Pharmaceutical compositions for preventing or treating inflammation and cosmetic compositions for improving inflammation or skin-wrinkle

Abstract

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammation comprising a specific peptide; and a cosmetic composition for the improvement of skin inflammation or skin wrinkles. The peptide acts as an antagonist to FAK and inhibits signaling pathways associated with FAK, thereby lowering the expression of MMP-1 and MMP-9 and inhibiting the degradation of collagen. The peptide can be usefully used for the prevention or treatment of inflammation; and for the improvement of skin inflammation or skin wrinkles.

Description

Technology Field

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

[0002] Focal adhesion kinases (FAKs) activated by inflammation-inducing molecules induce an inflammatory response 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; phosphorylated FAK enters the nucleus and binds to P53 and MDM2, inducing the ubiquitination of P53 and proteasome degradation to inhibit apoptosis, while FAK phosphorylated by TNF-α activates IκB (Nuclear Factor-κB) and JNK to induce 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 et al.). When the phosphorylation of FAK decreases, the activity of MMPs decreases 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, substances capable of inhibiting FAK-associated signaling pathways can not only act as active substances capable of suppressing inflammation, but also act as functional cosmetic substances to improve skin inflammation or skin aging (e.g., skin wrinkles).

[0004] The inventors have disclosed that peptides derived from the p65 subunit of NF-κB and peptides 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 No. 10-1594032 and No. 10-2041803). The problem to be solved

[0005] The inventors conducted various studies to develop an active substance based on small peptides that can act as an antagonist to FAK. As a result, they discovered that a specific peptide, namely a 3-mer peptide composed of Glu-Leu-Cys, acts as an antagonist to FAK and inhibits FAK-associated signaling pathways, thereby lowering the expression of MMP-1 and MMP-9 and inhibiting the degradation of collagen, which can be usefully applied to the prevention or treatment of inflammation; and the improvement of skin inflammation or skin wrinkles.

[0006] Accordingly, the present invention aims to provide a pharmaceutical composition for the prevention or treatment of 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 comprising the specific peptide. means of solving the problem

[0008] According to one aspect of the present invention, a pharmaceutical composition for the prevention or treatment of inflammation is provided, comprising a peptide of the following 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 a peptide of Formula 1 or a pharmaceutically acceptable salt thereof. Effects of the invention

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

[0013] Figure 1 shows the results of measuring the signal reduction of the peptide (VE-Fakanin)-AMC and FAK-FITC of the present invention composed of Glu-Leu-Cys by FAK siRNA treatment. Figure 2 is the result of analyzing the change in FAK-P53 interaction by peptide treatment of the present invention. Figure 3 is the result of analyzing the change in MDM2-P53 interaction by peptide treatment of the present invention. Figure 4 shows the results of analyzing changes in FAK(Y397) phosphorylation by peptide treatment of the present invention. Figure 5 is the result of analyzing the change in IκB expression levels by peptide treatment of the present invention. Figure 6 shows the results of analyzing the change in JNK(T183 / Y185) phosphorylation by peptide treatment of the present invention. Figure 7 is the result of analyzing the change in MMP-1 and MMP-9 expression levels by peptide treatment of the present invention. Figure 8 shows the results of analyzing changes in MMP-1 and collagen expression levels by peptide treatment of the present invention. Figure 9 is the result of analyzing the change in the amount of senescent cells produced by the peptide treatment of the present invention. Figure 10 shows the results of analyzing changes in tissue state caused by peptide treatment of the present invention in a 3D human skin model. Specific details for implementing the invention

[0014] The present invention provides a pharmaceutical composition for the prevention or treatment of inflammation, comprising a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0015] <Chemical Formula 1>

[0016]

[0017] In addition, the present invention provides a cosmetic composition for improving skin inflammation or skin wrinkles, comprising a peptide of Formula 1 or a pharmaceutically acceptable salt thereof.

[0018] In the pharmaceutical composition or cosmetic composition of the present invention, the peptide of Formula 1 may also be denoted as "Glu-Leu-Cys". The amino acids constituting the peptide of Formula 1 may independently be in the form of L-amino acids or D-amino acids. Pharmaceutically acceptable salts of the peptide derivative of Formula 1 include, for example, acid addition salts, but are not limited thereto.

[0019] In the pharmaceutical composition or cosmetic composition of the present invention, the "inflammation" includes skin inflammation caused by various causes (i.e., dermatitis), and preferably includes skin inflammation caused by ultraviolet rays.

[0020] "Skin aging" refers to skin aging, including the formation of skin wrinkles caused by intrinsic and external factors, and preferably may be skin photoaging accompanied by the formation of skin wrinkles, and more preferably includes skin photoaging caused by ultraviolet stimulation accompanied by the formation of skin wrinkles. In one embodiment, the skin aging includes wrinkle formation on the skin, i.e., skin wrinkles.

