Pharmaceutical composition for preventing or treating inflammation, and cosmetic composition for alleviating inflammation or skin aging

Abstract

The present invention provides: a pharmaceutical composition for preventing or treating inflammation, the composition comprising a specific peptide; and a cosmetic composition for alleviating skin inflammation or skin wrinkles. The peptide acts as an antagonist for FAK and inhibits a signaling pathway associated with FAK, thereby lowering the expression of MMP-1 and MMP-9 and inhibiting the degradation of collagen. The peptide can be effectively used for preventing or treating inflammation and alleviating skin inflammation or skin wrinkles.

Description

Pharmaceutical composition for preventing or treating inflammation and cosmetic composition for improving inflammation or skin aging

[0001] The present invention relates to a pharmaceutical composition for preventing or treating inflammation, comprising a specific peptide; and a cosmetic composition for improving inflammation or skin aging. The peptide acts as an antagonist for focal adhesion kinase (FAK), inhibiting signaling pathways associated with FAK, thereby reducing the expression of matrix metalloproteinase-1 and MMP-9 and inhibiting collagen degradation.

[0002] Focal adhesion kinase (FAK), activated by inflammatory molecules, induces inflammatory responses by activating JNK, NF-κB, etc. through downstream signaling pathways (Zhi-Min Wu, et al., Journal of Neuro-Oncology volume 77, pages 117-123 (2006); Hyunho Yoon, et al., Journal of Histochemistry & Cytochemistry Volume 63, Issue 2, February 2015, Pages 114-128, etc.).

[0003] FAK is phosphorylated and activated by H2O2 or TNF-α, and the phosphorylated FAK enters the nucleus and binds to P53 and MDM2, inducing ubiquitination and proteasomal degradation of P53, thereby inhibiting apoptosis. In addition, FAK phosphorylated by TNF-α activates IκB (Nuclear Factor-κB) and JNK, thereby causing an inflammatory response (Murphy JM, et al., Inflammation, 44(3), 1130-44; Takasuke Harada, et al., Arteriosclerosis, Thrombosis, and Vascular Biology. 2017 | Volume 37, Issue 1: 156-165, etc.). When the phosphorylation of FAK is reduced, the activity of MMPs is reduced and collagen degradation is inhibited (Peng Zhang, et al., European Journal of Oral Sciences, 123, 249-253; Yan-Ning Yang, et al., Ophthalmic Research, 48(4), 165-170; MIN YAO, et al., Molecular Medicine Reports, 15, 915-921, etc.). Therefore, a substance that can inhibit the FAK-related signaling pathway can not only act as an active substance that can suppress inflammation, but also act as a functional cosmetic substance for improving skin inflammation or skin aging (e.g., skin wrinkles).

[0004] The present inventors have disclosed that a peptide derived from the p65 subunit of NF-κB and a peptide derived from TNFR have anti-inflammatory activity by inhibiting the expression of various inflammatory mediators whose expression is induced by NF-κB activity, and have skin photoaging inhibitory activity by inhibiting the expression of inflammatory mediators MMP-1, TNF-α, and IL-1α that are transcriptionally regulated by NF-κB (Korean Patent Registration Nos. 10-1594032 and 10-2041803).

[0005] The present inventors conducted various studies to develop active substances based on small peptides that can act as FAK antagonists. As a result, we discovered that a specific peptide, a 3-mer peptide composed of Glu-Leu-Cys, acts as an FAK antagonist, inhibiting the FAK-related signaling pathway, thereby reducing the expression of MMP-1 and MMP-9 and inhibiting collagen degradation, and thus can be usefully applied to prevent or treat inflammation and improve skin inflammation or wrinkles.

[0006] Accordingly, the present invention aims to provide a pharmaceutical composition for preventing or treating inflammation comprising the specific peptide as an active ingredient.

[0007] In addition, the present invention aims to provide a cosmetic composition for improving skin inflammation or skin wrinkles, which comprises the specific peptide.

[0008] According to one aspect of the present invention, a pharmaceutical composition for preventing or treating inflammation is provided, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] <Chemical Formula 1>

[0010]

[0011] According to another aspect of the present invention, a cosmetic composition for improving skin inflammation or skin wrinkles is provided, comprising the peptide of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0012] The present invention has revealed that the peptide according to the present invention (i.e., a peptide composed of Glu-Leu-Cys) acts as an antagonist for FAK. That is, the present invention has revealed that the peptide according to the present invention effectively suppresses inflammatory responses by binding to FAK and inhibiting FAK-related signaling pathways, thereby reducing the expression of MMP-1 and MMP-9 and inhibiting collagen degradation. Furthermore, the present invention has revealed that senescent cells are effectively reduced by UVB irradiation by the peptide according to the present invention, and that in a 3D human skin model, the epidermis and dermis damaged by UVB irradiation are restored by the peptide according to the present invention and collagen degradation is inhibited. Therefore, the peptide according to the present invention can be usefully applied to a pharmaceutical composition for wound healing or promoting wound healing; and a cosmetic composition for improving skin aging, including skin inflammation or skin wrinkles.

