Peptides with anti-aging activity and uses thereof

Abstract

The present invention relates to a peptide having an anti-aging activity; and a composition for skin regeneration or inhibition of skin aging, the composition comprising the peptide as an active ingredient. The peptide comprising the amino acid sequence of SEQ ID NO: 1 according to the present invention has an anti-aging activity, and thus can be used as a raw material for a cosmetic for skin regeneration or inhibition of skin aging, or can be used as a raw material for a pharmaceutical preparation for prevention, improvement or treatment of photoaging.

Description

Technical Field

[0001] This invention relates to peptides with anti-aging activity, and compositions containing the peptide as an active ingredient for inhibiting skin aging or regenerating skin. Background Technology

[0002] Skin cells age for various reasons, and wrinkles result from this aging. Aging can be divided into age-related aging (intrinsic aging) and photoaging (photochemical aging). Age-related aging is the natural aging process that occurs over time based on biological processes, while photoaging involves degenerative changes in areas exposed to sunlight. Photoaging and age-related aging can occur in combination. Natural cellular aging can occur when the expression or activity of various factors related to cell growth is inhibited, and skin cells can undergo photoaging due to light with wavelengths between 280 nm and 400 nm contained in sunlight. In particular, exposure to UVB ultraviolet radiation, such as that in the wavelength range of 280 nm to 320 nm, can damage the skin or fibers and cause tanning, thus darkening the skin tone. When UVB irradiates skin cells, it promotes the accumulation of ROS and free radicals in the cells and stimulates intracellular signal transduction systems induced by free radicals. This can induce oxidative stress in biomolecules such as DNA, proteins, and lipids, thus causing damage to skin tissue.

[0003] When oxidative stress in skin cells increases, keratinocytes in the epidermis or fibroblasts in the dermis are stimulated. The expression of genes acting as collagen-degrading enzymes, such as matrix metalloproteinases (MMPs), increases through a series of intracellular signal transduction processes, leading to a reduction in collagen. Collagen, a major component of the skin (comprising 90% of the dermis), protects the skin from external stimuli and forces by providing strength and tension. This results in skin aging or wrinkles. Therefore, regulating the expression of genes involved in the synthesis or degradation of fibroblasts such as collagen is expected to prevent skin cell aging and reduce wrinkles.

[0004] The cellular aging process caused by ultraviolet radiation from sunlight not only leads to cosmetic problems due to skin damage, but also to serious health problems, such as cancer caused by DNA damage to cells, damage to the central and peripheral nervous systems, disruption of the immune system, reproductive organ abnormalities, developmental abnormalities in infants and young children, and skin diseases such as chloracne. Therefore, it is urgent to address these issues.

[0005] To address these issues, various materials have been developed, and attempts have been made to overcome them using extracts obtained from various plants or microorganisms. However, in many cases, the exact composition of the extract is unknown because it is impossible to specifically identify which substances in extracts obtained from natural products are effective in protecting skin cells or reversing signs of aging. Therefore, extract components containing unknown substances have also been used. Given that this substance is intended for direct contact with or application to the human body, its side effects must be minimized, and human safety must be ensured. Therefore, there is a need to accurately identify single substances with the effects required to address the aforementioned problems and apply them to human skin cells.

[0006] [Existing technical documents]

[0007] [Non-patent literature]

[0008] (Non-patent document 1) S Edgar et al., Effects of collagen-derived bioactivepeptides and natural antioxidant compounds on proliferation and matrixprotein synthesis by cultured normal human dermal fibroblasts, Scientific Reports, 2018; 8:10474 Summary of the Invention

[0009] Technical issues

[0010] The purpose of this invention is to provide a peptide with anti-aging activity.

[0011] Another object of the present invention is to provide a composition for inhibiting skin aging or regenerating skin, comprising a peptide having anti-aging activity as an active ingredient.

[0012] Another object of the present invention is to provide a cosmetic composition for inhibiting skin aging or regenerating skin, comprising a peptide having anti-aging activity as an active ingredient.

[0013] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of photoaging, comprising a peptide having anti-aging activity as an active ingredient.

[0014] Technical solution

[0015] To achieve the above objectives, one aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0016] Another aspect of the present invention provides a composition for inhibiting skin aging or regenerating skin, comprising a peptide having anti-aging activity as an active ingredient.

[0017] Another aspect of the present invention provides a cosmetic composition for inhibiting skin aging or regenerating skin, comprising a peptide having anti-aging activity as an active ingredient.

[0018] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of photoaging, comprising a peptide having anti-aging activity as an active ingredient.