[0021] The pharmaceutical composition of the present invention may include excipients such as lactose and corn starch, lubricants such as magnesium stearate, known and usable emulsifiers, suspending agents, buffers, isotonic agents, etc., and may be formulated in a parenteral administration form, preferably a parenteral administration form including a topical preparation for the skin. In the case of intramuscular, intraperitoneal, subcutaneous, and intravenous administration forms, a sterile solution of the active ingredient is typically prepared, and a buffer capable of appropriately adjusting the pH of the solution may be included; in the case of intravenous administration, an isotonic agent may be included to impart isotonicity to the preparation. Furthermore, the pharmaceutical composition of the present invention may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier, such as saline solution with a pH of 7.4, and may be introduced topically into the intramuscular bloodstream of a patient in the form of a solution. Additionally, it may be formulated into transdermal administration formulations such as topical solutions, emulsions, ointments, and patches according to conventional pharmaceutical methods. The pharmaceutical composition of the present invention may be administered to patients with various inflammations at a dose of about 1 to 50 mg / kg per day. The appropriate dosage may generally be changed depending on the patient's age, weight, and symptoms.

[0022] The cosmetic composition of the present invention may be in the form of a functional cosmetic composition comprising the above-described peptide as an active ingredient. The cosmetic composition may be prepared in various forms according to conventional cosmetic manufacturing methods. For example, the cosmetic composition may be prepared in the form of cosmetic products, toners, creams, lotions, etc., containing the above-described peptide, and may be used by diluting with conventional cleansing liquids, astringent liquids, and moisturizing liquids. 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 above-described peptide is an amount effective for achieving an effect of improving skin inflammation or skin wrinkles, for example, 1 x 10⁻⁶ with respect to the total weight of the composition. -5 ~ 1 x 10 -2 It may be contained in a weight % content, preferably about 1 x 10 -4 ~ 1 x 10 -3 It may be contained in a weight % content.

[0023] The present invention will be explained in more detail below 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.

[0024] Example 1. Synthesis of Peptides

[0025] A peptide composed of Glu-Leu-Cys was synthesized using the FMOC solid-phase method with an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, South Korea). The synthesized peptide was purified and analyzed using reverse-phase HPLC (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, Roseville, USA).

[0026] Example 2. Preparation of a composition containing a peptide

[0027] The peptide prepared in Example 1 (a peptide composed of Glu-Leu-Cys) was dissolved in triple-distilled water to a concentration of 1000 ppm. The obtained peptide solution was used in the following test examples.

[0028] Test Example 1: FAK binding evaluation

[0029] 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.

[0030] (1) Test materials

[0031] - Preparation of test substances

[0032] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0033] - Test

[0034] 1) Cell line: Human keratinocyte (HaCaT, CLS)

[0035] 2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0036] 3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0037] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: GIBCO

[0038] - Test materials

[0039] 1) Peptide-AMC of the present invention

[0040] Storage Conditions: Store frozen at -20℃ / Manufacturer: Peptron

[0041] 2) Anti-FAK(C-20) antibody

[0042] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-558

[0043] 3) Goat anti-Rabbit IgG-FITC (Fluorescein isothiocyanate) antibody

[0044] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: INVITROGEN, A27034

[0045] 4) Phalloidin-Rhodamin

[0046] Storage Conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, R415

[0047] 5) siRNA transfection

[0048] 5-1) FAK siRNA

[0049] Storage Conditions: Store frozen at -20℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-29310

[0050] 5-2) Lipofectamine™ RNAiMAX Transfection Reagent

[0051] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: INVITROGEN, 13778075

[0052] 5-3) Opti-MEM ® Medium

[0053] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: GIBCO, 31985062

[0054] 6) Rh TNF-α protein

[0055] Storage Conditions: Store frozen at -20℃ / Manufacturer: R&D System, 210-TA-005

[0056] (2) Test method

[0057] - Composition of the test group

[0058] Sample throughput n per test group Control siRNA 1 mL 5 TNF-α 25 ng / ml + Control siRNA 1 mL 5 TNF-α + FAK siRNA 30 nM 1 mL 5

[0059] - Examination process

[0060] 1) Place a 12 mm circular microscope cover glass in a 24-well culture plate and 2.5 x 10 4 After dispensing 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 cell confluence was 50% or higher.

[0061] 2) For siRNA treatment, the medium was replaced with a dedicated medium (DMEM + 10% FBS).

[0062] 3) siRNA + Opti-MEM ® Media and transfection reagent + Opti-MEM ® The culture medium was prepared in a 1:1 ratio, slowly mixed, and then reacted at room temperature for 5 minutes.

[0063] 4) The solution of 3) was added to cultured cells and treated for 48 hours to conduct the test.

[0064] 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).

[0065] 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).

[0066] 7) FAK antibody (1:100) was treated and reacted at room temperature for 40 minutes, then washed three times with washing solution (PBS).

[0067] 8) The secondary antibody (1:1000) was treated and reacted at room temperature for 40 minutes, then washed three times with a washing solution (PBS).

[0068] 9) Mounting was performed using a mounting solution.

[0069] 10) The fluorescence signal (AMC) detected in the cells was observed and captured using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0070] - Observation and judgment of results

[0071] The luminescence levels of the peptide-AMC of the present invention in the TNF-α treated group and the FAK siRNA treated group were compared and analyzed based on the control siRNA negative control group.

