Novel senescence treatment peptide and composition comprising same

Abstract

The present disclosure relates to a novel peptide for senescence treatment and a use thereof. Specifically, the peptide according to the present disclosure has a binding force with p53 superior to FOXO4 and inhibits the intracellular interaction between p53 and FOXO4, thereby enabling p53 to be released outside the nucleus and moved to mitochondria, inducing apoptosis of aged cells, and selectively inducing apoptosis of aged cells in skin cells.

Description

Technical Field

[0001] The present disclosure relates to a novel aging therapeutic peptide and compositions comprising the same. Background Art

[0002] It is well known that cell aging not only contributes to tissue aging, but also plays an important role in causing diseases related to weakened tissue function. As aging progresses, the number of senescent cells with weakened functions increases, and the increased senescent cells secrete inflammatory factors called senescence-associated secretory factors (senescence-associated secretory phenotype, SASP), thereby causing tissue inflammation and disease.

[0003] Recently, there have been reports that senotherapeutics such as senolytics that selectively remove senescent cells or senomorphics that restore the functions of senescent cells to normal and inhibit the secretion of SASP can inhibit tissue aging, thereby potentially extending lifespan or treating age-related diseases. As a result, attention has focused on the discovery of more effective and safe senotherapeutics.

[0004] The development of such substances can be widely used in inhibiting aging, preventing the decline of aging-related functions, treating senile diseases, etc., and is therefore considered the most important goal in the development of substances for treating aging. Recently, reports have shown that various compounds can be used to improve aging tissues and treat some diseases, and for this reason, attention to aging therapeutic agents including senescent cell lysate preparations has further increased.

[0005] However, the substances reported so far are drugs discovered by screening using existing compound libraries, and are already known as anticancer agents or have been used as other drugs in the past. When administered to humans for a long time, they may cause side effects and are restricted, or as substances with strong antioxidant effects, no substances have been developed to investigate the direct aging treatment effects and mechanisms. Summary of the invention

[0006] Technical issues

[0007] The present disclosure aims to provide a novel aging therapeutic peptide that induces apoptosis of skin aging cells by inhibiting the interaction between p53 and FOXO4 (Forkhead box protein 4).

[0008] Technical Solution

[0009] To achieve the above object, one embodiment of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0010] Another embodiment of the present invention provides a composition for improving skin wrinkles comprising a peptide including the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0011] Another embodiment of the present invention provides a composition for treating aging comprising a peptide including the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0012] Effects of the Invention

[0013] The peptide disclosed herein has a better binding affinity with p53 than FOXO4 and inhibits the intranuclear interaction between p53 and FOXO4, thereby allowing p53 to be released outside the nucleus and move to the mitochondria, inducing apoptosis of aged cells, and can selectively induce apoptosis of aged cells in skin cells.

[0014] In addition, a composition containing the peptide according to the present disclosure as an active ingredient can effectively improve skin wrinkles due to its excellent MMP-1 inhibitory activity.

[0015] In addition, the composition containing the peptide according to the present disclosure as an active ingredient can have a significant effect of selectively causing apoptosis of aging cells in skin cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1a , Figure 1b and Figure 1c This is a diagram showing the results of confirming that FOXO4 expression increases in skin aging cells;

[0017] Figure 2 This is a diagram showing the results of confirming the efficacy of the peptide according to one embodiment of the present invention on apoptosis of skin aging cells;

[0018] Figure 3a and Figure 3b This is a diagram showing the results of confirming the apoptotic effect of the peptide according to one embodiment of the present invention on skin aging cells by B-gal staining and microscopic observation;

[0019] Figure 4 is a diagram showing the results of observing the movement of p53 in the cytoplasm based on the peptide according to one embodiment of the present invention;

[0020] Figure 5 It is a graph showing the results of confirmation of the MMP-1 inhibitory activity of the peptide according to an embodiment of the present invention. DETAILED DESCRIPTION

[0021] Hereinafter, the present invention will be described in detail.

