Peptides having anti-aging activity and their uses

Abstract

The present invention relates to a peptide having anti-aging activity and a composition for inhibiting skin aging or regenerating skin, which contains the peptide as an active ingredient. The peptide containing the amino acid sequence of SEQ ID NO: 1 according to the present invention has anti-aging activity, and can be used as a raw material for cosmetics for inhibiting skin aging or regenerating skin, or for pharmaceuticals for preventing, improving, or treating photoaging.

Description

Technical Field

[0001] The present invention relates to a peptide having anti-aging activity and a composition for inhibiting skin aging or promoting skin regeneration containing the peptide as an active ingredient.

Background Art

[0002] Skin cells may undergo aging due to various causes, and aging of skin cells may cause wrinkles. It can be classified into chronological aging (intrinsic aging), which is natural aging that appears over time through biological processes, and photoaging (photo-induced aging), in which degenerative changes occurring in areas exposed to sunlight and the chronological aging appear complexly. When the expression or activity of various factors related to cell growth is inhibited, natural cell aging may occur, and photoaging of skin cells may progress due to light with wavelengths in the range of 280 nm to 400 nm contained in sunlight. Among them, in particular, irradiation with ultraviolet rays such as UVB having wavelengths in the range of 280 nm to 320 nm may cause damage to the skin and fibers, and there may be a phenomenon of skin darkening and burning. When irradiated with UVB, the accumulation of ROS and free radicals in skin cells is promoted, and the radicals may stimulate the intracellular signal transduction system, inducing oxidative stress on biomolecules such as DNA, proteins, and lipids, which may cause damage to skin tissue.

[0003] When the oxidative stress of skin cells increases, it stimulates keratinocytes in the epidermis and fibroblasts in the dermis, and through a series of intracellular signal transduction processes, the expression of genes such as MMP (matrix metalloproteinase), which is a collagen-degrading enzyme, may increase. Collagen, which accounts for 90% of the dermis as a major component of the skin and plays a role in imparting strength and tension to the skin and protecting the skin from external stimuli and forces, may be induced to decrease, so skin aging and wrinkles may be formed. Therefore, it is possible to prevent skin cell aging and expect an effect of improving wrinkles by regulating the expression of genes related to the synthesis and decomposition of fibrous proteins such as the collagen.

[0004] The aging effect of cells caused by the irradiation of ultraviolet rays contained in sunlight not only causes problems in terms of beauty due to skin damage, but also may induce cancer due to DNA damage of cells, damage to the central and peripheral nervous systems, destruction of the immune system, disorders of reproductive organs, disorders of infant and child development, and skin diseases such as chloracne, and serious health problems may also occur. Therefore, a solution to this problem is an urgent situation.

[0005] In order to solve the above problems, various types of substances have been developed, and there have been attempts to overcome the above problems using extracts obtained from various types of plants and microorganisms. However, in many cases, it is not confirmed which specific substances in the extracts obtained from natural products can derive the protective effect on skin cells and the recovery effect from the aging phenomenon, and the exact composition is unknown. Therefore, it is the actual situation to use extract compositions that also contain unknown substances. Due to the characteristics that must be directly contacted with or applied to the human body, side effects must be minimized and the safety for the human body must be ensured. Therefore, it is necessary to accurately investigate a single substance having an effect of solving the above problems and apply it to human skin cells and the like.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] One object of the present invention is to provide a peptide having anti-aging activity.

[0008] Another object of the present invention is to provide a composition for inhibiting skin aging or promoting skin regeneration, which contains the peptide having the above-mentioned activity as an active ingredient.

[0009] Still another object of the present invention is to provide a cosmetic composition for inhibiting skin aging or promoting skin regeneration, which contains the peptide having the above-mentioned activity as an active ingredient.

[0010] Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating photoaging, which contains the peptide having the above-mentioned activity as an active ingredient.

Means for Solving the Problems

[0011] To achieve the above object, one aspect of the present invention provides a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0012] Another aspect of the present invention provides a composition for inhibiting skin aging or promoting skin regeneration, which contains the peptide as an active ingredient.

[0013] Still another aspect of the present invention provides a cosmetic composition for inhibiting skin aging or promoting skin regeneration, which contains the peptide as an active ingredient.

[0014] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating photoaging, which contains the peptide as an active ingredient.

Effects of the Invention

[0015] The peptide containing the amino acid sequence of SEQ ID NO: 1 according to the present invention has anti-aging activity, and thus can be used as a raw material for cosmetics aimed at inhibiting skin aging or promoting skin regeneration, or for pharmaceuticals aimed at preventing, improving, or treating photoaging.

