Peptides with skin condition improving activity and their uses

Abstract

The present invention relates to a peptide having skin condition improving activity. The peptide of the present invention increases the expression levels of extracellular matrix components and skin barrier strengthening factors, decreases the expression of skin cell damage factors that were increased by ultraviolet irradiation, and restores the expression levels of extracellular matrix components and skin barrier strengthening factors that were decreased by ultraviolet irradiation or heat shock.

Description

[Technical Field]

[0001] [Cross-citation with related applications] This application claims priority based on Korean Patent Application No. 10-2023-0106435, filed on 14 August 2023, and all content disclosed in said Korean Patent Application is incorporated herein by reference.

[0002] [Technical field] This invention relates to a novel peptide having skin condition improving activity and its uses. [Background technology]

[0003] The skin is the organ that encloses all organs and tissues of the human body, playing a vital role in protecting the body from external invaders and regulating homeostasis by maintaining internal moisture and temperature. Histologically, the skin is composed of three layers: the epidermis, dermis, and subcutaneous fat. Skin aging can be divided into aging due to intrinsic factors and aging due to extrinsic factors. Aging due to intrinsic factors occurs naturally with age and is caused by a decrease in skin cell activity due to genetic factors such as telomere shortening. Aging due to extrinsic factors is caused by environmental factors such as sunlight, cold, wind, and fine dust, with ultraviolet and infrared rays from sunlight being the most important cause.

[0004] As skin ages, collagen and elastic fibers, which make up the fibrous tissue that supports the skin's structure, harden. On the other hand, the matrix (ground substance) that fills the spaces between cells and between collagen and elastic fibers has a very strong water-retaining capacity, but as such matrix substances decrease, skin elasticity declines and wrinkles form. Representative substances in this matrix are hyaluronic acid and mucopolysaccharides, and as skin ages, hyaluronidase increases, and the amount of hyaluronic acid in the dermis gradually decreases. In addition, reactive oxygen species (free radicals) induced by ultraviolet light or generated by intracellular respiration react with unsaturated fatty acids in the cell membrane to form new radicals called peroxyradicals, which then undergo a chain reaction in the presence of oxygen, inducing lipid peroxidation again. Lipid peroxides can further amplify lipid peroxidation reactions and react with proteins to damage the function of enzymes and receptor systems. Thus, reactive oxygen species are a major cause of damage to skin cells and tissues. Furthermore, reactive oxygen species disrupt the skin's antioxidant defense network, which consists of antioxidant enzymes and non-enzymatic antioxidants, thereby shifting the balance between oxidizers and antioxidants towards the oxidative side. Continuous oxidative stress damages biological components through processes such as lipid peroxidation, protein oxidation, activation of proteolytic enzymes that destroy interstitial components, chain severance and abnormal cross-linking of the elastic fibers collagen and elastin, hyaluronic acid chain severance, promotion of melanin production, and DNA oxidation.

[0005] Hemidesmosomes are adhesions that attach epithelial tissue to the basal layer tissue directly beneath it. Hemidesmosome adhesion is mediated by hemidesmosome integrin proteins, which bind to inner keratin filaments while simultaneously binding to laminin in the extracellular basal layer, thereby attaching the cell to the basal layer. In skin structure, hemidesmosomes appear at the dermoepidermal junction and serve as the region where keratinocytes in the epidermal basal layer attach to the extracellular matrix of the dermis. Through this structure, the density of the skin barrier is firmly maintained.

[0006] The following are patent documents relating to peptide-active substances having activity to improve skin aging or wrinkles: Korean Registered Patent No. 10-2486996, Korean Registered Patent No. 10-2507392, and International Patent Application PCT-KR2021-011278. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Korean Registered Patent No. 10-2486996 [Patent Document 2] Korean Registered Patent No. 10-2507392 [Patent Document 3] International Patent Application PCT-KR2021-011278 [Overview of the project] [Problems that the invention aims to solve]

[0008] The inventors have made research efforts to develop effective active substances that can be used to suppress skin aging and improve skin conditions such as wrinkle reduction. As a result, the inventors have completed the present invention by experimentally confirming that the novel peptide they synthesized exhibits excellent skin condition improving activity, preventing skin aging and wrinkles caused not only by endogenous factors but also by exogenous factors.

[0009] Accordingly, it is an object of the present invention to provide a novel peptide having skin condition improving activity.

[0010] Another object of the present invention is to provide a composition for improving skin conditions, which comprises the peptide as an active ingredient. [Means for Solving the Problem]

[0011] In order to achieve the above-mentioned object of the present invention, One aspect of the present invention provides a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1.

[0012] Another aspect of the present invention provides a composition for improving skin conditions, which comprises the peptide as an active ingredient.

[0013] The present invention is described in detail below.

[0014] Peptides and their activity According to one aspect of the present invention, there is provided a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1.

[0015] [Amino acid sequence of SEQ ID NO: 1] EIVRKKPI

[0016] As used herein, the term "peptide" refers to a linear molecule formed by amino acid residues linked to each other via peptide bonds.