[0022] In one aspect, the present invention relates to a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0023] In another aspect, the present invention relates to a composition for improving skin wrinkles, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0024] In another aspect, the present invention relates to a method for improving skin wrinkles, comprising the step of administering an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject in need thereof.

[0025] In another aspect, the present invention relates to use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for preparing a composition for improving skin wrinkles.

[0026] In another aspect, the present invention relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1 for use in improving skin wrinkles.

[0027] In still another aspect, the present invention relates to a composition for inducing apoptosis of aging skin cells, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an effective ingredient.

[0028] In another aspect, the present invention relates to a method for inducing apoptosis of aging skin cells, comprising the step of administering an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject in need thereof.

[0029] In another aspect, the present invention relates to use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for preparing a composition for inducing apoptosis of skin aging cells.

[0030] In another aspect, the present invention relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1 for use in inducing apoptosis in aging skin cells.

[0031] In still another aspect, the present invention relates to a composition for treating aging, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0032] In another aspect, the present invention relates to a method for treating aging, comprising the step of administering an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject in need thereof.

[0033] In another aspect, the present invention relates to use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for preparing a composition for treating aging.

[0034] In yet another aspect, the present invention relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1 for use in the treatment of aging.

[0035] In one embodiment, the senotherapy may include inducing apoptosis in senescent skin cells.

[0036] In one embodiment, the peptide may include the amino acid sequence of AYSQNGWANRRRRGGRRRRRR (SEQ ID NO: 1).

[0037] In one embodiment, the peptide may consist of an amino acid sequence of AYSQNGWANRRRRGGRRRRRR (SEQ ID NO: 1).

[0038] In one embodiment, the peptide can inhibit the activity of MMP-1.

[0039] In one embodiment, the composition for improving skin wrinkles may contain the peptide in an amount of 0.001 to 1% by weight.

[0040] Specifically, the peptide may be contained in the skin wrinkle improving composition in an amount of 0.001% by weight or more, 0.002% by weight or more, 0.003% by weight or more, 0.004% by weight or more, 0.005% by weight or more, 0.006% by weight or more, 0.007% by weight or more, 0.008% by weight or more, 0.009% by weight or more, 0.01% by weight or more, 0.02% by weight or more, 0.03% by weight or more, 0.04% by weight or more, 0.05% by weight or more, 0.06% by weight or more, 0.07% by weight or more, 0.08% by weight or more, 0.09% by weight or more, 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight or more, 0.4% by weight or more, 0.5% by weight or more, 0.6% by weight or more, 0.7% by weight or more, 0.8% by weight or more, or 0.9% by weight or more. Furthermore, the peptide may be contained in the skin wrinkle improving composition in an amount of 1% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, 0.6% by weight or less, 0.5% by weight or less, 0.4% by weight or less, 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.09% by weight or less, 0.08% by weight or less, 0.07% by weight or less, 0.06% by weight or less, 0.05% by weight or less, 0.04% by weight or less, 0.03% by weight or less, 0.02% by weight or less, 0.01% by weight or less, 0.009% by weight or less, 0.008% by weight or less, 0.007% by weight or less, 0.006% by weight or less, 0.005% by weight or less, 0.004% by weight or less, 0.003% by weight or less, or 0.002% by weight or less.

[0041] In one embodiment, the peptide may be contained in the composition for treating aging or the composition for inducing apoptosis of skin aging cells in an amount of 0.001 to 1% by weight.

