resulting in an effect similar to retinol.

[0041]

[0042] <Example 3> Confirmation that the peptide of Example 1 activates the retinoic acid receptor and increases the physical binding between P53 and POLII.

[0043]

[0044] 1. To confirm whether the peptide of Example 1 increases the transcriptional activity of P53, which is related to cell anti-aging, the peptide of Example 1 was treated to cells, and then the effect on the physical binding of P53 and POLII (RNA Polymerase II) was evaluated through the in situ PLA (proximity ligation assay) method.

[0045] 2. Specifically, 4.5 × 10 in each well of a 24-well microplate 4HaCaT cells (human keratinocytes) of dogs were seeded in DMEM serum medium and cultured for 24 hours in a 37℃, 5% CO2 incubator, and then treated with each peptide of Example 1 and incubated for 30 minutes under the same conditions. The concentration of each peptide in the medium was 1 and 10 μM, and untreated cells were used as a control group. Afterwards, the cells in each well were washed with PBS and fixed by treating with 2% formaldehyde for 15 minutes, and then treated with 0.1% TritonX-100 for 5 minutes to increase antibody permeability into the cells. Anti-P53 polyclonal antibody (CST, USA) and anti-POLII monoclonal antibody (Santa Cruz, USA) were added, and PLA probes were added using an in situ PLA kit (Sigma-Aldrich), followed by hybridization, ligation, amplification, and mounting steps.

[0046] 3. Afterwards, the luminescence signals (PLA signals) detected in each cell were measured using a fluorescence microscope (logosbio / CelenaS, Korea) to quantify the physical interaction between P53 and POLII antibodies, and the results are shown in Figure 2.

[0047] 4. Referring to Figure 2, it can be confirmed that when Raragin A to B of Example 1 are treated, the physical binding between P53 and POLII is increased compared to the untreated control group. In other words, it can be seen that Raragin A to B prevent cell aging by increasing the transcriptional activity of P53.

[0048]

[0049] <Example 4> Evaluation of the effect of the peptide of Example 1 on intracellular collagen synthesis

[0050]

[0051] 1. Immunofluorescence analysis was performed to evaluate the effect of the peptide of Example 1 on intracellular collagen synthesis.

[0052] 2. Specifically, 4.5 X 10 in each well of a 24-well microplate 4 Human dermal fibroblasts were seeded with medium (CEFOgro Human MSC Growth Medium) and cultured in a 37℃, 5% CO2 incubator for 24 hours, and then treated with each peptide of Example 1 and incubated for 24 hours under the same conditions. The concentration of each peptide in the medium was 1 and 10 μM, and the control group was untreated and the group treated with 1 μM Retinol. The cells in each well were washed with PBS, fixed by treating with 4% formaldehyde for 15 minutes, and then treated with 0.1% TritonX-100 for 5 minutes to increase antibody permeability into the cells. The cells were washed with PBS solution and treated with anti-COL1A2 polyclonal antibody (Santa Cruz, CA, USA) for 1 hour, and then treated with mouse anti-goat IgG-FITC for 40 minutes. For cytoskeletal staining, phalloidin-rhodamine was used for 40 minutes. After mounting with a reagent containing DAPI for nuclear staining, the cells were observed under a fluorescence microscope (Logosbio / CelenaS, Korea). The results were quantified using a program (NIS-Eliments-3.1), and are shown in Figure 3.

[0053] 3. Looking at Figure 3, it can be confirmed that when Raragin A to B of Example 1 were treated, collagen synthesis was significantly increased compared to the untreated control group.

[0054]

[0055] <Example 5> Evaluation of whether the peptide of Example 1 inhibits melanin synthesis in cells

[0056]

[0057] 1. In order to evaluate the effect of the peptide of Example 1 on melanin synthesis in cells, it was evaluated whether the peptide of Example 1 inhibits melanin synthesis under conditions in which melanocytes were stimulated with α-MSH to promote melanin synthesis.

[0058] 2. Specifically, 1×10 in each well of a 6-well microplate 5 B16F10 (mouse melanocytes) cells were seeded in DMEM serum medium and cultured for 24 hours in a 37°C, 5% CO2 incubator. After treatment with 100 nM α-Melanocyte stimulating hormone (α-MSH) and each of the peptides of Example 1 in the medium, the cells were incubated under the same conditions for 72 hours. The concentration of each peptide in the medium was 1 and 10 μM. As a control group, the group without any treatment, the group treated with only α-MSH, and the group treated with 1 μM Retinol were used. Afterwards, to measure the amount of extracellular melanin, the supernatant was taken, and the melanin released in the medium was measured for absorbance at 400 nm. To measure the amount of intracellular melanin, the cells were washed with PBS after removing the medium, and then lysed with 1 M NaOH at 60°C for 30 minutes and the absorbance was measured at 400 nm. The amount of free melanin and the amount of intracellular melanin were quantified by measuring the absorbance, and the results are shown in Table 2.

[0059] 3. Looking at Table 2, it can be confirmed that when Raragin A to B of Example 1 were treated, the increase in melanin induced by α-MSH stimulation was reduced compared to the untreated control group. In other words, it can be confirmed that the peptide of Example 1 has a whitening effect.

[0060]

[0061]

[0062] <Example 6> Skin patch test of cosmetic composition containing peptide of Example 1

[0063]

[0064] 1. Each of the peptides manufactured in Example 1 was added to a commercially available solubilized ampoule base at a concentration of 50 ppm to manufacture a cosmetic composition. Cosmetic composition 1 represents the solubilized ampoule base to which the peptide of Example 1 was not added, cosmetic composition 2 represents the case where Raragin A was added, and cosmetic composition 3 represents the case where Raragin B was added.

[0065] 2. Nine adults in their 20s to 60s were instructed to apply the same amount of the above cosmetic composition to their faces every morning and evening for six weeks. After six weeks, a sensory evaluation was conducted on skin improvement, and the results are shown in Table 3. The sensory evaluation items included whether there was skin irritation, the degree of wrinkle improvement, the degree of skin color improvement (whitening), and the degree of skin regeneration (comprehensively considering increased elasticity and reduced scars). The condition was evaluated as good with a score of 5, and bad with a score of 0.

[0066] 3. Looking at Table 3, it can be confirmed that cosmetic compositions 2 and 2 receive higher scores than cosmetic composition 1, indicating that the peptide of Example 1 can be used in a cosmetic composition to improve wrinkle improvement, skin whitening, and skin regeneration effects.

[0067]

[0068]

[0069] In the above, the applicant has described preferred embodiments of the present invention, but such embodiments are only examples of implementing the technical idea of ​​the present invention, and any change or modification that implements the technical idea of ​​the present invention should be interpreted as falling within the scope of the present invention.

Claims

1. A peptide consisting of the amino acid sequence of sequence number 1 or 2.

2. In paragraph 1, The above peptide is a peptide characterized by activating a retinoic acid receptor.

3. In paragraph 1, The above peptide is characterized in that it is used for skin whitening, wrinkle improvement or skin regeneration.

4. A cosmetic composition characterized by comprising a peptide consisting of an amino acid sequence of sequence number 1 or 2.

5. A composition for skin whitening, wrinkle improvement or skin regeneration, characterized in that it comprises a peptide consisting of an amino acid sequence of sequence number 1 or 2.

6. A cosmetic composition for skin whitening, characterized in that it comprises a peptide consisting of an amino acid sequence of sequence number 1 or 2.

7. A cosmetic composition for improving skin wrinkles, characterized in that it comprises a peptide consisting of an amino acid sequence of sequence number 1 or 2.

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