[0017] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention can be used without modification, but variants or fragments of amino acids having different sequences resulting from deletion, insertion, substitution of amino acid residues, or a combination thereof may be used within a range that does not affect the intrinsic activity of the peptide, for example, the skin condition improving activity.

[0018] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., within a range that does not alter the activity thereof.

[0019] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to a peptide comprising the amino acid sequence of SEQ ID NO: 1, a variant thereof, or an active fragment thereof. The said substantially identical amino acid sequence means an amino acid sequence having at least 75%, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1, respectively. In addition, the peptide may additionally comprise a targeting sequence, a tag, a labeling residue, and an amino acid sequence produced for a specific purpose to increase half-life or peptide stability.

[0020] The peptide of the present invention may be one in which N-terminal and / or C-terminal modifications are induced in order to select a partial site of the amino acid sequence and increase the activity thereof. Through such N-terminal and / or C-terminal modifications, the stability of the peptide of the present invention can be remarkably improved, for example, the half-life of the peptide when administered in vivo can be increased. The term "stability" is meant to include not only in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo, but also storage stability (e.g., room temperature storage stability).

[0021] The N-terminal modification may involve the attachment of 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) to the N-terminus of the peptide. The C-terminal modification may also involve the attachment of a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), etc., to the C-terminus of the peptide, and is not limited to these.

[0022] The peptides of the present invention can be produced by various methods widely known in the art to which the present invention pertains. For example, the peptides of the present invention can be produced according to chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis techniques (U.S. Patent No. 5,516,891).

[0023] The peptide of the present invention has the activity to improve skin condition.

[0024] In one embodiment, the peptide of the present invention has the following activity related to the improvement of skin condition: (i) Increase the gene expression levels of collagen Iα1, fibronectin, and elastin, which are components of the extracellular matrix. (ii) Increase the expression of procollagen Iα1 and hyaluronic acid, which are components of the extracellular matrix. (iii) Increase the expression levels of the SIRT1 (Sirtuin 1) gene and the AQP3 (Aquaporin 3) gene, which are skin barrier strengthening factors. (iv) Reduce the level of reactive oxygen species increased by ultraviolet irradiation. (v) Increase the gene expression levels of collagen Iα1, fibronectin, and elastin, which are components of the extracellular matrix that have been reduced by UV irradiation. (vi) Increase the expression levels of skin barrier strengthening factors that are reduced by UV irradiation, such as SIRT1, AQP3, COL17A1, ITGB1, ITGB4, ITGA6, and Plectin. (vii) Reduce the expression level of MMP-1 that has increased due to UV irradiation and heat shock.

[0025] Composition for improving skin condition According to another aspect of the present invention, a composition for improving skin condition is provided, comprising a peptide containing the amino acid sequence disclosed in Sequence ID No. 1 as an active ingredient.

[0026] The peptide of the present invention, having the activity described above, exhibits excellent efficacy in improving skin condition and is used for skin condition improvement applications.

[0027] In one embodiment, the improvement of skin condition in the skin condition improving composition may be improvement of skin aging, promotion of skin regeneration, improvement of skin elasticity, improvement of skin wrinkles, or improvement of the skin barrier.

[0028] In one embodiment, the skin condition improving composition of the present invention has one or more of the following activities: (i) Increase the expression levels of the genes for collagen Iα1, fibronectin, and elastin, which are components of the extracellular matrix. (ii) Increase the expression of procollagen Iα1 and hyaluronic acid, which are components of the extracellular matrix. (iii) Increase the expression levels of the SIRT1 (Sirtuin 1) gene and the AQP3 (Aquaporin 3) gene, which are skin barrier strengthening factors. (iv) Reduce the level of reactive oxygen species increased by ultraviolet (UV) irradiation. (v) Increase the gene expression levels of collagen Iα1, fibronectin, and elastin, which are components of the extracellular matrix that have been reduced by UV irradiation. (vi) Increase the expression levels of skin barrier strengthening factors that are reduced by UV irradiation, such as SIRT1, AQP3, COL17A1, ITGB1, ITGB4, ITGA6, and Plectin. (vii) Reduce the expression level of MMP-1 that has increased due to UV irradiation and heat shock.

[0029] The skin condition improving composition of the present invention may contain the peptide of the present invention at a concentration of 0.01 μM to 1000 μM. Specifically, the peptide of the present invention may be present in concentrations of 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 6 It may be contained in concentrations of 00 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1 μM to 200 μM; 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM, but is not limited to these.

[0030] In one example, the above-mentioned composition for improving skin condition may be a cosmetic composition.

[0031] The cosmetic composition may be manufactured in any dosage form commonly produced in the art to which the present invention belongs, and may be a topical skin preparation. For example, it can be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto.

[0032] The aforementioned cosmetic composition can be manufactured in various forms such as solutions, sol-gels, emulsions, oils, waxes, and aerosols, including softening lotions, nourishing lotions, nourishing creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, packs, sprays, powders, hair tonics, hair creams, hair lotions, hair shampoos, hair rinses, hair conditioners, hair sprays, hair aerosols, pomades, and gels. However, it is not limited to these forms.

[0033] The cosmetic composition of the present invention may contain other additives such as excipients and carriers, and may also incorporate common ingredients used in general skin cosmetics as needed.

