AMPK-activating peptide and uses thereof

Abstract

The present invention relates to a novel AMPK-activating peptide and uses thereof. The novel peptide of the present invention exhibits excellent anti-aging, antioxidant, and anti-inflammatory effects, and can reduce wrinkles by promoting collagen synthesis. Accordingly, the peptide is highly applicable not only to a cosmetic composition and a food composition, but also to a pharmaceutical composition for treating / alleviating AMPK-related diseases.

Description

AMPK active peptide and its uses

[0001] The present invention relates to a novel AMPK-active peptide and its uses.

[0002] The skin is an organ continuously exposed to external stimuli (ultraviolet rays, fine dust, changes in temperature or humidity, microorganisms, mechanical friction, etc.), and the resulting inflammation, oxidative stress, impaired barrier function, and dermal matrix imbalance (changes in collagen / elastin) lead to various skin diseases and cosmetic problems.

[0003] Currently, widely used treatments include topical steroids, calcineurin inhibitors, retinoids, antibiotics, keratolytics, and moisturizers; however, steroids present safety issues such as skin atrophy and telangiectasia with long-term use, while calcineurin inhibitors frequently cause burning and irritation, and retinoids often lead to skin compatibility problems such as dryness, erythema, and photosensitivity. Antibiotics carry limitations regarding resistance and the risk of recurrence.

[0004] Furthermore, while antioxidants, moisturizing / barrier-strengthening ingredients, and plant extracts are widely applied, it is difficult to ensure consistent efficacy on the human body due to factors such as the oxidation and instability of raw materials and insufficient skin permeability. Although immediate effects such as short-term hydration or increased radiance are present, these often dissipate over time. Additionally, significant variability in individual responses leads to a recurring limitation where individual perceived efficacy is lower than the average.

[0005] Furthermore, in modern living environments, factors such as imbalances in sleep and dietary habits and stress act simultaneously to manifest complex conditions including inflammation, oxidative stress, and abnormalities in sebum or keratinization; consequently, there are limitations in improving such multiple pathological conditions through a single-efficacy-focused approach.

[0006] Accordingly, there is a continuous demand for the development of cosmetic ingredients capable of improving multi-target indicators.

[0007] The objective of the present invention is to provide a novel peptide.

[0008] Another objective of the present invention is to provide an anti-inflammatory composition comprising the novel peptide.

[0009] Another objective of the present invention is to provide a cosmetic composition comprising the novel peptide.

[0010] Another objective of the present invention is to provide a food composition comprising the novel peptide.

[0011] Another objective of the present invention is to provide a pharmaceutical composition comprising the novel peptide.

[0012] Another objective of the present invention is to provide a method and / or use for improving skin by applying a composition containing the novel peptide to the skin or administering it to an individual, and a method and / or use for preventing or treating AMPK-related diseases.

[0013] One aspect of the present invention for achieving the above-mentioned purpose relates to a peptide composed of an amino acid sequence of any one of SEQ ID NOs 1 to 10.

[0014] Specifically, the peptide may activate AMP-activated protein kinase (AMPK).

[0015] In the present invention, 'AMP activated protein kinase (AMPK)' refers to a serine / threonine kinase composed of α-β-γ subunits that is involved in regulating the energy homeostasis of cells.

[0016] In the present invention, the activation of the AMP-activated protein kinase can be achieved by transmitting or operating a signal that phosphorylates AMPK by upstream kinases such as LKB1 or CaMKKβ (CAMKK2), or by AMP / ADP promoting the phosphorylation of AMPK under conditions of energy depletion. The AMPK thus activated can act to restore or maintain cellular energy balance, such as by reorganizing metabolic pathways like phosphorylating acetyl-CoA carboxylase (ACC), thereby promoting the inhibition and oxidation of fatty acid synthesis, inhibiting protein synthesis, or promoting glucose absorption.

[0017] In the present invention, the peptide comprises an amino acid sequence of any one of SEQ ID NOs 1 to 10, or comprises a variant that maintains the AMPK activity of the present invention while having sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more with these. Here, the 'variant' may further include chemical modifications such as amino acid substitution, insertion, deletion, acetylation, amidation, lipidation, etc.

[0018] Additionally, specifically, the peptide may have one or more activities selected from the group consisting of anti-aging, antioxidant, anti-inflammatory, and wrinkle improvement.

