Vitamin D receptor activating peptide and Cosmetic composition including the same

Abstract

The present invention relates to a peptide that activates vitamin D receptors and a cosmetic composition using the same.

Description

Technology Field

[0001] The present invention relates to a peptide that activates vitamin D receptors and a cosmetic composition using the same. Background Technology

[0002] Vitamin D is an important hormone for bone growth and maintenance, as well as for maintaining mineral homeostasis. It binds to vitamin D receptors (VDRs) present in various tissues and cells within the human body, thereby regulating gene expression involved in cell growth, differentiation, and immune function. Furthermore, research is underway regarding vitamin D and vitamin D receptors to improve skin issues (hair loss, inflammation, acne, etc.) and skin conditions (skin regeneration, etc.).

[0003] Accordingly, as described in the following patent document, an activator for activating the vitamin D receptor is being developed.

[0004] <Patent Literature>

[0005] Patent Publication No. 10-2010-0099181 (Published Sep. 10, 2010) "Novel Vitamin D Receptor Activator and Method for Manufacturing the Same"

[0006] However, conventional vitamin D receptor activators are not sufficiently effective, or most are chemically synthesized, which poses a problem as they may cause side effects during use. The problem to be solved

[0007] The present invention has been devised to solve the above-mentioned problems,

[0008] The present invention aims to provide a peptide that activates vitamin D receptors, which can be used for the alleviation of acne, the alleviation of hair loss, the alleviation of inflammation, or the regeneration of skin, and a cosmetic composition using the same. means of solving the problem

[0009] The present invention is implemented by an embodiment having the following configuration to achieve the aforementioned objective.

[0010] According to one embodiment of the present invention, the peptide according to the present invention is characterized by being composed of any one of the amino acid sequences of SEQ ID NOs 1 to 3.

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

[0012] According to another embodiment of the present invention, the peptide according to the present invention is characterized by being used for alleviating skin troubles or for skin regeneration.

[0013] According to another embodiment of the present invention, the peptide according to the present invention is characterized by being used for the alleviation of atopy, the alleviation of acne, the alleviation of hair loss, the alleviation of inflammation, or the regeneration of skin.

[0014] According to another embodiment of the present invention, the cosmetic composition according to the present invention is characterized by comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 3.

[0015] According to another embodiment of the present invention, a composition for alleviating skin troubles or regenerating skin according to the present invention is characterized by comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 3.

[0016] According to another embodiment of the present invention, the cosmetic composition according to the present invention is characterized by being used for the alleviation of atopy, the alleviation of acne, the alleviation of hair loss, the alleviation of inflammation, or the regeneration of skin. Effects of the invention

[0017] The present invention can achieve the following effects through the previously described embodiment.

[0018] The present invention has the effect of alleviating acne, alleviating hair loss, alleviating inflammation, or promoting skin regeneration by activating a vitamin D receptor using a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 3. Brief explanation of the drawing

[0019] FIG. 1 is a graph showing the effect of a peptide according to one embodiment of the present invention on the physical binding of VDR and RXR. FIG. 2 is a graph showing the effect of a peptide according to one embodiment of the present invention on the intracellular expression of S100A3. FIG. 3 is a graph showing the effect of a peptide according to one embodiment of the present invention on the inhibition of increased NFkB nuclear translocation induced by LPS. Figure 4 is a graph showing the effect of a peptide according to one embodiment of the present invention on the increase in transcriptional activity of P53. Specific details for implementing the invention

[0020] Hereinafter, a vitamin D receptor activating peptide according to the present invention and a cosmetic composition containing the same will be described in detail with reference to the attached drawings. Unless otherwise specifically defined, all terms in this specification have the same general meaning as 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 definition used in this specification shall prevail. Furthermore, detailed descriptions of known functions and configurations that may unnecessarily obscure the essence of the present invention are omitted. Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0022] One embodiment of the present invention relates to a peptide that activates a vitamin D receptor, wherein the peptide is characterized by being composed of the amino acid sequence of SEQ ID NO. 1 (Glu-Val-Phe-Gly), 2 (Val-Ile-Gly-Phe), or 3 (Ile-Gly-Phe-Ala). The peptide can improve skin troubles and skin conditions by activating a vitamin D receptor. Specifically, the peptide can prevent excessive sebum secretion and provide an anti-inflammatory effect to improve inflammatory skin conditions such as acne and atopic dermatitis, prevent or stop hair loss, and promote skin regeneration. The peptide may be used in cosmetic compositions, therapeutic compositions, etc.

[0024] Another embodiment of the present invention relates to a cosmetic composition comprising a peptide having an amino acid sequence of any one of SEQ ID NOs 1 to 3, wherein the cosmetic composition can alleviate skin troubles such as acne, hair loss, and skin inflammation, and promote skin regeneration. Various known ingredients that can provide formulation stability and additional skin condition improvement effects may be added to the cosmetic composition.