[0034] If the dosage form of the cosmetic composition is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide can be used as the carrier component.

[0035] If the dosage form of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, propellants such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included, but are not limited thereto.

[0036] If the dosage form of the cosmetic composition is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as the carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan may be used.

[0037] If the dosage form of the cosmetic composition is a suspension, the carrier component may be 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, or microcrystalline cellulose, aluminum methhydroxyl, bentonite, agar, or tracant.

[0038] When the dosage form of the cosmetic composition is a surfactant-containing cleanser, the carrier component may be an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinate monoester, isethionate, imidazolinium derivative, methyltauric acid, sarcosinic acid, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

[0039] If the dosage form of the cosmetic composition is a hair shampoo, base components for forming the shampoo, such as a thickener, surfactant, viscosity modifier, humectant, pH adjuster, preservative, and essential oil, may be mixed in. CDE may be used as the thickener, LES, an anionic surfactant, and cocobetaine, an amphoteric surfactant, as the surfactant, polyquaternium may be used as the viscosity modifier, glycerin may be used as the humectant, and citric acid and sodium hydroxide may be used as the pH adjuster. Pomelo extract may be used as the preservative, and essential oils such as cedarwood, peppermint, and rosemary, as well as silk amino acids, pentanol, or vitamin E may also be added.

[0040] The components included in the cosmetic composition may, in addition to the peptide and carrier component of the present invention as active ingredients, further include, but are not limited to, components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0041] Another aspect of the present invention provides a functional food composition for improving skin condition, comprising a peptide containing the amino acid sequence disclosed in Sequence ID No. 1 as an active ingredient.

[0042] The aforementioned improvement in skin condition includes improvement of skin aging, promotion of skin regeneration, improvement of skin elasticity, improvement of skin wrinkles, or improvement of the skin barrier.

[0043] In the functional food composition of the present invention, the activity related to improving skin condition is the same as that described in the above-mentioned composition for improving skin condition, so we will not use that information to provide a redundant explanation.

[0044] In the functional food composition of the present invention, the peptide, which is the active ingredient, may be included in an amount of 10% by weight or less of the total weight of the composition, specifically in an appropriate amount selected within the range of 0.000001% by weight to 10% by weight.

[0045] The functional food composition of the present invention may contain a food-grade effective amount of peptide and a food-grade acceptable carrier.

[0046] The functional food composition of the present invention contains not only peptides as active ingredients, but also ingredients that are normally added during food production, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of carbohydrates include monosaccharides, such as glucose and fructose; disaccharides, such as maltose, sucrose, and oligosaccharides; and polysaccharides, such as common sugars like dextrin and cyclodextrin, and sugar alcohols like xylitol, sorbitol, and erythritol. As flavorings, natural flavorings, thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (saccharin, aspartame, etc.) can be used.

[0047] In addition to the components described above, the functional food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and enhancers (for cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, it may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. For example, when the functional food composition of the present invention is manufactured as a drink, in addition to the peptide which is the active ingredient of the present invention, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, and the like.

[0048] There are no particular restrictions on the types of functional foods mentioned above. Examples of functional foods and foods include meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen noodles, other noodle products, gums, dairy products including ice cream, various soups, drinking water, tea drinks, alcoholic beverages and vitamin complexes, dairy products, fermented milk, etc., and can include all functional foods and foods in the usual sense.

[0049] According to another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of skin aging is provided, comprising a peptide containing the amino acid sequence disclosed in Sequence ID No. 1 as an active ingredient.

[0050] In one specific example, the skin aging may be photoaging of the skin.

[0051] In one example, the photoaging of the skin may be photoaging caused by ultraviolet light.

[0052] In one example, the photoaging of the skin may be photoaging caused by infrared radiation.

[0053] In one embodiment, the pharmaceutical composition for the prevention or treatment of skin aging of the present invention, or the peptide containing the amino acid sequence of SEQ ID NO: 1 contained in the composition, has one or more of the following activities: (i) Increase the expression levels of the genes for collagen Iα1, fibronectin, and elastin, which are components of the extracellular matrix. (ii) Increase the expression of procollagen Iα1 and hyaluronic acid, which are components of the extracellular matrix. (iii) Increase the expression levels of the SIRT1 (Sirtuin 1) gene and the AQP3 (Aquaporin 3) gene, which are skin barrier strengthening factors. (iv) Reduce the level of reactive oxygen species increased by ultraviolet (UV) irradiation. (v) Increase the expression levels of collagen Iα1, fibronectin, and elastin genes, which are components of the extracellular matrix that have been reduced by UV irradiation. (vi) Increase the expression levels of skin barrier strengthening factors that are reduced by UV irradiation, such as SIRT1, AQP3, COL17A1, ITGB1, ITGB4, ITGA6, and Plectin. (vii) Reduce the expression level of MMP-1 that has been increased by ultraviolet irradiation and heat shock (infrared irradiation).

[0054] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of the peptide comprising the amino acid of SEQ ID NO: 1 of the present invention.

[0055] The term "therapeutically effective amount" means an amount sufficient to achieve the activity or efficacy of the peptide, which is the active ingredient of the pharmaceutical composition of the present invention, for example, an amount sufficient to achieve the efficacy of treating or preventing skin aging as described above.