[0042] Specifically, the peptide may be contained in the aging treatment composition or the composition for inducing apoptosis of skin aging cells in an amount of 0.001% by weight or more, 0.002% by weight or more, 0.003% by weight or more, 0.004% by weight or more, 0.005% by weight or more, 0.006% by weight or more, 0.007% by weight or more, 0.008% by weight or more, 0.009% by weight or more, 0.01% by weight or more, 0.02% by weight or more, 0.03% by weight or more, 0.04% by weight or more, 0.05% by weight or more, 0.06% by weight or more, 0.07% by weight or more, 0.08% by weight or more, 0.09% by weight or more, 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight or more, 0.4% by weight or more, 0.5% by weight or more, 0.6% by weight or more, 0.7% by weight or more, 0.8% by weight or more, or 0.9% by weight or more. Furthermore, the peptide may be contained in the aging treatment composition or the composition for inducing apoptosis of skin aging cells in an amount of 1% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, 0.6% by weight or less, 0.5% by weight or less, 0.4% by weight or less, 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.09% by weight or less, 0.08% by weight or less, 0.07% by weight or less, 0.06% by weight or less, 0.05% by weight or less, 0.04% by weight or less, 0.03% by weight or less, 0.02% by weight or less, 0.01% by weight or less, 0.009% by weight or less, 0.008% by weight or less, 0.007% by weight or less, 0.006% by weight or less, 0.005% by weight or less, 0.004% by weight or less, 0.003% by weight or less, or 0.002% by weight or less.

[0043] In one embodiment, the composition may be a cosmetic or a non-therapeutic oral composition. Specifically, the composition may be a cosmetic composition for improving skin wrinkles, a non-therapeutic oral composition for improving skin wrinkles, a cosmetic composition for treating aging, a non-therapeutic oral composition for treating aging, a cosmetic composition for inducing apoptosis of aged skin cells, or a non-therapeutic oral composition for inducing apoptosis of aged skin cells.

[0044] The dosage form of the non-therapeutic oral composition is not particularly limited, and can be, for example, in the form of tablets, granules, pills, powders, liquids such as beverages, caramels, gels, sticks, tea bags, etc. For each dosage form of the non-therapeutic oral composition, in addition to the active ingredient, those skilled in the art can easily select and prepare the ingredients commonly used in the art according to the dosage form or purpose of use, which can produce a synergistic effect when used simultaneously with other raw materials.

[0045] The composition can be administered by various methods such as simple ingestion, drinking, injection, spraying or squeezing.

[0046] According to one embodiment of the present invention, the non-therapeutic oral composition can be, for example, various foods including chewing gum, caramel products, sugars, ice cream, biscuits, etc., beverage products such as soft drinks, mineral water, alcoholic beverages, vitamins or minerals, etc.

[0047] In the non-therapeutic oral composition according to one embodiment of the present invention, in addition to its active ingredients, additives may be included. Such additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, thickeners, etc. according to their functions.

[0048] Sweeteners are used to impart a suitable sweet taste, and both natural sweeteners and synthetic sweeteners can be used in the non-therapeutic oral composition according to one aspect of the present invention. Natural sweeteners are preferably used, and as natural sweeteners, sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose can be cited.

[0049] Flavoring agents are used to enhance flavor or fragrance, and both natural flavoring agents and synthetic flavoring agents can be used. Natural flavoring agents are preferably used. When natural flavoring agents are used, in addition to flavor, the purpose of increasing nutrition can also be achieved at the same time. As natural flavoring agents, they can be obtained from apples, lemons, citrus, grapes, strawberries, peaches, etc., or from green tea leaves, wilting, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. In addition, natural flavoring agents obtained from ginseng (red ginseng), bamboo shoots, aloe, ginkgo, etc. can be used. Natural flavoring agents can be liquid concentrates or solid extracts. Depending on the situation, synthetic flavoring agents can be used. As synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, etc. can be used.

[0050] As preservatives, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, calcium benzoate, EDTA (ethylenediaminetetraacetic acid) etc. can be used. In addition, as emulsifiers, gum arabic, carboxymethyl cellulose, xanthan gum, pectin etc. can be used. As acidulants, citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid etc. can be used. In addition to the purpose of improving taste, as the purpose of inhibiting the proliferation of microorganisms, acidulants can be added so that the non-therapeutic oral composition maintains appropriate acidity. As tackifiers, suspending agents, precipitants, gel forming agents, swelling agents etc. can be used.