[0026] The present invention will be explained in more detail below through examples. However, these examples are intended only to explain the invention in more detail and do not limit the scope of the invention.

[0028] <Example 1> Synthesis of Peptides

[0030] The peptides of SEQ ID NOs 1 to 3 listed in Table 1 below were synthesized using the FMOC solid-phase method with an automated synthesizer (PeptrEx-R48, Peptron, South Korea). The synthesized peptides were purified and analyzed using reverse-phase high-speed liquid chromatography (Prominence LC-20AB, Shimadzu, Japan) with a C18 analysis RP column (Shiseido capcell pak), and identified using a mass spectrometer (HP 1100 Series LC / MSD, Hewlett-Packard, USA).

[0031] Peptide names Sequence number amino acid sequence VDR Pep 1 Sequence No. 1 Glu-Val-Phe-Gly VDR Pep 2 Sequence No. 2 Val-Ile-Gly-Phe VDR Pep 3 Sequence No. 3 Ile-Gly-Phe-Ala

[0033] <Example 2> Verify whether the peptide of Example 1 activates the vitamin D receptor to increase the physical binding of VDR and RXR.

[0035] 1. Vitamin D receptor (VDR) binds to retinoid X receptor (RXR) to regulate the transcription of target DNA. To determine whether the peptide of Example 1 activates VDR, the effect on the physical binding of VDR and RXR was evaluated through an in situ PLA (proximity ligation assay) method.

[0036] 2. Specifically, 4.5 × 10⁻⁶ in each well of the 24-well microplate 4HaCaT cells (human keratinocytes) were seeded into DMEM serum medium and cultured for 24 hours in a 37°C, 5% CO2 incubator. Then, each of the peptides of Example 1 (VDR Pep 1 to 3) was treated and incubated for 30 minutes under the same conditions. The concentration of each peptide in the medium was set to 1 and 10 μM, and a control group with no treatment was used. Subsequently, the cells in each well were washed with PBS, fixed by treatment 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-VDR monoclonal antibody (Santa Cruz, USA) and anti-RXR polyclonal antibody (Santa Cruz, USA) were added, and a PLA probe was added using an in situ PLA kit (Sigma-Aldrich), followed by hybridization, ligation, amplification, and mounting steps.

[0037] 3. Subsequently, the luminescence signals (PLA signals) detected in each cell were measured using a confocal laser microscope (Olympus fluoview FW1000; Olympus, Tokyo, Japan) to quantify the physical interaction between the VDR and RXR antibodies, and the results are shown in Figure 1.

[0038] 4. Looking at Figure 1, it can be seen that when VDR Pep 1 to 3 of Example 1 are applied, the physical bonding between VDR and RXR is increased compared to the untreated control group. That is, VDR Pep 1 to 3 activate VDR, thereby increasing the physical bonding between VDR and RXR, which can alleviate skin diseases (hair loss, skin inflammation, acne) and promote skin regeneration.

[0040] <Example 3> The peptide of Example 1, which promotes hair differentiation, Ca 2+Evaluation of the effect on the intracellular expression level of the binding protein S100A3

[0042] 1. To confirm the effect of the peptide of Example 1 on preventing hair loss, Ca, which promotes hair differentiation, was used with the Western blot technique. 2+ The effect on the expression of the binding protein S100A3 was examined.

[0043] 2. Specifically, 5 × 10 HaCaT cells per well 6 After preparing the cells, each of the peptides of Example 1 (VDR Pep 1 to 3) was treated and incubated for 24 hours under the same conditions. The concentration of each peptide in the medium was set to 1 and 10 μM, and untreated cells were used as a control. After washing the HaCaT cells three times with PBS, they were lysed in a 1% NP40 lysis buffer (1% Nonidet P40, 0.1 M NaCl, 0.05 M tris (pH 8.0), 5 mM EDTA) containing 0.1 μM PMSP (phenylmethylsulfonyl fluoride), 1 μg / ml pepstatin A, 10 μg / ml leupeptin, 1 μg / ml aprotinin, and 1 mM Na3VO4.

[0044] 3. Cell lysates were quantified using the Bradford assay method, and samples were prepared for electrophoresis. Immunoprecipitates were subjected to electrophoresis on a 15% polyacrylamide gel, and the separated proteins were transferred to a nitrocellulose membrane. The samples were then treated with a blocking solution (Tris-buffered saline (TBS) containing 0.05% Tween 20 and 3% bovine serum albumin) at room temperature for approximately 1 hour. Subsequently, the samples were incubated for 2 hours in TBS buffer containing anti-S100A3 polyclonal antibody (Santa Cruz, CA, USA), followed by washing with TBS buffer containing 0.05% Tween 20. After treatment with horseradish peroxidase conjugated anti-rabbit IgG (Santa Cruz, CA, USA) at room temperature for 1 hour, the samples were washed 5 times with TBS containing 0.05% Tween 20 and developed using an antibody detection kit (Ab Frontier, Korea), and the results are shown in Figure 2. To confirm that an equal amount of cell lysate was electrophoresed, actin was recognized using an anti-beta actin monoclonal antibody (Santa Cruz, CA, USA).