[0019] In the present invention, 'anti-aging' refers to preventing or slowing down the progression of aging-related metabolism, structure, or function at the cellular or tissue level, and includes preserving or restoring function by repairing cells or tissues that have fallen into an inefficient or overloaded state due to aging.

[0020] In the present invention, "antioxidant" refers to the action of inhibiting oxidation. Although the human body maintains a balance between oxidation-promoting substances and oxidation-inhibiting substances, if this balance is lost due to various factors and the system shifts toward promoting oxidation, oxidative stress is induced within the body, which can lead to cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of oxidative stress, are chemically unstable and highly reactive, easily reacting with various biomaterials such as DNA, proteins, lipids, and carbohydrates. They attack macromolecules within the body, causing irreversible damage to cells and tissues, leading to mutations, cytotoxicity, and cancer, and also serve as a direct cause of aging. By eliminating or reducing the aforementioned reactive oxygen species, an antioxidant effect can be obtained to prevent aging and maintain health.

[0021] In the present invention, "inflammation" refers to a phenomenon that occurs for a series of defensive purposes to minimize the reaction and restore the damaged area to its original state when cells or tissues are damaged by any cause. It can collectively refer to functional disorders that trigger nerve, blood vessel, lymphatic, fluid, and cellular reactions, resulting in pain, swelling, redness, fever, etc. In the present invention, "anti-inflammation" encompasses the reduction of the expression of inflammation-inducing factors and the improvement of symptoms, and includes the suppression, alleviation, and healing of inflammation.

[0022] In the present invention, "skin wrinkles" refers to fine lines formed by the deterioration of the skin, which may be caused by genetic factors, a decrease in collagen and elastin present in the dermis of the skin, or external environmental factors. In the present invention, "wrinkle improvement" refers to suppressing or inhibiting the formation of wrinkles on the skin, or alleviating wrinkles that have already formed.

[0023] Another aspect of the present invention relates to a nucleic acid encoding a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

[0024] The above 'nucleic acid encoding a peptide' refers to a polynucleotide that includes a codon corresponding to a target peptide, thereby causing the peptide to be generated or synthesized upon translation. The above nucleic acid may be DNA (genomic DNA, cDNA, synthetic DNA) or RNA (mRNA, etc.) and includes double-stranded or single-stranded forms, and circular or linear forms.

[0025] The nucleic acid encoding any one of the peptides in SEQ ID NOs 1 to 20 includes not only sequences that encode the amino acid sequence as is, but also all nucleic acids capable of generating or synthesizing the same amino acid sequence through codon substitution or codon optimization. Additionally, nucleic acids encoding modified peptides having substitutions, insertions, or deletions within a range that substantially maintains the biochemical properties of the original sequence may also be included.

[0026] The above nucleic acid may be operably linked with an expression modulation sequence and may include a promoter suitable for transcription initiation and stable expression, a 5' / 3' UTR, a translation initiation sequence, a termination codon for protein synthesis termination, and a transcription termination signal. Additionally, secretion of the generated peptide may be regulated by adding a secretion signal peptide or an organelle targeting sequence as needed, and a tag sequence may be fused for purification and detection processes.

[0027] Another aspect of the present invention relates to an anti-inflammatory composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10. “Anti-inflammatory” is as described above.

[0028] Another aspect of the present invention relates to a cosmetic composition for skin improvement or anti-inflammatory purposes comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

[0029] In the present invention, "skin improvement" comprehensively refers to the process of treating, alleviating, or mitigating skin damage caused by intrinsic or extrinsic factors of the skin, or the effects thereof.

[0030] Specifically, the skin improvement described above may be one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement, but is not limited thereto. Anti-aging, antioxidant, and wrinkle improvement are as described above.

[0031] The cosmetic composition of the present invention may be prepared in a formulation selected from the group consisting of a solution, an external ointment, a cream, a foam, a nourishing lotion, a softening lotion, a pack, a softening water, a lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing product, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray, but is not limited thereto.

[0032] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate, for example, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., as conventional ingredients, but is not limited thereto.

[0033] Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

[0034] The composition of the present invention can be used by a transdermal administration method, such as by direct application or spraying onto the skin, and the administration route of the composition of the present invention can be administered through any general route as long as it can reach the target tissue.

