Peptide activating vitamin d receptor, and cosmetic composition comprising same

Abstract

The present invention relates to a peptide activating a vitamin D receptor, and a cosmetic composition using same.

Description

Vitamin D receptor activating peptide and cosmetic composition containing the same

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

[0002] Vitamin D is a hormone crucial for bone growth and maintenance, as well as mineral homeostasis. It binds to the vitamin D receptor (VDR), present in various tissues and cells in the body, regulating gene expression involved in cell growth, differentiation, and immune function. Furthermore, research is being conducted on vitamin D and its receptor to improve skin conditions (e.g., hair loss, inflammation, acne) and skin regeneration.

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

[0004] <Patent Document>

[0005] Patent Publication No. 10-2010-0099181 (published on September 10, 2010) "Novel Vitamin D Receptor Activator and Preparation Method Thereof"

[0006] However, conventional vitamin D receptor activators are not sufficiently effective, and most are 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 vitamin D receptor and a cosmetic composition using the same, which can be used for alleviating acne, alleviating hair loss, alleviating inflammation, or regenerating skin.

[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 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 in that it is used for alleviating skin troubles or regenerating skin.

[0013] According to another embodiment of the present invention, the peptide according to the present invention is characterized in that it is used for alleviating atopy, alleviating acne, alleviating hair loss, alleviating inflammation, or regenerating skin.

[0014] 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 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 trouble or regenerating skin according to the present invention is characterized in that it comprises 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 in that it is used for alleviating atopy, alleviating acne, alleviating hair loss, alleviating inflammation, or regenerating skin.

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

[0018] The present invention has the effect of promoting alleviation of acne, alleviation of hair loss, alleviation of inflammation, or regeneration of skin by activating a vitamin D receptor using a peptide consisting of any one of the amino acid sequences of sequence numbers 1 to 3.

[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 VDR and RXR.

[0020] Figure 2 is a graph showing the effect of a peptide according to one embodiment of the present invention on the intracellular expression of S100A3.

[0021] Figure 3 is a graph showing the effect of a peptide according to one embodiment of the present invention on inhibiting the increase in NFkB nuclear translocation induced by LPS.

[0022] Figure 4 is a graph showing the effect of a peptide according to one embodiment of the present invention on increasing the transcriptional activity of P53.

[0023] Hereinafter, a vitamin D receptor activating peptide according to the present invention and a cosmetic composition comprising 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 configurations 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.

[0024]

[0025] One embodiment of the present invention relates to a peptide that activates a vitamin D receptor, wherein the peptide is characterized by having an 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 the vitamin D receptor. Specifically, the peptide can improve inflammatory skin conditions such as acne and atopy by preventing excessive secretion of sebum and providing an anti-inflammatory effect, and can prevent or prevent hair loss and promote skin regeneration. The peptide can be used in cosmetic compositions, therapeutic compositions, etc.

[0026]

[0027] Another embodiment of the present invention relates to a cosmetic composition comprising a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 3, wherein the cosmetic composition can alleviate skin problems 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.

[0028]

[0029] 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.

[0030]

[0031] <Example 1> Synthesis of peptides

[0032]

[0033] Peptides of sequence numbers 1 to 3 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).

[0034]

[0035]

[0036] <Example 2> Confirmation that the peptide of Example 1 activates the vitamin D receptor and increases the physical binding between VDR and RXR.

[0037]

[0038] 1. Vitamin D receptor (VDR) binds to retinoid X receptor (RXR) to regulate transcription of target DNA. To confirm whether the peptide of Example 1 activates VDR, the effect on physical binding of VDR and RXR was evaluated using the in situ PLA (proximity ligation assay) method.

[0039] 2. Specifically, 4.5 × 10 in each well of a 24-well microplate 4HaCaT cells (human keratinocytes) were seeded in DMEM serum medium and cultured for 24 hours in a 37℃, 5% CO2 incubator, and then treated with each of the peptides of Example 1 (VDR Pep 1 to 3) 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-VDR 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.

[0040] 3. Afterwards, the luminescence signals (PLA signals) detected from each cell were measured using a confocal laser microscope (Olympus fluoview FW1000; Olympus, Tokyo, Japan) to quantify the physical interaction between VDR and RXR antibodies, and the results are shown in Fig. 1.

[0041] 4. Referring to Figure 1, it can be confirmed that when VDR Pep 1 to 3 of Example 1 are treated, the physical binding 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 binding between VDR and RXR, thereby alleviating skin diseases (hair loss, skin inflammation, acne) and promoting skin regeneration.