[0019] Beneficial effects

[0020] The peptide containing 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 that inhibit skin aging or regenerate skin, or as a raw material for pharmaceutical products that prevent, improve or treat photoaging.

[0021] The effects of the present invention are not limited to those described above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description. Attached Figure Description

[0022] Figure 1a The diagram illustrates the expression of Col1a1, fibronectin, and elastin when fibroblasts are treated with the peptides of the present invention, where Con represents the untreated group and TGF-β represents the positive control.

[0023] Figure 1b The figure illustrates the effect of treating fibroblasts with the peptides of the present invention on the expression of Col1a1, where Con represents the untreated group and TGF-β represents the positive control.

[0024] Figure 1c The figure illustrates the effect of treating fibroblasts with the peptides of the present invention on fibronectin expression, where Con represents the untreated group and TGF-β represents the positive control.

[0025] Figure 1d The figure illustrates the effect of treating fibroblasts with the peptides of the present invention on elastin expression, where Con represents the untreated group and TGF-β represents the positive control.

[0026] Figure 2a A view showing the expression of AQP3 and SIRT1 when keratinocytes are treated with the peptides of the present invention is provided, where Con represents the untreated group and EGF represents the positive control.

[0027] Figure 2bThe figure illustrates the effect of treating keratinocytes with the peptides of the present invention on the expression of AQP3, where Con represents the untreated group and EGF represents the positive control.

[0028] Figure 2c The figure illustrates the effect of treating keratinocytes with the peptides of the present invention on SIRT1 expression, where Con represents the untreated group and EGF represents the positive control.

[0029] Figure 3a The figure illustrates the effect of the peptides of the present invention on the increased levels of intracellular reactive oxygen species (ROS) in fibroblasts due to ultraviolet (UV) irradiation, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox.

[0030] Figure 3b The figure illustrates the effect of the peptides of the present invention on the increased levels of intracellular reactive oxygen species (ROS) in keratinocytes due to ultraviolet (UV) irradiation, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox.

[0031] Figure 4a To illustrate the effect of the peptides of the present invention on the expression of Col1a1 and fibronectin genes reduced by ultraviolet (UV) irradiation in fibroblasts, a view is provided, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox.

[0032] Figure 4b The figure illustrates the effect of the peptide of the present invention on the reduced expression of the Col1a1 gene in fibroblasts caused by ultraviolet (UV) irradiation, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptide of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox.

[0033] Figure 4cThe diagram illustrates the effect of the peptides of the present invention on fibronectin gene expression reduced by ultraviolet (UV) irradiation in fibroblasts, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox.

[0034] Figure 5a The figure illustrates the effect of the peptide of the present invention on MMP-1 expression increased by ultraviolet (UV) irradiation in fibroblasts, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptide of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox.

[0035] Figure 5b The figure illustrates the effect of the peptides of the present invention on increased MMP-1 expression in keratinocytes due to ultraviolet (UV) irradiation, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox.

[0036] Figure 6a The figure illustrates the effect of the peptides of the present invention on the increased MMP-2 activity in fibroblasts due to ultraviolet (UV) irradiation, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox.

[0037] Figure 6b The figure illustrates the effect of the peptides of the present invention on the increased MMP-9 activity in keratinocytes due to ultraviolet (UV) irradiation, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 µM, 50 µM, and 100 µM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and Trolox represents the positive control irradiated with UV and treated with Trolox. Detailed Implementation

[0038] The invention will be described in more detail below.

[0039] One aspect of the present invention provides a peptide comprising the amino acid sequence described in SEQ ID NO: 1.

[0040] In this specification, the term "peptide" refers to a linear molecule composed of amino acid residues, and more specifically, the peptides of the present invention may include the amino acid sequence described in SEQ ID NO: 1.

[0041] Within the scope of not affecting functionality, a "peptide" can be a variant or fragment of amino acids that have a different sequence through the deletion, insertion, substitution, or combination of amino acid residues. Amino acid exchanges that do not completely alter the activity of the peptide are known in the art. In some cases, peptides can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Therefore, the peptides of the present invention include peptides containing an amino acid sequence substantially identical to that of a peptide comprising the amino acid sequence of SEQ ID NO: 1, its variants, or its active fragment.

[0042] The term "essentially identical protein" refers to an amino acid sequence that has 75% or more, preferably 80% or more, such as 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the amino acid sequence of SEQ ID NO: 1. However, it is not limited to this. As long as the protein has 75% or more of the amino acid sequence homology and has the same activity, it is included within the scope of this invention.

[0043] Furthermore, the peptides of the present invention may also include targeting sequences, tags, labeled residues, or amino acid sequences prepared for a specific purpose to increase the half-life or stability of the peptide.