[0072] (3) Test results

[0073] Compared to the negative control group, it was observed that the peptide-AMC signal (blue fluorescence) of the present invention increased upon TNF-α treatment, and it was observed that the peptide-AMC signal of the present invention decreased upon simultaneous treatment with FAK siRNA. A similar signal pattern was also observed in the same test group treated with FAK antibodies (Fig. 1). These results suggest that the test substance specifically binds to FAK, the target protein.

[0074] (4) Conclusion

[0075] As a result of evaluating the degree of FAK binding of the test substance, the test substance bound to FKA in the TNF-α treated group and showed an AMC (blue fluorescence) signal, and when FAK expression was inhibited by treatment with FAK siRNA, the AMC signal decreased. Therefore, it is determined that the peptide of the present invention (a peptide composed of Glu-Leu-Cys) specifically binds to FAK.

[0076] Test Example 2: Evaluation of FAK signaling inhibition efficacy

[0077] FAK is phosphorylated and activated by H2O2 or TNF-α, and phosphorylated FAK is known to enter the nucleus and bind to P53 and MDM2, thereby inducing ubiquitination of P53 and proteasome degradation to inhibit apoptosis. To confirm whether the peptide of the present invention inhibits FAK activation, the effect on the degree of FAK phosphorylation was investigated through Western blot analysis, and the interactions between FAK, P53, and MDM2 in the nucleus were confirmed through in situ PLA.

[0078] (1) Test materials

[0079] - Preparation of test substances

[0080] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0081] - Test

[0082] 1) in situ PLA

[0083] 1-1) Cell line: Human keratinocyte (HaCaT, CLS)

[0084] 1-2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0085] 1-3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0086] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: GIBCO

[0087] 2) Western blot analysis

[0088] 2 - 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0089] 2-2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0090] 2-3) Medium: CEFOgro Human MSC Growth Medium

[0091] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: CEFObio

[0092] - Test materials

[0093] 1) in situ PLA

[0094] 1-1) Anti-FAK(B-8) Antibody

[0095] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-271195

[0096] 1-2) Anti-P53 Antibody

[0097] Storage Conditions: Store frozen at -20℃ / Manufacturer: CELL SIGNALING, 2527S

[0098] 1-3) Anti-MDM2(D-7) Antibody

[0099] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-13161

[0100] 1-4) Hydrogen peroxide (H2O2)

[0101] Storage Conditions: Store frozen at -20℃ / Manufacturer: JUNSEI, 23150S0350

[0102] 1-5) NaveniFlex 100RM

[0103] Storage Conditions: Store frozen at -20℃ / Manufacturer: NaveniFlex, NV C-NF MR.100

[0104] 1-6) Prolong™ diamond antifade mountant with DAPI

[0105] Storage Conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, P36962

[0106] 1-7) Digital Fluorescence Imaging System

[0107] Manufacturer: LOGOS BIOSYSTEMS, CS20002

[0108] 2) Western blot analysis

[0109] 2-1) Anti-FAK-phospho(Y397) Antibody

[0110] Storage Conditions: Store frozen at -20℃ / Manufacturer: CELL SIGNALING, 3283

[0111] 2-2) Goat Anti-Rabbit IgG Fc-HRP

[0112] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0113] 2-3) NP40 Cell Lysis Buffer

[0114] Storage Conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, FNN0021

[0115] 2-4) Bovine serum albumin (BSA)

[0116] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0117] 2-5) Protein assay dye reagent concentrate

[0118] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0119] 2-6) Immuno-bolt for Protein Blotting ® PVDF membrane

[0120] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0121] 2-7) WEST SAVE GOLD,

[0122] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0123] 2-8) Da Vinci Western Imaging System

[0124] Manufacturer: DAVINCH-K, CAS-400SM

[0125] 2-9) Rh TNF-α protein

[0126] Storage Conditions: Store frozen at -20℃ / Manufacturer: R&D System, 210-TA-005

[0127] (2) Test method

[0128] (2-1) in situ PLA

[0129] - Composition of the test group

[0130] Sample throughput n per test group Control 1 mL 5 H2O2200 μM 1 mL 5 H2O2 + peptide of the present invention 2.5 ppm 1 mL 5 H2O2 + peptide of the present invention 5 ppm 1 mL 5 H2O2 + peptide of the present invention 10 ppm 1 mL 5

[0131] - Examination process

[0132] 1) Place a 12 mm microscope ring cover glass in a 24-well culture plate and 4.5 x 10 4 1 mL of cells were dispensed into each well.

[0133] 2) After culturing for 1 day, the monolayer culture status of the cells was checked, and the test substance was applied when the cell confluence was 60% or higher.

[0134] 3) After preparing H2O2 at a concentration of 200 μM mixed with test substances at 1, 5, and 10 ppm, respectively, the negative control and test substances were treated for 30 minutes.

[0135] 4) After fixing the cells using 4% paraformaldehyde, the cells were perforated with 0.1% Triton X-100 to perform a pretreatment that increases the permeability of cell antibodies.

[0136] 5) Subsequent tests were conducted using an In situ PLA Kit (NaveniFlex 100RM) and were performed according to the manufacturer's instructions.

[0137] 6) After washing once with PBS, block with blocking solution at 37°C for 30 minutes.

[0138] 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).

[0139] 8) After adding the PLA probe and reacting at 37°C for 1 hour, the mixture was washed three times with TTBS.

