Peptides with anti-aging activity and uses thereof

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 regenerating skin, which contains the peptide as an active ingredient. [Background technology]

[0002] Skin cells can undergo aging for various reasons, which can lead to wrinkles. It can be divided into chronological aging (intrinsic aging), which occurs naturally over time due to biological processes, and photoaging (photoinduced aging), which occurs when degenerative changes occur in areas exposed to sunlight and chronological aging are combined. Natural cellular aging can occur when the expression or activity of various factors related to cell growth is inhibited, and photoaging of skin cells can be accelerated by light with wavelengths of 280 to 400 nm contained in sunlight. In particular, exposure to ultraviolet rays, such as UVB with wavelengths in the 280 to 320 nm range, can cause damage to skin and fibers, resulting in a darkening of the skin. UVB exposure promotes the accumulation of ROS and free radicals in skin cells, which can stimulate intracellular signaling pathways and induce oxidative stress in biomolecules such as DNA, proteins, and lipids, potentially resulting in skin tissue damage.

[0003] Increased oxidative stress in skin cells stimulates epidermal keratinocytes and dermal fibroblasts, potentially increasing the expression of genes such as matrix metalloproteinase (MMP), a collagen-degrading enzyme, through a series of intracellular signaling processes. This can lead to a decrease in collagen, a major component of the skin that accounts for 90% of the dermis and provides strength and tension to the skin, protecting it from external stimuli and forces, potentially resulting in skin aging and wrinkles. Therefore, regulating the expression of genes related to the synthesis and degradation of fibrous proteins such as collagen can be expected to prevent skin cell aging and improve wrinkles.

[0004] The aging of cells caused by exposure to ultraviolet rays contained in sunlight not only causes cosmetic problems due to skin damage, but can also cause serious health problems such as cancer due to DNA damage in cells, damage to the central and peripheral nervous systems, destruction of the immune system, disorders of reproductive organs, impaired development in infants and babies, and skin diseases such as chloracne, making a solution to this problem urgently needed.

[0005] To solve the above problems, various types of substances have been developed, and attempts have been made to overcome the above problems using extracts obtained from various types of plants and microorganisms. However, in many cases, it is not known which specific substances in extracts obtained from natural substances can protect skin cells or restore them from aging, and the exact composition is unknown, so extract compositions containing unknown substances are used. Because they must be used by direct contact with or application to the human body, side effects must be minimized and safety for the human body must be ensured. Therefore, there is a need to accurately identify a single substance that has the effect of solving the above problems and apply it to human skin cells, etc. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] S Edgar et al., Effects of collagen-derived bioactive peptides and natural antioxidant compounds on proliferation and matrix protein synthesis by cultured normal human dermal fibroblasts, Scientific Reports, 2018;8:10474 Summary of the Invention [Problem to be solved by the invention]

[0007] An 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 regenerating skin, which contains a peptide having the above-mentioned activity as an active ingredient.

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

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

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

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

[0013] Yet another aspect of the present invention provides a cosmetic composition for inhibiting skin aging or regenerating skin, which comprises the peptide as an active ingredient.

[0014] Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating photoaging, comprising the peptide as an active ingredient. [Effects of the Invention]

[0015] The peptide comprising the amino acid sequence of SEQ ID NO: 1 according to the present invention has anti-aging activity and can therefore be used as a raw material for cosmetics intended to inhibit skin aging or regenerate skin, or for pharmaceuticals intended to prevent, improve, or treat photoaging.