[0044] Furthermore, to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad bioactivity spectrum), and reduced antigenicity, the N-terminus or C-terminus of the peptides of the present invention may be modified. "Stability" includes not only in vivo stability for protecting the peptides of the present invention from attack by proteases in vivo, but also storage stability (e.g., storage stability at room temperature).

[0045] N-terminal modification can be, but is not limited to, binding of a protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristoyl, stearoyl, and polyethylene glycol (PEG) to the N-terminus of the peptide.

[0046] The C-terminus can be modified by the binding of hydroxyl (-OH), amino (-NH2), hydrazine (-NHNH2), etc., to the C-terminus of the peptide, but is not limited to these.

[0047] The peptides of this invention can be synthesized, for example, using apparatus or genetic engineering techniques. When synthesizing peptides using an apparatus, the desired peptides can be synthesized using the Fmoc solid-phase method in an automated peptide synthesizer.

[0048] In a specific embodiment of the present invention, the peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention is synthesized and identified using the Fmoc solid-phase method, and selection is made by verifying the efficacy of the identified peptide.

[0049] In a specific embodiment of the present invention, in order to verify the efficacy of the identified peptide, the peptide of the present invention is selected by verifying the following effects of the peptide: the effect of increasing the expression levels of extracellular matrix (ECM) components and skin barrier strengthening factors, the effect of reducing the level of reactive oxygen species increased by ultraviolet radiation, and the effect of inhibiting the expression and activity of collagen degradation-related proteins increased by ultraviolet radiation.

[0050] Therefore, the peptide of the present invention containing the amino acid sequence of SEQ ID NO: 1 has skin aging inhibitory activity or skin regeneration activity.

[0051] Another aspect of the present invention provides a composition for inhibiting skin aging or regenerating skin, having a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0052] Skin aging can be the natural aging of cells over time, or it can be photoaging caused by sunlight. Furthermore, aging can be induced by intracellular oxidative stress, which can be caused by various factors. Therefore, cell growth inhibition or apoptosis may occur, the synthesis of various fibrous proteins that make up skin cells may be suppressed, and the expression of degradative enzymes may increase. In particular, when exposed to ultraviolet radiation, the expression of genes such as Col1a1, fibronectin, and elastin in skin cells is suppressed, which can lead to photoaging and wrinkles.

[0053] "Skin regeneration" can promote the growth and vitality of skin cells to prevent damage to cells from external and / or internal stimuli, reduce skin wrinkles, improve elasticity, and strengthen the skin barrier. Skin regeneration also includes preventing skin damage caused by external environmental factors (such as pigmentation) or improving or treating photoaging of the skin.

[0054] Compositions for inhibiting skin aging or promoting skin regeneration enhance resistance to cell damage caused by aging or external stimuli.

[0055] "External stimuli" can be physical stimuli, chemical stimuli caused by the application of cosmetics or other external preparations, stimuli caused by ultraviolet rays, or stimuli caused by infrared rays.

[0056] Damaged cells can be skin fibroblasts or keratinocytes.

[0057] The peptide of this invention can induce an increase in the expression of fibrin synthesis genes in skin cells. Therefore, this peptide can reduce wrinkles and improve skin elasticity by increasing fibrin synthesis, and can also increase the resistance and vitality of skin cells by improving the skin barrier, and enhance the regenerative capacity of skin damaged by external stimuli or aging. The skin aging inhibition and skin regeneration effects of the peptide of this invention can be confirmed by measuring the expression levels of proteins involved in cell proliferation or anti-aging genes, as well as fibrin, whose expression levels change within skin cells as skin aging progresses.

[0058] Compositions for inhibiting skin aging or promoting skin regeneration can increase the expression or secretion of collagen, fibronectin, or elastin in skin cells.

[0059] Compositions for inhibiting skin aging or promoting skin regeneration can increase the expression of AQP3 or SIRT1, which are skin barrier strengthening factors.

[0060] Compositions for inhibiting skin aging or promoting skin regeneration can suppress the production of reactive oxygen species (ROS) caused by ultraviolet radiation.

[0061] Compositions for inhibiting skin aging or promoting skin regeneration can increase the expression of collagen or fibronectin that is reduced by ultraviolet radiation.

[0062] Compositions for inhibiting skin aging or promoting skin regeneration can inhibit the expression of MMP-1 increased by ultraviolet radiation, or can inhibit the activity of MMP-2 and MMP-9 increased by ultraviolet radiation.

[0063] In a specific embodiment of the invention, it is shown that the peptide of the present invention comprising the amino acid sequence SEQ ID NO: 1 increases the expression of genes encoding extracellular matrix proteins such as Col1a1, fibronectin, and elastin in fibroblasts.