[0016] The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

Brief Description of the Drawings

[0017]

Fig. 1a

Fig. 1b

Fig. 1c

Fig. 1d

Fig. 2a

Fig. 2b

Fig. 2c

Fig. 3a

Fig. 3b

Fig. 4a

Fig. 4b

Fig. 4c

Fig. 5a

Fig. 5b

Fig. 6a

Fig. 6b

Mode for Carrying Out the Invention

[0018] Hereinafter, the present invention will be described in detail.

[0019] One aspect of the present invention provides a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1.

[0020] As used herein, the term "peptide" means a linear molecule composed of amino acid residues. Specifically, the peptide of the present invention may comprise the amino acid sequence set forth in SEQ ID NO: 1.

[0021] The "peptide" may be a variant or fragment of an amino acid having a different sequence due to deletion, insertion, substitution, or a combination thereof of amino acid residues within a range that does not affect the function, or an amino acid exchange that does not globally change the activity of the peptide is known in the art. In some cases, the peptide may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Therefore, the peptide of the present invention includes a peptide having an amino acid sequence substantially identical to the peptide containing the amino acid sequence of SEQ ID NO: 1, and variants or active fragments thereof.

[0022] The "substantially identical protein" means an amino acid sequence having a sequence homology of 75% or more, preferably 80% or more, for example, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more with the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. If it has a homology of 75% or more of the amino acid sequence and has the same activity, it is included in the scope of the present invention.

[0023] In addition, the peptide of the present invention may further include a targeting sequence, a tag, a labeled residue, a half-life, or an amino acid sequence produced for a specific purpose of increasing peptide stability.

[0024] In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, water absorption, titer, efficacy, etc.), altered specificity (for example, a wide range of biological activity spectra), and reduced antigenicity, the N-terminus or C-terminus of the peptide of the present invention may be modified. The "stability" is used to mean not only the in vivo stability that protects the peptide of the present invention from the attack of proteolytic enzymes in the living body, but also the storage stability (for example, room temperature storage stability).

[0025] The N-terminal modification may be one in which a protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and a polyethylene glycol (PEG) is bonded to the N-terminus of the peptide, but is not limited thereto.

[0026] The C-terminal modification may be one in which a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), etc. is bonded to the C-terminus of the peptide, but is not limited thereto.

[0027] The peptide of the present invention may be synthesized, for example, using equipment or genetic engineering techniques. When synthesizing using equipment, the desired peptide may be synthesized by an automatic peptide synthesizer by the Fmoc solid-phase method.

[0028] In a specific embodiment of the present invention, the peptide containing the amino acid sequence of SEQ ID NO: 1 of the present invention was synthesized and identified using the Fmoc solid-phase method, and the peptides were selected through verification of the efficacy of the identified peptides.

[0029] In a specific embodiment of the present invention, in order to verify the efficacy of the identified peptides, the peptides of the present invention were selected through verification of the effectiveness against the extracellular matrix (ECM) components, the effect of increasing the expression level of skin barrier strengthening factors, the effect of decreasing the reactive oxygen species level increased by ultraviolet irradiation, and the effect of suppressing the expression and activity of collagen degradation-related proteins increased by ultraviolet rays.

[0030] Therefore, the peptide containing the amino acid sequence of SEQ ID NO: 1 of the present invention has skin aging inhibitory activity or skin regeneration activity.

[0031] Another aspect of the present invention provides a composition for inhibiting skin aging or regenerating skin, which contains a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0032] The "skin aging" may be aging that occurs naturally from cells over time, or photoaging that occurs due to sunlight. Further, the aging may be induced by an intracellular oxidative stress phenomenon that can occur due to various causes, whereby cell growth inhibition and cell death may progress, and the synthesis of various fibrous proteins that make up skin cells may be inhibited, and the expression of degrading enzymes may increase. In particular, due to ultraviolet irradiation, in skin cells, the expression of genes such as Col1a1, fibronectin, and elastin may be suppressed, which may induce photoaging and wrinkle formation of skin cells.

[0033] The "skin regeneration" may promote the growth of skin cells, enhance viability, prevent cell damage from external and / or internal stimuli, improve skin wrinkles, enhance elasticity, and strengthen the skin barrier. The skin regeneration may include the meaning of preventing skin damage induced by the external environment, for example, preventing pigmentation or improving and / or treating skin photoaging.