[0051] The non-therapeutic oral composition according to one embodiment of the present invention may include, in addition to the additives described above, physiologically active substances or minerals with guaranteed stability as additives known in the art for the purpose of supplementing / enhancing functionality and nutrition.

[0052] Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, vitamin B12, tocopherol, dibenzoylthiamine, etc.; examples of minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferric citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc.

[0053] In the non-therapeutic oral composition according to one embodiment of the present invention, the aforementioned additives may be contained in appropriate amounts according to the product type so as to achieve the purpose of addition.

[0054] The cosmetic composition according to one embodiment of the present invention may contain a medium or matrix acceptable in cosmetics or dermatology. As a full dosage form suitable for topical use, it can be provided in the following forms, such as in the form of a solution, gel, solid, pasty anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a fine particle ball, or an ionic (liposome) and non-ionic vesicle dispersant, or a cream, a lotion, a skin lotion, a powder, an ointment, a spray or a concealer stick. These compositions can be manufactured according to conventional methods in the art. In addition, the cosmetic composition can also be used in the form of an aerosol composition further containing a propellant compressed in the form of foam.

[0055] The dosage form of the cosmetic composition is not particularly limited and can be appropriately selected according to the purpose. For example, it can be prepared into dosage forms such as skin care lotion, skin softener, toner, lotion, milky lotion, moisturizing lotion, nourishing lotion, massage cream, nutrient cream, moisturizing cream, hand cream, foundation, essence, nourishing essence, facial mask, soap, facial foam, facial cleanser, facial cream, facial water, powder, moisturizing lotion, moisturizing cream, moisturizing oil, body cleanser and moisturizing essence.

[0056] When the dosage form of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicon, bentonite, silicon dioxide, talc or zinc oxide can be used as a carrier component.

[0057] When the dosage form of the present invention is a powder or a spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component. In particular, when the dosage form is a spray, a propellant such as chlorofluorocarbons, propane / butane or dimethyl ether can be further included.

[0058] When the dosage form of the present invention is a solution or an emulsion, a solvent, a solubilizer or an emulsifier is used as a 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 sorbitan fatty acid ester.

[0059] When the dosage form of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth gum can be used as a carrier component.

[0060] When the dosage form of the present invention is a surfactant-containing cleanser, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazoline derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glyceryl fatty acid esters, etc. can be used as carrier components.

[0061] In addition to the peptide, the cosmetic composition may also contain functional additives and ingredients contained in general cosmetic compositions. As the functional additives, the ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular polysaccharides, sphingolipids and seaweed juice may be included.

[0062] In addition to the functional additives, the composition may further contain ingredients contained in a general cosmetic composition as required. Examples of the ingredients contained in addition to the functional additives include oil components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH regulators, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, and the like.

[0063] The present invention can provide the following embodiments as one embodiment.

[0064] In a first embodiment, a peptide comprising the amino acid sequence of SEQ ID NO: 1 can be provided.

[0065] A second embodiment provides a peptide, in the first embodiment, wherein the peptide consists of the amino acid sequence of SEQ ID NO:1.

[0066] A third embodiment provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for preparing a composition for improving skin wrinkles.

[0067] A fourth embodiment provides a use according to the third embodiment, wherein the peptide consists of an amino acid sequence of SEQ ID NO:1.

[0068] A fifth embodiment provides the use according to any one of the third and fourth embodiments, wherein the peptide inhibits MMP-1 activity.

[0069] A sixth embodiment provides a use according to any one of the third to fifth embodiments, wherein the peptide is contained in an amount of 0.001 to 1% by weight.

[0070] A seventh embodiment provides a use according to any one of the third embodiment to the sixth embodiment, wherein the composition is a cosmetic or a non-therapeutic oral composition.