[0140] 9) Reactions A, B, and C were treated sequentially and reacted at 37°C for 1 hour, 30 minutes, and 90 minutes, respectively. Finally, the samples were washed twice with TBS (0.01 M Tris, 0.15 M NaCl) and mounted using a mounting solution containing DAPI (nuclear staining).

[0141] 10) PLA signals detected in cells were observed and captured using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0142] - Observation and judgment of results

[0143] The PLA fluorescence signal was evaluated by quantitative analysis using NIS-Elements BR3.1. The luminescence signal resulting from the interaction between FAK-P53 and MDM2-P53 in the test substance treatment group was compared and analyzed against the negative control group.

[0144] (2-1) Western Blot Analysis

[0145] - Composition of the test group

[0146] Sample throughput n per test group Control 1 mL 3 TNF-α 25 ng / ml 1 mL 3 TNF-α + peptide of the present invention 1 ppm 1 mL 3 TNF-α + peptide of the present invention 5 ppm 1 mL 3 TNF-α + peptide of the present invention 10 ppm 1 mL 3

[0147] - Examination process

[0148] 1) 5 x 10⁶ in a 6-well culture plate 6 Cells were dispensed into each well. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was conducted when the cell confluence was 80% or higher.

[0149] 2) The negative control and test substance were treated for 10 minutes according to the concentration of each treatment group.

[0150] 3) TNF-α was administered at a concentration of 25 ng / ml for 5 minutes.

[0151] 4) Cells were lysed using NP40 cell lysis buffer, and a cell extract for electrophoresis was prepared through quantification using the Bradford assay method.

[0152] 5) 20 μg of the quantified cell extract was loaded into each well of a sodium dodecyl sulfate-polyacrylamide gel and electrophoresis was performed.

[0153] 6) Proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0154] 7) A blocking solution (3% BSA, 0.05% Tween 20, TBS) was applied to a PVDF membrane and reacted at room temperature for 1 hour.

[0155] 8) The primary antibody was reacted at room temperature for 2 hours and washed 3 times with a washing solution (0.05% Tween 20, TBS).

[0156] 9) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with the washing solution.

[0157] 10) After exposure using an antibody detection kit, the results were confirmed through a Western blot imaging system.

[0158] - Observation and judgment of results

[0159] The expression levels of each protein resulting from treatment with the test substance were evaluated by quantitative analysis using ImageJ, based on the expression level of β-Actin used as a loading control, which was captured using a Western blot imaging system. Changes in FKA phosphorylation in the test substance treatment group were observed based on the negative control.

[0160] (3) Test results

[0161] (3-1) in situ PLA

[0162] Compared to the negative control group, the interaction between FAK-P53 and MDM2-P53 increased in the H2O2-treated group, and the interaction between FAK-P53 and MDM2-P53 decreased in a concentration-dependent manner in the treatment group in which FAK activation was inhibited by treatment with the test substance (Figs. 2 and 3).

[0163] (3-2) Western Blot Analysis

[0164] Compared to the negative control group, FAK (Y397) phosphorylation increased in the TNF-α treated group, and FAK (Y397) phosphorylation decreased in a concentration-dependent manner in the test substance treated group (Fig. 4).

[0165] (5) Conclusion

[0166] As a result of evaluating the FAK signaling inhibitory ability of the test substance, it was confirmed that the test substance inhibits FAK activation and inactivates 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 an FAK antagonist.

[0167] Test Example 3: Evaluation of efficacy in suppressing inflammatory response

[0168] FAK phosphorylated by TNF-α is known to cause an inflammatory response by activating IκB (Nuclear Factor-κB) and JNK. 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.

[0169] (1) Test materials

[0170] - Preparation of test substances

[0171] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0172] - Test

[0173] 1) Western blot analysis (IκB)

[0174] 1-1) Cell line: Human keratinocyte (HaCaT, CLS)

[0175] 1-2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0176] 1-3) Medium: DMEM (Dulbecco's Modified Eagle Medium)

[0177] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: GIBCO

[0178] 2) Western blot analysis (JNK-phospho)

[0179] 2 - 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0180] 2-2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0181] 2-3) Medium: CEFOgro Human MSC Growth Medium

[0182] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: CEFObio

[0183] - Test materials

[0184] 1) Western blot analysis (IκB)

[0185] 1-1) Anti-IκBα(L35A5) antibody

[0186] Storage Conditions: Store frozen at -20℃ / Manufacturer: CELL SIGNALING, 4814

[0187] 1-2) Goat Anti-Mouse IgG Fc-HRP

[0188] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0189] 1-3) NP40 Cell Lysis Buffer

[0190] Storage Conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, FNN0021

[0191] 1-4) Bovine serum albumin (BSA)

[0192] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0193] 1-5) Protein assay dye reagent concentrate

[0194] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0195] 1-6) Immuno-bolt for protein blotting ® PVDF membrane

[0196] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0197] 1-7) WEST SAVE GOLD,

[0198] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0199] 1-8) Da Vinci Western Imaging System

[0200] Manufacturer: DAVINCH-K, CAS-400SM

[0201] 1-9) Rh TNF-α protein

[0202] Storage Conditions: Store frozen at -20℃ / Manufacturer: R&D System, 210-TA-005