[0034] The composition for inhibiting skin aging or regenerating skin enhances resistance to cell damage caused by aging or external stimuli.

[0035] The "external stimuli" may be physical stimuli, chemical stimuli by the application of cosmetics or other external agents, stimuli by ultraviolet rays, or stimuli by infrared rays.

[0036] The cells to be damaged may be skin fibroblasts or keratinocytes.

[0037] The peptide of the present invention can induce an increase in the expression of genes for fibrous protein synthesis in skin cells. Therefore, the peptide can improve skin wrinkles and elasticity by increasing the synthesis of fibrous proteins, increase the resistance and viability of skin cells from external stimuli by improving the skin barrier, and enhance the regenerative capacity of skin damaged by external stimuli or aging. By confirming the expression levels of proteins related to cell proliferation, anti-aging genes, and fibrous proteins whose expression levels change in skin cells as skin aging progresses, the effects of the peptide of the present invention in suppressing skin aging and promoting skin regeneration can be confirmed.

[0038] The composition for suppressing skin aging or promoting skin regeneration can increase the expression or secretion of collagen, fibronectin, or elastin in skin cells.

[0039] The composition for suppressing skin aging or promoting skin regeneration can increase the expression of AQP3 or SIRT1, which are skin barrier strengthening factors.

[0040] The composition for suppressing skin aging or promoting skin regeneration can suppress the generation of reactive oxygen species (ROS) by ultraviolet rays.

[0041] The composition for suppressing skin aging or promoting skin regeneration can increase the expression of collagen or fibronectin decreased by ultraviolet rays.

[0042] The composition for suppressing skin aging or promoting skin regeneration can suppress the expression of MMP-1 increased by ultraviolet rays, or suppress the activities of MMP-2 and MMP-9 increased by ultraviolet rays.

[0043] In a specific embodiment of the present invention, the peptide containing the amino acid sequence of SEQ ID NO: 1 of the present invention was shown to increase the expression of genes encoding extracellular matrix proteins such as Col1a1, fibronectin, and elastin in fibroblasts.

[0044] In addition, the peptide was shown to increase the expression of skin barrier strengthening genes such as AQP3 and SIRT1 in fibroblasts and / or keratinocytes.

[0045] In addition, the peptide was shown to reduce the intracellular reactive oxygen species (ROS) level in fibroblasts and / or keratinocytes damaged by ultraviolet rays.

[0046] In addition, the peptide was shown to increase the decreased expression of Col1a1 and fibronectin genes in fibroblasts damaged by ultraviolet rays.

[0047] In addition, the peptide was shown to suppress the increased expression of MMP-1 or the increased activities of MMP-2 and / or MMP-9 in fibroblasts and / or keratinocytes damaged by ultraviolet rays.

[0048] Therefore, it is clear that the peptide of the present invention has skin aging inhibitory or skin regeneration activity by regulating the expression of genes related to the aging phenomenon of skin cells, and the peptide of the present invention can be usefully used as an active ingredient of a composition for skin aging inhibition or skin regeneration.

[0049] Another aspect of the present invention provides a cosmetic composition for skin aging inhibition or skin regeneration, comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0050] The skin aging inhibition or skin regeneration may be prevention or improvement of skin wrinkles, enhancement of skin elasticity, strengthening of skin barrier, prevention of pigmentation, or improvement of skin photoaging.

[0051] Since the peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the item of "composition for skin aging inhibition or skin regeneration", specific descriptions refer to the foregoing content, and hereinafter only the configurations specific to the cosmetic composition for skin aging inhibition or skin regeneration will be described.

[0052] The cosmetic composition may be manufactured into any dosage form that is commonly manufactured in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, etc., and may be formulated into these, but is not limited thereto.

[0053] The cosmetic composition may be manufactured in various forms such as a softening lotion, nutritive lotion, nutritive cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, pomade, gel, etc., such as solutions, sol-gels, emulsions, oils, waxes, aerosols, etc., but is not limited thereto.

[0054] The cosmetic composition of the present invention may contain excipients, carriers, and other additives, and it is possible to apply and blend as much of the ordinary components formulated in general skin cosmetics as necessary.

[0055] When the dosage form of the cosmetic composition is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide, etc. may be used as the carrier component.

[0056] When the dosage form of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as the carrier component. Particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may additionally be included, but is not limited thereto.

[0057] When the dosage form of the cosmetic composition is a solution or an emulsion, a solvent, a solubilizer or an emulsifier may be used as the carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol fatty acid ester, polyethylene glycol, or fatty acid ester of sorbitan may be used.