[0056] In this specification, the term “prevention” means reducing the risk of contracting a disease or disorder, and includes all actions that suppress or delay the onset of a disease by preventing the progression of the disease or one or more of its clinical signs.

[0057] In this specification, the term “treatment” means alleviating a disease or disorder, and includes all actions that prevent or reduce the progression of a disease or one or more of its clinical signs, or improve or beneficially alter the symptoms of a disease.

[0058] The pharmaceutical compositions of the present invention may contain a pharmaceutically acceptable carrier.

[0059] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil.

[0060] The pharmaceutical composition of the present invention may further contain, but is not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above-mentioned components.

[0061] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, (21st ed., 2005, Lippincott Williams & Wilkins).

[0062] The pharmaceutical composition of the present invention may be administered by any appropriate route for treating skin aging, for example, orally or parenterally. In the case of parenteral administration, it may be administered by intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, local administration, transdermal administration, or topical application to the skin.

[0063] The dosage of the pharmaceutical composition may be 0.0001 μg to 1000 mg, 0.001 μg to 1000 mg, 0.01 μg to 1000 mg, 0.1 μg to 1000 mg, 0.1 μg to 500 mg, or 1.0 μg to 1000 mg per day, but is not limited thereto. It may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, administration route, excretion rate, and response sensitivity.

[0064] The pharmaceutical compositions of the present invention can be manufactured in unit volume form or in multi-volume containers by formulation using pharmaceutically acceptable carriers and / or excipients in a manner readily available to a person with ordinary skill in the art to which the invention pertains. The dosage form may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may further contain a dispersant or stabilizer.

[0065] The pharmaceutical composition of the present invention may contain the peptide of the present invention at a concentration of 0.01 μM to 1000 mM. Specifically, the peptide of the present invention may be present at concentrations of 0.01 μM to 900 mM, 0.01 μM to 500 mM, 0.01 μM to 300 mM, 0.01 μM to 100 mM, 0.01 μM to 50 mM, 0.01 μM to 30 mM, 0.01 μM to 10 mM, 0.01 μM to 1 mM, 0.01 μM to 0.1 mM, 0.01 μM to 0.01 mM, 1.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM. It may be included in concentrations of M, 0.05 μM to 300 μM, 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1 μM to 200 μM; 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM, but is not limited to these.

[0066] Applications of peptides Another aspect of the present invention provides applications for using a peptide comprising the amino acid sequence disclosed in Sequence ID No. 1 to improve skin conditions.

[0067] Another aspect of the present invention provides an application for using a peptide comprising the amino acid sequence disclosed in Sequence ID No. 1 for the manufacture of cosmetics or functional foods for improving skin conditions.

[0068] Another aspect of the present invention provides a method for improving a skin condition, comprising the step of administering a peptide containing the amino acid sequence disclosed in Sequence ID No. 1 or a composition containing the peptide as an active ingredient to a patient or subject in need of improvement of their skin condition.

[0069] As used herein, the terms “patient” or “subject” refer to humans or non-human animals, such as humans, primates, mammals, and vertebrates. Another aspect of the present invention provides applications for using peptides comprising the amino acid sequence disclosed in Sequence ID No. 1 for the treatment or prevention of skin aging.

[0070] Another aspect of the present invention provides an application for using a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 for the manufacture of a pharmaceutical for treating or preventing skin aging. Another aspect of the present invention provides a method for treating skin aging, comprising the step of administering a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 to a patient or subject in need of treatment for skin aging.

[0071] As used herein, the terms “patient” or “subject” refer to humans or other animals, such as humans, primates, mammals, and vertebrates.

[0072] The aforementioned uses, methods for improving skin conditions, and methods for treating skin aging may be applied in the same manner to the peptides of the present invention, their activity, and compositions described above, and these descriptions will not be repeated to avoid excessive complexity in the specification. [Effects of the Invention]

[0073] The peptide of the present invention has skin condition improving activity. The peptide of the present invention increases the expression levels of extracellular matrix components and skin barrier strengthening factors, decreases the expression of skin cell damage factors that increased due to UV irradiation, and restores the expression levels of extracellular matrix components and skin barrier strengthening factors that decreased due to UV irradiation or heat shock.

[0074] However, the effects of the present invention are not limited to those mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description. [Brief explanation of the drawing]

