PEPTIDE HAVING ACTIVITY OF IMPROVING SKIN CONDITION AND USE THEREOF

Abstract

Provided are a peptide having an activity of improving skin condition and use thereof. In particularly, provided are a peptide consisting of an amino acid sequence of SEQ ID NO: 1, a cosmetic composition including the peptide for improving skin condition, and a pharmaceutical composition including the peptide for preventing or treating an inflammatory skin disease.

Claims

1. A peptide consisting of an amino acid sequence of SEQ ID NO: 1.

2. The peptide of claim 1, wherein an N-terminus of the peptide is bound to any one protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG).

3. The peptide of claim 1, wherein a C-terminus of the peptide is bound to any one protecting group selected from the group consisting of an amino group (—NH.sub.2), a tertiary alkyl group, and an azide group (—NHNH.sub.2).

4. The peptide of claim 1, wherein the peptide exhibits any one or more characteristics selected from the following: (a) inhibition of apoptosis of fibroblasts and keratinocytes; (b) promotion of collagen synthesis; (c) inhibition of expression of matrix metalloproteases; (d) restoration of activity of fibroblasts and keratinocytes; and (e) inhibition of expression of inflammatory cytokines.

5. A cosmetic composition for improving skin condition, the composition comprising the peptide of any one of claims 1 to 4 as an active ingredient.

6. The cosmetic composition of claim 5, wherein the improving of the skin condition includes wrinkle relief, skin elasticity improvement, wound regeneration, skin aging inhibition, or alleviation of an inflammatory skin disease.

7. The cosmetic composition of claim 6, wherein the skin aging is caused by ultraviolet light.

8. A pharmaceutical composition for preventing or treating an inflammatory skin disease, the composition comprising the peptide of any one of claims 1 to 4 as an active ingredient.

9. The pharmaceutical composition of claim 7, wherein the inflammatory skin disease includes acne, atopic dermatitis, psoriasis, seborrheic dermatitis, contact dermatitis, lupus erythematosus, or papular urticarcia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG. 1 shows a result confirming the increased expression of procollagen 1α after adding a peptide consisting of an amino acid sequence of SEQ ID NO: 1 to fibroblasts.

[0060] FIG. 2 shows a result confirming the increased expression of PPAR-γ, PPAR-δ, and PGC-1α after adding the peptide to fibroblasts.

[0061] FIG. 3 shows a result confirming the decreased expression of cleaved caspase-3, which is increased by ultraviolet irradiation, after adding the peptide to fibroblasts.

[0062] FIG. 4 shows a result confirming the decreased expression of cleaved PARP-1 and cleaved caspase-3, which are increased by ultraviolet irradiation, after adding the peptide to keratinocytes.

[0063] FIG. 5 shows a result confirming decreased expression of MMP-1 and the decreased activity of MMP-2, which are increased by ultraviolet irradiation, after adding the peptide to fibroblasts.

[0064] FIG. 6 shows a result confirming the increased expression of Coll al, Fibronectin, and Elastin, which are decreased by ultraviolet irradiation, after adding the peptide to fibroblasts.

[0065] FIG. 7 shows a result confirming the increased expression of SIRT1 and AQP3, which are decreased by ultraviolet irradiation, after adding the peptide to keratinocytes.

[0066] FIG. 8 shows a result confirming the decreased expression of TNF-α, COX-2, IL-1β, and IL-6, which are increased by ultraviolet irradiation, after adding the peptide to keratinocytes.

MODE OF DISCLOSURE

[0067] Hereinafter, the present disclosure will be described in detail with reference to Examples below. However, these Examples are provided for illustrative purposes only, and the scope of the present disclosure is not limited thereto.

Example 1. Synthesis of Peptide

[0068] A peptide having an amino acid sequence of SEQ ID NO: 1 of Table 1 was synthesized by using an automatic peptide synthesizer Milligen 9050 (Millipore, USA). Then, C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA) was performed thereon to purely separate the synthesized peptide. Here, ACQUITY UPLC BEH300 C18 column (2.1 mm×100 mm, 1.7 μm, Waters Co, USA) was used.