[0058] When the dosage form of the cosmetic composition is a suspension, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth may be used as the carrier component.

[0059] When the dosage form of the cosmetic composition is surfactant-containing cleansing, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionic acid, an imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl amide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as the carrier component.

[0060] When the dosage form of the cosmetic composition is a hair shampoo, base components for composing a shampoo, such as a thickening agent, a surfactant, a viscosity modifier, a humectant, a pH adjuster, a preservative, and an essential oil, may be mixed with the peptide of the present invention. As the thickening agent, CDE may be used. As the surfactant, LES which is an anionic surfactant and coco betaine which is an amphoteric surfactant may be used. As the viscosity modifier, polyquaternium may be used. As the humectant, glycerin may be used. As the pH adjuster, citric acid and sodium hydroxide may be used. As the preservative, grapefruit extract or the like may be used. In addition, essential oils such as siderwood, peppermint, and rosemary and silk amino acids, pentaol, or vitamin E may be added.

[0061] Components contained in the cosmetic composition may further include, in addition to the peptide of the present invention and the carrier component as active ingredients, components usually used in cosmetic compositions, for example, ordinary adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, but are not limited thereto.

[0062] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating photoaging, which contains a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0063] Since the peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the item of "composition for suppressing skin aging or skin regeneration", specific descriptions refer to the above-mentioned content, and hereinafter only the configurations specific to the pharmaceutical composition for preventing or treating photoaging will be described.

[0064] In the present specification, the "photoaging" means a phenomenon in which skin damage, pigmentation, wrinkle formation, or a decrease in skin elasticity occurs when exposed to sunlight repeatedly and for a long period of time.

[0065] As used herein, "prevention" means all actions that suppress or delay the occurrence, spread, and recurrence of photoaging by the peptide of the present invention or a composition containing the same.

[0066] As used herein, "improvement or treatment" means all actions that favorably modify photoaging, such as delaying, interrupting, or reversing the progression of photoaging by the peptide of the present invention or a composition containing the same.

[0067] In a specific embodiment of the present invention, the peptide can prevent, improve, or treat photoaging by increasing the expression or secretion of extracellular matrix (ECM) proteins such as collagen, fibronectin, or elastin, and increasing the expression of skin barrier strengthening factors such as AQP3 or SIRT1.

[0068] In addition, the peptide can prevent, improve, or treat photoaging by suppressing the production of reactive oxygen species (ROS) in cells damaged by aging or external stimuli, increasing the expression of decreased collagen or fibronectin, and suppressing the expression of increased MMP-1 or the activities of increased MMP-2 and MMP-9.

[0069] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier.

[0070] Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose, and glycols, and the like. Stabilizers and preservatives may also be further included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutically acceptable carriers may be referred to with reference to those described in Remington’s Pharmaceutical Sciences (19th ed., Mack Publishing Company, Easton, PA, 1995).

[0071] The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it may be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0072] The pharmaceutical composition of the present invention may be formulated into a preparation for oral administration or a preparation for parenteral administration according to the administration route as described above. Specifically, the preparation for parenteral administration may be formulated and used in the form of an injection preparation or a topical preparation.

[0073] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient for the treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective dose can be determined according to factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route and excretion rate, the treatment period, the drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered simultaneously with, separately from, or sequentially to conventional therapeutic agents, and may be administered in single or multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects considering all the above factors, which can be easily determined by those skilled in the art.

[0074] The effective amount of the pharmaceutical composition may vary according to the age, sex, condition, weight of the patient, the absorption degree, inactivation rate, excretion rate of the active ingredient in the body, the type of disease, and the drugs used concurrently, and may be increased or decreased according to the administration route, severity, sex, weight, age, etc. For example, the pharmaceutical composition may be administered at about 0.0001 μg to 500 mg per kg of the patient's body weight per day, preferably 0.01 μg to 100 mg.

[0075] As described above, since the peptide of the present invention is excellent in skin aging inhibition and skin regeneration effects, it can be used as a raw material for cosmetics aimed at skin aging inhibition or skin regeneration, or pharmaceuticals aimed at the prevention, improvement, or treatment of photoaging.

[0076] Yet another aspect of the present invention provides a method for inhibiting skin aging or regenerating skin, which comprises administering to a subject a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0077] Yet another aspect of the present invention provides a method for preventing or treating photoaging, which comprises administering to a subject a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0078] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the item of "Composition for Inhibiting Skin Aging or Skin Regeneration", so the specific description refers to the content described above.