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

[0017] [Figure 1a] FIG. 1 shows the expression of collagen, fibronectin, and elastin when fibroblasts were treated with the peptide of the present invention: Con is the untreated group, and bFGF is the positive control group. [Figure 1b] 1 is a graph showing the effect on collagen expression when fibroblasts are treated with the peptide of the present invention: Con is the untreated group, and bFGF is the positive control group. [Figure 1c] 1 is a graph showing the effect of treating fibroblasts with the peptide of the present invention on fibronectin expression: Con is the untreated group, and bFGF is the positive control group. [Figure 1d] 1 is a graph showing the effect on elastin expression when fibroblasts are treated with the peptide of the present invention: Con is the untreated group, and bFGF is the positive control group. [Figure 2a] 1 is a graph showing the effect of treating fibroblasts with the peptide of the present invention on the secretion of collagen and hyaluronic acid proteins: Con is the untreated group, and TGF-b1 is the positive control group. [Figure 2b] 1 is a graph showing the effect of the peptide of the present invention on the secretion of collagen and hyaluronic acid protein when keratinocytes are treated with the peptide: Con is the untreated group, and TGF-b1 is the positive control group. [Figure 3a] FIG. 1 shows the expression of SIRT1 and AQP3 when keratinocytes were treated with the peptide of the present invention: Con is the untreated group, and EGF is the positive control group. [Figure 3b] 1 is a graph showing the effect of treating keratinocytes with the peptide of the present invention on the expression of SIRT1: Con is the untreated group, and EGF is the positive control group. [Figure 3c] 1 is a graph showing the effect of the peptide of the present invention on the expression of AQP3 when keratinocytes are treated with the peptide: Con is the untreated group, and EGF is the positive control group. [Figure 4] 1 is a graph showing the effect of the peptide of the present invention on intracellular reactive oxygen species (ROS) levels increased by ultraviolet (UV) irradiation in keratinocytes: Con is the non-UV irradiated group, NC is the UV irradiated group, 10 μM, 50 μM, and 100 μM are groups treated with UV irradiation and the peptide of the present invention at various concentrations, and Trolox is the positive control group treated with UV irradiation and Trolox. [Figure 5a] This figure confirms the effect of the peptide of the present invention on the expression of SIRT1 and AQP3, which was reduced by ultraviolet (UV) irradiation in keratinocytes: Con is the non-UV irradiated group, NC is the UV irradiated group, 10 μM, 50 μM, and 100 μM are groups treated with UV irradiation and the peptide of the present invention at various concentrations, and NaC is the positive control group treated with UV irradiation and N-acetylcysteine. [Figure 5b] This is a graph showing the effect of the peptide of the present invention on the expression of SIRT1 reduced by ultraviolet (UV) irradiation in keratinocytes: Con is the non-UV irradiated group, NC is the UV irradiated group, 10 μM, 50 μM, and 100 μM are groups treated with UV irradiation and the peptide of the present invention at various concentrations, and NaC is the positive control group treated with UV irradiation and N-acetylcysteine. [Figure 5c] This is a graph showing the effect of the peptide of the present invention on the expression of AQP3 reduced by ultraviolet (UV) irradiation in keratinocytes: Con is the non-UV irradiated group, NC is the UV irradiated group, 10 μM, 50 μM, and 100 μM are groups treated with UV irradiation and the peptide of the present invention at various concentrations, and NaC is the positive control group treated with UV irradiation and N-acetylcysteine. [Figure 6a] This figure confirms the effect of the peptide of the present invention on the expression of cleaved PARP-1 and cleaved caspase-3, which was increased by ultraviolet (UV) irradiation in keratinocytes: Con is the non-UV-irradiated group, NC is the UV-irradiated group, 10 μM, 50 μM, and 100 μM are groups treated with UV irradiation and the peptide of the present invention at various concentrations, and NaC is the positive control group treated with UV irradiation and N-acetylcysteine. [Figure 6b]1 is a graph showing the effect of the peptide of the present invention on the expression of cleaved PARP-1, which is increased by ultraviolet (UV) irradiation in keratinocytes: Con is the non-UV-irradiated group, NC is the UV-irradiated group, 10 μM, 50 μM, and 100 μM are groups treated with UV irradiation and the peptide of the present invention at various concentrations, and NaC is the positive control group treated with UV irradiation and N-acetylcysteine. [Figure 6c] This is a graph showing the effect of the peptide of the present invention on the expression of Cleaved Caspase-3, which was increased by ultraviolet (UV) irradiation in keratinocytes: Con is the non-UV-irradiated group, NC is the UV-irradiated group, 10 μM, 50 μM, and 100 μM are groups treated with UV irradiation and the peptide of the present invention at various concentrations, and NaC is the positive control group treated with UV irradiation and N-acetylcysteine. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below.