[0035] Another aspect of the present invention relates to a food composition for skin improvement or anti-inflammatory purposes, comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs. 1 to 10. Specifically, the skin improvement may be one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement, but is not limited thereto. Anti-aging, antioxidant, and wrinkle improvement are as described above.

[0036] There are no restrictions on the types of the above foods, and they are not limited to any products that may contain or be added to the peptide of the present invention, such as sausages, meat, bread, chocolates, snacks, candies, confectionery, ramen, pizza, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0037] Specifically, the above food composition may be a health functional food. The above health functional food is a food that emphasizes the biological regulatory function of food and is a food that has been given added value to act and manifest for a specific purpose using physical, biochemical, or biotechnological methods. The components of such health functional food are designed and processed to fully exert biological regulatory functions on the body related to biological defense, regulation of body rhythms, and prevention and recovery of diseases, and may contain food additives, sweeteners, or functional ingredients that are acceptable as food.

[0038] The above-mentioned health functional food may be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills for the purpose of improving skin and anti-inflammatory effects.

[0039] When the peptide of the present invention is used as a health functional food (or as an additive to a health functional beverage), it may be added as is, used together with other foods or food ingredients, or used appropriately according to conventional methods. The amount of the peptide can be appropriately determined according to its intended use (prevention, health or improvement, therapeutic treatment).

[0040] Additionally, specifically, the above food composition may be for inner beauty. The term "inner beauty" collectively refers to "edible cosmetics" that solve skin problems through dietary habit improvement in the form of food while serving the purpose of cosmetics, and is not limited to those that may contain, add, or mix the peptide of the present invention.

[0041] Another aspect of the present invention relates to a method for improving skin aesthetically, comprising the step of applying the peptide of the present invention to the skin. This can be administered via a transdermal method, such as by direct application or spraying onto the skin, or ingested in the form of food for inner beauty purposes. In the method for improving skin aesthetically, the administration route can be administered through any general route as long as it can reach the target tissue.

[0042] Another aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of AMPK-related diseases, comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

[0043] Specifically, the above AMPK-related diseases may be one or more selected from the group consisting of dermatitis, atopic dermatitis, psoriasis, acne, and keloid, but are not limited thereto.

[0044] In the present invention, 'dermatitis' refers to any inflammatory skin disease occurring in the epidermis and / or dermis, and typically includes symptoms such as erythema, edema, scaling, papules / vesicles, exudation, and itching. Depending on the cause or clinical course, it may include atopic dermatitis, contact dermatitis (allergic or irritant), seborrheic dermatitis, etc.

[0045] AMPK is activated through phosphorylation, and the activated AMPK downregulates NF-κB / AP-1 signaling to reduce the secretion of inflammatory cytokines such as IL-6, IL-8, and TNF-α. In addition, it can eliminate inflammation persistence factors by promoting autophagy through mTORC1 inhibition and ULK1 activation, and alleviate ROS-induced inflammation by reducing oxidative stress through Nrf2 pathway activation. The peptide of the present invention can exhibit anti-inflammatory effects through AMPK activation and can prevent, treat, and / or improve related dermatitis.

[0046] In the present invention, 'psoriasis' is a chronic, relapsing inflammatory skin disease characterized by erythematous plaques accompanied by silvery-white scales, accompanied by itching and stinging, as well as epidermal thickening and hyperkeratosis. In psoriasis, AMPK activity remains low due to reduced energy regulation and autophagy; as a result, mTORC1 signaling is upregulated and NF-κB / STAT3-mediated inflammatory responses (TNF-α, IL-6, IL-17 / IL-23, etc.)) become excessive. When AMPK is activated by phosphorylation through the peptide of the present invention, autophagy is restored through mTORC1 inhibition and ULK1 activation, thereby alleviating the overproliferation and hyperkeratosis of keratinocytes, and simultaneously, inflammatory cytokines can be reduced through the downregulation of NF-κB / STAT3.

[0047] In the present invention, 'acne' is a disease characterized by excessive sebum, follicular hyperkeratosis, and inflammation, in which inflammatory signals are heightened. The peptide of the present invention can exhibit an anti-inflammatory effect through AMPK activation, and when AMPK is activated by phosphorylation, lipid synthesis in sebaceous gland cells is lowered, which reduces sebum secretion and stabilizes the pore environment.