[0079] As used herein, the term "therapeutically effective amount" means the amount of the peptide that achieves the purpose of improving the incidence of the disease during the treatment of the peptide and usually avoids harmful side effects related to other treatment methods.

[0080] Still another aspect of the present invention provides the use of the amino acid sequence of SEQ ID NO: 1 for use in the manufacture of a medicament for the prevention or treatment of photoaging.

[0081] Hereinafter, the present invention will be described in detail by way of examples.

[0082] However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.

[0083] [Synthesis Example 1] Peptide synthesis containing the amino acid sequence of SEQ ID NO: 1 Using an automatic peptide synthesizer (Liberty, CEM Corporation, USA), a peptide having the amino acid sequence of SEQ ID NO: 1 described in [Table 1] below was synthesized, and these synthesized peptides were purified by C18 reverse-phase high-performance liquid chromatography (HPLC) (U-3000, Thermo fisher scientific, USA). The column used was Pursuit XRs C18 (250*4.65mm 100Å, Agilent, USA).

[0084]

Table 1

[0085] [Experimental Example 1] Confirmation of Inhibition of Natural Aging by Peptide Treatment 1-1. Confirmation of increased expression of genes encoding components of the extracellular matrix (ECM, extracellular matrix) (collagen, fibronectin, elastin) To confirm whether the peptide of the present invention can suppress the cell aging phenomenon that naturally progresses in cells, targeting fibroblasts, the expression levels of genes related to natural aging were confirmed.

[0086] First, NIH3T3 cells (mouse fibroblast cell line) were seeded in a 6-well plate at a cell density of 3×10 5 cells / well, and then cultured in DMEM medium containing 10% Fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum free DMEM media, and the peptide of the present invention was mixed with 1 mL of the serum-free DMEM medium at concentrations of 10, 50, and 100 μM respectively, and then subcultured to the cells. PC (positive control) was treated with TGF-β 5 ng / ml instead of the peptide. The cells were cultured in a CO2 incubator at 37°C for 24 hours.

[0087] The cultured cells were washed twice with PBS, and RNA was extracted from the cells using easy blue (iNtRON, Cat.No.: 17061, Korea). The amount of the extracted RNA was quantified, and after adding 1000 ng of RNA per tube, the RT reaction was carried out using a kit (enzynomics, Cat.No.: PT200, Korea). RT-PCR was carried out using a PCR kit (enzynomics, Cat.No.: P581T, Korea). The sequences of the primers for collagen, fibronectin, elastin used in RT-PCR and the primers for GAPDH used for comparison of the total RNA amount of each treatment group are shown in [Table 2].

[0088]

Table 2

[0089] As a result, as shown in FIGS. 1a to 1d, when the peptide was treated, it was confirmed that the expression of collagen, fibronectin, and elastin genes gradually increased as the concentration of the peptide increased. From this, it can be seen that as the expression of collagen, fibronectin, and elastin, which are extracellular matrix proteins that make up the dermis of fibroblasts, increases due to the peptide containing the amino acid sequence of SEQ ID NO: 1, there is an effect of suppressing or improving natural aging such as skin wrinkles.

[0090] 1-2. Confirmation of increased expression of skin barrier function-related genes (SIRT1 and AQP3) In order to confirm whether the peptide of the present invention can suppress the cell aging phenomenon that naturally progresses in cells using keratinocytes as a target, the expression level of genes related to natural aging was confirmed.

[0091] First, after seeding HaCaT cells in a 6-well plate at a cell density of 3×10 5 cells / well, they were cultured in DMEM medium containing 10% Fetal bovine serum (FBS) for 24 hours. After culturing the cells once with serum free DMEM media, the peptide of the present invention was mixed with 1 mL of the serum free DMEM media at concentrations of 10, 50, and 100 μM, respectively, and subcultured to the cells. PC (positive control) was treated with 100 nM of EGF instead of the peptide. The cells were cultured in a CO2 incubator at 37° C. for 24 hours.

[0092] After washing the cultured cells twice with PBS, RNA was extracted from the cells using easy blue (iNtRON, Cat. No.: 17061, Korea). The extracted RNA was subjected to an RT reaction using a kit (enzynomics, Cat. No.: RT200, Korea). Subsequently, RT-PCR was performed using a PCR kit (enzynomics, Cat. No.: R581T, Korea). The sequences of the AQP3 and SIRT1 primers used during RT-PCR and the sequence of the GAPDH primer used for comparison of the total RNA amount in each treatment group are shown in the above [Table 2].