[0013] Figure 1 shows the results of measuring the signal reduction of the peptide (VE-Fakanin)-AMC of the present invention composed of Glu-Leu-Cys and FAK-FITC by FAK siRNA treatment.

[0014] Figure 2 shows the results of analyzing changes in FAK-P53 interaction due to peptide treatment of the present invention.

[0015] Figure 3 shows the results of analyzing changes in MDM2-P53 interaction due to peptide treatment of the present invention.

[0016] Figure 4 shows the results of analyzing changes in FAK (Y397) phosphorylation by peptide treatment of the present invention.

[0017] Figure 5 shows the results of analyzing the change in IκB expression level due to peptide treatment of the present invention.

[0018] Figure 6 shows the results of analyzing changes in JNK (T183 / Y185) phosphorylation by peptide treatment of the present invention.

[0019] Figure 7 shows the results of analyzing changes in the expression levels of MMP-1 and MMP-9 by peptide treatment of the present invention.

[0020] Figure 8 shows the results of analyzing changes in MMP-1 and collagen expression levels by peptide treatment of the present invention.

[0021] Figure 9 shows the results of analyzing the change in the amount of senescent cells produced by the peptide treatment of the present invention.

[0022] Figure 10 shows the results of analyzing the change in tissue condition due to peptide treatment of the present invention in a 3D human skin model.

[0023] The present invention provides a pharmaceutical composition for preventing or treating inflammation, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0024] <Chemical Formula 1>

[0025]

[0026] In addition, the present invention provides a cosmetic composition for improving skin inflammation or skin wrinkles, comprising the peptide of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0027] In the pharmaceutical composition or cosmetic composition of the present invention, the peptide of Chemical Formula 1 may also be expressed as "Glu-Leu-Cys". The amino acids constituting the peptide of Chemical Formula 1 may independently be in the form of L-amino acids or D-amino acids. Pharmaceutically acceptable salts of the peptide derivative of Chemical Formula 1 include, but are not limited to, acid addition salts, for example.

[0028] In the pharmaceutical composition or cosmetic composition of the present invention, the “inflammation” includes skin inflammation (i.e., dermatitis) caused by various causes, and preferably includes skin inflammation caused by ultraviolet rays.

[0029] "Skin aging" refers to aging of the skin, including wrinkle formation caused by intrinsic and extrinsic factors, and may preferably be photoaging of the skin accompanied by wrinkle formation, and more preferably includes photoaging of the skin caused by ultraviolet ray stimulation accompanied by wrinkle formation. In one embodiment, the skin aging includes wrinkle formation on the skin, i.e., skin wrinkles.

[0030] The pharmaceutical composition of the present invention may include excipients such as lactose and corn starch, lubricants such as magnesium stearate, and known and usable emulsifiers, suspending agents, buffers, isotonic agents, etc., and may be formulated as a parenteral dosage form, preferably a parenteral dosage form including a topical skin preparation. In the case of intramuscular, intraperitoneal, subcutaneous, and intravenous administration forms, a sterile solution of the active ingredient is usually prepared, and a buffer capable of suitably adjusting the pH of the solution may be included, and in the case of intravenous administration, a isotonic agent may be included to impart isotonicity to the preparation. In addition, the pharmaceutical composition of the present invention may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier such as saline having a pH of 7.4, and may be locally introduced into the intramuscular bloodstream of a patient in the form of a solution. In addition, it may be formulated as a transdermal dosage form such as a topical solution, emulsion, ointment, or patch according to a conventional pharmaceutical method. The pharmaceutical composition of the present invention can be administered to various inflammatory patients at a daily dose of about 1 to 50 mg / kg. The appropriate dosage may generally vary depending on the patient's age, weight, and symptoms.

[0031] The cosmetic composition of the present invention may be in the form of a functional cosmetic composition containing the above-described peptide as an active 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, which may be diluted with a conventional cleansing solution, astringent solution, or moisturizing solution and used. In addition, the cosmetic composition may include conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions. In the cosmetic composition, the content of the peptide is an amount effective to achieve an effect of improving skin inflammation or skin wrinkles, for example, 1 x 10 based on the total weight of the composition. -5 ~ 1 x 10 -2 It may be contained in a content of weight %, preferably about 1 x 10 -4 ~ 1 x 10 -3 It can be contained in a content of weight%.

[0032] 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.

[0033] Example 1. Synthesis of peptides

[0034] A peptide consisting of Glu-Leu-Cys 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).

[0035] Example 2. Preparation of a composition containing a peptide

[0036] The peptide (peptide composed of Glu-Leu-Cys) prepared in Example 1 was dissolved in triple-distilled water to a concentration of 1000 ppm. The obtained peptide solution was used in the following test examples.