[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" refers to a linear molecule consisting of amino acid residues, and specifically, the peptide of the present invention may comprise the amino acid sequence shown in SEQ ID NO:1.

[0021] The "peptide" may be an amino acid variant or fragment having a different sequence due to deletion, insertion, substitution, or a combination thereof of amino acid residues, provided that the function is not affected. Amino acid replacements that do not overall alter the activity of the peptide are known in the art. Optionally, the peptide may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Thus, the peptide of the present invention includes a peptide having substantially the same amino acid sequence as a peptide comprising the amino acid sequence of SEQ ID NO: 1, as well as variants thereof or active fragments thereof.

[0022] The term "substantially identical protein" refers to an amino acid sequence having 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 sequence homology with the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. Proteins having 75% or more amino acid sequence homology and the same activity are included within the scope of the present invention.

[0023] Additionally, the peptides of the present invention may further comprise targeting sequences, tags, labeled residues, or specifically engineered amino acid sequences to increase half-life or peptide stability.

[0024] Furthermore, the N-terminus or C-terminus of the peptide of the present invention may be modified to obtain better chemical stability, enhanced pharmacological properties (half-life, water absorption, potency, efficacy, etc.), altered specificity (e.g., broader biological activity spectrum), or reduced antigenicity. The term "stability" is used to mean not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protease enzymes, but also storage stability (e.g., room temperature storage stability).

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

[0026] The C-terminal modification may be, but is not limited to, a hydroxyl group (-OH), an amino group (-NH), an azide (-NHNH), or the like attached to the C-terminus of the peptide.

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

[0028] In a specific embodiment of the present invention, a peptide comprising 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 efficacy of the identified peptide was verified and selected.

[0029] In a specific embodiment of the present invention, to verify the efficacy of the identified peptides, the peptides of the present invention were selected through verification of their effectiveness in increasing the expression levels of ECM components and skin barrier strengthening factors, reducing the level of reactive oxygen species increased by UV irradiation, restoring the expression of skin barrier strengthening genes decreased by UV, and suppressing the expression of cell death-inducing factors increased by UV.

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

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

[0032] The "skin aging" may refer to natural aging that occurs in cells over time, or photoaging caused by sunlight. Furthermore, aging may be induced by intracellular oxidative stress, which can occur for a variety of reasons, leading to cell growth inhibition and cell death, inhibiting the synthesis of various fibrous proteins that make up skin cells and increasing the expression of degradative enzymes. In particular, UV exposure may suppress the expression of genes such as Col1a1, fibronectin, and elastin in skin cells, potentially inducing photoaging and wrinkle formation in skin cells.

[0033] The "skin regeneration" may mean promoting the growth of skin cells, strengthening their viability, preventing cell damage from external and / or internal stimuli, improving skin wrinkles, increasing skin elasticity, and strengthening the skin barrier, and the skin regeneration also includes the prevention of skin damage induced by the external environment, such as pigmentation, or improving and / or treating skin photoaging.

[0034] In one embodiment, the composition for anti-aging or regenerating skin promotes resistance to cell damage caused by aging or external stimuli.

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

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

[0037] The peptide of the present invention can induce increased expression of fiber protein synthesis genes in skin cells. Therefore, the peptide can improve skin wrinkles and elasticity by increasing fiber protein synthesis, and can improve the skin barrier, thereby increasing the resistance and viability of skin cells against external stimuli and promoting the regeneration ability of skin damaged by external stimuli or aging. The skin aging inhibition and skin regeneration effects of the peptide of the present invention can be confirmed by examining the expression levels of cell proliferation-related proteins, anti-aging genes, and fiber proteins, whose expression levels change in skin cells as skin aging progresses.

[0038] In one embodiment, the composition for inhibiting skin aging or regenerating skin may increase the expression of collagen, fibronectin, or elastin in skin cells.

[0039] In one embodiment, the composition for inhibiting skin aging or regenerating skin may increase the expression of SIRT1 or AQP3, which are skin barrier strengthening factors.