[0071] An eighth embodiment provides a use of a peptide comprising the amino acid sequence of SE Q ID NO: 1 for preparing a composition for treating aging.

[0072] A ninth embodiment provides a use according to the eighth embodiment, wherein the peptide consists of an amino acid sequence of SEQ ID NO:1.

[0073] A tenth embodiment provides the use in any one of the eighth embodiment and the ninth embodiment, wherein the peptide is contained in an amount of 0.001 to 1% by weight.

[0074] An eleventh embodiment provides a use according to any one of the eighth to tenth embodiments, wherein the composition is a cosmetic or a non-therapeutic oral composition.

[0075] Below, the content of the present invention is further described in detail by embodiments and test examples. However, these embodiments and test examples are only provided for understanding the content of the present invention, and the scope of rights of the present invention is not limited by these embodiments and test examples, and deformation, substitution and insertion etc. commonly known in the art can be carried out, and these are also included in the scope of the present invention.

[0076] [Test Example 1] Confirmation of FOXO4 expression in senescent cells

[0077] In order to confirm whether FOXO4 expression also increases in aged skin cells, immunofluorescence (IF) and polymerase chain reaction (PCR) experiments were performed to confirm whether FOXO4 is expressed in normal skin fibroblasts and aged skin fibroblasts. Aged skin fibroblasts were prepared by treating normal skin fibroblasts with doxorubicin (Dox) and insulin-like growth factor-1 (IGF-1) to induce early aging (DI model) and by inducing aging by continuous subculture of skin fibroblasts (RS model).

[0078] Specifically, in the DI model, normal human dermal fibroblasts (Normal human dermal fibroblasts, Lonza, Switzerland) were cultured in DMEM medium (DMEM Medium, Lonza, Switzerland) containing 10% FBS, and then 1×10 3 The number of cells was inoculated and cultured at 37°C in a 5% CO2 medium for 24 hours. Doxorubicin hydrochloride (Sigma) and IGF-1 (Sigma) were treated at a concentration of 100 ng / ml for four days to the cultured wells to induce early aging of skin fibroblasts. As a control group, normal skin fibroblasts that were not treated with Dox and IGF-1 were prepared. Then, for immunofluorescence, the cell culture medium was washed 3 times with PBS and fixed in 4% paraformaldehyde for 30 minutes. Then, the skin fibroblasts were washed 3 times with PBS containing 0.2% Triton X-100, added to PBS containing 3% BSA, and left for 1 hour. Next, the sample was incubated in 1% BSA containing rabbit anti-FOXO4 polyclonal antibody (SAB4501887, Sigma) (diluted 1:500) at 4°C overnight to allow the antibody to bind to the antigen contained in the skin fibroblasts. The sample was then washed three times with PBS and incubated with Alexa Fluor. After incubation at room temperature for 1 hour, the cells were washed three times with PBS and 488-labeled goat polyclonal anti-rabbit IgG-H&L secondary antibody (A32731, ThermoFisher Scientific) (diluted at 1:1000). Next, a drop of DAPI-containing mounting solution was dripped onto a microscope slide, and the cover glass was placed on the slide for fixation. The fixed samples were observed using a confocal microscope (LSM 980, Zeiss, Germany). Figure 1a The results are shown in .