[0042]

[0043] <Example 3> The peptide of Example 1 promotes hair differentiation, Ca 2+Evaluation of the effect on the intracellular expression of S100A3, a binding protein

[0044]

[0045] 1. To confirm the effect of the peptide of Example 1 on preventing hair loss, Ca, which promotes hair differentiation, was used as a Western blot technique. 2+ The effect on the expression of S100A3, a binding protein, was confirmed.

[0046] 2. Specifically, HaCaT cells were seeded at 5×10 per well. 6 After preparation, each of the peptides (VDR Pep 1 to 3) of Example 1 was treated and incubated under the same conditions for 24 hours. The concentration of each peptide in the medium was 1 and 10 μM, and untreated cells were used as a control group. The HaCaT cells were washed three times with PBS and then lysed with 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.

[0047] 3. After quantifying the cell lysate using the Bradford assay, samples for electrophoresis were prepared. The immunoprecipitates were electrophoresed on a 15% polyacrylamide gel, and the developed proteins were transferred to a nitrocellulose membrane, which was 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. Afterwards, the membrane was reacted for 2 hours in TBS buffer containing anti-S100A3 polyclonal antibody (Santa Cruz, CA, USA), and then washed 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 cells were washed five times with TBS containing 0.05% Tween 20 and developed using an antibody detection kit (Ab frontier, Korea). The results are shown in Fig. 2. To confirm that the same amount of cell lysate was electrophoresed, actin was recognized using an anti-beta actin monoclonal antibody (Santa Cruz, CA, USA).

[0048] 4. Referring to Figure 2, when treating VDR Pep 1 to 3 of Example 1, Ca that promotes hair differentiation 2+ It can be confirmed that the expression of S100A3, a binding protein, is promoted. In other words, it can be seen that VDR Pep 1 to 3 activate VDR and alleviate hair loss.

[0049]

[0050] <Example 4> Evaluation of whether the peptide of Example 1 inhibits the increase in NFkB nuclear translocation induced by LPS

[0051]

[0052] 1. When cells are stimulated with LPS, NFkB (P50, P65) moves into the nucleus and initiates an inflammatory response. To confirm whether the peptide of Example 1 suppresses this, cells were treated with LPS and the peptide of Example 1, and the degree of movement of P50 and P65 into the nucleus was evaluated using the in situ PLA (proximity ligation assay) method.

[0053] 2. Specifically, 4.5 × 10 in each well of a 24-well microplate 4 HaCaT cells (human keratinocytes) were seeded in DMEM serum medium and cultured for 24 hours in a 37℃, 5% CO2 incubator, and then treated with 10 ug / ml of LPS in the medium and each of the peptides of Example 1 (VDR Pep 1 to 3) and incubated under the same conditions for 30 minutes. The concentration of each peptide in the medium was 1 and 10 μM, and untreated and treated with LPS alone were used as controls. The cells in each well were washed with PBS, 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-P65 polyclonal antibody (CST, USA) and anti-P50 monoclonal antibody (Santa Cruz, CA, USA) were added, and PLA probes were added using an In situ PLA kit (Sigma-Aldrich), followed by hybridization, ligation, amplification, and mounting steps.

[0054] 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.

[0055] 4. Referring to Figure 3, it can be confirmed that when VDR Pep 1 to 3 of Example 1 were treated, the increase in nuclear migration of NFkB induced by LPS stimulation was reduced compared to the untreated control group. In other words, it can be seen that VDR Pep 1 to 3 activate VDR and alleviate inflammation.

[0056]

[0057] <Example 5> Evaluation of whether the peptide of Example 1 increases the transcriptional activity of P53

[0058]

[0059] 1. In order 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 degree of binding between P53 and POLII (RNA Polymerase II) was confirmed through the in situ PLA (proximity ligation assay) method.

[0060] 2. Specifically, 4.5 × 10 in each well of a 24-well microplate 4HaCaT cells (human keratinocytes) were seeded in DMEM serum medium and cultured for 24 hours in a 37℃, 5% CO2 incubator. Then, each of the peptides of Example 1 (VDR Pep 1 to 3) in the medium was treated 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. The cells in each well were washed with PBS, 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, CA, USA) were added, and PLA probes were added using an In situ PLA kit (Sigma-Aldrich), followed by hybridization, ligation, amplification, and mounting steps.

[0061] 3. The luminescence signals (PLA signals) detected in each cell were measured using a fluorescence microscope (Logosbio / CelenaS, Korea) to quantify the interaction between P65 and P50 antibodies in the nucleus, and the results are shown in Figure 4.

[0062] 4. Referring to Figure 4, it can be confirmed that when VDR Pep 1 to 3 of Example 1 were treated, the binding of P53 and RNApolymerase II was increased compared to the untreated control group. In other words, it can be seen that VDR Pep 1 to 3 activate VDR, increase the transcriptional activity of P53, and prevent cellular aging.