[0040] In one embodiment, the composition for inhibiting skin aging or regenerating skin may inhibit the generation of reactive oxygen species (ROS) caused by ultraviolet rays.

[0041] In one embodiment, the composition for inhibiting skin aging or regenerating skin may increase the expression of SIRT1 or AQP3, which is decreased by ultraviolet rays.

[0042] In one embodiment, the composition for inhibiting skin aging or regenerating skin is a composition for inhibiting cleaved PAR increased by ultraviolet rays. P It can suppress the expression of -1 or Cleaved Caspase-3.

[0043] In a specific embodiment of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention has been shown to increase the expression of Col1a1, fibronectin, and elastin in fibroblasts and keratinocytes.

[0044] The peptide has also been shown to increase the expression of skin barrier strengthening genes such as SIRT1 and AQP3 in keratinocytes.

[0045] The peptide has also been shown to reduce intracellular reactive oxygen species (ROS) levels in UV-damaged keratinocytes.

[0046] The peptide was also shown to increase the expression of skin barrier strengthening genes such as SIRT1 and AQP3 in UV-damaged keratinocytes.

[0047] The peptide was also shown to reduce the expression of apoptotic-inducing factors such as cleaved PARP-1, Bax, and cleaved caspase-3 in keratinocytes damaged by UV rays.

[0048] Therefore, it is clear that the peptide of the present invention has skin aging inhibition 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 inhibiting skin aging or regenerating skin, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0050] In one embodiment, the skin aging inhibition or skin regeneration may be prevention or improvement of skin wrinkles, improvement of skin elasticity, strengthening of the skin barrier, prevention of pigmentation, or improvement of skin photoaging.

[0051] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the "Composition for inhibiting skin aging or regenerating skin" section above, so the specific description will be referred to above, and only the components unique to the cosmetic composition for inhibiting skin aging or regenerating skin will be described below.

[0052] The cosmetic composition may be prepared in any dosage form commonly prepared in the art, such as, but not limited to, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, and spray.

[0053] The cosmetic composition may be prepared in various forms such as a solution, sol-gel, emulsion, oil, wax, aerosol, etc., including, but not limited to, a softening lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, a powder, a hair tonic, a hair cream, a hair lotion, a hair shampoo, a hair rinse, a hair conditioner, a hair spray, a hair aerosol, a pomade, a gel, etc.

[0054] The cosmetic composition of the present invention may contain other additives such as excipients and carriers, and it is possible to apply and blend in the required amounts of common ingredients blended in general skin cosmetics.

[0055] When the cosmetic composition is in the form of a paste, cream, or gel, the carrier component may be animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or the like.

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

[0057] When the cosmetic composition is in the form of a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, and examples of such solvents include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, and sorbitan fatty acid esters.

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

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

[0060] When the cosmetic composition is in the form of a hair shampoo, the peptide of the present invention may be mixed with base ingredients for forming a shampoo, such as a thickener, surfactant, viscosity modifier, moisturizer, pH adjuster, preservative, essential oil, etc. The thickener may be CDE, the surfactant may be LES, an anionic surfactant, or cocobetaine, an amphoteric surfactant, the viscosity modifier may be polyquaternary, the moisturizer may be glycerin, and the pH adjuster may be citric acid or sodium hydroxide. The preservative may be grapefruit extract, and in addition, essential oils such as cedarwood, peppermint, and rosemary, silk amino acids, pentaol, or vitamin E may be added.

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

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

[0063] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the "Composition for inhibiting skin aging or regenerating skin" section above, so the above content will be referred to for specific explanations, and only the components specific to the pharmaceutical composition for preventing or treating photoaging will be described below.

[0064] As used herein, the term "photoaging" refers to the phenomenon of skin damage, pigmentation, wrinkle formation, or loss of skin elasticity caused by repeated and prolonged exposure to sunlight.

[0065] As used herein, "prevention" refers to any action of suppressing or delaying the onset, spread, and recurrence of photoaging using the peptide of the present invention or a composition containing the same.