[0048] In the present invention, 'keloid' is a lesion resulting from excessive fibroblast proliferation, where metabolic and inflammatory regulation is disrupted, leading to a decrease in AMPK activity. When AMPK is activated by phosphorylation through the peptide of the present invention, autophagy is restored through mTORC1 inhibition and ULK1 activation, thereby removing damaged proteins and excessive extracellular matrix (ECM) and inhibiting fibroblast overproliferation, which can induce flattening of the keloid.

[0049] For administration, the pharmaceutical composition of the present invention may include, in addition to the peptide of the present invention, a pharmaceutically acceptable carrier, excipient, or diluent. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0050] In addition, the pharmaceutical composition of the present invention can be applied in any formulation, and more specifically, may be a parenteral formulation. Parenteral formulations may be injectable, topical, or spray forms such as aerosols. Even more specifically, it may be in the form of an injectable.

[0051] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0052] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Furthermore, the pharmaceutical composition of the present invention may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0053] The term "individual" in the present invention includes animals or humans having a skin pigmentation disorder whose symptoms can be improved by the administration of a pharmaceutical composition according to the present invention. By administering the therapeutic composition according to the present invention to an individual, the skin pigmentation disorder can be effectively prevented and treated.

[0054] The term "administration" in the present invention means introducing a specific substance into a human or animal by any appropriate method, and the route of administration of the therapeutic composition according to the present invention may be oral or parenteral administration through any general route as long as it can reach the target tissue, and may also be applied to the skin. Additionally, the therapeutic composition according to the present invention may be administered by any device capable of delivering the active ingredient to target cells.

[0055] The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art.

[0056] Another aspect of the present invention relates to a method for cosmetic skin improvement comprising the step of applying to the skin a composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10. The skin improvement is as described above, and specifically, the skin improvement may be one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement, but is not limited thereto.

[0057] Another aspect of the present invention relates to a method for improving skin, comprising the step of administering to an individual a composition comprising a peptide having an amino acid sequence of any one of SEQ ID NOs 1 to 10. The skin improvement is as described above, and specifically, the skin improvement may be one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement, but is not limited thereto.

[0058] Another aspect of the present invention relates to a method for preventing or treating AMPK-related diseases, comprising the step of administering to an individual a composition comprising a peptide having an amino acid sequence of any one of SEQ ID NOs 1 to 10. AMPK-related diseases, etc. are as described above, and specifically, the AMPK-related diseases may be one or more selected from the group consisting of dermatitis, atopic dermatitis, psoriasis, acne, and keloid, but are not limited thereto.

[0059] Another aspect of the present invention relates to a skin improvement use comprising the step of administering to an individual a composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10. The skin improvement is as described above, and specifically, the skin improvement may be one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement, but is not limited thereto.

[0060] Another aspect of the present invention relates to a use for the prevention or treatment of AMPK-related diseases, comprising the step of administering to an individual a composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10. AMPK-related diseases, etc. are as described above, and specifically, the AMPK-related diseases may be one or more selected from the group consisting of dermatitis, atopic dermatitis, psoriasis, acne, and keloid, but are not limited thereto.

[0061] The novel peptide of the present invention phosphorylates and activates AMPK, exhibits excellent anti-aging, antioxidant, and anti-inflammatory effects, and can improve wrinkles by promoting collagen synthesis. Accordingly, it has high utility not only in cosmetic compositions and food compositions but also in pharmaceutical compositions for the treatment or improvement of AMPK-related diseases.

[0062] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

[0063] Figure 1 shows the results confirming the AMPK activation effect of the peptide of the present invention.

[0064] Figures 2 and 3 show the results confirming the collagen synthesis-promoting effect of the peptide of the present invention.

[0065] Figures 4 and 5 show the results confirming the anti-aging effect of the peptide of the present invention.

[0066] Figures 6 and 7 show the results confirming the antioxidant effect of the peptide of the present invention.

[0067] Figures 8 and 9 show the results confirming the anti-inflammatory effect of the peptide of the present invention.

[0068] The present invention will be explained in detail below by way of examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

[0069] Example 1. Synthesis of Peptides

[0070] The synthesized peptide was synthesized using the FMOC solid-phase method with an automated synthesizer (WellPep, Incheon, South Korea). The synthesized peptide was purified and analyzed by reverse-phase high-speed liquid chromatography (reverse-phase HPLC) using a C18 analysis RP column (Shiseido capcellpak) (1260 Infinity II Agilent, CA, USA), and identified using a mass spectrometer (LC-MS / MS; LTQ Orbitrap XL, Thermo Fisher Scientific, Waltham, MA, USA).