[0037] Test Example 1: FAK binding evaluation

[0038] In order to confirm whether the peptide of the present invention (a peptide composed of Glu-Leu-Cys) binds to the target protein FAK, the peptide-AMC of the present invention labeled with the fluorescent substance AMC (7-amino-4-methyl coumarin) was treated to cells, and whether it binds to FAK was confirmed through immunofluorescence.

[0039] (1) Test materials

[0040] - Preparation of test substances

[0041] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0042] - Test system

[0043] 1) Cell line: Human Keratinocyte (HaCaT, CLS)

[0044] 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.

[0045] 3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0046] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage Conditions: Refrigerated / Manufacturer: GIBCO

[0047] - Test materials

[0048] 1) Peptide-AMC of the present invention

[0049] Storage conditions: -20℃ frozen storage / Manufacturer: Peptron

[0050] 2) Anti-FAK (C-20) antibody

[0051] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-558

[0052] 3) Goat anti-rabbit IgG-FITC (Fluorescein isothiocyanate) antibody

[0053] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, A27034

[0054] 4) Phalloidin-Rhodamin

[0055] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, R415

[0056] 5) siRNA transfection

[0057] 5-1) FAK siRNA

[0058] Storage conditions: -20℃ frozen storage / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-29310

[0059] 5-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0060] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0061] 5-3) Opti-MEM ® Medium

[0062] Storage conditions: Refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0063] 6) Rh TNF-α protein

[0064] Storage conditions: -20℃ frozen storage / Manufacturer: R&D System, 210-TA-005

[0065] (2) Test method

[0066] - Composition of the test group

[0067]

[0068] - Examination process

[0069] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4 After dispensing the cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the confluency of the cells was 50% or higher.

[0070] 2) The medium was replaced with a dedicated medium (DMEM + 10% FBS) for siRNA treatment.

[0071] 3) siRNA + Opti-MEM ® Media and transfection reagent + Opti-MEM ® The mixture was mixed in a 1:1 ratio and slowly mixed, then left to react at room temperature for 5 minutes.

[0072] 4) 3) The solution was added to cultured cells and treated for 48 hours to conduct the test.

[0073] 5) The peptide-AMC (1:100) of the present invention and TNF-α (25 ng / ml) were reacted at 4°C for 16 hours, and then washed three times with a washing solution (PBS).

[0074] 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).

[0075] 7) FAK antibody (1:100) was treated, reacted at room temperature for 40 minutes, and then washed three times with washing solution (PBS).

[0076] 8) After treating with secondary antibody (1:1000), reacting at room temperature for 40 minutes, the cells were washed three times with washing solution (PBS).

[0077] 9) Mounting was performed using a mounting solution.

[0078] 10) The fluorescence signal (AMC) detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0079] - Observe and judge results

[0080] The luminescence levels of the peptide-AMC of the present invention were compared and analyzed in the TNF-α treatment group and the FAK siRNA treatment group based on the control siRNA negative control group.

[0081] (3) Test results

[0082] Compared to the negative control group, an increase in the peptide-AMC signal (blue fluorescence) of the present invention was observed upon TNF-α treatment, and a decrease in the peptide-AMC signal of the present invention was observed upon simultaneous treatment with FAK siRNA. A similar signal pattern was observed in the same test group treated with FAK antibody (Fig. 1). These results suggest that the test substance specifically binds to the target protein, FAK.

[0083] (4) Conclusion

[0084] As a result of evaluating the degree of FAK binding of the test substance, the test substance bound to FKA in the TNF-α treatment group and displayed an AMC (blue fluorescence) signal, and when FAK expression was suppressed by treating with FAK siRNA, the AMC signal was reduced. Therefore, it is determined that the peptide of the present invention (a peptide composed of Glu-Leu-Cys) specifically binds to FAK.

[0085] Test Example 2: Evaluation of FAK signaling inhibition efficacy

[0086] FAK is phosphorylated and activated by H2O2 or TNF-α, and the phosphorylated FAK is known to enter the nucleus, bind to P53 and MDM2, and induce ubiquitination and proteasomal degradation of P53, thereby inhibiting apoptosis. To confirm whether the peptide of the present invention inhibits FAK activation, the effect on the phosphorylation level of FAK was investigated through Western blot analysis, and the interaction between FAK, P53, and MDM2 in the nucleus was confirmed through in situ PLA.

[0087] (1) Test materials

[0088] - Preparation of test substances

[0089] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0090] - Test system

[0091] 1) in situ PLA

[0092] 1-1) Cell line: Human Keratinocyte (HaCaT, CLS)

[0093] 1-2) 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 2-3 days.

[0094] 1-3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0095] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage Conditions: Refrigerated / Manufacturer: GIBCO

[0096] 2) Western blot analysis

[0097] 2-1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0098] 2-2) 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 2-3 days.