[0066] As used herein, "amelioration or treatment" refers to any action that favorably alters photoaging, such as slowing, stopping, or reversing the progression of photoaging, using 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 inhibiting the production and accumulation of reactive oxygen species in keratinocytes damaged by UV rays, increasing the expression of skin barrier strengthening factors such as SIRT1 and AQP3, and inhibiting the expression of cell death inducers such as cleaved PARP-1 and cleaved caspase-3.

[0068] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

[0069] The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may include water, a suitable oil, saline, aqueous glucose, glycol, etc. Stabilizers and preservatives may also be 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 found in Remington's Pharmaceutical Sciences (19th ed., Mack Publishing Company, Easton, PA, 1995).

[0070] The pharmaceutical compositions of the present invention can be administered to mammals, including humans, by any method, for example, orally or parenterally, including, but not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0071] The pharmaceutical composition of the present invention may be formulated into a preparation for oral administration or a preparation for parenteral administration depending on the administration route as described above, and the preparation for parenteral administration may be specifically formulated into an injection preparation or an external preparation for use.

[0072] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the patient's disease, the activity of the drug, its sensitivity, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, 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. It may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum efficacy at the minimum dose without side effects, which can be easily determined by one skilled in the art.

[0073] The effective amount of the pharmaceutical composition may vary depending on the patient's age, sex, condition, and weight, the degree of absorption of the active ingredient into the body, the inactivation rate, the excretion rate, the type of disease, and concomitant drugs, and may be increased or decreased depending on the administration route, severity, sex, weight, age, etc. For example, the pharmaceutical composition may be administered in an amount of about 0.0001 μg to 500 mg, preferably 0.01 μg to 100 mg, per kg of patient body weight per day.

[0074] As described above, the peptide of the present invention has excellent effects of inhibiting skin aging and regenerating skin, and therefore can be used as a raw material for cosmetics aimed at inhibiting skin aging or regenerating skin, or for pharmaceuticals aimed at preventing, improving, or treating photoaging.

[0075] Yet another aspect of the present invention provides a method for inhibiting skin aging or regenerating skin, comprising administering a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject.

[0076] Yet another aspect of the present invention provides a method for preventing or treating photoaging, comprising administering a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject.

[0077] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in the "Composition for inhibiting skin aging or regenerating skin" section above, and therefore the above content is used for the detailed description.

[0078] As used herein, the term "therapeutically effective amount" refers to an amount of peptide that achieves the goal of improving the incidence of disease during treatment with the peptide and avoids adverse side effects usually associated with other therapies.

[0079] Yet another aspect of the present invention provides the use of the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for the prevention or treatment of photoaging.

[0080] The present invention will be described in more detail below with reference to examples.

[0081] However, the following examples are merely for the purpose of illustrating the present invention, and the content of the present invention is not limited to the following examples.

[0082] [Synthesis Example 1] Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 1 The peptides having the amino acid sequence of SEQ ID NO: 1 shown in Table 1 were synthesized using an automated peptide synthesizer (Liberty, CEM Corporation, USA). These synthesized peptides were purified and separated using a C18 reverse-phase high-performance liquid chromatography (HPLC) (U-3000, Thermo Fisher Scientific, USA). The column used was a Pursuit XRs C18 (250*4.65 mm 100 Å, Agilent, USA).

[0083] [Table 1]

[0084] [Experimental Example 1] Confirmation of natural aging inhibition by peptide treatment 1-1. Confirmation of increased expression of extracellular matrix (ECM) components (collagen, fibronectin, elastin) To confirm whether the peptide of the present invention can inhibit the cellular senescence phenomenon that naturally occurs in cells, the expression levels of genes related to natural aging were examined using fibroblasts.

[0085] First, 5 × 10 NIH3T3 cells (mouse fibroblast cell line) were cultured. 5 After seeding into a 6-well plate at a cell density of 100 cells / 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 peptides of the present invention were mixed with 1 mL of the serum-free DMEM media at concentrations of 10, 50, and 100 μM and then cultured in the cells. PC (positive control) was treated with 100 nM bFGF instead of the peptides. The cells were cultured in a CO2 incubator at 37°C for 24 hours.