[0093] As a result, as shown in FIGS. 2a to 2c, it was confirmed that when the peptide was treated, the mRNA levels of AQP3 and SIRT1, which are skin barrier strengthening factors, increased.

[0094] From this, it can be seen that as the expression levels of AQP3 and SIRT1, which are skin barrier strengthening factors, increase in keratinocytes due to the peptide containing the amino acid sequence of SEQ ID NO: 1, the effect of suppressing or improving natural aging is present.

[0095] [Experimental Example 2] Confirmation of suppression of exogenous aging by peptide treatment 2-1. Confirmation of the effect of suppressing reactive oxygen species increased by ultraviolet rays When UV is irradiated, the level of reactive oxygen species increases. Therefore, when the peptide of the present invention is treated, it was confirmed whether the level of reactive oxygen species decreases again and the aging phenomenon due to oxidative stress of cells can be suppressed.

[0096] First, NIH3T3 cells and HaCaT cells were each 5×10 5After seeding in a 6-well plate at the cell density of cell / well, the cells were cultured in DMEM medium containing 10% Fetal bovine serum (FBS) for 24 hours. After washing the cultured cells once with serum free DMEM media, the peptide of the present invention was mixed with 1 mL of the serum free DMEM media at the concentrations of 10, 50, and 100 μM respectively and subcultured to the cells. PC (Positive control) was treated with 50 μM of Trolox instead of the peptide.

[0097] The cells were cultured in a CO2 incubator at 37 °C for 1 hour. The medium of the cultured cells was transferred to a tube, 1 ml of PBS was added, and then, using a UV irradiator (VILBER LOURMAT, Cat No.: 3102 - BSU, France), NIH3T3 cells were irradiated with UVA of 8 J / cm 2 and HaCaT cells were irradiated with UVB of 15 mJ / cm 2 After removing the PBS, 900 μL of the medium transferred to the tube was subcultured and cultured in a CO2 incubator at 37 °C for 24 hours. After treating with 10 μM of DCFH - DA (dichloro - dihydro - fluorescein diacetate), it was wrapped with foil and cultured in a CO2 incubator at 37 °C for 30 minutes. After washing twice with PBS, 500 μL of 1XTE was subcultured to separate the cells. After centrifugation, the cells were washed with PBS, and then the fluorescence value was measured via FACS.

[0098] As a result, as shown in FIGS. 3a and 3b, it was confirmed that when the peptide of the present invention was treated on NIH3T3 cells and HaCaT cells, the intracellular reactive oxygen species level increased by UV irradiation decreased.

[0099] From this, it can be seen that the peptide containing the amino acid sequence of SEQ ID NO: 1 reduces the reactive oxygen species level increased by UV irradiation in keratinocytes again, and by inhibiting oxidative stress, it has the effect of preventing the photoaging phenomenon of cells.

[0100] Confirmation of increased expression of extracellular matrix components (collagen, fibronectin) genes suppressed by ultraviolet rays When irradiated with UV, the expression of genes related to extracellular matrix components is inhibited. Therefore, when treating the peptide of the present invention, it was confirmed whether the cell photoaging phenomenon can be suppressed by confirming whether the expression levels of Col1a1 and fibronectin genes recover and increase again.

[0101] First, NIH3T3 cells were seeded in a 6-well plate at a cell density of 5×10 5 cells / well and cultured in DMEM medium containing 10% Fetal bovine serum (FBS) for 24 hours. After washing the cultured cells once with serum free DMEM media, the peptide of the present invention was mixed with 1 mL of the serum free DMEM media at concentrations of 10, 50, and 100 μM, respectively, and subcultured to the cells. PC (positive control) was treated with 2.5 mM N-Acetylcysteine or 50 μM Trolox instead of the peptide. The cells were cultured in a CO2 incubator at 37°C for 1 hour.

[0102] After transferring the medium of the cultured cells to a tube (e-tube), 1 ml of PBS was added, and 6 J / cm was irradiated using a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France). 2Irradiated with UVA. After removing PBS, 900 μL of the medium transferred to the tube was subcultured and cultured in a 37 °C CO2 incubator for 6 hours. After washing twice with PBS, RNA was extracted from the cells using easy blue (iNtRON, Cat. No.: 17061, Korea). The amount of the extracted RNA was quantified, 1000 ng of RNA per tube was added, and an RT reaction was carried out using a kit (enzynomics, Cat. No.: RT200, Korea). Then, RT-PCR was carried out using a PCR kit (enzynomics, Cat. No.: R581T, Korea). The sequences of the Col1a1, fibronectin primers used for RT-PCR and the sequence of the GAPDH primer used for comparison of the total RNA amount in each treatment group are shown in the above [Table 2].