[0063]

[0064] <Example 6> Confirmation of the sebum suppression effect through a skin patch test of a cosmetic composition containing the peptide of Example 1.

[0065]

[0066] 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 is not 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.

[0067] 2. The subjects were 21 men in their 40s with excessive sebum secretion. The forehead area was wiped with 70% ethanol and then dried. The detailed tape was attached to the forehead and then removed for confirmation. Afterwards, the same amount of each of the above cosmetic compositions 1 to 4 was applied to the forehead area every morning and evening for 3 weeks (by soaking a sheet with the above cosmetic composition and then attaching the sheet to the forehead for a certain period of time). After 3 weeks, the detailed tape was attached to the forehead and then removed for confirmation. The detailed tape before and after application of the cosmetic composition was checked to confirm the relative reduction in sebum.

[0068] 3. As a result of the experiment, when the forehead skin was checked after using the cosmetic composition for 3 weeks, no irritation occurred, and when the relative decrease in sebum before and after using the cosmetic composition was confirmed, no significant decrease in sebum was confirmed when the control cosmetic composition 1 was used, but a significant decrease in sebum was confirmed when cosmetic compositions 2 to 4 were used. That is, it can be seen that VDR Pep 1 to 3 of Example 1 suppress the generation of sebum.

[0069]

[0070] <Example 7> Confirmation of the anti-inflammatory effect of the cosmetic composition containing the peptide manufactured in Example 1 through a skin patch test.

[0071]

[0072] 1. 21 adults suffering from inflammatory skin diseases such as atopy and acne were instructed to apply the same amount of each of the cosmetic compositions 1 to 4 prepared in Example 6-1 to the affected area of ​​the skin every morning and evening for 3 weeks. Then, on the day after 3 weeks, each participant was asked to subjectively evaluate the improvement in skin condition, and the results are shown in Table 3. In the evaluation, 5 points were given for significant improvement, 4 points for improvement, 3 points for average, 2 points for deterioration, and 1 point for significant deterioration.

[0073] 2. As shown in Table 2, cosmetic compositions 2 to 4 exhibit remarkable skin improvement effects. That is, VDR Pep 1 to 3 can be used in cosmetic compositions to alleviate inflammatory skin diseases such as atopy and acne, thereby improving skin condition.

[0074]

[0075]

[0076] <Example 8> Confirmation of skin regeneration effect through skin patch test of cosmetic composition containing peptide manufactured in Example 1

[0077]

[0078] 1. 21 elderly people in their 60s were subjected to a study in which the same amount of cosmetic compositions 1 to 4 prepared in Example 6-1 were applied to the face every morning and evening for 10 weeks. After 10 weeks, the skin regeneration effect was subjectively evaluated, taking into account wrinkle improvement, skin elasticity, and scar reduction.

[0079] 2. As a result of the experiment, it was evaluated that there was no skin regeneration effect when the control cosmetic composition 1 was used, but when cosmetic compositions 2 to 4 were used, it was evaluated that the skin regeneration effect was recognized due to the relative improvement in wrinkles, improvement in skin elasticity, and reduction in scars.

[0080]

[0081] <Example 9> Confirmation of hair loss alleviation effect through skin patch test of cosmetic composition containing peptide manufactured in Example 1

[0082]

[0083] 1. A cosmetic composition to confirm the hair loss alleviation effect was prepared with the composition ratio shown in Table 2 below.

[0084]

[0085] 2. 21 men suffering from alopecia were subjected to applying the same amount of cosmetic compositions 1 to 4 prepared in Example 9-1 to the hair loss area every morning and evening for 10 weeks, and then the condition of the hair was visually checked after 10 weeks.

[0086] 2. As a result of the experiment, when the control group, cosmetic composition 1, 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.

[0087]

[0088] 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 any one of the amino acid sequences of sequence numbers 1 to 3.

2. In paragraph 1, The above peptide is a peptide characterized by activating a vitamin D receptor.

3. In paragraph 1, The above peptide is characterized as being used for alleviating skin troubles or regenerating skin.

4. In paragraph 1, The above peptide is characterized in that it is used for alleviating atopy, alleviating acne, alleviating hair loss, alleviating inflammation, or regenerating skin.

5. A cosmetic composition characterized by comprising a peptide composed of any one of the amino acid sequences of sequence numbers 1 to 3.

6. A composition for alleviating skin trouble or regenerating skin, characterized in that it comprises a peptide composed of any one of the amino acid sequences of sequence numbers 1 to 3.

7. In paragraph 5, The above cosmetic composition is characterized in that it is used for alleviating atopy, alleviating acne, alleviating hair loss, alleviating inflammation, or regenerating skin.

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