[0086] The cultured cells were washed twice with PBS, and RNA was extracted from the cells using Easy Blue (IntRON, Cat. No. 17061, Korea). RT-PCR was performed on the extracted RNA using an RNA kit (Enzynomics, Cat. No. P581T, Korea). The sequences of the primers for collagen (Col1a1), fibronectin, and elastin used in RT-PCR, as well as the GAPDH primer used to compare the total RNA levels in each treatment group, are shown in Table 2.

[0087] [Table 2]

[0088] As a result, as shown in Figures 1a to 1d, it was confirmed that when treated with the peptide, the expression of Col1a1, fibronectin, and elastin genes gradually increased as the peptide concentration increased. This indicates that the peptide containing the amino acid sequence of SEQ ID NO: 1 has the effect of suppressing or improving natural aging such as skin wrinkles as it increases the expression of collagen, fibronectin, and elastin, which are extracellular matrix proteins that make up the dermis of fibroblasts.

[0089] 1-2. Confirmation of increased protein levels of extracellular matrix (ECM) components (collagen and hyaluronic acid) To confirm whether the peptide of the present invention can inhibit the cellular aging phenomenon that naturally occurs in cells, the amount of proteins related to natural aging was examined using fibroblasts and keratinocytes.

[0090] First, NIH3T3 cells and HaCaT cells (human keratinocyte cell line) were cultured at 1 × 10 4Cells were seeded into 24-well plates at a cell density of 100 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 medium, 2 mL of the serum-free DMEM medium was mixed with the peptides of the present invention at concentrations of 50, 100 μM, and 5 ng / ml, and 500 μL was distributed into three wells per condition. For NIH3T3 cells, 5 ng / mL of TGF-β1 was used as a positive control (PC). For HaCaT cells, 100 nM of IGF-1 was used as a PC instead of the peptide. The cells were cultured in a CO2 incubator at 37°C for 72 hours.

[0091] The culture medium of the cultured NIH3T3 and HaCaT cells was centrifuged to sediment the supernatant, and the supernatant was transferred to an e-tube, which was used for collagen ELISA (Abcam, Cat No.: ab210579, UK) and hyaluronic acid (HA) ELISA (ECHELON biosciences, Cat No.: K-1200, USA), respectively.

[0092] As a result, as shown in Figures 2a and 2b, it was confirmed that when treated with the peptide, the amount of collagen and hyaluronic acid proteins gradually increased as the concentration of the peptide increased.

[0093] This shows that as the amount of collagen and hyaluronic acid proteins, which are extracellular matrix proteins that make up the dermis of fibroblasts and keratinocytes, increases by peptides containing the amino acid sequence of Sequence No. 1, natural aging such as skin wrinkles can be suppressed or improved.

[0094] 1-3. Confirmation of increased expression of skin barrier function-related genes (SIRT1 and AQP3) To confirm whether the peptide of the present invention can inhibit the cellular aging phenomenon that naturally occurs in cells, the expression levels of genes related to natural aging were examined using keratinocytes.

[0095] First, 3 × 10 HaCaT cells were 5 After seeding into a 6-well plate at a cell density of 100 cells / 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 then 1 mL of the serum-free DMEM media was mixed with the peptides of the present invention at concentrations of 10, 50, and 100 μM and cultured in the cells. PC (positive control) was treated with 100 nM EGF instead of the peptides. The cells were cultured in a CO2 incubator at 37°C for 24 hours.

[0096] The cultured cells were washed twice with PBS, and RNA was extracted from the cells using Easy Blue (IntRON, Cat. No. 17061, Korea). The extracted RNA was subjected to RT reaction using a kit (enzynomics, Cat. No. RT200, Korea). RT-PCR was then performed using a PCR kit (enzynomics, Cat. No. R581T, Korea). The sequences of the SIRT1 and AQP3 primers used for RT-PCR are shown in Table 3. The sequence of the GAPDH primer used to compare the total RNA levels of each treatment group is shown in Table 2.