[0099] 2-3) Medium: CEFOgro Human MSC Growth medium

[0100] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage Conditions: Refrigerated / Manufacturer: CEFObio

[0101] - Test materials

[0102] 1) in situ PLA

[0103] 1-1) Anti-FAK (B-8) antibody

[0104] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-271195

[0105] 1-2) Anti-P53 antibody

[0106] Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 2527S

[0107] 1-3) Anti-MDM2 (D-7) antibody

[0108] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-13161

[0109] 1-4) Hydrogen peroxide (H2O2)

[0110] Storage conditions: -20℃ frozen storage / Manufacturer: JUNSEI, 23150S0350

[0111] 1-5) NaveniFlex 100RM

[0112] Storage conditions: -20℃ frozen storage / Manufacturer: NaveniFlex, NV C-NF MR.100

[0113] 1-6) Prolong™ diamond antifade mountant with DAPI

[0114] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, P36962

[0115] 1-7) Digital Fluorescence Imaging System

[0116] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0117] 2) Western blot analysis

[0118] 2-1) Anti-FAK-phospho(Y397) antibody

[0119] Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 3283

[0120] 2-2) Goat anti-rabbit IgG Fc-HRP

[0121] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0122] 2-3) NP40 cell lysis buffer

[0123] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0124] 2-4) Bovine serum albumin (BSA)

[0125] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0126] 2-5) Protein assay dye reagent concentrate

[0127] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0128] 2-6) Immuno-bolt for protein blotting ® PVDF membrane

[0129] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0130] 2-7) WEST SAVE GOLD,

[0131] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0132] 2-8) DaVinci Western Imaging System

[0133] Manufacturer: DAVINCH-K, CAS-400SM

[0134] 2-9) Rh TNF-α protein

[0135] Storage conditions: -20℃ frozen storage / Manufacturer: R&D System, 210-TA-005

[0136] (2) Test method

[0137] (2-1) in situ PLA

[0138] - Composition of the test group

[0139]

[0140] - Examination process

[0141] 1) Place 12 mm microscope cover glass in a 24-well culture plate and add 4.5X10 4 1 mL of cells were dispensed into each well.

[0142] 2) After culturing for 1 day, the monolayer culture status of the cells was checked, and the test substance was treated when the confluency of the cells was 60% or higher.

[0143] 3) After mixing 1, 5, and 10 ppm of each test substance with 200 μM H2O2, the negative control and test substance were treated for 30 minutes.

[0144] 4) After fixing the cells using 4% paraformaldehyde, pretreatment was performed to increase the permeability of the cell antibody by perforating the cells with 0.1% Triton X-100.

[0145] 5) Subsequent tests were conducted using the In situ PLA Kit (NaveniFlex 100RM) and the tests were conducted according to the manufacturer's instructions.

[0146] 6) After washing once with PBS, blocking was performed with blocking solution at 37°C for 30 minutes.

[0147] 7) 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).

[0148] 8) After adding the PLA probe and reacting at 37°C for 1 hour, it was washed three times with TTBS.

[0149] 9) Reactions A, B, and C were processed in sequence and reacted at 37°C for 1 hour, 30 minutes, and 90 minutes, respectively. Finally, the cells were washed twice with TBS (0.01 M Tris, 0.15 M NaCl) and mounted using a mounting solution containing DAPI (nuclear stain).

[0150] 10) The PLA signal detected in the cells was observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0151] - Observe and judge results

[0152] The PLA fluorescence signal was quantitatively analyzed and evaluated using NIS-Elements BR3.1. The luminescence signals resulting from the interaction between FAK-P53 and MDM2-P53 in the test substance-treated group were compared and analyzed based on the negative control group.

[0153] (2-1) Western blot analysis

[0154] - Composition of the test group

[0155]

[0156] - Examination process

[0157] 1) 5X10 in a 6-well culture plate 6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 80% or higher.

[0158] 2) The negative control and test substances were treated for 10 minutes at the appropriate concentration for each treatment group.

[0159] 3) TNF-α was treated at a concentration of 25 ng / ml for 5 minutes.

[0160] 4) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0161] 5) Electrophoresis was performed by loading 20 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0162] 6) The proteins analyzed by SDS-PAGE were transferred to a PVDF membrane.

[0163] 7) The PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.

[0164] 8) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0165] 9) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0166] 10) After exposure using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0167] - Observe and judge results

[0168] The expression levels of each protein after treatment with the test substance were evaluated by quantitative analysis using ImageJ based on the expression levels of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system. Changes in FKA phosphorylation in the test substance-treated group were observed based on the negative control group.

[0169] (3) Test results

[0170] (3-1) in situ PLA

[0171] Compared to the negative control group, the interaction between FAK-P53 and MDM2-P53 increased in the H2O2 treatment group, and in the treatment group in which FAK activation was inhibited by treatment with the test substance, the interaction between FAK-P53 and MDM2-P53 decreased in a concentration-dependent manner (Figs. 2 and 3).

[0172] (3-2) Western blot analysis

[0173] Compared to the negative control group, FAK (Y397) phosphorylation increased in the TNF-α treatment group, and it was observed that FAK (Y397) phosphorylation decreased in a concentration-dependent manner in the test substance treatment group (Fig. 4).