[0203] 2) Western blot analysis (JNK-phospho)

[0204] 2-1) Anti-JNK-phospho antibody

[0205] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-6254

[0206] 2-2) Goat Anti-Mouse IgG Fc-HRP

[0207] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0208] 2-3) NP40 Cell Lysis Buffer

[0209] Storage Conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, FNN0021

[0210] 2-4) Bovine serum albumin (BSA)

[0211] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0212] 2-5) Protein assay dye reagent concentrate

[0213] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0214] 2-6) Immuno-bolt for Protein Blotting ® PVDF membrane

[0215] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0216] 2-7) WEST SAVE GOLD,

[0217] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0218] 2-8) Da Vinci Western Imaging System

[0219] Manufacturer: DAVINCH-K, CAS-400SM

[0220] 2-9) Rh TNF-α protein

[0221] Storage Conditions: Store frozen at -20℃ / Manufacturer: R&D System, 210-TA-005

[0222] (2) Test method

[0223] (2-1) Western blot analysis (IκB)

[0224] - Composition of the test group

[0225] Sample throughput n per test group Control 1 mL 3 TNF-α 25 ng / ml 1 mL 3 TNF-α + peptide of the present invention 1 ppm 1 mL 3 TNF-α + peptide of the present invention 5 ppm 1 mL 3 TNF-α + peptide of the present invention 10 ppm 1 mL 3

[0226] - Exam process

[0227] 1) 5 x 10⁶ in a 6-well culture plate 6 Cells were dispensed into each well. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was conducted when the cell confluence was 80% or higher.

[0228] 2) TNF-α was prepared by mixing it with test substances at concentrations of 1, 5, and 10 ppm, respectively, at a concentration of 25 ng / ml, and then the negative control and test substances were treated for 1 hour or 30 minutes.

[0229] 3) Cells were lysed using NP40 cell lysis buffer, and a cell extract for electrophoresis was prepared through quantification using the Bradford assay method.

[0230] 4) 20 μg of the quantified cell extract was loaded into each well of a sodium dodecyl sulfate-polyacrylamide gel and electrophoresis was performed.

[0231] 5) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0232] 6) A blocking solution (3% BSA, 0.05% Tween 20, TBS) was applied to a PVDF membrane and reacted at room temperature for 1 hour.

[0233] 7) The primary antibody was reacted at room temperature for 2 hours, and washed 3 times with a washing solution (0.05% Tween 20, TBS).

[0234] 8) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with the washing solution.

[0235] 9) After exposure using an antibody detection kit, the results were confirmed through a Western blot imaging system.

[0236] - Observation and judgment of results

[0237] The expression levels of each protein resulting from treatment with the test substance were evaluated by quantitative analysis using ImageJ, based on the expression level of β-Actin used as a loading control, which was captured using a Western blot imaging system. Changes in IκB expression levels in the test substance treatment group were observed based on the negative control.

[0238] (2-2) Western blot analysis (JNK-phospho)

[0239] - Composition of the test group

[0240] Sample throughput n per test group Control 1 mL 3 TNF-α 25 ng / ml 1 mL 3 TNF-α + peptide of the present invention 1 ppm 1 mL 3 TNF-α + peptide of the present invention 5 ppm 1 mL 3 TNF-α + peptide of the present invention 10 ppm 1 mL 3

[0241] - Examination process

[0242] 1) 5 x 10⁶ in a 6-well culture plate 6 Cells were dispensed into each well. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was conducted when the cell confluence was 80% or higher.

[0243] 2) The negative control and test substance were treated for 10 minutes according to the concentration of each treatment group.

[0244] 3) TNF-α was administered at a concentration of 25 ng / ml for 5 minutes.

[0245] 4) Cells were lysed using NP40 cell lysis buffer, and a cell extract for electrophoresis was prepared through quantification using the Bradford assay method.

[0246] 5) 20 μg of the quantified cell extract was loaded into each well of a sodium dodecyl sulfate-polyacrylamide gel and electrophoresis was performed.

[0247] 6) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0248] 7) A blocking solution (3% BSA, 0.05% Tween 20, TBS) was applied to a PVDF membrane and reacted at room temperature for 1 hour.

[0249] 8) The primary antibody was reacted at room temperature for 2 hours and washed 3 times with a washing solution (0.05% Tween 20, TBS).

[0250] 9) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with the washing solution.

[0251] 10) After exposure using an antibody detection kit, the results were confirmed through a Western blot imaging system.

[0252] - Observation and judgment of results

[0253] The expression levels of each protein treated with the test substance were evaluated by quantitative analysis using ImageJ, based on the expression level of β-Actin used as a loading control, which was captured using a Western blot imaging system. Changes in JNK phosphorylation in the test substance treatment group were observed based on the negative control.

[0254] (3) Test results

[0255] (3-1) Western blot analysis (IκB)

[0256] Compared to the negative control group, the expression level of IκB decreased due to TNF-α treatment, and in the test substance treatment group, the expression level of IκB increased in a concentration-dependent manner (Fig. 5).

[0257] (3-2) Western blot analysis (JNK-phospho)

[0258] Compared to the negative control group, JNK(T183 / H185) phosphorylation increased in the TNF-α treated group and decreased in a concentration-dependent manner in the test substance treated group (Fig. 6).