[0064] Furthermore, the peptide was shown to increase the expression of skin barrier strengthening genes such as AQP3 and SIRT1 in fibroblasts and / or keratinocytes.

[0065] Furthermore, the peptide was shown to reduce the level of intracellular reactive oxygen species (ROS) in fibroblasts and / or keratinocytes damaged by ultraviolet radiation.

[0066] Furthermore, it was shown that the peptide increased the expression of Col1a1 and fibronectin genes, which were reduced in fibroblasts damaged by ultraviolet radiation.

[0067] Furthermore, it was shown that the peptide inhibited the increase in MMP-1 expression or the increase in MMP-2 and / or MMP-9 activity in UV-damaged fibroblasts and / or keratinocytes.

[0068] Therefore, it is obvious that the peptides of the present invention have skin aging inhibition or skin regeneration activity by regulating the expression of genes related to skin cell aging, and the peptides of the present invention can be effectively used as active ingredients in compositions for skin aging inhibition or skin regeneration.

[0069] Another aspect of the present invention provides a cosmetic composition for inhibiting skin aging or regenerating skin, having a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0070] Skin aging inhibition or skin regeneration can prevent or reduce skin wrinkles, improve skin elasticity, strengthen the skin barrier, prevent pigmentation, or improve photoaging of the skin.

[0071] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in "Composition for Inhibiting or Regenerating Skin Aging," and therefore its detailed description is provided above. Hereinafter, only the specific formulation of the cosmetic composition for inhibiting or regenerating skin aging will be described.

[0072] Cosmetic compositions can be prepared into any formulation commonly prepared in this field, such as solutions, suspensions, lotions, creams, gels, lotions, toners, powders, soaps, surfactant-containing cleansers, oils, powder foundations, lotion foundations, wax foundations, and sprays, but are not limited thereto.

[0073] Cosmetic compositions can be prepared in various forms, such as solutions, sols and gels, emulsions, oils, waxes, and aerosols, including, but not limited to, toners, nourishing lotions, nourishing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, face masks (packs), sprays, powders, hair growth creams, hair pastes, hair conditioners, shampoos, hair conditioners, hair gels, hair aerosols, hair oils, gels, etc.

[0074] The cosmetic compositions of the present invention may contain other additives, such as excipients and carriers, and can be applied and mixed as needed with commonly used ingredients that can be used with ordinary skin cosmetics.

[0075] When the cosmetic composition of the present invention is formulated as a cream, lotion, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0076] When the cosmetic composition of the present invention is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component, and in particular, when the cosmetic composition is formulated as a spray, it may additionally contain a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether, but is not limited thereto.

[0077] When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol aliphatic esters, polyethylene glycol, fatty acid esters of sorbitol, etc.

[0078] When the cosmetic composition is formulated as a suspension, liquid diluents such as water, ethanol, or propylene glycol can be used, and suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester or polyoxyethylene dehydrated sorbitan ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, tragacanth gum, etc.

[0079] When the cosmetic composition is formulated as a detergent containing surfactants, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazolonium derivatives, methyl taurine, sarcosinates, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, etc., can be used as carrier components.

[0080] When the cosmetic composition is formulated as a shampoo, basic ingredients used to form the shampoo, such as thickeners, surfactants, viscosity modifiers, humectants, pH adjusters, preservatives, or essential oils, can be mixed with the peptides of the present invention. CDE can be used as a thickener, LES as an anionic surfactant or cocobetaine as an amphoteric surfactant can be used as a surfactant, polyquaternium salts can be used as viscosity modifiers, glycerin can be used as a humectant, and citric acid or sodium hydroxide can be used as a pH adjuster. Grapefruit extract and the like can be used as preservatives, and essential oils such as cedarwood, peppermint, or rosemary, serine amino acids, pentanol, or vitamin E can also be added.

[0081] In addition to the peptides and carrier components of the present invention, the cosmetic composition may also contain, but is not limited to, conventionally used components in cosmetic compositions such as conventional adjuvants, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances as active ingredients.

[0082] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of photoaging having a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0083] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in "Compositions for Inhibiting or Regenerating Skin Aging," and therefore its detailed description is provided above. Hereinafter, only the specific formulation of the pharmaceutical composition for preventing or treating photoaging will be described.

[0084] In this specification, "photoaging" refers to skin damage, pigmentation, wrinkles, or decreased skin elasticity caused by repeated or prolonged exposure to sunlight.

[0085] In this specification, "prevention" means any action by which the peptide of the present invention or a composition containing the peptide inhibits or delays the occurrence, spread, and recurrence of photoaging.