[0174] (4) Conclusion

[0175] As a result of evaluating the FAK signaling inhibitory ability of the test substance, it was confirmed that the test substance inhibited FAK activation and inactivated the downstream signaling pathway. Therefore, it is determined that the peptide of the present invention (a peptide composed of Glu-Leu-Cys) can act as a FAK antagonist.

[0176] Test Example 3: Evaluation of Inflammatory Response Inhibition Efficacy

[0177] FAK phosphorylated by TNF-α is known to activate IκB (Nuclear Factor-κB) and JNK, thereby inducing an inflammatory response. To confirm whether the peptide of the present invention exhibits anti-inflammatory efficacy by inhibiting FAK, the expression of IκB and JNK phosphorylation was confirmed through Western blot analysis.

[0178] (1) Test materials

[0179] - Preparation of test substances

[0180] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0181] - Test system

[0182] 1) Western blot analysis (IκB)

[0183] 1-1) Cell line: Human Keratinocyte (HaCaT, CLS)

[0184] 1-2) 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 2-3 days.

[0185] 1-3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0186] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated storage / Manufacturer: GIBCO

[0187] 2) Western blot analysis (JNK-phospho)

[0188] 2-1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0189] 2-2) 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 2-3 days.

[0190] 2-3) Medium: CEFOgro Human MSC Growth medium

[0191] Composition: 10% fetal bovine serum, 1% antibiotic / Storage conditions: Refrigerated / Manufacturer: CEFObio

[0192] - Test materials

[0193] 1) Western blot analysis (IκB)

[0194] 1-1) Anti-IκBα (L35A5) antibody

[0195] Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 4814

[0196] 1-2) Goat anti-mouse IgG Fc-HRP

[0197] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0198] 1-3) NP40 cell lysis buffer

[0199] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0200] 1-4) Bovine serum albumin (BSA)

[0201] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0202] 1-5) Protein assay dye reagent concentrate

[0203] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0204] 1-6) Immuno-bolt for protein blotting ® PVDF membrane

[0205] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0206] 1-7) WEST SAVE GOLD,

[0207] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0208] 1-8) DaVinci Western Imaging System

[0209] Manufacturer: DAVINCH-K, CAS-400SM

[0210] 1-9) Rh TNF-α protein

[0211] Storage conditions: -20℃ frozen storage / Manufacturer: R&D System, 210-TA-005

[0212] 2) Western blot analysis (JNK-phospho)

[0213] 2-1) Anti-JNK-phospho antibody

[0214] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-6254

[0215] 2-2) Goat anti-mouse IgG Fc-HRP

[0216] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0217] 2-3) NP40 cell lysis buffer

[0218] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0219] 2-4) Bovine serum albumin (BSA)

[0220] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0221] 2-5) Protein assay dye reagent concentrate

[0222] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0223] 2-6) Immuno-bolt for protein blotting ® PVDF membrane

[0224] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0225] 2-7) WEST SAVE GOLD,

[0226] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0227] 2-8) DaVinci Western Imaging System

[0228] Manufacturer: DAVINCH-K, CAS-400SM

[0229] 2-9) Rh TNF-α protein

[0230] Storage conditions: -20℃ frozen storage / Manufacturer: R&D System, 210-TA-005

[0231] (2) Test method

[0232] (2-1) Western blot analysis (IκB)

[0233] - Composition of the test group

[0234]

[0235] - Exam process

[0236] 1) 5X10 in a 6-well culture plate 6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 80% or higher.

[0237] 2) After mixing TNF-α at a concentration of 25 ng / ml with each of 1, 5, and 10 ppm of the test substance, the negative control and test substance were treated for 1 hour or 30 minutes.

[0238] 3) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0239] 4) Electrophoresis was performed by loading 20 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0240] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0241] 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.

[0242] 7) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0243] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0244] 9) After exposure using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0245] - Observe and judge results

[0246] The expression levels of each protein after treatment with the test substance were evaluated by quantitative analysis using ImageJ based on the expression levels of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system. Changes in the expression levels of IκB in the test substance-treated group were observed based on the negative control group.

[0247] (2-2) Western blot analysis (JNK-phospho)

[0248] - Composition of the test group

[0249]

[0250] - Examination process

[0251] 1) 5X10 in a 6-well culture plate 6 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 80% or higher.

[0252] 2) The negative control and test substances were treated for 10 minutes at the appropriate concentration for each treatment group.

[0253] 3) TNF-α was treated at a concentration of 25 ng / ml for 5 minutes.

[0254] 4) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0255] 5) Electrophoresis was performed by loading 20 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0256] 6) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0257] 7) The PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.

[0258] 8) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0259] 9) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0260] 10) After exposure using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0261] - Observe and judge results

[0262] The expression levels of each protein after treatment with the test substance were evaluated by quantitative analysis using ImageJ based on the expression levels of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system. Changes in JNK phosphorylation in the test substance-treated group were observed based on the negative control group.