[0259] (5) Conclusion

[0260] It was confirmed that the test substance controlled the activation of IκB by increasing IκB, which is reduced by TNF-α, in a concentration-dependent manner, and inhibited the activation of JNK by decreasing JNK (T183 / H185) phosphorylation, which is increased by TNF-α. Therefore, the peptide of the present invention (a peptide composed of Glu-Leu-Cys) is considered to have an anti-inflammatory effect.

[0261] Test Example 4: Evaluation of Collagen Degradation Inhibitory Efficacy

[0262] 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.

[0263] (1) Test materials

[0264] - Preparation of test substances

[0265] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0266] - Test

[0267] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0268] 2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0269] 3) Medium: CEFOgro Human MSC Growth Medium

[0270] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: CEFObio

[0271] - Test materials

[0272] 1) Western blot analysis

[0273] 1-1) Anti-MMP-1(3B6) Antibody

[0274] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-21731

[0275] 1-2) Anti-MMP-9(C-20) Antibody

[0276] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-6840

[0277] 1-3) Goat Anti-Mouse IgG Fc-HRP

[0278] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0279] 1-4) Rabbit Anti-Goat IgG Fc-HRP

[0280] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: ABFRONTEIR

[0281] 1-5) NP40 Cell Lysis Buffer

[0282] Storage Conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, FNN0021

[0283] 1-6) Bovine serum albumin (BSA)

[0284] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0285] 1-7) Protein assay dye reagent concentrate

[0286] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: BIO_RAD, #5000006

[0287] 1-8) Immuno-bolt for protein blotting ® PVDF membrane

[0288] Storage conditions: Store at room temperature / Manufacturer: BIO-RAD, #1620177

[0289] 1-9) WEST SAVE GOLD,

[0290] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: AB FRONTIER, LF-QC0103

[0291] 1-10) Da Vinci Western Imaging System

[0292] Manufacturer: DAVINCH-K, CAS-400SM

[0293] 1-11) Rh TNF-α protein

[0294] Storage Conditions: Store frozen at -20℃ / Manufacturer: R&D System, 210-TA-005

[0295] 2) Immunofluorescence

[0296] 2-1) Anti-MMP-9(C-20) Antibody

[0297] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-6840

[0298] 2-2) Anti-COL1A2(C-19) Antibody

[0299] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: SANTA CRUZ BIOTECHNOLOGY, sc-8786

[0300] 2-3) Rabbit anti-goat IgG-FITC (Fluorescein isothiocyanate) antibody

[0301] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: INVITROGEN, A11078

[0302] 2-4) Phalloidin-Rhodamin

[0303] Storage Conditions: Store frozen at -20℃ / Manufacturer: INVITROGEN, R415

[0304] 2-5) Rh TNF-α protein

[0305] Storage Conditions: Store frozen at -20℃ / Manufacturer: R&D System, 210-TA-005

[0306] 2-6) Bovine serum albumin (BSA)

[0307] Storage Conditions: Keep refrigerated at 4℃ / Manufacturer: CELLCONIC, FNN0021

[0308] (2) Test method

[0309] (2-1) Western Blot Analysis

[0310] - Composition of the test group

[0311] Sample throughput n per test group Control 1 mL 3 TNF-α 25 ng / ml 1 mL 3 TNF-α + peptide of the present invention 1 ppm 1 mL 3 TNF-α + peptide of the present invention 5 ppm 1 mL 3 TNF-α + peptide of the present invention 10 ppm 1 mL 3

[0312] - Examination process

[0313] 1) 5 x 10⁶ in a 6-well culture plate 6 Cells were dispensed into each well. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was conducted when the cell confluence was 80% or higher.

[0314] 2) The negative control and test substance were treated for 30 minutes according to the concentration of each treatment group.

[0315] 3) TNF-α was administered at a concentration of 25 ng / ml for 24 hours.

[0316] 4) Cells were lysed using NP40 cell lysis buffer, and a cell extract for electrophoresis was prepared through quantification using the Bradford assay method.

[0317] 5) 20 μg of the quantified cell extract was loaded into each well of a sodium dodecyl sulfate-polyacrylamide gel and electrophoresis was performed.

[0318] 6) The proteins developed on SDS-PAGE were transferred to a PVDF membrane.

[0319] 7) A blocking solution (3% BSA, 0.05% Tween 20, TBS) was applied to a PVDF membrane and reacted at room temperature for 1 hour.

[0320] 8) The primary antibody was reacted at room temperature for 2 hours and washed 3 times with a washing solution (0.05% Tween 20, TBS).

[0321] 9) The secondary antibody was reacted at room temperature for 1 hour and washed 5 times with the washing solution.

[0322] 10) After exposure using an antibody detection kit, the results were confirmed using a Western blot imaging system.

[0323] - Observation and judgment of results

[0324] The expression levels of each protein resulting from treatment with the test substance were evaluated by quantitative analysis using ImageJ, based on the expression level of β-Actin used as a loading control, which was captured using a Western blot imaging system. Changes in the expression levels of MMP-1 and MMP-9 in the test substance treatment group were observed relative to the negative control.