[0086] In this specification, "improvement or treatment" means any beneficial alteration of photoaging behavior, such as delaying, stopping, or reversing the progression of photoaging by means of the peptide of the present invention or a composition containing the peptide.

[0087] In a specific embodiment of the present invention, the peptide increases the expression or secretion of extracellular matrix (ECM) proteins such as collagen, fibronectin, or elastin, and increases the expression of skin barrier strengthening factors such as AQP3 or SIRT1, thereby preventing, improving, or treating photoaging.

[0088] In addition, this peptide inhibits the production of reactive oxygen species (ROS) in cells damaged by aging or external stimuli, increases the expression of reduced collagen or fibronectin, and inhibits the expression of increased MMP-1 or the activity of increased MMP-2 and MMP-9, thereby preventing, improving or treating photoaging.

[0089] The pharmaceutical compositions of the present invention may also contain a pharmaceutically acceptable carrier.

[0090] The pharmaceutical composition may also contain, for example, a carrier for oral administration or a carrier for parenteral administration as a pharmaceutically acceptable carrier. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Furthermore, carriers for parenteral administration may include water, suitable oils, salt solutions, aqueous glucose solutions, ethylene glycol, etc. In addition, the pharmaceutical composition may contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, and ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. For a description of suitable pharmaceutically acceptable carriers, refer to Remington's Pharmaceutical Sciences (19th edition, Mack Publishing Company, Easton, PA, 1995).

[0091] The pharmaceutical compositions of the present invention can be administered to mammals, including humans, by any method. For example, the pharmaceutical compositions of the present invention can be administered orally or parenterally. Parenterally administration can be (but is not limited to) intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, local, sublingual, or rectal administration.

[0092] The pharmaceutical compositions of the present invention can be formulated into oral or parenteral formulations according to the administration routes described above, and the parenteral formulations can be specifically formulated and used in the form of injectable or topical formulations.

[0093] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts. In this invention, a "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment. Effective dosage levels can be determined based on factors including the patient's disease type and severity, drug activity, drug sensitivity, timing of administration, route of administration and excretion rate, duration of treatment, and concurrently used drugs, as well as other factors known in the medical field. The pharmaceutical compositions can be administered as a single therapeutic agent or in combination with other therapeutic agents; the pharmaceutical compositions can be administered simultaneously, separately, or sequentially with conventional therapeutic agents; or the pharmaceutical compositions can be administered alone or multiple times. Importantly, considering all the foregoing factors, the pharmaceutical composition can be administered in an amount that yields maximum effect with minimal dosage and without side effects, and the amount of pharmaceutical composition to be administered can be readily determined by those skilled in the art.

[0094] The effective amount of the drug composition can vary depending on the patient's age, sex, condition, weight, the extent of absorption, inactivation rate, excretion rate of the active ingredient in the body, disease type, and concomitant drugs. Furthermore, the effective amount of the drug composition can be increased or decreased depending on the route of administration, severity, sex, weight, age, etc. For example, the drug composition can be administered at a dose of approximately 0.0001 μg to 500 mg per kg of patient body weight per day, preferably 0.01 μg to 100 mg.

[0095] As described above, the peptides of the present invention have excellent skin aging inhibition and skin regeneration effects, and therefore can be used as raw materials for cosmetics for skin aging inhibition or skin regeneration, or as raw materials for pharmaceutical products for preventing, improving or treating photoaging.

[0096] Another aspect of the present invention provides a method for inhibiting skin aging or promoting skin regeneration, the method comprising administering to a subject a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0097] Another aspect of the present invention provides a method for preventing or treating photoaging, the method comprising administering to a subject a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0098] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in “Composition for Inhibiting or Regenerating Skin Aging”, and therefore its detailed description is given above.

[0099] In this specification, the term "therapeutic effective amount" refers to the amount of peptide that achieves the goal of reducing the frequency of disease attacks during peptide therapy, while avoiding the harmful side effects typically involved in other treatments.

[0100] Another aspect of the present invention provides the use of the amino acid sequence of SEQ ID NO: 1 for preparing a medicine for preventing or treating photoaging.

[0101] The present invention will be described in detail below with reference to embodiments.

[0102] However, the following embodiments are merely illustrative of the present invention, and the present invention is not limited to the following embodiments.