[0263] (3) Test results

[0264] (3-1) Western blot analysis (IκB)

[0265] Compared to the negative control group, the expression level of IκB was decreased by TNF-α treatment, and in the test substance treatment group, the expression level of IκB was observed to increase in a concentration-dependent manner (Fig. 5).

[0266] (3-2) Western blot analysis (JNK-phospho)

[0267] Compared to the negative control group, JNK (T183 / H185) phosphorylation increased in the TNF-α treatment group and decreased in a concentration-dependent manner in the test substance treatment group (Fig. 6).

[0268] (4) Conclusion

[0269] It was confirmed that the test substance controlled the activation of IκB by increasing IκB decreased by TNF-α in a concentration-dependent manner, and suppressed the activation of JNK by reducing the phosphorylation of JNK (T183 / H185) increased by TNF-α. Therefore, it is judged that the peptide of the present invention (peptide composed of Glu-Leu-Cys) has an anti-inflammatory effect.

[0270] Test Example 4: Evaluation of collagen decomposition inhibition efficacy

[0271] It is known that when the phosphorylation of FAK decreases, the activity of MMP decreases and collagen degradation is inhibited. In order to confirm whether the peptide of the present invention inhibits the activity of collagen-degrading enzymes MMP-1 and MMP-9 and collagen degradation, the expression levels of MMP-1, MMP-9, and COL2B1 were observed through Western blot analysis, and the expression of intracellular MMP-9 and COL2B1 was observed through immunofluorescence staining.

[0272] (1) Test materials

[0273] - Preparation of test substances

[0274] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0275] - Test system

[0276] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0277] 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.

[0278] 3) Medium: CEFOgro Human MSC Growth medium

[0279] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage Conditions: Refrigerated / Manufacturer: CEFObio

[0280] - Test materials

[0281] 1) Western blot analysis

[0282] 1-1) Anti-MMP-1 (3B6) antibody

[0283] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-21731

[0284] 1-2) Anti-MMP-9 (C-20) antibody

[0285] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-6840

[0286] 1-3) Goat anti-mouse IgG Fc-HRP

[0287] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0288] 1-4) Rabbit anti-goat IgG Fc-HRP

[0289] Storage conditions: Refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0290] 1-5) NP40 cell lysis buffer

[0291] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, FNN0021

[0292] 1-6) Bovine serum albumin (BSA)

[0293] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0294] 1-7) Protein assay dye reagent concentrate

[0295] Storage conditions: Refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0296] 1-8) Immuno-bolt for protein blotting ® PVDF membrane

[0297] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0298] 1-9) WEST SAVE GOLD,

[0299] Storage conditions: Refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0300] 1-10) DaVinci Western Imaging System

[0301] Manufacturer: DAVINCH-K, CAS-400SM

[0302] 1-11) Rh TNF-α protein

[0303] Storage conditions: -20℃ frozen storage / Manufacturer: R&D System, 210-TA-005

[0304] 2) Immunofluorescence

[0305] 2-1) Anti-MMP-9 (C-20) antibody

[0306] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-6840

[0307] 2-2) Anti-COL1A2 (C-19) antibody

[0308] Storage conditions: Refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-8786

[0309] 2-3) Rabbit anti-goat IgG-FITC (Fluorescein isothiocyanate) antibody

[0310] Storage conditions: Refrigerated at 4℃ / Manufacturer: INVITROGEN, A11078

[0311] 2-4) Phalloidin-Rhodamin

[0312] Storage conditions: -20℃ frozen storage / Manufacturer: INVITROGEN, R415

[0313] 2-5) Rh TNF-α protein

[0314] Storage conditions: -20℃ frozen storage / Manufacturer: R&D System, 210-TA-005

[0315] 2-6) Bovine serum albumin (BSA)

[0316] Storage conditions: Refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0317] (2) Test method

[0318] (2-1) Western blot analysis

[0319] - Composition of the test group

[0320]

[0321] - Examination process

[0322] 1) 5X10 in a 6-well culture plate 6Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 80% or higher.

[0323] 2) The negative control and test substances were treated for 30 minutes at the appropriate concentration for each treatment group.

[0324] 3) TNF-α was treated at a concentration of 25 ng / ml for 24 hours.

[0325] 4) Cells were lysed using NP40 cell lysis buffer, and cell extracts for electrophoresis were prepared through quantification using the Bradford assay method.

[0326] 5) Electrophoresis was performed by loading 20 μg of cell extracts quantified on a sodium dodecyl sulfate-polyacrylamide gel into each well.

[0327] 6) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0328] 7) The PVDF membrane was treated with a blocking solution (3% BSA, 0.05% Tween 20, TBS) and reacted at room temperature for 1 hour.

[0329] 8) The primary antibody was reacted at room temperature for 2 hours, and washed three times with washing solution (0.05% Tween 20, TBS).

[0330] 9) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with washing solution.

[0331] 10) After photosensitization using an antibody detection kit, it was confirmed using a Western blot imaging system.