[0071] The synthesized peptides are as shown in Table 1 below.

[0072] Sequence Number Peptide Name Amino Acid Sequence (NC)1Pep1Trp-Leu-LysWLK2Pep2Ala-Asp-Phe-GlyADFG3Pep3Gly-Leu-Ser-AsnGLSN4Pep4Asp-Gly-Glu-PheDGEF5Pep5Phe-Leu-Arg-ThrFLRT6Pep6Gly-Ser-Pro-AsnGSPN7Pep7Leu-Ser-AsnLSN8Pep8Ac-Leu-Ser-AsnAc-LSN9Pep9Cys-Leu-Ser-AsnCLSN10Pep10Cys-Asn-Ser-LeuCNSL

[0073] "Ac" in Table 1 above indicates that the N-terminus of the peptide is acetylated.

[0074] Experimental Example 1. Confirmation of AMPK activation

[0075] AMPK activation occurs through phosphorylation. The effect of the peptide of the present invention on the phosphorylation of AMPK was confirmed using the Western blotting technique.

[0076] 5 x 10 HaCaT cells (human keratinocytes) per well 6 After preparing the cells, they were treated with the peptide of Example 1 and incubated for 24 hours. The concentration of the peptide in the medium was set to 10 μM, and untreated cells were used as a control. After washing the HaCaT cells three times with PBS, they were lysed in a 1% NP40 lysis buffer (1% Nonidet P40, 0.1 M NaCl, 0.05 M tris (pH 8.0), 5 mM EDTA) containing 0.1 μM PMSF (phenylmethylsulfonyl fluoride), 1 μg / ml pepstatin A, 10 μg / ml leupeptin, 1 μg / ml aprotinin, and 1 mM Na3VO4.

[0077] After quantifying the cell lysate using the Bradford assay method, samples were prepared for electrophoresis. The immunoprecipitate was subjected to electrophoresis on a 10% polyacrylamide gel, and the separated proteins were transferred to a nitrocellulose membrane and treated with a blocking solution (Tris-buffered saline, TBS) containing 0.05% Tween 20 and 5% bovine serum albumin for about 1 hour at room temperature. Subsequently, the samples were reacted for 2 hours in TBS buffer containing anti-AMPK-p(T172) polyclonal antibody (Cell Signaling Technology, USA), followed by washing with TBS buffer containing 0.05% Tween 20. After treatment with horseradish peroxidase conjugated anti-rabbit or mouse IgG (Santa Cruz, CA, USA) for 1 hour at room temperature, the samples were washed 5 times with TBS containing 0.05% Tween 20 and developed using an antibody detection kit (Ab Frontier, Korea). To confirm that an equal amount of cell lysate was electrophoresed, actin was recognized using an anti-beta actin monoclonal antibody (Santa Cruz Biotechnology, USA). The expression level of phosphorylated AMPK (AMPK-p) relative to actin was quantified using the Image J program.

[0078] As a result, as shown in Figure 1, it was confirmed that peptides 1 to 10 of the present invention all mediate AMPK activation by inducing phosphorylation of AMPK.

[0079] Experimental Example 2. Confirmation of collagen synthesis-promoting effect

[0080] Immunofluorescence analysis was performed to confirm the collagen synthesis-promoting effect of the peptide of the present invention. Specifically, 4.5 x 10⁶ in each well of a 24-well microplate4 Human dermal fibroblasts were seeded with CEFOgro Human MSC Growth Medium and cultured for 24 hours in a 37°C, 5% CO2 incubator. Subsequently, H2O2 and the peptide of the present invention were added to the medium, respectively, and the cells were incubated under the same conditions for 24 hours. Cells in each well were washed with PBS and fixed by treatment with 4% formaldehyde for 15 minutes, followed by treatment with 0.1% TritonX-100 for 5 minutes to increase antibody permeability into the cells. Cells were washed with PBS solution and treated with the anti-COL1A2 polyclonal antibody (Santa Cruz, CA, USA) for 1 hour, followed by treatment with mouse anti-goat IgG-FITC for 40 minutes. For cell nucleus staining, cells were mounted with a reagent containing DAPI, observed using a fluorescence microscope (Logosbio / CelenaS, Korea), and quantified using the program (NIS-Eliments-3.1). The control group (control in the figure) included both H2O2 and peptide. This is the untreated group.