[0103] Examples of the present invention

[0104] [Synthesis example 1]

[0105] Synthesis of a peptide containing the amino acid sequence of SEQ ID NO:1

[0106] Peptides containing the amino acid sequence of SEQ ID NO:1 [Table 1] were synthesized using an automated peptide synthesizer (Liberty, CEM Corporation, USA), and then the synthesized peptides were separated and purified using C18 reversed-phase high-performance liquid chromatography (HPLC) (U-3000, Thermofisher Scientific, USA). Pursuit XRs C18 (250...) (4.65 mm 100 Å, Agilent, USA) was used as a chromatographic column.

[0107] [Table 1]

[0108] [Experimental Example 1] Confirmation of the inhibitory effect of peptide treatment on natural aging

[0109] 1-1. Confirmation of increased expression of extracellular matrix (ECM) component genes (collagen, fibronectin, and elastin)

[0110] The expression levels of genes involved in the natural aging of fibroblasts were measured to confirm whether the peptides of the present invention inhibited the naturally occurring cellular aging phenomenon.

[0111] First, NIH3T3 cells (mouse fibroblast line) were thromboplasted at a rate of 3 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates 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 medium, and the peptides of the present invention were mixed into 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 5 ng / mL TGF-β instead of the peptides. Cells were cultured in a CO2 incubator at 37°C for 24 hours.

[0112] 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 extracted RNA was quantified by adding 1,000 ng of RNA to each tube and performing RT-PCR using a kit (Enzynomics, Cat. No.: PT200, Korea). RT-PCR was performed using a PCR kit (Enzynomics, Cat. No.: P581T, Korea). The primer sequences for collagen, fibronectin, and elastin used in RT-PCR, as well as the primer sequences for GAPDH used to compare the total RNA amount in each treatment group, are shown in [Table 2].

[0113] [Table 2]

[0114] As a result, such Figures 1a to 1d As shown, it was confirmed that when treated with peptides, the expression of collagen, fibronectin, and elastin genes gradually increased with increasing peptide concentration. Therefore, it can be concluded that peptides containing the amino acid sequence of SEQ ID NO: 1 increase the expression of collagen, fibronectin, and elastin, which are extracellular matrix proteins constituting dermal fibroblasts, thus achieving an effect of inhibiting or improving natural aging such as skin wrinkles.

[0115] 1-2. Confirmation of increased expression of skin barrier function-related genes (SIRT1 and AQP3)

[0116] The expression levels of genes involved in the natural aging of keratinocytes were measured to confirm whether the peptides of the present invention inhibited the naturally occurring cellular aging process.

[0117] First, HaCaT cells were loaded at a rate of 3 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates 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 medium, and then the peptides of the present invention were mixed at concentrations of 10 µM, 50 µM, and 100 µM in 1 mL of serum-free DMEM medium and dispensed into the cells. Positive controls (PCs) were treated with 100 nM EGF instead of the peptides. Cells were cultured in a CO2 incubator at 37°C for 24 hours.

[0118] 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 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 primers for AQP3 and SIRT1 used in RT-PCR, and the sequences of the primers for GAPDH used to compare the total RNA amount in each treatment group, are shown in [Table 2].

[0119] As a result, such Figures 2a to 2c As shown, it was confirmed that when treated with peptides, the mRNA levels of AQP3 and SIRT1, which are skin barrier strengthening factors, increased.

[0120] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO: 1 increases the expression levels of AQP3 and SIRT1, which are skin barrier strengthening factors in keratinocytes, thus achieving the effect of inhibiting or improving natural aging.

[0121] [Experimental Example 2]

[0122] Confirmation of the inhibitory effect of peptide treatment on extrinsic aging

[0123] 2-1. Confirmation of the effect of inhibiting reactive oxygen species increased by ultraviolet radiation.

[0124] UV irradiation increases the level of reactive oxygen species (ROS). Therefore, it was confirmed whether treatment with the peptides of the present invention reduced ROS levels again and whether it inhibited aging caused by oxidative stress in cells.

[0125] First, NIH3T3 cells and HaCaT cells were respectively loaded with 5×10 5 Cells were seeded at a density of 10 cells / well in 6-well plates 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 medium, and then the peptides of the present invention were mixed in 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 50 µM Trolox instead of the aforementioned peptides.

[0126] Cells were cultured in a CO2 incubator at 37°C for 1 hour. The culture medium was transferred to tubes, 1 ml of PBS was added, and then the cells were irradiated with a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France) at 8 J / cm². 2 NIH3T3 cells were irradiated with UVA at 15 mJ / cm². 2 HaCaT cells were irradiated with UVB. After removing PBS, 900 µL of culture medium was transferred to tubes and cultured in a CO2 incubator at 37°C for 24 h. After treatment with 10 µM dichlorodihydrofluorescein diacetate (DCFH-DA), the cells were wrapped in foil and cultured in a CO2 incubator at 37°C for 30 min. The cells were washed twice with PBS and 500 μL of 1XTE was allocated for cell separation. After centrifugation, the cells were washed with PBS, and fluorescence values ​​were measured by FACS.