[0103] As a result, as shown in FIGS. 4a to 4c, it was confirmed that when the peptide of the present invention was treated on NIH3T3 cells, the mRNA levels of Col1a1 and fibronectin decreased by UV irradiation increased again.

[0104] From this, it can be seen that the peptide containing the amino acid sequence of SEQ ID NO: 1 has the effect of suppressing the photoaging phenomenon of cells by restoring the expression levels of Col1a1 and fibronectin decreased by UV irradiation in fibroblasts.

[0105] 2-3. Confirmation of suppression of MMP-1 expression increased by ultraviolet rays Since the expression of MMP-1, which encodes a protein related to collagen degradation, increases by ultraviolet irradiation, when the peptide of the present invention is treated, whether the expression of the MMP-1 protein is suppressed again and the cell aging phenomenon can be suppressed was confirmed.

[0106] First, 5×10 NIH3T3 cells and HaCaT cells were each 5After seeding in a 6-well plate at the cell density of cell / well, the cells were cultured in DMEM medium containing 10% Fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum free DMEM media, and the peptide of the present invention was mixed with 1 mL of serum free DMEM media at concentrations of 10, 50, and 100 μM, respectively, and then subcultured to the cells. PC (positive control) was treated with 25 and 50 μM of Trolox instead of the peptide.

[0107] The cells were cultured in a 37 °C CO2 incubator for 1 hour. The medium of the cultured cells was transferred to a tube (e-tube), 1 mL of PBS was added, and then NIH3T3 cells were irradiated with UVA of 6 J / cm 2 by a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France), and HaCaT cells were irradiated with 15 mJ / cm 2Irradiated with UVB. After removing PBS, 900 μL of the medium that was transferred back to the tube was added again and cultured in a 37 °C CO₂ incubator for 24 hours. The medium of the cultured cells was transferred to a tube, washed twice with PBS, and then cell lysis buffer was added to lyse the cells. After preparing the sample by treating with 5X sample buffer, SDS-PAGE was performed using a 10% SDS-PAGE gel. The proteins separated via SDS-PAGE were transferred to a PVDF membrane. Blocking was carried out at room temperature for 1 hour using 5% skim milk. The anti-MMP-1 antibody (Abcam, Cat.No.: ab137332, UK) was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. Subsequently, it was washed 3 times for 10 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS). Goat anti-rabbit IgG (Jackson Immune Research, Cat.No.: 111-035-033, USA) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 2 hours. Then, detection was performed using a film with an ECL solution (GE Healthcare, Cat.No.: RPN2232, USA). β-actin was confirmed using an anti-β-actin antibody (Santacruz biotechnology, USA) to compare the total amount of proteins used in the experiment.

[0108] As a result, as shown in FIGS. 5a and 5b, it was confirmed that the peptide of the present invention can suppress the expression of MMP-1 increased by UV irradiation in NIH3T3 cells and HaCaT cells.

[0109] From this, since the peptide containing the amino acid sequence of SEQ ID NO: 1 can reduce the expression of MMP-1 increased by UV irradiation in fibroblasts and keratinocytes again, it can be seen that there is an effect of improving skin wrinkles by suppressing the aging phenomenon of cells.

[0110] 2-4. Confirmation of Inhibition of Activities of MMP-2 and MMP-9 Increased by Ultraviolet Rays Since MMP-2 and MMP-9, which encrypt proteins related to collagen degradation, increase their activity upon ultraviolet irradiation, when treating the peptide of the present invention, it was confirmed whether the activities of the MMP-2 and MMP-9 proteins could be suppressed again to suppress the cell aging phenomenon.

[0111] First, NIH3T3 cells and HaCaT cells were seeded in a 6-well plate at a cell density of 5×10 5 cells / well and then cultured in DMEM medium containing 10% Fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum free DMEM media, and the peptide of the present invention was mixed with 1 mL of serum free DMEM media at concentrations of 10, 50, and 100 μM, respectively, and then subcultured to the cells. PC (positive control) was treated with 25 and 50 μM of Trolox instead of the peptide.