[0332] - Observe and judge results

[0333] The expression levels of each protein after treatment with the test substance were evaluated by quantitative analysis using ImageJ based on the expression levels of β-Actin, which was used as a loading control, by photographing using a Western blot imaging system. Changes in the expression levels of MMP-1 and MMP-9 in the test substance-treated group were observed based on the negative control group.

[0334] (2-2) Immunofluorescence staining

[0335] - Composition of the test group

[0336]

[0337] - Examination process

[0338] 1) Place 12 mm microscope round cover glasses in a 24-well culture plate and add 2.5X10 4 After dispensing the cells into each well, the monolayer culture status of the cells was checked after 24 hours of culture, and the test was conducted when the confluency of the cells was 50% or higher.

[0339] 2) The negative control and test substances were treated for 15 minutes at the appropriate concentration for each treatment group.

[0340] 3) TNF-α was treated at a concentration of 25 ng / ml for 24 hours.

[0341] 4) After fixing the cells using 4% paraformaldehyde, pretreatment was performed to increase the permeability of the cell antibody by perforating the cells with 0.1% Triton X-100.

[0342] 5) After washing once with PBS, blocking was performed with blocking solution (5% BSA in PBS) at 37°C for 10 minutes.

[0343] 6) To stain the cytoskeleton, phalloidin-rhodamine (1:500) was reacted at room temperature for 40 minutes, and then washed three times with washing solution (PBS).

[0344] 7) The antibody for confirmation was diluted 1:100, reacted at 4°C for 16 hours, and washed three times with PBS.

[0345] 8) The FITC-conjugated secondary antibody was diluted 1:500 and incubated at 37°C for 40 minutes, then washed 5 times with PBS.

[0346] 9) Mounting was performed using a mounting solution.

[0347] 10) The fluorescent signals detected in the cells were observed and photographed using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0348] - Observe and judge results

[0349] The expression levels of MMP-9 and COL1A2 (collagen) in the TNF-α treatment group and the test substance treatment group were compared and analyzed based on the control group.

[0350] (3) Test results

[0351] (3-1) Western blot analysis

[0352] Compared to the negative control group, the expression levels of MMP-1 and MMP-9 increased in the TNF-α treatment group, and decreased in a concentration-dependent manner in the test substance treatment group (Fig. 7).

[0353] (3-2) Immunofluorescence staining

[0354] Compared to the negative control group, the expression level of MMP-1 increased in the TNF-α treatment group and decreased in a concentration-dependent manner in the test substance treatment group, whereas the expression level of COL1A2 decreased in the TNF-α treatment group and increased in the test substance treatment group (Fig. 8).

[0355] (4) Conclusion

[0356] The test substance was confirmed to inhibit the activity of collagen-degrading enzymes MMP-1 and MMP-9 and increase collagen expression. Therefore, the peptide of the present invention (a peptide composed of Glu-Leu-Cys) is believed to have the effect of inhibiting collagen degradation by inhibiting the MMP reaction.

[0357] Test Example 5: Evaluation of Cell Aging Inhibition Efficacy

[0358] Ultraviolet (UV) light activates matrix metalloproteinases (MMPs) to degrade ECM (extracellular matrix) components, thereby inducing aging. As cells age, the expression of beta-galactosidase increases. The peptide of the present invention was examined to determine whether it inhibits cellular aging using a senescence beta-galactosidase staining assay.

[0359] (1) Test materials

[0360] - Preparation of test substances

[0361] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0362] - Test system

[0363] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0364] 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.

[0365] 3) Medium: CEFOgro Human MSC Growth medium

[0366] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage Conditions: Refrigerated / Manufacturer: CEFObio

[0367] - Test materials

[0368] 1) Senescence beta-galactosidase Staining Kit

[0369] Storage conditions: -20℃ frozen storage / Manufacturer: CELL SIGNALING, 9860S

[0370] 2) VLX-3W research radiometer

[0371] Manufacturer: VILVER

[0372] (2) Test method

[0373] - Composition of the test group

[0374]

[0375] - Examination process

[0376] 1) 5X10 in a 24-well culture plate 5 Cells were seeded into each well. After 24 hours of culture, the monolayer culture status of the cells was confirmed, and testing was performed when the cell confluency was 100% or higher.

[0377] 2) After removing the medium, add PBS and use a VLX-3W research radiometer to irradiate UVB (312 nm) at 0.06 J / cm 2 The intensity of the test was investigated.

[0378] 3) After removing PBS, the negative control and test substances were treated for 24 hours at the appropriate concentration for each treatment group.

[0379] 4) After rinsing with PBS, fixation was performed by treating with a fixing solution for 15 minutes.

[0380] 5) After rinsing with PBS, the cells were treated with β-galactosidase staining solution using the Senescence β-Galactosidase Staining Kit and cultured at 37°C for more than 16 hours.

[0381] 6) Observed and photographed using a microscope (OLYMPUS, CKX53).