[0079] In the RS model, skin fibroblasts (Normal human dermal fibroblasts, Lonza, Switzerland) were cultured for more than 30-40 times in DMEM medium (DMEM Medium, Lonza, Switzerland) containing 10% FBS to prepare aged skin fibroblasts. For immunofluorescence assay, chamber slides (Thermo Fisher Scientific, USA) were plated with 1×10 3 Aged skin fibroblasts p41 and normal skin fibroblasts were inoculated with cells and cultured in a 5% CO2 medium at 37°C for 48 hours. Then, the cell culture medium was washed three times with PBS and fixed in 4% paraformaldehyde for 30 minutes. Next, the skin fibroblasts were washed three times with PBS containing 0.2% Triton X-100, added to PBS containing 3% BSA, and left for 1 hour. Next, the sample was incubated overnight at 4°C in 1% BSA containing a rabbit anti-FOXO4 polyclonal antibody (SAB4501887, Sigma) (diluted 1:500) to allow the antibody to bind to the antigen contained in the skin fibroblasts. Then, the sample was washed three times with PBS and incubated with Alexa Fluor. After incubation at room temperature for 1 hour, the cells were washed three times with PBS and 488-labeled goat polyclonal anti-rabbit IgG-H&L secondary antibody (A32731, ThermoFisher Scientific) (diluted at 1:1000). Next, a drop of DAPI-containing mounting solution was dripped onto a microscope slide, and the cover glass was placed on the slide for fixation. The fixed samples were observed using a confocal microscope (LSM 980, Zeiss, Germany). Figure 1b The results are shown in .

[0080] Furthermore, the expression level of FOXO4 was confirmed by PCR test according to the number of subcultures of skin fibroblasts. 6 Skin fibroblasts that had been subcultured for 6, 35, and 42 times were inoculated with the number of cells, and cultured at 37°C in a 5% CO2 medium for 48 hours. The cell culture medium was then washed three times with PBS and TRIzol solution (TRIzol TM Reagent, Invitrogen) extraction method to isolate total RNA (total RNA). Using 1ug of isolated RNA, after synthesizing into cDNA by reverse transcriptase (RevertAid Firist strand cDNA synthesis kit, Thermofisher), dilute it to a final concentration of 200ul. In order to perform PCR, mix 1ul of FOXO4 primer (Hs00172973_m1, ThermoFisher Scientific), 10ul of TaqManUniversal premix II (Taqman universal master mix II with UNG, AppliedBiosystems), and 5ul of distilled water, and finally make a mixed kit diluted to 16ul. Mix 16ul of the mixed kit and 4ul of cDNA, and perform qPCR under the following reaction conditions. The reaction conditions were set at 95°C, initialization reaction for 10 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 60 seconds, extension reaction at 50°C for 20 seconds, and repeated 40 times in a PCR device (7500Fast Real-time PCR system, Applied Biosystems). The GAPDH gene was corrected to indicate the expression level of the targeted gene, and Figure 1c The results are shown in .

[0081] like Figure 1a , Figure 1b and Figure 1c As shown in Figure 2, it was confirmed that FOXO4 expression increased in aged skin fibroblast models (DI (Doxorubicin / IGF-1) treatment model and RS (replicative senescence) model). Figure 1c The results showed that the expression of FOXO4 increased significantly in the RS model from passage 35 to passage 42.

[0082] [Test Example 2] Confirmation of the effect of selective apoptosis of skin aging cells

[0083] As shown in Experimental Example 1, skin fibroblasts were treated with doxorubicin and IGF-1 at a concentration of 100 ng / ml for four days to induce aging (DI model), or skin fibroblasts were cultured to passage 40 to obtain aged fibroblasts (RS model), and then these aged fibroblasts were treated with peptide 21a-D consisting of the amino acid sequence of AYSQNGWANRRRRGGRRRRRR (SEQ ID NO: 1), peptide 22a-L consisting of the amino acid sequence of QRKKRRQRRRPRRNAWG NQSYA (SEQ ID NO: 2), peptide 22a-D consisting of the amino acid sequence of AYSQNGWANRRPRRRQRRKKRG (SEQ ID NO: 3) and peptide FOXO4-DRI consisting of the amino acid sequence of LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG (SEQ ID NO: 4) at a concentration of 100 ug / ml for three days, and the survival rate of the aged cells was measured. The survival rate of aged cells was determined using a CCK-8 quantitative kit (cell counting kit-8, Dojindo) according to the proposed protocol. Specifically, after treating DMEM medium diluted 1 / 100 with CCK-8, the absorbance was measured at 450 nm using a microplate reader (spectrostar nano, BMG labtech) after 1 hour. Figure 2 The results are shown in .