[0112] The cells were cultured in a 37°C, CO2 incubator for 1 hour. The medium of the cultured cells was transferred to a tube (e-tube), 1 ml of PBS was added, and then NIH3T3 cells were irradiated with 6 J / cm 2 of UVA by a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France), and HaCaT cells were irradiated with 15 mJ / cm 2UVB irradiation was performed. After removing PBS, 900 μL of the medium transferred to the tube again was added and cultured in a CO2 incubator at 37 °C for 6 hours. The medium of the cultured cells was transferred to a tube, washed twice with PBS, and then cell lysis buffer was added to lyse the cells. After preparing the sample by treating with 5X sample buffer, SDS-PAGE was carried out using a 10% SDS-PAGE gel. The proteins separated via SDS-PAGE were transferred to a PVDF membrane. Blocking was carried out at room temperature for 1 hour using 5% skim milk. The anti-β-actin antibody (Santacruz biotechnology, USA) was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. Subsequently, it was washed 3 times for 10 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS). Goat anti-rabbit IgG (Jackson Immune Research, Cat. No.: 111-035-033, USA) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 2 hours. Then, detection was carried out using a film with an ECL solution (GE Healthcare, Cat. No.: RPN2232, USA). Next, to analyze the amounts of MMP-2 and MMP-9, after preparing the sample by treating the cell culture medium transferred to the tube with 4X zymography sample buffer, SDS-PAGE was carried out using an 8% SDS-PAGE gel. The gel was washed with a 0.2% tween-20 solution for 30 minutes and then with distilled water (DW) for 30 minutes. After putting the gel into TNCB buffer, it was reacted in a 37 °C incubator for 16 hours. The treated gel was stained with Coomassie brilliant blue R250 (Sigma) and treated with a decolorizing buffer (5% ethanol, 7.5% acetic acid, and distilled water). Then, the activities of MMP-2 and MMP-9 were confirmed by observing the gel-like empty bands that appeared due to the hydrolysis of gelatin.

[0113] As a result, as shown in FIGS. 6A and 6B, it was confirmed that the peptide of the present invention suppresses the activities of MMP-2 and MMP-9 proteins whose activities were increased by ultraviolet rays in fibroblasts and keratinocytes.

[0114] From this, since the activities of MMP-2 and MMP-9 proteins increased by ultraviolet rays in fibroblasts and keratinocytes are decreased and restored again by the peptide containing the amino acid sequence of SEQ ID NO: 1, and the amount of collagen can be increased again, it can be seen that the improvement effect of skin wrinkles is achieved by suppressing the aging phenomenon of cells.

[0115] As described above, the present invention has been described in detail only for the described embodiments, but it is obvious to those skilled in the art that various modifications and corrections are possible within the scope of the technical idea of the present invention, and such modifications and corrections naturally belong to the appended claims.

Claims

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

1.

2. The peptide according to claim 1, which has skin aging inhibitory activity or skin regenerative activity.

3. A composition for inhibiting skin aging or regenerating skin, comprising the peptide according to claim 1 as an active ingredient.

4. The composition for inhibiting skin aging or regenerating skin according to claim 3, which enhances resistance to cell damage caused by aging or external stimuli.

5. The composition for inhibiting skin aging or regenerating skin according to claim 4, wherein the external stimulus is ultraviolet light or infrared light.

6. The composition for inhibiting skin aging or regenerating skin according to claim 4, wherein the cell is a skin fibroblast or a keratinocyte.

7. The composition for inhibiting skin aging or regenerating skin according to claim 3, which i) increases the expression or secretion of collagen, fibronectin, or elastin, and ii) increases the expression of SIRT1 or AQP3.

8. The composition for inhibiting skin aging or regenerating skin according to claim 3, which i) inhibits the generation of reactive oxygen species (ROS) by ultraviolet light, ii) increases the expression of collagen, fibronectin, or elastin decreased by ultraviolet light, and iii) inhibits the expression of MMP-1 or the activities of MMP-2 and MMP-9 increased by ultraviolet light.

9. A cosmetic composition for inhibiting skin aging or regenerating skin, comprising the peptide according to claim 1 as an active ingredient.

10. The cosmetic composition for inhibiting skin aging or regenerating skin according to claim 9, wherein the inhibition of skin aging or skin regeneration is prevention or improvement of skin wrinkles, enhancement of skin elasticity, strengthening of the skin barrier, prevention of pigmentation, or improvement of skin photoaging.

11. A pharmaceutical composition for preventing or treating photoaging, comprising the peptide according to claim 1 as an active ingredient.

Images