[0045] 4. Referring to FIG. 2, when VDR Pep 1 to 3 of Example 1 are treated, Ca that promotes hair differentiation 2+ It can be confirmed that the expression of the binding protein S100A3 is promoted. In other words, it can be seen that VDR Pep 1 to 3 activate VDR to alleviate hair loss.

[0047] <Example 4> Evaluate whether the peptide of Example 1 inhibits the increase in NFkB nuclear translocation induced by LPS

[0049] 1. When cells are stimulated with LPS, NFkB (P50, P65) migrate into the nucleus to initiate an inflammatory response. To determine whether the peptide of Example 1 inhibits this, LPS and the peptide of Example 1 were applied to cells, and the extent of migration of P50 and P65 into the nucleus was evaluated using the in situ PLA (proximity ligation assay) method.

[0050] 2. Specifically, 4.5 × 10⁻⁶ in each well of the 24-well microplate 4 HaCaT cells (human keratinocytes) were seeded into DMEM serum medium and cultured for 24 hours in a 37°C, 5% CO2 incubator. Then, 10 μg / ml of LPS and each of the peptides of Example 1 (VDR Pep 1 to 3) were added to the medium, and the cells were incubated under the same conditions for 30 minutes. The concentration of each peptide in the medium was set to 1 μM and 10 μM. Controls were used, including no treatment and treatment with LPS alone. Cells in each well were washed with PBS and fixed by treatment with 2% formaldehyde for 15 minutes, followed by treatment with 0.1% TritonX-100 for 5 minutes to increase antibody permeability into the cells. Anti-P65 polyclonal antibody (CST, USA) and anti-P50 monoclonal antibody (Santa Cruz, CA, USA) were added, and a PLA probe was added using an In situ PLA kit (Sigma-Aldrich), followed by hybridization, ligation, amplification, and mounting steps.

[0051] 3. The luminescence signals (PLA signals) detected in each cell were measured using a confocal laser microscope (Olympus fluoview FW1000; Olympus, Tokyo, Japan) to quantify the interaction between P65 and P50 antibodies in the nucleus, and the results are shown in Fig. 3.

[0052] 4. Looking at Figure 3, it can be seen that when VDR Pep 1 to 3 of Example 1 are treated, the increase in nuclear translocation of NFkB induced by LPS stimulation is reduced compared to the untreated control group. In other words, it can be seen that VDR Pep 1 to 3 activate VDR to alleviate inflammation.

[0054] <Example 5> Evaluation of whether the peptide of Example 1 increases the transcriptional activity of P53

[0056] 1. To determine whether the peptide of Example 1 increases the transcriptional activity of P53, which is associated with anti-aging of cells, the peptide of Example 1 was treated to cells, and then the degree of binding between P53 and POLII (RNA Polymerase II) was confirmed through the in situ PLA (proximity ligation assay) method.

[0057] 2. Specifically, 4.5 × 10⁻⁶ in each well of the 24-well microplate 4 HaCaT cells (human keratinocytes) were seeded into DMEM serum medium and cultured for 24 hours in a 37°C, 5% CO2 incubator. Afterward, each of the peptides of Example 1 (VDR Pep 1 to 3) was treated into the medium, and incubated under the same conditions for 30 minutes. The concentration of each peptide in the medium was set to 1 and 10 μM, and a control group without any treatment was used. Cells in each well were washed with PBS and fixed by treatment 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, CA, USA) were added, and a PLA probe was added using an In situ PLA kit (Sigma-Aldrich), followed by hybridization, ligation, amplification, and mounting steps.

[0058] 3. The interaction between P65 and P50 antibodies in the nucleus was quantified by measuring the luminescence signals (PLA signals) detected in each cell using a fluorescence microscope (Logosbio / CelenaS, Korea), and the results are shown in Figure 4.

[0059] 4. Looking at Figure 4, it can be seen that when treated with VDR Pep 1 to 3 of Example 1, the binding of P53 and RNApolymerase II increased compared to the untreated control group. In other words, it can be seen that VDR Pep 1 to 3 activate VDR, thereby increasing the transcriptional activity of P53 and preventing cell aging.