[0075] [Figure 1a] The peptide of the present invention increases the mRNA expression levels of the genes for collagen Iα1 (COLA1), fibronectin (FN1), and elastin (ELN) in NIH3T3 cells. [Figure 1b] The peptide of the present invention increases the mRNA expression levels of the genes for collagen Iα1 (COLA1), fibronectin (FN1), and elastin (ELN) in NIH3T3 cells. [Figure 2] The present invention demonstrates that the peptide increases the secretion of pro-collagen Iα1 and hyaluronic acid in NIH3T3 cells and HaCaT cells. [Figure 3] The peptide of the present invention increases the mRNA expression levels of the SIRT1 (Sirtuin 1) gene and the AQP3 (Aquaporin 3) gene in HaCaT cells. [Figure 4a] The peptide of the present invention demonstrates that it reduces the intracellular ROS (reactive oxygen species) levels that were increased by UV irradiation in NIH3T3 cells and HaCaT cells. [Figure 4b]The peptide of the present invention demonstrates that it reduces the intracellular ROS (reactive oxygen species) levels that were increased by UV irradiation in NIH3T3 cells and HaCaT cells. [Figure 5a] The peptide of the present invention increases the mRNA expression levels of collagen Iα1 (COLA1), fibronectin (FN1), and elastin (ELN) genes, which were reduced by UV irradiation, in NIH3T3 cells. [Figure 5b] The peptide of the present invention increases the mRNA expression levels of collagen Iα1 (COLA1), fibronectin (FN1), and elastin (ELN) genes, which were reduced by UV irradiation, in NIH3T3 cells. [Figure 6a] This invention demonstrates that the peptide increases the mRNA levels of SIRT1 and AQP3, which were reduced by UV irradiation, in HaCaT cells. [Figure 6b] This invention demonstrates that the peptide increases the mRNA levels of SIRT1 and AQP3, which were reduced by UV irradiation, in HaCaT cells. [Figure 6c] The peptide of the present invention increases the mRNA levels of COL17A1 (Collagen XVIIα1), ITGB1 (Integrin β1), ITGB4 (Integrin β4), ITGA6 (Integrin α6), and Plectin in HaCaT cells, which were reduced by UV irradiation. [Figure 6d] The peptide of the present invention increases the mRNA levels of COL17A1 (Collagen XVIIα1), ITGB1 (Integrin β1), ITGB4 (Integrin β4), ITGA6 (Integrin α6), and Plectin in HaCaT cells, which were reduced by UV irradiation. [Figure 7a]The peptide of the present invention demonstrates that it reduces the expression of MMP-1, which was increased by UV irradiation, in NIH3T3 cells and HaCaT cells. [Figure 7b] The peptide of the present invention demonstrates that it reduces the expression of MMP-1, which was increased by UV irradiation, in NIH3T3 cells and HaCaT cells. [Figure 8] The present invention demonstrates that the peptide reduces the expression of MMP-1, which was increased by heat shock, in NIH3T3 cells. [Modes for carrying out the invention]

[0076] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0077] Manufacturing Example 1: Peptide Production Using an automated peptide synthesizer (Milligen 9050, Millipore, USA), peptides having the amino acid sequence of SEQ ID NO: 1, as described in Table 1, were synthesized. These synthesized peptides were then separated into pure form using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). An ACQUITY UPLC BEH300C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA) was used.

[0078] [Table 1]

[0079] The efficacy of the peptide of Sequence ID No. 1, which was manufactured as described above, was evaluated through the following experiments.

[0080] Experimental Example 1: Expression analysis of genes that make up the extracellular matrix The effect of the peptides produced in Production Example 1 on the expression of components of the extracellular matrix (ECM) was evaluated by measuring the mRNA expression levels of the genes of each component.

[0081] NIH3T3 cells (mouse fibroblast cell line) 3 × 10 5 Cells were seeded into 6-well plates at a cell / well density and cultured for 24 hours. The cultured cells were washed once with serum-free DMEM medium. Peptides prepared in Preparation Example 1 were mixed with serum-free DMEM medium at different concentrations to prepare 10 μM, 50 μM, and 100 μM peptide medium solutions, which were then added to the cells. The cells were cultured for 24 hours in a 37°C CO2 incubator, washed twice with PBS, and then RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). The amount of RNA was quantified, and 1000 ng of RNA was added per tube. After that, cDNA synthesis was performed using an RT kit (enzynomics, Cat. No.: RT200, Korea), and then PCR was performed using a PCR kit (enzynomics, Cat. No.: P581T, Korea). The primers used for PCR are shown in Table 2 below.

[0082] The experimental results confirmed that in NIH3T3 cells, the peptide of Sequence ID No. 1 increased the mRNA expression levels of collagen Iα1 (collagen, type I, alpha 1), fibronectin, and elastin, which are components of the extracellular matrix (ECM) (Figure 1).

[0083] Experimental Example 2: Expression Analysis of Protein Components of the Extracellular Matrix The effect of the peptide produced in Production Example 1 on the expression of components of the extracellular matrix (ECM) was evaluated by measuring the expression levels of Collagen Iα1 and HA (Hyaluronic acid).

[0084] NIH3T3 cells (mouse fibroblast cell line) and HaCaT cells (human keratinocyte cell line) were divided into 1 × 10⁻¹⁶ cells. 4 Cells were seeded into 24-well plates at a cell / well density and cultured for 24 hours. The cultured cells were washed once with serum-free DMEM medium. Peptides prepared in Preparation Example 1 were mixed with serum-free DMEM medium at different concentrations to prepare 10 μM, 50 μM, and 100 μM peptide medium solutions, which were then added to the cells. The cells were cultured for 72 hours in a 37°C CO2 incubator. The NIH3T3 cell culture medium and HaCaT cell culture medium were centrifuged to precipitate cells and impurities, and only the supernatant of the culture medium was collected and transferred to an Eppendorf tube. The obtained culture medium was used for Pro-Collagen ELISA (abcam, Cat No.: ab210579, UK) and HA (Hyaluronic acid) ELISA (ECHELON biosciences, Cat No.: K-1200, USA).