[0097] [Table 3]

[0098] As a result, as shown in Figures 3a to 3c, it was confirmed that treatment with the peptide increased the mRNA levels of SIRT1 and AQP3, which are factors that strengthen the skin barrier. This indicates that the peptide containing the amino acid sequence of SEQ ID NO: 1 has the effect of suppressing or improving natural aging by increasing the expression levels of SIRT1 and AQP3, which are factors that strengthen the skin barrier, in keratinocytes.

[0099] [Experimental Example 2] Confirmation of suppression of extrinsic aging by peptide treatment 2-1. Confirmation of the inhibitory effect of active oxygen increased by ultraviolet rays Since UV irradiation increases the level of reactive oxygen species, we investigated whether treatment with the peptide of the present invention would reduce the level of reactive oxygen species again and suppress the aging phenomenon caused by oxidative stress in cells.

[0100] First, 5 × 10 HaCaT cells were 5 Cells were seeded into 6-well plates at a cell density of 100 cells / well and 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 then the peptides of the present invention were mixed in 1 mL of serum-free DMEM media at concentrations of 10, 50, and 100 μM and transfected into the cells. PC (positive control) was treated with 25 μM Trolox instead of the peptides.

[0101] The cells were cultured for 1 hour at 37°C in a CO2 incubator. The culture medium for the cultured cells was transferred to a tube, 1 ml of PBS was added, and the cells were irradiated with 15 J / cm2 UV light using a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France). 2The cells were irradiated with 100 μL of UVB. After removing the PBS, 900 μL of the medium transferred to the tube was aliquoted and cultured in a CO2 incubator at 37°C for 24 hours. After treatment with 10 μM DCFH-DA (dichloro-dihydro-fluorescein diacetate), the cells were wrapped in foil and cultured in a CO2 incubator at 37°C for 30 minutes. After washing twice with PBS, the cells were separated by aliquoting 500 μL of 1XTE. After centrifugation, the cells were washed with PBS and then measured for fluorescence intensity via FACS.

[0102] As a result, as shown in FIG. 4, it was confirmed that when HaCaT cells were treated with the peptide of the present invention, the intracellular reactive oxygen levels increased by UV irradiation were reduced.

[0103] This indicates that the peptide containing the amino acid sequence of SEQ ID NO: 1 reduces the level of reactive oxygen species that increases in keratinocytes due to UV irradiation, inhibiting oxidative stress and thereby preventing cellular photoaging.

[0104] 2-2. Confirmation of increased expression of skin barrier function-related genes (SIRT1 and AQP3) suppressed by UV rays Since UV irradiation inhibits the expression of genes related to skin barrier function, we confirmed whether the expression levels of SIRT1 and AQP3 genes were restored and increased when the peptide of the present invention was treated, thereby confirming whether cellular photoaging could be suppressed.

[0105] First, 3 × 10 HaCaT cells were 5After seeding into a 6-well plate at a cell density of 100 cells / 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 then the peptides of the present invention were mixed with 1 mL of serum-free DMEM media at concentrations of 10, 50, and 100 μM and transfected into the cells. PC (positive control) was treated with 2.5 mM N-acetylcysteine ​​(NaC) instead of the peptides. The cells were cultured for 1 hour in a CO2 incubator at 37°C.

[0106] The cultured cells were washed twice with PBS, and then 1 ml of PBS was added. The cells were then irradiated with 15 J / cm using a UV irradiator (VILBER LOURMAT, Cat. No.: 3102-BSU, France). 2 The cells were irradiated with 1000 μL of UVB. After removing the PBS, 900 μL of the medium transferred to the tube was aliquoted and cultured in a CO2 incubator at 37°C for 24 hours. After washing twice with PBS, RNA was extracted from the cells using Easy Blue (IntRON, Cat. No.: 17061, Korea). The extracted RNA was subjected to RT reaction using an RNA kit (enzynomics, Cat. No.: P581T, Korea). RT-PCR was then performed using a PCR kit (enzynomics, Cat. No.: R581T, Korea). The sequences of the SIRT1 and AQP3 primers used in RT-PCR and the GAPDH primer sequence used to compare the total RNA levels in each treatment group are listed in Tables 2 and 3.

[0107] As a result, as shown in Figures 5a to 5c, it was confirmed that when HaCaT cells were treated with the peptide of the present invention, the mRNA levels of skin barrier-related factors, which had decreased due to UV irradiation, increased again.