TABLE-US-00001 TABLE 1 SEQ ID NO. Sequence (5′->3′) 1 ECEELEEK

Example 2. Confirmation of Effect of Increasing Expression of Procollagen 1α

[0069] NIH3T3 mouse fibroblasts were seeded in a 6-well plate at a density of 5×10.sup.3 cells/well, and cultured for 16 hours. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 24 hours. Then, the amount of procollagen 1α in the medium was measured by using a Procollagen 1α ELISA kit (Us biological lifescience, USA). Meanwhile, a non-treated group (Con) was used as a control group, and a group to which 100 nM of bFGF was added and a group to which 5 ng/ml of TGF-β1 was added were used as positive control groups.

[0070] As a result, as shown in FIG. 1, it was confirmed that the expression of procollagen 1 a, which is a factor related to collagen synthesis in fibroblasts, was increased by the addition of the peptide consisting of the amino acid sequence of SEQ ID NO: 1.

Example 3. Confirmation of Effect of Enhancing Activity of Fibroblasts

[0071] In this example, an effect the peptide according to an embodiment on the cellular activity of fibroblasts was to be confirmed by measuring the expression levels of PPAR-γ, PPAR-δ, and PGC-1α, which are mitochondrial biogenesis-related genes. In detail, NIH3T3 mouse fibroblasts were seeded in a 6-well plate at a density of 3×10.sup.5 cells/well, and cultured for 16 hours. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 24 hours. Then, mRNA was extracted from the cultured cells, and reverse-transcribed into cDNA by using a cDNA synthesis kit and PCR pre-mix (Intron, Korea). Afterwards, a polymerase chain reaction (PCR) was performed by using the cDNA and primers of PPAR-γ, PPAR-δ, and PGC-1α. Meanwhile, a control group and positive groups were used in the same manner as in Example 2, and the nucleotide sequences of the primers used herein are shown in Table 2.

TABLE-US-00002 TABLE 2 SEQ Primer Sequence (5′->3′) ID NO. PPAR-γ Foward ACGATCTGCCTGAGGTCTGT 2 Reverse CATCGAGGACATCCAAGACA 3 PPAR-δ Foward CTGAAGGGAAGGGGGTAGAG 4 Reverse CAGTCTGGATGCTGCTACA 5 PGC-1α Foward ACTGAGCTACCCTTGGGATG 6 Reverse TAAGGATTTCGGTGGTGACA 7 GAPDH Foward GGTGTGAACGGATTTGGCCGTATTG 8 Reverse CCGTTGAATTTGCCGTGAGTGGAGT 9

[0072] As a result, as shown in FIG. 2, it was confirmed that the expression of PPAR-γ, PPAR-δ, and PGC-1α, which are factors related to the cellular activity of fibroblasts, was increased by the addition of the peptide consisting of the amino acid sequence of SEQ ID NO: 1. Referring to these results, it was confirmed that the peptide according to an embodiment contributed to improving skin conditions including wrinkle relief, skin elasticity improvement, wound regeneration, and the like by increasing the cellular activity of fibroblasts and promoting collagen synthesis.

Example 4. Confirmation of Inhibitory Effect on Apoptosis of Fibroblasts and Keratinocytes

[0073] NIH3T3 mouse fibroblasts or HaCaT human keratinocytes were respectively seeded in a 6-sell plate at a density of 3×10.sup.5 cells/well, and cultured for 16 hours. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 1 hour. Each well containing the cultured cells was washed with phosphate buffered saline (PBS), and the NIH3T3 cells or HaCaT cells were respectively irradiated with ultraviolet light of 6 J/cm.sup.2 or 15 J/cm.sup.2, so as to induce an increase in the expression of apoptosis proteins. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 24 hours. Cell lysates were obtained by adding a lysis buffer to the cultured cells, and then, western blotting was performed thereon by using cleaved PARP-1 and cleaved caspase-3 antibodies (Santacruz biotechnology, USA). Meanwhile, a non-treated group (Con) was used as a control group. For use as a negative control group and a positive control group, a non-treated group after ultraviolet irradiation (NC), and a group to which 2.5 mM of NaC was added after ultraviolet irradiation were used, respectively

[0074] As a result, as shown in FIGS. 3 and 4, it was confirmed that the expression of cleaved caspase-3 which was increased by ultraviolet irradiation in the fibroblasts was reduced by the addition of the peptide having the amino acid sequence of SEQ ID NO: 1. As such, it was confirmed that the expression of cleaved PARP-1 and cleaved caspase-3 which were increased by ultraviolet irradiation in the keratinocytes was significantly reduced. Referring these results, it was confirmed that the peptide according to an embodiment was able to inhibit apoptosis of the skin cells.