[0085] The experimental results confirmed that in NIH3T3 cells, the peptide of Sequence ID No. 1 increased the secretion of pro-collagen Iα1, one of the components of the extracellular matrix (Figure 2). Furthermore, in HaCaT cells, the peptide of Sequence ID No. 1 increased the secretion of hyaluronic acid (HA), another component of the extracellular matrix (Figure 2).

[0086] Experiment Example 3: Expression Analysis of Skin Barrier-Related Genes We evaluated the effect of the peptide produced in Production Example 1 on the expression of genes related to the skin barrier.

[0087] HaCaT cells (human keratinocyte cell line) 3 × 10 5Cells were seeded into 6-well plates at a cell / well density and cultured for 24 hours. The cultured cells were washed once with serum-free DMEM medium. Peptides prepared in Preparation Example 1 were mixed with serum-free DMEM medium at different concentrations to prepare 10 μM, 50 μM, and 100 μM peptide medium solutions, which were then added to the cells. The cells were cultured for 24 hours in a 37°C CO2 incubator, washed twice with PBS, and then RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). After quantifying the amount of RNA and adding 1000 ng of RNA per tube, cDNA synthesis was performed using an RT kit (enzynomics, Cat. No.: RT200, Korea), followed by PCR using a PCR kit (enzynomics, Cat. No.: P581T, Korea). The primers used for PCR are shown in Table 2.

[0088] SIRT1 (Sirtuin 1) and AQP3 (Aquaporin 3) are known factors that influence the maintenance of the skin barrier by regulating aging and moisture retention in epidermal cells. Experimental results confirmed that the peptide of Sequence ID No. 1 increases the mRNA expression levels of the SIRT1 (Sirtuin 1) and AQP3 (Aquaporin 3) genes, which are skin barrier strengthening factors, in HaCaT cells (Figure 3).

[0089] Experimental Example 4: Suppression of UV-induced reactive oxygen species (ROS) generation We evaluated whether the peptide produced in Production Example 1 suppresses the generation of UV-induced reactive oxygen species (ROS).

[0090] NIH3T3 cells (mouse fibroblast cell line) and HaCaT cells (human keratinocyte cell line) were divided into 5 × 10⁻¹⁰ cells. 5Cells were seeded into 6-well plates at a cell / well density and cultured for 24 hours. The cultured cells were washed once with serum-free DMEM medium. The peptides prepared in Preparation Example 1 were mixed with serum-free DMEM medium at different concentrations to prepare 10 μM, 50 μM, and 100 μM peptide medium solutions, which were then added to the cells. The peptide-added cells were cultured for 1 hour in a 37°C CO2 incubator. After culturing, the medium was removed from the cells and PBS was added. Using a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France), NIH3T3 cells were exposed to UVA at 6 J / cm². 2 For HaCaT cells, UVB is applied at 15 mJ / cm². 2 Irradiation was performed. After removing PBS, the cells were again given the peptide medium solutions of the respective concentrations. The peptide-treated cells were cultured for 24 hours in a 37°C CO2 incubator. After being treated with 10 μM DCFH-DA (2',7'-dichlorofluorescein diacetate), the cells were covered with foil and cultured for 30 minutes in a 37°C CO2 incubator. Subsequently, the cells were washed twice with PBS, harvested, and their fluorescence values ​​were measured using FACS.

[0091] The experimental results confirmed that the peptide with SEQ ID NO: 1 reduced intracellular ROS levels that had increased due to UV irradiation in NIH3T3 cells (Figure 4a). Furthermore, it was confirmed that the peptide with SEQ ID NO: 1 again reduced intracellular ROS levels that had increased due to UV irradiation in HaCaT cells (Figure 4b).

[0092] Experimental Example 5: Recovery of extracellular matrix gene expression reduced by UV irradiation We evaluated whether the peptide of Sequence ID No. 1, produced in Production Example 1, could restore the expression levels of extracellular matrix genes that had decreased due to UV irradiation.

[0093] NIH3T3 cells (mouse fibroblast cell line) 5 × 10 5Cells were seeded into 6-well plates at a cell / well density and cultured for 24 hours. The cultured cells were washed once with serum-free DMEM medium. The peptides prepared in Preparation Example 1 were mixed with serum-free DMEM medium at different concentrations to prepare 10 μM, 50 μM, and 100 μM peptide medium solutions, which were then added to the cells. The cells with added peptides were cultured for 1 hour in a 37°C CO2 incubator. After culturing the cells, the medium was removed from the cells and PBS was added. UVA was applied at 8 J / cm using a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France). 2 The cells were irradiated. After removing PBS, the peptide medium solutions of the respective concentrations were added to the cells again. The cells with added peptides were cultured for 6 hours in a 37°C CO2 incubator. After washing twice with PBS, RNA was separated using easy blue (iNtRON, Cat. No.: 17061, Korea). The amount of RNA was quantified, and 1000 ng of RNA was added per tube, followed by cDNA synthesis using an RT kit (enzynomics, Cat. No.: RT200, Korea). Subsequently, PCR was performed using a PCR kit (enzynomics, Cat. No.: P581T, Korea). The primers used for PCR are shown in Table 2.