[0084] like Figure 2 As shown, it was confirmed that when the peptide 21a-D having the amino acid sequence of SEQ ID NO: 1 according to one embodiment of the present invention was treated, only the apoptosis of skin aged cells was selectively induced. It was confirmed that when the peptides 22a-L and 22a-D having the amino acid sequences of SEQ ID NO: 2 and 3 were treated, the apoptosis effect on aged cells was weak, and when the peptide FOXO4-DRI having the amino acid sequence of SEQ ID NO: 4 was treated, although the apoptosis effect on aged cells was confirmed, it also had an apoptosis effect on normal cells, and thus it was confirmed that the selective apoptosis effect on aged cells was weak. Thus, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 according to one embodiment of the present invention showed a selective apoptosis effect on skin aged cells.

[0085] [Test Example 3] Macroscopic observation of the selective apoptosis effect on skin aging cells

[0086] Using the same method as in Experimental Example 2, peptide 21a-D consisting of the amino acid sequence of AYSQNGWANRRRRGGRRRRRR (SEQ ID NO: 1) and peptide FOXO4-DRI consisting of the amino acid sequence of LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG (SEQ ID NO: 4) were treated to the DI model and RS model, and then SA-β-gal (Senescence-Associated β-galactosidase) staining and microscopic observation were performed to confirm the apoptotic effect of skin aging cells by naked eyes. SA-β-gal staining was performed using the Senescence Cell Histochemical Staining Kit (Senescence Cell Histochemical Staining Kit, CS0030, Sigma, USA) and stained according to the protocol. After 24 hours of treatment, it was confirmed by optical microscopy whether the cells were stained. Figure 3a and Figure 3b The results are shown in .

[0087] like Figure 3a and Figure 3b As shown, it was confirmed that when the peptide having the amino acid sequence of SEQ ID NO: 1 according to one embodiment of the present invention was treated, the number of senescent cells was significantly reduced.

[0088] [Test Example 4] Observation of cytoplasmic migration of p53

[0089] When FOXO4 (Forkhead box protein 4) is exposed to external stimuli such as ultraviolet rays, it binds to p53 at the site of DNA damage, thereby causing cell senescence through the expression of p21, a cell senescence regulatory factor. When FOXO4-p53 binding is inhibited, p53 moves to the mitochondria and induces the release of cytochrome c, causing apoptosis of senescent cells.

[0090] An immunofluorescence (IF) experiment was performed to confirm whether such a phenomenon was observed when the peptide having the amino acid sequence of SEQ ID NO: 1 according to one embodiment of the present invention was treated with skin aging cells. Using the same method as in Experimental Example 2, peptide 21a-D was treated with DI (Doxorubicin / IGF-1) to aging skin fibroblasts, and the expression and cytoplasmic migration of FOXO4 and p53 were observed.

[0091] Specifically, 1×10 3 Skin fibroblasts were inoculated with 100 ng / ml of Dox and IGF-1 for four days in a 5% CO2 medium, and cultured for 24 hours at 37°C. After inducing early senescence of skin fibroblasts, peptide 21a-D was treated at a concentration of 100 ug / ml for three days in the cultured wells. For immunofluorescence, the cell culture solution was washed three times with PBS and fixed with ice-cold methanol at -20°C for 10 minutes. Then, it was added to PBS containing 3% BSA and left for 1 hour. Then, the sample was incubated overnight at 4°C in 1% BSA containing rabbit anti-FOXO 4 polyclonal antibody (SAB4501887, Sigma, USA) (diluted at 1:500) and mouse anti-p53 antibody (#9286, Cell signaling technology, USA), so that the antibody binds to the antigen contained in the skin fibroblasts. Then, the samples were washed three times with PBS and 488-labeled goat polyclonal anti-rabbit IgG-H&L secondary antibody (A32731, ThermoFisher Scientific, USA) (diluted 1:1000) and Alexa After incubation at room temperature for 1 hour with goat polyclonal anti-mouse IgG-H&L (A11001, ThermoFisher, USA) labeled with 488, the samples were washed three times with PBS. Next, a drop of mounting solution containing DAPI was dripped onto a microscope slide, and the cover glass was placed on the slide for fixation. The fixed samples were observed using a confocal microscope (LSM 980, Zeiss, Germany). Figure 4 The results are shown in .