[0382] - Observe and judge results

[0383] The degree of senescent cell (green) production in the UVB treatment group and the test substance treatment group was compared and analyzed based on the negative control group.

[0384] (3) Test results

[0385] Compared to the control group, the UVB treatment group was observed to have an increased production of senescent cells, and the test substance treatment group was confirmed to have a concentration-dependent decrease in the production of senescent cells (Fig. 9).

[0386] (4) Conclusion

[0387] It was confirmed that the generation of senescent cells decreased in a concentration-dependent manner in the test substance-treated group. Therefore, it is believed that the peptide of the present invention (a peptide composed of Glu-Leu-Cys) has the effect of reducing the generation of senescent cells caused by UVB.

[0388] Test Example 6: Evaluation of Collagen Decomposition Inhibition Efficacy in a 3D Human Skin Model

[0389] It is known that UV irradiation of a 3D human skin culture model reduces collagen production. Using Verhoeff Van Gieson Staining, we observed whether the peptide of the present invention inhibits UV-induced collagen degradation in a 3D human skin model (Neoderm-ED) similar to human skin.

[0390] (1) Test materials

[0391] - Preparation of test substances

[0392] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple-distilled water to prepare a concentration of 1000 ppm.

[0393] - Test system

[0394] 1) 3D human skin model: Neoderm-ED

[0395] 2) Management: Cultured in an incubator (5% CO2, 37℃) and tested within 3 days of receipt.

[0396] 3) Badge: Maintenance medium

[0397] Composition: 10% fetal bovine serum / Storage conditions: Refrigerated storage / Manufacturer: TEGO SCIENCE

[0398] - Test materials

[0399] 1) Elastic stain Kit (Verhoeff Van Gieson / EVG Stain)

[0400] Storage conditions: Store at room temperature / Manufacturer: ABCAM, ab150667

[0401] 2) VLX-3W research radiometer

[0402] Manufacturer: VILVER

[0403] (2) Test method

[0404] - Composition of the test group

[0405]

[0406] - Examination process

[0407] 1) After receiving Neoderm-ED, a dedicated medium was added and cultured for 24 hours.

[0408] 2) Using a VLX-3W research radiometer on Neoderm-ED, UVB (312 nm) was applied at 0.06 J / cm 2 Investigated by the intensity of

[0409] 3) The negative control and test substances were treated at a concentration of 10 ppm for 48 hours.

[0410] 4) Neoderm-ED was separated from the insert well using a blade to create a paraffin block.

[0411] 5) Slides were prepared by cutting sections at 4 ㎛ thickness.

[0412] 6) Paraffin washing and water dehydration process using xylene (Et-OH 100% >95%>90%>80%>70%) was performed.

[0413] 7) After rinsing in DW, the Elastic Stain Kit (Verhoeff Van Gieson EVG Stain) (abcam) was used, and the test was performed according to the manufacturer's instructions.

[0414] 8) Mix hematoxyline solution (5%), ferric chloride solution (10%), and Lugol's iodine solution to make an Elastic Stain Solution, apply it to the slide for 15 minutes, and wash the slide in running water.

[0415] 9) Rinse the slide 20 times in ferric chloride (2%) differentiating solution and then wash it again in running water.

[0416] 10) After treating with sodium thiosulfate solution for 1 minute, the slides were washed in running water.

[0417] 11) After treating with Van Gieson's solution for 2 minutes, it was washed with Et-OH 95% > 100%.

[0418] 12) Mounting was performed using a mounting solution.

[0419] 13) The stained tissue was observed and photographed using a microscope (OLYMPUS, BX53F2).

[0420] - Observe and judge results

[0421] Based on the negative control group, the degree of skin structure damage and collagen decomposition caused by UVB in the test substance treatment group was compared and analyzed.

[0422] (3) Test results

[0423] Compared to the negative control group, the UVB treatment group showed damage to the shape of the epidermal layer and dermal layer, whereas the test substance treatment group showed recovery of the structure of the epidermal layer and dermal layer similar to the negative control group (Fig. 10).

[0424] (4) Conclusion

[0425] It was confirmed that the peptide of the present invention restored skin tissue damaged by UVB in a 3D human skin model. Therefore, it is believed that the peptide of the present invention (a peptide composed of Glu-Leu-Cys) has the effect of inhibiting collagen degradation not only at the cellular level but also in skin tissue, as well as the effect of protecting tissue from UVB or helping to restore damaged tissue.

Claims

1. A pharmaceutical composition for preventing or treating inflammation, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. <Chemical formula 1> 2. A pharmaceutical composition according to claim 1, characterized in that the inflammation is skin inflammation.

3. A pharmaceutical composition according to claim 1, characterized in that the inflammation is skin inflammation caused by ultraviolet rays.

4. A cosmetic composition for improving skin inflammation or skin wrinkles, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof. <Chemical formula 1> 5. A cosmetic composition according to claim 4, characterized in that the skin inflammation or skin wrinkle is skin inflammation or skin wrinkle caused by ultraviolet rays.

Images