[0094] The experiment showed that UV irradiation in NIH3T3 cells reduced the mRNA expression levels of the collagen Iα1, fibronectin, and elastin genes. However, treatment with the peptide of Sequence ID No. 1 increased the expression levels of these genes again (Figures 5a and 5b).

[0095] Experimental Example 6: Restoration of skin barrier-related gene expression reduced by UV irradiation. We evaluated whether the peptide of Sequence ID No. 1, produced in Production Example 1, could restore the expression levels of skin barrier-related genes that had decreased due to UV irradiation.

[0096] HaCaT cells (human keratinocyte cell line) 5 × 105 Cells were seeded in a 6-well plate at a density of cells / well and cultured for 24 hours. The cultured cells were washed once with serum-free DMEM medium. The peptide prepared in Preparation Example 1 was mixed into serum-free DMEM medium at different concentrations to prepare peptide medium solutions of 10 μM, 50 μM and 100 μM, which were then added to the cells. The peptide-treated cells were cultured for 1 hour in a CO2 incubator at 37°C. After culturing the cells, the medium was removed from the cells, and PBS was added. UVB was applied at 20 mJ / cm using a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France) 2 for irradiation. After removing PBS, the peptide medium solutions of each aforementioned concentration were added again to the cells. The peptide-treated cells were cultured for 4 hours in a CO2 incubator at 37°C. After washing twice with PBS, RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). The amount of RNA was quantified, after adding 1000 ng of RNA per tube, cDNA synthesis was performed using an RT kit (enzynomics, Cat. No.: RT200, Korea). Subsequently, PCR was performed using a PCR kit (enzynomics, Cat. No.: P581T, Korea). The primers used for PCR are shown in Table 2.

[0097] SIRT1 (Sirtuin 1) and AQP3 (Aquaporin 3) are known as factors that affect the maintenance of the skin barrier through the regulation of cell aging and moisturization in the epidermis. The experimental results showed that in HaCaT cells, the mRNA levels of SIRT1 and AQP3 were decreased by UV irradiation, but treatment with the peptide of SEQ ID NO: 1 restored the decreased mRNA expression levels of these genes (Figure 6a and Figure 6b).

[0098] Hemidesmosomes are regions through which keratinocytes in the epidermal basal layer attach to the extracellular matrix (ECM) of the dermis, thereby maintaining the density of the skin barrier. Experimental results showed that in HaCaT cells, UV irradiation reduced the mRNA levels of hemidesmosome components COL17A1 (Collagen XVIIα1), ITGB1 (Integrin β1), ITGB4 (Integrin β4), ITGA6 (Integrin α6), and Plectin. Treatment with the peptide of Sequence ID No. 1 restored the mRNA expression levels of these genes that had been reduced (Figures 6c and 6d).

[0099] Experimental Example 7: Suppression of UV-induced MMP-1 expression The effect of the peptide of Sequence ID No. 1, produced in Production Example 1, on UV-induced MMP-1 expression was evaluated.

[0100] NIH3T3 cells (mouse fibroblast cell line) and HaCaT cells (human keratinocyte cell line) were divided into 5 × 10⁻¹⁰ cells. 5 Cells were seeded into 6-well plates at a cell / well density and cultured for 24 hours. The cultured cells were washed once with serum-free DMEM medium. The peptides prepared in Preparation Example 1 were mixed with serum-free DMEM medium at different concentrations to prepare 10 μM, 50 μM, and 100 μM peptide medium solutions, which were then added to the cells. The cells with added peptides were cultured for 1 hour in a 37°C CO2 incubator. After culturing the cells, the medium was removed from the cells and PBS was added. Using a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France), NIH3T3 cells were exposed to UVA at 6 J / cm². 2 The cells were irradiated with UVB at a rate of 15 mJ / cm². 2The cells were irradiated. After removing PBS, the peptide medium solutions of the respective concentrations were added to the cells again. The peptide-added cells were cultured for 24 hours in a 37°C CO2 incubator. After washing twice with PBS, the cells were lysed with cell lysis buffer. After processing with 5X sample buffer, SDS-PAGE was performed using a 10% SDS-PAGE gel. The proteins separated via SDS-PAGE were transferred to a PVDF membrane. The cells were blocked at room temperature for 1 hour using 5% skim milk. Anti-MMP-1 antibody (Abcam, Cat No.: ab137332, UK) was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. The loading control group was reacted in the same manner using anti-α-tubulin antibody (Santa Cruz Biotechnology, Cat No.: sc69969, USA). The membranes were washed three times for 10 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS). Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson ImmunoResearch, Cat. No.: 111-035-003, USA) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 1 hour. The loading control group, α-tubulin, was treated with the same product and reacted in the same manner. After treatment with ECL solution (GE Healthcare, Cat. No.: RPN2232, USA), the expression levels were analyzed using an ImageQuant 800 instrument (Cytiva, Cat. No.: 29-3994-81, USA).