[0092] like Figure 4 As shown, it can be confirmed that the expression of FOXO4 and p53 in the nucleus of skin aging cells increases by DI treatment, and it can be confirmed that p53 moves from the nucleus to the cytoplasm by treatment with peptide 21a-D, thereby inhibiting the binding with FOXO4. Thus, it can be confirmed that when the peptide having the amino acid sequence of SEQ ID NO: 1 according to one embodiment of the present invention is treated, the binding of p53 and FOXO4 in the nucleus is inhibited, thereby inducing apoptosis of skin aging cells.

[0093] [Test Example 5] Confirmation of MMP-1 inhibitory activity

[0094] Using the same method as in Experiment 2, peptide 21a-D consisting of the amino acid sequence of AYSQNGWANRRRRGGRRRRRR (SEQ ID NO: 1) and peptide FOXO4-DRI consisting of the amino acid sequence of LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG (SEQ ID NO: 4) were treated with the DI model, and the ability to inhibit the expression of MMP-1 in aged cells was confirmed. After the cultured cells were collected, Trizol solution (TRIzol TM Total RNA was isolated by RT-PCR (RT-PCR) extraction with 1 μg of RNA. After being synthesized into cDNA by reverse transcriptase (RevertAid Firist strand cDNA synthesis kit, Thermofisher), the final concentration was diluted to 200 μl. In order to perform PCR, 1 μl of MMP-1 primer (Hs00899658_m1, ThermoFisher Scientific), 10 μl of Taq Man Universal premix II (Taqman universalmaster mix II with UNG, Applied Biosystems), and 5 μl of distilled water were mixed to produce a mixed kit diluted to 16 μl. 16 μl of the mixed kit and 4 μl of cDNA were mixed and qPCR was performed under the following reaction conditions. The reaction conditions were set at 95°C, initialization reaction for 10 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 60 seconds, extension reaction at 50°C for 20 seconds, and repeated 40 times in a PCR device (7500Fast Real-time PCR system, AppliedBiosystems). The GAPDH gene was corrected to indicate the expression level of the targeted gene, and Figure 5 The results are shown in .

[0095] like Figure 5 As shown, it was confirmed that the expression of MMP-1 increased in skin aging cells by DI treatment, and it was confirmed that when the peptide 21a-D having the amino acid sequence of SEQ ID NO: 1 according to one embodiment of the present invention was treated, the expression of MMP-1 was inhibited.

Claims

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

1.

2. The peptide according to claim 1, wherein: The peptide consists of the amino acid sequence of SEQ ID NO:

1.

3. Use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for preparing a composition for improving skin wrinkles.

4. The use according to claim 3, wherein: The peptide consists of the amino acid sequence of SEQ ID NO:

1.

5. The use according to claim 3, wherein: The peptide inhibits MMP-1 activity.

6. The use according to claim 3, wherein: The peptide is contained in an amount of 0.001 to 1% by weight.

7. The use according to claim 3, wherein: The composition is a cosmetic or a non-therapeutic oral composition.

8. Use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for preparing a composition for treating aging.

9. The use according to claim 8, wherein: The peptide consists of the amino acid sequence of SEQ ID NO:

1.

10. The use according to claim 8, wherein: The peptide is contained in an amount of 0.001 to 1% by weight.

11. The use according to claim 8, wherein: The composition is a cosmetic or a non-therapeutic oral composition.

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