Example 5. Confirmation of Inhibitory Effect on Matrix Metalloprotease Increased by Ultraviolet Light

[0075] NIH3T3 mouse fibroblasts were seeded in a 6-well plate at a density of 3×10.sup.5 cells/well, and cultured for 16 hours. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 1 hour. Each well containing the cultured cells was washed with PBS, and the NIH3T3 cells were irradiated with ultraviolet light of 6 J/cm.sup.2, so as to induce an increase in the expression of MMP-1 and MMP-2. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 24 hours. Afterwards, cell lysates were obtained by adding a lysis buffer to the cultured cells, and then, western blotting was performed thereon by using MMP-1 antibodies (cell signaling, USA) to measure the expression of MMP-1.

[0076] Meanwhile, the cell lysates were subjected to gelatin zymography to measure the activity of MMP-2. In detail, after protein electrophoresis (SDS-PAGE) was performed by using 2 mg/ml of gelatin as a substrate. Following the electrophoresis, the gel was treated with 2.5% Triton X-100 for 30 minutes, and then incubated in a buffer containing 50 mM Tris-HCl, 0.2 M NaCl, 5 mM CaCl.sub.2, and 1% Triton X-100 at 37° C. for 24 hours. Afterwards, the gel was stained with Coo-massie Brilliant Blue R250 (Sigma), and de-stained with a buffer containing 5% methanol, 7.5% acetic acid, and distilled water. Then, the bands formed by gelatin hydrolysis were observed with naked eyes. Meanwhile, a control group, a negative control group, and a positive control group were used in the same manner as in Example 4.

[0077] As a result, as shown in FIG. 5, it was confirmed that the expression of MMP-1 and the activity of MMP-2 which were increased by ultraviolet irradiation in the fibroblasts were reduced by the addition of the peptide having the amino acid sequence of SEQ ID NO: 1.

Example 6. Confirmation of Restoration Effect on Cellular Activity of Fibroblasts and Keratinocytes Inhibited by Ultraviolet Light

[0078] In this example, changes in the cellular activity of fibroblasts were evaluated through the expression of Col1a1, Fibronectin, and Elastin, which are known as components of the dermis. Also, changes in the cellular activity of keratinocytes were evaluated through the expression of SIRT1, which is an anti-aging gene, and AQP3, which is a component of the skin barrier. Based on the evaluation results, the effect of the peptide according to an embodiment on the restoration of the cellular activity of the fibroblasts and keratinocytes was confirmed. In detail, NIH3T3 mouse fibroblasts or HaCaT human keratinocytes were respectively seeded in a 6-sell plate at a density of 3×10.sup.5 cells/well, and cultured for 16 hours. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 1 hour. Each well containing the cultured cells was washed with PBS, and the NIH3T3 cells or the HaCaT cells were respectively irradiated with ultraviolet light of 6 J/cm.sup.2 or 20 J/cm.sup.2, so as to induce inhibition of the cellular activity. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 6 hours. Then, mRNA was extracted from the cultured cells, and reverse-transcribed into cDNA by using a cDNA synthesis kit and PCR pre-mix (Intron, Korea). Afterwards, PCR was performed by using: the cDNAs derived from the fibroblasts and primers of Col1 at Fibronectin, and Elastin; and the cDNAs derived from the keratinocytes and primers of SIRT1 and AQP3. Meanwhile, a control group, a negative group, and a positive group were used in the same manner as in Example 4, and the nucleotide sequences of the primers used herein are shown in Tables 3 and 4.