[0061] <Example 6> Confirmation of sebum-inhibiting effect of cosmetic composition containing the peptide of Example 1 through skin patch test

[0063] 1. Cosmetic compositions were prepared by adding each of the peptides prepared in Example 1 to a commercially available solubilized ampoule base at a concentration of 50 ppm. Cosmetic composition 1 represents a solubilized ampoule base without the peptide of Example 1 added, cosmetic composition 2 represents the case where VDR Pep 1 is added, cosmetic composition 3 represents the case where VDR Pep 2 is added, and cosmetic composition 4 represents the case where VDR Pep 3 is added.

[0064] 2. For 21 men in their 40s with excessive sebum secretion, the forehead area was wiped with 70% ethanol and dried. A detailed tape was applied to the forehead and then removed for verification. Subsequently, equal amounts of each of the above cosmetic compositions 1 to 4 were applied to the forehead area every morning and evening for 3 weeks (by soaking a sheet with the cosmetic composition and attaching the sheet to the forehead for a certain period of time). On the day following the 3 weeks, a detailed tape was applied to the forehead and then removed for verification. The relative reduction in sebum was confirmed by examining the detailed tapes before and after the application of the cosmetic compositions.

[0065] 3. As a result of the experiment, the forehead skin was examined after using the cosmetic composition for 3 weeks and no irritation occurred. When the relative reduction of sebum before and after using the cosmetic composition was examined, no significant reduction in sebum was observed when using the control cosmetic composition 1, but a significant reduction in sebum was observed when using cosmetic compositions 2 to 4. In other words, it can be seen that VDR Pep 1 to 3 of Example 1 inhibit the generation of sebum.

[0067] <Example 7> Confirmation of anti-inflammatory effect of the cosmetic composition containing the peptide prepared in Example 1 through a skin patch test

[0069] 1. Twenty-one adults suffering from inflammatory skin diseases such as atopic dermatitis and acne were subjected to the application of equal amounts of cosmetic compositions 1 to 4 prepared in Example 6, 1 to the affected skin areas every morning and evening for three weeks. After three weeks, each participant was asked to provide a subjective evaluation of the improvement in their skin condition on the day following the three weeks, and the results are shown in Table 3. In the evaluation, participants were asked to rate the results as follows: significant improvement was rated 5, improvement 4, average 3, worsening 2, and significant worsening 1.

[0070] 2. Looking at Table 2, it can be seen that cosmetic compositions 2 to 4 have a significant skin improvement effect. That is, it can be seen that VDR Pep 1 to 3 can be used in cosmetic compositions to alleviate inflammatory skin diseases such as atopic dermatitis and acne, thereby improving skin condition.

[0071] cosmetic composition 1 2 3 4 skin improvement 2.6 4.4 4.6 4.7

[0073] <Example 8> Confirmation of skin regeneration effect of the cosmetic composition containing the peptide prepared in Example 1 through a skin patch test

[0075] 1. Twenty-one elderly people in their 60s were subjected to the application of equal amounts of cosmetic compositions 1 to 4 prepared in Example 6-1 to their faces every morning and evening for 10 weeks, and after 10 weeks, they were asked to make a subjective evaluation of the skin regeneration effect, considering wrinkle improvement, skin elasticity, and scar reduction.

[0076] 2. As a result of the experiment, it was evaluated that there was no skin regeneration effect when using cosmetic composition 1, which was the control group, but when using cosmetic compositions 2 to 4, it was evaluated that there was a skin regeneration effect due to the relative improvement of wrinkles, improved skin elasticity, and reduction of scars.

[0078] <Example 9> Confirmation of hair loss alleviation effect of the cosmetic composition containing the peptide prepared in Example 1 through a skin patch test

[0080] 1. A cosmetic composition to confirm the hair loss alleviation effect was prepared with the composition ratios in Table 2 below.

[0081] cosmetic composition Content (wt%) 1 2 3 4 VDR Pep 1 0 0.04 0 0 VDR Pep 2 0 0 0.04 0 VDR Pep 3 0 0 0 0.04 antiseptic 0.02 0.02 0.02 0.02 spices 0.4 0.4 0.4 0.4 glycerin 2 2 2 2 purified water Remaining amount Remaining amount Remaining amount Remaining amount

[0082] 2. For 21 men suffering from alopecia, equal amounts of cosmetic compositions 1 to 4 prepared in Example 9, 1 were applied to the affected area every morning and evening for 10 weeks, and the condition of the hair was visually checked after 10 weeks.

[0083] 2. As a result of the experiment, when cosmetic composition 1, the control group, was used, the hair did not become thicker or regrowth occurred, but when cosmetic compositions 2 to 4 were used, it was confirmed that the hair became thicker and the number of hairs increased.

[0085] Although the applicant has described preferred embodiments of the present invention above, such embodiments are merely examples of implementing the technical concept of the present invention, and any modification or alteration that implements the technical concept of the present invention should be interpreted as falling within the scope of the present invention.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A cosmetic composition characterized by comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 3. Claim 6 delete Claim 7 delete

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