[0325] (2-2) Immunofluorescence staining

[0326] - Composition of the test group

[0327] Sample throughput n per test group Control 1 mL 5 TNF-α 25 ng / ml 1 mL 5 TNF-α + peptide of the present invention 1 ppm 1 mL 5 TNF-α + peptide of the present invention 5 ppm 1 mL 5 TNF-α + peptide of the present invention 10 ppm 1 mL 5

[0328] - Examination process

[0329] 1) Place a 12 mm circular microscope cover glass in a 24-well culture plate and 2.5 x 10 4 After dispensing 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 cell confluence was 50% or higher.

[0330] 2) The negative control and test substance were treated for 15 minutes according to the concentration of each treatment group.

[0331] 3) TNF-α was administered at a concentration of 25 ng / ml for 24 hours.

[0332] 4) After fixing the cells using 4% paraformaldehyde, the cells were perforated with 0.1% Triton X-100 to increase the permeability of the cell antibodies.

[0333] 5) After washing once with PBS, blocking was performed at 37°C for 10 minutes with a blocking solution (5% BSA in PBS).

[0334] 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).

[0335] 7) The antibody for confirmation was diluted 1:100, reacted at 4°C for 16 hours, and washed 3 times with PBS.

[0336] 8) The FITC-conjugated secondary antibody was diluted 1:500 and reacted at 37°C for 40 minutes, then washed 5 times with PBS.

[0337] 9) Mounting was performed using a mounting solution.

[0338] 10) Fluorescence signals detected in cells were observed and captured using a digital fluorescence imaging system (LOGOS BIOSYSTEMS, CS20002).

[0339] - Observation and judgment of results

[0340] The expression levels of MMP-9 and COL1A2 (collagen) in the TNF-α treated group and the test substance treated group were compared and analyzed based on the control group.

[0341] (3) Test results

[0342] (3-1) Western Blot Analysis

[0343] Compared to the negative control group, the expression levels of MMP-1 and MMP-9 increased in the TNF-α treated group, while the expression levels decreased in a concentration-dependent manner in the test substance treated group (Fig. 7).

[0344] (3-2) Immunofluorescence staining

[0345] Compared to the negative control group, the expression level of MMP-1 increased in the TNF-α treated group and decreased in a concentration-dependent manner in the test substance treated group, whereas the expression level of COL1A2 decreased in the TNF-α treated group and increased in the test substance treated group (Fig. 8).

[0346] (4) Conclusion

[0347] It was confirmed that the test substance inhibits the activity of collagen-degrading enzymes MMP-1 and MMP-9 and increases collagen expression. Therefore, it is determined that the peptide of the present invention (a peptide composed of Glu-Leu-Cys) has the effect of inhibiting collagen degradation by inhibiting the MMPs reaction.

[0348] Test Example 5: Evaluation of Cell Aging Inhibitory Efficacy

[0349] UV (Ultraviolet) activates MMPs to degrade ECM (Extracellular Matrix) components, thereby inducing aging, and as cells age, the expression of beta-galactosidase increases. We investigated whether the peptide of the present invention inhibits cell aging through a senescence beta-galactosidase staining assay.

[0350] (1) Test materials

[0351] - Preparation of test substances

[0352] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0353] - Test

[0354] 1) Cell line: Human dermal fibroblast (HDF Passage 5-10, CEFObio)

[0355] 2) Cell management: The cell line was thawed from cryopreservation and inoculated into a 100 cm² animal cell culture dish containing culture medium, cultured in an incubator (5% CO2, 37℃), and subcultured with fresh culture medium every 2 to 3 days.

[0356] 3) Medium: CEFOgro Human MSC Growth Medium

[0357] Composition: 10% Fetal Bovine Serum, 1% Antibiotic / Storage: Refrigerated / Manufacturer: CEFObio

[0358] - Test materials

[0359] 1) Senescence beta-Galactosidase Staining Kit

[0360] Storage Conditions: Store frozen at -20℃ / Manufacturer: CELL SIGNALING, 9860S

[0361] 2) VLX-3W research radiometer

[0362] Manufacturer: VILVER

[0363] (2) Test method

[0364] - Composition of the test group

[0365] Sample throughput n per test group Control 1 mL 3 UV 0.06 J / cm 2 1 mL 3 UV + 1 ppm of the peptide of the present invention 1 mL 3 UV + 5 ppm of the peptide of the present invention 1 mL 3 UV + 10 ppm of the peptide of the present invention 1 mL 3

[0366] - Examination process

[0367] 1) 5 x 10⁶ in a 24-well culture plate 5 Cells were dispensed into each well. After 24 hours of culture, the monolayer culture status of the cells was checked, and the test was conducted when the cell confluence was 100% or higher.

[0368] 2) After removing the culture medium, add PBS and apply UVB (312 nm) at 0.06 J / cm² using a VLX-3W research radiometer. 2 It was investigated with the intensity of.

[0369] 3) After removing PBS, the negative control and test substance were applied for 24 hours according to the concentrations of each treatment group.

[0370] 4) After rinsing with PBS, the fixation solution was applied for 15 minutes to fix the sample.