[0081] As a result, as shown in Figures 2 and 3, COL1A2 was reduced upon treatment with H2O2 alone ('-' in Figure 2), and when peptides 1 to 10 of the present invention were treated respectively, it was confirmed that the fluorescence intensity reduced by H2O2 was significantly restored, COL1A2 increased distinctly, and the cell morphology was maintained stably. This demonstrates that the peptides of the present invention have an effect of promoting collagen synthesis.

[0082] Experimental Example 3. Confirmation of anti-aging effect

[0083] After inducing aging in cells by treating them with H2O2, aged cells can be identified through Senescence β-Galactosidase Staining, thereby confirming the anti-aging effect of the peptide of the present invention.

[0084] Specifically, 1 x 10⁻⁶ in each well of a 24-well microplate 5 Human HDFs (HDFs) were seeded into DMEM serum medium and cultured for 24 hours in a 37°C, 5% CO2 incubator. Subsequently, H2O2 and the peptide of the present invention were added to the medium at various concentrations, followed by incubation under the same conditions for 24 hours. Cells in each well were washed with PBS, and fixation and staining steps were performed using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, USA) according to the manufacturer's instructions. The number of senescent cells was quantified by measuring the cyan signal detected in the senescent cells using an optical microscope (Olympus, Japan). The control group (control in the figure) was treated with both H2O2 and the peptide. This is the untreated group.

[0085] As a result, as shown in Figures 4 and 5, when treated with H2O₂ alone ('-' in Figure 4), the proportion of SA-β-gal positive cells increased significantly by more than three times compared to the control group, indicating that the cell population had entered a state of significant senescence due to oxidative stress. When treated with the peptide of the present invention, the proportion of SA-β-gal positive cells decreased in a concentration-dependent manner, and it was confirmed that it showed excellent anti-aging activity by significantly inhibiting cell senescence induced by oxidative stress.

[0086] Experimental Example 4. Confirmation of Antioxidant Effect

[0087] It was confirmed through the DCFH-DA assay whether the peptide of the present invention inhibits intracellular ROS (Reactive Oxygen Species) generated after treating skin cells with H2O2. Specifically, 1 x 10⁶ 5Human keratinocytes (HaCaT) were seeded with medium (DMEM) and cultured for 24 hours in a 37°C, 5% CO2 incubator. Afterward, H2O2 and the peptide of the present invention were treated, respectively, and incubated for 2 hours under the same conditions. Cells in each well were washed with PBS, and the DCFDA Cellular ROS Detection Assay Kit (abcam, USA) was used according to the manufacturer's instructions.

[0088] Intracellular DCFH is oxidized by ROS generation to emit fluorescence; the detected fluorescence signal was measured using an optical microscope (Logosbio / CelenaS, Korea) and quantified using the program (NIS-Eliments-3.1). The control group (control in the figure) included both H2O2 and peptide This is the untreated group.

[0089] As a result, as shown in Figures 6 and 7, when treated with H2O2 alone ('-' in Figures 6 and 7), the DCF fluorescence intensity increased significantly compared to the control group, indicating that oxidative stress was induced. When treated with the peptide of the present invention, oxidative stress was reduced in a concentration-dependent manner, and it was confirmed that intracellular reactive oxygen species were effectively inhibited.

[0090] Experimental Example 5. Confirmation of anti-inflammatory effect

[0091] When an inflammatory response is induced by H2O2, the expression of IL-6 and TNF-alpha is promoted; therefore, Western blotting was used to confirm whether the peptide of the present invention inhibits the promotion of IL-6 and TNF-alpha expression. Specifically, HaCaT cells (human keratinocytes) were 5 x 10 per well. 6After dispensing the cells to the desired number, they were treated with H2O2 and the peptide of the present invention, and then incubated under the same conditions for 24 hours. The HaCaT cells were washed three times with PBS and then lysed with 1% NP40 lysis buffer (1% Nonidet P40, 0.1M NaCl, 0.05M tris (pH 8.0), 5M EDTA) containing 0.1μM PMSF (phenylmethylsulfonyl fluoride), 1μg / mL pepstatin A, 10μg / mL leupeptin, 1μg / mL aprotinin, and 1mM Na3VO4.