[0127] As a result, as shown in Figures 3A and 3B, it was confirmed that when NIH3T3 cells and HaCaT cells were treated with the peptides of the present invention, the increased levels of intracellular reactive oxygen species due to ultraviolet irradiation were reduced.

[0128] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO: 1 reduces the level of reactive oxygen species in keratinocytes that are increased by UV irradiation and inhibits oxidative stress, thus preventing photoaging of cells.

[0129] 2-2. Confirmation of increased expression of extracellular matrix components (collagen and fibronectin) genes suppressed by ultraviolet radiation.

[0130] UV irradiation inhibits the expression of genes related to extracellular matrix components. Therefore, by confirming whether the expression levels of Col1a1 and fibronectin genes recovered and increased again, it was confirmed that photoaging of cells was inhibited when treated with the peptides of the present invention.

[0131] First, NIH3T3 cells were loaded at 5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 6-well plates 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 medium, and then the peptides of the present invention were mixed in 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 2.5 mM N-acetylcysteine ​​or 50 µM Trolox instead of the peptides. Cells were cultured in a CO2 incubator at 37°C for 1 hour.

[0132] Transfer the culture medium to a tube (e-tube), add 1 ml of PBS, and irradiate with a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France) at 6 J / cm². 2 The cells were irradiated with UVA. After removing PBS, 900 µL of culture medium was transferred to tubes and incubated in a CO2 incubator at 37°C for 6 hours. 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 extracted RNA was quantified by adding 1,000 ng of RNA to each tube and performing 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 primers for Col1a1 and fibronectin used in RT-PCR, as well as the sequences of the primers for GAPDH used to compare the total RNA amount in each treatment group, are shown in [Table 2].

[0133] As a result, such Figures 4a to 4c As shown, it was confirmed that when NIH3T3 cells were treated with the peptides of the present invention, the mRNA levels of Col1a1 and fibronectin, which were reduced by ultraviolet irradiation, increased again.

[0134] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO: 1 restores the expression levels of Col1a1 and fibronectin in fibroblasts that were reduced by UV irradiation, thus inhibiting the photoaging of cells.

[0135] 2-3. Confirmation of inhibition of MMP-1 expression increased by ultraviolet radiation

[0136] The expression of MMP-1, a protein associated with collagen degradation, increases with ultraviolet radiation. Therefore, it was confirmed whether treatment with the peptides of the present invention again inhibited MMP-1 protein expression and whether it suppressed cellular senescence.

[0137] First, NIH3T3 cells and HaCaT cells were respectively loaded with 5×10 5 Cells were seeded at a density of [number] cells / well in 6-well plates 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 medium, and the peptides of the present invention were mixed in 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 25 µM and 50 µM Trolox instead of the aforementioned peptides.

[0138] Cells were cultured in a CO2 incubator at 37°C for 1 hour. The culture medium was transferred to tubes (e-tubes), 1 ml of PBS was added, and then the cells were irradiated with UV light (VILBER LOURMAT, Cat No.: 3102-BSU, France) at 6 J / cm². 2 NIH3T3 cells were irradiated with UVA at 15 mJ / cm². 2HaCaT cells were irradiated with UVB. After removing PBS, 900 µL of culture medium was added to the tubes and the cells were incubated in a CO2 incubator at 37°C for 24 hours. The culture medium was transferred to tubes, and the cells were washed twice with PBS, followed by the addition of cell lysis buffer to the lysed cells. Samples were prepared by treating the cells with 5X sample buffer and then performing SDS-PAGE on a 10% SDS-PAGE gel. The proteins separated by SDS-PAGE were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. Anti-MMP-1 antibody (Abcam, Cat. No.: ab137332, UK) was diluted to 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. Subsequently, the membrane was washed three times with 0.1% PBS-T (PBS solution of 0.1% Tween-20) for 10 minutes each time. Goat anti-rabbit IgG (Jackson Immune Research, Cat. No.: 111-035-033, USA) was diluted to 1:3000 in 5% skim milk and reacted with the membrane for 2 hours. Subsequently, the membrane was used for detection with ECL solution (GE Healthcare, Cat. No.: RPN2232, USA). Anti-β-actin antibody (Santacruz biotechnology, USA) was used to identify β-actin to compare the total amount of protein used in the experiment.

[0139] As a result, such Figure 5a and Figure 5b As shown, the peptide of the present invention was confirmed to inhibit the expression of MMP-1 increased by UV irradiation in NIH3T3 cells and HaCaT cells.