[0108] This indicates that the peptide containing the amino acid sequence of SEQ ID NO: 1 has the effect of suppressing cellular photoaging by restoring the expression levels of skin barrier-related factors that were reduced in keratinocytes by UV irradiation.

[0109] 2-3. Confirmation of suppression of expression of cell death-inducing factors (cleaved PARP-1 and cleaved caspase-3) increased by UV rays Since UV irradiation increases the expression of cell death-inducing factors, we investigated whether the expression levels of the increased cell death-inducing factors, cleaved PARP-1 and cleaved caspase-3, decreased again when treated with the peptide of the present invention, thereby determining whether photoaging of cells could be suppressed and damaged cells could be regenerated.

[0110] First, 5 × 10 HaCaT cells were 5 After seeding into 6-well plates at a cell density of 100 cells / 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 then the peptides of the present invention were mixed in 1 mL of serum-free DMEM media at concentrations of 10, 50, and 100 μM and transfected into the cells. PC (positive control) was treated with 2.5 mM N-acetylcysteine ​​(NaC) instead of the peptides.

[0111] The cells were cultured for 1 hour at 37°C in a CO2 incubator, the culture medium was transferred to a tube, 1 ml of PBS was added, and the cells were then irradiated with UV light (VILBER LOURMAT, Cat No.: 3102-BSU, France) at 15 J / cm2. 2The cells were then irradiated with 1000μL of UVB. After removing the PBS, 900μL of the medium transferred to the tube was added back and cultured in a CO2 incubator at 37℃ for 24 hours. The cells were then washed twice with PBS and lysed in cell lysis buffer. Samples were prepared using 5X sample buffer and then subjected to SDS-PAGE using a 10% SDS-PAGE gel. The separated proteins were transferred to a PVDF membrane. After blocking with 5% skim milk for 1 hour at room temperature, anti-cleaved PARP-1 antibody (CST, Cat. No.: 9541S, USA), anti-cleaved Caspase-3 antibody (CST, Cat. No.: 9661S, USA), and anti-β actin antibody (Santa Cruz, Cat. No.: SC-47778, USA) were diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. The membrane was then washed three times with 0.1% PBS-T (0.1% Tween-20 in PBS) for 10 minutes each. Goat anti-rabbit IgG (Jackson Immunoresearch, Cat. No. 111-035-033, USA) and goat anti-mouse IgG (Jackson Immunoresearch, Cat. No. 111-035-003, USA) were diluted 1:3000 in 5% skim milk and reacted with the membrane for 2 hours. Detection was performed on the film using ECL solution (GE Healthcare, Cat. No. RPN2232, USA).

[0112] As a result, as shown in Figures 6a to 6c, it was confirmed that the peptide of the present invention inhibits the expression of apoptosis-inducing factors whose expression is increased in cells damaged by UV irradiation.

[0113] This indicates that the peptide containing the amino acid sequence of Sequence No. 1 reduces the expression of cell death-inducing factors that increased due to UV irradiation, suppressing cellular photoaging and promoting skin regeneration.

[0114] Although the present invention has been described in detail above only with respect to the embodiments described, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the technical concept of the present invention, and it is natural that such modifications and variations fall within the scope of 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 , wherein the peptide has skin aging inhibitory activity or skin regenerating 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, wherein the composition 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 cells are skin fibroblasts or keratinocytes.

7. The composition for inhibiting skin aging or regenerating skin according to claim 3, wherein the composition 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, wherein the composition i) inhibits the generation of reactive oxygen species (ROS) caused by ultraviolet rays, ii) increases the expression of SIRT1 or AQP3 that has been decreased by ultraviolet rays, and iii) inhibits the expression of cleaved PARP-1 or cleaved caspase-3 that has been increased by ultraviolet rays.

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 skin regeneration according to claim 9, wherein the inhibition of skin aging or skin regeneration is prevention or improvement of skin wrinkles, improvement of skin elasticity, strengthening of skin barrier, prevention of pigmentation, or improvement of skin photoaging.

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

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