[0092] After quantifying the cell lysate using the Bradford assay method, samples were prepared for electrophoresis. The immunoprecipitates were subjected to electrophoresis on a 12.5% ​​polyacrylamide gel, and the separated proteins were transferred to a nitrocellulose membrane. The samples were then treated with a blocking solution (Tris-buffered saline, TBS) containing 0.05% Tween 20 and 5% bovine serum albumin for approximately 1 hour at room temperature. Subsequently, the samples were incubated for 2 hours in TBS buffer containing anti-IL-6 polyclonal antibody (Santa Cruz Biotechnology, USA) and anti-TNF-alpha polyclonal antibody (Cell Signaling Technology, USA), respectively, followed by washing with TBS buffer containing 0.05% Tween 20. After treatment with horseradish peroxidase-conjugated anti-rabbit or mouse IgG (Santa Cruz, CA, USA) for 1 hour at room temperature, the samples were washed 5 times with TBS containing 0.05% Tween 20. Development was performed using an antibody detection kit (Ab Frontier, Korea). To confirm the electrophoresis of equal volumes of cell lysates, actin was recognized using an anti-beta actin monoclonal antibody (Santa Cruz Biotechnology, USA). The expression levels of IL-6 and TNF-alpha relative to actin were quantified using the Image J program. The control group (control in the figure) included both H2O2 and peptides This is the untreated group.

[0093] As a result, as shown in Figures 8 and 9, when treated with H2O2 alone ('-' in Figures 8 and 9), the expression of IL-6 and TNF-alpha was significantly increased compared to the control group, inducing an inflammatory response.

[0094] When treated with the peptide of the present invention, the expression of IL-6 and TNF-alpha was inhibited in a concentration-dependent manner; in particular, TNF-alpha was normalized to the control level or lower, and IL-6 was also significantly reduced in a concentration-dependent manner, significantly alleviating overexpression. This demonstrates that the peptide of the present invention effectively blocks pro-inflammatory cytokine signals amplified by oxidative stress and can improve skin inflammation and the inflammatory aging environment.

[0095] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0096] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A peptide composed of any one of the amino acid sequences of sequence numbers 1 to 10.

2. In claim 1, the peptide is a peptide that activates AMP-activated protein kinase (AMPK).

3. The peptide of claim 1, wherein the peptide has one or more activities selected from the group consisting of anti-aging, antioxidant, anti-inflammatory, and wrinkle improvement.

4. A nucleic acid encoding a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

5. An anti-inflammatory composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

6. A cosmetic composition for skin improvement or anti-inflammatory purposes comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

7. A cosmetic composition according to claim 6, wherein the skin improvement is one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement.

8. A food composition for skin improvement or anti-inflammatory purposes, comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

9. A food composition according to claim 8, wherein the skin improvement is one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement.

10. A pharmaceutical composition for the prevention or treatment of AMPK-related diseases, comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

11. A pharmaceutical composition according to claim 10, wherein the AMPK-related disease is one or more selected from the group consisting of dermatitis, atopic dermatitis, psoriasis, acne, and keloid.

12. A method for cosmetic skin improvement comprising the step of applying to the skin a composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

13. A method for improving skin, comprising the step of administering to an individual a composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

14. A skin improvement method according to claim 13, wherein the skin improvement is one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement.

15. A method for preventing or treating an AMPK-related disease, comprising the step of administering to an individual a composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

16. A method for the prevention or treatment of an AMPK-related disease according to claim 15, wherein the AMPK-related disease is one or more selected from the group consisting of dermatitis, atopic dermatitis, psoriasis, acne, and keloid.

17. A skin improvement use comprising the step of administering to an individual a composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

18. In paragraph 17, the skin improvement use is one or more selected from the group consisting of anti-aging, antioxidant, and wrinkle improvement.

19. Use for the prevention or treatment of AMPK-related diseases, comprising the step of administering to an individual a composition comprising a peptide composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

20. Use for the prevention or treatment of AMPK-related diseases according to claim 19, wherein the AMPK-related disease is one or more selected from the group consisting of dermatitis, atopic dermatitis, psoriasis, acne, and keloid.

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