TABLE-US-00003 TABLE 3 SEQ Primer Sequence (5′->3′) ID NO. Col1a1 Foward CACCCTCAAGAGCCTGAGTC 10 Reverse AGACGGCTGAGTAGGGAACA 11 Fibronectin Foward CCAGGAACCGAGTACACCAT 12 Reverse ATACCCAGGTTGGGTGATGA 13 Elastin Foward GGACCCCTGACTCGCGACCT 14 Reverse GGGGAGGTGGGACTGCCCAA 15 GAPDH Foward GGTGTGAACGGATTTGGCCGTATTG 8 Reverse CCGTTGAATTTGCCGTGAGTGGAGT 9

TABLE-US-00004 TABLE 4 SEQ Primer Sequence (5′->3′) ID NO. SIRT1 Foward TCAGTGGCTGGAACAGTGAG 16 Reverse TCTGGCATGTCCCACTATCA 17 AQP3 Foward CCTTCTTGGGTGCTGGAATA 18 Reverse ACACGATAAGGGAGGCTCTG 19 GAPDH Foward GGTGTGAACGGATTTGGCCGTATTG 8 Reverse CCGTTGAATTTGCCGTGAGTGGAGT 9

[0079] As a result, as shown in FIGS. 6 and 7, it was confirmed that the expression of Col1 a1, Fibronectin, and Elastin which were inhibited by ultraviolet irradiation in fibroblasts was restored back to normal levels by the addition of the peptide consisting of the amino acid sequence of SEQ ID NO: 1. As such, it was also confirmed that the expression of SIRT1 and AQP3 which were reduced by ultraviolet irradiation in keratinocytes was restored. Based on these results, it was found that the peptide according to an embodiment contributed to improving the pathological environment of the skin, including decreased density of the dermal layer and decreased skin barrier, by restoring the cellular activity of the damaged skin cells.

Example 7. Confirmation of Inhibitory Effect on Expression of Inflammatory Factor Increased by Ultraviolet Rays

[0080] HaCaT human keratinocytes were seeded in a 6-well plate at a density of 3×10.sup.5 cells/well, and cultured for 16 hours. Afterwards, the culture medium was replaced with a serum-free medium, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 was added thereto at a concentration of 10 μM, 50 μM, or 100 μM, and the cells were cultured for 1 hour. Each well containing the cultured cells was washed with PBS, and the HaCaT cells were irradiated with ultraviolet light of 15 J/cm.sup.2, so as to induce an increase in the expression of inflammatory cytokine which is an inflammatory factor. Then, mRNA was extracted from the cultured cells, and reverse-transcribed into cDNA by using a cDNA synthesis kit and PCR pre-mix (Intron, Korea). Afterwards, PCR was performed by using the cDNA and primers of TNF-α, COX-2, IL-1β, and IL-6. Meanwhile, a control group and a positive group were used in the same manner as in Example 4, and the nucleotide sequences of the primers used herein are shown in Table 5.

TABLE-US-00005 TABLE 5 SEQ Primer Sequence (5′->3′) ID NO. TNF-α Forward CGTCAGCCGATTRTGCTATCT 20 Reverse CGGACTCCGCAAAGTCTAAG 21 Forward ATCATTCACCAGGCAAATTGC 22 Reverse GGCTTCAGCATAAAGCGTTTG 23 IL-1β Forward TTCGACACATGGGATAACGA 24 Reverse TCTTTCAACACGCAGGACAG 25 IL-6 Forward AAAGAGGCACTGCCAGAAAA 26 Reverse ATCTGAGGTGCCCATGCTAC 27 GAPDH Forward GGTGTGAACGGATTTGGCCGTATTG 8 Reverse CCGTTGAATTTGCCGTGAGTGGAGT 9

[0081] As a result, as shown in FIG. 8, it was confirmed that the expression of TNF-α, COX-2, IL-1β, and IL-6 which were increased by ultraviolet irradiation in the keratinocytes was reduced by the addition of the amino acid sequence SEQ ID NO: 1. Referring these results, it was confirmed that the peptide according to an embodiment was able to alleviate or improve the inflammatory response of the skin.

[0082] The foregoing descriptions are only for illustrating the present disclosure, and it will be apparent to a person having ordinary skill in the art to which the present invention pertains that the embodiments disclosed herein can be easily modified into other specific forms without changing the technical spirit or essential features. Therefore, it should be understood that Examples described herein are illustrative in all respects and are not limited.