[0140] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO:1 reduces the expression of MMP-1 in fibroblasts and keratinocytes that is increased by UV irradiation and inhibits cellular aging, thus achieving the effect of reducing skin wrinkles.

[0141] 2-4. Confirmation of the inhibition of MMP-2 and MMP-9 activity increased by ultraviolet radiation.

[0142] The activity of MMP-2 and MMP-9 proteins, which are associated with collagen degradation, increases with ultraviolet radiation. Therefore, it was confirmed whether treatment with the peptides of the present invention again inhibited the activity of MMP-2 and MMP-9 proteins, and whether it inhibited cellular aging.

[0143] First, NIH3T3 cells and HaCaT cells were mixed at a ratio of 5 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / well in 6-well plates 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 medium, and the peptides of the present invention were mixed in 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 25 µM and 50 µM Trolox instead of the aforementioned peptides.

[0144] Cells were cultured in a CO2 incubator at 37°C for 1 hour. The culture medium was transferred to tubes (e-tubes), 1 ml of PBS was added, and then the cells were irradiated with UV light (VILBER LOURMAT, Cat No.: 3102-BSU, France) at 6 J / cm². 2 NIH3T3 cells were irradiated with UVA at 15 mJ / cm². 2HaCaT cells were irradiated with UVB. After removing PBS, 900 µL of culture medium was added to the tubes and the cells were incubated in a CO2 incubator at 37°C for 6 hours. The culture medium was transferred to tubes, and the cells were washed twice with PBS, followed by the addition of cell lysis buffer to the lysed cells. Samples were prepared by treating the cells with 5X sample buffer and then performing SDS-PAGE using a 10% SDS-PAGE gel. The proteins separated by SDS-PAGE were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. Anti-β-actin antibody (Santacruz biotechnology, USA) was diluted to 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. Subsequently, the membrane was washed three times with 0.1% PBS-T (PBS solution of 0.1% Tween-20) for 10 minutes each time. Goat anti-rabbit IgG (JacksonImmune Research, Cat. No.: 111-035-033, USA) was diluted to 1:3000 in 5% skim milk and reacted with a membrane for 2 hours. Subsequently, detection was performed using the membrane with ECL solution (GE Healthcare, Cat. No.: RPN2232, USA). Next, to analyze the amounts of MMP-2 and MMP-9, cell culture medium transferred to tubes was treated with 4X enzyme spectrum sample buffer to prepare samples, and the samples were SDS-PAGEd using an 8% SDS-PAGE gel. The gel was washed with 0.2% Tween-20 solution for 30 minutes, followed by washing with distilled water (DW) for 30 minutes. The gel was placed in TNCB buffer and incubated at 37°C for 16 hours. The treated gel was stained with Coomassie Brilliant Blue R250 (Sigma) and treated with destaining buffer (5% ethanol, 7.5% acetic acid, and distilled water). Subsequently, the activity of MMP-2 and MMP-9 was confirmed by observing the empty bands that appeared on the gel during gelatin hydrolysis.

[0145] As a result, such Figure 6a and Figure 6b As shown, the peptides of the present invention have been shown to inhibit the activity of MMP-2 and MMP-9 proteins in fibroblasts and keratinocytes that are increased by UV irradiation.

[0146] It can be concluded that because the peptide containing the amino acid sequence of SEQ ID NO:1 causes the activity of MMP-2 and MMP-9 proteins in fibroblasts and keratinocytes, which are increased by ultraviolet radiation, to decrease and then recover, the amount of collagen can be increased again, thereby inhibiting the aging of cells and thus achieving the effect of reducing skin wrinkles.

[0147] As described above, although the present invention has been described in detail with reference only to the embodiments described herein, it will be apparent to those skilled in the art that various modifications and alterations can be made without departing from the technical spirit of the present invention, and such modifications and alterations are obviously within the scope of the claims.

Claims

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

1.

2. A composition comprising the peptide according to claim 1 as an active ingredient.

3. Use of the composition according to claim 2 in the preparation of a product for inhibiting skin aging.

4. Use of the composition according to claim 2 in the preparation of products for improving resistance to cell damage caused by aging or external stimuli.

5. The use according to claim 4, wherein the external stimulus is ultraviolet or infrared radiation.

6. The use according to claim 4, wherein the cells are skin fibroblasts or keratinocytes.

7. A cosmetic composition comprising the peptide according to claim 1 as an active ingredient.

8. Use of the cosmetic composition according to claim 7 in the preparation of a product for inhibiting skin aging.

9. Use of the peptide according to claim 1 in the preparation of a medicament for the prevention or treatment of photoaging.

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