[0101] The experimental results showed that in NIH3T3 cells, UV irradiation increased MMP-1 expression, and the peptide with SEQ ID NO: 1 suppressed the UV-induced increase in MMP-1 expression (Figure 7a). Furthermore, in HaCaT cells, UV irradiation increased MMP-1 expression, and the peptide with SEQ ID NO: 1 suppressed the UV-induced increase in MMP-1 expression (Figure 7b).

[0102] Experimental Example 8: Suppression of thermally induced MMP-1 expression The effect of the peptide of Sequence ID No. 1, produced in Production Example 1, on heat-induced MMP-1 expression was evaluated.

[0103] NIH3T3 cells (mouse fibroblast cell line) 5 × 10 5Cells were seeded into a 6-well plate at a cell / well density and cultured for 24 hours. The cultured cells were washed once with serum-free DMEM medium. Peptides prepared in Preparation Example 1 were mixed with serum-free DMEM medium at different concentrations to prepare 10 μM, 50 μM, and 100 μM peptide medium solutions, which were then added to the cells. Cells with added peptides were cultured for 1 hour in a 37°C CO2 incubator. After sealing the lid and lower plate of the 6-well plate with paraffin film, the lower plate was immersed in a 44°C water bath and cultured for 30 minutes. After removing the existing medium, the culture medium solutions containing the peptides at the aforementioned concentrations were added again, and the cells were cultured for 8 hours in a 37°C CO2 incubator. After washing the cells twice with PBS, the cells were lysed with cell lysis buffer. After processing with 5X sample buffer, SDS-PAGE was performed using a 10% SDS-PAGE gel. Proteins isolated via SDS-PAGE were transferred to a PVDF membrane. Blocking was performed at room temperature for 1 hour using 5% skim milk. Anti-MMP-1 antibody (Abcam, Cat No.: ab137332, UK) was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. The loading control group was reacted in the same manner using anti-α-tubulin antibody (Santa Cruz Biotechnology, Cat No.: sc69969, USA). Washing was performed three times for 10 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS). Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson ImmunoResearch, Cat No.: 111-035-003, USA) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 1 hour. The secondary antibody against α-tubulin, which served as the loading control, was also prepared using the same product and reacted in the same manner.After processing with ECL solution (GE Healthcare, Cat. No.: RPN2232, USA), the expression levels were analyzed using an ImageQuant 800 (Cytiva, Cat. No.: 29-3994-81, USA) instrument.

[0104] The experimental results showed that heat shock increased MMP-1 expression in NIH3T3 cells, and the peptide of Sequence ID No. 1 suppressed the heat shock-induced increase in MMP-1 expression (Figure 8).

[0105] [Table 2-1] [Table 2-2]

[0106] While the above has provided illustrative examples of typical embodiments of this application, the scope of this application is not limited to such specific embodiments, and can be appropriately modified within the scope of the claims of this application by anyone with ordinary skill in the relevant art.

Claims

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

1.

2. A composition for improving skin condition, comprising the peptide described in claim 1 as an active ingredient.

3. The skin condition improving composition according to claim 2, wherein the improvement of the skin condition is improvement of skin aging, promotion of skin regeneration, improvement of skin elasticity, improvement of skin wrinkles, or improvement of the skin barrier.

4. The composition for improving skin condition according to claim 2, wherein the composition has skin condition improving activity through one or more of the following activities: (i) Activity that increases the expression levels of collagen Iα1, fibronectin, and elastin genes; (ii) Activity that increases the expression of procollagen Iα1 and hyaluronic acid; (iii) Activity that increases the expression levels of the SIRT1 (Sirtuin 1) gene and the AQP3 (Aquaporin 3) gene, which are skin barrier strengthening factors; (iv) Activity to reduce the level of reactive oxygen species increased by ultraviolet (UV) irradiation; (v) Activity that increases the expression levels of collagen Iα1, fibronectin, and elastin genes that have been reduced by UV irradiation; (vi) Activity that increases the expression levels of skin barrier strengthening factors SIRT1, AQP3, COL17A1, ITGB1, ITGB4, ITGA6, and Plectin, which are reduced by UV irradiation; and (vii) Activity to reduce the expression level of MMP-1 increased by UV irradiation and heat shock.

5. The composition for improving skin condition according to claim 2, wherein the composition is a cosmetic composition.

6. The skin condition improving composition according to claim 5, wherein the cosmetic composition is one dosage form selected from the group consisting of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, and spray.

7. A functional food composition for improving skin condition, containing a peptide with the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

8. The functional food composition for improving skin condition according to claim 7, wherein the improvement of skin condition is improvement of skin aging, promotion of skin regeneration, improvement of skin elasticity, improvement of skin wrinkles, or improvement of the skin barrier.

9. A pharmaceutical composition for the prevention or treatment of skin aging, comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

10. The pharmaceutical composition for preventing or treating skin aging according to claim 9, wherein the skin aging is photoaging of the skin.

11. The pharmaceutical composition for preventing or treating skin aging according to claim 10, wherein the aforementioned photoaging of the skin is photoaging caused by ultraviolet light.

12. The pharmaceutical composition for preventing or treating skin aging according to claim 10, wherein the aforementioned photoaging of the skin is photoaging caused by infrared radiation.

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