[0371] 5) After rinsing with PBS, the sample was treated with a β-galactosidase staining solution using the Senescence β-Galactosidase Staining Kit and incubated at 37°C for at least 16 hours.

[0372] 6) Observation and imaging were performed using a microscope (OLYMPUS, CKX53).

[0373] - Observation and judgment of results

[0374] Based on a negative control group, the degree of senescent cell (green) generation in the UVB-treated group and the test substance-treated group was compared and analyzed.

[0375] (3) Test results

[0376] Compared to the control group, an increase in the production of senescent cells was observed in the UVB-treated group, while a concentration-dependent decrease in the production of senescent cells was confirmed in the test substance-treated group (Fig. 9).

[0377] (4) Conclusion

[0378] It was confirmed that the production of senescent cells in the test substance treatment group decreased in a concentration-dependent manner. Therefore, it is determined that the peptide of the present invention (a peptide composed of Glu-Leu-Cys) has the efficacy to reduce the production of senescent cells caused by UVB.

[0379] Test Example 6: Evaluation of collagen degradation inhibitory efficacy in a 3D human skin model

[0380] It is known that irradiating a 3D human skin culture model with UV light reduces collagen production. Verhoeff Van Gieson staining was used to observe whether the peptide of the present invention inhibits the degradation of collagen caused by UV light in a 3D human skin model (Neoderm-ED) similar to human skin.

[0381] (1) Test materials

[0382] - Preparation of test substances

[0383] The peptide of the present invention (a peptide composed of Glu-Leu-Cys) was dissolved in triple distilled water to produce a concentration of 1000 ppm.

[0384] - Test

[0385] 1) 3D Human Skin Model: Neoderm-ED

[0386] 2) Management: Incubated in an incubator (5% CO2, 37℃) and tested within 3 days of receipt.

[0387] 3) Medium: Maintenance medium

[0388] Composition: 10% Fetal Bovine Serum / Storage: Refrigerated / Manufacturer: TEGO SCIENCE

[0389] - Test materials

[0390] 1) Elastic stain Kit (Verhoeff Van Gieson / EVG Stain)

[0391] Storage conditions: Store at room temperature / Manufacturer: ABCAM, ab150667

[0392] 2) VLX-3W research radiometer

[0393] Manufacturer: VILVER

[0394] (2) Test method

[0395] - Composition of the test group

[0396] Test group Sample throughput n per test group Control 1 mL 5 UV 0.06 J / cm 2 1 mL 5 UV + 10 ppm of the peptide of the present invention 1 mL 5

[0397] - Examination process

[0398] 1) After receiving Neoderm-ED, dedicated medium was added and cultured for 24 hours.

[0399] 2) UVB (312 nm) at 0.06 J / cm² using a VLX-3W research radiometer on Neoderm-ED 2 It was investigated with the intensity of.

[0400] 3) The negative control and test substance were treated at a concentration of 10 ppm for 48 hours.

[0401] 4) Neoderm-ED was separated from the insert well using a blade to produce a paraffin block.

[0402] 5) Slides were prepared by sectioning them to a thickness of 4 µm.

[0403] 6) Paraffin washing and hydration processes using xylene (Et-OH 100% >95%>90%>80%>70%) were carried out.

[0404] 7) After rinsing with DW, the test was performed using the Elastic Stain Kit (Verhoeff Van Gieson EVG Stain) (abcam), and the test was conducted according to the manufacturer's instructions.

[0405] 8) Hematoxyline solution (5%), ferric chloride solution (10%), and Lugol's iodine solution were mixed to make an Elastic Stain Solution, treated on a slide for 15 minutes, and then washed under running water.

[0406] 9) Rinse the slide 20 times in a Ferric Chloride (2%) Differentiating Solution and wash it again under running water.

[0407] 10) After treating with sodium thiosulfate solution for 1 minute, the slide was washed under running water.

[0408] 11) After treatment with Van Gieson's Solution for 2 minutes, washed with Et-OH 95% > 100%.

[0409] 12) Mounting was performed using a mounting solution.

[0410] 13) The stained tissue was observed and photographed using a microscope (OLYMPUS, BX53F2).

[0411] - Observation and judgment of results

[0412] The degree of skin structure damage and collagen degradation caused by UVB in the test substance treatment group was compared and analyzed based on a negative control group.

[0413] (3) Test results

[0414] Compared to the negative control group, the shape of the epidermal and dermal layers in the UVB-treated group was damaged, whereas in the test substance-treated group, the structure of the epidermal and dermal layers was restored similarly to the negative control group (Fig. 10).

[0415] (4) Conclusion

[0416] 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 determined that the peptide of the present invention (a peptide composed of Glu-Leu-Cys) has the efficacy to inhibit the degradation of collagen not only at the cellular level but also in skin tissue, as well as the efficacy to protect tissue from UVB or help restore damaged tissue.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of skin inflammation, comprising as an active ingredient a peptide of the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof. <Chemical Formula 1> Claim 2 delete Claim 3 A pharmaceutical composition according to claim 1, characterized in that the skin inflammation is skin inflammation caused by ultraviolet rays. Claim 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> Claim 5 A cosmetic composition according to claim 4, characterized in that the skin inflammation or skin wrinkles are skin inflammation or skin wrinkles caused by ultraviolet rays.

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